The Genetic Material Lecture Note ppt.ppt
- 1. 2-1 The Nature of Genetic Material Historical Background • Miescher isolated nuclei from pus (white blood cells) in 1869 – Found a novel phosphorus-bearing substance = nuclein – Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins • End of 19th century – DNA and RNA separated from proteins • Levene, Jacobs, et al. characterized basic composition of DNA and RNA
- 2. 2-2 Transformation in Bacteria • Key experiments done by Frederick Griffith in 1928 • Observed change in Streptococcus pneumoniae — from virulent (S) smooth colonies where bacterial had capsules, to avirulent (R) rough colonies without capsules • Heat-killed virulent colonies could transform avirulent colonies to virulent ones
- 3. 2-3 Outline of Griffith’s Transformation Experiments
- 4. 2-4 DNA: The Transforming Material In 1944 a group used a transformation test similar to Griffith’s procedure taking care to define the chemical nature of the transforming substance – Techniques used excluded both protein and RNA as the chemical agent of transformation – Other treatments verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent
- 5. 2-5 Analytical Tools Physical-chemical analysis has often used: 1. Ultracentrifugation Used to estimate size of material 2. Electrophoresis Indicated high charge-to-mass ratio 3. Ultraviolet Absorption Spectrophotometry Absorbance of UV light matched that of DNA 4. Elementary Chemical Analysis Nitrogen-to-phosphorus ratio of 1.67, not found in protein
- 6. 2-6 DNA Confirmation • In 1940s geneticists doubted use of DNA as it appeared to be monotonous repeats of 4 bases • By 1953 Watson & Crick published the double-helical model of DNA structure and Chargaff had shown that the 4 bases were not present in equal proportions • Hershey and Chase demonstrated that bacteriophage infection comes from DNA
- 7. 2-7 Procedure for the Hershey-Chase Transformation Experiments
- 8. 2-8 Summary • Genes are made of nucleic acid, usually DNA • Some simple genetic systems such as viruses have RNA genes
- 9. 2-9 The Chemical Nature of Polynucleotides • Biochemists determined the components of nucleotides during the 1940s • The component parts of DNA –Nitrogenous bases: • Adenine (A) • Cytosine (C) • Guanine (G) • Thymine (T) –Phosphoric acid –Deoxyribose sugar
- 10. 2-10 Nucleotides and Nucleosides • RNA component parts – Nitrogenous bases • Like DNA except Uracil (U) replaces Thymine – Phosphoric acid – Ribose sugar • Bases use ordinary numbers • Carbons in sugars are noted as primed numbers • Nucleotides contain phosphoric acid • Nucleosides lack the phosphoric acid
- 11. 2-11 Purines and Pyrimidines • Adenine and guanine are related structurally to the parent molecule purine • Cytosine, thymine and uracil resemble pyrimidine
- 12. 2-12 DNA Linkage • Nucleotides are nucleosides with a phosphate group attached through a phosphodiester bond • Nucleotides may contain one, two, or even three phosphate groups linked in a chain
- 13. 2-13 A Trinucleotide The example trinucleotide has polarity – Top of molecule has a free 5’-phosphate group = 5’ end – Bottom has a free 3’- hydroxyl group = 3’ end
- 14. 2-14 Summary • DNA and RNA are chain-lie molecules composed of subunits called nucleotides • Nucleotides contain a base linked to the 1’-position of a sugar and a phosphate group • Phosphate joins the sugars in a DNA or RNA chain through their 5’- and 3’- hydroxyl groups by phosphodiester bonds
- 15. 2-15 DNA Structure The Double Helix • Rosalind Franklin’s x-ray data suggested that DNA had a helical shape • The data also indicated a regular, repeating structure • DNA was believed to require an irregular sequence • Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned to the interior
- 16. 2-16 DNA Helix • Structure compared to a twisted ladder – Curving sides of the ladder represent the sugar- phosphate backbone – Ladder rungs are the base pairs – There are about 10 base pairs per turn • Arrows indicate that the two strands are antiparallel
- 17. 2-17 Summary • The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside • The bases pair in a specific way: – Adenine (A) with thymine (T) – Guanine (G) with cytosine (C)
- 18. 2-18 Genes Made of RNA Hershey & Chase investigated bacteriophage, virus particle by itself, a package of genes – This has no metabolic activity of its own – When virus infects a host cell, the cell begins to make viral proteins – Viral genes are replicated and newly made genes with viral protein assemble into virus particles Some viruses contain DNA genes, but some viruses have RNA genes, either double- or single-stranded
- 19. 2-19 Physical Chemistry of Nucleic Acids DNA and RNA molecules can appear in several different structural variants – Changes in relative humidity will cause variation in DNA molecular structure – The twist of the DNA molecule is normally shown to be right-handed, but left-handed DNA was identified in 1979
- 20. 2-20 A Variety of DNA Structures • High humidity DNA is called the B-form • Lower humidity from cellular conditions to about 75% and DNA takes on the A-form – Plane of base pairs in A- form is no longer perpendicular to the helical axis – A-form seen when hybridize one DNA with one RNA strand in solution • When wound in a left- handed helix, DNA is termed Z-DNA • One gene requires Z- DNA for activation
- 21. 2-21 Variation in DNA between Organisms • Ratios of G to C and A to T are fixed in any specific organism • The total percentage of G + C varies over a range to 22 to 73% • Such differences are reflected in differences in physical properties
- 22. 2-22 DNA Melting • With heating, noncovalent forces holding DNA strands together weaken and break • When the forces break, the two strands come apart in denaturation or melting • Temperature at which DNA strands are ½ denatured is the melting temperature or Tm • GC content of DNA has a significant effect on Tm with higher GC content meaning higher Tm
- 23. 2-23 DNA Denaturation • In addition to heat, DNA can be denatured by: – Organic solvents – High pH – Low salt concentration • GC content also affects DNA density – Direct, linear relationship – Due to larger molar volume of an A-T base pair than a G-C base pair
- 24. 2-24 Summary • GC content of a natural DNA can vary from less than 25% to almost 75% • GC content has a strong effect on physical properties that increase linearly with GC content – Melting temperature, the temperature at which the two strands are half-dissociated or denatured – Density – Low ionic strength, high pH and organic solvents also promote DNA denaturation
- 25. 2-25 DNA Renaturation • After two DNA strands separate, under proper conditions the strands can come back together • Process is called annealing or renaturation • Three most important factors: – Temperature – best at about 25 C below Tm – DNA Concentration – within limits higher concentration better likelihood that 2 complementary will find each other – Renaturation Time – as increase time, more annealing will occur
- 26. 2-26 Polynucleotide Chain Hybridization Hybridization is a process of putting together a combination of two different nucleic acids – Strands could be 1 DNA and 1 RNA – Also could be 2 DNA with complementary or nearly complementary sequences
- 27. 2-27 DNA Sizes DNA size is expressed in 3 different ways: – Number of base pairs – Molecular weight – 660 is molecular weight of 1 base pair – Length – 33.2 Å per helical turn of 10.4 base pairs Measure DNA size either using electron microscopy or gel electrophoresis
- 28. 2-28 DNAs of Various Sizes and Shapes • Phage DNA is typically circular • Some DNA will be linear • Supercoiled DNA coils or wraps around itself like a twisted rubber band
- 29. 2-29 Summary • Natural DNAs come in sizes ranging from several kilobases to thousands of megabases • The size of a small DNA can be estimated by electron microscopy • This technique can also reveal whether a DNA is circular or linear and whether it is supercoiled
- 30. 2-30 Relationship between DNA Size and Genetic Capacity How does one know how many genes are in a particular piece of DNA? – Can’t determine from DNA size alone – Factors include: • How DNA is devoted to genes? • What is the space between genes? – Can estimate the upper limit of number genes a piece of DNA can hold
- 31. 2-31 DNA Size and Genetic Capacity How many genes are in a piece of DNA? – Start with basic assumptions • Gene encodes protein • Protein is abut 40,000 D – How many amino acids does this represent? • Average mass of an amino acid is about 110 D • Average protein – 40,000 / 110 = 364 amino acids • Each amino acid = 3 DNA base pairs • 364 amino acids requires 1092 base pairs
- 32. 2-32 DNA Genetic Capacity How large is an average piece of DNA? – E. coli chromosome • 4.6 x 106 bp • ~4200 proteins – Phage (infects E. coli) • 4.85 x 104 bp • ~44 proteins – Phage x(one of smallest) • 5375 bp • ~5 proteins
- 33. 2-33 DNA Content and the C-Value Paradox • C-value is the DNA content per haploid cell • Might expect that more complex organisms need more genes than simple organisms • For the mouse or human compared to yeast this is correct • Yet the frog has 7 times more per cell than humans
- 34. 2-34 C-Value Paradox • The observation that more complex organisms will not always need more genes than simple organisms is called the C-value paradox • Most likely explanation for the paradox is that DNA that does not code for genes is present when the less complex organism has more DNA
- 35. 2-35 Summary • There is a rough correlation between DNA content and number of genes in a cell or virus • This correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widely • C-value paradox is probably explained not by extra genes, but by extra noncoding DNA in some organisms