Download Chem*4570 Applied Biochemistry Lecture 11 Conjugation and

Chem*4570 Applied Biochemistry Lecture 11
Conjugation and Recombination
Stryer pp. 819-828. Lehninger pp. 949-967
Bacterial conjugation allows for the transfer of genetic characters within a species,
from genomic DNA to genomic DNA in the gene’s normal context.
Alleles - alternative variants of a gene, e.g. wild type versus mutant, normal versus Thr supersensitive
etc.
In contrast vectors are agents that allow transfer of genes between organisms, not necessarily the same
species, but the transferred genes do not usually end up in their normal genomic context.
Plasmids are small autonomously replicating DNA circles that lie outside the main chromosome, placing
genes in an episomal context. Plasmids can be set up to express the genes they carry but represent an
extra cost to their host so can be lost.
Genes can also be integrated into the genome via phage such as phage λ and its derivatives, but will
not be in their normal genetic context.
Recombination plays a major role in role certain kinds of gene transfer.
Bacterial conjugation
E. coli K12 exists in several different mating types.
F+ cells contain the F plasmid or fertility factor, and can transfer it by
conjugation to F– cells which lack the factor. Recipients of the F factor
then become F+. F+ cells may spontaneously lose the F factor and revert
to F– .
The F plasmid includes the genes required for formation of the sex pilus,
responsible for attaching to F– cells, which express an appropriate
receptor.
The F plasmid may incorporate a section of the bacterial genome, allowing transfer of genes
to another cell. Recombination events may then integrate the genes into the recipient’s genome.
F plasmid may also itself become incorporated into the genome, in which case tranfer of the F
factor to the recipient can carry the entire genome into the recipient. Since this creates far more
opportunities for recombination, strains with genomic F factor are designated Hfr cells, for high
frequency of recombination. Integration occurs spontaneously at a frequency of about 10–5 .
Reversion of Hfr to F+ occurs at a similar frequency. In reverting to F+, the F plasmid may acquire a
small portion of the host genome, and will pass this on in future conjugation events.
Conjugation in yeasts.
Wild type yeast, like many eukaryotes, is a diploid (two copies of each chromosome) although lab
strains for convenience are maintained as haploids (single chromosomes). Yeast exists in two mating
types a and α which are heritable, i.e. progeny of simple cell fission have the mating type of their
parent.
The mating type is maintained by an
expressible copy of one of two
separate silent genes, one for each
mating type. Cells can undergo
mating type switch, e.g. induced
by starvation, in which one of the
silent genes places a new copy of
itself in the expressed locus. This
drop-in replacement is known
as a gene cassette.
Mixed mating types will then
undergo conjugation, allowing
exchange of genetic
characters.
The mated cells undergo
meiosis and then sporulate,
forming a tetrad containing
the haploid progeny of the
conjugation.
After conjugation, selection and screening techniques are used to recover cells which have acquired
the desired characters.
Conjugation allows strains selected for one mutation, e.g. Hom– to be combined with genes from
another strain e.g. AECR (and also to replace the lexA– with a normal regulated SOS repair system).
Recombination
Recombination is the process whereby sequences from one DNA molecule can exchange with
sequences in another molecule.
Homologous recombination may occur where where there are regions of sequnce match between the
incoming and the target DNA. Incoming DNA may be DNA transferred by conjugation, by
transformation with a plasmid, or bare DNA inserted by a technique such as electroporation.
Site specific recombination occurs as a mechanism to integrate external DNA into a host genome.
This requires the existence of specific target sequence in the host.
For example the attB locus in E.coli acts as the attchment site for insertion of phage λ DNA into the
host genome.
Homologous recombination involves several proteins. The existence of such an elaborate process is
probably a consequence of recombination being an important DNA repair mechanism..
RecBCD then provides the
helicase/nuclease complex, or
unwindase that generates single
stranded DNA. A a sequence
called the chi site, GCTGGTGG,
the nuclease cuts near its 3’, end,
releasing a single strand of DNA
with a 3’ end. Chi sites are widely
distributed in E.coli DNA, about 4
kbp apart.
RecA binds the single stranded
DNA, and promotes invasion of
the single strand into homologous
double stranded DNA, displacing a
loop.
The displaced loop can also be nicked, leaving another 3’ end, and it too will invade the opposite
strand, creating a crossover or Holliday junction. The junction then migrates along the DNA,
mediated by the proteins RuvA and RuvB in an ATP dependent process. Thus the final crossover
site need not be closely associated with the original chi site.
Cleavage of the Holliday junction is followed by religation so that two independent duplex strands
form once more. Depending on the plane of cleavage, this may result in a minimal transfer of DNA
between strands, or alternatively opposite strands may religate, so that a cross-over event has occurred.
These crossover events allow transfer of sequences from the incoming DNA to the genomic DNA of
the cell.
Nick and strand displacement
RecB, RecC
RecA binds ssDNA
RecA promotes strand invasion into homologous
duplex
second nick and displacement
of strand on 2nd duplex
junction formed by ligation
branch migration, RuvA, RuvB
plane of cleavage determines
extent of recombination