Chapter 2 DNA: The Genetic Material

1. Chapter 2DNA: The Genetic Material

2. The Search for the Genetic Material • 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. • 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.

3. Griffith’s Transformation Experiment • 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. • He called this agent the transforming principle, but did not know what it was or how it worked.

4. Griffith’s Transformation Experiment • The experiment

5. Avery’s Transformation Experiment • In 1944, Avery, MacLeod, and McCarty published results of a study that identified the transforming principle from S. pneumoniae. • Only the nucleic acid fraction was capable of transforming the bacteria.

6. Hershey and Chase’s Bacteriophage Experiment • In 1953, more evidence for DNA as the genetic material resulted from Alfred Hershey and Martha Chase’s work on E. coli infected with bacteriophage T2.

7. Hershey and Chase’s Bacteriophage Experiment • How was the experiment done? • T2 proteins were labeled with 35S, • T2 DNA was labeled with 32P. • Each group of labeled viruses was mixed separately with the E. coli host. • Phage attachment was disrupted with a kitchen blender, and the location of the label determined

8. RNA as Genetic Material • All known cellular organisms and many viruses have DNA as their genetic material. • Some viruses, however, use RNA instead. • Bacteriophages such as MS2 and Qb. • Animal viruses such as poliovirus and human immunodeficiency virus (HIV). • Plant viruses such as tobacco mosaic virus (TMV) and barley yellow dwarf virus.

9. The Composition and Structure of DNA and RNA • DNA and RNA are polymers composed of monomers called nucleotides. • Each nucleotide has three parts: • pentose (5-carbon) sugar. • nitrogenous base. • phosphate group. Purines Pyrimidines

10. Nucleotides • 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 9 nitrogen in purines and the 1 nitrogen in pyrimidines). • 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. • Nucleotide naming conventions are given in Table 2.1.

12. DNA and RNA Polynucleotides • Polynucleotides of both DNA and RNA are formed by stable covalent bonds (phosphodiester bonds) 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. • The asymmetry of phosphodiester bonds creates 3’-to-5’ polarity within the nucleic acid chain.

13. DNA Double Helix • Base Composition Studies • 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. • X-Ray Diffraction Studies • 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

14. DNA Double Helix Model 5’ 3’ • Two polynucleotide chains wound around each other in a right-handed helix. • The two chains are antiparallel. • 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. 5’ 3’

15. DNA Double Helix Model cont. • The bases of the two strands are held together by hydrogen bonds between complementarybases (two for A-T pairs and three for G-C pairs). • Complementary base pairing means that the sequence of one strand dictates the sequence of the other strand. • 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.

16. DNA Double Helix Model cont. • 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.

17. Different DNA Structures • A-DNA and B-DNA • A-DNA is the dehydrated form, and so it is not found in cells. It is a right-handed helix with 11 bp/turn and a diameter of 2.2 nm. A-DNA has a deep and narrow major groove as well as a wide and shallow minor groove. • B-DNA is the hydrated form of DNA, the kind normally found in cells. It is also a right-handed helix with only 10 bp/turn and a diameter of 2 nm. B-DNA has a wide major groove and a narrow minor groove, and its major and minor grooves are of about the same depth.

18. Different DNA Structures • Z-DNA • Z-DNA is a left-handed helix with a zigzag sugar–phosphate backbone that gives it its name. It has 12.0 bp/turn and a diameter of 1.8 nm. Z-DNA has a deep minor groove and a very shallow major groove. • 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. Some organisms show evidence of Z-DNA, but its physiological role, if any, is unknown.

19. The People Behind the DNA Structure Discovery

20. RNA Structure • 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, C) while it contains U rather than T. • Functional RNA in a cell is single-stranded, but internal base pairing can produce secondary structure in the molecule. • Some viruses use either dsRNA or ssRNA for their genomes. Double-stranded RNA is structurally very similar to dsDNA.

21. DNA and Chromosomes • Cellular DNA is organized into chromosomes. • A genome is the chromosome or set of chromosomes that contains all the DNA of an organism. • In prokaryotes the genome is usually a single circular chromosome. • In eukaryotes, the genome is one complete haploid set of nuclear chromosomes. • Mitochondrial and sometimes chloroplast DNA are also present.

22. Viral Chromosomes • Viral nucleic acid may be dsDNA, ssDNA, dsRNA, or ssRNA, linear or circular, a single molecule or several segments. • T-even bacteriophages, herpesviruses, and gemini virus have dsDNA genomes with one linear DNA molecule. • Parvovirus and bacteriophage FX174 are viruses with ssDNA chromosomes. • Reoviruses virus group with dsRNA genomes. • Picornoviruses (e.g., poliovirus) single ssRNA genome, • Influenza virus segmented ssRNA genome

23. Prokaryotic Chromosomes • The typical prokaryotic genome is one circular dsDNA chromosome, • 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. • 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.

24. Bacterial Chromosome Organization and Twisting • When E. coli is gently lysed, it releases one 4.6-Mb circular chromosome, highly supercoiled. • 4.6-Mb double helix is about 1 mm in length, about 103 times longer than an E. coli cell. DNA supercoiling helps it fit into the cell.

25. Bacterial Chromosome Organization and Twisting • In addition prokaryotes organize their DNA into looped domains, with the ends of the domains held so that each is supercoiled independently. • In E. coli there are about 400 domains of varying lengths.

26. Genomes and Chromosomes • The genome of most prokaryotes consists of one chromosome. • 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.

27. Eukaryotic Genomes and Chromosomes • Most eukaryotes have a diploid number of chromosomes. • The structural complexity and the C-value of an organism are not related, creating the C-value paradox.

28. Chromatin Structure • Chromatin is a DNA and protein complex in the nucleus. Its structure is the same in all eukaryotes. • The proteins in this complex are the histones • Histones are abundant, small proteins with a net (+) charge. • Histones organize DNA, condensing it and preparing it for further condensation by nonhistone proteins.

29. Nucleosomes and DNA condensation • Two molecules each of histones H2A, H2B, H3, and H4 associate to form a nucleosome core, and DNA wraps around it 1.65 times for a six-fold condensation factor. Nucleosome cores are about 11 nm in diameter. • H1 further condenses the DNA to create chromatin with a diameter of 30 nm, additional six-fold condensation. The solenoid model is proposed to be formed of 6 nucleosomes per turn of a spiral.

30. Beyond the Solenoid • Beyond the 30-nm filament stage, 30–90 loops of DNA attach to a protein scaffold. 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. • Fully condensed chromosome is 10,000-fold shorter and 400-fold thicker than DNA alone.

31. More Illustrations

32. Euchromatin and Heterochromatin • Euchromatin is actively transcribed and lacks repetitive sequences. • Heterochromatin remains condensed throughout the cell cycle. It replicates later than euchromatin and is transcriptionally inactive. There are two types based on activity: • Constitutive heterochromatin occurs at the same sites in both homologous chromosomes of a pair and consists mostly of repetitive DNA (e.g., centromeres). • 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).

33. Centromeres and Telomeres • Centromeres and telomeres are eukaryotic chromosomal regions with special functions. • Centromeres are required for accurate segregation of chromatids. • 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.

34. Centromeres • Yeast (Saccharomyces cerevisiae) centromeres • 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

35. Telomeres • Simple telomeric sequences are short, species-specific, and tandemly repeated. (Tetrahymena 5’-TTGGGG-3’) ( Human 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

36. Unique and Repetitive DNA Sequences • Sequences vary widely in how often they occur within a genome. • a. Unique-sequence DNA, present in one or a few copies. • b. Moderately repetitive DNA, present in a few to 105 copies. • c. Highly repetitive DNA, present in about 105–107 copies.

37. Types of Repetitive Sequences • LINEs (long interspersed repeated sequences) with sequences of 1,000–7,000 bp or more. • LINE-1 in mammals , sequences up to 7 kb in length, that can act as transposons. • SINEs (short interspersed repeated sequences) with sequences of 100–500 bp. • Alu repeats found in some primates, including humans, 200–300 bp make up 9% of the genome. • SINEs are also transposons but are dependent on LINES for transposase genes. • Tandemly repetitive sequences range from very short sequences (1–10 bp) to genes and longer sequences. • Centromere and telomere sequences as well as rRNA and tRNA genes.