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CLONING AND CHARACTERIZATION OF A NOVEL
KELCH-LIKE GENE IN ZEBRAFISH
WU YI LIAN
NATIONAL UNIVERSITY OF SINGAPORE
2003
CLONING AND CHARACTERIZATION OF A NOVEL
KELCH-LIKE GENE IN ZEBRAFISH
BY
WU YI LIAN (BSc. Hons)
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
DEPARTMENT OF BIOLOGICAL SCIENCES
NATIONAL UNIVERSITY OF SINGAPORE
2003
Acknowledgements
ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my supervisor, A/P Gong Zhiyuan, for his
invaluable guidance, unwavering patience and mentorship in the course of my research. I
am especially grateful for the many opportunities that has been given to me to explore in
both the research and management fields, that has made my experience in the lab a
enriching and rewarding one.
I am also thankful to past and present members of the laboratory, Chen Mingru, Ju
Bensheng, Ke Zhiyuan, Kee Peck Wai, Liu Xingjun, Pan Xiufang, Safia SR, Shan
Tao, Simon Lim, Sudha PM, Tay Tuan Leng, Tong Yan, Wan Haiyan, Wang Hai,
Wang Xukun, Yan Tie, Zeng Sheng, Zeng Zhiqiang for their invaluable advice and
help. Life-long friendships have been forged even though we’re no longer working
together and I enjoy our little get-togethers every few months.
I also want to thank Aaron, Ka-leng, Sandra Tan, Chen Sufen, Jacqueline Tan for your
friendship and all the laughter that we’ve shared. Especially to Ka-Leng, for always
providing a listening ear, even when you’re miles away. Special thanks also goes to
Sandra, my first “Shifu” in the laboratory, for all the patience and guidance over the years.
Special thanks also goes to Lay Hua, for her support and all the great times spent.
To my parents, thank you for your unconditional love and support in all the decisions and
paths that I have chosen to take in my life. For your prayers and also for always reminding
me to look to the Lord Jesus.
Most of all, I am eternally grateful to God, without whom nothing would be possible. For
all the many blessings in my life, and for being my unfailing source of strength and help
and hope throughout these years and this thesis.
i
Table of Contents
TABLE OF CONTENTS
Acknowledgements
i
Table of Contents
ii
List of Figures and Tables
iv
List of Abbreviations
v
Summary
vii
1
Chapter I. Introduction
1. Beyond the Genome: Turning Data into Knowledge
1.1 The Human Genome Unveiled
1.2 Gene Annotation
1.3 Comparative Genomics
1.4 Expressed Sequence Tags (ESTs) and In Silico Analysis
1.5 Generation of Functional Data using Model Organisms
2. Zebrafish in the Context of the Human Genome Project
2.1 Zebrafish as an Experimental System
2.2 Mutagenesis Screens
2.3 Genomic Infrastructure
2.4 The Syntenic Relationship of the Zebrafish and Human Genomes
2.5 Experimental Tractability
2.6 Zebrafish: From Disease Modelling to Drug Discovery
3. Rationale of the Project
2
2
2
4
8
11
13
13
14
19
21
24
26
28
Chapter II. Materials and Methods
30
1. Cloning of Full Length Zebrafish klhl cDNA
1.1 Rapid Amplification of cDNA Ends (RACE)-PCR
1.2 Recovery of DNA Fragments from Agarose Gel
1.3 Ligation
1.4 Transformation
1.4.1 Preparation of Competent Cells
1.4.2 Transformation
1.5 Colony Screening
1.6 Isolation and Purification of Plasmid DNA
1.7 Automated Sequencing
1.8 Sequence Homology Search
2. Characterization of Zebrafish klhl Expression
2.1 Northern Hybridisation
2.1.1 Isolation of Total RNA
2.1.2 Formaldehyde RNA Gel Electrophoresis and Blotting
31
31
33
33
35
35
35
36
36
37
38
ii
39
39
39
39
Table of Contents
2.1.3 Labelling of Radioactive Probe
2.1.4 Hybridisation
2.1.5 Washes and Autoradiography
2.1.6 Membrane Stripping
2.2 Whole-Mount In Situ Hybridisation on Zebrafish Embryos
2.2.1 Probe synthesis
2.2.2 Preparation of staged zebrafish embryos
2.2.3 In Situ Hybridisation
2.2.4 Incubation with Preabsorbed Antibodies
2.2.5 Staining
2.2.6 Mounting and photography
2.3 Two-Colour Whole Mount In Situ Hybridisation
3. Characterization of Human Ortholog KLHL
3.1 Identification of Human Orthologous Gene KLHL
3.2 Cloning of KLHL Fragment
3.3 Northern Blot Analysis
40
41
42
43
43
43
44
45
45
46
46
47
49
49
49
49
Chapter III. Results
51
1. Identification of ES34 as a Putative Kelch Repeat Protein
2. Molecular Cloning of Zebrafish klhl
3. Sequence Analysis of Zebrafish klhl
4. klhl is Conserved Across Zebrafish, Human, Mouse and Rat
5. Genome Mapping of klhl
6. Developmental Accumulation of klhl
7. Tissue Distribution Analysis of klhl in Adult Zebrafish
8. Expression of klhl is Similar in Human, Rat and Zebrafish
9. Ontogenetic Expression of klhl during Somitogenesis
10. Expression of klhl in Fast and Slow Muscle
11. Expression of klhl during Cardiac Morphogenesis
12. Expression of klhl in Cranial Muscle Development
52
53
53
59
62
67
69
69
72
77
79
79
Chapter IV. Discussion
84
1. Zebrafish as a Model for Vertebrate Biology
2. klhl is a Member of the Kelch Family of Proteins
3. klhl is Expressed in the Somites and Cardiac Muscles
4. Role of klhl in Muscle Structure and Function
5. Comparative Genomics, a Look into Evolutionary History
6. Rapid In Silico Cloning of Genes
7. Future Directions
85
86
88
90
93
95
97
References
99
iii
List of figures and tables
LIST OF FIGURES AND TABLES
Fig. 1
Map of pBK-CMV vector
32
Fig. 2
Map of pT7Blue vector
34
Fig. 3
Nucleotide and predicted amino acid sequence of zebrafish klhl cDNA
54
Fig. 4
Alignment of the kelch repeats of zebrafish klhl and human KLHL
58
Fig. 5
Amino acid sequence alignment of zebrafish klhl, Fugu klhl, human
60
KLHL, mouse (m) Klhl and rat ® Klhl proteins
Fig. 6
Genome mapping of klhl
63
Fig. 7
Expression of klhl in developing zebrafish embryos in comparison to
68
two other MSP genes, tpma and mylz2
Fig. 8
Tissue distribution of klhl mRNAs in comparison with tpma and mylz2
70
mRNAs in adult zebrafish
Fig. 9
Northern blot analysis of KLHL mRNA in human tissues
71
Fig. 10
Expression of klhl and tpma in zebrafish embryos
74
Fig. 11
Ontogenetic expression of klhl, tpma and mylz2 during the various
76
stages of somitogenesis
Fig. 12
Comparison of expression of klhl, tpma, desmin and smbpc in 36 hpf
78
embryos
Fig. 13
klhl expression during cardiac morphogenesis
80
Fig. 14
Localization of klhl transcripts in 72 hp embryos
83
Fig. 15
A schematic overview of cytoskeletal linkages in striated muscle
90
Fig. 16
Schematic model of the cytoskeletal filament linkages at the
92
sacrolemma of striated muscle
Table 1
Summary of EST clones homologous to klhl
iv
73
List of abbreviations
LIST OF ABBREVIATIONS
aa
amino acid
AP
alkaline phosphatase
arp
acidic ribosomal protein gene
BAC
bacterial artificial chromosome
BCIP
5-bromo-3-chloro-3-indolyl phosphate
bp
base pair
BTB
broad-complex, tramtrack, bric-a-brac
cDNA
DNA complementary to RNA
cmlc2
cardiac myosin light chain 2
cpm
counts per minute
DEPC
diethyl pyrocarbonate
DIG
digoxygenin
DNA
deoxyribonucleic acid
dNTP
deoxyribonucleotide triphosphate
EDTA
ethylene diaminetetraacetic acid
ENU
ethylnitrosourea
ES
embryonic subtractive
EST
expressed sequence tag
FCS
fetal calf serum
GFP
green fluorescent protein
HGP
human genome project
hpf
hours post fertilization
kb
kilo base pair
klhl
kelch-like gene
LB
Luria-Bertani medium
LG
linkage group
MA
maleic acid
MGI
Merck Gene Index
MOPS
3-(N-morpholino)propanesulfonic acid
mRNA
messenger ribonucleic acid
MSP
muscle specific protein
MTN
multiple tissue blot
mya
million years ago
mylz2
myosin, light polypeptide 2, fast skeletal muscle gene
NBT
nitroblue terazolium
nt
nucleotide
v
List of abbreviations
ORF
open reading frame
PAC
P1-derived artificial chromosome
PBS
phosphate buffered saline
PBST
PBS, 0.1% Tween 20
PCR
polymerase chain reaction
PFA
paraformaldehyde
POZ
poxvirus and zinc finger
RACE
rapid amplication of cDNA ends
RAPD
randomly amplified polymorphic DNA
RH
radiation hybrid
RNA
ribonucleic acid
SAGE
serial analysis of gene expression
SDS
sodium dodecyl sulfate
smbpc
slow myosin binding protein C
SSC
sodium chloride-trisodium citrate solution
SSCT
sodium chloride-trisodium citrate solution, 0.1% Tween 20
tpma
alpha tropomyosin gene
UTR
untranslated region
vhmc
ventricular myosin heavy chain
YAC
yeast artificial chromosome
vi
Summary
SUMMARY
The completion of the human genome project brings with it the task of deciphering
and interpreting the sequence, carrying it from sequence to function. The zebrafish has
rapidly emerged as the forerunner for scientists riding the next wave of genome
exploration, being uniquely positioned to study vertebrate development. In the study,
zebrafish was used as the model to isolate and characterize a novel gene, kelch-like, klhl
that we had identified in an earlier screen for important genes involved in embryogenesis.
klhl was found to be a member of the kelch-repeat superfamily, containing two
evolutionary conserved domains- BTB/POZ domain and six kelch repeats. Many members
of the kelch-repeat superfamily have been shown to be involved in the organization of cell
shape and function. Database mining revealed the presence of putative orthologues of klhl
in human, mouse, rat and pufferfish. klhl was determined to map to zebrafish linkage 13
and was found to be syntenic with the proposed ortholog of klhl in human, mouse and rat.
In an effort to elucidate the function of klhl, klhl gene expression was compiled by
northern and in situ hybridization. klhl is specifically expressed in the fast skeletal and
cardiac muscle. Comparisons of klhl with previously identified muscle genes, tpma and
mlyz2, indicated that klhl is expressed around 10 hpf and is one of the earliest genes to be
expressed in the somitogenic pathway. Northern blot analyses show that the human
ortholog, KLHL, is also specifically expressed in the skeletal muscles and heart. In silico
analyses of rat EST clones corresponding to rat Klhl ortholog also indicate that its
expression pattern in rat is also conserved, suggesting the evolutionary conserved role of
klhl. The expression pattern of klhl as well as the presence of the kelch repeats indicate a
possible role for klhl in the organization of striated muscle cytoarchitecture.
vii
Introduction
Chapter I
Introduction
1
Introduction
1. Beyond the Genome: Turning Data into Knowledge
1.1 The Human Genome Unveiled
April 2003 marked the fiftieth anniversary of the discovery of the double helix by
James Watson and Francis Crick. A momentous event in the history of biology, the 1953
breakthrough marked a new chapter in science, opening the door to the exploration of
many avenues which has become the occupation of researchers all over the world. April
2003 also marked the completion of one of the most important and ambitious scientific
projects in history: the sequencing of the human genome (Pennisi, 2003), that fittingly
may prove to be an appropriate close to the chapter opened some fifty years before.
Involving the coordinated effort of 20 laboratories and hundreds of people around the
world, the human genome project (HGP) was an impressive technical and logistical feat
with the sequence representing an enormous opportunity to understand biology and
accelerate biomedical research. However this represents just the data acquisition phase.
Faced with an avalanche of sequence data, researchers are now faced with the daunting
task of deciphering and interpreting the data and get more biology from the sequences.
Indeed, as well put by the paper on the draft genome of the International Human Genome
Sequencing Consortium (Lander et al., 2001), “the human genome project is but the latest
increment in a remarkable scientific program whose origins stretch back a hundred years
to the rediscovery of Mendel’s laws and whose end is nowhere in sight.”
1.2 Gene Annotation
Whilst the human genome was not the first to be sequenced, with over 45
completely sequenced genomes including those of the worm Caenorhabditis elegans and
fly Drosophila melanogaster completed by the time the draft sequence was released in
2
Introduction
February 2001 (Bernel et al., 2001), it represented a new challenge to researchers with the
ultimate goal to compile a complete list of all human genes and their encoded proteins
(Lander et al, 2001; Shoemaker et al., 2001). Gene identification is particularly difficult in
human DNA owning to the large size of its genome. One of the reasons for the increase in
genome size in human as compared to the worm or fly is due to the introns becoming
much longer (about 50 kb versus 5 kb). The exons, on the other hand, are roughly the
same size (Birney et al., 2001; Lander et al., 2001). Thus, the density of the genes in the
human genome was much lower than for any other genome sequenced back in 2001
(Bork and Copley, 2001).
For the most part, gene prediction is done computationally. A combination of three
basic approaches was employed in the sequencing projects to predict the genes (Lander et
al., 2001, Venter et al., 2001). The first approach is based on ab initio prediction of exons
based on compositional signals found in the DNA sequence. Groups of exons are
identified based on certain computational algorithms that gather statistical information
about splice junctions, exon and intron lengths for example (Birney et al., 2001; Lander et
al., 1998;). While these ab initio predictions were quite accurate in the fly (Reese et al.,
2000) and worm, they would not be so reliable for the human draft sequence. The low
signal (exon) to noise (intron) ratio leads to misprediction by computational gene finding
strategies. In addition, gaps and errors within the draft sequence would give rise to frameshifts, when the reading frame of the gene is disrupted by the addition or removal of bases
(Birney et al., 2001). The second approach is based on direct experimental evidence of
transcription provided by expressed sequence tags (ESTs), short sequences of DNA
corresponding to a fragment of a complementary DNA (cDNA). Analysing genomic
sequences in the context of ESTs provides a more accurate resource for resolving gene
3
Introduction
structure against the vast genomic background. This method is however subjected to
artefactual and contaminant sequences from heterogeneous nuclear RNA, genomic DNA
and vector sequences. Estimation of gene number based on EST numbers have led to
varying estimates from 35,000 to 120,000 genes (Ewing and Green, 2000; Liang et al.,
2000). The third approach uses indirect evidence based on sequence similarity to
previously identified genes and proteins in humans and other organisms. This approach,
while effective in identifying genes, cannot differentiate between a functional or nonfunctional (pseudogene) gene. A pseudogene is a non-functional copy that is very similar
to a normal gene but that has been altered slightly so that it is not expressed. Also, novel
genes cannot be identified by this method. Following the release of the draft sequence, the
gene number was put at 30,000 to 40,000 (Lander et al., 2001; Venter et al., 2001), a far
cry from the 80,000 – 100,000 genes thought to exist at one time (Gardiner-Garden and
Frommer, 1987; Levin, 1990). Of these, ~15,000 were known genes and the remaining
10,000- 20,000 gene predictions of lower confidence, possessing evidence derived only
from the bioinformatics approaches of sequence homology and ab initio predictions
(Lander et al., 2001; Saha et al., 2002). Even today, following the completion of the
human genome sequence, the number of human genes have not been determined
conclusively, with Francis Collins, director of the National Human Genome Research
Institute (NHGRI) putting it at a little under 30,000 (Pennisi, 2003).
1.3 Comparative Genomics
One tool for gene identification that will become more powerful with the
completion of more genome projects is comparative genomics. The science of
comparative genomics has a long and fruitful history in biology. It has its roots in
4
Introduction
Aristotle, who understood that the commonalities among species would facilitate
comprehension of the underlying “differentiae” that distinguish animals with common
features. Comparing the human genome with those of other species would not only help us
understand what makes us genetically different, it may also help us understand our genes,
their regulation and expression and their complex interactions (Murphy et al., 2001).
One of the most startling things to emerge from the draft sequence was the fact that
the human genome, despite being about 30 times larger than the fly and worm genomes,
contained only about twice the number of genes (Lander et al., 2001; Venter et al., 2001).
It was clear that physical and behavioural differences between species were not simply a
consequence of gene number. Comparative studies between human and the fly, and
between human and the worm revealed that the biggest difference laid in the complexity
of the proteins: more domains per protein and novel combinations of domains (Baltimore,
2001). About 60% of fly proteins and 40% of worm proteins have sequence similarity to
predicted human proteins. Yet more than 90% of the domains identified in human proteins
were also present in the fly or worm proteins (Lander et al, 2001; Venter et al., 2001). The
story is one of new architectures built from old pieces, with shuffling of domains, creating
new permutations.
While the value of comparative analysis of distantly related organisms is beyond
dispute, comparison of closely related genomes would be more important in resolving the
issue at hand – identifying the genes and their functions. Comparing conserved sequence
regions between two related organisms would allow us to identify genes and other
important regions in both organisms with no previous knowledge of either gene content.
This is because thanks to natural selection, genes are more likely to retain their sequences
through evolution than the DNA surrounding them. However, there are limitations to
5
Introduction
functional interferences based on interspecies comparisons of anciently diverged coding
sequences (Makalowski and Boguski, 1998). Furthermore, gene regulatory elements are
not amenable to comparisons across vast evolutionary distances as they are more
divergent (Makalowski and Boguski, 1998). As succinctly put by Rubin (2001), “the ideal
species for comparison are those whose form, physiology and behaviour are as similar as
possible, but whose genomes have evolved sufficiently that non-functional sequences have
had time to diverge”. However, he also warns that in practice, there is no ideal species,
because different genes and regulatory sites evolve at different rates.
In what is seen as a pilot project to evaluate which genome sequences would be the
best appropriate to aid in the annotation of the human genome and the understanding of
vertebrate genome evolution (phylogenomics), the National Institute of Health (NIH)
Intramural Sequencing Centre is mapping and sequencing segments of 11 vertebrate
genomes orthologous to six regions on human chromosome 7. (http://www.nisc.nih.gov)
(Thomas and Touchman, 2002). (The 11 genomes are mouse, rat, pig, cow, dog, cat,
baboon, chimpanzee, chicken, zebrafish and pufferfish.) The power of comparative
sequence analysis with related organisms at suitable evolutionary distances to identify
genes have been exemplified in many cases. Crollius and colleagues (2000) reported
successes in comparisons between the human genome and that of pufferfish Tetraodon
nigroviridis. With a genome eight times more compact than that of human, the pufferfish
proved valuable in identifying potential exons in the human genome (Crollius et al., 2000).
Through alignment of mouse DNA related to human chromosome 19, Stubbs and her
group identified exons, regulatory elements, and candidate genes that were missed by
other predictive methods (Dehal et al., 2001).
6
Introduction
Recently, the draft sequences of the Fugu and mouse genome and the comparative
analyses with the human sequence were published in August 2002 and December 2002
respectively (Aparicio et al., 2002; Waterston et al., 2002). Preliminary analysis of the
pufferfish genome by Aparicio and colleagues suggest that the Fugu gene dataset may
help uncover as many as 1000 novel human genes in the human genome. Conserved gene
order or synteny was also discovered between the human and Fugu genes. Findings from
the mouse genome support the notion that there are only about 30,000 genes in a typical
mammalian genome, 99% of which have a sequence match in the human genome. 96% of
these genes lie with syntenic regions of mouse and human chromosomes (Waterston et al.,
2002).
The comprehensive conservation of linkage between the human and mouse
genome (http://www.ncbi.nlm.nih.gov/Homology) has several practical applications. First,
the comparative maps allow the rapid identification of gene orthologs. Two genes are
orthologous if they diverged after a speciation event, when a new species forms from an
existing one; two genes are paralogous if they diverged after a gene duplication event. The
identification of orthologs is particularly useful when investigating disease phenotypes
(Watkins-Chow et al., 1997; Lander et al., 2001), allowing the correlation of mouse
models and human disease. This also facilitates the positional cloning of disease genes.
Second, the study of conserved segments among genomes provides insights into the rates
and patterns of chromosomal evolution, as well as into the forces that help to shape the
genomes of modern-day animals (O’Brien et al., 1999; Lander et al., 2001; Murphy et al.,
2001). Third, cross-referencing of human and mouse genomes aids in the assembly of the
mouse sequence using the human sequence as a scaffold (Lander et al., 2001).
7
Introduction
Indeed, it seems that for the immediate future, the most dramatic developments in
eukaryotic genome biology are likely to be in comparative genomics (Taylor, 2001).
Advanced technologies of the HGP have been harnessed to describe the complexities of
genome organization not only in the mammalian species (mouse, rat, dog, chimp) but also
in other vertebrates such as the pufferfish and zebrafish.. Each of these whole genome
shotgun sequences is expected to fill in a piece of the evolutionary history, providing us
with a better insight into the laboratory notebook of evolution.
1.4 Expressed Sequence Tags (ESTs) and In Silico Analysis
Playing a complementary role to the genome sequencing projects is the EST
sequencing projects. In the 1990s, Brenner (1990) and other investigators advocated the
large-scale sequencing of transcription products of genes, in the form of cDNAs, as a
prelude to genomic DNA sequencing. The rationale for this was that it would be more
useful and cost effective as the protein-coding regions of our genes only make up ~3% of
the entire genome. The remaining 97% is of unknown function and often referred to as
“junk DNA”. The era of high-throughput cDNA sequencing was initiated in 1991 by a
landmark paper by Adams and colleagues (1991) demonstrating the richness of data that
could be derived from an EST sequencing project. The basic strategy involved the random
selection of cDNA clones after which single-pass sequencing was performed. This
sequencing could be from either the 5’ and/or 3’ end of the clone, and the sequence is not
checked for errors or artefacts. In their article, they generated partial sequences from 609
randomly selected cDNA clones from a human brain library. Of these 609 sequences, 197
(32%) matched to human sequences, 48 (8%) matched to entries of other organisms and
230 (38%) had no significant matches. The results demonstrated that sufficient
8
Introduction
information was contained in 150 to 400 bases of a nucleotide sequence from one
sequencing run for preliminary identification of the cDNA. In addition, it revealed the
utility of ESTs for novel gene discovery.
The use of ESTs in the identification of genes has been exemplified in numerous
studies. Most recently however was the use of ESTs in the prediction of genes on human
chromosome 21 (Hattori et al., 2000). Of the 225 genes identified on chromosome 21, 42
genes were only identified with the use of ESTs (Yuan et al., 2001). This represented
18.7% of the gene identification process that relied on EST sequences. Besides its use in
gene identification and annotation of genomic sequences, ESTs have assumed important
roles in the construction of gene-based physical maps of several genomes, including that
of human (Schuler et al., 1996). In this application, PCR or hybridisation assays
developed from ESTs can be used to identify bacterial artificial chromosomes (BACs), or
other types of large insert clones from which genome physical maps are constructed.
Placement of ESTs onto a physical map immediately identifies the genomic intervals that
contain the sequences for the gene (Marra et al., 1998).
Since then, EST projects have been initiated on a diverse collection of organisms
that include C. elegans, D. melanogaster, rat, mouse and zebrafish. For many of these
organisms, the ESTs could be subdivided further into tissue types. The EST database,
dbEST, is the fastest growing division of the GenBank (Pandey and Lewitter, 1999). To
date, over 18,762,324 sequences from 594 species have been reported in the database
(dbEST
release
3
October
2003,
http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html). While this large dataset of
DNA sequences is data rich, it is unfortunately information poor with absence of
additional correlative data. The sequence generated is generally of poor quality with
9
Introduction
misreads and filled with library construction and sequencing artefacts (Yuan et al., 2001).
Such a situation thus led to the development of EST gene indices such as the UniGene
(Boguski and Schuler, 1995; Schuler et al., 1996), Merck Gene Index (MGI) (Eckman et
al., 1998) and TIGR Gene Index (Quakenbush et al., 2000). The goal of all gene indices is
to reduce the vast amount of data into a organized catalogue from which one can
determine how many unique transcripts exist and whether a new sequence falls into any of
the
existing
ESTs
cluster
(Yuan
et
al.,
2001).
The
UniGene
database
(http://www.ncbi.nlm.nih.gov/UniGene) categorizes GenBank sequences into a nonredundant set of gene-oriented clusters, where a single cluster represents all the ESTs that
correspond to a unique gene. Related information, such as the tissue types in which the
gene is expressed and its location is also provided. Currently, the UniGene database
contains 13 data sets, eight of which belong to animals. The eight organisms are human,
mouse, rat, fly, zebrafish, clawed frog, cow and mosquito. Large-scale sequence
comparisons have also been used to cross-reference the sequence clusters of the various
organisms. The HomoloGene database (http://www.ncbi.nlm.nih.gov/HomoloGene)
displays curated and calculated orthologs and homologs for nucleotide sequences
represented in UniGene. The advent of such databases ushers in a new era in which
classical biological analyses that were once performed at the bench are now performed
rapidly in silico (Pandey and Lewitter, 1999). Since one gene is often represented by
multiple ESTs, it is possible to generate a contiguous sequence by assembling ESTs that
overlap. Such in silico cloning methods are nowadays used regularly to complete the
mRNA sequence or to identify novel gene orthologs and homologs. In addition, in silico
expression data, which is obtained by simply counting the frequency of ESTs, is often
seen accompanying a paper reporting the cloning of a new gene (Ko, 2001).
10
Introduction
1.5 Generation of Functional Data using Model Organisms
With the large amount of data accumulating from the genome project, it is no
surprise that in silico analysis is very much in evidence. There is a heightened expectation
that the increasingly powerful computer analyses of computer databases today would be
sufficient to take us from sequence to function. Indeed much of what we know about the
function of human genes is inferred computationally. To rectify this problem, studies are
underway to generate functional data in model organisms.
Annotation by sequence similarity or domain structure is usually the first step
performed in many studies, but such predictions can sometimes be unreliable and
misleading. Genes of similar sequences may have acquired new functions during
evolution. This is particularly true for duplicated genes. In their study of the triplicate
Drosophila genes paired, gooseberry and gooseberry-neuro, Li and Noll (1994) suggested
that following duplication, genes acquire new functions by changes in their regulatory
regions generating an altered expression. Adaptation of the protein is “secondary and a
necessary consequence of its expression in the newly acquired context of this function”
(Xue et al., 2001). Further studies by Xue et al. (2001) also implied that while the Cterminal portions of paired and gooseberry are divergent in their primary sequences, they
were qualitatively the same. Such results led Noll’s groups to question the validity of
amino acid similarity as a general measure of functional equivalence in homologous
proteins (Xue and Noll, 1996; Xue et al., 2001). Thus information in databases is not by
itself, sufficient to determine biological function but serve as a foundation for the design
of detailed experimental studies to establish the actual function of the molecules.
Much more information about gene function can be obtained from knowing
expression patterns and gain- or loss-of function studies and model organisms would
11
Introduction
feature heavily in this respect. Such studies can realistically be done only in model
organisms not only because of ethical and social issues, but more importantly because the
sophisticated genetic and transgenic experimentation needed to resolve the complex
biological networks are not available in humans. Genome-wide initiatives in assessing
expression and function are underway for all model organisms. The Berkeley Drosophila
genome project, for one, is surveying the expression of all Drosophila genes by wholemount in situ hybridisation in embryos and creating a catalogue of gene mutations by
insertions of P elements or Gal4 activation domains into many different sites in the
genome (Spradling et al., 1995; 1999; Kopczynski et al., 1998).
The question to be asked at this point however would be the extent of functional
interchangeability of the genes among the different organisms. Over the years, it has
emerged from studies in many animal models, not only individual protein domains and
proteins, but entire biochemical pathways are conserved throughout evolution (Miklos and
Rubin, 1996). In the Ras and Notch signalling cascade, for example, many of the protein
components are conserved between yeasts, flies, worms, and humans (Artavanis-Tsakonas
et al., 1995, Wasserman et al., 1995). Knowledge of the biological role of a shared protein
in one organism can then be transferred to other organisms. The extent to which a disease
or biological process can reasonably be modelled in an organism phylogenetically
different from us must be critically examined otherwise we run the risk of creating
interesting but useless information which might confound the issue (Margolin, 2001). The
genome projects in each of the model organisms would greatly facilitate this work and
with the human genome sequence, allow the speedy transfer of knowledge to human
biology.
12
Introduction
2. Zebrafish in the Context of the Human Genome Project
One of the most promising model organisms to emerge in light of the HGP is the
zebrafish (Danio rerio), a small tropical freshwater teleost fish. It is “a dream system for
scientists riding the next wave for genome-wide exploration” (Fishman, 2001). A
combination of various factors ensures that the zebrafish will have an important role in the
functional analysis of the human genome. Some of these factors include its tractability in
mutagenesis screens to the availability of genomic resources which will be elaborated in
the next sections.
2.1 Zebrafish as an Experimental System
Originating from the Ganges river in India, the zebrafish first emerged as a model
system for the study of developmental biology in the 1980s. Pioneering the use of this
inexpensive fish was George Streisinger and colleagues (1981) at the University of
Oregon who recognized the many virtues of this experimental system for genetic analyses.
Some of these virtues include its short generation time, the large brood size and the
external development of clear, transparent embryos, which makes the zebrafish embryos
experimentally accessible. Development is rapid and with 12 hours after fertilization one
can visualize the establishment of a body plan that is typically vertebrate (Westerfield,
1989). By 5 days after fertilization, most organs, or at least their primordia are in place
(Kimmel et al., 1995). Laboratory methods for its husbandry are well established
(Westerfield, 1994) and the stages of embryonic development thoroughly described and
characterized (Kimmel et al., 1995). While the significance of Streisinger’s work with
zebrafish was not widely recognized at that time, it marked the birth of a new animal
model system that has since risen to become a pre-eminent model in biomedical research
13
Introduction
(Beier, 1998; Grunwald and Eisen, 2002; for recent reviews, see Shin and Fishman, 2002,
Ackermann and Paw, 2003 and Rubinstein, 2003).
2.2 Mutagenesis Screens
The ability to carry out classical forward genetic analyses with zebrafish was
largely responsible for its rise in prominence. Since its early days as a research organism,
the appeal of the zebrafish has relied on its potential use in genetic screens which was
unique among vertebrate model organisms. Today, no other vertebrate can rival the
repertoire of zebrafish mutagenesis tools, breeding strategies and screening methods
(Malicki et al., 2002). Previously, saturation mutagenesis of Drosophila had been used
successfully by Nüsslein-Volhard and Eric Wieschaus to uncover more than 200 genes
involved in pattern formation and unravel the regulatory cascade of molecular events
(Nüsslein-Volhard and Wieschaus, 1980; Kalthoff, 1996). The results of such studies had
been extrapolated successfully to vertebrates with mutations in the vertebrate homologue
of the gene having profound developmental consequences. This demonstrated the
conservation of pathways even in highly divergent organisms like Drosophila and the
mouse. Despite this, several new features characterize the vertebrate which are not present
in invertebrates, specifically with respect to organ form and function. Some examples
include the development and function of the notochord, kidneys and multi-chambered
heart, which are unique in vertebrates (Driever and Fishman, 1996; Fishman, 1999;
Dooley and Zon, 2000). Within vertebrates, these processes have been well conserved.
Little, however, was known about them. A similar analysis was thus proposed in
vertebrates to uncover loci of developmental importance, especially those important in
14
Introduction
organ form and function, which were not scored in Drosophila screens (Nüsslein-Volhard,
1994).
Saturation mutagenesis screening had previously been applied only to invertebrates
as the large number of animals needed for screens deemed them prohibitively expensive
for vertebrates other than the zebrafish. The zebrafish possessed some advantages over the
other more established vertebrate models such as the mouse and Xenopus, both of which
do not breed prolifically and the embryos are not readily observable, making them
unsuitable for the long, laborious screening process (Kahn, 1994). All these factors led to
the zebrafish becoming the vertebrate of choice for random, genome-wide, large-scale
mutagenesis of genes crucial for vertebrate development (Driever et al., 1996; Haffter et
al., 1996; Schulte-Merker, 2000).
The first large-scale genetic screens in vertebrates were carried out in zebrafish in
1996 using the chemical mutagen ethylnitrosourea (ENU). Undertaken by groups in
Massachusetts General Hospital, Boston and Max Planck Institute, Tüebingen, the two
screens, conducted in parallel, identified more than 2,000 mutants involved in embryonic
development (Driever et al., 1996; Haffter et al., 1996). The basis of the screens was an
outgrowth of the work that had previously been done in Drosophila (Nüsslein-Volhard
and Wieschaus, 1980). Random mutations were induced by treating the male fish with
ENU, which was known to be an efficient germ-line mutagen in mice. ENU generates
single-nucleotide mutations in the germ-line principally by alkylating guanine residues
with consequent GC→AT transitions (Solnica-Krezel et al., 1994). The levels of ENU
administered had been titered to generate one to two mutations per haploid genome
(Mullins et al., 1994; Solnica-Krezel et al., 1994). The mutants were then bred to
homozygosity in a three-generation scheme (Driever et al., 1996; Haffter et al., 1996).
15
Introduction
The main tool for identification of mutant phenotypes was detailed visual inspection of the
embryos under the dissecting microscope (Driever et al., 1996; Haffter et al., 1996). This
inspection was performed at five different stages during embryonic and early larval
development. By the time the studies were performed, the development of the zebrafish
embryo had been studied in detail, from the pre-gastrula and gastrula stages to the
pharyngula stages through to the early larval period (Kimmel et al., 1995), lending to a
strong base of knowledge for the identification of mutant phenotypes. The mutations are
believed to have affected more than 500 genetic loci, affecting an impressive range of
targets: eye, pigment, kidney, notochord, muscle, brain and fins, just to name a few
(Warren and Fishman, 1998). The screens and the mutants uncovered were the subject of
an entire issue of the journal Development (December 1996 volume 123) and the study
was described in Science as “an accomplishment of historic proportions” (Grunwald,
1996).
However, these first screens were not saturating, and concentrated on the
identification of genes involved in early development (Driever et al., 1996; Haffter et al.,
1996). The Tüebingen group has undertaken a second saturation mutagenesis screen of the
zebrafish, Tüebingen 2000, in collaboration with Artemis Pharmaceuticals and this second
screen is aiming more at the later stages of organogenesis (Schulte-Merker, 2000).
The expectation that the zebrafish model will introduce screens as a standard tool
of vertebrate genetics has been fulfilled. In addition to the large-scale screens, a number of
smaller screens have been conducted in zebrafish, identifying numerous other loci
required for different physiological processes. The utility of zebrafish in such screens is
due largely to the establishment of techniques allowing the manipulation of the ploidy and
parental origin of genes in zebrafish (Streisinger et al., 1981; Kimmel, 1989). The ability
16
Introduction
to generate haploid embryos, for example, facilitates genetic screens by eliminating a
generation or more from crossing schemes (Kimmel, 1989; Walker, 1999). Such genetic
screens, based on analysis of zebrafish haploid or parthenogenetic diploid embryos, have
been used to identify genes required during embryogenesis (Henion et al., 1996;
Alexander et al., 1998; Beattie et al., 1999).
Besides the different screening methods, there are also several means by which
mutations can be induced in the zebrafish germ-line, mainly chemical mutagenesis,
radiation methods and insertional mutagenesis (Knapik, 2000). Chemical mutagenesis
using ENU is by far the most widely employed method in zebrafish as it is effective and
easily administered by incubating the fish in ENU. Other chemicals that have been used
include EMS and TMP which cause small deletions. Radiation methods using X-rays and
gamma rays are routinely performed in zebrafish laboratories to induce genome-wide
mutations. Causing large multigene lesions, this method is not useful for the annotation of
genes by functions. The last method of insertional mutagenesis involves the insertion and
integration of exogenous DNA sequences into the genome, disrupting the genes at the site
of insertion. While insertional mutagens have been shown to be less efficient than
chemicals (Spradling et al., 1995; Schier et al., 1996), this system shows extraordinary
potential as the inserted DNA serves as a tag to clone the mutated gene. This greatly
speeds up the normally laborious process inherent with the use of chemical mutagens. The
average time taken to clone a gene responsible for a ENU-induced mutation is about 1.5
years, although it is expected to decrease to 9 months following completion of the
zebrafish genome project (Chen et al., 2002). At the moment, the genes underlying only
about 50 mutants have been reported out of the hundreds of mutants uncovered in the
mutagenesis screens (Golling et al., 2002). Many of these genes have been previously
17
Introduction
described as important developmental genes in other species. Efficient methods of
insertional mutagenesis would thus contribute significantly to the task of assigning
functions to genes.
Several advances have been made towards the use of insertional mutagenesis in
zebrafish with the use of retroviruses. In 1994, Nancy Hopkins and her group identified a
pseudotyped retroviral vector that could infect the zebrafish germ-line (Lin et al., 1994).
The pseudotyped retrovirus system was found to be able to generate a large number of
insertions at different loci very efficiently (Gaiano et al., 1996a) and this has made it
possible for large-scale insertional mutagenesis to be performed (Gaiano et al., 1996b;
Amsterdam et al., 1999; Golling et al., 2002). Several genes have been identified using
this technology (Allende et al., 1996; Becker et al., 1998; Kawakami et al., 2000a;
Golling et al., 2002). More noteworthy is the fact that it takes as little as two weeks to
identify the retrovirally mutated gene (Golling et al., 2002). In addition, many of the genes
identified using insertional mutatgenesis are novel genes without known biological or
biochemical functions. The number of genes cloned by insertional mutagenesis is
expected to rise quickly with the development of a high-titer retrovirus producer cell line,
circumventing the problem of reproducibly making high-titer, non-toxic virus preparations
(Chen et al., 2002). According to Chen et al. (2002), preparations from this line allowed
the generation of about 500,000 germ-line-transmissible insertions in a population of
25,000 founder fish in about 2 months.
Transposons have also been evaluated for their efficacy and use in insertional
mutagenesis system in zebrafish (Ivics et al., 1999). While still in its infancy, several
transposon systems show great potential as a tool to develop insertional mutagenesis.
Some examples include the Tol2 element from medaka (Kawakami et al., 2000b) and the
18
Introduction
synthetic Sleeping Beauty (SB) transposon systems (Ivics et al., 1997; Hackett et al.,
2001). In particular, the SB system has been used for insertional mutagenesis employing
both gene-traps and enhancer-traps (Hackett et al., 2001).
2.3 Genomic Infrastructure
Another virtue of the zebrafish lies in the wide availability of zebrafish genetic and
genomic resources. Zebrafish mutations identified in the screens define the function of
hundreds of essential genes in the vertebrate genome. For these mutants to be useful,
cloning of the mutated genes is essential to allow the elucidation the molecular
mechanisms underlying cellular function (reviewed in Postlethwait and Talbot, 1997). The
two main approaches of cloning mutated genes, positional cloning and candidate gene
approach, have benefited greatly from the recent advances in zebrafish genomic
infrastructure (reviewed in Talbot and Hopkins, 2000; Malicki et al., 2002).
The efficient identification of genes disrupted by mutation in zebrafish requires
dense maps of the genome. Prior to 1994, there was no genetic map for zebrafish and the
paucity of resources such as large-insert genomic libraries rendered the task virtually
impossible (Malicki et al., 2002). Today, a full array of genomic and molecular genetic
tools is available. Large-insert genomic libraries needed for positional cloning have been
generated. To date, two zebrafish yeast artificial chromosome (YAC) libraries, one
bacterial artificial chromosome (BAC) library, and one P1-derived artificial chromosome
(PAC) library have been constructed (Zhong et al., 1998; Amemiya et al., 1999) and used
successfully to isolate known genes and/or genomic regions (Amemiya et al., 1999).
Several genetic linkage maps have been developed which cover essentially the entire
genome (see Talbot and Hopkins, 2000) in which each chromosome is represented by a
19
Introduction
single linkage group (Johnson et al., 1996). Among vertebrates, only human, mouse, rat,
and zebrafish have closed linkage maps. More than 3845 microsatellite (CA) repeats have
been meiotically mapped since the last update in July 2001, providing an average
resolution
sufficient
to
initiate
positional
cloning
(Shimoda
et
al.,
1999;
http://zebrafish.mgh.harvard.edu). Published genetic linkage maps have also localized
~1500 cloned genes and ESTs (Postlethwait et al., 1998; Gates et al., 1999; Kelly et al.,
2000; Woods et al., 2000). Radiation hybrid (RH) maps with markers which include
simple sequence length polymorphisms (SSLPs), cloned genes and ESTs, have been
developed for zebrafish (Kwok et al., 1998; Geisler et al., 1999; Hukriede et al., 1999,
2001). The two zebrafish RH maps, LN54 and Goodfellow T51, together cover >90% of
the zebrafish genome (Talbot and Hopkins, 2000) and will provide a framework for the
EST sequencing and mapping projects currently underway. As of dbEST release 3
October 2003, the zebrafish EST sequences deposited in GenBank number 362,362,
making it the eight highest species in a list of 594 species.
Efforts have also been initiated to obtain the complete sequence of the zebrafish
genome, a feat that will undoubtedly increase the usefulness of the genetic and genomic
tools in the fish. While the finished zebrafish genome is expected to be completed only in
2005 by the Sanger Institute, sequences from the whole genome shotgun and clone
sequencing project are made available online (http://www.sanger.ac.uk/Projects/D_rerio/).
Zebrafish sequences are also available through the ensembl website which features the
zebrafish whole genome shotgun assembly sequence version 2 as released on the 3rd April
2003 (http://www.ensembl.org/Danio_rerio/).
Last but not least, the utility of the genomic infrastructure to the community of
zebrafish investigators is heavily dependent upon the existence of mechanisms that
20
Introduction
facilitate access to this information. As more labs started working with the zebrafish, the
Zebrafish Information Network (ZFIN) (http://zfin.org) was set up as to cope with the
phenomenal rate of increase of information. The ZFIN is a centralized database for
zebrafish researchers, providing links and information about zebrafish genes, mutations,
genetic maps etc (Westerfield et al., 1999a,b; Sprague et al., 2003). In addition, zebrafish
resources
are
also
available
from
the
NCBI
site
(http://www.ncbi.nlm.nih.gov/genome/guide/D_rerio.html).
2.4 The Syntenic Relationship of the Zebrafish and Human Genomes
The third virtue of the system is the conservation of synteny between zebrafish and
human genomes. Besides facilitating the identification of mutants by positional cloning
and the candidate gene approach, the genetic maps have been useful in comparative
studies between zebrafish and other vertebrate genomes. By comparing the map positions
of zebrafish genes and their mammalian orthologs, Postlewait et al. (1998) discovered that
a significant fraction of genes show synteny between the genomes, conserved
chromosome segments. In general, the likelihood that a syntenic relationship will be
disrupted correlates with the physical distance between the loci and the evolutionary
distance between the species. Despite the 450 million years of evolutionary distance
between zebrafish and human (Kumar and Hedges, 1998), analyses have identified 167
conserved syntenies involving two or more putatively orthologous genes (Gates et al.,
1999; Woods et al., 2000). Furthermore, the analyses also identified 136 orthologus pairs
that were not members of conserved syntenies. While this may reflect errors in mapping or
in orthology determination, they may also nucleate additional synteny groups as additional
genes are mapped. A minimum estimate of ~300 conserved synteny groups was thus
21
Introduction
estimated between the zebrafish and human genomes (Wood et al., 2000). Similar results
were obtained in another study done at the same time (Barbazuk et al., 2000). Analyses of
mouse and human, as well as zebrafish and human synteny groups have also led to the
conclusion that mouse and human, which diverged ~112 million years ago (mya), have
greater conservation than zebrafish and human (Gates et al., 1999; Woods et al., 2000).
Despite the current gaps in the zebrafish-human comparative map, conservation of
synteny between the two has had several uses. First, such analyses have been valuable in
defining candidate genes for zebrafish mutant (Karlstrom et al., 1999; Schmid et al., 2000).
For example, the yot locus was mapped to linkage group 9 (LG9) which had been shown
to be syntenic to human chromosome 2. A survey of genes on human chromosome 2,
together with an inference that yot mutations affected Hedgehog signalling led to the
identification of gli2 as a candidate for yot (Karlstrom et al., 1999). Second, the
correspondence between the zebrafish and human genome may be used to predict
orthologous gene relationships (Barbazuk et al., 2000). While orthologs are best identified
by branching patterns on phylogenetic trees, this approach is not feasible for many of the
ESTs (Woods et al., 2000). The sequence-based prediction of gene orthology is however
sometimes not reliable, particularly in the case of multigene families. A synteny-based
approach might be useful in resolving the issue. Based on the syntenic correspondence of
zebrafish and human genomes, Barbazuk et al. (2000) suggested human orthologs for 20
genes or ESTs out of 32 whose ortholog relationships could not be confidently identified
by BLAST. Third, zebrafish comparative maps can help in the understanding of the
vertebrate genome, particularly as a valuable outgroup, distinguishing shared features of
mammalian genomes and those derived from ancestral genomes. (Postlethwait et al., 1998,
2000; Gates et al., 1999; Woods et al., 2000).
22
Introduction
Comparative mapping data suggests that a genome duplication event occurred
early in the lineage leading to zebrafish following its divergence from the tetrapods.
Numerous studies reveal that teleosts gene families often contain more members than the
equivalent families in mammals (reviewed in Wittbrodt et al., 1998). For example, there
are four engrailed genes in zebrafish while tetrapods have only two members (Force et al.,
1999). Mapping studies also suggest that these events were the result of whole-genome
duplication instead of tandem duplications as zebrafish has two copies of large
chromosome segments surrounding the engrailed genes syntenic to mammalian genomes.
The findings of the engrailed genes were corroborated by similar studies (Amores et al.,
1998; Postlethwait et al., 1998; Gates et al., 1999). Evidence in other teleosts like medaka
and pufferfish, suggests that this event occurred early in the evolution of the teleost
lineage (Wittbrodt et al., 1998; Smith et al., 2002). The data from such studies can also
help clear up the origin of the human genome. In their analysis of zebrafish comparative
maps, Postlethwait et al. (2000) have thrown up some intriguing hypotheses addressing
whether certain mammalian chromosomes may have been part of larger composite
chromosomes that subsequently underwent chromosome fission in different mammalian
lineages. Following the whole genome duplication of zebrafish after divergence with the
tetrapods, zebrafish should have twice as many chromosomes as humans in the absence of
chromosome rearrangements. Zebrafish, however, only has 25 chromosomes in the
haploid set, 2 more than humans. By examining the loci in zebrafish and the various
tetrapods, human, mouse and cat, Postlethwait et al. (2000) suggests that tetrapods and
fish both had a low-numbered ancestral vertebrate karyotype, possibly 12 or 13
chromosomes in the haploid set. In the single round of duplication leading to the teleost
lineage, these would have doubled to the 24 or so chromosomes characterizing most fish
23
Introduction
genomes while in mammals, these would have broken apart into the high numbered
karyotypes defining many mammalian genomes.
2.5 Experimental Tractability
Another virtue of the zebrafish is the array of cellular, molecular and genetic
techniques available in the zebrafish system. Methods of introducing DNA into zebrafish
embryos have included microinjection, electroporation and the use of microprojectiles.
The microinjection of plasmid DNA has proven to be the most reliable method of
producing transgenic zebrafish. Transgenic zebrafish carrying the green fluorescent
protein (GFP) derivatives have been successfully generated for many studies including
cell lineage tracing experiments, promoter studies and tissue-specific transgene expression
for example (reviewed in Gong et al., 2001). Such GFP transgenic fishes under the control
of tissue-specific promoters may come in useful in future mutagenesis studies targeting
specific tissues and organs. There has also been the development of other types of
transgenics in zebrafish, including the GAL4-UAS (Sheer and Campos-Ortega, 1999) and
cre-loxP system, which allows one to express a gene product in a directed stage- and
tissue-specific manner. Such systems allow the function of a gene product to be
determined in any given process, particularly in cases where its function in later stages is
obscured by phenotypic consequences accrued in the early stages of embryogenesis. More
recently, Ando et al. (2001) reported a new method of conditional gene expression in
zebrafish involving photo-mediated activation of caged mRNA. This method is simple,
rapid and economical, not requiring the generation of any transgenic lines. It involves the
chemical modification of RNA by a synthetic compound 6-bromo-4-diazomethyl-7hydroxycoumarin (Bhc-diazo) which forms a covalent bond with the phosphate group on
24
Introduction
the backbone of RNA, inactivating or caging the RNA. This Bhc-caged mRNA is
reactivated by photoillumination with long-wave ultraviolet (UV) light (350-365 nm) as
Bhc undergoes photolysis, uncaging the RNA. Using this method, Ando et al. (2001)
showed the Bhc-caged Gfp mRNA had severely reduced translational activity in vitro,
whereas illumination of Bhc-caged mRNA with UV light led to partial recovery of
translational activity.
Besides gain-of-function analyses using the ectopic expression of genes, loss-offunction analyses are also important to fully determine the function of a gene in vivo.
While such reverse genetics approaches such as gene knockouts used to be severely
lacking in zebrafish, or rather in all vertebrate systems other than the mouse, recent
advances have improved the prospects in zebrafish. Ma et al. (2000) demonstrated that
zebrafish cells obtained from short-term cell cultures could generate germ-line chimeras
following their introduction into a host embryo. Shuo Lin and colleagues reported the
nuclear transfer in zebrafish using long-term-cultured donor cells (Lee et al., 2002),
holding promise for gene targeting in zebrafish. Recently, Wienholds and colleagues
reported the first successful report of generation of a fish mutant for rag-1 by reverse
genetics (Wienholds et al., 2002). In this method, male fish were first mutagenized by
ENU and crossed with wild type females. Sperm was then collected from individual F1
fish. After nested PCR amplification screening for a mutation in a gene of interest, they
recovered and bred "target-selected" zebrafish. Although further steps are still required to
develop the gene knockout methodology, the work reported in these studies shows
promise in the future for introducing targeted mutations into zebrafish.
While the gene knockout technology is still not available, the advent of translationblocking morpholino oligonucleotides has led to a method of sequence-specific gene
25
Introduction
inactivation in zebrafish (Nasevicius and Ekker, 2000; Ekker and Larson, 2001; Malicki et
al., 2002). Morpholinos have been shown to effectively and specifically induce
phenotypes similar to that of chemically induced loss-of-function genes (Nasevicius and
Ekker, 2000). More recently, a new reverse genetics tool was described in zebrafish using
modified peptide nucleic acids (MPNA) to selectively shut down the production of
individual proteins (Jesuthasan, 2002; Urtishak, 2002). A variant of a reverse genetic
screen, large-scale whole-mount in situ hybridisation screens are feasible in zebrafish
owning to the transparency of the embryos. Such screens have been used successfully to
identify important genes involved in embryonic development (Meng et al.,1999; Kudoh et
al., 2001).
2.6 Zebrafish: From Disease Modelling to Drug Discovery
The repertoire of techniques available in zebrafish has added to its sheer elegance
as a model organism and the zebrafish is uniquely positioned to bridge the gap between its
vertebrate and invertebrate counterparts in studies of development and genetics. In
addition to its developmental advantages, recent studies indicate that the zebrafish has a
great potential to serve as a model for human disease that range from heart failure and
vascular disease to fields as diverse as osteoporosis, renal failure, Parkinson’s disease,
diabetes and cancer (for recent reviews, see Shin and Fishman, 2002; Ackermann and Paw,
2003). Many of the mutant phenotypes identified in the mutagenesis screens are
reminiscent of human clinical disorders. The validity of using the zebrafish as a model for
human disease is illustrated by the various examples of zebrafish mutant phenotypes with
clinical relevance in the various fields of haematopoiesis (Brownlie et al., 1998; Wang et
al., 1998), cardiac and renal development (reviewed in Dooley and Zon, 2000; Ward and
26
Introduction
Lieschke, 2002) among others. The study of the biology of the phenotypes has provided
new insights into the pathophysiology of the disease. For example, the work of Brownlie
et al (1998) in identifying the sauternes (sau) mutant represented the first animal model of
congenital sideroblastic anaemia (CSA) in humans. The sau mutant is characterized by
delayed erythroid maturation and abnormal globin gene expression, resulting in a
microcytic, hypochromic anaemia. Positional cloning identified the mutant gene as
encoding for a erythroid-specific enzyme δ-aminolevulinate synthase (ALAS2), required
for haem biosynthesis. In humans, mutations in ALAS2 cause CSA.
More recently, Langenau et al. (2003) reported the induction of clonally derived T
cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c-myc under
control of the zebrafish Rag2 promoter. Such transgenic oncofish may be used in drug
screens for prevention and treatment of tumours as well as in genetic screens for
identifying mutations that suppresses or enhance tumorigenesis. The current momentum
behind the zebrafish as a model organism augurs well not only for developmental
biologists, but also for those dissecting the genetic components of human disease.
The ex utero development of transparent zebrafish embryos also lends its hands to
the search for drugs and novel therapeutic approaches in a ‘chemical genetic’ approach
(Peterson et al., 2000; Shin and Fishman, 2002; Kid and Weinstein, 2003; Langheinrich,
2003). The zebrafish embryo is permeable to many small molecules. This feature, together
with the small size of the zebrafish embryo allows for the simultaneous screening of large
number of drugs following exposure of the embryos to a library of low molecular weight
compounds in 96 well plates. In an elegant study by Peterson et al. (2000), the effect of
~1000 small molecules on zebrafish development were screened simultaneously by
monitoring whole zebrafish embryos for anatomic alterations at frequent intervals.
27
Introduction
Peterson and his colleagues were able to identify several small molecules that modulated
various aspects of vertebrate ontogeny. In particular, their results allowed them to dissect
the logic of melanocyte and otolith development and identify the critical periods for the
events. Such results indicate the unexplored potential of chemical screening to dissect
developmental processes and identify novel genes in vertebrate development. Thus, such
studies hold promise for preclinical drug discovery as well as toxicological evaluation.
3. Rationale of the Project
With the aim to identify novel zebrafish genes important in embryonic
development, we had previously performed a small-scale in situ hybridisation screen in
zebrafish embryos with 75 unidentified clones derived from a subtracted embryonic
cDNA library (Wu, 1999). Our focus was on genes whose expression is spatially and
temporally regulated during development as many genes with developmental regulatory
function are expressed in a regionalized fashion. Screens of this nature have been carried
out in Xenopus, Drosophila, mouse and zebrafish embryos, yielding a large selection of
genes with highly regulated expression patterns (Gawantka et al., 1998; Kopczynski et al.,
1998; Neidhardt et al., 2000; Kudoh et al., 2001). Such studies supplement mutagenesis
screens which requires laborious processes, moving from mutant to gene. Moreover, as
mutagenesis screens relies heavily on “phenotype first” approach, genes with subtle lossof-function phenotypes or genes whose function can be compensated for by other genes or
pathways are unlikely to be found.
In our screen, we found that 19 out of the 75 (25.3%) clones presented a restricted
expression pattern. Six of these clones were sequenced completely and we found two of
them encoding novel proteins. In particular, one clone ES34, was expressed specifically in
28
Introduction
the somites and it possessed an evolutionary conserved protein domain known as the kelch
motif.
The kelch motif was first discovered as a sixfold tandem element in the Drosophila
kelch protein that is essential for oogenesis (Xue and Cooley, 1993). It is a segment of 4456 amino acids in length and multiple sequence alignment reveals eight key conserved
residues, including four hydrophobic residues followed by a double glycine element,
separated from two characteristically spaced aromatic residues (Adams et al., 2000).
Proteins containing kelch repeats appear to play fundamental roles in cellular activities as
evident by the pathological consequences of mutations in kelch repeats that have been
found in humans and mouse (Bomont et al., 2000; Nemes et al., 2000; Bradybrook et al.,
2001; VanHouten et al., 2001). For example, Bomont et al. (2000) found that a kelch
protein, gigaxonin, is mutated in giant axonal neuropathy which corresponds to a
generalized disorganization of the cytoskeletal intermediate filaments. This report is in
agreement with other studies in which kelch proteins are emerging as key links between
microfilaments and a variety of cellular structures and functions (reviewed in Adams et al.,
2001).
Considering the roles this family of proteins may play in human health and disease,
it is of interest to isolate the full-length cDNA clone of this gene from zebrafish. This
would allow us to deduce the complete amino acid sequence for comparison with its
human ortholog. Further study of its expression pattern in zebrafish will predict the
expression
and
function
of
the
29
novel
human
orthologous
gene.
Materials and methods
Chapter II
Materials and Methods
30
Materials and methods
1. Cloning of Full Length Zebrafish klhl cDNA
1.1 Rapid Amplification of cDNA Ends (RACE)-PCR
Polymerase chain reaction (PCR) is a powerful tool to amplify DNA fragments
millions of times by a thermostable DNA polymerase and a pair of primers. The RACE
procedure or one-sided PCR is a method by which the PCR technique can be used to
amplify the 3’ and 5’ ends of a cDNA using a small stretch of known sequence within
the gene. ES34 full-length 5’ cDNA sequence was obtained using the RACE-PCR
method from a cDNA library made from 24 hpf embryos (generously provided by Dr
Valdimir Korzh, Fish Developmental Biology, Institute of Molecular Agrobiology)
constructed in pBK-CMV (Fig. 1) using the Lambda Uni-Zap XR cloning system
(Stratagene, USA). The cDNAs were cloned uni-directionally between the EcoRI and
XhoI
sites
(5'Æ3')
of
pBK-CMV.
Two
gene-specfic
primers
KR1
(5’-
CAGCATCTAGGGACTTCCAT-3’) and KR2 (5’-TTTGCCACTGGTTTGAGGAT3’) and a vector antisense primer T3, were used for amplification. The components of
this polymerase chain reaction (PCR) (50 µl) included 5 µl of 10X PCR buffer (0.5 M
KCl; 0.1 M Tris-HCl, pH 8.8; 15 mM MgCl2; 1% Triton X-100), 2.5 µl of 2 mM
dNTP, 0.5 µl of 0.2 µg/µl sense primer, 0.5 µl of 0.2 µg/µl antisense primer, 0.2 µl of
5 U/µl Taq polymerase and 1 µl template DNA. The cycling condition was as follows:
94 °C/5 min, 30 cycles of 94 °C/30 sec, 55 °C/1 min, and 72 °C/1 min, and finally 72
°C/5 min. The amplification was carried out in a Hybaid PCR Express thermal cycler.
All PCR products were run on 1% agarose gel with 0.5 µg/ml ethidium bromide in 1x
TAE buffer and visualized on 312 nm UV box (Model TF-35M UV transilluminator
Villber Lourmat, France).
31
Materials and methods
Fig. 1. Map of pBK-CMV vector (reproduced from Stratagene catalogue)
32
Materials and methods
1.2 Recovery of DNA Fragments from Agarose Gel
The QIAquick Gel Extraction Kit (Qiagen, USA) was used to recover the DNA
fragments of interest from the agarose gel according to the manufacturer instructions.
Briefly, the gel slice containing the DNA band was cut from the gel and melted at
50°C in Buffer QX1 for 10 minutes and then loaded into a QIAquick spin column. The
volume of Buffer QX1 used was approximately three times of the gel slice volume.
The column was centrifuged at 14,000 rpm for 1 minute, washed by adding 0.75 ml of
Buffer PE, and spun again. After removing residual Buffer PE by spinning at 14,000
rpm for 1 minute, 30 µl of H2O was added to the centre of the column. The column
was incubated at room temperature for 1 minute and the DNA fragment was eluted
into a 1.5-ml centrifuge tube by centrifugation at 14,000 rpm for 1 minute.
1.3 Ligation
The recovered PCR products were cloned into the pT7Blue T-vector system
(Novagen, USA) (Fig. 2). The pT7 Blue T-vector was prepared by the manufacturer by
cutting the vector with EcoRV and adding a 3’ terminal thymidine to both ends. These
single 3’-T overhangs at the insertion site greatly improve the efficiency of ligation of
PCR products into the vector because the Taq DNA polymerase generates a 3’ adenine
overhang in the PCR products. The ligation reaction was carried out in 20 µl reaction
volume, containing 2 µl of 10X ligation buffer (0.3 M Tris-HCl, pH 7.8; 0.1 M MgCl2;
0.1 M DTT and 5 mM ATP), insert DNA, vector DNA and 1 unit T4 DNA ligase
(Gibco BRL, USA). The molar ratio of insert-to-vector DNA was usually 3:1. Ligation
reaction was incubated at 16°C overnight.
33
Materials and methods
Fig. 2. Map of pT7Blue vector (reproduced from Novagen catalogue)
34
Materials and methods
1.4 Transformation
1.4.1 Preparation of Competent Cells
For the preparation of competent bacteria cells, 2 ml of LB broth was
inoculated with a single fresh colony of Escherichia coli strain DH5α and incubated at
37°C with 250 rpm shaking overnight. The following morning, 0.5 ml of the culture
was re-inoculated into a 250 ml flask containing 50 ml of LB broth and shaken at 250
rpm at 37°C until OD600 reached around 0.5. The culture was transferred into 50 ml
Falcon 2070 tubes and chilled on ice for 15 minutes. Cells were pelleted by
centrifugation at 1,000 g at 4°C for 15 minutes. The pelleted cells were drained
thoroughly and resuspended in 1/3 starting culture volume of RF1 (100 mM RbCl; 50
mM MnCl2; 30 mM potassium acetate; 10 mM CaCl2 and 15% glycerol). After
incubation on ice for 15 minutes, the cells were spun down and resuspended in 1/12.5
of the original volume of RF2 (10 mM MOPS; 10 mM RbCl; 75 mM CaCl2; 15%
glycerol). After another 15 minutes incubation on ice, the competent cells were
transferred into 1.5-ml microcentrifuge tubes in aliquots of 100 µl and fast-frozen in
liquid nitrogen. These aliquots can be stored at -80°C for several months.
1.4.2 Transformation
Normally 10 µl of ligation reaction was added into 100 µl of E.coli DH5α
competent cells and incubated on ice for 30 minutes. This was followed by a heat
shock at 37°C for 90 seconds after which the tube was immediately placed on ice for 2
minutes. 900 µl of LB medium was added to the transformation mixture and incubated
at 37°C for 1 hour with shaking at 200 rpm. 1/10 and 9/10 of the transformation
reaction mixture was spread onto two separate LB plates supplemented with ampicillin
35
Materials and methods
(50 µg/ml) in order to produce proper density of transformant colonies. The plates
were incubated at 37°C overnight.
1.5 Colony Screening
PCR can be applied to screen for correct recombinant DNA directly using the
bacteria colonies, as DNA would be effectively released from bacteria cells under the
repeated high temperature during PCR. One pair of vector primers flanking cloned
insert will define the size of insert by a PCR reaction.
For PCR screening, colonies to be examined were marked in numerical order.
A toothpick was used to touch the colony and the attached bacteria were spread to the
bottom of a PCR tube, which was preloaded with 20 µl of PCR mixture, containing 0.6
units of Taq DNA polymerase, 2 µl of 10X PCR buffer, 1 µl of 2 mM dNTP mix and
0.2 µg of each sense and antisense primers. T7 and U19 primers were used for the PCR.
The PCR program includes initial denaturation at 94°C for 5 minutes, followed by 30
cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds and
elongation at 72°C for 1.5 minutes. PCR product was examined in 1% agarose gel.
Colonies that yielded PCR products with expected size were inoculated for plasmid
DNA preparation.
1.6 Isolation and Purification of Plasmid DNA
Small-scale preparation of plasmid DNA was carried out using the Wizard Plus
SV Miniprep Kit (Promega, USA) according to the centrifugation protocol as
described by the manufacturer. This protocol involved alkaline lysis, binding of
plasmid to a spin column, followed by elution of DNA with water.
36
Materials and methods
3 ml of overnight bacteria culture in LB-ampicillin (50 µg/ml) medium was
harvested by centrifugation at 10,000 g for one minute using the 5417C centrifuge
(Eppendorf, Germany). The bacterial pellet was resuspended in 250 µl of Cell
Resuspension Solution (50 mM Tris-HCL, pH 7.5; 10 mM EDTA; 100 µg/ml RNase
A). 250 µl of Cell Lysis solution (0.2M NaOH, 1% SDS) was added to the bacterial
suspension and mixed by gently inverting the tube several times. 10 µl of alkaline
protease (25 µg/µl) was then added and incubated for 5 minutes at room temperature.
350 µl of neutralisation solution (4.09 M guanidine hydrochloride; 0.759 M potassium
acetate; 2.12 M glacial acetic acid) was added to neutralize the mixture. After
centrifugation at 14,000 rpm for 10 minutes, the clear lysate was transferred to a spin
column in a collection tube and centrifuged for 1 minute at 14,000 g. The flow-through
was discarded and the column was re-inserted into the collection tube. 750 µl of wash
solution (60 mM potassium acetate; 10 mM Tris-HCl, pH 7.5; 60% ethanol) was added
to the spin column and centrifuged at 14,000 rpm for 1 minute. This step was repeated
with 250 µl wash solution and centrifuged at 14,000 rpm for 2 minutes. The spin
column was next transferred to a sterile 1.5-ml microcentrifuge tube and 50 µl of
sterile water was applied, and left to stand for one minute. Plasmid DNA was eluted by
centrifugation at 14,000g for one minute.
1.7 Automated Sequencing
Automated sequencing reactions were carried out using the ABI Prism Big Dye
Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems,
USA). The gene specific primers used to obtain the full-length sequence were KR-int
5'-GACCCTGTCTCTATACACCA-3’
and
ES34-int
5'-
CGGTCAGCGCAGGCCGTCCG-3’. Each sequencing reaction (10 µl) contained 4 µl
37
Materials and methods
of Terminator Ready Reaction Mix (Perkin Elmer, USA), 200-500 ng of double strand
DNA, and 3.2 pmol of primer. PCR was performed using the GeneAmp PCR System
9600 (Perkin Elmer) with 25 cycles of 96°C/10 seconds, 50°C/5 seconds and 60°C/4
minutes, and finally hold at 4°C. Ethanol precipitation was carried out to purify the
extension products. 2 µl of 3 M NaOAc (pH4.6) and 50 µl of 95% ethanol was mixed
with the 20 µl reaction mix, and incubated at room temperature for 15 minutes. The
tube was spun for 20 minutes at 14,000 rpm, 4°C. The pellet was rinsed with 250 µl of
70% ethanol and air-dried. The DNA pellet was dissolved in 4 µl of loading dye (50
ml contains 1 ml of 25 mM EDTA, pH8.0; 10 ml of deionised formamide; 50 mg
Dextran blue and 39 ml of H2O) and heated to 92°C for 3 minutes. Samples were then
chilled on ice for 2 minutes before being loaded into the wells of 6% polyacrylamide
sequencing gel (50 ml of gel mix contains 5 ml of long ranger gel solution; 5ml of 10x
TBE; 26 ml of H2O; 18 g of urea; 250 µl 10%APS and 35 µl TEMED). The
electrophoresis was carried out at 1,690 volts for 5-9 hours. The sequencing ladders
were analysed automatically by an ABI PRISM 377 DNA sequencer system and
software.
1.8 Sequence Homology Search
DNA sequences were submitted to FASTA (http://www2.ebi.ac.uk/fasta3/) for
sequence homology search. The search was against all DNA entries in EMBL database
(recent release + new releases). Motif searches were conducted using Pfam
(http://www.sanger.ac.uk/Software/Pfam/search.shtml). Sequence alignments were
performed using CLUSTAL program.
38
Materials and methods
2. Characterization of Zebrafish klhl Expression
2.1 Northern Hybridisation
2.1.1 Isolation of Total RNA
Total RNA from zebrafish embryos and different adult tissues were extracted
using TRIzol reagent (Gibco BRL). Briefly, about 200 embryos or 100 mg of tissues
were frozen in liquid nitrogen and homogenized in 1 ml of TRIzol reagent. The
homogenate was incubated at room temperature for 5 minutes to allow nucleoproteins
to dissociate before chloroform was added. The mixture was shaken vigorously by
hand for 15 seconds and incubated for another 5 minutes. This was followed by
centrifugation at 12,000 g for 15 minutes at 4oC to separate the aqueous and organic
phases. 500 µl of aqueous phase was then transferred to a new tube and an equal
amount of isopropanol was added. The RNA was precipitated by incubation at room
temperature for 10 minutes, following which it was pellet by centrifugation at 12,000 g
for 10 minutes at 4oC and washed with 1 ml of 70% ethanol. The RNA pellet was then
dissolved in 15 µl of DEPC (diethyl pyrocarbonate) treated water. RNA was quantified
by optical density reading at 260 nm and 280 nm using UV-1601 spectrophotometer
(SHIMADZU, Japan). One unit of OD260 is equivalent to 40 µg/ml of RNA,
OD260:OD280 ratios >2.0 indicates good quality of RNA products.
2.1.2 Formaldehyde RNA Gel Electrophoresis and Blotting
10 µg of total RNA was fractionated on a 1.2% denaturing agarose gel (100 ml
of gel contains 1.2 g agarose, 10 ml of 10X MOPS, 73 ml of H2O and 17 ml of 37%
formaldehyde). Each RNA sample contained 50% formamide, 1X MOPS, 7%
formaldehyde and 0.1 mg/ml ethidium bromide; and was heated at 65°C for 10
minutes before loading with 1X loading buffer (0.4% bromophenol blue; 6% sucrose
39
Materials and methods
in water). The gel was run at 75 volts in running buffer containing 1X MOPS and 3%
formaldehyde until the dye was near the end. After electrophoresis, the gel was rinsed
in distilled water and a picture was taken with a ruler to show the distance among the
bands. The RNA was transferred to Hybond™-N nylon membrane (Amersham, USA)
overnight using 20X SSC (3 M NaCl; 0.3 M sodium citrate, pH7.0) as transfer buffer.
The membrane was then air-dried and cross-linked by UV irradiation on a 312 mm UV
box for 3 minutes.
2.1.3 Labelling of Radioactive Probe
The cDNA fragments was amplified by PCR using the vector primers and used
as templates for probe labelling with the Random Primers DNA Labelling System
(Gibco BRL, USA). Random priming involved the use of random hexamers that
anneal at random sites along the template. Klenow polymerase was used to extend the
hexamers and at the same time to incorporate labeled mucleotide, [α-32P] dATP (Spp.
Act. 3000 Ci/mmole; 10 mCi/ml in aqueous solution) (Amersham, USA) into the
probe.
25 ng of DNA in 10.5 µl of MilliQ water was denatured at 100°C for 5 minutes
and cooled in ice immediately. 1 µl each of dGTP, dTTP and dCTP with 7.5 µl of
Random Primers Buffer Mixture and 2.5 µl of
32
P-dATP was added to make up to a
total volume of 25 µl after the addition of 0.5 µl Klenow Fragment. The reaction was
incubated at room temperature for 1 hour and terminated by adding 2.5 µl of the STOP
solution to the reaction mixture.
After labelling, the nick column (Pharmacia, Sweden) was used to remove
unincorporated radiolabelled nucleotides as this produces high signal-to-background
ratios on hybridisation results. The column contains Sephadex® G-50 DNA Grade
40
Materials and methods
which serves as a gel filtration matrix. Large fragments that do not interact with the
matrix are eluted first while unincorporated nucleotides that are trapped in the pores of
the matrix are eluted later. First, the cover of the column was removed to allow the
column buffer to drain out. The column was then washed with 2 ml TE buffer (10 mM
Tris; 10 mM EDTA, pH 8.0). After that, the labelling reaction was loaded to the top of
the column. The incorporated DNA fragments were eluted with 400 µl of TE buffer
each for three times. The elutions were collected with three individual Eppendorf tubes
and monitored for radioactivity by liquid scintillation counting. Normally, the second
elution with the highest radioactivity was used as the probe for the hybridisation. 2 µl
of the eluate was mixed with 3 ml of scintillation fluid (BCS, Amersham) and
monitored for radioactivity. The counting was carried out on a Wallace Guardian 1414
Liquid Scintillation Counter (Biolaboratories, USA) for 120 seconds and 1 x 106
cpm/ml of hybridisation buffer was used for the hybridisation.
2.1.4 Hybridisation
Prehybridisation was conducted to prevent non-specific hybridisation of the
probe. The denatured salmon sperm DNA acted as a blocking reagent that helped to
reduce the background signal. The membranes were placed in a hybridisation-rolling
bottle (HB-OV-BS, Hybaid) with the DNA side facing inwards. The bottle contains 5
ml of hybridisation buffer (50% formamide; 5X Denhardt’s solution; 4X SET; 0.2%
NaPPi; 25 mM phosphate buffer; 0.5% SDS, 100 µg/ml denatured salmon sperm DNA
and 10% w/v dextran sulfate) (for 20X SET solution, 3 M NaCl; 0.6 M Tris, pH 8.0
and 40 mM EDTA). Salmon sperm DNA stock (10 mg/ml) was denatured in boiling
water for 5 minutes and then kept on ice for 5 minutes. The bottle was then transferred
41
Materials and methods
to a hybridisation incubator (Mini Oven MKII, Hybrid). Prehybridisation was carried
out at 42°C for more than 2 hours with a spinning speed of 7 rpm.
Labelled probe was denatured at 92°C for 5 minutes in a heat-block (type
17600, Thermolyne, U.S.A) and then immediately chilled on ice for 5 minutes. The
hybridisation bottle was taken out of the incubator and probe was added to the buffer
to the final concentration of 1 x 106 cpm/ml. Hybridisation was performed at 42°C for
16 hours.
2.1.5 Washes and Autoradiography
After hybridisation, the buffer was discarded and 20 ml of washing solution
(2X SET; 0.2% NaPPi and 0.5% SDS) was added. The hybridisation bottle was
agitated by gentle shaking at room temperature for 15 minutes with two changes of
solution after which the following two washing steps were performed at 65°C for 20
minutes each. Pre-warmed wash solution was used and the incubator was preset at
65°C. A final stringent wash was carried out using a final wash solution (0.2X SET;
0.5% SDS) at 65°C for 20 minutes. This was conducted only if the radioactivity count
was still too high.
The membrane was wrapped with a Saran polyethylene to keep the membrane
moist. An X-omat (Kodak) film was placed and autoradiographed at -80°C for
overnight. The autoradiogram was developed using the M35 X-omat developer (Kodak,
USA).
42
Materials and methods
2.1.6 Membrane Stripping
The probe hybridised on the membrane was stripped away by washing the
membrane in striping solution (0.05x SET, 0.1% SDS) at 80 °C for 30 minutes. The
membrane was air-dried and ready for reprobing.
2.2 Whole-Mount In Situ Hybridisation on Zebrafish Embryos
2.2.1 Probe synthesis
5 µg of plasmid DNA was linearized at the 5’ end of the cDNA insert by SmaI
digestion at 37 °C for 45 minutes. The digestion reaction was stopped by
phenol/chloroform extraction, followed by ethanol precipitation. The linearized DNA
was resuspended in 20 µl of water. 1 µg of linearized DNA was used to synthesis the
digoxygenin (DIG)/Fluorescein probe. The reaction was performed at 37 °C for 2
hours in a total volume of 20 µl containing 4 µl of 5x transcription buffer (Stratagene),
2 µl of DIG/Fluorescein-NTP mix [10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM
UTP, and 3.5 mM DIG/Fluorescein-UTP (Boehinger, Germany)], 1 µl of RNAse
inhibitor (40 U/µl) (Promega) and 1 µl of T7 RNA polymerase (50 U/µl) (Promega).
Following the reaction, 2 µl of RNAse free DNAse I (Promega) was used to digest the
DNA template at 37 °C for 15 minutes. Digestion was stopped by adding 1 µl of 0.5 M
EDTA (pH 8.0). 2.5 µl of 4 M LiCl and 75 µl of cold 100% ethanol was added to
precipitate the RNA. After washing with 70% ethanol, the RNA probe was
resuspended in 100 µl of DEPC treated water. The probe was purified using Chroma
spin 100 DEPC H2O columns (Clontech, USA) by spinning at 700 g for 5 minutes to
remove the impurity and small RNA fragments.
43
Materials and methods
2.2.2 Preparation of staged zebrafish embryos
Zebrafish (Danio rerio) were purchased from local aquarium fish farms and
their embryos were staged according to 'The Zebrafish Book' (Westerfield, 1995) and
presented as hours post fertilization (hpf) at 28.5oC. To avoid pigment development in
later stage (>30 hpf) embryos, 0.003% PTU (1-phenyl-2-thiourea, Sigma) was added
when the embryos were 10-16 hpf. Staged embryos were fixed in 4% PFA
(paraformaldehyde)/ PBS (phosphate buffered saline - 0.8% NaCl; 0.02% KCl;
0.0144% Na2HPO4; 0.024% KH2PO4, pH7.4) for 12 to 24 hours at room temperature
or 4°C. Embryos younger than 16 hpf were fixed before dechorionization and the
chorion was removed afterwards. Embryos older than 16 hpf were dechorionated
before fixation. Older embryos with tails were hibernated on ice for 15 minutes before
fixation to prevent curling of tails. After fixation, the embryos were washed in PBST
(PBS, 0.1% Tween 20) twice for one minute, four times for 30 minutes at room
temperature on a nutator (Clay Adams, Becton Dickinson, USA). Embryos at 24 hpf
and older were treated with proteinase K (10 µg/ml, Boehringer, Germany). The time
of exposure depended upon the embryonic stages and the specific activity of proteinase
K, which varied from batch to batch. For most cases, the time below was adopted.
16-24 hpf
3-4 minutes
24-32 hpf
5-6 minutes
32-50 hpf
10-20 minutes
50-72 hpf
20-40 minutes
To stop the proteinase K reaction, the proteinase K solution was removed
completely, and the embryos were fixed again in 4% PFA/PBS for 20 minutes at room
temperature. Embryos were then washed in PBST twice for 1 minute each and four
times for 20 minutes each.
44
Materials and methods
2.2.3 In Situ Hybridisation
Embryos were prehybridised in 500 µl PBST + 500 µl hybridisation buffer (50%
formamide, 5X SSC, 50 µg/ml heparin, 500 µg/ml yeast tRNA, 0.1% Tween 20, 10 mM
citric acid, pH6.0) at room temperature for an hour. The solution was replaced with 1 ml
of fresh hybridisation buffer and the embryos were prehybridised at 70oC for 2-5 hours. 1
µl of DIG/Fluorescein probe was diluted in 200 µl of hybridisation buffer and denatured at
80oC for 5 minutes, followed by 5 minutes of ice bath. Embryos of different stages were
selected and mixed together and buffer containing the probe was added. Hybridisation was
performed at 70oC in a circulating water bath overnight.
The following morning, the probe solution was removed and replaced with
prewarmed 5X SSC. This was left to wash at 70oC for two hours, followed by 0.2X SSC
at 70oC for two hours. The embryos were then washed at room temperature twice for five
minutes in PBS.
2.2.4 Incubation with Preabsorbed Antibodies
Commercial
DIG
and
fluorescein-alkaline
phosphatase
(AP)
antibodies
(Boehringer) should be preincubated with biological tissues, preferably of the same stage
as the sample that later will be used for detection of signals in order to decrease the
staining background and to increase signal-to-noise ratio. In the study, anti-DIG and
Fluor-AP was diluted to 1:500 and 1:50 in 10% FCS/PBS (fetal calf serum in PBS)
respectively and incubated with 50 zebrafish embryos of any stages on a nutator at 4°C
overnight. After that, the antibodies solution was transferred to a new tube and diluted to
1:5000 and 1:500 with 10% FCS/PBS. 10 µl of 0.5 M EDTA (pH 8.0) and 5 µl of 10%
45
Materials and methods
sodium azide were added to a volume of 10 ml antibody solution to prevent bacterial
growth. The preabsorbed antibody was stored at 4°C and can be used repeatedly.
Following hybridisation, the embryos were incubated in 10% FCS/PBS for two
hours at room temperature to block non-specific binding sites for antibody. The blocking
solution was replaced with alkaline-phosphatase (AP)-coupled anti-DIG Fab fragments,
and the embryos were incubated at 4oC overnight.
2.2.5 Staining
The next day, embryos were washed in PBST twice for one minute, four times for
30 minutes, followed by twice for five minutes in PBS, once for 30 seconds and twice for
10 minutes in buffer 9.5 (0.1 M Tris-HCl, pH 9.5, 50 mM MgCl2, 10 mM NaCl, 0.1%
Tween 20) on a nutator at room temperature. For staining, 4.5 µl of NBT (50 mg/ml
nitroblue terazolium in 70% dimethyl formamide, Boehringer Mannhein, Germany) and
3.5 µl of BCIP (5-bromo-3-chloro-3-indolyl phosphate, Boehringer Mannhein, Germany)
were added into 1 ml of buffer 9.5 and placed in the dark. Staining was stopped by
washing the embryos twice for 10 minutes in PBS. Embryos were then transferred to 4%
PFA/PBS and kept at 4oC.
2.2.6 Mounting and photography
Selected embryos were washed with PBS twice for 10 minutes each and
transferred to 50% glycerol/PBS, equilibrated at room temperature for a couple of hours.
For whole-mount, a single chamber was made by placing stacks of 2-3 small cover
glasses on both sides of a 25.4 x 76.2 mm microscope slide. Small cover glasses in the
46
Materials and methods
stacks will be perfectly solid 1 hour after placing a drop of Permount between them.
Selected embryo was transferred to the chamber in a small drop of 50% glycerol/PBS and
oriented by a needle. A 22 x 44 mm cover glass with a small drop of the same buffer was
superimposed onto the embryo. The orientation of the embryo can be adjusted by gently
moving the cover glass.
For flat specimen, the yolk of selected embryo was removed completely by needles.
The embryo without yolk was then placed onto a slide with a small drop of 50%
glycerol/PBS and adjusted to a proper orientation by removing excess of liquid and by
needles. A small fragment of cover glass (as small as possible) was covered onto the
embryo. Care was taken to avoid bubbles and a drop of 50% glycerol/PBS was added to
fill the space under the cover glass. This specimen was sealed with nail polish along the
edge of the cover glass to prevent it from drying.
For cross-section, some of the stained embryos were embedded in a 1.5%
agarose/sucrose block and equilibrated in 30% sucrose overnight. On the following day,
embedded embryos were mounted on a holder using tissue freezing medium (Jung), and
sectioned with a cryostat microtome (Leica CM 1900) in transverse orientation (15 µm).
Sections were placed on Fisherbrand Superfrost/Plus microscope slides, and mounted
using glycerol/PBS (1:1) solution. The slides were sealed with nail varnish.
Photos were taken using a camera mounted to an Olympus AX-70 microscope
(Olympus, Japan). The film used was Kodak Gold 200 ASA.
2.3 Two-Colour Whole-mount In Situ Hybridisation
In two-color whole-mount in situ hybridisation, two different RNA probes labelled
with DIG and Fluorescein respectively are used for the same embryos. Fluorescein
47
Materials and methods
labeling is performed following the same procedure as in DIG labelling. However, instead
of using DIG-UTP, Fluor-UTP is used instead. For hybridisation, the two probes are
added to the same tube in a ratio of 2:1 Fluorescein to DIG (Fluorescein is less sensitive to
be detected than DIG). After incubation at 68ºC for overnight, probes were removed by
washing in 2XSSCT (2XSSC + 0.1% Tween 20) at 68ºC for 2 hours, followed by another
wash in 0.2XSSCT (0.2XSSC + 0.1% Tween 20) at 68ºC for 2 hours. The DIG detection
was first carried out as described in the previous sections (see 2.2.4 and 2.2.5).
Following the DIG staining with NBT/BCIP, the embryos were washed with MA
buffer (0.15 M maleic acid; 0.1 M NaCl; pH7.5) twice for 10 minutes each. To remove the
phosphatase activity of first antibody, the embryos were incubated with 0.1 M glycine (pH
2.2) for 30 minutes at room temperature. After that, the embryos were washed in PBST
four times for 10 minutes each and then incubated in blocking buffer (5% Blocking
Reagent in MA buffer, Boehringer) for 2 hours at room temperature. Subsequently,
embryos were incubated with Anti-Fluorescein-AP (see section 2.2.4) at 4ºC for overnight.
To detect the fluorescein signal, the embryos were first washed with MA buffer 4
times for 1 hour each, followed by wash with Buffer 8.2 (0.1 M Tris-HCl, pH 8.2; 50 mM
MgCl2; 10 mM NaCl; and 0.1% Tween 20) three times for 5 minutes each at room
temperature. Embryos were then stained in staining buffer, a 1:1 mixture of Fast Red
solution (made by dissolving ½ Fast Red tablet [Boehringer] in 1 ml Buffer 8.2, spinning
down undissolved particles and transferring the supernatant to a new tube) and NAMP
solution (a 1:100 dilution of NAMP stock [50 mg/ml Naphthol As-MX, Sigma] in Buffer
8.2) for 1-3 hours. The stained embryos were washed in PBST twice for 10 minutes each
and can be stored in 4% PFA/PBS for several months.
3. Characterization of Human Ortholog KLHL
48
Materials and methods
3.1 Identification of Human Orthologous Gene KLHL
The putative human homolog of zebrafish klhl was identified by searching the
Fasta databases using the DNA and amino acid sequence of klhl. This orthology was
confirmed by synteny analysis of both the zebrafish and human genomes. Mapping
positions of zebrafish ESTs were identified from the publicly available RH panel maps
found
at
http://zfin.org,
http://wwwmap.tuebingen.mpg.de
and
http://www.ncbi.nlm.nih.gov/genome/guide/D_rerio.html. The map positions of human
genes
were
obtained
from
http://www.ncbi.nlm.nih.gov/LocusLink
and
http://www.ensembl.org.
3.2 Cloning of KLHL Fragment
Based on the sequence annotated in Ensembl (http://www.ensembl.org), we
designed two gene-specific primers that spanned the second exon of the gene, HC34F (5’ACAGATGCTTCTTATGGCCC-3’) and HC34R (5’-GAAATCCATCCATCACAGCC3’) to amplify a 0.9 kb fragment of KLHL from human DNA. The template genomic DNA
was extracted from the leucocytes of peripheral blood and was provided generously by a
member of the laboratory (Tong, 2001). The conditions of the reaction were as previously
described in section 1.1. The fragment cloned was sequenced as described in 1.7 to
ascertain the desired DNA fragment was cloned.
3.3 Northern Blot Analysis
A MTN blot containing 2 µg of poly A+ RNA isolated from a variety of human
tissues (Clontech #7760-1, USA) was hybridised to a
32
P-radiolabelled KLHL probe
according to the manufacturer instructions. The probe was generated as previously
49
Materials and methods
described in 2.1.3. Briefly, the MTN bolt was prehybridised in 10 ml of ExpressHyb
Solution (Clontech) for 30 minutes at 68oC with rolling in a hybridisation incubator.
Radioactively labelled probe was heat-denatured at 95-100oC for 5 minutes and
immediately chilled on ice. The prehybridisation solution was replaced with fresh
ExpressHyb solution containing the probe to a final concentration of 1 x 106 cpm/ml and
hybridisation was carried out at 68oC for 1 hour.
Following hybridisation, the solution was discarded and 10 ml of washing solution
1 (2x SSC, 0.05% SDS) was added. The blot was washed for 40 minutes with continuous
agitation at room temperature with 2 changes of solution. A further two washes were
performed with wash solution 2 (0.1x SSC, 0.1% SDS) at 50oC. The blot was removed
from the bottle with forceps and immediately covered with Saran wrap. It was
subsequently exposed to X-ray film at –80oC overnight.
The probe was stripped away by washing the blot with 0.5% SDS preheated to 90100oC for 10 minutes following the blot was left to air-dry. The blot was subsequently
hybridised with a β-actin cDNA probe provided by the manufacturer of the MTN blot
(Clontech) used as an RNA loading and transfer control.
50
Results
Chapter III
Results
51
Results
1. Identification of ES34 as a Putative Kelch Repeat Protein
We identified ES34 through a systematic search for novel genes with tissuespecific expression patterns during zebrafish embryogenesis (Wu, 1999). The ES34
clone, selected for its somite-specific expression pattern, contained an insert of 948 bp
with a major open reading frame (ORF) encoding a putative polypeptide of 188 amino
acids. At the DNA level, apart from identifying a number of ESTs and genomic
sequences, sequence homology searches did not reveal any significant matches to any
known gene in the database. Analysis of the translated amino acid, however, revealed
that it contained a kelch motif and showed ~25% homology to members of a protein
family called the kelch-repeat superfamily (reviewed in Adams et al., 2000).
The kelch motif is an ancient and evolutionarily conserved sequence motif of
44-56 amino acids in length. First identified in Drosophila kelch (Xue and Cooley,
1993), over 28 kelch- containing proteins have so far been isolated and characterized
(Adams et al., 2002) in organisms as diverse as viruses, fungi to mouse and human.
These proteins typically contain a series of four to seven motifs that form kelch repeats
and have been grouped together into the kelch-repeat superfamily. Members of this
superfamily have diverse range of biological roles that range from actin-binding to cell
regulation and are structurally diverse but are grouped together based on the presence
of the kelch repeat.
Search against motif database PFAM (http://www.sanger.ac.uk/Software/Pfam/)
analysis of the putative polypeptide encoded by ES34 revealed that the protein
contained only 3 kelch repeats. In addition, the ORF of the predicted gene sequence
did not have an in-frame stop codon upstream of the first ATG. Thus, ES34 may be a
partial cDNA clone and additional cloning sequences were sought to obtain the fulllength clone of this gene. This gene will be termed klhl henceforth after the various
52
Results
human kelch-like genes belonging to the same familiy (Solysik-Espanola et al., 1999;
Lai et al., 2000; Nemes et al., 2000; Braybrook et al., 2001; Wang et al., 2001).
2. Molecular Cloning of Zebrafish klhl
Vector- and gene-specific primers were used to amplify the missing 5’ cDNA
from a cDNA library made from 24 hpf embryos constructed using the vector pBKCMV (Fig. 1). T3 vector primer and KR1 5’-CAGCATCTAGGGACTTCCAT-3’ were
used for the primary PCR. The products were then subjected to secondary PCR with
T3 and nested gene-specific primer KR2 5’-TTTGCCACTGGTTTGAGGAT-3’. The
size of the PCR product was estimated to be about 1.5 kb. The PCR products were gel
extracted for direct ligation into pT7-Blue vector (Fig. 2). Clones carrying the correct
insert size (~1.5 kb) were sequenced from both ends using vector primers U19 and T7.
As the sequences obtained were relatively short and had no overlapping regions,
internal primers were designed for further sequencing. The sequences were aligned
using the DNAMAN program and were found to be continuous with the original ES34
clone. The assembly of the sequences from the two clones is shown in Fig. 3 and the
total length of the nucleotide sequence is 2326 bp long.
3. Sequence Analysis of Zebrafish klhl
The 2326-bp sequence was translated and found to contain an ORF encoding a
635 amino acid product (Fig. 3). We infer the ATG codon at nucleotide (nt) residue 76
to be the true start site of translation because it begins the longest reading frame and is
preceded by numerous stop codons in the 5’ untranslated region (UTR) for all three
reading frames. In addition, the putative methionine initiation codon occurs in the
53
Results
Fig. 3. Nucleotide and predicted amino acid sequence of zebrafish klhl cDNA. The
2326 bp sequence was assembled from the 5’ RACE fragment and ES34 clone. The
start nucleotide of ES34 is indicated by an arrowhead. The proposed start codon is
highlighted in bold. The stop codon is indicated by an asterisk, and the potential
polyadenylation signal AATAAA is doubly underlined. The BTB/POZ domain is
underlined, and the six kelch repeats are boxed up. Gene-specific PCR primers used
for 5’ RACE and sequencing are shown in bold and indicated by overhead arrows.
54
Results
1
GAGGTGCAAGTCTGTCTTTCTCCTGCACCCACTTCATCTCCACATCGGTCTTGGCTGTGA
61
1
CCCATCACTACAGTCATGGCACCCAAAAAGAACAAGGCGGCTAAGAAGAGCAAAGCCGAT
M A P K K N K A A K K S K A D
121
16
ATCAACGAGATGACGATCATGGTCGAGGACAGCCCCTCCAACAAAATCAACGGGCTCAAC
I N E M T I M V E D S P S N K I N G L N
181
36
ACGCTCCTGGAGGGCGGAAACGGCTTTAGTTGCATCTCCACCGAAGTCACCGACCCCGTC
T L L E G G N G F S C I S T E V T D P V
241
56
TATGCACCAAACCTCCTGGAGGGTCTGGGCCACATGAGGCAGGACAGCTTCCTCTGTGAC
Y A P N L L E G L G H M R Q D S F L C D
301
76
CTCACGGTAGCAACCAAATCCAAGTCCTTCGACGTTCACAAAGTAGTGATGGCATCCTGC
L T V A T K S K S F D V H K V V M A S C
361
96
481
136
AGCGAGTACATCCAAAACATGCTCCGGAAGGATCCGTCTCTAAAGAAGATTGAGCTCAGT
S E Y I Q N M L R K D P S L K K I E L S
KR-int
GATTTATCCCCAGTTGGTTTGGCTACAGTCATCACTTATGCCTATTCTGGAAAACTGACC
D L S P V G L A T V I T Y A Y S G K L T
KR-int
CTGTCTCTATACACCATCGGCAGCACTATATCTGCAGCCTTGCTCCTCCAGATCCACACT
L S L Y T I G S T I S A A L L L Q I H T
541
156
TTGGTGAAAATGTGTAGTGATTTCTTAATGCGGGAGACTAGCGTGGAGAATTGCATGTAT
L V K M C S D F L M R E T S V E N C M Y
601
176
GTGGTCAACATTGCCGACACGTACAATCTAAAAGAGACGAAGGAAGCTGCTCAGAAGTTC
V V N I A D T Y N L K E T K E A A Q K F
661
196
ATGCGAGAGAACTTCATTGAGTTCTCCGAGATGGAGCAGTTCCTCAAACTCACCTACGAG
M R E N F I E F S E M E Q F L K L T Y E
721
216
CAAATCAACGAGTTCCTCACAGACGACTCACTTCAGTTGCCTTCAGAGCTCACGGCTTTC
Q I N E F L T D D S L Q L P S E L T A F
781
236
CAGATCGCAGTCAAGTGGTTGGATTTTGATGAAAAGAGGTTGAAGTACGCTCCTGATCTG
Q I A V K W L D F D E K R L K Y A P D L
841
256
CTGTCCAACATCCGTTTTGGCACCATCACCCCCCAGGATCTTGTTAGTCACGTGCAGAAC
L S N I R F G T I T P Q D L V S H V Q N
901
276
GTTCCCAGGATGATGCAGGATGCCGAGTGCCACCGTTTGCTGGTCGACGCCATGAATTAC
V P R M M Q D A E C H R L L V D A M N Y
961
296
CACCTGCTGCCGTTCCAGCAGAACATCCTTCAATCCCGGAGGACCAAAGTTCGTGGAGGT
H L L P F Q Q N I L Q S R R T K V R G G
ES34-int
CTCCGAGTTCTGCTTACTGTTGGCGGACGGCCTGCGCTGACCGAAAAGTCTCTCAGCAAG
L R V L L T V G G R P A L T E K S L S K
Kelch R1
GACATTCTCTACAGGGACGAGGATAATGTCTGGAACAAGCTGACGGAGATGCCTGCTAAG
D I L Y R D E D N V W N K L T E M P A K
421
116
1021
316
1081
336
1141
356
1201
376
AGCTTCAATCAGTGTGTGGCCGTTTTGGACGGTTTCCTTTACGTGGCTGGAGGAGAAGAC
S F N Q C V A V L D G F L Y V A G G E D
Kelch R2
CAGAATGATGCAAGAAACCAGGCAAAGCATGCAGTCAGCAATTTCAGCAGATACGACCCC
Q N D A R N Q A K H A V S N F S R Y D P
55
Results
1261
396
CGATTCAACACGTGGATCCACCTAGCCAACATGATTCAGAAGCGTACTCATTTCAGCCTC
R F N T W I H L A N M I Q K R T H F S L
1321
416
1501
476
AACACCTTCAATGGTCTGCTCTTCGCCGTCGGGGGCCGTAATTCTGACGGCTGCCAGGCG
N T F N G L L F A V G G R N S D G C Q A
ES34
Kelch R3
KR2
KR1
TCTGTCGAGTGCTACGTCCCATCCTCAAACCAGTGGCAAATGAAAGCCCCAATGGAAGTC
S V E C Y V P S S N Q W Q M K A P M E V
KR1
CCTAGATGCTGCCATGCCAGCTCAGTTATCGATGGCAAGATCTTGGTTAGCGGTGGTTAC
P R C C H A S S V I D G K I L V S G G Y
Kelch R4
ATTAACAACGCCTACTCTCGAGCCGTCTGTTCCTACGACCCATCCACTGATAGCTGGCAG
I N N A Y S R A V C S Y D P S T D S W Q
1561
496
GATAAAAACAGCCTGAGCAGCCCGAGAGGATGGCACTGTTCGGTGACCGTCGGAGATCGT
D K N S L S S P R G W H C S V T V G D R
1621
GCTTACGTGCTCGGCGGCAGTCAACTGGGCGGACGTGGAGAGAGAGTAGACGTCTTGCCT
516
A Y V L G G S Q L G G R G E R V D V L P
Kelch R5
GTTGAATGCTATAACCCTCACTCTGGCCAGTGGAGCTACGTTGCCCCCTTGCTGACGGGA
V E C Y N P H S G Q W S Y V A P L L T G
1381
436
1441
456
1681
536
1741
556
1801
576
GTGAGCACTGCAGGCGCTGCCACCTTGAATAACAAGATCTACCTCTTGGGCGGCTGGAAT
V S T A G A A T L N N K I Y L L G G W N
Kelch R6
GAGATTGAGAAGAAGTACAAGAAATGCATTCAGGTTTATAATCCTGATCTTAACGAATGG
E I E K K Y K K C I Q V Y N P D L N E W
1861
596
ACTGAAGATGACGAATTGCCAGAGGCTACGGTTGGTATCTCGTGTTGTGTCGTCACCATC
T E D D E L P E A T V G I S C C V V T I
1921
616
CCCACACGCAAAACACGAGAGTCGAGGGCCAGCTCGGTGTCATCCGCACCAGTTAGTATA
P T R K T R E S R A S S V S S A P V S I
1981
TAAGCAGAGAGAGAGAGAGTGGTGGGTAAATGTATTTGAGTTGCTAAAGGTCAATTTATA
*
2041
CTTCTGCGTCAAGTAGGTAGCACAGATCCGGCAAAGCTTCATCACACACTTTGGTCGTGC
2101
ACACTTCACCATACCAAATAAATGCAACTACATATTTCCGCGATGTGGAATGCAAGGTCT
2161
GTGATTGGTCAGATTTGGTAGAGATGACAAAATGTGGGCGGGGCCGACAGTTGTGAGAGA
2221
GAGGCAAGTGTTTAACAAGTGTCAAGTCCTATGGAGGAGCACTGTATGGATACGTTTGTT
2281
TTGTTTACTCTGTGATTAAAGTTATTAAACGTTAGAAAAAAAAAAA
56
Results
context (5’-GTCATGG-3’) of a nearly perfect Kozak sequence (5’-A/GCCATGG-3’)
(Kozak, 1991). An AATAAA sequence at nt 2116-2122 may serve as a
polyadenylation signal.
Analysis of the amino acid sequence of klhl with protein domain identification
software revealed the presence of two conserved domains, kelch repeats and the
BTB/POZ domain (Fig. 3). The kelch repeat domain (amino acids 318-615) is
contained within the carboxy-terminal half of klhl and consists of six repeats of
approximately 50 amino acids in length. Comparison of the various kelch repeats in
klhl reveal that while the sequence identity between individual repeats is low, multiple
alignment of the kelch repeats from klhl shows a conserved pattern of residues,
including a double glycine element (GG) and a tyrosine (Y) separated from a
tryptophan (W) by precisely six residues, suggesting that the sequence and tandem
arrangement of kelch repeats in klhl is similar to those found in the kelch protein
family (Fig. 4). Towards the amino-terminal portion of the clone lies the BTB (broadcomplex, tramtrack, bric-a-brac) (Godt et al., 1993) or POZ (poxvirus and zinc finger)
domain (Bardwell and Treisman, 1994; Albagli et al., 1995). The BTB/POZ domain is
a ~120 aa motif that has been identified in several actin binding proteins having a
kelch motif, as well as in several C2H2-type zinc finger transcription factors (Albagli et
al., 1995; Collins et al., 2001). This domain is believed to be important for proteinprotein interaction and has been shown to mediate both homo- and heterodimeric
protein-protein interactions in vitro and the formation of multimeric complexes in vivo
(Bardwell and Treisman, 1994; Robinson and Cooley, 1997).
57
Results
klhl
klhl
klhl
klhl
klhl
klhl
R1
R2
R3
R4
R5
R6
318
367
421
468
515
567
KLHL
KLHL
KLHL
KLHL
KLHL
KLHL
R1
R2
R3
R4
R5
R6
317
366
420
467
514
566
**
*
366
420
467
514
566
615
365
419
466
513
565
614
Fig. 4. Alignment of the kelch repeats of zebrafish klhl and human KLHL.
Conserved residues are highlighted in dark blue and three / two identical residues in
four sequences are highlighted in mauve and light blue respectively. Dots represent
gaps inserted for maximal alignment. The beginning and end residues for the kelch
repeats are indicated. The invariant glycine doublet and tryptophan residues are
identified by asterisks. Sequence alignment was performed using the multiple
alignment program in DNAMAN software package. The multiple alignment of the six
repeats from Klhl and KLHL was based on the same criteria used by Lai et al (2000).
58
Results
4. klhl is Conserved Across Zebrafish, Human, Mouse and Rat
The blast search of klhl against the non-redundant GenBank protein database
revealed that klhl showed remarkable homology (72% identity) to a hypothetical
protein on human chromosome 6 (accession number CAC16284) and also to
hypothetical mouse (NP_766513) and rat proteins (XP_236428), sharing about 71%
identity to both. Of lower significance were hits to known members of the kelch repeat
superfamily of proteins, such as human kelch-like 5 protein AAL08584 and rat
actinfillin (AAM74154), sharing only about 26% identity. The low conservation
between klhl and the reported kelch-like proteins suggests that klhl is most likely a
novel member of the kelch-repeat superfamily rather than a zebrafish ortholog of the
various known kelch-like proteins (Kelch-like protein 1-6, X and ENC-1 etc)
(Hernandez et al., 1997; Soltysik-Espanola et al., 1999; Lai et al., 2000; Nemes et al.,
2000; Bradybrook et al., 2001; Wang et al., 2001).
Multiple alignment of our predicated klhl protein and the hypothetical
mammalian proteins identified in the BLAST search showed remarkable conservation
spanning the entire length of the proteins (Fig. 5), with klhl sharing around 74-76%
identity with the various mammalian Klhl proteins. Human KLHL shares around 92%
identity with the two rodent Klhl while the mouse and rat Klhl sequences share a close
97% identity. The high sequence identity between klhl and the other mammalian
proteins strongly suggests that the genes encoding for these putative proteins could
well be the mammalian orthologs of klhl. We designated the human, mouse and rat
hypothetical proteins as KLHL, m-Klhl and r-Klhl respectively.
A putative ortholog of klhl was also identified in pufferfish (Ensembl peptide
ID SINFRUP00000164410) using the blast search against Ensembl peptides
(http://www.ensembl.org/Multi/blastview). The Ensembl database provides up-to-date
59
Results
Fig. 5. Amino acid sequence alignment of zebrafish klhl, Fugu klhl, human KLHL,
mouse (m) Klhl and rat (r) Klhl proteins. Conserved residues are highlighted in light
blue and three/ two identical residues in four sequences are highlighted in purple and
pink respectively. Dots represent gaps inserted for maximal alignment. Sequence
alignment was performed using the multiple alignment program in DNAMAN
software
package.
The
sequence
of
Fugu
klhl
(Ensembl
peptide
ID
SINFRUP00000164410) was obtained from Ensembl and sequences of KLHL
(GenBank accession number CAC16284), m-Klhl (NP_766513) and r-Klhl
(XP_236428) obtained from GenBank.
60
Results
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
MAPKK K KK K DINEMTIMVEDSP NKINGLN LLEGGNGFSCIS EVTD YAPNL
MAPKKNKAAKKSKADINEMTIMVEDSPSNKINGLNTLLEGGNGFSCISTEVTDPVYAPNL
MAPKKNKTAKKSKGDINEMTIMVEDSPVNKINGLNTLLEGGNGFNCISTEVTDSVYAPNL
M
APKK KT KK K DINEMTIMVEDSPVNKINGLN LLEGGNGF CIS EVTD YAPNL
MAPKK.KIVKKNKGDINEMTIIVEDSPLNKLNALNGLLEGGNGLSCISSELTDASYGPNL
M
APKK K KKNK DINEMTIIVEDSPLNKLNALNGLLEGGNGLSCISSELTD SYGPNL
MAPKK.KTIKKNKAEINEMTIIVEDSPLSKLNALNGLLEGSNSLSCVSSELTDTSYGPNL
M
APKK KT KKNK EINEMTIIVEDSPL KLNALNGLLEGSNSLSCVSSELTD SYGPNL
MAPKK.KTLKKNKPEINEMTIIVEDSPLNKLNALNGLLGGENSLSCVSSELTDTSYGPNL
M
APKK KT KKNK EINEMTIIVEDSPLNKLNALNGLLGGENSLSCVSSELTD SYGPNL
60
60
59
59
59
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
LEGLG MRQDSFLCDL VATK KSFDVHK VMASCSEYI NMLRKDPS KKIEL DLSPV
LEGLGHMRQDSFLCDLTVATKSKSFDVHKVVMASCSEYIQNMLRKDPSLKKIELSDLSPV
LEGLSNMRQESFLCDLTVATKSKSFDVHRVVMASCSEYIRNILKKDPTLQKIDLNELSPV
L
EGLS MRQESFLCDL VATK KSFDVHR VMASCSEYI NILKKDP
KIDLNELSPV
LEGLSKMRQENFLCDLVIGTKTKSFDVHKSVMASCSEYFYNILKKDPSIQRVDLNDISPL
L
EGLSKMRQE FLCDLVIGTKTKSFDVHKSVMASCSEYFYNILKKDPS RVDLNDISPL
LEGLSKMRQESFLCDLVIGTKTKSFDVHKSVMASCSEYFYNILKNDPSTKRVDLNDIAPL
L
EGLSKMRQESFLCDLVIGTKTKSFDVHKSVMASCSEYFYNILK DPS KRVDLNDI PL
LEGLSKMRQESFLCDLVIGTKTKSFDVHKSVMASCSEYFYNILKNDPSTKRVDLNDIAPL
L
EGLSKMRQESFLCDLVIGTKTKSFDVHKSVMASCSEYFYNILK DPS KRVDLNDI PL
120
120
119
119
119
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
GLATVI YAY GKLTLSLYTIGS ISAAL LQIHTLVKMCSDFLMRE SVENCMYVVNIA
GLATVITYAYSGKLTLSLYTIGSTISAALLLQIHTLVKMCSDFLMRETSVENCMYVVNIA
GLATAITYAYSGKLTLSLYGIGSTIAAAMLLQIGTLVKMCSDFLMQELSVENCMYVANIA
G
LAT I YAY GKLTLSLY IGS I AAM LQI TLVKMCSDFLM E SVENCMYV NIA
GLATVIAYAYTGKLTLSLYTIGSIISAAVYLQIHTLVKMCSDFLIREMSVENCMYVVNIA
G
LATVIAYAYTGKLTLSLYTIGSIISAAVYLQIHTLVKMCSDFLIRE SVENCMYVVNIA
GLATVIAYAYTGKLTLSLYTIGSIISAAVYLQIHTLVKMCSDFLIREISVENCMYVVNIA
G
LATVIAYAYTGKLTLSLYTIGSIISAAVYLQIHTLVKMCSDFLIRE SVENCMYVVNIA
GLATVIAYAYTGKLTLSLYTIGSIISAAVYLQIHTLVKMCSDFLIREISVENCMYVVNMA
G
LATVIAYAYTGKLTLSLYTIGSIISAAVYLQIHTLVKMCSDFLIRE SVENCMYVVNMA
180
180
179
179
179
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
DTY LKE K AAQKFMRENFIEF E EQFLKLTYEQINEFL DD LQLPSEL AFQIAVK
DTYNLKETKEAAQKFMRENFIEFSEMEQFLKLTYEQINEFLTDDSLQLPSELTAFQIAVK
DAYALKETKKAAQKFMRENFIEFSEMEQFLKLTFEQISDFLSDDSLSLPSELTAFQIAMK
D
Y LKE K AAQKFMRENFIEF E EQFLKLTFEQI DFL DD L LPSEL AFQIAMK
ETYSLKNAKAAAQKFIRDNFLEFAESDQFMKLTFEQINELLIDDDLQLPSEIVAFQIAMK
E
TY LKNAKAAAQKFIRDNFLEFAESDQFMKLTFEQINELL DDDLQLPSEIVAFQIAMK
ETYSLKNAKATAQKFIRDNFIEFAESEQFMKLTFEQINELLVDDDLQLPSELVAFQIAMK
E
TY LKNAKA AQKFIRDNFIEFAESEQFMKLTFEQINELL DDDLQLPSELVAFQIAMK
ETYCLKNAKATAQKFIRDNFIEFADSEQFMKLTFEQINELLIDDDLQLPSELVAFQIAMK
E
TY LKNAKA AQKFIRDNFIEFADSEQFMKLTFEQINELL DDDLQLPSELVAFQIAMK
240
240
239
239
239
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
WLDFDEKRLKYAPDLLSNIRFGTI PQDLV VQ VPRMMQDAECHRLLVDAMNYHLLPF
WLDFDEKRLKYAPDLLSNIRFGTITPQDLVSHVQNVPRMMQDAECHRLLVDAMNYHLLPF
WLDFDEKRLKYAADLLTHIRFGTISAQELVNHVQSVPRMMQDAECHRLLVDAMNYHLLPY
W
LDFDEKRLKYAADLL HIRFGTISAQELVN VQ VPRMMQDAECHRLLVDAMNYHLLPY
WLEFDQKRVKYAADLLSNIRFGTISAQDLVNYVQSVPRMMQDADCHRLLVDAMNYHLLPY
W
LEFDQKRVKYAADLLSNIRFGTISAQDLVNYVQ VPRMMQDADCHRLLVDAMNYHLLPY
WLEFDQKRVKHAADLLSNIRFGTISAQDLVNYVQTVPRMMQDADCHKLLVDAMNYHLLPY
W
LEFDQKRVK AADLLSNIRFGTISAQDLVNYVQ VPRMMQDADCHKLLVDAMNYHLLPY
WIEFDQKRVKHAADLLSNIRFGTISAQDLVNYVQTVPRMMQDADCHKLLVDAMNYHLLPY
W
IEFDQKRVK AADLLSNIRFGTISAQDLVNYVQ VPRMMQDADCHKLLVDAMNYHLLPY
300
300
299
299
299
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
QQN LQSRRTKVRGG RVL TVGGRPALTEKSLSKDILYRD DN W KLTEMPAKSFNQC
QQNILQSRRTKVRGGLRVLLTVGGRPALTEKSLSKDILYRDEDNVWNKLTEMPAKSFNQC
QQNILQSRRTKVRDGLKVILTVGGRPALTEKSLSKDVLYRDTDNLWNKLTELPAKSFNQC
Q
QN LQSRRTKVRDG KVI TVGGRPALTEKSLSKDVLYRD DN W KLTELPAKSFNQC
HQNTLQSRRTRIRGGCRVLVTVGGRPGLTEKSLSRDILYRDPENGWSKLTEMPAKSFNQC
H
QNTLQSRRTRIRGGCRVL TVGGRPGLTEKSLSRDILYRDPENGWSKLTEMPAKSFNQC
HQNTLQSRRTRIRGGCRVLITVGGRPGLTEKSLSRDILYRDPENGWSKLTEMPAKSFNQC
H
QNTLQSRRTRIRGGCRVL TVGGRPGLTEKSLSRDILYRDPENGWSKLTEMPAKSFNQC
HQNTLQSRRTRIRGGCRVLITVGGRPGLTEKSLSRDVLYRDPENGWSKLTEMPAKSFNQC
H
QNTLQSRRTRIRGGCRVL TVGGRPGLTEKSLSRDVLYRDPENGWSKLTEMPAKSFNQC
360
360
359
359
359
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
VAVLDGFLYVAGGEDQNDARNQAKHAVSNF RYDPRFNTWIHL M QKRTHFSL FNG
VAVLDGFLYVAGGEDQNDARNQAKHAVSNFSRYDPRFNTWIHLANMIQKRTHFSLNTFNG
VAVLDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHLTNMSQRRTHFSLNTFNG
V
AVLDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHL M QRRTHFSL FNG
VAVMDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHLASMNQKRTHFSLSVFNG
V
AVMDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHL SMNQKRTHFSLSVFNG
VAVMDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHLGSMNQKRTHFSLSVFNG
V
AVMDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHL SMNQKRTHFSLSVFNG
VAVMDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHLGSMNQKRTHFSLSVFNG
V
AVMDGFLYVAGGEDQNDARNQAKHAVSNFCRYDPRFNTWIHL SMNQKRTHFSLSVFNG
420
420
419
419
419
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
LLFAVGGRN DG ASVECYVPS NQWQ KAPMEVPRCCHAS V DGKILV GGYI NAY
LLFAVGGRNSDGCQASVECYVPSSNQWQMKAPMEVPRCCHASSVIDGKILVSGGYINNAY
LLFAVGGRNADGVQASLECYVPSSNQWQMKAPMDVPRCCHASSVIDGKILVSGGYINNAY
L
LFAVGGRNADG ASLECYVPS NQWQ KAPMDVPRCCHAS V DGKILV GGYI NAY
LVYAAGGRNAEGSLASLECYVPSTNQWQPKTPLEVARCCHASAVADGRVLVTGGYIANAY
L
VYA GGRNAEGSLASLECYVPSTNQWQPK PLEVARCCHASAVADGRVLVTGGYI NAY
LLYAVGGRNSEGSLASLECYVPSTNQWQPKAPLEVARCCHASAVADGRVIVTGGYIGSAY
L
LYAVGGRN EGSLASLECYVPSTNQWQPKAPLEVARCCHASAVADGRVIVTGGYI AY
LLYAVGGRNAEGSLASLECYVPSTNQWQPKAPLEVARCCHASAVADGRVIVTGGYIGSAY
L
LYAVGGRNAEGSLASLECYVPSTNQWQPKAPLEVARCCHASAVADGRVIVTGGYI AY
480
480
479
479
479
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
SR VC YDP D WQD
SRAVCSYDPSTDSWQDKNSLSSPRGWHCSVTVGDRAYVLGGSQLGGRGERVDVLPVECYN
LS PRGWHC V VGDR YVLGGSQLG RGERVDVL VE Y
SRAVCSYDPSTDTWQDKSSLSTPRGWHCAASMGDRAYVFGGSQLGGRGERVDVLAVESYN
S
R VC YDP D WQD
LSTPRGWHCA MGDR YV GGSQLG RGERVDVL VESY
SRSVCAYDPASDSWQELPNLSTPRGWHCAVTLSDRVYVMGGSQLGPRGERVDVLTVECYS
S
RSVCAYDPA D WQELP LSTPRGWHCAV LSDR YVMGGSQLGPRGERVDVLTVE YS
SRSVCAYDPALDAWQELPQLSTPRGWHCAVALGDRLYVMGGSQLGPRGERVDVLTVESFS
S
RSVCAYDPA D WQELP LSTPRGWHCAV LGDR YVMGGSQLGPRGERVDVLTVESFS
SRSVCAYDPALDAWQELPGLSTPRGWHCSVALGDRVYVMGGSQLGPRGERVDVLTVESFS
S
RSVCAYDPA D WQELP LSTPRGWHC V LGDR YVMGGSQLGPRGERVDVLTVESFS
540
540
539
539
539
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
P GQWSYVAPL GVSTAG
PHSGQWSYVAPLLTGVSTAGAATLNNKIYLLGGWNEIEKKYKKCIQVYNPDLNEWTEDDE
LN K YLLGGWNE EKKYKKCIQ YNPDLNEWTEDDE
PHSGQWSYCTPLHTGVSTAGISLLNNKIYLLGGWNEGEKKYKKCIQVYNPDLNEWTEDDE
P
GQWSY PL GVSTAGIS LN K YLLGGWNEGEKKYKKCIQ YNPDLNEWTEDDE
PATGQWSYAAPLQVGVSTAGVSALHGRAYLVGGWNEGEKKYKKCIQCFSPELNEWTEDDE
P
GQWSY APL VGVSTAGVSALHGRAYLVGGWNEGEKKYKKCIQCF PELNEWTEDDE
PAARQWSFVAPLPVGVSTAGVSALHGRAYLLGGWNEGDKKYKKCIQCFNPELNEWTEDDE
P
QWSFVAPL VGVSTAGVSALHGRAYLLGGWNEGDKKYKKCIQCFNPELNEWTEDDE
PVARQWSFVAPLPVGVSTAGVSALHGRAYLVGGWNEGEKKYKKCIQCFNPELNEWMEDDE
P
QWSFVAPL VGVSTAGVSALHGRAYLVGGWNEGEKKYKKCIQCFNPELNEW EDDE
600
600
599
599
599
klhl
fugu-klhl
KLHL
m-Klhl
r-Klhl
LPEATVGISCC V IP
LPEATVGISCCVVTIPTRKTRESRASSVSSAPVS
TRESRASSVSS PVS
LPEATVGISCCIITVPTRKTRESRASSVSSAPVS
L
PEATVGISCC I VP
TRESRASSVSS PVS
LPEATVGVSCCTLSMPNNVTRESRASSVSSVPVS
L
PEATVGVSCCTL MPN VTRESRASSVSSVPVS
LPEATVGVSCCTLAMPNSVSRESRASSVSSVPVS
L
PEATVGVSCCTL MPN V RESRASSVSSVPVS
LPEATVGVSCCTLAMPNSVSRESRASSVSSVPVS
L
PEATVGVSCCTL MPN V RESRASSVSSVPVS
634
634
633
633
633
61
Results
and comprehensive sequence data from several metazoan organisms including human,
mouse, rat, zebrafish and the pufferfish Fugu rubipes (Clamp et al.,2003). Zebrafish
and Fugu klhl share about 87% identity as compared to the 74-76% identity shared
between zebrafish and the mammalian sequences, demonstrating the closer
evolutionary relationship between the two teleosts fish. Fugu klhl share 73-75%
identity with the mammalian Klhls (Fig. 5)
5. Genome Mapping of klhl
It is well known that the correspondence between the two genomes may be
used for the prediction of gene orthologs (Barbazuk et al., 2000). We thus sought to
confirm the gene orthology relationship through conserved synteny analysis. Findings
from the release of the draft sequence of mouse show that as much as 96% of genes
present in both the mouse and human genome lie in syntenic regions. Work by
Postlewait and others (1998) have revealed that extensive contiguous blocks of
synteny exist between the zebrafish and human genomes.
A
search
of
the
zebrafish
EST
database
(http://www.genetics.wustl.edu/fish_lab/frank/cgi-bin/fish/) identified several clones
that are identical to klhl. Among them was clone fb95e08 that had previously been
mapped to linkage group (LG) 13 using both LN54 and T51 radiation hybrid (RH)
panels and in a meiotic mapping heat shock (HS) panel (Kwok et al., 1998; Geisler et
al., 1999; Geisler and Jorg, unpublished; Hukriede et al., 1999; Kelly et al., 2000) (Fig.
6A).
The gene encoding for the putative human KLHL protein, KLHL (Genbank
accession no. Q9H511) was found to map to human 6p12.2 (http://www.ensembl.org).
The genes encoding for mouse (Genbank accession no. NM_172925) and rat Klhl
62
Results
Fig. 6. Genome mapping of klhl. (A) The partial linkage map of zebrafish LG13,
indicating the position of klhl gene in relation to other markers. Map positions of klhl
(shown in bold) and markers on the T51 and LN54 radiation hybrid panels, and HS
meiotic panel were obtained from ZFIN (http://zfin.org/ZFIN) and the Tüebingen
zebrafish genome map web page (http://wwwmap.tuebingen.mpg.de). (B) Map
location of klhl, gsta3, bmp5, and bpag1 on zebrafish LG13 and their orthologs on
human chromosome 6 (Hsa6). Intrachromosomal rearrangements have altered the gene
order in fish and mammalian chromosomes. The relative chromosomal locations for
the
human
orthologs
were
(http://www.ncbi.nlm.nih.gov/LocusLink),
obtained
and
the
from
Ensembl
Locuslink
database
(http://www.ensembl.org). The maps are not drawn to scale. (C) Conservation of
synteny between zebrafish LG13, Hsa 6, mouse chromosome 9 (Mmu9) and rat
chromosome 8. The apparent orthologs are arranged in the same column.
63
A
LG13
T51
LN54 PANEL
cM
cR
56.0
7.4
z11918
76.0
110.0
12
12
4
8
7
11
4
4
8
28
cR
fa09h08 z5643
fa25h01
z11918
fk08d06 (gsta3)
z4176 z10513 fc45e02
fc38a05 (bpag1)
zehl0669 (bmp5)
z9564
z5643
fa09h08
40.04
49.22
56.26
z9564
z13611
3.2
zf-es31 (gsta)
5.9
fb95e08 (klhl)
9.1
fc79g12.x1
fj16a03 fc83g12
fj21h11
fa09h08
7.89
17.0
z5366
13
4
8
4
17
237.0
18.25
25.7
fb95e08 (klhl)
41
25.2
cM
fe36e11
150.0
191.0
HS PANEL
fj97f06
z6104
fj99f10
chunp1029
5 cM
20 cR
z9564 z13611
fb26d02
64
fb95e08 (klhl)
Results
B
Hsa 6
LG13
T51
LN54
80 cR
HS
3.2 cM
gsta
95 cR
bpag1
106 cR
bmp5
110 cR
40 cR
17.0 cM
klhl
GSTA
6p12.2
KLHL
6p12.2
BMP5
6p12.1
BPAG1
6p12-p11
C
Zebrafish
LG13
gsta3
bpag1
bmp5
klhl
fk08d06
fc38a05
zehl0669
fb95e08
BPAG1
BMP5
KLHL
Bmp5
Klhl
zf-es31
Human
Hsa6p12-11
GSTA3
Mouse
Mmu9
Gsta3
Rat
Rn8
Gsta3
Klhl
65
Results
(Ensembl gene ID ENSRNOG00000006224) proteins were found to be present on
mouse chromosome 9 and rat chromosome 8 respectively (http://www.ensembl.org).
A syntenic relationship between human chromosome 6 and mouse chromosome 9 had
previously been described (http://www. ncbi.nlm.nih.gov/Homology) (Fig. 6C).
Woods et al. (2000) had previously identified a syntenic relationship between
zebrafish LG13 and human chromosome 6. Both zebrafish LG13 and human
chromosome contain at least 5 pairs of orthologous genes, mekk4/MEKK4,
kiaa0796/KIAA0796, gsta3/GSTA3, fbx5/FBX5 and dld/DLL1. However, many of
these genes are not located in the same region as klhl. Only one gene, glutathione Stransferase 3 gsta3 (fk08d06 and zf-es31), is located close to klhl (Fig. 6A). Consistent
with
this
data,
GSTA3
maps
close
to
KLHL
(6p12.2)
(http://www.ncbi.nlm.nih.gov/LocusLink) (Fig. 6B,C). The region containing klhl and
gsta3 in zebrafish LG13 might thus be syntenic to the region containing KLHL and
GSTA3 in human chromosome 6. In mouse, Gsta3 has also been mapped close to Klhl
on chromosome 9 (Fig. 6C).
To further strengthen this relationship, ESTs mapped close to klhl and gsta3
were examined and an EST (zehl0669) coding for bone morphogenetic protein 5
(bmp5) gene was identified. The ortholog BMP5 has been mapped near the locus of
KLHL,located at position 6p12.1 (Fig. 6B,C) while the mouse Bmp5 has also been
mapped in a nearby region on chromosome 9 (Fig. 6C). We have also found a
zebrafish bulbous pemphigoid antigen 1 gene, bpag1 (fc38a05), near klhl, similar to
the presence of a human ortholog gene, BPAG1 (6p12-p11), near KLHL (Fig. 6B,C).
These results confirms that zebrafish LG13 is syntenic to human chromosome 6 and
mouse chromosome 9 and further supports that KLHL, m-Klhl and r-Klhl are orthologs
of klhl in human, mouse and rat respectively.
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Results
However, it is noteworthy that the genes are not arranged in the same manner
in the zebrafish and human chromosome. As seen in Fig. 6B, the gene order is
rearranged between the two chromosomes. This result is not unexpected and is
consistent with Postlethwait et al. (2000) where it was noted that while large blocks of
conserved syntenies were observed between zebrafish and humans, gene orders were
frequently inverted and transposed.
6. Developmental Accumulation of klhl
To examine the temporal expression of klhl, northern blot hybridization was
carried out using total RNA extracted from different stages of zebrafish embryos, and
also from adult fish. As klhl had previously been found to be expressed specifically in
the somites in developing zebrafish embryos (Wu, 1999), the stages of embryos chosen
ranged from 12 hpf (6 somite stage), the beginning of somitogenesis, to hatched fry
(72 hpf). To position klhl in the somitogenic pathway, we compared its expression
pattern with two muscle specific protein (MSP) cDNA clones, α-tropomyosin (tpma)
and fast skeletal muscle myosin light polypeptide 2 (mylz2), which had previously been
characterized in our laboratory (Xu et al., 2000).
The expression pattern of tpma and mylz2 obtained in this study was consistent
with that of Xu et al. (2000), with tpma transcripts first becoming detectable at ~12 hpf,
and mylz2 ~18 hpf (Fig. 7). The expression of the two genes increased rapidly during
development and was maintained at high levels through to 72 hpf. Both genes were
also expressed in the adult fish, albeit at a lower level than in developing embryos.
As shown in Fig. 7, klhl was expressed at about the same time as mylz2 with its
transcripts detectable at ~18 hpf. Its expression pattern was similar to that of the other
67
Results
Fig. 7. Expression of klhl in developing zebrafish embryos in comparison to two
other MSP genes, tpma and mylz2. A northern blot containing 10 µg/lane of total
RNA isolated from zebrafish embryos of various stages from beginning of
somitogenesis (12 hpf) to hatched fry (72 hpf) was hybridized with individual cDNA
probes as indicated on the left of each panel. The stages of embryos are indicated at the
top of each lane; adult, RNA prepared from whole fish. The sizes of major hybridized
transcripts are indicated on the right. The same blot was hybridized with a ubiquitously
expressed acidic ribosomal phosphoprotein (arp) probe to monitor the quantity and
quality of the RNAs.
68
Results
two genes with an increase in expression level during development, and a weaker
expression in the adult fish. The size of the klhl transcript detected was about 4.0 kb.
The disparity in size observed between our predicted 2.4 kb full length clone and the
4.0 kb mRNA suggests that the klhl gene might have an unusual long 5’ UTR or polyA
tail. A ubiquitous acidic ribosomal phosphoprotein P0 (arp) cDNA probe was used to
hybridize to the same RNA blot to monitor the quality of RNA and to ensure the even
loading of all RNA samples (Ju et al., 1999).
7. Tissue Distribution Analysis of klhl in Adult Zebrafish
To examine if the restricted pattern of expression was maintained in the adult
zebrafish, northern blot hybridization was carried out using total RNA extracted from
several adult tissues. The two MSP cDNA clones, tpma and mylz2 were again included
in the study. As shown in Fig. 8, a single transcript was detected in the heart and
skeletal muscle lanes. In adult tissues, klhl mRNA was expressed strongly in the trunk
skeletal muscle and weakly in the heart. This expression pattern was similar to that of
tpma mRNA which was also detected in the heart and skeletal muscle. mylz2 is
expressed specifically in the trunk skeletal muscles. arp was used as a control to
monitor the quality and quantity of the RNAs.
8. Expression of klhl is Similar in Human, Rat and Zebrafish
To determine if KLHL expression is similar to that of klhl, we designed KLHLspecific primers to amplify a 0.9 kb DNA fragment from human DNA. The fragment
was radiolabeled and used as a probe in northern blot analysis of poly (A+) RNA from
human adult tissue. As seen in Fig. 9, a single transcript of ~7.0 kb was detected in the
heart and skeletal muscle tissues.
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Results
Fig. 8. Tissue distribution of klhl mRNAs in comparison with tpma and mylz2
mRNAs in adult zebrafish. A northern blot containing 10 µg/lane of total RNA
isolated from eight different zebrafish tissues was hybridized with individual cDNA
probes as indicated on the left of each panel. The names of tissues are indicated at the
top of the lanes. Abbreviations: B, brain; E, eyes; G, gill; H, heart; I, intestine; L, liver;
M, trunk skeletal muscle; O, ovary. The same blot was hybridized with a ubiquitously
expressed acidic ribosomal phosphoprotein (arp) probe to monitor the quantity and
quality of the RNAs.
70
Results
Fig. 9. Northern blot analysis of KLHL mRNA in human tissues. The human blot
was hybridized with a radiolabelled KLHL-specific probe and with control human βactin probe (bottom panel). Positions of molecular size makers are indicated on the left.
71
Results
Based on the results of searches in public human, mouse and rat EST databases,
we were also able to deduce that klhl is expressed in similar tissues in human and rat
(Table 1). We were able to identify two human EST clones that were identical to
KLHL. One was isolated from a cDNA library constructed using mRNA from a cDNA
head library and the other from a fetal heart cDNA library, indicating that KLHL is
expressed in the fetal heart. We also identified three mouse and 10 rat EST clones that
showed amino acid sequence homology with zebrafish klhl. While we were not able to
extract much information about Klhl expression in the mouse, all the rat EST clones
identified were isolated from cDNA libraries constructed from fetal heart or muscle
mRNA (Table 1). Thus, it is likely that klhl is expressed in a similar fashion in human,
rat and zebrafish and its function is conserved among the different species.
9. Ontogenetic Expression of klhl during Somitogenesis
To reveal further the temporal and spatial expression pattern of klhl, whole
mount in situ hybridization was carried out with embryos of various somitogenesis
stages. In zebrafish, somite formation begins at about 10 hpf, and ends at 24 hpf, with
one pair of somites forming approximately every half hour (Hanneman and
Westerfield, 1989; Kimmel et al., 1995). Probes for tpma and mylz2 were also included
for comparison.
Xu et al. (2000) had previously studied the expression of MSP genes in
zebrafish embryos by whole mount in situ hybridization. Based on the timing and
pattern of their expression during somitogenesis, Xu et al. (2000) had classified the
MSP genes into three groups: early, intermediate and late. tpma belonged to the early
gene group. First detected around 10 hpf, tpma is expressed in the adaxial cells prior to
somite formation (Fig. 10B). From 10 to 12 hpf, when the first six somites are formed,
72
Results
Table 1. Summary of EST clones homologous to klhl
Organism Unigene
Cluster
Human
Mouse
Mm.208843
Genbank
Library Source
Accession No
BF824983
head
R57328
fetal heart
AA672987
myotubules
AV174355
8-day embryo
BB618579
BB528731
15 days embryo head
BB663561
Rat
Rn. 22511
BB369350
16 days embryo head
AW254032
rat atrium at 16.5 dpc, ventricle at 16.5
dpc, AV canal at 16.5 dpc, atrium at 15
dpc, ventricle at 15 dpc, AV canal at 15
AW525772
dpc and ventrile at 13 dpc.
rat atrium at 15 dpc, ventricle at 16.5
BF408492
dpc, atrium at 16.5 dpc, ventricle at 13
dpc, ventricle at 15 dpc, AV canal at 15
BF414303
dpc.
BF543966
Normalized rat atrium
AA849356
Normalized rat muscle
AI171017
AI171183
73
Results
Fig. 10. Expression of klhl (A, C) and tpma (B, D) in zebrafish embryos. (A, B)
Dorsal views of 2-somite stage embryos, anterior to the left. (C, D) Dorsal views of
flat mounted 12 hpf (6-somite) embryos, anterior to the left.
74
Results
the tpma transcript signal intensifies and extends posteriorly as the gene is activated in
a rostral-caudal manner (Fig. 10B, D, 11F, G). This signal becomes stronger in older
embryos as the amount of transcripts starts to increase with the increase in somites
number (Fig. 11H-J). tpma is expressed in all the formed somites. mylz2, on the other
hand, is activated later than tpma, with its transcripts first detected at 16 hpf (Xu et al.,
2000) (Fig. 11K). As an example of an intermediate gene, mylz2 is not expressed in all
formed somites, with its transcripts only detected in the older somites. For example, by
16 hpf, when 14 somites were formed, expression of mylz2 transcripts were found only
in the anterior 10 somites (Fig. 11K) while tpma transcripts were detected in all 14
somites. This appears to be the case for the intermediate and late genes group in which
expression is absent in the last 2-6 formed somites (Xu et al., 2000).
Based on the result of the northern hybridization, we would expect klhl to be
expressed in a similar fashion with mylz2. On examination of klhl expression in 16 hpf
embryos, we found strong expression in all 14 formed somites (Fig. 11D), similar to
that of tpma (Fig. 11I). In addition, another domain of expression was found in
bilateral stripes of cells, just adjacent to the future hindbrain (boxed up in Fig. 11D,
Fig 13A). We went on to investigate the expression of klhl in younger embryos and
found that it displayed a similar expression pattern as that of tpma. First detected at the
2-somite stage, klhl expression lags about 30 minutes behind that of tpma. Expression
of klhl first occurs in the adaxial cells on both sides of the notochord (Fig. 10A). By 12
hpf (6-somite), klhl expression was detected in the formed somites and in the posterior
adaxial cells in the unsegmented region (Fig. 10C), with its level of expression
increasing with the development of somites (Fig. 11B-E). Expressed in all formed
somites, klhl expression is a typical early MSP gene as defined by Xu et al. (2000).
The inconsistency between the results of the northern blot hybridization and in situ
75
Fig. 11. Ontogenetic expression of klhl (A-E), tpma (F-J) and mylz2 (K-L) during the various stages of somitogenesis. Vertical panel
columns shown embryos at the same stage: 11hpf (A, F), 12 hpf (B, G), 14 hpf (C, H), 16 hpf (D, I, K) and 18 hpf (E, J, K). Additional domain
of expression of klhl is boxed up in (D). All panels show lateral views of embryos, anterior to the left.
76
Results
hybridization could be accounted for by the fact that the initial expression of klhl was
too low to be detected by northern. This explanation could be borne out from in situ
hybridization results where a much longer staining time was required for detection of
klhl expression as compared to tpma, despite the fact that similar probe concentrations
were used.
10. Expression of klhl in Fast and Slow muscle
To verify the muscle specificity of klhl, trunk-level tissue sections of 36 hpf
embryos were examined. In the adult, zebrafish muscle fibers can be subdivided into
slow and fast muscle fibers and precursor to these two fiber types can be identified
very early in development. By 24 hpf, fast muscle fibers are found in the deep cells of
myotome, whereas slow muscle fibers form a superficial monolayer on the surface of
the myotome (Devoto et al., 1996; Stickney et al., 2000).
To determine gene expression in fast and slow muscles, two-color in situ
hybrididization was carried out using the klhl probe and a slow muscle myosin probe,
smbpc. smbpc, encoding a slow myosin binding protein C, is expressed only in the
slow muscle (Fig. 12J-K) (Xu et al., 2000). Compared with the specific staining of
smbpc in slow muscles, klhl transcripts were fast-muscle-specific (Fig. 12A-C). No
expression was detected in the superficial slow muscle cells defined by smbpc mRNA
expression. This was confirmed by the comparison of expression of tpma and smbpc
mRNAs, and desmin and smbpc mRNAs. tpma mRNA had previously been shown to
be expressed only in the fast skeletal muscle (Xu et al., 2000) and its expression is
similar to that of klhl (Fig. 12D-F). desmin, on the other hand, is expressed in both fast
and slow muscles (Xu et al., 2000) (Fig. 12G-I).
77
Results
Fig. 12. Comparison of expression of klhl (A-C), tpma (D-F), desmin (G-I) and
smbpc (J-L) in 36 hpf embryos. (A, D, G, J) Lateral view of whole mount 36 hpf
embryos. (B, E, H, K) Cross sections of embryos at the trunk position as indicated by
arrowheads in A, D, G, and J respectively. (C, F, I) Cross sections of embryos doublystained with fluorescein labeled smbpc antisense riboprobe (magenta) and digoxigenin
labeled klhl (C), tpma (F) and desmin (I) antisense riboprobe (purple). (L) Cross
section of embryo stained with smbpc antisense riboprobe only.
78
Results
11. Expression of klhl during Cardiac Morphogenesis
We went on to study in more detail the additional domain of expression of klhl
that we had observed earlier. First appearing at the 13-somite stage (15.5 hpf), klhl
transcripts are present as bilateral stripes of cells, lying in close proximity to the yolk
and adjacent to the future hindbrain (Fig. 13A and data not shown). As development
proceeds, klhl-expressing cells rapidly migrates towards the midline, appearing as a
butterfly-shaped configuration at 18 hpf (Fig. 13B) and finally fusing to become a ring
structure by 19.5 hpf (Fig. 13C) which is transformed into the heart tube by 26 hpf (Fig.
13D). The ring structure is actually a shallow cardiac cone formed as the cells posterior
to the bridge fuse followed by an anterior closure that creates a central lumen. The
apex of the cone is raised dorsally around the lumen (Yelon et al., 1999; Yelon, 2001).
Cardiac morphogenesis continues to be highly dynamic after the heart tube has formed.
By 30 hpf, klhl transcripts are present as a curved tube as cardiac looping takes place
(Lee et al., 1996; Yelon et al., 1999; Yelon, 2001) (Fig. 13E-G). Anatomic chamber
differentiation also takes place at the same time and constriction is evident at the
atrioventricular boundary by 48 hpf (Fig. 13H).
klhl transcripts persist in the
embryonic heart at least through 48 hpf and are also present in adult heart (Fig. 8). klhl
is expressed uniformly throughout the myocardium, and its expression reveals the
entire progression of heart formation.
12. Expression of klhl in Cranial Muscle Development
We also analyzed klhl expression in late embryogenesis to determine if klhl
was expressed similarly to tpma in the cranial skeletal muscles. Like trunk skeletal
muscle, cranial skeletal muscles in vertebrates are derived from paraxial mesoderm
and express myogenic regulatory genes that activate structural genes that give muscles
79
Results
Fig. 13. klhl expression during cardiac morphogenesis. (A-F) Dorsal views of embryos
at (A) 16 hpf, (B) 18 hpf, (C) 19.5 hpf, (D) 24 hpf, (E) 26 hpf , (F) 30 hpf, anterior at the
top. (G, H) Head-on views of 36 hpf (F) and 48 hpf embryos (G). The embryo’s left side is
to the right of the figure. The ventricle is indicated with an arrowhead and the atrium is
indicated with an arrow in (H).
80
81
Results
their contractile function (Schilling and Kimmel, 1997). As seen in Fig. 14, klhl, like
MSP genes, is expressed in cranial muscles. The pattern of expression observed is in
essential similar to that of tpma (Schilling and Kimmel, 1997) and mylz2 (Xu et al.,
2000).
82
Results
Fig. 14. Localization of klhl transcripts in 72 hpf embryos. (A) Ventral view of embryo,
anterior to the left. (B) Lateral view of left side of the embryo. Abbreviations: ah, adductor;
am, adductor mandibulae; ao, adductor operculi; do, dilator operculi; hh, hyohyoideus; ih,
interhyal; ima, intermadibularis anterior; imp, intermendibularis posterior; io, inferior
oblique; lap, levator arcus palatini; mr, medial rectus; PFB, pectoral fin bud; psm,
presomite muscle; so, superior oblique; sr, superior rectus; vam, ventral abdominal muscle.
The nomenclature for cranial skeletal and muscle elements is based on Schilling and
Kimmel (1997).
83
Discussion
Chapter IV
Discussion
84
Discussion
1. Zebrafish as a Model for Vertebrate Biology
The zebrafish has become a model of choice for many scientists seeking to
understand vertebrate development. This has largely been a result of the successes of the
large-scale mutagenesis screens as well as the advances in zebrafish genome research. My
study here has focused on the isolation and characterization of a novel zebrafish gene, klhl,
expressed in skeletal and cardiac muscles. This study is an offshoot of an earlier project to
identify novel genes important in zebrafish embryogenesis using the method of whole
mount RNA in situ hybridisations (Wu, 1999). Identification of the expression pattern of
novel genes can often shed light on their function. Such applications are especially useful
in light of the millions of mouse, rat and human ESTs that have accumulated in the
genome projects. However, large-scale expression studies using higher vertebrates are
both cumbersome and cost-prohibitive for such a vast collection of genes. Zebrafish
provide an alternative approach to this problem. Large number of zebrafish embryos can
be produced inexpensively. Hundreds of whole mount in situ hybridisations to staged
embryos using zebrafish EST sequences can be performed simultaneously to reveal their
expression patterns. Such an approach is promising in light of the fact that many zebrafish
genes have mammalian orthologs in EST databases. This is exemplified by the
identification of human, mouse and rat orthologs of klhl in my study. Furthermore, many
zebrafish mutant phenotypes have been found to resemble certain human diseases. Thus,
the zebrafish could present an alternative vertebrate model of human diseases (Dodd et al.,
2000). The molecular and functional characterization of genes in zebrafish might be useful
for assigning functions to human genes known only by sequences that are identified by the
HGP.
85
Discussion
2. klhl is a Member of the Kelch Family of Proteins
To this end, I report here the cloning and developmental analysis of a novel
zebrafish gene, klhl. The 1905 bp ORF of klhl is predicted to encode a 635 aa protein that
possesses two evolutionary conserved domains– an N-terminal BTB/POZ domain and
followed by six kelch repeats. BTB/POZ is found in two main contexts: one is in zinc
finger-containing proteins, in which it mediates a transcriptional repression activity
(Chang et al., 1996); the second context, in association with 4-6 kelch repeats (Adams et
al., 2000). The BTB-POZ domain has been shown to mediate homodimerization or
heterodimerization of proteins that contain it (Bardwell and Treisman, 1994). Database
mining and sequence analyses of various genomes available (from human, Drosophila,
C.elegans, S, cerevisiae etc) using the kelch motif consensus (Pam01344) as a query
sequence identify the domain as an evolutionary conserved one, found throughout
phylogeny (Adams et al., 2000; Prag and Adams, 2003). Besides the BTB-POZ domain,
the kelch motif has been found to associate with other protein motifs such as discoidin, Fbox, coiled-coil but can also been found alone as well, with the number of repeats ranging
from four to seven. Whatever the association, the sets of repeated kelch motifs are
predicted to form a β-propeller structure based on the crystal structure determined for a
single kelch-repeat protein, fungal galactose oxidase (PDB 1GOF) (Bork and Doolittle,
1994; Adams et al., 2000). Considering that the kelch motif appears in so many different
contexts, it is no surprise that kelch-repeat proteins have multiple potential binding
interactions and play functionally diverse activities in the cell (For a list of the interactions
and cellular functions of kelch repeat proteins, see Adams et al., 2000).
Besides klhl, both the BTB/POZ and kelch domains have been found in a number
of proteins that constitute a subfamily of the kelch superfamily, termed the N-dimer, C86
Discussion
propeller proteins (Adams et al., 2000). Members of this family contain an N-terminal
BTB/POZ domain and four to six kelch motifs located within the C-terminal region.
Examples of proteins belonging to this subgroup include Drosophila kelch (Robinson and
Cooley, 1997; Kelso et al., 2002), Calicin (von Bulow et al., 1995), ENC-1 (Hernandez et
al., 1997), Keap1 (Itoh et al., 1999) and Mayven (Soltysik-Espanola et al., 1999). One
recurrent function among this group of proteins concerns sub-cellular organization, in
particular actin-binding. Drosophila kelch is a structural component of actin-rich ring
canals that regulate the nutrient transport from the nurse cells to the oocyte. Mayven and
ENC-1 are predominantly expressed in the human brain and the mouse neural system
respectively and are predicted to be important in the organization of the actin cytoskeleton
by functioning as actin-binding proteins (Hernandez et al., 1997; Soltysik-Epanola et al.,
1999). These proteins have been shown to bind directly with actin through their kelch
repeats. There are however other kelch-repeat proteins that affect cell morphology and
organization but do not themselves bind directly, or colocalize with actin. Calicin is one
example of this, responsible for the organization of an actin-negative structure, the sperm
calyx (von Bulow et al., 1995). Even more functionally diverse, Keap1 plays a role in
gene expression by sequestering a transcription factor Nrf2 in the cytoplasm under normal
conditions. This interaction is downregulated in the presence of electrophilic agents which
stimulate translocation of Nrf2 to the nucleus to counterattack this agents (Itoh et al.,
1999). A recent study however indicates that Keap1 might also have a function in cell
morphology and organization. Velichkova and colleagues (2002) has found that Keap1
binds to the SH3 domain of myosin-VIIa and associates with it in specialized adhesion
junctions. At the heart of this association was the kelch repeat domain. It is apparent from
87
Discussion
the above that we cannot assign a specific function to klhl based upon its sequence
similarity to the kelch family, as different members appear to have diverse functions.
3. klhl is Expressed in the Somites and Cardiac Muscles
Northern blot analyses showed that klhl and its human homolog KLHL are both
specifically expressed in the skeletal muscles and heart. We also noted that the rat Klhl
EST clones were isolated from a cDNA library constructed using mRNA from fetal heart
or muscle. This suggests that the role klhl plays has been conserved through evolution.
Previously, another kelch protein gene, human Sarcosin, had been found to be expressed
in the muscles as well (Taylor et al., 1998). Detailed analysis using a blot containing
various human muscle tissues revealed that expression of Sarcosin was restricted to
sarcomeric muscle, skeletal muscle and heart muscle (Taylor et al., 1998). The rat
ortholog of Sarcosin, Kelch-related protein 1 (Krp1) was also found to express in the heart
and skeletal muscle (Spence et al., 2000). Expression of klhl and Sarcosin mRNAs thus
appear to be limited to the striated muscles (i.e. skeletal and cardiac muscles). While
cardiac muscle can form a third class of fiber besides smooth and striated muscles, it
resembles striated muscles in many aspects (Darnell et al., 1990).
To gain more insight about the functional role of klhl, we examined the expression
of klhl during zebrafish development. klhl was first detected in the embryos at the 2somite stage, around 10.5 hpf. At this stage, it is expressed in the first two somites and
also in the adaxial cells adjacent to the notochord. These adaxial cells would later migrate
and differentiate into slow muscle fibers of the adult fish (Devoto et al., 1996). Striated
muscle fibers in zebrafish, like most vertebrates, can be broadly classified into either fast
or slow muscle depending on contraction speeds and metabolic activities (Darnell et al.,
88
Discussion
1990; Devoto et al., 1996). Slow muscle fibers contract and relax slowly, and they can
create and maintain tension for long periods of time. Fast muscle fibers contract fast and
fatigue fast and are used to generate rapid movements by sudden bursts of contraction.
However, a survey of klhl expression in zebrafish formed somites showed that it was not
expressed in the slow muscles. It was expressed only in the fast muscle. Similar results
have been obtained with a few other fast muscle genes like tpma and troponin C (Xu et al.,
2000) that are also expressed in adaxial cells (Xu et al., 2000). Such observations indicate
that the differentiating slow muscles may cease the expression of genes during or after cell
migration to the superficial layer (Xu et al., 2000).
The expression of klhl in the early segmentation period embryos makes it one of
the earliest genes to be expressed in the somitogenic pathway. Examples of MSP genes
expressed at this early stage include desmin and tpma (Xu et al., 2000). In fact, the
expression pattern of klhl closely correlates with that of tpma in the skeletal muscles. Like
tpma, klhl expression increases with the number of somites and is expressed throughout
somitogenesis and in the adult muscles. Expression of klhl in other skeletal muscles such
as head muscles is also similar to tpma.
We also detected expression of klhl in myocardial precursors. While skeletal and
cardiac tissues have a similar sarcomeric organization and function, they have distinct
cellular origins arising from separate populations of mesodermally derived progenitor
cells (Gregorio and Antin, 2000). klhl mRNA was first detected at the 13-somite stage in
bilateral stripes of myocardial cells. The initiation of klhl expression at this stage
corresponds to the expression of two muscle-specific contractile protein genes, cmlc2 and
ventricular myosin heavy chain (vhmc) in zebrafish (Yelon et al., 2000). In the chick
embryo, muscle-specific contractile protein genes are first detected in cardiac progenitors
89
Discussion
at the one-to-four-somite embryo stage, just as the most-anterior regions of the two heart
primordia begin to fuse (Gregorio and Antin, 2000). This suggests that the expression of
MSP genes takes place at approximately the same time in both zebrafish and chicks.
Cardiac fusion in zebrafish takes place shortly after the first detection of cmlc2 and vhmc
at the 17-somite stage (Yelon et al., 2000).
4. Role of klhl in Muscle Structure and Function
The striated muscle machinery contains a complex interconnected cytoskeletal
network critical for its contractile activity (Clark et al., 2002) (Fig. 15). The basic
contractile unit of the striated muscle, the sarcomere, contains four filament systems:
actin-containing thin filaments that span the I-band and overlap with myosin-containing
thick filaments in the A-band, titin and nebulin. The individual sarcomeres are bordered
by Z-lines, where the thin filaments, titin and nebulin are anchored (Fig. 15). The precise
Fig. 15. A schematic
overview of cytoskeletal
linkages in striated
muscle (Adapted from
Clark et al., 2002).
assembly and alignment of the various filament systems is critical for muscular
contraction. While the filament systems has been intensely studied and the molecular
interactions between actin and myosin generating the motion of contraction well known,
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Discussion
the mechanisms by which they become organized during myofibril assembly are still
poorly understood as evident by the continual discovery of novel proteins that are
associated with the contractile apparatus (Clark et al., 2002). Deciphering the precise
relationships among striated muscle components is important considering the diverse and
complex number of muscle myopathies that result directly from mutations in contractile
and associated proteins. In all likelihood, the domain organization of klhl, its evolutionary
conserved restricted expression pattern in the striated muscles in zebrafish and the various
mammalian species suggests that it plays an important role in the assembly of the striated
muscle. The spatial and temporal expression pattern of klhl as well as its domain
organization suggests that klhl may play a role in the organization of the striated muscle
cytoarchitecture.
Some insights into the function of klhl in striated muscle can be obtained from
examining another kelch-related protein that is also specifically expressed in the striated
muscle, sarcosin (Taylor et al., 1998), also called Krp1 (Spence et al., 2000; Lu et al.,
2003). klhl and Krp1 share only about 20% sequence identity. The work of Spence and
colleagues (2000) indicate the possible role of Krp1 in defining the structure and processes
that occur at cell tips. Krp1 was found to colocalize with F-actin at the membrane-rufflelike structures in the tips of pseudopodia of rat fibroblasts although Krp1 and actin were
not found to interact. In addition, overexpression of Krp1 in transformed rat fibroblasts
were found to dramatically elongated pseudopodia while the expression of truncated Krp1
polypeptides, BTB/POZ domain or kelch repeats only, resulted in the reduction of
pseudopodia length, presumably by acting as dominant negative mutants. The entire
protein is thus required for the interaction with the necessary processes.
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Discussion
More recently, Krp1 was picked up in another screen by Lu and colleagues for NRAP binding partners (2003). N-RAP is an actin-binding LIM protein, concentrated at
myotendinous junctions (MTJ) in skeletal muscle and intercalated disks in cardiac muscle
(Fig. 16). The MTJ and intercalated disks are the sites of mechanical coupling between the
myofibrils and the cell membrane, necessary to effectively transmit force. N-RAP serves
as a link between the terminal actin and protein complexes at the cell membrane,
Fig. 16. Schematic model of the cytoskeletal filament linkages at the
sarcolemma of striated muscle.(Adapted from Clark et al., 2002).
interacting with actin with its C-terminus and binding to talin through its N-terminus LIM
domain. The C-terminus of talin may also associate with vinculin. The multiple potential
interactions with talin, actin and vinculin at the MTJ provide a stable mechanical link
between the contractile cytoskeleton and the sacrolemma because it is the focal point for
much of the force generated during contraction (Clark et al., 2002). Like the earlier study
by Spence et al. (2000), Krp1 was found at the periphery of cells, this time at the
periphery of mature myofibrils that appeared to be joining laterally with narrow myofibrils
in cultured chicken cardiomyocytes. This lateral fusion transforms myofibril precursors
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Discussion
into mature myofibrils with broad Z-lines. Krp1 is postulated to be involved late in
myofibril assembly, catalysing the lateral fusion of myofibril precursors.
It has been suggested that the order of expression of MSP genes may reflect the
assembly sequence of the myofibril. One prominent model of myofibrillogenesis proposes
that thick and thin filaments assemble independently in muscle cells. In this model, thick
filaments appear later and are incorporated into preformed structures containing α-actinin,
sarcomeric actin and titin (Holtzer et al., 1997; Gregorio and Antin, 2000). In their study
of zebrafish MSP genes, Xu et al. (2000) also noted that that the genes for thin filament
proteins are expressed earlier than the genes for thick filament proteins. klhl, like tpma, is
expressed earlier than zebrafish skeletal α-actin which is the predominant isoform of actin
found in adult striated muscles (Xu et al., 2000). The early expression of klhl in the
somitogenic pathway and in myocardial precursors suggests its importance for the
assembly of the myofibril structure. A search of the Ensembl database with the human
sarcosin sequence (Genbank accession number (AAH06534) indicates there might be a
zebrafish ortholog of sarcosin in zebrafish (ENSDARP00000012238) too, sharing about
60% identity. It would be interesting to isolate this proposed ortholog of Krp1 in zebrafish
and characterize its temporal expression pattern to determine if the timing of activation of
the gene is later than klhl.
5. Comparative Genomics, a Look into Evolutionary History
The usefulness of the zebrafish as a model to elucidate the function of human
genes has heightened with the construction of genetic linkage and RH maps by various
groups. Despite the 450 million years of evolutionary distance between zebrafish and
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Discussion
human (Kumar and Hedges, 1998), analyses of human and zebrafish gene maps reveal
extensive conservation of synteny between the two species, i.e. genes that are on the same
chromosome in human tend to be located in the same chromosome fragment in zebrafish
(Postlethwait et al., 1998).
In the study, we were able to determine the chromosomal location of klhl to LG13.
In humans, the proposed ortholog of KLHL was mapped to the short arm of chromosome
at position 6p12.2 while in mouse, the gene is located on the long arm of chromosome 9.
The proposed rat ortholog has been mapped to chromosome 8. Comparative mapping
suggests that zebrafish klhl is located in a similar genomic organization as in the human,
mouse and rat genome, being clustered together with the bmp5 and gsta3 gene. The
membership of the klhl genes in the LG13-Hsa6-Mmu9-Rn8 conserved synteny group
adds support to the predicted orthology. We were also able to identify another pair of
orthologous genes that are part of the LG13-Hsa6 conserved synteny group,
bpag1/BPAG1. Interestingly, however, the mouse ortholog of this gene, dld, is part of
another conserved synteny group between Hsa6 and Mmu1. Postlethwait et al. (2000) in
their study of vertebrate chromosomes suggested that mammalian genomes derive from
the fission of large ancestral chromosomes and these broken up differentially in different
tetrapod lineages. Taken together, the data suggest that klhl, gsta3, bmp5 and bpag1 were
located on a single chromosome in the last common ancestor of mammals and zebrafish.
However, this hypothesized chromosome broke apart differently in different lineages in
human and mouse. Our study of gene order between LG13 and Hsa6 also reveals that
while LG13 and Hsa6 are syntenic to each other, multiple intrachromosomal
rearrangements have altered gene orders in zebrafish and humans.
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Discussion
Besides being used for the prediction of gene orthology, such comparative gene
maps are useful for the identification of mutant genes, facilitating both positional cloning
and the candidate gene approach. Comparing the map locations of zebrafish genes and
their human counterparts, for example, could suggest candidates for zebrafish mutations.
Unfortunately, we have not been able to identify any disease locus associated with either
klhl or its mammalian homologs.
6. Rapid In Silico Cloning of Genes
Our study also reflects the emerging trend in the use of nucleotide sequence
databases to clone or identify a relevant gene. Nowadays, many analyses, previously
limited to the bench, can be performed with a computer. Such in silico analyses reduce the
time and effort needed to identify or clone a gene.
The cornerstone of molecular biological approaches in the genome project is the
identification of expressed genes by brute force sequencing – the generation of ESTs
(Schimenti, 1998). Despite their fragmentary nature, ESTs have proven to be useful in
analysing gene expression, cloning of genes and as markers on chromosomes (Schuler,
1997; Gong, 1999). This has led to the development of EST gene indices like Unigene.
Using automated procedures, ESTs are partitioned into sets or clusters that are very likely
to represent distinct genes. In addition to strong sequence similarity, clone identifiers can
be used to group ESTs derived from the same cDNA even when their sequences do not
overlap. Such a database has proven useful in our study in the identification of the partial
sequence for rat Klhl from various ESTs that belong to Unigene cluster Rn. 22511. We
had earlier used the sequence of zebrafish klhl to query the rat ESTs database to source out
rat ESTs clones similar to klhl. The result of this search was similar to the clones in the
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Discussion
Unigene cluster, indicating the utility of the database in assembly of individual ESTs into
a consensus sequence (Zhuo et al., 2001). Besides the identification of orthologous genes,
in silico analyses provide a fast and inexpensive way of obtaining information about gene
expression. For example, by looking at the cDNA libraries from which the ESTs are
obtained, we suggest that rat Klhl is expressed in a similar fashion to its orthologs in
zebrafish and human. Such a computational approach to expression analyses can even be
extended to determine the transcriptional profile of tissues as performed by Bortoluzzi and
colleagues (2000).
Another genome database programme that was useful in our study was the
Ensembl genome database project (Hubbard et al., 2000;Clamp et al., 2003). Ensembl
annotates known genes and predicts novel genes, providing a database of human genome
annotation. According to Hubbard et al. (2002), “Ensembl genes are regarded as being
accurate predicted gene structures with a low false positive rate” as they are supported by
experimental evidence of at least one form via sequence homology. Using Ensembl, we
were able to identify the putative human homolog of klhl located on chromosome 6.
Besides identifying two ESTs that match to this putative gene, we also have experimental
evidence that this gene is expressed in human skeletal muscles and heart. In addition to
annotating the human genome, the Ensembl database provides up-to-date sand
comprehensive sequence data from several metazoan organisms including human, mouse,
rat, zebrafish and the pufferfish Fugu rubipes (Clamp et al.,2003). We were able to
identify the putative mouse, rat and pufferfish ortholog of klhl through this function of
Ensembl. Again, we have also managed to identify several ESTs matching to the predicted
mouse and rat gene, providing evidence that it is expressed. The advent and demonstrated
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Discussion
utility of such databases suggests that the rigorous cloning of genes might be a thing of the
past.
7. Future Directions
With the increasing information on gene sequences from genome projects, the
problem of elucidating the function of genes has spawned a new area of research that is
being called “functional genomics”. At the forefront of this new era is a small tropical
freshwater fish originating from India, the zebrafish. A variety of factors have made the
zebrafish an excellent system for the analysis of the vertebrate genome. In this study, a
novel zebrafish kelch gene with orthologs in human, mouse and rat as well as pufferfish
was identified and characterized. Elucidation of the role klhl plays in the zebrafish would
have implications in the human system. The analysis of the expression pattern of klhl as
well as its domain organization generates many implications about its possible function.
Continuous effort is still required thereafter to determine the exact role this protein might
play in muscle structure and function.
To gain more insight into the function of klhl, functional analyses to perturb klhl
gene expression must be performed. There are two broad classes of functional analysis,
namely gain-of-function and loss-of-function analyses. Gain-of-function studies involve
the overexpression of a gene while loss-of-function analyses are carried out by destroying
or inhibiting the gene. Gain-of-function study can be easily achieved in zebrafish by the
microinjection of klhl RNA into the zebrafish embryo or by transgenesis. Loss-of-function
analyses are however not as straightforward. The nature of zebrafish enables the
application of chemical, deletional and insertional mutagenesis approaches to knock out
genes. However, these methods are unspecific. In recent years much effort has been
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Discussion
devoted to the development of new methods for loss-of-function study, including
dominant negative and RNA interference experiments (Hunter, 2000). While the efficacy
of such methods in zebrafish are still under debate (Li et al., 2000, Oates et al., 2000), the
recent development of morpholino-based gene knock-down technology opens the door to
the genome-wide assignment of function based on sequence in zebrafish (Nasevicius and
Ekker, 2000). Morpholinos are chemically modified oligonucleotides with similar basestacking abilities as natural genetic material. In zebrafish, they have been used widely and
have proven effective in the blocking of gene function (refer to Genesis special
morpholino, Vol. 30, Issue 3, 2001). Morpholinos work by interfering with the translation
initiation process.
In additional to the functional analyses, it will be necessary to identify the subcellular localization and also potential binding partners of klhl in zebrafish. GFP-tagged
klhl cDNA construct can be transfected into zebrafish embryos or muscle cell lines to
determine its cellular localization while in vitro coprecipitations assays can be performed
with glutathione S-transferase (GST) fusions of klhl and potential binding partners such as
talin. It would also be interesting to isolate and characterize the zebrafish ortholog of krp1
and determine its temporal pattern of expression in zebrafish embryos. Such a study would
provide us with a better understanding of myofibrillar assembly and their associated
myopathic disorders, and hopefully this will oneday translate into therapeutics treatments.
As we move into the near future where studies of the relationship of gene function and
protein structure will become increasingly important, further insights can be obtained from
the determination of klhl crystal structure.
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Discussion
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Discussion
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[...]... the zebrafish, the Zebrafish Information Network (ZFIN) (http://zfin.org) was set up as to cope with the phenomenal rate of increase of information The ZFIN is a centralized database for zebrafish researchers, providing links and information about zebrafish genes, mutations, genetic maps etc (Westerfield et al., 199 9a, b; Sprague et al., 2003) In addition, zebrafish resources are also available from... Postlethwait and Talbot, 1997) The two main approaches of cloning mutated genes, positional cloning and candidate gene approach, have benefited greatly from the recent advances in zebrafish genomic infrastructure (reviewed in Talbot and Hopkins, 2000; Malicki et al., 2002) The efficient identification of genes disrupted by mutation in zebrafish requires dense maps of the genome Prior to 1994, there was no genetic... bridge the gap between its vertebrate and invertebrate counterparts in studies of development and genetics In addition to its developmental advantages, recent studies indicate that the zebrafish has a great potential to serve as a model for human disease that range from heart failure and vascular disease to fields as diverse as osteoporosis, renal failure, Parkinson’s disease, diabetes and cancer (for... Relationship of the Zebrafish and Human Genomes The third virtue of the system is the conservation of synteny between zebrafish and human genomes Besides facilitating the identification of mutants by positional cloning and the candidate gene approach, the genetic maps have been useful in comparative studies between zebrafish and other vertebrate genomes By comparing the map positions of zebrafish genes... years ago (mya), have greater conservation than zebrafish and human (Gates et al., 1999; Woods et al., 2000) Despite the current gaps in the zebrafish- human comparative map, conservation of synteny between the two has had several uses First, such analyses have been valuable in defining candidate genes for zebrafish mutant (Karlstrom et al., 1999; Schmid et al., 2000) For example, the yot locus was mapped... genetic map for zebrafish and the paucity of resources such as large-insert genomic libraries rendered the task virtually impossible (Malicki et al., 2002) Today, a full array of genomic and molecular genetic tools is available Large-insert genomic libraries needed for positional cloning have been generated To date, two zebrafish yeast artificial chromosome (YAC) libraries, one bacterial artificial chromosome... groups as additional genes are mapped A minimum estimate of ~300 conserved synteny groups was thus 21 Introduction estimated between the zebrafish and human genomes (Wood et al., 2000) Similar results were obtained in another study done at the same time (Barbazuk et al., 2000) Analyses of mouse and human, as well as zebrafish and human synteny groups have also led to the conclusion that mouse and human,... single linkage group (Johnson et al., 1996) Among vertebrates, only human, mouse, rat, and zebrafish have closed linkage maps More than 3845 microsatellite (CA) repeats have been meiotically mapped since the last update in July 2001, providing an average resolution sufficient to initiate positional cloning (Shimoda et al., 1999; http:/ /zebrafish. mgh.harvard.edu) Published genetic linkage maps have also... mutagenesis (Knapik, 2000) Chemical mutagenesis using ENU is by far the most widely employed method in zebrafish as it is effective and easily administered by incubating the fish in ENU Other chemicals that have been used include EMS and TMP which cause small deletions Radiation methods using X-rays and gamma rays are routinely performed in zebrafish laboratories to induce genome-wide mutations Causing large... see Shin and Fishman, 2002; Ackermann and Paw, 2003) Many of the mutant phenotypes identified in the mutagenesis screens are reminiscent of human clinical disorders The validity of using the zebrafish as a model for human disease is illustrated by the various examples of zebrafish mutant phenotypes with clinical relevance in the various fields of haematopoiesis (Brownlie et al., 1998; Wang et al., 1998), ... and Talbot, 1997) The two main approaches of cloning mutated genes, positional cloning and candidate gene approach, have benefited greatly from the recent advances in zebrafish genomic infrastructure... represents just the data acquisition phase Faced with an avalanche of sequence data, researchers are now faced with the daunting task of deciphering and interpreting the data and get more biology... cytoskeletal filament linkages at the 92 sacrolemma of striated muscle Table Summary of EST clones homologous to klhl iv 73 List of abbreviations LIST OF ABBREVIATIONS aa amino acid AP alkaline phosphatase