Recombination and Genetic Engineering

1. Recombination and Genetic Engineering Microbiology

2. Eucaryotic recombination • Recombination • 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 • General recombination • can be reciprocal or nonreciprocal • Site-specific recombination • Replicative recombination

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

10. Inversions • 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) • Exogenote • DNA that is transferred to recipient • Endogenote • genome of recipient • Merozyogote • recipient cell that is temporarily diploid as result of transfer process

13. Bacterial Plasmids • Small, double-stranded, usually circular DNA molecules • Are replicons • have their own origin of replication • can exist as single copies or as multiple copies • Curing • elimination of plasmid • can be spontaneous or induced by treatments that inhibit plasmid replication but not host cell reproduction

14. Bacterial plasmids… • Episomes • plasmids that can exist either with or without integrating into chromosome • Conjugative plasmids • have genes for pili • can transfer copies of themselves to other bacteria during conjugation

16. Fertility Factors • conjugative plasmids • 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 • Encode colicin • 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 • Virulence plasmids • carry virulence genes • e.g., genes that confer resistance to host defense mechanisms • e.g., genes that encode toxins • Metabolic plasmids • carry genes for metabolic processes • e.g., genes encoding degradative enzymes for pesticides • e.g., genes for nitrogen fixation

21. Transposable Elements • Transposition • 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 • Transposase • Composite transposons( Tn) • Carry genes in addition to those needed for transposition • Conjugative transposons • 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.

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 • Class 1 • GCTNAGC - Not AT rich • Sites found approximately every 100 bases in the E. coli genome • Class 2 • 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

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 • resistance 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 • F+ = donor • contains F factor • F– = recipient • 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 • Hfr strain • 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 chromosomal gene • F plasmid • formed by incorrect excision from chromosome • contains  1 genes from chromosome • F cell can transfer F plasmid to recipient 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 • Competent cell • capable of taking up DNA • May be important route of genetic exchange in nature