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Genetics Chapter 9.docx

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University of Guelph
Molecular Biology and Genetics
MBG 2040
Mark Baker

Genetics Chapter 9: DNA and the Molecular Structure of Chromosomes Functions of the Genetic Material: - In 1865, Mendel showed that “Merkmalen” (now genes) transmitted genetic information. Many studies were one that demonstrated that the genetic material must perform three essential functions: (1) The genotypic function, replication. The genetic material must store genetic information and accurately transmit that information from parents to offspring, generation after generation. (2) The phenotypic function, gene expression. The genetic material must control the development of the phenotype of the organism. That is, the genetic material must dictate the growth of the organism from the single-celled zygote to mature adult. (3) The evolutionary function, mutation. The genetic material must undergo changes to produce variations that allow organisms to adapt to modifications in the environment so evolution can occur. - Other early genetic studies established correlation between patterns of transmission of genes and the behaviour of chromosomes during sexual production, providing strong evidence that genes are usually located on chromosomes. - Chromosomes are composed of 2 types of large organic molecules called proteins and nucleic acids. - Nucleic acids are of 2 types: Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). - During 1950’s results of elegant experiments established that genetic information is stored in nucleic acids, not in proteins. - In most organisms genetic information s encoded in structure of DNA, however in small viruses’ genetic information is encoded in RNA. Proof that Genetic Information is Stored in DNA: - Most of DNA cells is located in chromosomes, while RNA and proteins are also abundant in cytoplasm suggested that DNA harbors the genetic information of living organisms. In addition to the fact that a correlation exists between amount of DNA per cell and number of sets of chromosomes per cell.  Most somatic cells of diploid organisms contain twice the amount of DNA as haploid germ cells of same species.  Molecular composition of DNA is same in all cells of organism, whereas composition of both RNA and proteins is variable from one cell type to another  DNA is more stable than RNA or proteins. Since genetic material must store and transmit info from parents to offspring, expect it to be stable, like DNA. However, none of the above PROVES it!  Proof that DNA Mediates Transformation: - Frederick Griffith’s discovery of transformation in Streptococcus Pneumoniae  When Griffith injected both heat-killed Type IIIS bacteria (virulent when alive) and live Type IIR bacteria (avirulent) into mice, many of the mice developed pneumonia and died, and live Type IIIS cells were recovered from their carcasses. Something from the heat killed cells- the “transforming principle”- had converted the live Type IIR cells to Type IIIS. - Sia and Dawson performed same experiment in vitro, showing that mice played no role in transformation process. Sia and Dawson set stage for Avery, MacLeod and McCartys demonstration that the “transforming principle” in S. Pneumoniae is DNA. Avery and colleagues showed that DNA is the only component of the Type IIIS cells required to transform Type IIR cells to Type IIIS. - The most definitive experiments in Macleod, Avery and McCarty’s proof involved us of enzymes that degrade DNA, RNA, or protein. - Purified DNA from Type IIIS cells were treated with enzymes (1) DNase, which degrades DNA, (2) RNase, which degrades RNA, or (3) Protease, which degrades proteins. DNA was then tested for its ability to transform Type IIR cells to Type IIIS. Only DNase treatment had an effect- it eliminated all transforming activity. - Work of Avery and coworkers established that genetic information in Streptococcus is present in DNA.  Proof that DNA Carries Genetic Information in Bacteriophage - Alfred Hershey and Martha Chase demonstrated that DNA is genetic material in 1952. Results showed that the genetic info of a bacterial virus (bacteriophage T2) was present in its DNA. - Viruses are smallest living organisms and their reproduction if controlled by genetic info stored in nucleic acids, same process as cellular organisms. They can only produce in appropriate host cells. - Bacteriophage T2 is composed of about 50% DNA and 50% protein. Hershey and Chase showed that the DNA of the virus particle entered the cell, whereas most of the protein of the virus remained absorbed to outside of cell. Implication was that the genetic info necessary for viral reproduction was present in DNA. - DNA contains phosphorus but not sulfur, whereas protein contains sulfur but no phosphorus. Thus, they were able to label either (1) the phage DNA by growth in a medium containing radioactive isotopes of Phosphorus or (2) the phage protein coats by growth in a medium containing radioactive sulfur in place of normal isotope. - When T2 phage particles with sulfur (35S) were mixed with E. coli cells, most of the radioactivity (thus proteins) could be removed. When T2 particles labeled phosphorus were used the DNA was not subject to removal by shearing in a blender. - These results indicated that DNA of the virus enters host cell, whereas protein coat remains outside cell. The Structures of DNA and RNA  Nature of the Chemical Subunits in DNA and RNA: - Nucleic acids are macromolecules composed of repeating subunits called nucleotides. Each nucleotide is composed of : (1) Phosphate group (2) A five-carbon sugar (3) A cyclic nitrogenous base - In DNA the sugar is 2-deoxyribose and RNA is ribose. - There are 4 major bases in DNA: adenine (A), guanine (G), thymine (T), and cytosine (C). RNA uses uracil (U) instead of thymine. - Adenine and guanine are double ring bases called purines, while cytosine, thymine and uracil are single-ring bases called pyrimidines. - RNA usually exists as a single-stranded polymer composed of long sequence of nucleotides. DNA is usually a double-stranded molecule.  DNA Structure: The Double Helix - In 1952 James Watson and Francis Crick deduced the correct structure of DNA. Watson and Crick’s double-helix structure was based on two kinds of evidence: (1) When Erwin Chargaff and colleagues analyzed composition of DNA they found that the concentration of thymine was always equal to concentration of adenine and concentration of cytosine was always equal to guanine. Results suggested that thymine and adenine and cytosine and guanine in DNA had some fixed interrelationship. Data also showed that total concentration of pyrimidines was always equal to purines. (2) When x rays are focused though fibers the rays are deflected by atoms of molecules in special patterns, called diffraction patterns, which provides info about organization of components of molecules. Watson and Crick used X-ray diffraction data on DNA structure provided by Wilkins and Franklin. These data indicated DNA was a highly ordered, two stranded structure with repeating substructures every 0.34 nanometers. - Watson and Crick proposed that DNA exists as a right handed double helix in which two polynucleotide chains are coiled in a spiral. - Nucleotides are linked together by phosphodiester bonds, joining adjacent deoxyribose moieties. The two polynucleotide strands are held together by hydrogen bonding between bases in opposing stands. Adenine always pairs with thymine and guanine is always paired with cytosine, thus all base pairs consist of one purine and one pyrimidine. - The two strands of a DNA double helix are said to be complementary. This makes DNA uniquely suited to store and transmit genetic info from generation to generation. - The sugar-phosphate backbones of the two complementary strands are said to be antiparallel. The stability of DNA is due to hydrogen bonds and hydrophobic bonding between adjacent base pairs. - The two grooves of DNA are not identical; one, the major grove is much wider than the minor groove. Some proteins bind to major, while others bind to minor groove.  Supercoiling: - Supercoils are when one or both strands are cleaved and when complementary strands at one end are rotated or twisted around each other with other end in place not allowed to spin - Supercoiling causes DNA to collapse into a tightly coiled structure similar to coiled telephone cord. They are introduced/taken away from DNA by enzymes that play essential role in DNA replication. - Supercoiling occurs only in DNA molecules with fixed ends that are not able to rotate. Chromosome Structure in Prokaryotes and Viruses: - Prokaryotes are typically monoploid; only one set of genes. In most viruses and prokaryotes, the single set of genes is stored in a single chromosome, which in turn contains a single molecule of nucleic acid. - The large DNA molecule present in E. coli cell must exist in highly condensed configuration. This structure is called the folded genome and is the functional state of a bacterial chromosome. Within folded genome the DNA molecule is organized into 50 to 100 domains or loops, each independently negatively supercoiled. RNA and protein are components of folded genome, which can be partially relaxed by treatment with either DNase or RNase. RNase treatment will not affect supercoiling - Bacterial chromosomes contain circular molecules of DNA Chromosome Structure in Eukaryotes: - Most eukaryotes are diploid, having two sets of genes. They contain much more DNA than prokaryotes and it’s packed into several chromosomes, which is present in two or more copies.  Chemical Composition of Eukaryotic Chromosomes: - Chemical analysis, electron microscopy, and x-ray diffraction studies of isolated chromatin have provided valuable info about structure of eukaryotic chromosomes. - Chemical analysis of chromatin shows that it consists primarily of DNA and proteins with lesser amounts of RNA. Proteins are two major classes: (1) Basic (positively charged) proteins called histones (2) Heterogeneous, largely acidic (negatively charged) group of proteins called nonhistone chromosomal proteins. - Histones are present in chromatin of all eukaryotes in amount equivalent to amounts of DNA. The histones consist of five classes of proteins: H1, H2a. H2b, H3 and H4 and are present in almost all cell types. - 4 of the 5 types of histones are complexed with DNA to produce the basic structural subunits of chromatin, nucleosomes. They are highly conserved during evolution - Most of the 20 amino acids in proteins are neutral in charge , they have no charge at pH 7. However, a few are basic and a few are acidic. The histones are basic because they contain 20-30 % arginine and lysine, two positively charged amino acids. The positively charged side groups on histones are important in their interaction with DNA, which is polyanionic because of the negatively charged phosphate groups. - Histones are important in chromatin structure (DNA packaging) and are only nonspecifically involved in regulation of gene expression. Chemical modifications of histones can alter chromosome structure which can enhance or decrease level of expression of genes located in the modified chromatin. - The nonhistone portion of chromatin consists of large number of heterogeneous proteins. The nonhistone proteins probably don’t play central roles in the packaging of DNA into chromosomes. Instead they’re likely to regulate expression of specific genes or sets of genes.  Three Levels of DNA Packaging in Eukaryotic Chromosomes: - Chromatin is found to consist of a serious of ellipsoidal beads joined by thin threads. The bead or chromatin subunit is called the nucleosome. The complete chromatin subunit consists of the nucleosome core, the linker DNA and the associated nonhistone chromosomal proteins, all stabilized by the binding of one molecule of histone H1 to 3 outside of the structure. Three levels of condensation are required to package the 10 to 10 um of DNA: (1) First level involves packaging DNA as a negative supercoil into nucleosomes, to produce 11nm diameter interphase chromatin fiber. This involves an octamer of histone molecules, two each of histones H2a, H2b, H3 and H4. (2) Second level involved an additional folding or supercoiliing of the 11 nm nucleosome fiber to produce 30nm chromatin fiber. Histone H1 is involved in this supercoiling. (3) Finally, nonhistone chromosomal proteins form a scaffold that is involved in condensing the 30 nm chromatin into the tightly packed metaphase chromosomes. Thi third level appears to involved the separation of segments of the giant DNA molecules present in eukaryotic chromosomes into independently supercoiled domains or loop, this mechanism which third level occurs is unknown.  Centromeres and Telomeres: - The two homologous chromosomes of each chromosome pair separate to opposite poles of meiotic spindle during anaphase 1 of meiosis. During anaphase II of meiosis the sister chromatids of each chromosome move to opposite spindle poles and become daughter chromosomes. These movements depend on the attachment of spindle microtubules to specific regions of the chromosomes, the centromeres. - The centromere of a metaphase chromosome can be recognized as a constricted region. The production of two functional centromeres I a key step in transition from metaphase to anaphase and a functional centromere must be present on each daughter chromosome to avoid the deleterious effects of nondisjunction. - The structure of centromeres in multicellular plants and animals vary greatly from species to species. One feature they have in common is the presence of specific DNA sequences that are repeated many times. - It’s been known that telomeres (meaning end and part), or ends of eukaryotic chromosomes, have unique properties. Muller who introduced the term telomere in 1983demonstrated that Drosophila chromosomes without natural ends were not transmitted to progeny. - In study of maize chromosomes Barbara McClintock demonstrated that new ends of broken chromosomes are sticky and tend to fuse with each other. In contrast, the natural ends of normal (unbroken) chromosomes are stable and show no tendency to fuse with other broken ends. McClintock’s results indicated that telomeres must have special structures different from the ends produced by breakage of chromosomes. - Telomere also have unique structures because replication of DNA does not permit duplication of both strands of DNA at the ends of the molecules. Telomeres must provide 3 functions: (1) Prevent deoxyribonucleases from degrading the ends of linear DNA molecules (2) Prevent fusion of the ends with other DNA molecules (3) Facilitate replication of the ends of the linear DNA molecules without loss of material - Telomeres have basic pattern “T-A-G”. Most terminate with a G-rich single-stranded region. Telomeres in humans have been shown to form structures called t-loops, in which the single strand at the 3’ terminus invades an upstream telomeric repeat (TTAGGG) and pairs with the complementary strand, displacing the equivalent strand. DNA in these t- loops is protected from degradation by DNA repair processes by a protein complex called shelterin. Shelterin is composed of six different proteins. TRF1 and TRF2 bind to double stranded repeat sequences, and POT1 binds to single stranded repeat sequences. Chapter 10: Replication of DNA and Chromosomes - Synthesis of DNA involves three steps: (1) chain initiation, (2) chain extension or elongation and (3) chain termination.  Semiconservative Replication: - Watson and Crick proposed that the 2 complementary strands of double helix unwind and separate, and that each strand guides the synthesis of a new complementary strand. The sequence of bases in each parental stand is used as a template and the base-pairing restrictions within the double helix dictate the sequence of bases in the newly synthesized strand. This mechanism is called semiconservative replication. - In conservative replication, the parental double helix would be conserved, and a new progeny double helix would be synthesized. - In dispersive replication, segments of both strands of the parental DNA molecule would be conserved and used as templates for the synthesis of complementary segments that would subsequently be joined to produce progeny DNA strands. - Results presented by Cairns, Meselson and Stahl showed that DNA replicates semiconservatively in E. coli. - Meselson and Stahl grew E. coli cells for many generations in a medium in which the heavy isotope of nitrogen, N , had been substituted for normal, light isotope, N . Thus, the DNA of cells grown on medium containing N will have a greater density than N . 14 Molecules with different densities can be separated by equilibrium density-gradient centrifugation. They were able to distinguish between the three models by following changes in density of DNA grown on 15 medium then transferred to 14.  All DNA isolated from cells after one generation had a density halfway between the densities of “heavy” and “light” DNA, this intermediate is referred to as a hybrid. After two generations of growth half of the DNA was hybrid and half was light.  Conservative replication would not produce and DNA molecules with hybrid density; after one generation of conservative replication of heavy DNA in light medium, half of the DNA still would be heavy and other half would be light.  If replication were dispersive they would have observed a shift of DNA from heavy toward light in each generation (that is half heavy or hybrid after one generation, quarter heavy after two generations and so forth.  Unique Origins of Replication: - In prokaryotes and bacteria there is usually only one unique origin per chromosome. In large chromosomes of eukaryotes, multiple origins control replication. The single origin of replication, called oriC, in the E. coli chromosome has been characterized in detail. These three repeats are rich in A:T base pairs, facilitating the formation of a localized region referred to as the replication bubble. The two strands of AT-rich regions of DNA come apart more easily, input of less energy.  Visualization of Replication Forks by Autoradiography: - Gross structure of replicating bacterial chromosomes was first determined by John Cairns in 1963 by autoradiography. - The autoradiography indicated that that the unwinding of the two complementary parental strands and their semiconservative replication occur simultaneously or are closely coupled. Some kind of “swivel” must exist since they must rotate 360 to unwind each gyre of the helix. IT’s now known that this swivel is a transient single strand break produced by action of enzymes called topoisomerases. - Replication of E. coli chromosomes occur bidirectionally from unique origin of replication. Each Y-shaped structure is a replication fork and the 2 replication forks move in opposite directions.  Bidirectional Replication: - The phage (ʎ) chromosome has a single stranded region, 12 nucleotides long, at the 5’ end of each complementary strand. These single stranded ends, called “cohesive” or “sticky ends” are complementary to each other. The cohesive ends of a lambda chromosome can thus base-pair to form a hydrogen-bonded circular structure. - One of the first events to occur after a lambda chromosome is injected into a host cell is its conversion to a covalently closed circular molecule.  This conversion is catalyzed by DNA ligase, an enzyme that seals single strand breaks in DNA double helices. - Like the E. coli chromosome, the lambda chromosome replicates in its circular form - When DNA molecules are exposed to high temps or pH, the hydrogen and hydrophobic bonds that hold the complementary strands together in double-helix form are b
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