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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

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Chapter 8

Chapter 8

Microbial Genetics


Where we re going some simple learning some tough concepts
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.


Advantages of bacteria as a model system
Advantages of bacteria as a conceptsmodel system

  • Haploid

  • Grow quickly!


Ii types of mutations
II. Types of mutations concepts

  • 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)


Gene transfer in bacteria
Gene transfer in bacteria concepts

  • Important:

  • 1) antibiotic resistance spreading

  • 2) way of studying genes

    • 3 ways of transfer- conjugation, transduction, and transformation.


Announcements
announcements concepts

  • Natural Sciences Fall Symposium is Dec 5


Conjugation
CONJUGATION: concepts

  • 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!


The typical conjugation, out in nature, involves conceptsonly 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


Hfr f is integrated into the host chromosome
Hfr: F is integrated into the host chromosome: concepts

  • 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!


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.





Finding gene transfer
Finding gene transfer: gene(s) it carries: F’

  • 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?


Complementation
Complementation gene(s) it carries: F’

  • 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!


trpA-, trpB+

trpA+, trpB-


trpA-, trpB+

trpA-, trpB+


Transformation
Transformation: will

  • 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.


Transduction
Transduction: will

  • 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.



Hi everybody
Hi Everybody! phage head!

  • Exam on Friday

  • Review tomorrow!

  • MMS- this one was bad

  • EMS

  • NQO- High and Low

  • Azide- High and low


Mapping with transduction
Mapping with transduction: phage head!

  • 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----------


Donor phage head!-----------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.


  • 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.


  • 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?


Things to know
Things to know transduce genes to an X-Y- Z- recipient. Selection is for X+, and screening for Y+ and Z+. Results:

  • 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.


Not a quiz but if it were
Not a quiz, but if it were: transduce genes to an X-Y- Z- recipient. Selection is for X+, and screening for Y+ and Z+. Results:

  • 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)


A transduce genes to an X-Y- Z- recipient. Selection is for X+, and screening for Y+ and Z+. Results:

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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