process in which one or more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence
In eucaryotes usually occurs as the result of crossing-over during meiosis
Figure 13.1 3 Bacterial Recombination General Principles
Several types of recombination
can be reciprocal or nonreciprocal
4 Reciprocal general recombination
Most common type of recombination
A reciprocal exchange between pair of homologous chromosomes
Results from DNA strand breakage and reunion leading to crossing-over
5 Reciprocal general recom-bination Figure 13.2 6 Figure 13.2 7 Nonreciprocal general recombination
Incorporation of single strand of DNA into chromosome forming a stretch of heteroduplex DNA
Proposed to occur during bacterial transformation
Figure 13.3 8 Site-specific recombination
Insertion of nonhomologous DNA into a chromosome
often occurs during viral genome integration into host chromosome
enzymes responsible are specific for virus and its host
9 Site Specific Recombination
If the two sites undergoing recombination are oriented in the same direction this may result in a deletion
Recombination at inverted repeats causes and inversion
11 Replicative recombination
Accompanies replication of genetic material
Used by genetic elements that move about the genome
12 Horizontal gene transfer
Transfer of genes from one mature independent organism (donor) to another (recipient)
DNA that is transferred to recipient
genome of recipient
recipient cell that is temporarily diploid as result of transfer process
13 Bacterial Plasmids
Small double-stranded usually circular DNA molecules
have their own origin of replication
can exist as single copies or as multiple copies
elimination of plasmid
can be spontaneous or induced by treatments that inhibit plasmid replication but not host cell reproduction
14 Bacterial plasmids
plasmids that can exist either with or without integrating into chromosome
have genes for pili
can transfer copies of themselves to other bacteria during conjugation
15 (No Transcript) 16 Fertility Factors
e.g. F factor of E. coli
many are also episomes
Figure 13.5 17 F plasmid integration mediated by insertion sequences (IS) Figure 13.7 18 Resistance Factors
R factors (plasmids)
Have genes for resistance to antibiotics
Some are conjugative
usually do not integrate into chromosome
19 Col plasmids
kills E. coli
a type of bacteriocin
protein that destroys other bacteria usually closely related species
Some are conjugative
Some carry resistance genes
20 Other Types of Plasmids
carry virulence genes
e.g. genes that confer resistance to host defense mechanisms
e.g. genes that encode toxins
carry genes for metabolic processes
e.g. genes encoding degradative enzymes for pesticides
e.g. genes for nitrogen fixation
21 Transposable Elements
the movement of pieces of DNA around the genome
Transposable elements (transposons)
segments of DNA that carry genes for transposition
Widespread in bacteria eucaryotes and archaea
22 Types of transposable elements
Insertion sequences (IS elements)
Contain only genes encoding enzymes required for transposition
Composite transposons( Tn)
Carry genes in addition to those needed for transposition
Carry transfer genes in addition to transposition genes
23 IS sequences
Insertion elements are mobile genetic elements that occasionally insert into chromosomal sequences often disrupting genes .
Insertion elements are characterized by inverted terminal repeats . These terminal repeats likely are recognition sites for an enzyme responsible for the insertion.
Mobility of the element depends only on the element itself it is an autonomous element. Thus it must carry the coding ability for the transposase recognizing the inverted terminal repeats.
The direct repeats externally flanking the inverted repeats are not part of the insertion sequence. Instead they are chromosomal sequences that become duplicated upon insertion with one copy at each end this is called target-site duplication.
24 Characteristics of IS elements
The majority of IS elements are between 0.7 and l.8 kb in size and the termini tend to be l0 to 40 base pairs in length with perfect or nearly perfect repeats.
These sequences also tend to have RNA termination signals as well as nonsense codons in all three reading frames and are therefore polar.
Typically they encode one large open reading frame of 300 to 400 amino acids and by definition the protein encoded by this reading frame is involved in the transposition event.
Two exceptions to the size range given above should be noted The first is 5.7 kb and the other IS101 is a scant 0.2 kb in size. Although there are exceptions insertion sequences tend to be present in a small number of copies in the genome.
For example IS1 is present in 6 to l0 copies in E. coli chromosome while IS2 and 3 are typically present in about five copies.
25 IS actions
Insertion sequences mediate a variety of DNA rearrangements. One of the first recognitions of this fact was the involvement of insertion sequences in the integration of F and R plasmids into the host chromosome. This event gives rise to Hfr strains.
The initial DNA rearrangement mediated by IS elements is the insertional duplication that they tend to generate at the site of insertion.
IS1 generates an 8 or 9 base pair duplication while IS2 generates a 5 base pair duplication.
26 (No Transcript) 27 Transposons
As defined above a transposon is a mobile genetic element containing additional genes unrelated to transposition functions. In general there are known to be two general classes
Class l or compound Tns encode drug resistance genes flanked by copies of an IS in a direct or indirect repeat. A direct repeat exists when the two sequences at either end are oriented in the same direction while an indirect (or inverted) repeat exists when they are in opposite directions. In this class of transposons the IS sequence supplies the transposition function.
The second class of transposons are known as complex or Class 2. With these the element is flanked by short (30-40 bp) indirect repeats with the genes for drug resistance and transposition encoded in the middle (see figure of Tn3 below).
28 Preferential sites for transposition
GCTNAGC - Not AT rich
Sites found approximately every 100 bases in the E. coli genome
AT rich regions are preferable sites
Homology at ends of region
29 The transposition event
Usually transposon replicated remaining in original site while duplicate inserts at another site
Insertion generates direct repeats of flanking host DNA
30 IR inverted repeats Figure 13.8 31 Tn3 trans-position Class 2 Transpoison Complex Transposon 32 (No Transcript) 33 Generation of direct repeats 34 Effects of transposition
Mutation in coding region
-deletion of genetic material
Arrest of translation or transcription
Activation of genes
Generation of new plasmids
35 The U-tube experiment after incubation bacteria plated on minimal media no prototrophs demonstrated that direct cell to cell contact was necessary Figure 13.13 36 RTF resistance transfer factor a conjugative plasmid R1 plasmid sources of resistance genes are transposons 37 Bacterial Conjugation
transfer of DNA by direct cell to cell contact
discovered 1946 by Lederberg and Tatum
38 F x F Mating
contains F factor
does not contain F factor
F factor replicated by rolling-circle mechanism and duplicate is transferred
recipients usually become F
donor remains F
39 F factor
The F factor can exist in three different states
F refers to a factor in an autonomous extrachromosomal state containing only the genetic information described above.
The Hfr (which refers to high frequency recombination) state describes the situation when the factor has integrated itself into the chromosome presumably due to its various insertion sequences.
The F or (F prime) state refers to the factor when it exists as an extrachromosomal element but with the additional requirement that it contain some section of chromosomal DNA covalently attached to it. A strain containing no F factor is said to be F-.
40 F x F mating
In its extrachromosomal state the factor has a molecular weight of approximately 62 kb and encodes at least 20 tra genes. It also contains three copies of IS3 one copy of IS2 and one copy of a À sequence as well as genes for incompatibility and replication.
41 Hfr Conjugation
donor having F factor integrated into its chromosome
both plasmid genes and chromosomal genes are transferred
42 Hfr x F mating Figure 13.14b 43 F Conjugation integrated F factor
formed by incorrect excision from chromosome
contains 1 genes from chromosome
F cell can transfer F plasmid to recipient
chromosomal gene Figure 13.15a 44 F x F mating 45 Tra Y
Characterization of the Escherichia coli F factor traY gene product and its binding sites
WC Nelson BS Morton EE Lahue and SW Matson Department of Biology University of North Carolina Chapel Hill 27599.
46 Tra Genes
Tra Y gene codes for the protein binds to the Ori T
Initiates the transfer of plasmid across the bridge between the two cells
Tra I Gene is a helicase responsible for the conjugation
strand-specific transesterification (relaxase)
47 Conjugative Proteins
Key players are the proteins that initiate the physical transfer of ssDNA the conjugative initiator proteins
They nick the DNA and open it to begin the transfer
Working in conjunction with the helicases they facilitate the transfer of ss RNA to the F- cell
48 DNA Transformation
Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form
capable of taking up DNA
May be important route of genetic exchange in nature
49 (No Transcript) 50 (No Transcript) 51 Streptococcus pneumoniae nuclease nicks and degrades one strand DNA binding protein competence-specific protein 52 Artificial transformation
Transformation done in laboratory with species that are not normally competent (E. coli)
Variety of techniques used to make cells temporarily competent
calcium chloride treatment
makes cells more permeable to DNA
Transfer of bacterial genes by viruses
reproduce using lytic life cycle
reproduce using lysogenic life cycle
54 Lysogenic Phage 55 Lambda
In order for the lambda prophage to exist in a host E. coli cell it must integrate into the host chromosome which it does by means of a site-specific recombination reaction.
56 Attachment site
The E. coli chromosome contains one site at which lambda integrates. The site located between the gal and bio operons is called the attachment site and is designated attB since it is the attachment site on the bacterial chromosome.
The site is only 30 bp in size and contains a conserved central 15 bp region where the recombination reaction will take place.
he structure of the recombination site was determined originally by genetic analyses and is usually represented as BOB where B and B represent the bacterial DNA on either side of the conserved central element
57 Recombination site
The bacteriophage recombination site - attP - is more complex. It contains the identical central 15 bp region as attB.
The overall structure can be represented as POP. However the flanking sequences on either side of attP are very important since they contain the binding sites for a number of other proteins which are required for the recombination reaction. The P arm is 150 bp in length and the P arm is 90 bp in length.
Integration of bacteriophage lambda requires one phage-encoded protein - Int which is the integrase - and one bacterial protein - IHF which is Integration Host Factor.
Both of these proteins bind to sites on the P and P arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned.
The integrase enzyme carries out all of the steps of the recombination reaction which includes a short 7 bp branch migration.
59 Enzymes and Recombination
There are two major groups of enzymes that carry out site-specific recombination reactions one group - known as the tyrosine recombinase family - consists of over 140 proteins.
These proteins are 300-400 amino acids in size they contain two conserved structural domains and they carry out recombination reactions using a common mechanism involving a the formation of a covalent bond with an active site tyrosine residue.
The strand exchange reaction involves staggered cuts that are 6 to 8 bp apart within the recognition sequence.
All of the strand cleavage and re-joining reactions proceed through a series of transesterification reactions like those mediated by type I topoisomerases.
60 Excision of bacteriophages
Excision of bacteriophage lambda requires two phage-encoded proteins
Int (again!) and Xis which is an excisionase. It also requires several bacterial proteins.
In addition to IHF a protein called Fis is required.
All of these proteins bind to sites on the P and P arms of attL and attR forming a complex in which the central conserved 15 bp elements of attL and attR are properly aligned to promote excision of the prophage.
61 Generalized Transduction
Any part of bacterial genome can be transferred
Occurs during lytic cycle
During viral assembly fragments of host DNA mistakenly packaged into phage head
gene locations expressed in minutes reflecting time transferred
made using numerous Hfr strains
Figure 13.23 71 Transformation mapping
used to establish gene linkage
expressed as frequency of cotransformation
if two genes close together greater likelihood will be transferred on single DNA fragment
72 Generalized transduction mapping
used to establish gene linkage
expressed as frequency of cotransduction
if two genes close together greater likelihood will be carried on single DNA fragment in transducing particle
73 Specialized transduction mapping
provides distance of genes from viral genome integration sites
viral genome integration sites must first be mapped by conjugation mapping techniques
74 Recombination and Genome Mapping in Viruses
viral genomes can also undergo recombination events
viral genomes can be mapped by determining recombination frequencies
physical maps of viral genomes can also be constructed using other techniques
75 Recombination mapping
recombination frequency determined when cells infected simultaneously with two different viruses
Figure 13.24 76 Physical maps
genomes of two different viruses denatured mixed and allowed to anneal
regions that are not identical do not reanneal
allows for localization of mutant alleles
77 Physical maps
restriction endonuclease mapping
compare DNA fragments from two different viral strains in terms of electrophoretic mobility
determine nucleotide sequence of viral genome
identify coding regions mutations etc.
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