Chapter 8 Microbial Genetics

1. Chapter 8 Microbial Genetics • Evidences of DNA & RNA to be genetic materials • Mutations and Mutants • Gene Recombination • Transformation • Transduction • Conjugation • Transposons and Insertion Sequences • Plasmids • Preservation of type culture • Genetic Breeding

2. Chapter 8 Microbial Genetics Genetics: It is the science of heredity; it includes the study of what genes are, how they carry information, how they are replicated and pass to subsequent generations of cells or passed between organisms, and how the expression of their information within an organism determines the particular characteristics of that organism. Heredity: The inherited traits of microbes include their morphology, metabolism, and the ability to move and to interact with other organisms, perhaps causing disease. The phenomenon or the process that the parents transmit their genomes (controlling or influencing these characteristics ) to the offspring is called Heredity.

3. Variation and Mutation: A suddenly change or an alteration in the base sequence of DNA is called Mutation, which can be transmitted to the progenies. The changes or the alterations of the characteristics (controlled or influenced by mutants) of the offspring are called Variations. Genotype: Refer to the total genetic factors: the entire genome of the organism. ( YyRr × YYRR) Phenotype: Refer to the extra or intro characters expressed in organisms. Phenotype = Genotype + environments Breeding: to breed a new type or form of organism according to the genetic principles.

5. Section A Fundamental Materials of Genetics • Evidences of DNA & RNA to be genetic materials • Classical transformation experiment: The initial experiment was performed by Frederick Griffith in England in 1928. Two strains of S. pneumoniae were used in the test of immunity of mice against pneumonia that Griffith was interested in. One of the two was S (smooth) form, the other was R (rough). Capsule S. pneumoniae, S form

7. The results of Avery ’ s experiment in 1944: R cell+purifiede S cell DNA S colonies

8. Coliphage infective test • The coliphage infective test was published in 1952 by A. D. Hershey & M. Chase. The result of the test shown that the DNA was the carrier of genetic information. The experiment was performed as follows: Labeled by P32

9. Labeled by S35

10. Recombination test of RNA viruses TMV HRV TMV:tobacco mosaic virus; HMV: holmes ribgrass mosaic virus

11. 2. The Discovery and Debate of Prions In 1982, American neurobiologist Stanley Prusiner proposed that infectious proteins caused a neurological disease in sheep called scrapie. Experiments suggested that the infectious agent is pure protein. Prusiner coined the name PRION for proteinaceous infectious particle. Here is one hypothesis for how an infectious agent can lack nucleic acid . The major prion protein (PrP) and its gene are found in normal host, and the PrP gene is located on chromosome 20 in humans. An abnormal PrP designated PrPSc is found in brains of animals with scrapie. PrPSc may cause a change in PrP to refold into PrPSc . Anyway, its genetic mechanism is in debate.

12. Prusiner (1982)discoverd proteinaceous infectious particle and named it Prion or Virion--朊病毒 1997年，Stanley B. Prusiner owned Nobelpris

15. 羊搔痒症(scrapie) 牛海绵状脑病(spongiform encephalopathy) 人的库鲁病(kuru)、克雅氏病(Creutzfeldt Jakob disease, CJD)等

16. 引起人与动物的致死性中枢神经系统疾病

17. 3. Forms and Locations of Genetic materials Nucleic DNA Forms RNA Plasmid & organelle DNA Cell level Nucleic level Chromosome Location and levels Nucleic acid Gene level Genetic code Nucleotide Some important plasmids: F (fertility) factor; R (resistance)factor; Col factor (colicnogenic); Ti (tumor inducing) plasmid; Degrading plasmid; Cryptic plasmid

20. Section B Gene Mutation • A mutation is a change in the base sequenceof DNA. Such a change in the base sequence of a gene or the aberration in a chromosome will sometimes cause a change of the encoded phenotype by the related gene. We can regard them separately as *gene mutation or point mutation (alterations occurred inside the gene at certain points with one or several base pares ), and *chromosomal aberrations (large or long segment aberration in chromosome). • Gene mutation Types of gene mutation: Gene mutation can be separated into deferent groups according to their senses, phenotypes and mechanisms.

21. according to the sense (massage): Original sequence: 5 ’ - AUG CCC UCA AGA UGU GGG CAA-3 ’ Met Pro Ser Arg Cys Gly Gln Same-sense mutation: 5’-AUG CCC UCA AGA UGU GGA CAA-3 ’ Met Pro Ser Arg Cys Gly Gln Mis-sense mutation: 5’-AUG CCC UCA GGA UGU GGG CAA-3 ’ Met Pro Ser Gly Cys Gly Gln Nonsense mutation: 5’-AUG CCC UCA AGA UGA GGG CAA-3 ’ Met Pro Ser Arg stop Frameshift mutation: 5’-AUG CCC UCA AGU GUG GGC AA-3 ’ Met Pro Ser Ser Val Gly etc. Reverse mutation: 5’-AUG CCC UCA AGA UGU GGG CAA-3 ’

22. According to the phenotype: • Selective mutant • Non-selective mutant • According to the mechanism Aoxotroph: hisC+ hisC- tryA+ tryA- Resistant mutant: strr strs Conditional lethal mutant: ts(42 ℃ E.coli) Morphological mutant: R / S or color Antigenic mutant Yield mutant Spontaneous mutation Induced mutation

23. 2. Characters of Mutation • Spontaneous: do not know the reason • Non-corresponding: the result do not correspond to reason • Rarity: low mutation rate, generally 10-6 --10-9 • Independent: each mutation occurs independentlyA× B • Inducing: the mutation rate can be raised by mutagen • Stability: the mutation can be transmitted to offspring • Reversible: the mutation returned to its original type • ** Evidences of spontaneous and non-corresponding: • The Fluctuation and Replica Plating test

24. fluctuation analysis （变量实验）Salvador Luria and Max Delbruck（1943） Salvador Luria Max Delbruck The Nobel Prize in Physiology or Medicine 1969

25. E.coli 50 50 … ： · ． Anti-coliphage colonies Anti-coliphage colonies Fluctuation Test: 1943, S.E.Luria & M.Delbruck Conclusion: anti-coliphage colonies formed without the contact with phages.

26. replica plating（影印实验）Joshua Lederberg and Esther Lederberg（1952） Joshua Lederberg J. Lederberg was awarded the Noble Prize in Medicine and Physiology in 1958

28. strs No str ：． ：．． ． . ． ：． ． Str contain media strr strr strr Replica Plating Test: 1952, Lederberg.

29. 3. The mechanism of Gene Mutation • Mechanism of Induced Gene Mutation • Mutagen: Any factor, either physical or chemical, that can raise the rate of mutation could be called mutagen. • Such as: • X rays, gamma( γ ) rays, ultraviolet( UV) light are forms of physical radiation mutgens; • Nitrous acid, Nitrosoguanidine, base analog are forms of chemical mutagens. • Base Substitution: include transition and transversion • Transition: refer to the purine is substituted by • another purine. Such as A → G or G → A • Transversion: refer to the purine (pyrimidine) is • substituted by a pyrimidine (purine). • Such as A→T or C→G

30. A:T T:A A T C G G C C G ds DNA ss DNA … … Substitution of Base pair Transition Transversion ．． Hk C G C Transition induced by Nitrous acid Hk A He Hk C T T T A T ．． ．． ．． ．. … ．．

31. Transition induced by 5-BU (5-bromouracil): A : T G C A : T A : Buk G Bue G C A : T G Bue G C A : Buk G C A : BU A : T A : BU … … … … … … • Frameshift Mutation: Normal: ABC ABC ABC ABC ABC ABC … • Insertion: ABC ABC AB+ CAB CAB CAB … • AB+ CAB+ CA B+ C ABC ABC … • Deletion : ABC ABC- BCA BCA BCA BCA … • ABC BCA BCA CAB CAB ABCABC …

34. Chromosomal Aberration: • Normal: • Deletion: • Duplication: • Insertion: • Tranlocation: • Inversion: • Gene Transposition: In 1940s, American geneticist Barbara Mc Clintock first found transposition factor in the corn. The DNA segment which is transposable or changeable of its position within the genome or inter-genome is called trans~ or jumping gene.

36. There are 3 forms of the transposition elements in procaryotes: * Insertion sequence (IS) * Transposon (Tn) * Mutator phage (Mu) All of the 3 have transpposase and its gene, and both IS and Tn have a inverted terminal repeat sequences at the ends of DNA. Mu contains host ’ s DNA sequences at the ends of its DNA instead of the inverted terminal repeat sequences. The genetic effects of transposition are insertion, chromosomal aberration, and gene rearrangement.

37. Mechanism of Spontaneous Mutation: Spontaneous mutation is a term contrast to the induced mutation. It do has reasons that unknown or without the use of mutangens. Accumulation of inducing effects of low dose and for a long period including background radiation, harmful metabolic products, and environmental elements. Tautomeric effect, such as the base changing from keto-form to an enol-form which can cause a substitution of the bases. Ring out effect DNA is injured by UV and the reparation.

38. Ames test

40. Section 3 Gene Recombination • Genes come from different cells or individuals recombine and form a new cell or individual which gains additional characteristics is called gene recombination. • There are 3 forms of gene recombination in bacteria: • Transformation , Conjugation and Transduction. • Transformation • The recipient or receptor (a kind of bacterial cell) directly take up the free DNA segment of the donor and conformed it within its genome, and new characters coded by the genes are so obtained.

41. Requirements for the Transformation: • A receptor cell should be in its competence • A competent cell has competent factor extant on its cell surface which is a kind of protein. • DNA segments from the donor must be provided. • Procedures of Transformation: • ds DNA segment combined with competent factor • DNA is degraded into small segments(4-5 × 106) • ss DNA formed and enter the receptor cell • The DNA conformed with the receptor ’ s DNA and transformant formed after duplication.

42. Transformation mode of B. subtilis DNase DNA binding Pr. competent factor donor recipient DNase degrade donor’s SS DNA Competent Pr. conform nucleotides

43. 2. Transduction Transduction is performed by the defective phages carrying donor ’ s DNA which can be conformed with the receptor ’ s DNA forming a recombinant DNA. The cell ( receptor) is so called a transductant. Depends on the results of transduction, we can separate them into 2 types: generalized transduction restricted or specialized ~ Complete transduction Abortive transduction LFT ( low frequency ~) HFT ( high frequency ~)

44. [-] [-] [-] transduction J.Lederberg（1952）discovered in Salmonella typhimurium Try-his+ Try+his- A B mixed Try+his+

45. Try+his- Try-his+ A B [-] [-] Try+his+ Try-his+ F– What’s the reason for Try+his+ forming ?

47. Generalized transduction: Any gene or DNA segment of the donor may be carried by a complete defective phage, which is formed by mistaken package, into the receptor and lead to recombination between the DNA of donor and the DNA of the recipient cell. Complete transduction for P22 of Salmonella

48. Abortive transduction

49. Restricted or Specialized transduction: only some certain gene can be transferred by a lysogenic phage which should be a partial defective phage. Such as λdg or λdb infective to E.coli K12 . gal λ bio prophage UV λdgal λdbio bio- gal- λ gal+ bio+

50. LFT and HFT: LFT: the frequency of transductant formation is low with a rate of 10-6 . HFT: the frequency of transductant formation is higher with a percentage of 50% theoretically. Double lysogen λdg transductants [ E.coli K12 (λ / λdg ) F]