bacterial genetics Flashcards
what are three common misconceptions about bacterial genetics?
that bacteria don’t have genes/quantized portions of hereditary substance
that bacteria don’t have sex
that bacterial genetics has nothing to do with prokaryotes
when the truth is ‘everything that is true for E.coli is true for the elephant???’
what is transformation (and relevant experiment)?
its when exogenous DNA is imported and incorporated into a genome
demonstrated by the smooth vs rough bacteria experiment by Avery and McCarthy, when they discovered the dead smooth bacteria could pass DNA to the live rough bacteria
why do harsh conditions encourage transformation?
transformation is risky business - few bacteria have the ‘competence’ to take up this exogenous DNA
the bacteria has no indication as to whether or not the DNA will be beneficial, so harsh conditions increase likelihood that the bacteria ‘risks’ it
how does transformation work?
1) DNA binding proteins on the bacterial membrane bind to the extracellular DNA
2) a nuclease on the membrane nicks the DNA, one strand is degraded and the other is internalised using a translocase enzyme
3) The internalised strand is bound by RecA - a protein that prevents degradation
4) RecA also binds to the bacterial ell’s dsDNA, stretching it to increase it’s complementarity and allow for recombination
how does transduction work?
its transfer of DNA from one bacterium to another via a viral vector
a phage infects a bacterium and inserts DNA into the host’s, makes copies etc…
but in viral assembly of new phages, some/one idk take up some of the host DNA, and when they infect another bacterium and inject their DNA it now contains some of the previous host bacterium’s genes which get inserted into the new host’s genome
these phages that take up host DNA are called transducing particles
who discovered bacterial sex/conjugation?
Lederberg and Tatum
how did Lederberg and Tatum discover conjugation, including their controls?
use two strains of E.coli - A and B - deficient in producing different amino acids/molecules so that neither could survive alone - but when together it turned out they could grow
to ensure this wasn’t any kind of transformation, a U-tube was used with a filter that only allowed DNA through the middle - growth was not observed for whether strain leading to the conclusion that contact is required
how does conjugation work?
the donor cell requires a fertility plasmid - F+ cell, which has the transfer genes required for conjugation
the recipient is F - meaning it doesn’t have the fertility plasmid
the plasmid has genes required to form a sex pilus - like a grappling hook that pulls two cells closer together and forms a channel between them
the F+ cell transfers a single strand of the F+ plasmid to the recipient cell, where it is converted into dsDNA (using the rolling circle I think) the OG single strand is also made back into dsDNA, giving two F+ cells
what is an Hfr cell?
Hfr means nigh frequency recombination
the F+ plasmid can have insertion genes that result in the transferred DNA being integrated into the recipient cell chromosome instead of remaining as a plasmid
the recipient cell is considered Hfr if integration occurs
if the donor cell in conjugation is Hfr what usually happens?
If the donor is Hfr, it has all the genes needed for conjugation and will form a sex pillus etc… but the F+ genes are now integrated into the donor cell’s chromosome
The origin of transfer - where the nick in the donor DNA is - is in the middle of the integrated F+ genes, so in order for the entire plasmid of F+ genes to be passed on now, the entire chromosome will have to be transferred (single strand, then replicated but still)
This is very unlikely as it takes much longer and any mechanical disruption separates the cells, so when the donor is Hfr the recipient doesn’t usually get all the F+ genes and is therefore considered F-
However the cell has managed to transfer some of it’s chromosomal genes
how has the wide variety of Hfr strains allowed for the identification of arrangement and orientation of almost all genes before the genome sequence of E.coli became known?
Hfr strains transfer genes in different sequences
Genes further along on the chromosome taker longer to transfer
Experiments mixing bacteria that for example cant make tryptophan with those that can, and measuring how long it takes for the transfer to occur by seeing when tryptophan can be produced by the cells that originally could not,
have been used to map out where lots of genes are on the chromosome
eukaryotic vs prokaryotic DNA?
prokaryotes don’t ‘waste space’, almost all the sequence codes for proteins
eukaryotes have introns and VNTRs
what is a psuedogene?
a gene that has mutated/decayed into an inactive form/cannot produce a protein
what is an operon?
what is a regulon?
used by bacteria, it’s a group of genes coding for proteins often needed at the same time/for the same thing and so are grouped and transcribed together
same principle just a step up - a group of operons often needed at the same time/for same process, controlled by a common regulatory protein
do bacteria have monocistronic genes?
yes, but it’s rare, usually polycistronic
how is DNA stored in prokaryotes? how is coiling undone?
often as one circular chromosome/nucleoid?
histone-like proteins are used to condense the DNA
then it supercoils, negatively
topoisomerases make a double stranded nick through the circle of DNA, passing another part of the circle through it, then sealing the nick, to coil
some say topoisomerases are also involved in relaxing the supercoiling
in prokaryotes, briefly escribe how DNA replication works?
it is bidirectional and semiconservative
a replication fork forms at origin of replication, leading strand added to continuously, lagging is made discontinuously in Okazaki fragments
there can be more than one replication fork?
what is a catenane and how is it separated?
the original and new chromosomes form a catenane, which is like a Russian wedding ring
topoisomerase IV can be used to separate the two
OR
XerC and XerD, each recognise a dif site on each molecule and catalyse the cutting and re-joining of the two chromosomes that can now separate two opposite poles
how is DNA replication terminated in bacteria?
termination sequences, E.coli has roughly 8, which have similarities but are not identical
E.coli has around 8 termination sequences and 4288 genes, why is this not exact?
definition of gene can be subjective e.g. super small proteins may not be counted all the time
same thing for termination sequences - the definition for what classes as one is not crystal clear
what are two ways to ensure a gene is transcribed a lot?
a strong promotor sequence (viruses have the strongest)
being nearer to the origin of replication means there ends up being more copied of that gene and it is inevitably transcribed more
when E.coli is lysed, what happens to the cell?
electron microscopy shows the DNA spews out, it is 1500 times longer than the cell
explain what the plasmid name pBR322 means
p = plasmid
B and R stand for discoverers Bolivar and Rodriguez
322 = discovered on the 322nd attempt
MUST BE ITALICS IF IT’S GENETICS
what makes pBR322 a suitable vector for cloning/transformation? how are restriction sites named?
has one origin of replication
has two antibiotic resistance genes later used for identification of successful transformation
has six unique restriction sites e.g. HindIII (named after the bacteria it came from)
explain plasmid replication using the rolling circle
1) repA must make a nick in order to open the circle
2) DNAPol replicates the outer +ve strand using the inner -ve strand as a template
3) this results in a plasmid with the original inner -ve strand and a new +ve strand, while the original +ve strand has been misplaced
4) the single +ve strand is then used as a template and a complementary -ve strand is made for it, giving two ‘daughter’? plasmids
what is the measurement used for location on a plasmid?
minutes - how many minutes it takes for a gene at that location to be transferred in conjugation
what do Shine-Dalgarno sequences do?
complementary to RNA on the ribosome, helping it attach
they vary in their strength of how well they do this, so a ribosome is more likely to attach to and translate stronger Shine-Dalgarno sequences
the promotor sequences of translation/mRNA
what kind of phenotypes do plasmids encode?
non-housekeeping
things like antibiotic resistance/production
virulence
additional metabolic functions
production of gas vesicles
F+ plasmids for conjugation etc…
things not actually needed for survival, but that increase chances of survival
what function in a plasmid is often removed in laboratories?
transfer function, so your engineered plasmid won’t escape into other colonies of bacteria
what are typical features of mutations in plasmids? how can you select for them?
small, rare, heritable
good/bad/neutral
antibiotic resistance practical
what is the velveteen method?
girlboss discovered it, husband stole credit
Grow cells on agar containing e.g. an antibiotic to select bacteria with plasmids
Then you want to investigate individual mutants in any of these plasmids
Use the thin fibre cloth velveteen to kind of stamp bacteria from this agar plate to another fresh plate e.g. with minimal media/something that would prevent growth of your mutant
what are some common phenotypes to isolate in bacteria?
drug resistant, temperature sensitive, pigmented, phage resistant etc…
what are some common phenotypes to isolate in bacteria?
drug resistant, temperature sensitive, pigmented, phage resistant etc…
what are some common phenotypes to isolate in bacteria?
drug resistant, temperature sensitive, pigmented, phage resistant etc…
what are some common phenotypes to isolate in bacteria?
drug resistant, temperature sensitive, pigmented, phage resistant etc…
what are some common phenotypes to isolate in bacteria?
drug resistant, temperature sensitive, pigmented, phage resistant etc…
how does genetic nomenclature work (what are the rules?)
gene = three or four letters all in italics with only the fourth letter being a capital
the WT is written with a superscript like a +, mutants are not
multiple alleles are just numbered
phenotype = no italics or capitals, just superscript after the name, + or -, to indicate presence or absence of that phenotype
the encoded protein - no italics or superscript, the first letter is the only capital
what are mutagens and what does UV radiation cause?
agents that increase mutation rates and their mode of action
UV radiation can cause point mutations in DNA that are heritable
