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Trends in Biotechnology Molecules of Genetics 1. Concept 15 - DNA and proteins are key molecules of the cell nucleus. Miescher discovered DNAas a major.

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Presentation on theme: "Trends in Biotechnology Molecules of Genetics 1. Concept 15 - DNA and proteins are key molecules of the cell nucleus. Miescher discovered DNAas a major."— Presentation transcript:

1 Trends in Biotechnology Molecules of Genetics 1

2 Concept 15 - DNA and proteins are key molecules of the cell nucleus. Miescher discovered DNAas a major chemical of the nucleus around In the early 1900s, many people thought that proteins were the molecules able to carry large amounts of hereditary information from generation to generation. 2

3 DNA was known to be a very large molecule. At that time, no specific cellular function had yet been found for DNA. 3

4 Proteins were known to be important as enzymes and structural components of living cells. Proteins are polymers of numerous amino acids. These polymers are called polypeptides. The 20 amino acids of proteins could be made into more unique structures than the four-letter alphabet of DNA. 4

5 A nucleotide is made of three elements: phosphate, deoxyribose sugar, and a nitrogenous base. 5

6 The carbons of the deoxyribose sugar are numbered 1-5. In a nucleotide, the noitrogenous base is always bound to carbon#1, a hydroxyl group (OH) is bound to carbon#3 and the phosphate group is bound to carbon#5. 6

7 Each of the four nucleotides has a distinct nitrogenous base. 7

8 The sugars are connected to the phosphate group through a phosphodiester bond. 8

9 The phosphodiester bonds give the molecule a direction; from carbon#5 to carbon#3. [ 5 prime (5’) to 3 prime (3’) ] 9

10 Animation at The review problem is at 10

11 Concept 16 - One gene makes one protein Garrod described the inherited disorder alkaptonuria as an "inborn error of metabolism." He said that a gene mutation causes a defect in the biochemical pathway for eliminating liquid wastes. The phenotype of the disease shows dark urine. 11

12 Beadle and Tatum, used the bread mold Neurospora. 1 - molds exposed to radiation lose the ability to produce essential nutrients, and this slowed, even stopped the growth of the mold. 2 - growth can be restored by providing the mutated mold with a specific supplement. 12

13 Each mutation must inactivate the enzyme (protein) needed to synthesize the nutrient. So, one gene carries the directions for making one protein. 13

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15 Animation at The review problem is at 15

16 Concept 17 - A gene is made of DNA. 1920s - experiment - a harmless strain of bacteria can become infectious when mixed with a dead virulent strain of bacteria. The dead bacteria provide some chemical that "transforms" the harmless bacteria to infectious ones. This "transforming principle" appeared to be a gene. 16

17 1940’s – Avery found that a pure extract of the "transforming principle" was unaffected by treatment with protein-digesting enzymes but was destroyed by a DNA-digesting enzyme. So, the transforming principle is DNA. So, gene is made of DNA. 17

18 Animation at The review problem is at 18

19 Concept 18 - Bacteria and viruses have DNA too. 1940's, - it was discovered that bacteria have sex. During the process of conjugation, genes are exchanged through a mating channel that links two bacteria. 19

20 Electron microscopy - A virus attaches to a host bacterium and injects its genes through its channel-like tail. 1952, Hershey - DNA, alone, is needed for the reproduction of new viruses within an infected cell. Support - a gene is made of DNA. Viruses, as well as bacteria, can be used as models for studying genetics. 20

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22 Animation at The review problem is at 22

23 Concept 19 - The DNA molecule is shaped like a twisted ladder. DNA is made of smaller pieces called nucleotides. These are made of deoxyribose sugar, a phosphate group, and one of four nitrogen bases — adenine (A), thymine (T), guanine (G), and cytosine (C). Phosphates and sugars link to form a long polymer. The ratios of A-to-T and G-to-C are constant in all living things. X-ray crystallography provided the final clue that the DNA molecule is a double helix, shaped like a twisted ladder. 23

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25 Watson and Crick showed that alternating deoxyribose and phosphate molecules form the main long chains of the DNA helix. The chains are joined by complementary pairs of nitrogen bases — A always paired with T and G always paired with C. 25

26 Animation at The review problem is at 26

27 Proofreading for Functional Proteins Genes are made up of nucleotides in a double helix of complementary nucleotide pairs. Transcription – the DNA "instructions" are used to make complementary strands of RNA. Transcription must be accurate - even 1 in 100,000 incorrect bases can give a mutated, nonfunctional protein, and possibly cell death. 27

28 To transcribe DNA to RNA, an enzyme moves along one half of an unwound DNA helix, adding nucleotides to a RNA strand. One enzyme, pol II, transcribes parts of DNA that encode proteins into messenger RNA. When an incorrect base is attached to a growing RNA chain, the distance of the DNA-RNA complex distorts the RNA–DNA helix. 28

29 Pol II goes into a "backtracked" state and stops. It won’t continue until the mismatched nucleotide is removed from the strand. This proofreading function plays an important role in minimizing transcription errors, speeding up protein production, and ensuring accuracy in the transition of the genetic code to the proteins. Understanding the structure of pol II in the backtracked state, helps us understand this proofreading function of the enzyme. 29

30 Concept 20 - A half DNA ladder is a template for copying the whole. Watson and Crick said that one half of the DNA ladder could be a template for making the other half during DNA replication, because of the pairing of adenine-to-thymine and guanine-to- cytosine first, an enzyme was discovered — DNA polymerase — that adds complementary nucleotides to the template provided by a half DNA molecule. 30

31 Second, nitrogen isotopes were used to follow the construction of new DNA molecules during successive generations of bacteria. One strand of each DNA molecule is passed along unchanged to each of two daughter cells. This "conserved" strand acts as the template for DNA polymerase to synthesize a second complementary strand, which completes each new DNA molecule. 31

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33 Animation at The review problem is at 33

34 The term proofreading is used in genetics. It is the error-correcting processes of various biochemical reactions. When an incorrect base pair is recognized, some DNA polymerase reverses its direction by one base pair of DNA and cuts out the mismatched base. The polymerase can re-insert the correct base and replication can continue. 34

35 Concept 21- RNA is an intermediary between DNA and protein. RNA is common in the cytoplasm. Watson and Crick suggested that RNA must copy the DNA message in the nucleus and carry it out to the cytoplasm, where proteins are synthesized. Crick suggested an "adaptor" molecule reads the genetic code and selects the appropriate amino acids to add to a growing polypeptide chain. Flow of genetic information from DNA to RNA to protein became known as the "Central Dogma." 35

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37 Several types of RNA are involved in using genetic information. In the nucleus, the DNA code is "transcribed," or copied, into a messenger RNA (mRNA) molecule. In the cytoplasm, the mRNA code is "translated" into amino acids. Translation happens at the ribosome. The ribosome is partly made of RNA. Transfer RNA attaches to the amino acids. 37

38 Animation at The review problem is at 38

39 Information flows between DNA, RNA and protein. DNA -> protein is another special transfer, but it is not found in nature. 39

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41 Concept 22 - DNA words are three letters long. Researchers thought a three-letter code called a codon could contain the information of the 20 known amino acids. 41

42 Researchers synthesized different RNA molecules of single repeated codons. Each type of synthetic RNA was added to a cell- free translation system containing ribosomes, transfer RNAs, and amino acids. Each type produced a polypeptide chain of repeated units of a single amino acid. Several codons are "stop" signals and many amino acids are specified by several different codons. All 64 three-letter combinations do something. 42

43 Nirenberg and Matthaei showed that a synthetic messenger RNA made of only uracils can direct protein synthesis. The polyU mRNA resulted in a poly- phenylalanine protein. They had the first piece of the genetic code. 43

44 The entire genetic code was found by matching amino acids to synthetic triplet nucleotides. There is redundancy (some amino acids are encoded by more than one codon) and some codons start or stop the translation of mRNA. The genetic code was the same for almost all organisms. 44

45 Animation at The review problem is at 45

46 Concept 23 - A gene is a discrete sequence of DNA nucleotides. The triplet genetic code gives the definition of a gene as a sequence of DNA encoding a protein — beginning with a "start" codon and ending with a "stop" codon. 46

47 Discovery of methods to determine the exact sequence of nucleotides that compose a specific gene. DNA sequencing was built upon earlier knowledge of DNA polymerases and cell-free systems for replicating DNA. The chain-termination method, which makes clever use of a "defective" DNA nucleotide, became very important for DNA sequencing technology. 47

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49 Animation at The review problem is at 49

50 The chain-termination method relies on a way to see the short, terminated pieces of DNA. They have to be labeled. The first way of labeling the pieces was by using radioactive nucleotides. Later, fluorescent labels were used. 50

51 Next-Generation DNA Sequencing (NGS) - also known as high-throughput sequencing - describes a number of different modern sequencing technologies eg.: – Illumina (Solexa) sequencing – Roche 454 sequencing – Ion torrent: Proton / PGM sequencing – SOLiD sequencing These allow us to sequence DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing. 51

52 Illumina sequencing In NGS, many numbers of short reads are sequenced at one time. First the input sample must be cut into bp sections. Fragments are ligated to adaptors which are stuck to slides. PCR amplifies each read, creating a spot with many copies of the same read. They are then separated into single strands to be sequenced. 52

53 Fluorescently labeled nucleotides and DNA polymerase are added. The nucleotides are labeled with the color corresponding to the base. The nucleotides also have a terminator, so that only one base is added at a time. 53

54 An image is taken of the slide. In each read location, there will be a fluorescent signal indicating the base that has been added. 54

55 The slide is then prepared for the next cycle. The terminators are removed, allowing the next base to be added, and the fluorescent signal is removed, preventing the signal from contaminating the next image. 55

56 The process is repeated, adding one nucleotide at a time and imaging in between. 56

57 Computers are then used to detect the base at each site in each image and these are used to construct a sequence. 57

58 All of the sequence reads will be the same length, as the read length depends on the number of cycles carried out. Here is a video showing Illumina sequencing 58

59 454 sequencing Roche 454 sequencing can sequence much longer reads than Illumina. It sequences multiple reads at once by reading optical signals as bases are added. The DNA or RNA is fragmented into shorter reads, in this case up to 1kb. Adaptors are added to the ends and these are stuck to beads, one DNA fragment per bead. The fragments are then amplified by PCR using adaptor-specific primers. 59

60 Each bead is then placed in a single well of a slide. So each well will contain a single bead, covered in many PCR copies of a single sequence. The wells also contain DNA polymerase and sequencing buffers. 60

61 One of four NTP types is added. Where this nucleotide is next in the sequence, it is added to the sequence read. If that single base repeats, then more will be added. So if we add Guanine bases, and the next in a sequence is G, one G will be added, however if the next part of the sequence is GGGG, then four Gs will be added. 61

62 The addition of each nucleotide releases a light signal. These locations of signals are detected and used to determine which beads the nucleotides are added to. 62

63 This NTP mix is washed away. The next NTP mix is now added and the process repeated, cycling through the four NTPs. 63

64 This kind of sequencing generates graphs for each sequence read, showing the signal density for each nucleotide wash. The sequence can then be determined computationally from the signal density in each wash. 64

65 All of the sequence reads we get from 454 will be different lengths, because different numbers of bases will be added with each cycle. 65

66 Ion Torrent: Proton / PGM sequencing The input DNA or RNA is fragmented, this time ~200bp. Adaptors are added and one molecule is placed onto a bead. The molecules are amplified on the bead by emulsion PCR. Each bead is placed into a single well of a slide. 66

67 A single type of dNTP, along with buffers and polymerase, is added to the slide. Ion torrent and Ion proton sequencing do not use optical signals. Adding a dNTP to a DNA polymer releases an H + ion. 67

68 The pH is detected is each of the wells, as each H + ion released will decrease the pH. The changes in pH shows how many bases were added to the sequence read. 68

69 The dNTPs are washed away, and the process is repeated cycling through the different dNTP species. 69

70 The pH change is used to determine how many bases were added with each cycle. 70

71 Here is a video of SOLiD sequencing 71

72 Concept 24 - The RNA message is sometimes edited. In bacterial cells there is an exact correspondence between mRNA sequence and DNA sequence. Recombinant-DNA techniques allowed researchers to explore the genes of higher cells (eukaryotes). 72

73 It was found that mRNA transcripts appeared to be shorter than their corresponding genes. This became obvious in electron micrographs of mRNA bound to its complementary DNA template — where regions of DNA without corresponding mRNA form loops. 73

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75 The protein coding information in genes is interrupted by non-coding sequences called introns, which results in "split genes." The entire DNA code is transcribed into a temporary form of RNA (pre-mRNA), but this is edited in the nucleus to yield a mature mRNA. The process of RNA splicing involves removing non-coding regions, introns, and splicing together adjacent coding regions, exons. 75

76 Animation at The review problem is at 76

77 Concept 25 - Some viruses store genetic information in RNA it was discovered that some viruses shift their genetic information from RNA to DNA. Even so, these viruses ultimately make proteins in the same way as higher organisms. 77

78 During infection, the RNA code is first transcribed "back" to DNA — then to RNA to protein, according to the accepted scheme. The initial conversion of RNA to DNA is called reverse transcription, and viruses that use this mechanism are classified as retroviruses. A specialized polymerase, reverse transcriptase, uses the RNA as a template to synthesize complementary and double- stranded DNA molecule. 78

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80 Animation at The review problem is at 80

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82 The Rous Sarcoma Virus (RSV) 1911, Peyton Rous – cancer could be induced in healthy chickens – inject them with a cell-free extract of the tumor of a sick chicken. Grind up samples of the tumor – Pass material through a filter with pores so fine that not even bacteria could get through. – tumor filtrate was able to induce cancer when injected into chickens. 82

83 first example of an oncogenic virus – a virus capable of causing cancer. – tumor was a sarcoma, a tumor of connective tissue. – virus was named the Rous sarcoma virus (RSV). This is a retrovirus (as is HIV, the virus that causes AIDS). RSV infects a cell – its reverse transcriptase synthesizes DNA copies of its genome. – These enter the nucleus of the cell – insert themselves randomly throughout the DNA of the host's chromosomes. 83

84 Normal gene transcription within the nucleus now produces an RSV messenger RNA (mRNA) that reenters the cytoplasm. Some copies of this mRNA are then translated by the normal machinery (e.g., ribosomes) of the host cell into protein products. Other copies of the RNA become incorporated into new virus particles. 84

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86 The Rous sarcoma virus has only 4 genes: gag, which encodes the capsid protein pol, which encodes the reverse transcriptase env, which encodes the envelope protein src, which encodes a tyrosine kinase, an enzyme that attaches phosphate groups to Tyr residues on a variety of host cell proteins. 86

87 Each end of the RNA molecule has a set of repeated sequences of nucleotides ("R" and "P") that perform at least two important functions: – they enable the DNA copies of RSV to insert into the host's DNA and – they act as enhancers, causing the host nucleus to transcribe the RSV genes at a rapid rate. 87

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89 The Rous sarcoma virus has only 4 genes: gag, which encodes the capsid protein pol, which encodes the reverse transcriptase env, which encodes the envelope protein src, which encodes a tyrosine kinase, an enzyme that attaches phosphate groups to Tyr residues on a variety of host cell proteins. 89

90 Concept 26 - RNA was the first genetic molecule. 1960s - messenger RNA has the ability to store genetic information, while transfer and ribosomal RNA have the ability to translate genetic information into proteins. 1980s - some RNAs can even act as an enzyme to self-edit their own genetic code. 90

91 These results raised two questions: 1) Why does RNA play so many roles in the flow of genetic information? 2) Why bother storing genetic information in DNA, if RNA alone could do the job? 91

92 It now seems certain that RNA was the first molecule of heredity, so it evolved all the essential methods for storing and expressing genetic information before DNA came onto the scene. Single-stranded RNA is unstable and is easily damaged by enzymes. By doubling the existing RNA molecule, and using deoxyribose sugar instead of ribose, DNA evolved as a much more stable form to pass genetic information with accuracy. 92

93 Animation at Last part The review problem is at 93

94 Concept 27 - Mutations are changes in genetic information. The DNA sequences from two individuals of the same species are highly similar — differing by only about one nucleotide in 1,000. Each DNA difference results from a mutation — ranging from single nucleotide changes, to small repeated units, to larger insertions and deletions. 94

95 Some mutations generate novel changes that are starting points of evolution, and some are responsible for disease. The great majority of mutations occur in DNA regions that do not encode proteins. Most of these are neutral in terms of evolution or health; they have no negative or positive effect. 95

96 1920s - DNA mutations were first induced in Drosophila using X-rays. Other types of ionizing radiation were also found to produce mutations. Ultraviolet radiation, a component of sunlight, causes specific kinds of DNA damage, including the linking of adjacent thymine nucleotides. Chemicals from a variety of man-made and natural sources are known mutagens. Also, DNA replication, itself, is not perfect and is a source of new mutations. 96

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99 Concept 28 - Some types of mutations are automatically repaired. Researchers were trying to explain the odd behavior of their microbes. Cultures that were seemingly killed by exposure to ultraviolet light would recover after sitting by a window, and mutants would curiously pop up long after exposure to a mutagen. 99

100 Investigations of organisms from bacteria to humans have uncovered a large number of enzymes which repair damage from environmental mutagens or errors in DNA replication. Without these enzymes, DNA damage would cause intolerable levels of mutation. 100

101 Diseases caused by defective repair enzymes shorten life span, illustrating the central role of DNA repair in survival. But the occasional failure to repair DNA or correct errors in replication is also central to survival because these failures exist as mutations. 101

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