1. Recombination and Genetic Engineering Microbiology

2. Eucaryotic recombination 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 In eucaryotes, usually occurs as the result of crossing-over during meiosis Figure 13.1

3. Bacterial Recombination: General Principles Several types of recombination Several types of recombination –General recombination can be reciprocal or nonreciprocal can be reciprocal or nonreciprocal –Site-specific recombination –Replicative recombination

4. Reciprocal general recombination Most common type of recombination Most common type of recombination A reciprocal exchange between pair of homologous chromosomes A reciprocal exchange between pair of homologous chromosomes Results from DNA strand breakage and reunion, leading to crossing-over Results from DNA strand breakage and reunion, leading to crossing-over

5. Figure 13.2 Reciprocal general recom- bination

6. Figure 13.2

7. Nonreciprocal general recombination Incorporation of single strand of DNA into chromosome, forming a stretch of heteroduplex DNA Incorporation of single strand of DNA into chromosome, forming a stretch of heteroduplex DNA Proposed to occur during bacterial transformation Proposed to occur during bacterial transformation Figure 13.3

8. Site-specific recombination Insertion of nonhomologous DNA into a chromosome Insertion of nonhomologous DNA into a chromosome often occurs during viral genome integration into host 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 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 Recombination at inverted repeats causes and inversion

11. Replicative recombination Accompanies replication of genetic material Accompanies replication of genetic material Used by genetic elements that move about the genome 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) Transfer of genes from one mature, independent organism (donor) to another (recipient) Exogenote Exogenote –DNA that is transferred to recipient Endogenote Endogenote –genome of recipient Merozyogote Merozyogote –recipient cell that is temporarily diploid as result of transfer process

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

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

16. Fertility Factors conjugative plasmids conjugative plasmids e.g., F factor of E. coli e.g., F factor of E. coli many are also episomes many are also episomes Figure 13.5

17. Figure 13.7 mediated by insertion sequences (IS) F plasmid integration

18. Resistance Factors R factors (plasmids) R factors (plasmids) Have genes for resistance to antibiotics Have genes for resistance to antibiotics Some are conjugative Some are conjugative usually do not integrate into chromosome usually do not integrate into chromosome

19. Col plasmids Encode colicin Encode colicin –kills E. coli –a type of bacteriocin protein that destroys other bacteria, usually closely related species protein that destroys other bacteria, usually closely related species Some are conjugative Some are conjugative Some carry resistance genes Some carry resistance genes

20. Other Types of Plasmids Virulence plasmids Virulence plasmids –carry virulence genes e.g., genes that confer resistance to host defense mechanisms e.g., genes that confer resistance to host defense mechanisms e.g., genes that encode toxins e.g., genes that encode toxins Metabolic plasmids Metabolic plasmids –carry genes for metabolic processes e.g., genes encoding degradative enzymes for pesticides e.g., genes encoding degradative enzymes for pesticides e.g., genes for nitrogen fixation e.g., genes for nitrogen fixation

21. Transposable Elements Transposition Transposition –the movement of pieces of DNA around the genome Transposable elements (transposons) Transposable elements (transposons) –segments of DNA that carry genes for transposition Widespread in bacteria, eucaryotes and archaea Widespread in bacteria, eucaryotes and archaea

22. Types of transposable elements Insertion sequences (IS elements) Insertion sequences (IS elements) –Contain only genes encoding enzymes required for transposition Transposase Transposase Composite transposons( Tn) Composite transposons( Tn) –Carry genes in addition to those needed for transposition –Conjugative transposons Carry transfer genes in addition to transposition genes 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 mobile genetic elements that occasionally insert into chromosomal sequences, often disrupting genes.mobile genetic elements disrupting genesmobile genetic elements disrupting genes Insertion elements are characterized by inverted terminal repeats. These terminal repeats likely are recognition sites for an enzyme responsible for the insertion. Insertion elements are characterized by inverted terminal repeats. These terminal repeats likely are recognition sites for an enzyme responsible for the insertion.inverted terminal repeatsinverted terminal repeats 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. 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.autonomous 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. 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. 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.repeat These sequences also tend to have RNA termination signals as well as nonsense codons in all three reading frames and are therefore polar. 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. 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. 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. 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. 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. 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. 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: 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:transposon 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. 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 ). 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 Class 1 GCTNAGC - Not AT rich GCTNAGC - Not AT rich Sites found approximately every 100 bases in the E. coli genome Sites found approximately every 100 bases in the E. coli genome Class 2 Class 2 AT rich regions are preferable sites AT rich regions are preferable sites Homology at ends of region Homology at ends of region

29. The transposition event Usually transposon replicated, remaining in original site, while duplicate inserts at another site Usually transposon replicated, remaining in original site, while duplicate inserts at another site Insertion generates direct repeats of flanking host DNA Insertion generates direct repeats of flanking host DNA

30. Figure 13.8 IR = inverted repeats

31. Tn3 trans- position Class 2 Transpoison Complex Transposon

33. Generation of direct repeats

34. Effects of transposition Mutation in coding region Mutation in coding region -deletion of genetic material Arrest of translation or transcription Arrest of translation or transcription Activation of genes Activation of genes Generation of new plasmids Generation of new plasmids –resistance plasmids

35. Figure 13.13 The U-tube experiment demonstrated that direct cell to cell contact was necessary after incubation, bacteria plated on minimal media no prototrophs

36. R1 plasmid RTF = resistance transfer factor a conjugative plasmid sources of resistance genes are transposons

37. Bacterial Conjugation transfer of DNA by direct cell to cell contact transfer of DNA by direct cell to cell contact discovered 1946 by Lederberg and Tatum discovered 1946 by Lederberg and Tatum

38. F + x F – Mating F + = donor F + = donor –contains F factor F – = recipient F – = recipient –does not contain F factor F factor replicated by rolling-circle mechanism and duplicate is transferred F factor replicated by rolling-circle mechanism and duplicate is transferred recipients usually become F + recipients usually become F + donor remains F + donor remains F +

39. F factor The F factor can exist in three different states: 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. 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 "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-". 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. 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 Hfr strain –donor having F factor integrated into its chromosome both plasmid genes and chromosomal genes are transferred both plasmid genes and chromosomal genes are transferred

42. Figure 13.14b Hfr x F – mating

43. F Conjugation F plasmid F plasmid –formed by incorrect excision from chromosome –contains  1 genes from chromosome F cell can transfer F plasmid to recipient F cell can transfer F plasmid to recipient Figure 13.15a integrated F factor chromosomal gene

44. F x F – mating

45. Tra Y Characterization of the Escherichia coli F factor traY gene product and its binding sites 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. 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 Tra Y gene codes for the protein binds to the Ori T Initiates the transfer of plasmid across the bridge between the two cells Initiates the transfer of plasmid across the bridge between the two cells Tra I Gene is a helicase responsible for the conjugation Tra I Gene is a helicase responsible for the conjugation strand-specific transesterification (relaxase) strand-specific transesterification (relaxase)

47. Conjugative Proteins Key players are the proteins that initiate the physical transfer of ssDNA, the conjugative initiator 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 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 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 Uptake of naked DNA molecule from the environment and incorporation into recipient in a heritable form Competent cell Competent cell –capable of taking up DNA May be important route of genetic exchange in nature May be important route of genetic exchange in nature

51. DNA binding protein nuclease – nicks and degrades one strand competence-specific protein Streptococcus pneumoniae

52. Artificial transformation Transformation done in laboratory with species that are not normally competent (E. coli) Transformation done in laboratory with species that are not normally competent (E. coli) Variety of techniques used to make cells temporarily competent Variety of techniques used to make cells temporarily competent –calcium chloride treatment makes cells more permeable to DNA makes cells more permeable to DNA

53. Transduction Transfer of bacterial genes by viruses Transfer of bacterial genes by viruses Virulent bacteriophages Virulent bacteriophages –reproduce using lytic life cycle Temperate bacteriophages Temperate bacteriophages –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. 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 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. 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 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 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. 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.

58. Integration Integration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is Integration Host Factor. 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. 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. 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. 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. tyrosine recombinase family tyrosine recombinase family 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. 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. 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. 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: Excision of bacteriophage lambda requires two phage-encoded proteins: Int (again!) and Xis, which is an excisionase. It also requires several bacterial 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. 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. 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 Any part of bacterial genome can be transferred Occurs during lytic cycle Occurs during lytic cycle During viral assembly, fragments of host DNA mistakenly packaged into phage head During viral assembly, fragments of host DNA mistakenly packaged into phage head –generalized transducing particle

62. Generalized transduction

63. Specialized Transduction also called restricted transduction also called restricted transduction carried out only by temperate phages that have established lysogeny carried out only by temperate phages that have established lysogeny only specific portion of bacterial genome is transferred only specific portion of bacterial genome is transferred occurs when prophage is incorrectly excised occurs when prophage is incorrectly excised

64. Figure 13.20 Specialized transduction

65. Figure 13.20

66. Mapping the Genome locating genes on an organism’s chromosomes locating genes on an organism’s chromosomes mapping bacterial genes accomplished using all three modes of gene transfer mapping bacterial genes accomplished using all three modes of gene transfer

67. Hfr mapping used to map relative location of bacterial genes used to map relative location of bacterial genes based on observation that chromosome transfer occurs at constant rate based on observation that chromosome transfer occurs at constant rate interrupted mating experiment interrupted mating experiment –Hfr x F - mating interrupted at various intervals –order and timing of gene transfer determined

68. Figure 13.22a Interrupted mating

69. Figure 13.22b

70. E. coli genetic map gene locations expressed in minutes, reflecting time transferred gene locations expressed in minutes, reflecting time transferred made using numerous Hfr strains made using numerous Hfr strains Figure 13.23

71. Transformation mapping used to establish gene linkage used to establish gene linkage expressed as frequency of cotransformation expressed as frequency of cotransformation if two genes close together, greater likelihood will be transferred on single DNA fragment if two genes close together, greater likelihood will be transferred on single DNA fragment

72. Generalized transduction mapping used to establish gene linkage used to establish gene linkage expressed as frequency of cotransduction expressed as frequency of cotransduction if two genes close together, greater likelihood will be carried on single DNA fragment in transducing particle 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 provides distance of genes from viral genome integration sites viral genome integration sites must first be mapped by conjugation mapping techniques 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 also undergo recombination events viral genomes can be mapped by determining recombination frequencies viral genomes can be mapped by determining recombination frequencies physical maps of viral genomes can also be constructed using other techniques 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 recombination frequency determined when cells infected simultaneously with two different viruses Figure 13.24

76. Physical maps heteroduplex maps heteroduplex maps –genomes of two different viruses denatured, mixed and allowed to anneal regions that are not identical, do not reanneal regions that are not identical, do not reanneal –allows for localization of mutant alleles

77. Physical maps… restriction endonuclease mapping restriction endonuclease mapping –compare DNA fragments from two different viral strains in terms of electrophoretic mobility sequence mapping sequence mapping –determine nucleotide sequence of viral genome –identify coding regions, mutations, etc.