21st Century Manufacturing Episode 2 Part 10 pot

37. Biotechnology Chap.9 Additionally, enzymes can be produced synthetically and are used in everyday situations-for example in laundry detergents to break down proteins that may have been spilled on clothing. 9.5.4 The Genome and Chromosomes The genome is the complete genetic information or total DNA content of an organism. It is packaged into chromosomes, which are genetic structures consisting of tightly coiled threads of deoxyribonucleic acid (DNA) and associated proteins. The genome functions as a blueprint for making an organism. It contains all instruc- tions for building cell structures and directing life processes throughout the lifetime of the organism. Different organisms have different numbers of chromosomes per nucleus. Humans have 46 chromosomes arranged in 22 pairs plus XX or XY -except in the sperm or the egg, which has 22 chromosomes plus X or Y. The nuclei of most human cells contain two sets of chromosomes, each set contributed by one parent. Chromosomes can be observed under a microscope; when stained with certain dyes, they reveal a distinctive pattern of light and dark bands. In karyotype analysis, observable differences in size and banding patterns distinguish the chromosomes from one another. It is also possible to detect major chromosomal abnormalities such as that of Down's syndrome, in which there is an extra copy of chromosome 21. 9.5.5 DNA l.DeoxyriboNucleic Acid) as the Carrier of Genetic Information The science and engineering of deoxyribonucleic acid (DNA) are central to most biotech industrial processes. Why is DNA so special? The answer is that DNA is the genetic material for probably all living organisms," (It should be pointed out that some viruses use RNA rather than DNA as their genetic materiaL) Specifically, DNA carries the genetic information that is vital for the cells in a person's body to grow, function, and divide normally. Thus, DNA has a universal role in defining and regulating life on the planet, from simple bacteria to complex organisms like us. It is also remarkable for other reasons, including the fact that DNA can replicate itself in a precise fashion over the lifetime of an organism. DNA directs the production of proteins necessary for all cellular functions including its own synthesis. Earlier in the text, the double-helical structure of DNA was compared with a twisted rope ladder. To further understand the structure of DNA, imagine two strands of "beads" that have been twisted together. This double-helical structure is made up as follows: • Each "bead" is a 5-carbon, sugar/phosphate molecule called a nucleotide. Each nucleotide contains a base shown on the right side of Figure 9.5. There are four possible bases: two purines (adenine lA] or guanine [G)), or one of two pynm- idines (thymine [T} or cytosine [Cn (Figures 9.6 and 9.7). 40. Mascarenhas (1999), in his lectures, makes an informal but pedagogically helpful analogy between the genetic information stored in DNA and the notes and music stored on a standard cassette tape. Each track of the cassette tape contains the information for one song, just like one length of DNA stores the information for one gene. Between each track (or gene) there is a break before the next song (or gene) starts. Abo, with the naked eye, nothing can be seen on the cassette tape-but the music is there ready fa be expressed. Similarly,in the DNA strand, the genetic information is there, ready to be expressed. 9.5 A Bioscience Review 375 5'end O· I -O~p=O I o HJ ~ ~o}f-1 h~H ?H -o-p=o 6 I H,C , 0 HH H" o H I -o-p=o 6 H2t.'H_OOH~ ~ Base ~H o H Base 3'end F1gun 9.5 Schematic representation of the nucleotide that makes up DNA. RNA only DNA llDd RNA DNA only 0 H"N/ H NX"'N/ H H-<~IH H-<N I NAN/H I I H H I H Guanine Adenine 0 H"N/ H 0 I NX:N~H I HX'w_ H H3C~ ~ H I I I I H N/'O H N/'O H /'0 I I I H H H Uracil Cytosine Thymine FiJune 9.6 The five nitrogenous bases of DNA, and RNA-see Section 9.7.2 378 Biotechnology Chap. 9 (a) Adenme Thymine H;:Q i)=' s""H ··H H H 0 Sugar • Carbon atom • Nitrogen atom (b) H H~ H-N/ H S,." HQ-H /N-H 0 Sugar H Guanine Cytosine Fipre 'J.7 Base-pairing arrangements in DNA. (From An Introduction to Genefic Engilluring by Desmond S.T.Nicholl, © 1994. Reprinted with the permission of Cambridge University Press.) • To create the links within one strand, the nucleotides are attached to each other by a phosphodieeter bond between the phosphate group at the 5' carbon atom of one nucleotide and the :\' carbon atom of the next nucleotide. These bonds are shown on the left of Figure 9.5. This creates a directionality of 5' ? 3' to the string of nucleotides . • To create the links between the two strands, the bases on one strand are loosely attached with hydrogen bonds to the bases on the opposite strand (Figure 9.7). Base pairing follows a fixed rule: A always pairs with T; G always pairs with C. Thus, a sequence of ATGG on one strand means the other strand will have a complementary sequence ofTACC 5' - ATGGCfACCAAGGTA - 3' 3' -TACCGATGGTICCAT - 5' The genome can be described in terms of the number of these base pairs. DNA in a simple bacterial cell such as E. coli is made up of about 4 X 10 6 base pairs: all of these are shown together in Figure 9.8.By contrast, a human genome is about 3 bil- lion base pairs. 9.5.6 DNA Replication and Its Relationship to Cell Division Before a cell in an organism divides, the DNA replicates, as illustrated in Figures 9.9,9.10, and 9.11. As shown in Figure 9.9, the double-stranded DNA molecule unwinds into two single strands separating the A-T and CG base pairs. Next, with Adenme Thymine Carbon atom Nitroeen atom 'Sugar Sugar' Sugar Sugar Guanine Cytosine 9.5 A Bioscience Review 377 Hgurt' 9.8 Electron micrograph of an Eschonctn« coli (E. wli) DNA molecule the help of certain enzymes, each strand picks up the bases of free nucleotides in the cell. This is shown schematically in Figure 9.10. Once again, the base-pairing rules apply in this pickup process. In this way, each new double helix becomes a duplicate of the original one because, as shown in both Figure 9.10 and Figure 9.11, the original strands act as the template, specifying which bases are to be added to the growing strands. Thus when the cell proceeds to divide, each of the new Figure 9.9 Untwisting of linear DNA strands during replication. The strands untwist by rotating about the axis of the unreplicated DNA double helix. Template strand Presence of DNA polymerase _\J 5' Pairs of DNA strands " Figure 9.10 DNA replication involves the addition (bottom left) offree nucleotides. The bases are seen in this diagram as the hexagon shapes, and the sugar/phosphate bonds that become the strands are the attached "bar lines." DNA polymerase and other proteins catalyze the process. 378 -Position of next nucleotide added 10growing chain I'reenucleo6des 9.6 Bioprocesses 379 3' FllJlll'e9.11 TWoDNA molecules being created from one, leading to two daughter cells on the upper right and left of the figure. daughter cells receives a set of DNA molecules identical to that of the original cell-as indicated in Figure 9.11. •.6 BIOPROCESSES 9.6.1 Gene Expre ••lon: Connection between Gen •••DNA. RNA, and Proteins Genes are the basic unit of heredity. Each gene is a specific sequence of DNA nuc1eotides that carries the information to direct synthesis of a protein. The process by which the genetic information encoded on DNA flows into the cell's "protein fac tories" is a complex set of biochemical reactions (Figure 9.12). To daughter ceu z rodaughter ceu i 380 Biotechnology Chap.9 The genetic material J1gure 9.12 Summary of gt:n", expression. (From An Introduction to Genetic Engineering by Desmond S.T. Nicholl, © 1994.Reprinted with the permission of Cambridge University Press.) 9.6.2 RNA tHiboN-ucleic Acid) A key substance in gene expression is ribonucleic acid (RNA). The synthesis offunc- tional proteins out of information encoded in DNA requires several types of RNA. These include ribosomal RNA (rRNA), messenger RNA (mRNA), and transfer RNA (tRNA). Gene expression also requires several proteins, for example, RNA polymerases. Is arranged as Arra'nged' as a ),. ~ ""peo" "~::,~",, ~ Requires In prokaryotes In eukaryctes Ttanscriptjcn R,'''''''Y I~~ I" I simple P By Translation Genes. Geneshave Whi,ha'''UP'd'' ~ Operons I~::::" ~ Which are ~i'h~:::,.:.:::'Y~~<d'" ~ (mRNA) ~ Cosmpo,,"",o,s,p,,,,,,, A::O.'::, Ribosomes Codons andamino acids 9.6 Btoproceeees 38' RNA resembles DNA, but there are some major differences: • RNA is a chainlike molecule but single stranded. • RNA also has four bases, but RNA contains the base uracil (U) instead of the base thymine (T) that is used in DNA. •The sugar in RNA is ribose, whereas the sugar in DNA is deoxyribose. Both are 5-carbon sugars (pentoses), but the DNA sugar lacks an oxygen atom at the 2' carbon atom. The RNA sugar is thus called ribose, while the DNA sugar is called deoxyribose-that is, a ribose without oxygen at the 2' carbon atom. The abbreviated terms RNA and DNA are derived from these descriptions of the sugar molecules. 9.6.3 Transcription The first step in gene expression is a process known as transcription, in which a DNA molecule carrying the genetic information isused as a template to synthesize an mRNA moleculef Ttanscription is similar to DNA replication in that a single strand of DNA is used as the template for the synthesis of a complementary nucleic acid sequence. In Figure 9.13a, the shaded ellipse represents the enzyme called RNA poly- merase. As implied by the arrow, the polymerase moves along the DNA. Over short regions, the polymerase temporarily unwinds the two DNA strands as it moves along the genetic information. RNA polymerase also plays a role in linking the proper ribonucleotides of mRNA to each other, in order to form the mRNA chain. The DNA acts as the template to specify which is the correct ribonucleotide to add. This proceeds according to the base-pairing rules except that uracil (U) in the RNA replaces thymine (T) in the DNA. Note that the newly formed mRNA strand has the same sequence as the nontemplate DNA strand. For any given gene, only one of the DNA strands is used as the template for mRNA synthesis. 9.6.4 Promoters How does this whole process get triggered? The answer is that in addition to these base-pairing rules, there are other important regulatory sequences and orderings specified in the DNA strands (see Nicholl, 1994; Okamura, 1998). Thus "upstream" from the specific gene being expressed on the top right of Figure 9.13a, there are DNA sequences to which the RNA polymerase can bind. These sites for starting transcription-that is,which can temporarily bind the RNA polymerase to the DNA strands-are known as promoters. These promoter sites can also be seen as clever switches. Thus, when certain food products enter our system, the switches of the pro- moter sites are activated, thereby setting off the desirable chain reaction consisting of (DNA + mRNA + protein synthesis + needs of the cell + needs of the body). Speaking colloquially, this is why one generation of parents should encourage the next generation to eat a good breakfast. For example, a group of genes known as the lac operon in the E. coli bacteria in our intestines codes for the enzyme that can "Transcription also refers to the synthesis of rRNA and tRNA, but it is tbe ItlRNA thai is trans- latedinloproteins. 382 Biotechnology Chap. 9 (a) 5' ~ATGGCTACCAAOGTA II 3'-TACCGATGGTTCCAT I 5' RNA polymerase (b) NH, F1pre 9.13 Transcription in the upper sketch and translation in the lower two sketches. In (a) the DNA strands are temporarily separated by RNA polymerase, so lhat the mRNA ClUI be formed during gene expression. (From An IntrodU{;tion to Gtnmc Engineering by Desmond S.T. Nicholl, C 1994. Reprinted with the permission of Cambridge University Pr~) break down lactose. or milk:sugar. However, the "upstream" operator, or promoter, for this group of genes is a site that can only get triggered in the presence of lactose. The E. coli bacteria and lactose thus begin a complex chain reaction for breaking down other food in the intestine. 9.8.5 Translation or Protein Synthesis 'D'anslation is the process by which proteins are synthesized using the information carried by mRNA. the product of transcription. Recall that proteins are made up of amino acids. The specific nucleotide sequence of the mRNA determines which amino acids are to be linked to create a particular protein. A combination of three nucleotides (e.g.tACO, GOO. CAO) is called a codon and specifies an amino acid. Figure 9.14 describes the correspondence between the codons and the amino acids 9.6 Bioprocesses 383 F1pft '.14 The "genetic code": nucleotide sequences for amino acids. A total of 64 three-letter oodons can be formed from the four letters (A, C. G, and T) of the DNA nucleotide bases. However, there exist oniy20 amiDo acids, not 64.This means that there is more than one codon for most of the amino acids: for example, Valine (Val) is shown in four entries at the bottom left of the table.1hree of the COltons(UAA. VAG, and UGA) signal "stop" and the end of an mRNA chain. that they specify.The process by which the mRNA sequence is "translated" into an amino acid sequence is described in the following paragraph. Translation requires ribosomes. which are composed of ribosomal RNA (rRNA) and ribosomal proteins. Ribosomes are the protein factories of cells and are depicted in Figures 9.13b and 9.13c as the indented elliptical sbapes'' The ribosome moves along the mRNA chain, and successive amino acids are linked into a growing protein chain, as shown in Figure 9.13c.1his process requires another type of RNA- transfer RNA, or tRNA. There is at least one type of tRNA for each amino acid.With the rules of base pairing coming into play again, the mRNA's codon sequence is rec- ognized by tRNA molecules that are (1) carrying an anticodon of complementary ribonucleotides and (2) carrying the amino acid that is specified by the codon. Thus, as the ribosome moves along the mRNA, tRNAs bring the appropriate amino acids to be added to the growing protein chain. 'Ibis process continues until the last codon, the "stop" codon, is reached. As shown in Figure 9.14, this last codon can be UAA, UAG, or UGA, which do not specify an amino acid but rather signal that the protein ~icholl (1994) describes the ribosome as a "complex structure that essentially acts as a 'iig'-{e.g., see Chapter 7,Figures 7.17 to 1.19}-which holds the mRNA in place 50 that the correct amino acid can be added to the growing chain." [...]... diameter of DNA's "twisted rope ladder" is only 2 nanometers, whereas the movie film might typically be 2 centimeters wide This represents a 107 difference in width For length comparisons, the helical DNA (best seen at the top of Figure 9.9) has a pitch of 3.4 nanometers.,spanning 10 base pairs per cycle, whereas one frame of the movie film is again approximately 2 centimeters The handling of these minute... can subsequently form base pairs with other DNA in the splicing procedures (from Undmt4ndillg DNA and Gene Ckming by Drlica, Copyright C 19 92. Reprinted by permission of John Wiley & Sons, Inc.) shown in Figure 9 .21 ;growing bacterial colonies-more in Figure 9 .22 or less at life size-are shown 9.7.6 Transgenic Plants and Animals A transgenic plant or animal is one that has been altered to contain a gene... by DNA ligase (from Understanding DNA and Gene Cloning by Drlica, Copyright © 19 92 Reprinted by permission of John Wiley & Sons, Inc.) FIpre 9.l9 An artist's impressions of (a) Circular plasmids and (b) an enlargement of a plasmid showing a short region becoming singlc-atranded, Chap 9 310 Biotechnology Chap 9 F'igure9 .22 Byspreadingadillltesuspension ofE coliceUs onto solid agar in a petri dish, colonies... synthesis inside its cells 9.7 .2 The Essential Steps Figure 9.15,by Nicholl (1994),provides a "snapshot" of manufacturing by gene cloning The original DNA of an organism, plant, or animal is first cut into fragments 2: It is then joined to a carrier, called a vehicle or a vector Step 3: This recombined DNA is then reintroduced into the cell of a host organism With the proper manufacturing controls, clones... further purified the DNA, which was syringed out for Step 2 9.8.4 Step 2: Packaging of Figure 9 .23 1 into Bacteriophages (Center Right Rabbit DNA and phage DNA are first individually cut by a restriction enzyme The fragments of rabbit DNA are then mixed with the phage DNA, and the enzyme DNA ligase is added to join the DNA fragments Btotechnoloav 3 92 Chap 9 HemoglobinmRNA I~ DNA synthesis _ Reverse... preparation of these probes, and doing so in such a way as to have enough material to test all the plaques Probe preparation is shown on the left of Figure 9 .23 9.8.5 Step 3: Preparing mRNA as a Template (Upper Left Side of Figure 9 .23 1 for Probes One potential source for radioactive probes is messenger RNA from the gene being sought Hemoglobin represents a special case, because the mRNA in red blood cells... Techniques The splicing of eDNA with a plasmid, followed by cloning in E coli hosts, took place as in Step 2 However, it was still necessary to isolate the particular E coli cells that had taken up plasmid DNA into which the rabbit cDNA had been inserted The procedure that allows this is described in Figure 9 .24 The procedure deliberately chose a plasmid with two genes for antibiotic resistance: one for tetracycline... the colonies that had been formed on the tetracycline-containing agar plate (Figure 9 .24 d) were tested to find ones that failed to grow on an ampicillin-containing eger.This distinguished E coli cells containing plasmids joined to rabbit eDNA from those that did not have rabbit DNA attached to them Figures 9 .24 e and 9 .24 f show the two types ... genetic engineering is the potential for excising or adding genetic information to correct mutant or missing genes 9.8 GENETIC ENGINEERING OF HEMOGLOBIN 9.8.1 n, A CASE STUOY ON GENE CLONING Introduction The previous section gave a summary of the cutting, joining, and cloning procedures in biotechnology The analogy with movie-film splicing and joining was presented from Drlica (19 92) This is a helpful analogy,... of frames would II gene is represented, be contained and in fact many Reprinted 9.7.4 by permission Step 2: Joining DNA nucleotide in the two "bracketed" (from Understllnding DNA and Gene of John Wiley Cloning & Sons, areas by pairs on the and right Drlica, Copyright movie-film of the figure © 19 92 Inc.}, Using the Enzyme DNA Ligase Once isolated a DNA fragment is ready to be joined to another DNA molecule . chromosomes per nucleus. Humans have 46 chromosomes arranged in 22 pairs plus XX or XY -except in the sperm or the egg, which has 22 chromosomes plus X or Y. The nuclei of most human cells contain. Drlica, Copyright C 19 92. Reprinted by permission of John Wiley & Sons,Inc.). shown in Figure 9 .21 ;growing bacterial colonies-more or less at life size-are shown in Figure 9 .22 . 9.7.6 Transgenic. region becoming singlc-atranded, Nick Nick