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Chapter 8

Chapter 8. Microbial Genetics. Where we’re going- some simple learning, some tough concepts. Mainly the highlights of microbial genetics A few mutant types Three ways of gene transfer and how we find them and distinguish them from each other

vanna-clayton
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Chapter 8

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  1. Chapter 8 Microbial Genetics

  2. Where we’re going- some simple learning, some tough concepts • Mainly the highlights of microbial genetics • A few mutant types • Three ways of gene transfer and how we find them and distinguish them from each other • Finding genes, mapping genes using these techniques- a little math may show up, perhaps as extra credit. • Lots that’s historical- much supplanted by DNA sequencing, but still important.

  3. Advantages of bacteria as a model system • Haploid • Grow quickly!

  4. II. Types of mutations • phenotype/genotype nomenclature e.g., Lac- (capital letter, + or -), lac (three letters, italics, other letters for specific genes, e.g., lacZYA) • Lac- Unable to utilize lactose lac: genes for lactose utilization. • Leu-: unable to make leucine (AA) leu: genes forleucine biosynthesis. • Almost all phenotypes are growth/no growth! • How do we find these mutants?? By selection or screening. • Nutritional- sugar fermentation, amino acid biosynthesis • Prototroph, Auxotroph- refer to AA or other biosynthetic pathways. • Antibiotic resistance: EASY to find! • Conditional- TS (and suppressible)

  5. Gene transfer in bacteria • Important: • 1) antibiotic resistance spreading • 2) way of studying genes • 3 ways of transfer- conjugation, transduction, and transformation.

  6. announcements • Natural Sciences Fall Symposium is Dec 5

  7. CONJUGATION: • NOT eukaryotic, paramecium, conjugation. • Involves plasmids: circular pieces of DNA, separate from the chromosome; usually do nice things for the cell- • We saw them as gene vectors- for cloning! • Some are conjugative, and others are mobilizable. • CAUTION: next slide is X-Rated!

  8. The typical conjugation, out in nature, involves only a conjugative or mobilizable plasmid; the replication is “rolling circle” Plasmid F is a classic conjugative plasmid. Note that in conjugation, the recipient becomes F+ Recipient To find this event, the recipient must be changed, and must have a phenotype that distinguishes it from the donor and from unchanged recipients. Donor

  9. Hfr: F is integrated into the host chromosome: • Chromosome transfer: Fig 8-10 • The closer to the start of transfer, the sooner and more frequent is the transfer ( fig 9-9, 10) Note the meaning of the arrow!

  10. We transfer only part of the host chromosome, and the recipient doesn’t usually become F+ F will occasionally integrate into the host chromosome, usually as some specific locations. Then, the whole chromosome behaves as if it is a giant F plasmid.

  11. F': Excision of Hfr, carrying gene(s) with the F plasmid. High frequency transfer of only a few genes. The FIRST type of cloning! Fig 8-11

  12. An F’ with a particular gene would be named after the gene(s) it carries: F’lac

  13. Where it gets practical- R plasmid carrying six resistances!

  14. Finding gene transfer: • Donor and recipient; plates that ONLY allow recombinants to grow. • Ex: Hfr transfer of leu, thr, trp, his, arg to recipient. Hfr is a prototroph, but Strs ; recipient is an auxotroph, but Strr. • Mix, plate on minimal plate + strep + 4 of the AA's. • Transfer of an F’lac?

  15. Complementation • Other cool things you can do with genes on plasmids: Study mutations by complementation: • Mutations on different genes will complement each other. • Complementation groups= numbers of genes. • A cloned gene will complement a defective gene. • Caution: intracistronic, or intragenic, complementation happens sometimes. • Use a RecA strain!

  16. You made two Trp- mutants, but mutated different genes- they will complement each other. Think two dead cars, but with different problems trpA-, trpB+ trpA+, trpB- 

  17. You made two Trp- mutants, but mutated the same genes- they will NOTcomplement each other. trpA-, trpB+ trpA-, trpB+ 

  18. Transformation: • Transformation: Griffith, Avery, et. al- dead smooth- live rough. • Cells capable of being transformed = competent • Cotransformation: linked genes, near each other. • In genetic engineering, the DNA that is used is usually plasmid DNA; the event is detected b/c the plasmid imparts resistance to an antibiotic.

  19. Transduction: • Transduction: moving a gene by a virus; generally a mistake by the virus. • Generalized: virus, in packaging its DNA, sometimes packages host DNA. Example: P1 • (Optional)Specialized: lambda sometimes, by mistake, picks up the DNA nearby; these are often defective phages, but can still carry the gene.

  20. Some viruses can, by mistake, stuff chromosomal DNA in their phage head!

  21. Hi Everybody! • Exam on Friday • Review tomorrow! • MMS- this one was bad • EMS • NQO- High and Low • Azide- High and low

  22. Mapping with transduction: • Nearby genes cotransduce. • The closer 2 genes are the greater the frequency of cotransduction! • Cotransduction frequency: # cotransductants /total transductants • Donor • --------------a+-------b+----------------------------------c+------ • Recipient • --------------a----------b-----------------------------------c----------

  23. Donor-----------a+--------b+----------------------------------c+------Recipient--------------a----------b-------------------------------------c----------Donor-----------a+--------b+----------------------------------c+------Recipient--------------a----------b-------------------------------------c---------- • For cotransduction experiments, you always select for one gene, and then look (screen)for transduction of the second or third gene as well. • Infect the donor with the phage; use the phage to infect the recipient (there are some tricky technical parts here); look for recipients that have acquired the a+, b+, or c+ genes from the donor; usually you select for one (say a+) and screen for the others. • When three genes are involved, the transduction that produces the fewest # of transductants will reveal which gene is in the middle. • e.g.: select for a+, screen for b+,c+. • a+ and a+b+c- are common. • a+b+c+ less common • a+b-c+ least common- this requires a double crossover.

  24. There’s a formula that allows you to convert frequency of cotransduction into the distance between two genes. • formula: x= [1-(d/L)]3: • x= frequency, of cotransduction, d= distance (in kb), L= size of phage (in kb). As the distance between two genes approaches the size of the phage, d/L-> 1, 1-(d/L)-> 0, cotransduction frequency -> 0.

  25. Problems: cotransduction: An X+ Y+ Z+ donor is used to transduce genes to an X-Y- Z- recipient. Selection is for X+, and screening for Y+ and Z+. Results: • X+Y-Z-: 103 • X+Y+Z-: 315 • X+Y+Z+: 57 • X+Y-Z+: 3 • TOTAL: 478 • What is the cotransduction frequency of X&Y? of X and Z? • What is the order of these genes? • Awk, Kat, Nrd- A+,K+, N+ donor used to transduce A-K-N- recipient, selection for A+, screening for K and N • A+K- N-: 638 • A+K- N+: 309 • A+K+ N+: 115 • A+K+ N-: 1 • TOTAL: 1063 • Cotransduction frequency of A & K? of A & N? • Gene order?

  26. Things to know • LEARNING OBJECTIVES! • How to find mutant phenotypes, how to find a gene transfer event • Conjugation, transformation, transduction: • Types of conjugation, compare/contrast these three. • Gene order from cotransduction frequency.

  27. Not a quiz, but if it were: • Type of gene transfer that could be eliminated by Dnase treatment of supernatant from bacteria? • The Y chromosome is ~60 million bp- how long is it in centimeters? • Genes a&b cotransduce at a frequency of 60% (0.6); the transducing phage is 100kb. How far apart are they? (3√.6= .84)

  28. A L P H • Order of transfer is L-P-H-A; where and in what orientation is the F plasmid? T

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