05 bio+210+fq+2014+ch+7+microbial+genetics

8/10/2019 05 BIO+210+FQ+2014+Ch+7+Microbial+Genetics

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Ch. 7. Microbial Genetics

Learning Objectives: You should be able to explain anddescribe the following:DNA structure and compositionPlasmidsDNA replicationHow DNA codes for proteins (transcription and translation)RNA structure and compositionTypes of mutations (point and frameshift)Mutagens

Methods for selecting mutantsThe Ames TestGenetic recombination and gene transfer (transformation,

conjugation, transduction, and transposition)


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DNA structure and composition

The basic unit of DNA is the nucleotide thatcontains a phosphate group, deoxyribose sugar,

and a nitrogenous base

In the helix the strands are orientedantiparallel, base-pairing occurs in the center(hydrophobic region), with a hydrophilicphosphate-sugar backbone on the outside


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The bidirectionality of DNA replication

Origin

Replicationproceeds in bothdirections

Replication forks

Terminationof replication


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Transcription

Translationby ribosomes

5

5

5

3

3

3

DNA(genotype)

mRNA

Polypeptide(made ofamino acids)

Phenotype

NH 2 Methionine Arginine Tyrosine Leucine

reading framefor theribosomes


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Genetic Change in Bacteria

A genetic change alters an organism

s genotype

Bacteria are haploid

- have only 1 copy of their DNA, therefore achange in DNA can easily alter thephenotype of the bacteria


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Mutations of Genes

Mutation change in the nucleotide base sequence

Almost always harmful to the bacterial cell


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Types of Mutations

Point mutations one base pair is affected

E.g.: insertions, deletions, and substitutions

Frameshift mutations nucleotide triplets afterthe mutation are displaced

E.g.: insertions and deletions


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Point mutations

Changes in one base pair can lead to one of 3 possibilities:1. silent mutation no change in amino acid sequence

2. missense mutation change in amino acid sequence

3. nonsense mutation premature termination of the

protein synthesis

usually defective protein


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Is the mutated protein s function likely to be altered?


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Is the mutated protein

s function likely to be altered?


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Is the mutated protein

s function likely to be altered?


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Frameshift mutations

Ribosomes

read

the mRNA in codons(groups of 3 nucleotides); this is called areading frame

When frameshift mutations occur, these lead toa shift in the reading frame used by theribosomes, often leading to changes in theamino acid sequence of the protein


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Is the mutated protein s function likely to be altered?


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Ultraviolet light

Thymine dimer

Mutagens

Ionizing radiation induces breaksin chromosomes

Nonionizingradiation induces

pyrimidine

dimers(e.g. thymine

dimers)

What are the effects of thymine dimers on DNA?


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Mutagens

One example of a chemical mutagen

Nucleotide analog (e.g. base analog)

structurally similar to a normal nucleotide, may be

incorporated into DNA in place of a normal

nucleotide; it disrupts DNA and RNA replication

and causes point mutations


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An example of a base analog is 5-bromouracil which incorporates in place of thymine and binds withguanine

This will change the complementary strand beingsynthesized during DNA replication


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i i l i


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Positive selectionof mutants

In this example weare looking forcells that afterexposure to amutagen, havemutated and

become resistantto penicillin, and

can grow inmedium with

penicillin


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Stamp replica plateswith velvet.

Bacteria

Identify auxotrophas colony growing oncomplete medium butnot on lacking medium.

Inoculate auxotrophcolony into completemedium.

Complete mediumcontaining tryptophan

Medium lackingtryptophan

Incubation

All colonies grow. Tryptophan auxotrophcannot grow.


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The Ames Test Liver extract is used to mimic what happens in the human

liver (our liver helps us detoxify compounds, sometimesleading to the production of carcinogenic compounds)

A particular type of Salmonella mutant is used. This mutant

is his-

which means it cannot grow without the addition ofthe amino acid histidine in the medium

The goal of this assay is to test whether a chosen chemical iscapable of causing a mutation (i.e. capable of being amutagen) and causes the Salmonella mutant to revert its his

-

mutation and become his+

(his + means that the Salmonella is able to grow in medium without the addition of the amino

acid histidine).

Th


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TheAmesTest

Liverextract

Liverextract

Suspectedmutagen

Experimentaltube

Controltube

Culture ofhis Salmo nella

Mediumlacking

histidine

Incubation

Colony of revertant ( h is + ) Salmonel la No growth


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Genetic Recombination and Transfer

Homologous recombination: exchange of nucleotide sequences (homologous sequences)

Recombinants cells with DNA molecules that contain newnucleotide sequences

Vertical gene transfer organisms replicate theirgenomes and provide copies to descendants

Genetic


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Geneticrecombination Homologous

sequences

Enzyme nicks one strand ofDNA at homologous sequence.

3

5

3

5

DNA A

DNA B

B

A

Recombination enzyme inserts the cut strand intosecond molecule, which is nicked in the process.

Ligase anneals nicked endsin new combinations.


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Molecules resolveinto recombinants.

Recombinant A

Recombinant B


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Horizontal Gene Transfer Among Prokaryotes

Three types of horizontal gene transfer:

Transformation Transduction Bacterial conjugation


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Transformation

Cells that take up DNA are competent

Transformation in


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Transformation inStrep tococ cus pneumo niae


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Transduction

A virus (phage) carries DNA segment from donor

cell to recipient cell


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Phage injectsits DNA.

Phage enzymesdegrade host DNA.

Phage DNA

Bacterialchromosome

Cell synthesizes new

phages that incorporatephage DNA and, mistakenly,some host DNA.

Transducing phage

Phage


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Transducing phage

Transducing phageinjects donor DNA.

Recipient host cell

Donor DNA is incorporatedinto recipient

s chromosomeby recombination.

Transduced cell

InsertedDNA


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Conjugation is another way to transfer DNA betweenneighboring cells (horizontal transfer)

Bacterial conjugation


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j g

F plasmid Origin oftransfer

Conjugation pilus

Chromosome

F+ cell F cell

Donor cell attachesto a recipient cellwith its pilus.

Pilus may drawcells together.


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Donor chromosome Pilus

Pilus

F+ cell

Hfr cell

F recipient

F plasmid integrates

into chromosome byrecombination.

Cells join via aconjugation pilus.

Formation of an Hfr (High frequency of recombination cell) and whathappens when it encounters a F- cell:


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F+ cell (Hfr)

F plasmidPart of F plasmid

Donor DNA Portion of F plasmid partiallymoves into recipient cell.

Conjugation ends with piecesof F plasmid and donor DNAin recipient cell.

Donor DNA and recipientDNA recombine, making a

recombinant F

cell.

Incomplete F plasmid;cell remains F

Recombinant cell (still F )

Does this process require cell cell contact? How does this affectthe size of the DNA that is transferred from the donor to therecipient cell?


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Transposons and Transposition

Transposons are segments of DNA that move from one

location to another in the same or different molecule Result is a frameshift insertion

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