Microbial Genetics

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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?
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Microbial Genetics Gene - unit of
inheritance structural gene - information for a
protein regulatory gene - control protein
formation operon structural genes plus
regulatory genes
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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
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Transformation Griffiths, 1928 - vaccine for
pneumonia Streptococcus pneumoniae smooth vs.
rough strains transforming principle Avery,
McCarty MacLeod, 1944
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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)
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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
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Heteroduplex DNA mediated by recA
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Conjugation Lederberg Tatum, 1946
auxotrophs in E. coli auxotrophs mixed
108 cells plated on minimal medium
reversion vs. ? shown cell contact
required
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Conjugation Cell contact required
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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
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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
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Conjugation Pilus needed for cell contact
DNA synthesis needed for transfer rolling
circle replication begins at origin (ori) one
strand nicked
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Conjugation F x F- Hfr x F-
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Conjugation Plasmid exit from genome Hfr ?
F Hfr ? F'
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Conjugation Plasmid exit from genome Hfr ?
F Hfr ? F' recipient is a
partial diploid
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Mapping the bacterial genome Hfr transfer of
bacterial genes
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Mapping the bacterial genome
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Transduction Bacteriophage or phage -
bacterial viruses lytic or virulent phage
lysogenic or temperate phage
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Transduction Bacteriophage or phage -
bacterial viruses lytic or virulent phage
lysogenic or temperate phage
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Transduction Bacteriophage or phage -
bacterial viruses lytic or virulent phage
lysogenic or temperate phage Bacterial DNA
transfer Generalized transduction Restricted
transduction
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Transduction Generalized transduction
lytic or lysogenic phage any suitably sized
piece of DNA packaged
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Transduction Restricted transduction only
genes adjacent to insertion site phage ?
- att locus gal or bio loci
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Transduction Restricted transduction only
genes adjacent to insertion site phage ?
- att locus gal or bio loci phage
defective ?dgal requires helper phage
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Transduction Restricted transduction only
genes adjacent to insertion site phage ?
- gal or bio loci phage defective ?dgal
requires helper phage
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Fates of DNA Incorporation of genes
transformation, conjugation, transduction
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Fates of DNA Incorporation of genes
transformation, conjugation, transduction Persist
and replicate - clone of cells with the DNA
self-replicating plasmids
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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
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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
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Fates of DNA
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Host restriction restriction
endonucleases cut at specific nucleotide
sequences 6-8 base pairs most generate sticky
ends
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Restriction enzymes EcoRI
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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-
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Genetic engineering Tools vectors - plasmids,
bacteriophage restriction enzymes Products pr
oteins - see Table 9.2 in text resistant
plants