Bakteriegenetikk Mutasjoner og rekombinasjon

Presentasjon om: "Bakteriegenetikk Mutasjoner og rekombinasjon"— Utskrift av presentasjonen:

1 Bakteriegenetikk Mutasjoner og rekombinasjon Horisontal overføring av genetisk materiale In vitro teknikker

2 Mutasjon: forandring i arvestoffet
(genotypen forandret men ikke nødvendigvis fenotypen) Seleksjon: isolere mutanter under forhold der bestemt egenskap gjør at kun mutanten overlever Screening: hele populasjonen må undersøkes (f.eks replikaplating)

3 Kolonier med antibiotika- resistente bakterier vokser innenfor sonen
Figure: 10-01a Caption: (a) Development of antibiotic-resistant mutants within the inhibition zone of an antibiotic assay disc.

4 Replikaplating for å isolere leucin krevende mutanter (leucin auxotrofe)
Figure: 10-02a Caption: (a) Replica plating method for detection of nutritional mutants.

5 Medium med leucin medium uten leucin
Figure: 10-02b Caption: (b) Nutritional mutants, as revealed by the replica plating method. The photograph on the left shows the master plate. The colonies not appearing on the replica plate are marked with an X. The replica plate lacked one nutrient (leucine) present in the master plate. Therefore, the colonies marked with an X are leucine auxotrophs.

6 Forandring i ett basepar
Figure: 10-03 Caption: Possible effects of base-pair substitution in a gene encoding a protein: three different protein products from changes in the DNA for a single codon.

7 Den genetiske koden leses 3 og 3 baser. Ved insersjon
eller delesjon forandres leserammen herfra og ut hele genet Val Pro Cys Val Pro Val Val Leu Figure: 10-04 Caption: Shifts in the reading frame of messenger RNA caused by insertion or deletion mutations in DNA. The reading frame in mRNA is established by the ribosome that begins at the end (toward the left in the figure) and precedes by units of three bases (codons, Sections 7.13 and 7.15). The normal reading frame is referred to as the 0 frame, that missing a base the frame, and that with an extra base the frame. To determine the effects of a frameshift, translate the codons using Table 7.3.

8 -spontan (bakgrunnstråling, replikasjon)
Mutasjon: -spontan (bakgrunnstråling, replikasjon) -indusert (stråling, kjemikalier) Mutasjoner kan induseres av: -mutagene stoff (baseanaloger) -Kjemikalier som reagerer med DNA (alkylerende agens, interkalerende agens) -Stråling (UV lys absorberes godt, fører særlig til tymin-dimere. Kortere bølgelengder gir ioniserende stråling og mutasjoner dannes indirekte av frie radikaler) Figure: 10-05a-b Caption: Structure of two common nucleotide base analogs used to induce mutations and the normal nucleic acid bases they substitute for. (a) 5-Bromouracil can base pair with guanine causing AT to GC substitutions. (b) 2-Aminopurine can base pair with cytosine, causing AT to GC substitutions.

9 Reparasjonssystemer:
Enzymer som gjenkjenner skade og klipper den vekk. Polymerase I syntetiserer nytt DNA. Derfor meget lav mutasjonsfrekvens i villtype celler. Massiv skade: SOS respons (global respons for a redde cellen, replikasjon med feil)

10 SOS responsen: Figure: 10-07 Caption:
Mechanism of the SOS response. DNA damage results in conversion of RecA protein into a protease that cleaves LexA protein. LexA protein normally represses the activities of the recA gene and the DNA repair genes uvrA and umuD. (The UmuD protein is part of DNA polymerase V.) Note, however, that repression is not complete. Some RecA protein is produced even in the presence of LexA protein. With LexA inactivated, these genes become active. As a protease, the RecA protein also cleaves the lambda repressor protein.

11 Brukes til å teste om et kjemikalium er mutagent og kan føre til kreft
Ames test: Brukes til å teste om et kjemikalium er mutagent og kan føre til kreft Bruker histidin krevende Salmonella enterica og måler frekvens av tilbakemutasjon til villtype. (stammen har også defekter i reparasjonssystemer) Leverekstrakt tilsettes for å fange opp stoffer som endres av enzymene i leveren. Figure: 10-08 Caption: The Ames test is used to evaluate the mutagenicity of a chemical. Both plates were inoculated with a culture of a histidine-requiring mutant of Salmonella enterica. The medium does not contain histidine, so only cells that revert back to wild type can grow. Spontaneous revertants appear on both plates, but the chemical on the filter paper disc in the test plate (bottom) has caused an increase in the mutation rate, as shown by the large number of colonies surrounding the disc. Revertants are not seen very close to the disc because the concentration of the mutagen is so high there that it is lethal.

12 Rekombinasjon: - Sete spesifikk
- Homolog rekombinasjon (krever homologe områder, RecA protein)

13 Figure: 10-09 Caption: A simplified version of one molecular mechanism of genetic recombination. Homologous DNA molecules pair and exchange DNA segments. The mechanism involves breakage and reunion of paired segments. Two of the proteins involved, a single-stranded binding (SSB) protein and the RecA protein, are shown. The other proteins involved are not shown. The diagram is not to scale: Pairing can occur over hundreds or thousands of bases. Resolution occurs by cutting and ligating the cross-linked DNA molecules. Note that there are two possible outcomes, depending on which strands are cut during the resolution process. In one outcome the recombinant molecules have patches, whereas in the other the two parental molecules appear to have been cut and then spliced together.

14 Horisontal genoverføring:
Transformasjon (bart DNA tas opp av kompetente bakterier) Transduksjon (v.hj.a. virus) Konjugasjon (v.hj.a. plasmid) Det overførte DNA kan: -bli degradert -bli en stabil del av kromosomet (ved rekombinasjon) -eksistere som plasmid

15 Transformasjon: Figure: 10-13a-d Caption: Mechanism of DNA transfer by transformation in a gram-positive bacterium. (a) Binding of free doubled-stranded DNA by a membrane-bound DNA binding protein. (b) Passage of one of the two strands into the cell while nuclease activity degrades the other strand. (c) The single strand in the cell is bound by specific proteins, and recombination with homologous regions of the bacterial chromosome mediated by RecA protein occurs. (d) Transformed cell. Note that if recombination does not occur, the incoming DNA cannot replicate and will be lost. Eksempler på naturlig kompetente bakterier: Streptococcus pneumoniae, Bacillus subtilis, Haemophilus influenzae

16 Transduksjon: (generell) Figure: 10-14 Caption:
Generalized transduction: one possible mechanism by which virus (phage) particles containing host DNA can be formed

17 Transduksjon: (spesialisert) Figure: 10-15 Caption:
Normal lytic events and the production of particles transducing the galactose genes in an Escherichia coli cell containing a lambda prophage.

18 Fag konversjon: forandringer i en bakteriecelle som følge av lysogeni
Eks: - Salmonella anatum lysogen med -fag produserer forandret polysakkarid -Corynebacterium dipheria lysogen med -fag blir virulent

19 Plasmider: frie sirkler, kontrollerer egen replikasjon, forskjellige typer kan være tilstede i samme bakterie har gjerne gener som bakterien trenger i spesielle situasjoner (virulens faktorer, antibiotika produksjon, antibiotika resistens, bacteriocin produksjon og resistens, resistens mot tungmetaller, forskjellige metabolske funksjoner)

20 Plasmid R100 Figure: 10-19 Caption:
Genetic map of the resistance plasmid R100. The inner circle shows the size of the plasmid in kilobase pairs. The outer circle shows the location of major antibiotic resistance genes and other key functions: cat, chloramphenicol resistance; oriT, origin of conjugative transfer; mer, mercuric ion resistance; sul, sulfonamide resistance; str, streptomycin resistance; tet, tetracycline resistance; tra, transfer functions. The locations of insertion sequences (IS) and the transposor Tn10 are also shown. Several genes related to plasmid replication are found in the region from 88–92 kilobase pairs.

21 Figure: 10-18 Caption: Plasmid transfer from cell to cell during conjugation.

22 Integrasjon som episom
F plasmid Integrasjon som episom Konjugasjon Figure: 10-17 Caption: Genetic map of the F (fertility) plasmid of Escherichia coli. The numbers on the interior show the size of the plasmid in kilobase pairs (the exact size is 99,159 bp). The region shown in dark green at the bottom of the map contains genes primarily responsible for the replication and segregation of the F plasmid in normally growing cells. The light green region, the tra region, contains the genes involved in conjugative transfer. The oriT sequence is the origin of transfer during conjugation. The arrow indicates the direction of transfer (the tra region would be transferred last). The regions shown in yellow on F are transposable elements where integration into identical elements on the bacterial chromosome can occur and lead to the formation of different Hfr strains (see Section 10.9). Replikasjon, segregasjon

23 Overføring av F plasmid ved konjugasjon
Figure: 10-21a Caption: Transfer of plasmid DNA by conjugation. (a) In this example, the F plasmid of an F+ cell is being transferred to an F- recipient cell. Note the mechanism of rolling circle replication (Figure 9.20 and Figure 16.4).

24 Figure: 10-21b Caption: Transfer of plasmid DNA by conjugation. (b) Details of the replication and transfer process.

25 Integrering av F plasmid i kromosomet (Hfr)
Figure: 10-22 Caption: Integration of an F plasmid into the chromosome with the formation of an Hfr. The insertion of the F plasmid occurs at a variety of specific sites where IS elements are located, the one here being an IS3 located between the chromosomal genes pro and lac. Some of the genes on the F plasmid are shown. The arrow indicates the origin of transfer, oriT, with the arrow as the leading end. Thus, in this Hfr pro would be the first chromosomal gene to be transferred and lac would be among the last.

26 Deler av kromosomet kan overføres fra Hfr stamme
Figure: 10-23 Caption: Breakage of the Hfr chromosome at the origin of transfer and the beginning of DNA transfer to the recipient. Replication occurs during transfer (see Figure 10.21). Please note that the figure is not drawn to scale. The inserted F plasmid is less than 3% of the size of the Escherichia coli chromosome.

27 Genetisk kart over Escherichia coli
Overføring av gener fra Hfr stammer ble brukt til å kartlegge rekkefølgen av gener i E. coli Figure: 10-48 Caption: Circular linkage map of the chromosome of Escherichia coli strain K-12. On the outer edge of the map, the locations of a few of the mapped genes are indicated. A few operons are also shown, along with the direction in which they are transcribed. Along the inner edge of the map, the numbers from 0 to 100 refer to map position in minutes. The origin of DNA replication is marked oriC (84.3 min), and replication proceeds bidirectionally from this point (Section 7.6 and Figures 7.16–7.20). The inner circle shows the locations, in kilobase pairs, of the sequences recognized by the restriction enzyme NotI. Note that 0 min and 0 kilobase pairs are both, by convention, at the thr locus. The origins and directions of transfer of a few Hfr strains are also shown (arrows). The positions where five copies of the transposable element IS3 have been located in a particular strain are shown in blue. This element is also found in two copies on the F plasmid and is involved in Hfr formation (see Section 10.9). The position of the site where the bacteriophage lambda prophage integrates is shown in red (Section 9.10). If the prophage were present, it would add an extra 48.5 kilobase pairs (slightly over 1 min) to the map. The genes of the maltose regulon (Section 8.6), which includes several operons, are shown in green. Although most genes in this regulon have an abbreviation beginning with mal, note that one of the genes is lamB. This gene encodes a membrane protein involved in maltose uptake by the cell, but the protein is also the receptor for bacteriophage lambda. The gene rpsL (73 min) encodes a ribosomal protein. The gene was once called str because mutations in this gene lead to streptomycin resistance.

28 Komplementasjons analyse
Figure: 10-27 Caption: Complementation analysis. The protein products of both genes (A and B) are required to synthesize tryptophan. Mutations 1, 2, and 3 each lead to the same phenotype, a requirement for tryptophan. Complementation analysis indicates that mutations 2 and 3 are in one gene and that mutation 1 is in another.

29 Transposable elementer (transposons)
Invertert 41 bp sekvens på hver side av transposase genet Figure: 10-28a-b Caption: Maps of the transposable elements IS2 and Tn5. The red arrows underneath each map indicate the inverted repeats. The arrows above the maps show the direction of transcription of any genes on the elements. Tnp is the gene encoding the transposase. The transposase genes of these two elements are not closely related. (a) IS2 is an insertion sequence of 1327 base pairs with inverted repeats of 41 base pairs at its ends. (b) Tn5 is a composite transposon of 5.7 kilobase pairs with the insertion sequences IS50L and IS50R at its left and right ends, respectively. IS50L is not capable of independent transposition because there is a nonsense mutation (see Section 10.2) marked by a blue cross in its transposase gene. Otherwise, the two IS50 elements are very nearly identical. Note that these two IS50 elements are inverted with respect to each other. The genes kan, str, and bleo, confer resistance to the antibiotics kanamycin (and neomycin), streptomycin, and bleomycin. Interestingly, streptomycin resistance is not expressed in Escherichia coli.

30 Figure: 10-29 Caption: Transposition. Insertion of a transposable element generates a duplication of the target sequence. Note the presence of inverted repeats (IRs) at the ends of the transposable element. Figure shows more detailed models of the mechanism of transposition.

31 Figure: 10-30a-f Caption: Mechanisms of transposition. (a) In both conservative and replicative transposition the transpose makes cuts (marked with arrows) in the DNA strands at the end of the transposable element (orange) and at the target site (red). The number and location of the cuts may vary depending on the mechanism. (b) The target site becomes ligated to the transposable element. The black dots indicate free ends of DNA strands at which replication can occur (Section 7.5). (c) In conservative tranposition further cuts are made before DNA replication/repair occurs, and the transposable element is lost from the donor DNA. (d) Repair leads to duplication of the target site and completion of transposition to the new site. (e) In replicative transposition, replication occurs without the cutting of the transposable element from the donor site leading to two copies of the transposable element as part of a cointegrate. Note, however, this has led to the joining of the donor (light green) and target (dark green) DNA molecules together. (f) These molecules are separated (resolved) in a further reaction. Resolution of cointegrates is shown in more detail in Figure

32 Figure: 10-31 Caption: Replicative transposition. After the formation of single-strand cuts, a cointegrate structure arises by association of the two molecules (see Figure 10.30). After recombination, resolution of the cointegrate structure leads to the release of the original transposon and duplication of the transposon in the target molecule.

33 Integroner fra Pseudomonas
Figure: 10-33 Caption: Structure of two naturally occurring integrons from Pseudomonas. The integron In0 has the basic set of genes: intI1, encodes integrase; attI, the site where site-specific integration can occur; P, a promoter; and sulI, a gene conferring sulfonamide resistance that contains its own promoter. The integron In7 contains all of these genes, but in addition, a gene cassette has been integrated. All cassettes contain a site (blue square) for site-specific recombination. This cassette contains aadB, which confers resistance to certain aminoglycoside antibiotics. Integrasen her ligner på integraser som fag bruker ved setespesifikk rekombinasjon til lysogeni

34 In vitro rekombinant DNA lages ved å: -”klippe” med restriksjonsenzym
-”lime” med ligase Eco RI restriksjons endonuklease methylase Figure: 10-34a-b Caption: Restriction and modification of DNA. (a) The sequence of DNA recognized by the restriction endonuclease EcoRI. The red arrows indicate the bonds cleaved by the enzyme. The dashed line indicates the axis of symmetry of the sequence. (b) The same sequence after modification by the EcoRI methylase. The methyl groups added by this enzyme are shown.

35 Agarose gelelekroforese av plasmid DNA kuttet med restriksjonsenzym
Figure: 10-35b Caption: Agarose gel electrophoresis of DNA. (b) A photograph of a stained agarose gel. The DNA has been loaded into wells toward the top of the gel as shown, and the positive pole of the electrical field is at the bottom. The sample in lane A is used as a standard where the size of the fragments was known. Using the standards, one can determine the sizes of the fragments in the other lanes. Although each band in a lane will contain the same number of fragmented molecules, the bands stain less intensely at the bottom of the gel because the fragments are smaller and chemically there is less DNA to stain.

36 Plasmider blir brukt som kloningsvektorer
pBR322 Figure: 10-41 Caption: The structure of plasmid pBR322, an early and widely used cloning vector, showing the essential features. The arrow indicates the direction of DNA replication from the origin.

37 Figure: 10-42 Caption: The use of plasmid pBR322 as a cloning vector, showing how insertion of foreign DNA causes inactivation of the tetracycline resistance gene, permitting easy identification of transformants containing the cloned DNA fragment.

38 Syntese av DNA fragment in vitro ved PCR (polymerase chain reaction)
Figure: 10-45a-e Caption: The polymerase chain reaction (PCR) for amplifying specific DNA sequences. (a) Target DNA is heated to separate the strands, and a large excess of two oligonucleotide primers, one complementary to the target strand and one to the complementary strand, is added along with DNA polymerase. (b) Following primer annealing, primer extension yields a copy of the original double-stranded DNA. (c) Further heating, primer annealing, and primer extension yields a second double-stranded DNA. (d) The second double-stranded DNA. (e) Two additional PCR cycles yield 8 and 16 copies, respectively, of the original DNA sequence.