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גנטיקה של חיידקים. מוטציות Normal DNA מוטציה 1 – שינוי בסיס ב-DNA C Missense Mutation.

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Presentation on theme: "גנטיקה של חיידקים. מוטציות Normal DNA מוטציה 1 – שינוי בסיס ב-DNA C Missense Mutation."— Presentation transcript:

1 גנטיקה של חיידקים

2 מוטציות Normal DNA

3 מוטציה 1 – שינוי בסיס ב-DNA C Missense Mutation

4 T Nonsense Mutation מוטציה 2 - שינוי בסיס ב-DNA

5 מוטציה 3 - תזוזת מסגרת הקריאה w ATG CAT GCA TGC ATT TCC TGC TTA AAA w 1. Addition Mutation w AAT GCA TGC ATG CAT TTT CCT GCT TAA w Reading Frame is Shifted w 2. Deletion Mutation w TGC ATG CAT GCA TTT CCT GCT TAA w Reading Frame is Shifted

6 העברת מטען גנטי בחיידקים העברת מטען גנטי – דרוש על מנת לשרוד. w העברת מטען גנטי בחיידקים יכול להתרחש ב- 3 דרכים : Transformation - טרנספורמציה Transduction- טרנסדוקציה Cunjugation - קוניוגציה

7 הניסוי של גריפין Griffith’s Experiment 2 Strains of Streptococcus pneumoniae 1. Virulent strain with a capsule – Pneumonia 2. Avirulent strain without a capsule - no disease

8 Griffith’s Experiment הניסוי של גריפין 1.חיידקים חיים בעלי קפסולה הוזרקו לעכברים 2.העברים מתו 3.מושבות חיידקים בעלי קפסולה בודדו מהחיידק המת.

9 Griffith’s Experiment 1. חיידקים חיים ללא קפסולה הוזרקו לעכברים 2. העכברים נשארו בריאים 3. מושבות של חיידקים ללא קפסולה בודדו מהעכברים.

10 Griffith’s Experiment 1.חיידקים בעלי קפסולה, שהומתו ע"י חימום, הוזרקו לעכברים 2.העכברים נשארו בריאים 3.לא בודדו מובות חיידקים מהעכברים

11 Griffith’s Experiment 1.חיידקים חיים ללא קפסולה, וחיידקים בעלי קפסולה, שהומתו ע"י חימום, הוזרקו לעכברים 2.העכברים מתו 3.מושבות של חיידקים בעלי קפסולה בודדו מהעכברים המתים.

12 A typical bacterial cell chromosome

13 Transformation טרנספורמציה גנים עוברים מחיידק אחד לאחר כ “DNA ערום". Frederick Griffith (1928)

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15 Transfer of genetic information Transformation and DNA Recombination Dead cells?

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17 Transductionטרנסדוקציה DNA מועבר מחיידק אחד לשני ע"י וירוס. בקטריופאז' – וירוס התוקף תאי חיידקים בלבד.

18 טרנסדוקציה בקטריופאג ' או פאג ' - Bacteriophage or phage = וירוס התוקף חיידק lytic or virulent phage lysogenic or temperate phage Bacterial DNA transfer Generalized transduction Restricted transduction

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21 Transduction Generalized transduction – lytic or lysogenic phage any suitably sized piece of DNA packaged

22 Transduction Bacteriophage or phage - bacterial viruses lytic or virulent phage lysogenic or temperate phage

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26 Transduction Restricted transduction only genes adjacent to insertion site phage λ - gal or bio loci phage defective λdgal requires helper phage

27 Transduction

28 Conjugation One bacterium passes some DNA (in a plasmid) to another bacterium

29 bacterial conjugation/bacterial “sex”

30 conjugation

31 Bacterial Conjugation

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36 Conjugation F + x F - Hfr x F -

37 Conjugation Plasmid exit from genome: Hfr → F + Hfr → F' recipient is a partial diploid

38 Mapping the bacterial genome Hfr transfer of bacterial genes

39 Mapping the bacterial genome

40 Conjugation Lederberg & Tatum, 1946 auxotrophs in E. coli auxotrophs mixed – 10 8 cells plated on minimal medium reversion vs. ? shown cell contact required

41 Conjugation Hayes, et. al., 1950s discovered family of mutants with a high frequency of recombination – Hfr mutants no conversion of F - in mating bacterial gene transfer high plasmid integrated into host genome specific locus ‘cross-over’

42 Conjugation Pilus needed for cell contact DNA synthesis needed for transfer rolling circle replication begins at origin (ori) one strand nicked

43 Conjugation Plasmid exit from genome: Hfr → F + Hfr → F'

44 Plasmid pBR322

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49 Transduction Bacteriophage or phage - bacterial viruses lytic or virulent phage lysogenic or temperate phage

50 Transduction Bacteriophage or phage - bacterial viruses lytic or virulent phage lysogenic or temperate phage Bacterial DNA transfer Generalized transduction Restricted transduction

51 Transduction Generalized transduction – lytic or lysogenic phage any suitably sized piece of DNA packaged

52 Evolution Survival/selection of the fittest Transfer of genetic information with selective advantage  survival and propagation of the recipients

53 Results of mutation and gene transfer antibiotic resistance

54 Basic genetic engineering

55 Microbial Genetics Genetic notation Three letter designation for each gene lac gal ara his lacZ, hisG, araC Genome sum total of all the genes of the organism all of the ‘chromosomes’ of the organism, the DNA bacteria are haploid Genotype vs. Phenotype Genotype: genetic makeup of the organism Phenotype: appearance; visible characteristics Does phenotype always reflect the genotype?

56 Microbial Genetics Gene - unit of inheritance structural gene - information for a protein regulatory gene - control protein formation operon – structural genes plus regulatory genes

57 Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange

58 Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange Prokaryotes - transfer of genes from one cell to another one-way transfer of genes DNA transferred: exogenote recipient DNA: endogenote partial diploid may be formed

59 Recombination bringing new gene combinations together Eukaryotes - crossing over during meiosis reciprocal exchange Prokaryotes - transfer of genes from one cell to another one-way transfer of genes DNA transferred: exogenote recipient DNA: endogenote partial diploid may be formed Transformation Conjugation Transduction Limited transfer: one gene to a few genes closely related cells

60 Transformation Griffiths, vaccine for pneumonia Streptococcus pneumoniae smooth vs. rough strains ‘transforming principle’ Avery, McCarty & MacLeod, 1944

61 Transformation naked DNA released by cell binds to competent recipient cell competence is physiological state competence requires protein synthesis DNA binding protein on cell surface – receptor DNA must be dsDNA of suitable size (450-15,000 bp) one strand degraded ssDNA enters cell pairs with genome heteroduplex formed by recA

62 Heteroduplex DNA mediated by recA

63 Transformation

64 Conjugation Hayes, et. al., 1950s transfer one-way, not reciprocal donor and recipient strains (+ and – strains) donor: F + recipient: F - mating of F + x F - → 2 F + F is F plasmid bacterial gene transfer rare

65 Transduction Restricted transduction only genes adjacent to insertion site phage λ - att locus gal or bio loci

66 Transduction Restricted transduction only genes adjacent to insertion site phage λ - att locus gal or bio loci phage defective λdgal requires helper phage

67 Transduction Restricted transduction only genes adjacent to insertion site phage λ - gal or bio loci phage defective λdgal requires helper phage

68 Fates of DNA Incorporation of genes transformation, conjugation, transduction

69 Fates of DNA Incorporation of genes transformation, conjugation, transduction Persist and replicate - clone of cells with the DNA self-replicating plasmids

70 Fates of DNA Incorporation of genes transformation, conjugation, transduction Persist and replicate - clone of cells with the DNA self-replicating plasmids Persist but not replicate abortive transduction

71 Fates of DNA Incorporation of genes transformation, conjugation, transduction Persist but not replicate abortive transduction Persist and replicate - clone of cells with the DNA self-replicating plasmids Degradation or host restriction

72 Fates of DNA

73 Host restriction restriction endonucleases cut at specific nucleotide sequences – 6-8 base pairs most generate ‘sticky ends’

74 Restriction enzymes EcoRI

75 Host restriction restriction endonucleases cut at specific nucleotide sequences – 6-8 base pairs most generate ‘sticky ends’ host DNA protected by methylation -G-A-A-T-T-C- -C-T-T-A-A-G-

76 Genetic engineering Tools:vectors - plasmids, bacteriophage restriction enzymes Products proteins - see Table 9.2 in text resistant plants

77 Conjugation w Definition: Gene transfer from a donor to a recipient by direct physical contact between cells w Mating types in bacteria Donor F factor (Fertility factor) –F (sex) pilus Donor Recipient Lacks an F factor

78 Physiological States of F Factor w Autonomous (F + ) Characteristics of F + x F - crosses F - becomes F + while F + remains F + Low transfer of donor chromosomal genes F+

79 Physiological States of F Factor w Integrated (Hfr) Characteristics of Hfr x F - crosses F - rarely becomes Hfr while Hfr remains Hfr High transfer of certain donor chromosomal genes F+F+ Hfr

80 Physiological States of F Factor w Autonomous with donor genes (F’) Characteristics of F’ x F - crosses F - becomes F’ while F’ remains F’ High transfer of donor genes on F’ and low transfer of other donor chromosomal genes Hfr F’

81 Mechanism of F + x F - Crosses w DNA transfer Origin of transfer Rolling circle replication Pair formation – Conjugation bridge F+F+ F-F- F+F+ F-F- F+F+ F+F+ F+F+ F+F+

82 Mechanism of Hfr x F - Crosses w DNA transfer Origin of transfer Rolling circle replication w Homologous recombination Pair formation – Conjugation bridge HfrF-F- F-F- F-F- F-F-

83 Mechanism of F’ x F - Crosses w DNA transfer Origin of transfer Rolling circle replication Pair formation – Conjugation bridge F’ F-F- F-F-

84 Conjugation w Significance Gram - bacteria Antibiotic resistance Rapid spread Gram + bacteria Production of adhesive material by donor cells

85 Transposable Genetic Elements w Definition: Segments of DNA that are able to move from one location to another w Properties “Random” movement Not capable of self replication (not a replicon) Transposition mediated by site-specific recombination Transposase Transposition may be accompanied by duplication

86 Types of Transposable Genetic Elements w Insertion sequences (IS) Definition: Elements that carry no other genes except those involved in transposition Nomenclature - IS1 Structure – Importance Mutation Plasmid insertion Phase variation Transposase ABCDEFG GFEDCBA

87 Phase Variation in Salmonella H Antigens IS H1 gene H2 gene H1 flagella H2 flagella

88 Types of Transposable Genetic Elements w Transposons (Tn) Definition: Elements that carry other genes except those involved in transposition Nomenclature - Tn10 Structure Composite Tns – Importance Antibiotic resistance IS Resistance Gene(s) IS Resistance Gene(s)

89 The insertion of the IS-element is accompanied by a duplication of a short sequence around the insertion site. A proposed mechanism known as the cut and paste model for the occurence of this effect is illustrated opposite. A staggered single-strand cut is made in the target sequence followed by an insertion of the respective IS-elements. The cuts are subsequently sealed and as a result the target sequence is duplicated.

90 Plasmids w Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) w Episome - a plasmid that can integrate into the chromosome

91 Classification of Plasmids w Transfer properties Conjugative Nonconjugative w Phenotypic effects Fertility Bacteriocinogenic plasmid Resistance plasmid (R factors)

92 Bacteriocins (proteins toxic to other bacteria lacking the same plasmid) Toxins Proteins that mediate plasmid transfer to uninfected strains (i.e. the tra genes) Genes which provide the cell with a pathogenic feature: such as the ability invade cells (Yersinia pestis) or produce toxins (E coli 0:157) plasmids of this type are often called virulence plasmids. Some Examples of Plasmid Encoded Genes Antibiotic resistance genes (enzymes that modify or degrade antibiotics) -- plasmids with these genes are generally called R factors Heavy metal resistance (enzymes that detoxify metals by redox reactions) Growth on unusual substrates (enzymes for hydrocarbon degradation, etc.) Restriction/modification enzymes (protect DNA, degrade unprotected DNA)


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