Introduction of Biotechnology No.19: Microbial Genetics Fundamentals

Transcription

1 Handai Cyber University Introduction of Biotechnology No.19: Microbial Genetics Fundamentals Graduate School of Engineering, Osaka University Graduate School of Information Science, Osaka University International Center for Biotechnology, Osaka University 1

2 Hello! My name is Satoshi Harashima. Scientific interest: Yeast Genetics, Yeast Genomics Hobby: Listening and playing Jazz music Date of birth: 21 May 1949 ; Birthplace: Ehime, Japan 2

3 No.19 Microbial Genetics Fundamentals I. Brief history of microbial genetics II. Principle of genetic analyses III. Yeast genetics IV. Bacterial genetics V. Fungal genetics 3

4 I. Brief history of microbial genetics What is genetics Why do we study genetics? Advantage of classical genetics Feature of microbial materials 4

5 What is genetics? Genetics is the study of heredity Heredity is the phenomenon whereby biological traits are transmitted from one generation to another. Why do children resemble their parents? Genetics is the scientific discipline to study physical and molecular organization underlying hereditary process. 5

6 Why do we study genetics? Genetics occupies an important position in modern biological sciences Genetics is absolutely necessary for breeding of organisms industrially used for biotechnology 6

7 Major advantages of classical genetic approach Mutants can be isolated and characterized without any a priori understanding of the molecular basis of the function. To determine how many genes are involved in a function? To find other genes whose products may interact either physically or functionally with the products of these genes. 7

8 1865: Genetics begun with Mendel s work Plant 1900: Rediscovery of Mendel s law Untill1940: Fly and Corn as experimental materials for genetic study 1940 :Microorganisms start to be used for genetic study 8

9 Microorganisms used for genetic studies Escherichia coli Neurospora crass Aspergillus nidulans Saccharomyces cerevisiae 9

10 Feature of microbial genetics Short generation time Accumulated knowledge for cultivation method (suitable for Biochemistry) Defined media can be used (suitable for Biochemical genetics) Easy mutant-hunting due to haploidy A large number of sample can be dealt with (suitable for statistical analysis) 10

11 Quiz 1 Do following characters give an advantage of microorganisms in genetic study? 1) Invisible cell by eyes 2) Short life span 3) Haploid vegitative cells 4) Large number of cells in small space 11

12 II. Principle of genetic analyses III. Yeast genetics Isolation of mutants Dominance-recessiveness test Complementation test Epistasis-hypostasis test Linkage analysis 12

13 Essential approaches for genetic analyses 1) Isolation of mutants 2) Dominance-recessiveness test 3) Complementation test(recombination test) 4) Epistatic-hypostatic test 5) Linkage analysis 13

14 Isolation of mutants The most important step in genetic research. Genetics starts with isolation of mutants. Gene nomenclature is different from organisms to organisms HIS3: wild type allele for Saccharomyces cereivisiae his3-1 : mutant allele: Gene name (Three letter) Locus number Allele number Recessive mutation: lower case Dominant mutation: upper case 14

15 Dominance-recessiveness test Mutant (His-) hisx Cross HISX Wild type (His+) Diploid hisx/hisx If diploid exhibits If His+ phenotype If His- phenotype Mutation is Recessive Loss of function? Mutation is Dominant Gain of function? 15

16 Complementation test (for recessive mutation) Mutant X (His-) Mutant Y (His-) Mutant X (His-) Mutant Y (His-) hisx Cross hisy hisx Cross hisy Diploid hisx hisy Diploid hisx + + hisy If diploid exhibits His- phenotype If diploid exhibits His+ phenotype Mutation occurs in the same gene hisx = hisy Mutation occurs in different gene hisx hisy We could infer how many genes are involved in a particular biological phenomenon. 16

17 Epistasis-hypostasis test phox Cross phoy Mutant (Uninducible rapase production) Diploid Mutant (Constitutive rapase production) Meiotic segregant harboring phox phoy double mutations phox phoy If phox phoys double mutant shows Uninducible phenotype phox is epistatic Constitutive phenotype phoy is epistatic 17

18 Linkage analysis through meiosis Deleted based on copyright concern. 18

19 Linkage analysis Cross: HIS3 LEU2 x his3 lue2 PD NPD T Spore 1 HIS3 LEU2 his3 LEU2 HIS3 LEU2 Spore 2 HIS3 LEU2 his3 LEU2 HIS3 leu2 Spore 3 his3 leu2 HIS3 leu2 his3 LEU2 Spore 4 his3 leu2 HIS3 leu2 his3 leu2 Random assortment 1 : 1 : 4 Linkage >1 : <1 Centromere linkage 1 : 1 : <4 HIS3 LUE2 his3 Recombination leu2 19

20 NPD type his3 LEU2 his3 LEU2 HIS3 leu2 HIS3 leu2 T type HIS3 LEU2 HIS3 leu2 his3 LEU2 his3 leu2 Map distance (cm) = 100 1/2(T) + NPD Total tetrad = 100 ( ) ( ) T + 2 NPD 2PD+ NPD + T 1/2[ T 2( NPD) ]+ 4(NPD) 100 =100 Total tetrads T + 6(NPD) 2(PD+ NPD + T) 20

21 Quiz 2 Which is a correct tetra-type tetrad from a cross between MATa his3 strain and MATα leu2 strain? Spore A Spore B Spore C Spore D 1)His- Leu+, His+ Leu-, His+ Leu-, His- Leu+ 2)His+Leu+, His- Leu+, His+Leu-, His- Leu- 3)His+ Leu+, His- Leu-, His+ Leu+, His- Leu- 4)His- Leu-, His- Leu+, His+ Leu-, His+ Leu- 21

22 Five main genetic approaches 1) Isolation of mutants 2) Dominance-recessiveness test 3) Complementation test(recombination test) 4) Epistatic-hypostatic test 5) Linkage analysis 22

23 Genetic processes to exchange genetic materials in microorganisms 1) Sexual process (Conjugation, Mating ) Bacteria: E. coli Yeast : S. cerevisiae, S. pombe Fungi: N.crassa, A. nidulans 2) Parasexual process (hyphal fusion) Fungi: N.crassa, A. oryzae 3) Asexual processes (Transduction, Transformation) Bacteria: B. subtilis 23

24 IV. Bacterial genetics Transformation Conjugation Transduction 24

25 Brief history on bacterial transformation 1928: Griffith Streptococcus pneumoniae Pathogenic phenotype conversion 1944:Avery, MacLeod, McCarty Transforming particle is DNA 1958: Spizizen Natural transformation in Bacillus subtilis 25

26 Bacterial transformation Bacterial cell having an ability to incorporate DNA fragments Competent cell B Recombination DNA b c a X B X B c a Uptake of DNA fragments into cell Transformant 26

27 Brief history on bacterial transformation 1928: Griffith Streptococcus pneumoniae Pathogenic phenotype conversion 1944:Avery, MacLeod, McCarty Transforming particle is DNA 1958: Spizizen Natural transformation in Bacillus subtilis 1970: Mandel, Higa Artificial transformation in Escherichia coli 27

28 Bacterial conjugation F factor 1946 (Lederberg and Tatum): E. coli cell transfers DNA segments to another cell by direct cell-to-cell contact. Escherichia coli B C A Transfer during conjugation, after integration b c a F factor origin B A C F factor terminus Recombination b B a c c a Transconjugant 28

29 F plasmid is transferred into recipient through a pore Donor E. coli chromosome F plasmid Pilus (A) Reciepient Conjugation bridge (B) 29

30 Transfer of chromosome is mediated by F Donor F plasmid Conjugation Recipient Integrated F Donor Hfr strain (High frequency of recombination) Recombination X X 30

31 Summary of E. coli conjugation cycle F- cells do not contain the F factor and cannot transfer DNA by conjugation. They are, however, recipients of DNA transferred from F+, F or Hfr cells by conjugation. F+ cells contain the F factor in the cytoplasm and can therefore transfer F in a highly efficient manner to F- cells during conjugation. Hfr cells have F integrated into the bacterial chromosome, not in the cytoplasm. F cells have F carrying a part of the bacterial chromosome, which is generated from Hfr cell by pop-out. 31

32 Transduction 1951 (Lederberg and Zinder): Salmonella typhimurium Bacteriophage 32

33 Mechanism of general transduction in P1 Phage Bacterial chromosome a+ a+ a- Infection a+ a- Recombination a+ a+ Transductant A phage containing a+ gene 33

34 Mechanism of specialized transduction in phage λ Phage λ gal bio gal λ bio Integration between gal and bio gene λ λ gal+ gal+ gal+ bio+ gal+ Normal outlooping bio+ bio+ Rare abnormal outlooping 34 bio+

35 Quiz 3 Gram-negative bacterium Agrobacterium tumefacience can introduce a part of its Ti plasmid DNA called T-DNA into plant cells with cell-to-cell contact. What do you call this phenomenon? (1) Transformation (2) Transduction (3) Conjugation (4) Transfection 35

36 V. Fungal genetics Sexual cycle (Mating) Parasexual cycle (Hyphal fusion) Complementation test using heterokaryon 36

37 Sexual and parasexual processes to exchange genetic materials in fungi Deleted based on copyright concern. 37

38 Deleted based on copyright concern. Life cycle of Neurospora crassa, the orange bread mold. 38

39 Hyphae fuse to form heterokaryon Hyphae Fusion (n) white (n) yellow Heterokaryon Spontaneous formation of diploid (Green sectored colony) (2n) parental 39

40 Diploid colony (w+/w y+/y) Yellow sectored colony White sectored colony Deleted based on copyright concern. (n or 2n) yellow (n or 2n) white (2n) parental Mitotic recombination or haploidization occurs 40

41 Complementation test using heterokaryon in fungi arg-1 arg-2 Fusion If two arg mutations complements heterokaryon grows without arginine 41

42 Handai Cyber University No.19 : Microbial Genetics Fundamentals END Thank you very much for your attention! Don t forget to check the next slide, please! Graduate School of Engineering, Osaka University Graduate School of Information Science, Osaka Universit International Center for Biotechnology, Osaka University International Center for Biotechnology, Osaka University 42

43 Report ( submition) Q1. What is parasexual cycle observed in fungi. Q2. What is the difference between generalized transduction and specialized transduction? Q3. What are advantages in using microorganisms as experimental materials for genetics study? Submit your report to 43