Presentation on theme: "台大農藝系 遺傳學 601 20000 Chapter 2 slide 1 CHAPTER 2 DNA: The Genetic Material Peter J. Russell edited by Yue-Wen Wang Ph. D. Dept. of Agronomy, NTU A molecular."— Presentation transcript:
台大農藝系 遺傳學 601 20000 Chapter 2 slide 1 CHAPTER 2 DNA: The Genetic Material Peter J. Russell edited by Yue-Wen Wang Ph. D. Dept. of Agronomy, NTU A molecular Approach 2 nd Edition
台大農藝系 遺傳學 601 20000 Chapter 2 slide 2 The Search for the Genetic Material 1.Some substance must be responsible for passage of traits from parents to offspring. For a substance to do this it must be: a. Stable enough to store information for long periods. b. Able to replicate accurately. c. Capable of change to allow evolution.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 3 The Search for the Genetic Material 2.In the early 1900s, chromosomes were shown to be the carriers of hereditary information. In eukaryotes they are composed of both DNA and protein, and most scientists initially believed that protein must be the genetic material.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 4 Griffith’s Transformation Experiment 1.Frederick Griffith’s 1928 experiment with Streptococcus pneumoniae bacteria in mice showed that something passed from dead bacteria into nearby living ones, allowing them to change their cell surface. 2.He called this agent the transforming principle, but did not know what it was or how it worked.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 6 Avery’s Transformation Experiment Animation: DNA as Genetic Material: The Avery Experiment 1. In 1944, Avery, MacLeod and McCarty published results of a study that identified the transforming principle from S. pneumoniae. Their approach was to break open dead cells, chemically separate the components (e.g., protein, nucleic acids) and determine which was capable of transforming live S. pneumoniae cells. 2. Only the nucleic acid fraction was capable of transforming the bacteria. 3. Critics noted that the nucleic acid fraction was contaminated with proteins. The researchers treated this fraction with either RNase or protease and still found transforming activity, but when it was treated with DNase, no transformation occurred, indicating that the transforming principle was DNA.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 8 The Hershey-Chase Bacteriophage Experiment Animation: DNA as Genetic Material: The Hershey-Chase Experiment 1. More evidence for DNA as the genetic material came in 1953 with Alfred Hershey and Martha Chase’s work on E. coli infected with bacteriophage T2. 2. In one part of the experiment, T2 proteins were labeled with 35 S, and in the other part, T2 DNA was labeled with 32 P. Then each group of labeled viruses was mixed separately with the E. coli host. After a short time, phage attachment was disrupted with a kitchen blender, and the location of the label determined. 3. The 35 S-labeled protein was found outside the infected cells, while the 32 P-labeled DNA was inside the E. coli, indicating that DNA carried the information needed for viral infection. This provided additional support for the idea that genetic inheritance occurs via DNA.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 12 The Discovery of RNA as Viral Genetic Material 1.All known cellular organisms have DNA as their genetic material. Some viruses, however, use RNA instead. 2.Tobacco mosaic virus (TMV) is composed of RNA and protein; it contains no DNA. In 1956 Gierer and Schramm showed that when purified RNA from TMV is applied directly to tobacco leaves, they develop mosaic disease. Pretreating the purified RNA with RNase destroys its ability to cause TMV lesions (Figure 2.7). 3.In 1957 Fraenkel-Conrat and Singer showed that in TMV infections with viruses containing RNA from one strain and protein from another, the progeny viruses were always of the type specified by the RNA, not by the protein.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 15 The Composition and Structure of DNA and RNA 1. DNA and RNA are polymers composed of monomers called nucleotides. 2. Each nucleotide has three parts: a. A pentose (5-carbon) sugar. b. A nitrogenous base. c. A phosphate group. 3. The pentose sugar in RNA is ribose, and in DNA it’s deoxyribose. The only difference is at the 2’ position, where RNA has a hydroxyl (OH) group, while DNA has only a hydrogen.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 17 The Composition and Structure of DNA and RNA 4.There are two classes of nitrogenous bases: a. Purines (double-ring, nine-membered structures) include adenine (A) and guanine (G). b. Pyrimidines (one-ring, six-membered structures) include cytosine (C), thymine (T) in DNA and uracil (U) in RNA.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 19 The Composition and Structure of DNA and RNA 5. The structure of nucleotides has these features: a. The base is always attached by a covalent bond between the 1′ carbon of the pentose sugar and a nitrogen in the base (specifically, the nine nitrogen in purines and the one nitrogen in pyrimidines). b. The sugar-base combination is a nucleoside. When a phosphate is added (always to the 5′ carbon of the pentose sugar), it becomes a nucleoside phosphate, or simply nucleotide. c. Nucleotide examples are shown in Figure 2.11, and naming conventions are given in Table 2.1. 6. Polynucleotides of both DNA and RNA are formed by stable covalent bonds (phosphodiester linkages) between the phosphate group on the 5′ carbon of one nucleotide, and the 3′ hydroxyl on another nucleotide. This creates the “backbone” of a nucleic acid molecule. 7. The asymmetry of phosphodiester bonds creates 3′-5′ polarity within the nucleic acid chain.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 21 The Discovery of the DNA Double Helix 1. James Watson and Francis Crick published the famous double-helix structure in 1953. When they began their work, it was known that DNA is composed of nucleotides, but how the nucleotides are assembled into nucleic acid was unknown. Two additional sources of data assisted Watson and Crick with their model: a. Erwin Chargaff’s ratios obtained for DNA derived from a variety of sources showed that the amount of purine always equals the amount of pyrimidine, and further, that the amount of G equals C, and the amount of A equals T. b. Rosalind Franklin’s X ray diffraction images of DNA showed a helical structure with regularities at 0.34 nm and 3.4 nm along the axis of the molecule (Figure 8.9).
台大農藝系 遺傳學 601 20000 Chapter 2 slide 25 The Discovery of the DNA Double Helix 2. Watson and Crick’s three- dimensional model (Figure 2.13) has these main features: a. It is two polynucleotide chains wound around each other in a right-handed helix. b. The two chains are antiparallel. c. The sugar-phosphate backbones are on the outside of the helix, and the bases are on the inside, stacked perpendicularly to the long axis like the steps of a spiral staircase.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 26 d. The bases of the two strands are held together by hydrogen bonds between complementary bases (two for A-T pairs and three for G-C pairs). Individual H-bonds are relatively weak and so the strands can be separated (by heating, for example). Complementary base pairing means that the sequence of one strand dictates the sequence of the other strand.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 27 e. The base pairs are 0.34 nm apart, and one full turn of the DNA helix takes 3.4 nm, so there are 10 bp in a complete turn. The diameter of a dsDNA helix is 2 nm. f. Because of the way the bases H-bond with each other, the opposite sugar-phosphate backbones are not equally spaced, resulting in a major and minor groove. This feature of DNA structure is important for protein binding. 3. The 1962 Nobel Prize in Physiology or Medicine was awarded to Francis Crick, James Watson and Maurice Wilkins (the head of the lab in which Franklin worked). Franklin had already died, and so was not eligible.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 28 Different DNA Structures X ray diffraction studies show that DNA can exist in different forms (Figure 2.15). a. A-DNA is the dehydrated form, and so it is not usually found in cells. It is a right-handed helix with 10.9 bp/turn, with the bases inclined 13° from the helix axis. A-DNA has a deep and narrow major groove, and a wide and shallow minor groove. b. B-DNA is the hydrated form of DNA, the kind normally found in cells. It is also a right-handed helix, with only 10.0 bp/turn, and the bases inclined only 2° from the helix axis. B-DNA has a wide major groove and a narrow minor groove, and its major and minor grooves are of about the same depth. c. Z-DNA is a left-handed helix with a zigzag sugar-phosphate backbone that gives it its name. It has 12.0 bp/turn, with the bases inclined 8.8° from the helix axis. Z-DNA has a deep minor groove, and a very shallow major groove. Its existence in living cells has not been proven.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 29 Fig. 2.15 Space-filling models of different forms of DNA. a) A-DNA b) B-DNA c) Z-DNA
台大農藝系 遺傳學 601 20000 Chapter 2 slide 30 DNA in the Cell All known cellular DNA is in the B form. A-DNA would not be expected because it is dehydrated and cells are aqueous. Z-DNA has never been found in living cells, although many organisms have been shown to contain proteins that will bind to Z-DNA.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 31 RNA Structure 1.RNA structure is very similar to that of DNA. a.It is a polymer of ribonucleotides (the sugar is ribose rather than deoxyribose). b.Three of its bases are the same (A, G, and C) while it contains U rather than T. 2.RNA is single-stranded, but internal base pairing can produce secondary structure in the molecule. 3.Some viruses use RNA for their genomes. In some it is dsRNA, while in others it is ssRNA. Double-stranded RNA is structurally very similar to dsDNA.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 33 The Organization of DNA in Chromosomes 1.Cellular DNA is organized into chromosomes. A genome is the chromosome or set of chromosomes that contains all the DNA of an organism. 2.In prokaryotes the genome is usually a single circular chromsome. In eukaryotes, the genome is one complete haploid set of nuclear chromosomes; mitochondrial and chloroplast DNA are not included.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 34 Viral Chromosomes 1.A virus is nucleic acid surrounded by a protein coat. The nucleic acid may be dsDNA, ssDNA, dsRNA or ssRNA, and it may be linear or circular, a single molecule or several segments. 2.Bacteriophages are viruses that infect bacteria. Three different types that infect E. coli are good examples of the variety of chromosome structure found in viruses.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 35 T-even phage 3.The T-even phages (T2, T4 and T6) have similar structures; all have dsDNA genomes composed of a one linear DNA molecule surrounded by a protein coat.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 36 ΦX174 phage 4.ΦX174 is a small, simple virus with one short ssDNA chromosome. In 1959, Robert Sinsheimer found that the DNA of ΦX174 has a base composition that does not fit the complementary base-pair-rules. single strand DNA rather than dsDNA
台大農藝系 遺傳學 601 20000 Chapter 2 slide 37 λ phage 5. Bacteriophage λ is somewhat like the T-even phages in structure. However, its chromosome changes form. A linear molecule of dsDNA is packaged inside the protein head (Fig. 2.17), but after the virus infects its host the chromosome becomes circular due to base-pairing of complementary 12-base single-stranded regions at the ends of the linear molecule (Fig. 2.18).
台大農藝系 遺傳學 601 20000 Chapter 2 slide 40 Prokaryotic Chromosomes 1. The typical prokaryotic genome is one circular dsDNA chromosome, but some prokaryotes are more exotic, with a main chromosome and one or more smaller ones. When a minor chromosome is dispensable to the life of the cell, it is called a plasmid. Some examples: a. Borrelia burgdorferi (Lyme disease in humans) has a 0.91-Mb linear chromosome, plus an additional 0.53-Mb of DNA in 17 different linear and circular molecules. b. Agrobacterium tumefaciens (crown gall disease of plants) has a 3.0-Mb circular chromosome and a 2.1-Mb linear one.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 41 2.Archaebacteria also vary in chromosomal organization, but only circular forms have been found. Examples: a. Methanococcus jannaschii has three chromosomes of 1.66-Mb, 58-kb and 16-kb. b. Archaeoglobus fulgidus has one 2.2-Mb circular chromosome. 3. Both Eubacteria and Archaebacteria lack a membrane- bounded nucleus, hence their classification as prokaryotes. Their DNA is densely arranged in a cytoplasmic region called the nucleoid. 4.In an experiment where E. coli is gently lysed, it releases one 4.6-Mb circular chromosome, highly supercoiled (Figures 2.19 and 2.20). A 4.6-Mb double helix is about 1mm in length, about 10 3 times longer than an E. coli cell. DNA supercoiling helps it fit into the cell. Animation: DNA supercoiling
台大農藝系 遺傳學 601 20000 Chapter 2 slide 43 5.A molecule of B-DNA, with 10bp/turn of the helix, is in relaxed conformation. If turns of the helix are removed and the molecule circularized, the DNA will form superhelical turns to compensate for the added tension. 6.A nick in supercoiled DNA will allow it to return to a relaxed DNA circle (Figure 2.21). 7.Either overwinding or underwinding DNA will create a structure where 10bp/turn of the helix is not the most energetically favored conformation, and supercoils will be induced. Both positive and negative supercoils will condense the DNA. 8.All organisms contain topoisomerase enzymes to supercoil their DNA. 9.Prokaryotes also organize their DNA into looped domains, with the ends of the domains held so that each is supercoiled independently (Figure 2.22). 10.The compaction factor for looped domains is about 10-fold. In E. coli there are about 100 domains of about 40kb each.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 45 Eukaryotic Chromosomes 1. The genome of most prokaryotes consists of one chromosome, while most eukaryotes have a diploid number of chromosomes. 2. A genome is the information in one complete haploid chromosome set. The total amount of DNA in the haploid genome of a species is its C value (Table 2.4). The structural complexity and the C value of an organism are not related, creating the C value paradox.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 46 C value paradox
台大農藝系 遺傳學 601 20000 Chapter 2 slide 47 3.The form of eukaryotic chromosomes changes through the cell cycle: a.In G1, each chromosome is a single structure b.In S, chromosomes duplicate into sister chromatids but remain joined at centromeres through G2 c.At M phase, sister chromatids separate into daughter chromosomes 4.In G1 eukaryotic chromosomes are linear dsDNA, and contain about twice as much protein as DNA by weight. The DNA-protein complex is called chromatin, and it is highly conserved in all eukaryotes.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 48 Chromatin Structure 1. Both histones and non-histones are involved in physical structure of the chromosome. 2. Histones are abundant, small proteins with a net (+) charge. The five main types are H1, H2A, H2B, H3 and H4. By weight, chromosomes have equal amounts of DNA and histones. 3. Histones are highly conserved between species (H1 less than the others). 4. Histones organize DNA, condensing it and preparing it for further condensation by nonhistone proteins. This compaction is necessary to fit large amounts of DNA (2m/6.5ft in humans) into the nucleus of a cell. 5. Non-histone is a general name for other proteins associated with DNA. This is a big group, with some structural proteins, and some that bind only transiently. Non-histone proteins vary widely, even in different cells from the same organism. Most have a net (-) charge, and bind by attaching to histones.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 49 6.Chromatin formation involves histones, and condenses the DNA so it will fit into the cell. Chromatin formation has two components: a.Two molecules each of histones H2A, H2B, H3 and H4 associate to form a nucleosome core, and DNA wraps around it 1 3⁄4 times for a 7-fold condensation factor. Nucleosome cores are about 11 nm in diameter
台大農藝系 遺傳學 601 20000 Chapter 2 slide 51 b.H1 further condenses the DNA by connecting nucleosomes to create chromatin with a diameter of 30nm, for an additional 6 ‑ fold condensation. The solenoid model proposes that the nucleosomes form a spiral with 6 nucleosomes per turn (Figures 2.24 and 2.25).
台大農藝系 遺傳學 601 20000 Chapter 2 slide 53 7.Beyond the 30-nm filament stage, electron microscopy shows 30–90 loops of DNA attached to a protein scaffold (Figure 2.26). Each loop is 180–300 nucleosomes of the 30-nm fiber. SARs (scaffold-associated regions) bind nonhistone proteins to form loops that radiate out in spiral fashion (Figure 2.27). 8.Fully condensed chromosome is 10,000-fold shorter and 400-fold thicker than DNA alone.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 55 Euchromatin and Heterochromatin 1. The cell cycle affects DNA packing, with DNA condensing for mitosis and meiosis, and decondensing during interphase. Chromosomes are most condensed at metaphase, when the looped domains are further coiled and the chromatic has a diameter of about 700 nm. Non-histone proteins form the scaffold for this additional condensation. 2. Staining of chromatin reveals two forms: a. Euchromatin condenses and decondenses with the cell cycle. It is actively transcribed, and lacks repetitive sequences. Euchromatin accounts for most of the genome in active cells. b. Heterochromatin remains condensed throughout the cell cycle. It replicates later than euchromatin, and is transcriptionally inactive. There are two types based on activity: i. Constituitive heterochromatin occurs at the same sites in both homologous chromosomes of a pair, and is mostly repetitive DNA (e.g., centromeres). ii. Facultative heterochromatin varies between cell types or developmental stages or even between homologous chromosomes. It contains condensed, and thus inactive, euchromatin (e.g., Barr bodies)
台大農藝系 遺傳學 601 20000 Chapter 2 slide 56 Centromeric and Telomeric DNA 1. Centromeres and telomeres are eukaryotic chromosomal regions with special functions. 2. Centromeres are the site of the kinetochore, where spindle fibers attach during mitosis and meiosis. They are required for accurate segregation of chromatids. 3. Yeast (Saccharomyces cerevisiae) centromeres are well- studied. Called CEN regions, their sequence and organization are similar, but not identical, between the chromosomes. Other eukaryotes have different centromere sequences, so while function is conserved, it is not due to a single type of DNA sequence. (Fig. 2-28)
台大農藝系 遺傳學 601 20000 Chapter 2 slide 60 5. Telomeres are needed for chromosomal replication and stability. Generally composed of heterochromatin, they interact with both the nuclear envelope and each other. All telomeres in a species have the same sequence. a. Simple telomeric sequences are short, species-specific and tandemly repeated. (Examples: Tetrahymena is 5’-TTGGGG-3’, and human is 5’-TTAGGG-3’.) A new model suggests that the single-stranded end of the chromosome folds back to form a t-loop and then invades the double-stranded region to form a D-loop (Figure 2.30). b. Telomere-associated sequences are internal to the simple telomeric sequences. These complex repetitive sequences may extend many kb into the chromosome. c. Drosophila is unusual in having telomeres composed of transposons, rather than the short repeats seen in most eukaryotes.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 62 Unique-Sequence and Repetitive-Sequence DNA 1. Sequences vary widely in how often they occur within a genome. The categories are: a. Unique-sequence DNA, present in one or a few copies. b. Moderately repetitive DNA, present in a few to 10 5 copies. c. Highly repetitive DNA, present in about 10 5 –10 7 copies. 2. Prokaryotes have mostly unique-sequence DNA, with repeats only of sequences like rRNAs and tRNAs. Eukaryotes have a mix of unique and repetitive sequences. 3. Unique-sequence DNA includes most of the genes that encode proteins, as well as other chromosomal regions. Human DNA contains about 65% unique sequences.
台大農藝系 遺傳學 601 20000 Chapter 2 slide 63 4. Repetitive-sequence DNA includes the moderately and highly repeated sequences. They may be dispersed throughout the genome, or clustered in tandem repeats. 5. Dispersed repetitive sequences occur in families that have a characteristic sequence. Often the same few sequences are highly repeated, and comprise most of the dispersed repeats in the genome. Little is known of their function, or indeed whether they actually serve a function. There are two types of interspersion patterns found in all eukaryotic organisms: a. SINEs (short interspersed repeated sequences) with 100–500 bp sequences. An example is the Alu repeats found in some primates, including humans, where these 200–300 bp repeats make up 9% of the genome. b. LINEs (long interspersed repeated sequences) with sequences of 5 kb or more. The common example in mammals is LINE-1, with sequences up to 7 kb in length. 6. Tandemly repetitive sequences are common in eukaryotic genomes, ranging from very short (1–10 bp) sequences to genes and even longer sequences. This group includes centromere and telomere sequences, and rRNA and tRNA genes.