Principles of microbial genetics
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The document provides a historical overview of key discoveries related to DNA as the genetic material: 1) In the early 1900s, chromosomes were shown to carry hereditary information. By the 1940s-1950s, experiments by Avery, Griffith, Hershey and Chase provided evidence that DNA - not protein - was the genetic material. 2) Watson and Crick proposed the double helix structure of DNA in 1953 based on Chargaff's rules and Franklin's X-ray crystallography photos. Their model explained how DNA replicates and hereditary information is passed from parents to offspring. 3) Subsequent work in the 1960s by Nirenberg, Matthaei and others cracked the
Principles of microbial genetics
- 1. • In the mid-1800s; Gregor Mendel; studied the inheritance of various traits in pea plants. • In 1920s; P. A. Levene; elucidated basic chemical composition of nucleic acids. • Fred Griffith; 1928; transfer of virulence in the pathogen Streptococcus pneumoniae (pneumococcus). • Between 1941 and 1944; Archer Martin and Richard Synge; invention of paper chromatography. • By 1948; Erwin Chargaff; had begun using paper chromatography to analyse the base composition of DNA from a number of species. • In 1951; Rosalind Franklin arrived at King’s College, London, and joined Maurice Wilkins in his efforts to prepare highly oriented DNA fibers and study them by X-ray crystallography. By the winter of 1952–1953, Franklin had obtained an excellent X-ray diffraction photograph of DNA. • 1953; Watson and Crick propose DNA double helix. • 1962; Watson, Crick, and Wilkins received the Nobel Prize in 1962 for their discoveries. Historical Highlights
- 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. DNA as Genetic Material
- 3. In the early 1900s, chromosomes were shown to be the carriers of hereditary information. DNA and Protein Most scientists initially believed that protein must be the genetic material.
- 4. • 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
- 7. Avery’s Transformation Experiment 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. 2. Only the nucleic acid fraction was capable of transforming the bacteria. 3. Critics noted that the nucleic acid fraction was contaminated with proteins.
- 8. Oswald Avery with his colleagues set out to discover which constituent in the heat-killed virulent pneumococci was responsible for Griffith’s transformation.
- 9. The Hershey-Chase Bacteriophage Experiment 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 35S, and in the other part, T2 DNA was labeled with 32P. Then each group of labeled viruses was mixed separately with the E. coli host. 3. The 35S-labeled protein was found outside the infected cells, while the 32P-labeled DNA was inside the E. coli.
- 10. The Hershey-Chase Experiment (a) When E. coli was infected with a T2 phage containing 35S protein, most of the radioactivity remained outside the host cell. (b) When a T2 phage containing 32P DNA was mixed with the host bacterium, the radioactive DNA was injected into the cell and phages were produced. Thus DNA was carrying the virus’s genetic information.
- 11. The Hershey-Chase Waring Blender Martha Chase and Alfred Hershey, 1953
- 12. The Composition of Nucleic Acids
- 13. The Composition and Structure of DNA and RNA • DNA and RNA are polymers composed of monomers called nucleotides. • Each nucleotide has three parts: a. A pentose (5-carbon) sugar. b. A nitrogenous base. c. A phosphate group. • 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. Structures of deoxyribose and ribose in DNA and RNA
- 14. 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. Structures of the nitrogenous bases in DNA and RNA
- 15. • 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. • 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. • The asymmetry of phosphodiester bonds creates 3′-5′ polarity within the nucleic acid chain.
- 16. Fig. 2.11 Chemical structures of DNA and RNA
- 17. The Discovery of the DNA Double Helix • 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).
- 18. Molecular structure of DNA
- 20. Watson and Crick’s three-dimensional model 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.
- 21. 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.
- 22. 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. • 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.
- 23. Assignment on – Different DNA Structures
- 24. The Genetic Code • The final step in the expression of genes that encode proteins is translation. • The mRNA nucleotide sequence is translated into the amino acid sequence of a polypeptide chain. • Protein synthesis is called translation because it is a decoding process. • The information encoded in the language of nucleic acids must be rewritten in the language of proteins. • There is code degeneracy. i.e., there are up to 6 different codons for a given amino acid. • Only 61 codons, the sense codons, direct amino acid incorporation into protein. • The remaining 3 codons (UGA, UAG, and UAA) are involved in the termination of translation and are called stop or nonsense codons.
- 25. • The 5′ nucleotide in the anticodon can vary, but generally, if the nucleotides in the second and third anticodon positions complement the first two bases of the mRNA codon, an aminoacyl-tRNA with the proper amino acid will bind to the mRNA-ribosome complex. • This pattern is evident on inspection of changes in the amino acid specified with variation in the third position (see table). • This loose base pairing is known as wobble and relieves cells of the need to synthesize so many tRNAs. • Wobble also decreases the effects of DNA mutations.
- 26. • Genetic codons Discovered by Marshall Nirenberg ,Heinrich Matthaei,philip,Leder and Har Govind Khorana in 1968. • Nirenberg and Khorana the Nobel prize. History and definition of Genetic Code • Genetic code is a triplet of neiutides that coded that coded the specific amino acid. • Ex. AUG-met • UUU-Phe The amino acids encoded by all 64 possible codons were determined.
- 27. The genetic code required determining how 4 nucleotides (A, T, G, C) could encode more than 20 amino acids. Francis Crick and Sydney Brenner determined that the DNA is read in sets of 3 nucleotides for each amino acid. Each codon consists of three bases (triplet). There are 64 codons. They are all written in the 5' to 3' direction. 61 codons code for amino acids. The other three (UAA, UGA, UAG) are stop codons (or nonsense codons) that terminate translation. There is one start codon (initiation codon), AUG, coding for methionine. Protein synthesis begins with methionine (Met) in eukaryotes, and formylmethionine (fmet) in prokaryotes. The code is unambiguous. The Genetic Code
- 28. • The code is degenerate. More than one codon can specify a single amino acid. • All amino acids, except Met and tryptophan (Trp), have more than one codon. • For those amino acids having more than one codon, the first two bases in the codon are usually the same. The base in the third position often varies. • The code is almost universal (the same in all organisms). Some minor exceptions to this occur in mitochondria and some organisms. • The code is commaless (contiguous). • There are no spacers or "commas" between codons on an mRNA. • Neighboring codons on a message are non-overlapping.
- 29. reading frame: the series of nucleotides read in sets of 3 (codon) • only 1 reading frame is correct for encoding the correct sequence UU of amino acids UUC GAG UUU AUG UCG AAU U UCU CGA UGU UUG AGA UU AU CUC GAU GUU UGA GAA U Reading frame 1 Reading frame 2 Reading frame 3
- 30. Genetic Code
- 31. stop codons: 3 codons (UUA, UGA, UAG) in the genetic code used to terminate translation. start codon: the codon (AUG) used to signify the start of translation. 31 Types of codons
- 32. Unambiguous Each codon specifies a particular amino acid, the codon ACG codes for the amino acid threonine, and only threonine. Non overlapping This means that successive triplets are read in order. Each nucleotide is part of only one triplet codon. Commanelss which mean there is no punctuation witbeen two codans 32 Characteristics of the Genetic Code
- 33. DNA Codon RNA Codon
- 34. • The first two bases of the codon make normal (canonical) H-bond pairs with the 2nd and 3rd bases of the anticodon • At the remaining position, less stringent rules apply and non-canonical pairing may occur • The rules: first base U can recognize A or G, first base G can recognize U or C, and first base I can recognize U, C or A (I comes from deamination of A) • Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis too The Wobble Hypothesis
- 35. • It is a triplet code. • – Each three-nucleotide codon in the mRNA specifies one amino acid • • It is comma free. • – mRNA is read three bases at a time without skipping any bases. • • It is non-overlapping/non-ambiguous. • • It is almost universal. • – In nearly all organisms, most codons have the same amino acid meaning. • – Of 20 amino acids, 18 are encoded by 2 or more codons. • • The code has start and stop signals. • – AUG is the usual start signal and defines the open reading frame. Points to remember:
- 36. • • Stop signals are codons with no corresponding tRNA • – the nonsense or chain-terminating codons. • – generally three stop codons: UAG, UAA, and UGA. • More than one codon can code for each amino acid. • Each amino acid can be coded for by more than one codon
- 37. UNIT 2 Organization and function of genetic material • Bacterial • Viral
- 38. 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.
- 40. 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.
- 41. 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. 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
- 42. 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, 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)
- 43. Fig. 2.18 chromosome structure varies at stages of lytic infection of E. coli
- 45. 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.
- 46. 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 2.20). A 4.6-Mb double helix is about 1mm in length, about 103 times longer than an E. coli cell. DNA supercoiling helps it fit into the cell.
- 47. Fig. 2.20 Illustration of DNA supercoiling
- 48. 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. 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. 7. All organisms contain topoisomerase enzymes to supercoil their DNA. 8. Prokaryotes also organize their DNA into looped domains, with the ends of the domains held so that each is supercoiled independently (Figure 2.22). 9. The compaction factor for looped domains is about 10-fold. In E. coli there are about 100 domains of about 40kb each.
- 49. Fig. 2.22 Model for the structure of a bacterial chromosome
- 50. UNIT 3 Brief account of plasmids- • Structure • Types
- 51. • In addition to the genetic material present in the nucleoid, many procaryotes (and some yeasts and other fungi) contain extrachromosomal DNA molecules called plasmids. • In some cases, numerous different plasmids within a single species have been identified. (B. burgdorferi, carries 12 linear and 9 circular plasmids) • Plasmids- small double-stranded DNA molecules can exist independently of the chromosome replicate autonomously mostly circular • Linear plasmids possess special structures or sequences at their ends to prevent their degradation and to permit their replication. • Plasmids have relatively few genes, generally less than 30. Their genetic information is not essential to the host, and cells that lack them usually function normally.
- 52. • However, many plasmids carry genes that confer a selective advantage to their hosts in certain environments. • Episomes: Plasmids which are able to integrate into the chromosome and are thus replicated with the chromosome. • Plasmids are not always equally apportioned into daughter cells and sometimes are lost. The loss of a plasmid is called curing. • Curing can be induced by acridine mutagens, UV and ionizing radiation, thymine starvation, antibiotics, and growth above optimal temperatures. • Conjugative plasmids have genes for the construction of hair-like structures called pili and can transfer copies of themselves to other bacteria during conjugation. • F plasmid (F factor or fertility factor) of E. coli, was the first conjugative factor to be described; contains genes that direct the formation of sex pili that attach an F+ cell (a cell containing an F plasmid) to an F- cell (a cell lacking an F plasmid). • F factor is also an episome.
- 53. • R plasmids (Resistance factors or R factors) give antibiotic resistance on the cells that contain them. • R factors typically have genes that code for enzymes capable of destroying or modifying antibiotics. • Bacteriocin-encoding plasmids may give the bacteria that harbor them a competitive advantage in the microbial world. Bacteriocins are bacterial proteins that destroy other bacteria. • Col plasmids contain genes for the synthesis of bacteriocins known as colicins, which are directed against E. coli. Example; cloacins kill Enterobacter species. • Virulence plasmids encode factors that make their hosts more pathogenic. For example, enterotoxigenic strains of E. coli cause traveler’s diarrhea because they contain a plasmid that codes for an enterotoxin. • Metabolic plasmids carry genes for enzymes that degrade substances such as aromatic compounds (toluene), pesticides (2,4-dichlorophenoxyacetic acid), and sugars (lactose). • Metabolic plasmids even carry the genes required for some strains of Rhizobium to induce legume nodulation and carry out nitrogen fixation.
- 55. UNIT 4 Replication of DNA- • Rolling circle model Replication of RNA- • Reverse transcriptase
- 57. A process of producing two identical copies from one original DNA molecule Three basic steps: i) Initiation ii) Elongation iii) Termination i) Initiation: During initiation, some enzymes like helicase, cut the hydrogen bonds or produce the nick in the strands of DNA to make it available for the enzymes to carry out further process of replication. Single strand binding proteins restrict the rebinding of hydrogen bonds. Enzymes & proteins reach to the nick; make the complex and replication starts. DNA Replication a general account
- 58. Real difference is present at the step of elongation. ii) Elongation: Polymerase proceeds on the DNA and formation or elongation starts. In Eukaryotes, the DNA is much larger so the replication forms a linear structure often. But in some Prokaryotes, specially in bacteria who possess the plasmid, a unique type of replication occurs known as “Rolling circle Replication”.
- 59. • Replication in eukaryotes is bidirectional, this type is unidirectional (i.e. in prokaryotes) • Ideal example of this type is the circular plasmid of bacteria, as it happens only in circular genomes. Initiation • Initiates by the production of nick on one of the two strands producing free 3́-OH and 5́ phosphate ends, by the action of: • Helicase • Topoisomerases • Single stranded binding proteins (SSBPs). Helicase Bacterial Plasmid SSBPs Rolling Circle Model of DNA Replication
- 60. Elongation • For Elongation, the DNA polymerase III binds to the 3́-OH group of broken strand, using the unbroken strand as a template. • The polymerase will start to move in a circle for elongation, due to which it is named as Rolling circle model. As the elongation proceeds, the 5́end will be displaced and will grow out like a waving thread. DNA Polymerase III Broken Strand Origin Point
- 61. Termination • At the point of termination, the linear DNA molecule is cleaved from the circle, resulting in a double stranded circular DNA molecule and a single-stranded linear DNA molecule. • The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule. Origin Point
- 62. Example of Rolling Circle Model: The conjugation between F+ and F- Bacteria
- 63. Simplified Diagram of Rolling Circle Model of DNA Replication