Biol 240: Genetics

Microbial genetics grew from:

Microbiology

What did Microbial Genetics require?

required the development of model systems for genetic investigations, such as E.coli or Salmonella typherium

Comparison of previously studied microbes to currently studied microbes:

prevously, only practically important bacteria were studies (e.g. pathogens), but now, bacteria are mostly studied for the knowledge of microbial genetics overall

Replicon

Both the genomic DNA and plasmid DNA of a bacteria (the entire collection of genetic information)

Why are bacteria ideal genetic research candidates?

because they only have one chromosome, so it is easy to detect mutants

wild type

the phenotype for a character most commonly observed in natural populations (source for deriving mutants)

Mutant

a strain that carries a mutation relative to the wild type

Mutation

A change in a gene or chromosome that halts or alters function

Allele

An alternative form of a gene. (may be a gain, loss, or change of function)

auxotroph

a mutant that is unable to make a particular compound, often because of a mutation in amino acid creation

prototroph

A normal version of a bacteria with the wild type allele. can make all its shit normally (often the auxotrophs parental strain)

Genotype

description of the alleles within an organism

Phenotype

observable properties of a strain

What does the + and - mean beside a gene name?

a + means that the bacteria has that gene, while a - means the gene does not function

Selection

Isolation of cells with a particular genotype on the basis of growth (e.g. burning a haystack to find a needle, can find a His+ bacteria on the basis of growth but cannot find His- bacteria because it will not grow)

Screening

identification of cells with a particular phenotype on the basis of colour, morphology, not growth (e.g. using a pitchfork to separate strands of hay to find the single blue one, cannot burn it all down in this case)

selectable mutations generally grant a:

growth advantage under specific conditions (e.g. conditions that kill wildtype cells)

Nonselectable mutations:

confer neither an advantage or disadvantage to growth

what is required to detect particular nonselectable mutations?

screening techniques

Phenotypic Selection

use of a growth medium that will inhibit microbes lacking the desired gene(s)

What technique can Phenotypic Selection be categorized as?

selection (usually uses antibiotic)

Why is screening more tedious than selection?

Because in selection, the work is done automatically (by antibiotics, medium, etc.) while in screening, cells need to be separated manually

Phenotypic Screening

duplicate plates are made on different agars (one lacks a nutrient). Allows ability to see mutants that cannot grow in absense of nutrient (auxotrophs)

Patching method

inoculate colonies from a regular agar plate onto a gridded plate. Uusally more accurate than stamp plating

4 types of mutations

Silent, Missense, Nonsense, and Frameshift

silent mutations

results in no change in the amino acid sequence of the protein; usually in the third position of a codon

Missense mutations

a change in a codon that results in coding for a different amino acid in the eventual protein

nonsense mutation

a change that forms a stop codon where one should not be found

Frameshift mutation

the result of insertions or deletions of nucleotides, changing how a ribosome "reads" an mRNA molecule and can alter amino acid sequences of proteins

What is a "reversion"

a mutation that corrects a metabolic abnormality (previous mutation) back to the wild type form

Why can reversions be problematic?

It can make it difficult when trying to determine mutation rates of a chemical or DNA exchange rates between two microbes

How is this problem avoided?

double and triple auxotroph mutant strains were utilized instead, which decreases the possibility of a spontaneous revision mutation

Who showed that spontaneous mutations were not caused by selective pressure through replica plating?

Ester Lederburg

Who showed that variable resistance to phage infection is not caused by selective pressure?

Luria and Delbruck

Who compared two tubes of E.coli (one with selective pressures and one without) to eachother in terms of envolutionary progress in 75 days?

Richard Lenski

What did the experiment show?

the ability for a microbe to grow in the culture was enhanced over time

What are restriction enzymes?

bacterial enzymes that cut DNA at specific palindrome sequences (recognition site)

What are modification enzymes?

enzymes that accompany restriction enzymes (called R/M Systems). They recognize the same site as the paired restriction enzyme. Methyltransferase activity protects DNA from endonuclease activity

What can Restriction Enzymes be used for?

They can be used to insert pieces of DNA into cells, and clone cells of interest.

What is significant about recombinant plasmids?

they have DNA from other cells and will be able to exhibit the genes in that DNA

5 desirable traits for easier gene cloning:

origin of replication, selectable marker gene, multiple cloning site, small size, high copy number

Origin of Replication

Site where the replication of a DNA molecule begins. You need to have this if a plasmid is going to be maintained in a cell. (OriV)

selectable marker gene

genes carried by plasmids for certain traits, often for antibiotic resistance

Cloning sites

facilitates cloning of DNA. Putting DNA in one convenient location, allows for creating multiple restriction-enzymes in succession. They do not show up anywhere else in the vector

small size

easier to cut gene you want

high copy number

useful for propogating DNA, you need many plasmids

describe steps of recombination

RE cut patch of DNA out of cell, insert into new cell. RE cut new cell plasmid and ligase inserts the new DNA

What colour are mutant bacteria while using the x-gal system?

blue

shuttle vector plasmids

Have multiple types of origins. This expands the range of host cell types the plasmids can be inserted into

phage vectors

mix viral DNA with fragment of interest, allow it to be inserted into cell and fit in to genome

Cosmids

phage genomes that omit nearly all the phage DNA, leaving more room for the fragment. Only the critical phage 'cos' packaging recognition sites remain. Other elements include a multiple cloning site and an antibiotic selection marker

cohesive end sites

areas at end of phage DNA

Transformation

introduction of extracellular DNA directly into an organism. THIS DOES NOT REQUIRE DIRECT CONTACT

What is the name of cells that can use transformation?

naturally competent

what are two ways cells can be induced to be competent?

treatment with calcium ions and electroporation

How does DNA come into the cell and why?

it comes in as a single strand as a safety measure

What enzyme cleaves the DNA and replaces damaged portions of the cells genome with it?

RecA

Conjugation

A temporary union of two organisms for the purpose of DNA transfer.

What is the connection between the two cells called?

sex pilus

What is the name of the plasmid that carries the gene that makes the sex pilus?

F plasmid

What happens when there is one F- and one F+ cell together?

the F+ cell will create a sex pilus with an F- cell and transfer a copy of the F plasmid to it, making it an F+ cell

What bodily appendage do sex pili resemble?

arms (not penis). They pull other cell towards it for a kiss

The F Plasmid can fully integrate into the host chromosome by ___________ ___________

homologous recombination

The F Plasmid as an ____________

episome

episome

A genetic element that can exist either as a plasmid or as part of the bacterial chromosome.

What is a High Frequency Recombination cell?

it is an F+ cell that has had its F Plasmid integrate into its genome

The High Frequency Recombination (HFR) of the F Plasmid can be used to do what?

it can be used to map the locations of genes in the host chromosome

What is an F' plasmid?

an F' plasmid is an F plasmid that leaves after being integrated into the bacterial genome. When it is excised from the genome, it takes a bit of genome with it because it is innacurate.

What is one use for conjugation?

triparental conjugation (a donor plasmid does not have a transfer gene, but another cell has a plasmid with a transfer gene, so it helps transfer that original plasmid gene into the recipient cell.

Transposition

movement of DNA via mobile genetic elements.

Transposable elements can move within and between genomes

Who discovered transposition in corn?

Barbara McClintock

What two things can transposition be divided into?

insertion sequences and transposons

insertion sequences

incode only the proteins needed for transposition

transposons

contain other genes in addition to those needed for transposition

What two genes are required for transposition?

transposase and resolvase

replicative transposition

copies the element and moves the copy to another location

non-replicative transposition

cuts and pastes the element into a new location

Which gene is responsible for the copy paste?

resolvase

transposition can be used to disrupt _______ ______ and observe ___________ ________

functional genes, phenotypic changes

What happens to the plasmid that is used in transposition?

plasmid goes into new cell but it cannot replicate, so it is lost

Transduction

A virus accidentally packaging genomic DNA instead of viral DNA, and delivering that genomic DNA to another bacterial cell.

Historically, what was co-transduction frequency used for?

used to map out bacterial genomes (genes closer to marker genes would be transduced while ones farther away would not be)

What else can transduction be used for?

it can be used to modify bacterial genomes (e.g. produce anti-HIV bacteria in pussy