From EST analysis to potential silk gene discovery 1

From EST analysis to potential silk gene discovery From EST analysis to potential silk gene discovery HUANG WEIDONG (B.Sc., M.Sc.) HUANG WEIDONG 2006 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements I would like to express my appreciation to my supervisor, Associate Professor Yang Daiwen and Professor Lam Toong Jin, for their invaluable guidance, patience, and trust throughout the project. Special thanks to my former supervisor, Associate Professor Sin Yoke Min. Without his encouragement it will not have been possible for me to complete this project. I would also like to express my sincere appreciation to Associate Professor Gong Zhiyuan, for his helpful advice and critical suggestions. I would also like to thank all my colleagues, including some former lab members, for their friendship, help and company. Special thanks to Ashok Hegde, Seng Eng Kuan, Lin Zhi, Zhang Wensheng and Wu Qiang. The financial assistance in the form of a research scholarship provided by NUS is gratefully acknowledged. I would also like to thank my parents for their sustaining love. Although words are not even enough to express my gratitude, I would still like to thank my husband Jiayi, without his constant love and support, I would not have been able to accomplish or even start this thesis. i Table of Contents Acknowledgements i Table of Contents ii List of Figures ix List of Tables xii List of Abbreviations xiii Reference Appendix Summary Chapter General introduction Chapter Literature review 2.1 Silks in the nature 2.2 Spider silks 2.2.1 Spider silk production organs 2.2.2 Spinning process of silks 10 2.2.3 Mechanical properties of spider silks 15 2.2.4 Summary 15 2.3 Spider silk genes 16 2.3.1 Identification of spider silk genes 16 2.3.2 Structure and organization of spider silk genes 23 2.3.2.1 Spider silk genes are large transcripts 23 ii 2.3.2.2 Spider silk genes contain internal repetitive sequences 24 2.3.2.3 Spider silk genes have a C-terminal conservative region 26 2.3.2.4 Codon usage analysis and bias of spider silk genes 27 2.3.3 Summary 28 2.4 Expression and structural analysis of spider silk proteins 29 2.4.1 Expression of spider silk proteins in vitro 29 2.4.2 Structural analysis of spider silk proteins 32 2.4.3 Summary 37 2.5 EST strategy: history, applications and further trend 37 2.5.1 Concept of EST 37 2.5.2 Characteristics and analysis of EST data 38 2.5.3 Applications of EST approach 41 2.5.3.1 Gene identification 41 2.5.3.2 Expression profiling analysis 42 2.5.3.3 Construction of physical maps 43 2.5.3.4 Gene annotation from genomic sequences 43 2.5.4 Future prospects 44 2.5.5 Summary 45 Chapter General materials and methods 47 3.1 Spider 47 3.2 DNA manipulation 47 3.2.1 Agarose gel eletrophoresis 47 iii 3.2.2 Preparation of plasmid DNA from E. coli 47 3.2.3 DNA digestion, DNA fragment purification and ligation 48 3.2.4 DNA sequencing 48 3.2.5 DNA sequence analysis 48 3.2.6 Polymerase chain reaction (PCR) 49 3.3 RNA manipulation 49 3.3.1 Total RNA purification 49 3.3.2 Poly A+ RNA purification 50 3.4 Protein manipulation 51 3.4.1 Quantitative protein assay 51 3.4.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 52 3.4.3 Coomassie-blue staining 53 3.4.4 Desalting and buffer exchange of proteins 53 3.5 Bacteria transformation 54 3.5.1 Preparation of E. coli competent cells used for heat-shock transformation 54 3.5.2 Transformation of E.coli competent cells 55 Chapter cDNA library construction, generation and analysis of EST clones 4.1 Abstract 56 4.2 Introduction 56 4.3 Materials and methods 59 4.3.1 cDNA library construction 59 iv 4.3.1.1 Zap- cDNA synthesis 59 4.3.1.2 Packaging and tittering 63 4.3.1.3 Amplification 63 4.3.1.4 In vivo Mass Excision 64 4.3.1.5 Preservation of Spider Silk cDNA library 64 4.3.2 Generation and analysis of cDNA clone 64 4.3.2.1 Generation of cDNA clones 64 4.3.2.2 Sequence analysis 66 4.3.3 RT-PCR reactions 67 4.3.4 Recombinant proteins expression 68 4.4 Results and discussion 69 4.4.1 Construction and characterization of spider silk glands cDNA library 70 4.4.2 Overview of EST clones in spider silk glands 71 4.4.3 Distribution of the identified clones in spider silk glands cDNA library 75 4.4.4 The most abundant clones and some implications 77 4.4.4.1 Silk genes 80 4.4.4.2 Translational factors 80 4.4.4.3 Ornithine decarboxylase 81 4.4.4.4. Sec 61 and ubiquitin 82 4.4.4.5. Heat shock proteins 83 4.4.5. Novel cDNA clones containing repetitive sequence 84 v 4.4.6. RT-PCR analysis 95 4.4.7. Recombinant protein production 97 4.5. Conclusive remarks 98 Chapter Identification and characterization of TuSp1 100 5.1 Abstract 100 5.2 Introduction 100 5.3 Materials and methods 103 5.3.1 Sequence analysis of TuSp1 103 5.3.2 Northern blot analysis of TuSpI 104 5.3.2.1. RNA sample fractionation 104 5.3.2.2. Membrane transfer 105 5.3.2.3. Probe preparation 105 5.3.2.4. Hybridization and immunological detection 106 5.3.2.5. Stripping of the membranes 106 5.3.2.6. Probe removal from Northern blots 107 5.3.3 Expression specificity analysis (mRNA level) of TuSp1 107 5.3.3.1 RT-PCR reaction analysis 107 5.3.3.2 In situ hybridization 108 5.3.3.2.1 Fixation 108 5.3.3.2.2 Dehydration and embedding 108 5.3.3.2.3 Sectioning 109 5.3.3.2.4 Probe preparation 109 vi 5.3.3.2.4.1 RNA probe synthesis 109 5.3.3.2.4.2 Carbonate hydrolysis 110 5.3.3.2.4.3 Probe quantification 111 5.3.3.2.5 In situ section pretreatment 111 5.3.3.2.5.1 Deparaffination and rehydration 111 5.3.3.2.5.2 Proteinase K treatment and post fixation 112 5.3.3.2.6 Hybridization 112 5.3.3.2.7 Post-hybridization wash 113 5.3.3.2.8 Colorimetric detection 113 5.3.4 Expression specificity analysis (protein level) of TuSp1 114 5.3.4.1 Recombinant protein expression of TuSp1 114 5.3.4.2 Generation of antiserum against TuSp1 115 5.3.4.3 Western blot analysis 116 5.3.4.4 Immunofluorescence staining analysis 117 5.3.5 Structural analysis of recombinant protein Tu-81 118 5.3.5.1 Circular dichroism experiments 118 5.3.5.2 1D NMR analysis 118 5.4 Results and discussion 119 5.4.1 Sequence analysis of TuSp1 119 5.4.2 Northern blot analysis of TuSp1 124 5.4.3 Expression specificity of TuSp1 (mRNA level) 126 5.4.3.1 RT-PCR analysis 126 5.4.3.2 In situ hybridization analysis 129 vii 5.4.4 Expression specificity of TuSp1 (protein level) 137 5.4.4.1 Expression of TuSp1 recombinant proteins 137 5.4.4.2 Western blot analysis 140 5.4.4.3 Immunofluorescence analysis 145 5.4.5 TuSp1 is the major component in tubuliform gland and it encodes a novel fibroin gene from Nephila antipodiana 147 5.4.6 Structural analysis of TuSp1 153 5.5 Discussion and conclusive remarks 155 Chapter General discussion and conclusion 160 Reference 169 Appendix 179 viii List of Figures Figure 2.1. The seven specialized glands and their different amino acid compositions of a typical Araneid orb weaver. Figure 2.2. Spider spinneret (silk secreted from piriform gland spigot, Spiny Back Spider, Castercantha sp.). 12 Figure 2.3A. Diagrammatic representation of spider spinneret. 13 Figure 2.3B. Single spider spinneret showing the internal anatomy (J.M. Palmer). 13 Figure 2.4. A spider’s dragline spinneret. 14 Figure 2.5. Predicted amino acid sequence for the Spidroin protein, rearranged to show repetitive elements. Figure 2.6. 24 The silk proteins from N. clavipes are depicted as generalized, or consensus, amino acid repeats. Subscripts indicate the number of times a sequence is tandemly repeated. Figure 4.1 Nucleotide and deduced amino acid sequences of EST clone B386. Figure 4.2 87 Nucleotide and deduced amino acid sequences of EST clone C622. Figure 4.4 86 Nucleotide and deduced amino acid sequences of the EST clone B6. Figure 4.3 25 88 Nucleotide and deduced amino acid sequences of EST clone C675. 89 ix from tubulifrom is mainly composed of β-sheet structure. This is the first report about the solution structure of truncated TuSp1 fibroin protein while further studies will be able to reveal more information for the contribution and conversion of solution fibroin proteins to the solid silk fiber formation. Combining our above observations and the comparison of amino acid composition, we conclude that TuSp1 is expressed in tubuliform gland and contributes the major component of the fibroin from this gland. We have thus identified from an EST clone a novel spider silk fibroin member of the silk gene family. Noticeably, in the last couple of months, when we begin to conclude our work, there appeared a number of papers reporting the isolation, identification and evolutional studies of TuSp1 gene from tubuliform gland from different species of spiders. Tian et al. (Tian and Lewis, 2005) constructed tubuliform-gland-specific cDNA libraries from three different spider families, Nephila clavipes, Argiope aurantia, and Araneus gemmoides. Through sequence analysis and amino acid composition comparison, they inferred that TuSp1 (protein, tubuliform spidroin 1) contains highly homogenized repeats and is the major component of tubuliform silk. Further more, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were applied by Hu et al. (Hu, et al., 2005) to isolate the egg case fibroin, named tubuliform spidroin (TuSp1), from the black widow spider, Latrodectus hesperus. Real- - 163 - time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk and that of the predicted amino acid composition from the primary sequence of TuSp1 supports the assertion that TuSp1 represents a major component of egg case fibers. The authors also deduced that TuSp1 is composed of highly homogeneous repeats of 184 amino acids in length. Additionally, through sequence analysis of cDNA molecules from gland-specific cDNA libraries, TuSp1 sequences from 12 spider species that represent the extremes of phylogenetic diversity were reported by Garb et al., (Garb and Hayashi, 2005). The authors demonstrate that TuSp1 encodes tandem arrays of an approximately 200-aa-long repeat unit. These repeats across species reveal the strong influence of concerted evolution, resulting in intragenic homogenization. Phylogenetic analyses of 37 spider fibroin sequences also support the monophyly of TuSp1 within the spider fibroin gene family, consistent with a single origin of this ortholog group. Very recently (Nov. 2005), CySp1 (TuSp1) from spider Nephila clavata was reported by Zhao et al., (Zhao, et al., 2005). The fine organization of CySp1 (TuSp1) gene was disclosed, including the central repetitive region as well as the non-repetitive N-terminal and C-terminal parts. The N-terminal sequence of TuSp1 was reported for the first time. Also it has been indicated for the first time that the internal repetitive region consists of more than one type of complexes and remarkably conserved polypeptide repeats (A1B1). - 164 - The repetitive region is characterized by alternating arrays of hydrophobic and hydrophilic blocks. Through sequence alignment analysis, we conclude that our TuSp1 from Nephila antipodiana is the same as the one from N. clavipes (Tian and Lewis, 2005), L. hesperus (Hu et al., 2005) and other spider species (Garb, et al., 2005), and the CySp1 from Nephila clavata (Zhao, et al., 2005). Generally, our result of isolation and identification of TuSp1 from Nephila antipodiana is therefore confirmed by the studies from other groups. On the other hand, our clone of TuSp1 is short compared with those published ones. This is mainly due to that we did not screen our cDNA library using the clone obtained from EST analysis. Actually, we have tried 5’-RACE approach, to try to obtain the Nterminal part of TuSp1, however, due to the repetitive characteristic of TuSp1, this experiment did not success. No exception, all the four published TuSp1 papers employ the data of the amino acid composition of the tubuliform silk fiber to compare with that of the deduced TuSp1 gene, to infer that TuSp1 contributes the major component of tubuliform silk. Additionally, realtime quantitative PCR was also carried out by Hu et al. (Hu, et al., 2005) to demonstrates that TuSp1 is selectively expressed in the tubuliform gland. In our work, except for RTPCR, in situ hybridization analysis, Western blot and immunofluorescence approach were applied, to demonstrate at both the mRNA and protein level that TuSp1 gene has the glandspecific expression pattern. Moreover, in situ hybridization and immunofluorescence - 165 - techniques are applied for the first time in spider silk gene expression analysis. Our results might therefore provide new methods/strategies for silks gene’s identification in the future. In the past few of years, spider silk proteins have been identified from different types of silk glands in various species. However, so far there is not adequate information for fully understanding of the process of silk formation. The structure and evolution of genes that encode silk fibroin proteins are also needed for further elucidation. Therefore, future research of spider silk genes might focus on following aspects: 1. Molecular isolation and characterization EST clones. Isolation and sequence analysis of more EST clones may help to obtain novel cDNAs/proteins that play important roles in spider silk protein synthesis, silk fibroin protein secretion and silk fiber formation. The clones of special organization or novel sequence could be candidates for further analysis. 2. Biological functional analysis of novel clones. EST clones containing novel/repetitive sequences could be applied for biological functional analysis, including determination of the cellular localization, gene structure and potential biological functions. Novel and/or less abundant fibroin gene might be obtained through functional analysis. - 166 - 3. Gene structure, genomic organization and protein structural analysis of known silk genes. The N-terminal parts of most known spider fibroin genes are not clear, nor are the genomic structures of these silk genes. Further analysis might be applied for clarification of the gene structure and genomic organization of these genes. Further protein structural analysis of known spider fibroin proteins will also help for the understanding of silk formation. 4. Fibroin gene expression pattern analysis. In the last couple of years, it has been found that some of the fibroin genes could express in more than one type of silk glands. For example, ADF-3 (=MaSp2) was found to be expressed mainly in major ampullate silk gland; but was also found in flagelliform and aggregate silk gland (Guerette, et al., 1996); MaSp and MaSp2 genes were confirmed to be expressed in both major ampullate and tubuliform silk gland (Garb, 2005). On the other hand, some of silk glands could have more than one type of fibroin genes expressed, as Five fibroin genes, including ADF-2, ECP-1, MaSp and 2, and TuSp1, are found to be expressed in tubuliform silk gland (Guerette, et al., 1996; Hu, et al., 2005 and Garb, et al., 2005). Fibroin gene expression pattern analysis might be of help to provide some clues for disclosure of the mystery of the differentiation of various types of silk glands and evolution of fibroin genes; more information related to the expression specificity of fibroin genes might also help to clarify if the less abundantly expressed fibroin genes could be incorporated into the formation of silk fibers from a specific gland or not. - 167 - Noticeably, some of the techniques used in our current study could also be applied for future fibroin gene expression pattern analysis. In summary, one cDNA library of seven types of silk glands from Nephila antipodiana has been constructed and a partial cDNA of TuSp1 has been identified and characterized. 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Probe—against TuSp1 from young female spiders 13 4 Figure S3 Probe—against MiSp1 from mature female spiders 13 5 Figure S4 Probe—against TuSp1 from mature female spiders 13 6 x Figure 5.6 SDS-PAGE to analyze the recombinant protein of 81- 1a and 81- 1b 13 8 Figure 5.7 FPLC analysis of recombinant protein 81- 1a 13 9 Figure 5.8 Westren blot analysis using recombinant protein 81- 1a and 81- 1b to compare the effect... 4 X 10 9 8 X 10 8 2 X 10 9 1 X 10 8 2 X 10 8 1 X 10 8 5 X 10 8 1 X 10 5 3 X 10 4 9 X 10 3 1 X 10 3 5 X 10 3 3 X 10 3 9 X 10 4 1 X 10 5 Modulus: measurement of materials’s stiffness indicated by the slope of the strees strain curve; Strength: the maximal stress achieved prior to fracture; Energy to break: a measure of the material’s toughness indicated by the area under s stress-strain curve 2.2.4 Summary In general,... employ up to seven types of abdominal glands to produce silks for various purposes Since 19 90, two silk genes MaSp1 and MaSp2 from the major ampullate silk gland (making dragline and frame silk, Xu, and Lewis, 19 90; Hinman and Lewis, 19 92; Beckwitt and Arcidiacono, 19 94; Guerette, et al., 19 96; Beckwitt, et al., 19 98; Gatesy, et al., 20 01; Tian, et al., 2004), two silk genes from the minor ampullate silk. .. Figure 5.9 14 1 Native Tubuliform proteins from Nephila antipodiana detected by antiserum against recombinant protein 81a 14 2 Figure S5 Original pictures of figure 5.8 and figure 5.9 14 4 Figure 5 .10 Immunofluorescence analysis of TuSp1 protein and its cellular localization 14 6 Figure 5 .11 Alignment of spider silk fibroin C-terminal amino acid sequences 15 0 Figure 5 .12 Phylogenetic tree 15 1 Figure 5 .13 The... transition is needed for further investigation 2.3 Spider silk genes 2.3 .1 Identification of spider silk genes Since 19 90, a number of spider silk cDNAs/genes have been identified The first spider silk gene was reported by Xu and Lewis in 19 90 (Xu, and Lewis, 19 90) In this report, dragline silk from the major ampullate silk gland was purified and digested The digested silk peptide was applied for amino... cDNA of TuSp1( clone 81) Figure 5.2 12 1 Schematic representation of the domain structure and internal repetitive sequence of TuSp1 Figure 5.3 97 12 3 Northern blot analysis to show the expression of TuSp1 in Nephila antipodiana 12 5 Figure 5.4 RT-PCR to analyze the expression of TuSp1 (clone 81) 12 8 Figure 5.5 In situ hybridization 13 0 Figure S1 Probe—against MiSp1 from young female spiders 13 3 Figure... study are: 1 To construct a cDNA library sourcing from the seven types of silk glands from Nephila antipodiana; 2 To create an EST database using this cDNA library, and to isolate clones that are potentially involved in silk formation; 3 To characterize and analyze cDNAs that might be novel silk genes, or related to silk formation Various suitable methods and techniques will be applied, to try to provide... mass spectrometry and reverse genetics to isolate novel silk genes (Hu, et al., 2005) Table 2.2 Summarizes the information of the identified silk genes (modified from Hayashi, 2002) Accession Species cDNA /Gene Reference M3 713 7 Nep.cla MaSp1 (Spidroin 1) Xu and lewis, 19 90 U03848 Nep.cla MaSp1 U37520 U20329 M92 913 U20328 U03847 Nep.cla Nep.cla Nep.cla Ara.bic Ara.bic MaSp1 MaSp1 MaSp2 MaSp2 MaSp2 AF027735... of EST clone D106 Figure 4.6 Nucleotide and deduced amino acid sequences of EST clone D1 21 Figure 4.7 93 Nucleotide and deduced amino acid sequences of EST clone A105 Figure 4-9 91 Nucleotide and deduced amino acid sequences of EST clone D33 Figure 4.8 90 RT-PCR to analyze the mRNA levels of EST clone B6 94 96 Figure 4 -10 SDS-PAGE to analyze the recombinant protein of A105, B386 and C622 Figure 5.1 . Tu- 81 118 5.3.5 .1 Circular dichroism experiments 11 8 5.3.5.2 1D NMR analysis 11 8 5.4 Results and discussion 11 9 5.4 .1 Sequence analysis of TuSp1 11 9 5.4.2 Northern blot analysis of TuSp1 12 4. From EST analysis to potential silk gene discovery HUANG WEIDONG 2006 From EST analysis to potential silk gene discovery HUANG WEIDONG (B.Sc.,. expression of TuSp1 11 4 5.3.4.2 Generation of antiserum against TuSp1 11 5 5.3.4.3 Western blot analysis 11 6 5.3.4.4 Immunofluorescence staining analysis 11 7 5.3.5 Structural analysis of recombinant