PCR, Viral and Bacterial Genetics Chps, 18 and 17

Learning Objectives Describe the process of PCR Explain the use of gel electrophoresis List the essential components of bacterial DNA Compare and contrast transduction, transformation and conjugation as a means of bacterial gene exchange Describe the process of replica plating Compare and contrast the lytic and lysogenic cyle of bacteriophages Describe transposons in eukaryotes

Polymerase Chain Reaction Polymerase chain reaction (PCR) Produces many sequence copies without host cloning Amplifies known DNA sequences for analysis Only copies sequence of interest Primers bracket sequence Agarose gel electrophoresis Separates fragments by size and charge Gel molecular sieve

Produces 4 molecules Produces 8 molecules Cycle 1 Cycle 2 Cycle 3 2 molecules produced Produces 4 molecules Produces 8 molecules Target sequence Template DNA primers DNA containing target sequence to be amplified DNA primer New DNA These 2 molecules match target DNA sequence DNA primer New DNA Target sequence Figure 18.6: Research Method. The Polymerase Chain Reaction (PCR) Target sequence Template Fig. 18-6, p. 378

Animation: Polymerase chain reaction (PCR)

Micropipettor adding marker DNA fragments to well – – Well in gel for placing DNA sample Agarose gel Buffer solution PCR products already loaded to wells Gel box Figure 18.7: Research Method. Separation of DNA Fragments by Agarose Gel Electrophoresis + + Fig. 18-7a, p. 380

Lane with marker DNA fragments Fig. 18-7b, p. 380 Figure 18.7: Research Method. Separation of DNA Fragments by Agarose Gel Electrophoresis Fig. 18-7b, p. 380

Bacterial and Viral Genetics Chapter 17

Bacterial Genetics One-celled prokaryotic organisms Only some are pathogenic (ie, causing diseases) Many are symbiotic (ie, E. coli) Some can be infected by viruses (bacteriophages)

Bacterial Genetics Single circular strand of DNA Bacteria are haploid Bacteria do not undergo true sexual reproduction However, gene exchange and recombination is important for survival and adaptation

Bacterial Genetics Three main ways to get DNA from one bacteria to another for recombination Conjugation Transduction Transformation

Bacterial Plasmids Bacteria can recombine DNA with other bacteria of similar strains (conjugation) The exchange involves plasmids (small circular pieces of DNA) F+ (fertility) bacteria contain plasmids that allow for transfer

Bacterial Plasmids To initiate transfer, a bacterium produces a “conjugation bridge”- a tube extends from the F+ (donor) bacterium to the F- (recipient) bacterium The donor’s plasmid separates, and a complimentary piece travels across the bridge to the recipient bacterium A complimentary strand is produced by the recipient The recipient becomes an F+ bacterium

a. Transfer of the F factor Bacterial chromosome An F+ cell conjugates with an F– cell. 1 F factor F+ F– One strand of the F factor breaks at a specific point and begins to move from F+ (donor) to F– (recipient) cell as the F factor replicates. 2 DNA replication of the F factor continues in the donor cell, and a complementary strand to the strand entering the recipient cell begin to be synthesized. 3 Figure 17.4: Transfer of genetic material during conjugation between E. coli cells. (a) Transfer of the F factor during conjugation between F+ and F− cells. When transfer of the F factor is complete, replication has produced a copy of the F factor in both the donor and recipient cells; the recipient has become an F+. No chromosomal DNA is transferred in this mating. 4 Fig. 17-4a, p. 356

Bacterial Plasmids Sometimes bacterial plasmids (the F factor) can integrate into the bacterial chromosome This bacterium is called Hfr (high frequency recombination) This bacterium can conjugate with recipient cells, allowing part of the bacterial DNA to enter the recipient cell The recipient cell is now partially diploid and double crossover rearrangement can occur

Bacterial Plasmids

b. Transfer of bacterial genes chromosome c+ b+ d+ a+ 1 The F+ cell. F factor c+ b+ d+ a+ F factor integrates into the E. coli chromosome in a single crossover event. 2 Figure 17.4: Transfer of genetic material during conjugation between E. coli cells. (b) Transfer of bacterial genes and the produce of recombinants during conjugation between Hfr and F− cells. Bacterial chromosome d– c– a– b– A cell with integrated F factor—an Hfr donor cell —and an F– cell conjugate. These two cells differ in alleles: the Hfr is a+ b+ c+ d+, and F– cell is a– b– c– d–. 3 c+ b+ d+ a+ Hfr cell F– cell Fig. 17-4b (1), p. 356

Bacterial Plasmids: closer look

Mapping Genes by Recombination Full DNA transfer by conjugation takes 90 to 100 minutes Partial DNA transfer when sex pilus breaks Timing of DNA transfer allows mapping of E. coli chromosome, map units are minutes Order and timing of DNA transfer show E. Coli has circular chromosome

Bacterial Plasmids Kinds of information carried on plasmids includes: Resistance to antibiotics (R) Ability to manufacture amino acids Fertility factor (F+) - proteins for the conjugation bridge

Bacterial Transformation Some bacteria have DNA-binding proteins on their cell walls They can integrate similar bacterial DNA into their own genome This can be natural or induced in the lab by heat or electroporation (electrical shock)

Bacterial Transduction DNA may also be carried by bacteriophages When a bacteriophage is being assembled in an infected cell, it may incorporate pieces of the bacterial DNA into its shell That DNA is injected along with bacteriophage DNA during the next infection cycle

Bacteriophages Virulent- always kill their hosts after replication. Temperate- can live inside host for generations, DNA being replicated in a controlled fashion until activated Lytic cycle- virus proteins cause viral assembly (both viral and cell DNA) and cell bursts Lysogenic cycle- quiescent bacterial replication with viral DNA integrated into bacterial chromosome

Replica Plating Replica plating identifies and counts genetic recombinations in bacterial colonies Master plate pressed onto sterile velveteen Velveteen pressed onto replica plates with different growth media Complete medium has full complement of nutrient substances Auxotrophic mutants will not grow on media missing particular nutrients

Master plate with complete medium Replica plate with minimal medium Colony growth Figure 17.5: Research Method. Replica Plating Fig. 17-5a, p. 359

Bacteriophages T even phages Lambda (λ) – temperate phage which reactivates easily with UV light Lambda phage is used

E. coli Lambda Bacteriophage Lamba (λ) E. coli bacteriophage Typical temperate phage with two paths Lytic cycle goes directly from infection to progeny virus release Lysogenic cycle integrates λ chromosome into host Insertion at specific sequences, then crosses over Prophage viral genome inactive until trigger Specialized transduction transfer of host genes near λ genome

Lysogenic Cycle Lytic Cycle Stepped Art Fig. 17-8, p. 362 Figure 17.8 The infective cycle of lambda, an example of a temperate phage, which can go through the lytic cycle or the lysogenic cycle. Stepped Art Fig. 17-8, p. 362

17.3 Transposable Elements Insertion sequence elements and transposons major types of bacterial transposable elements Transposable elements were first discovered in eukaryotes Eukaryotic transposable elements are classified as transposons or retrotransposons Retroviruses are similar to retrotransposons

Transposons and TEs Transposable genetic elements (TE) or jumping genes Two major types of bacterial TEs: insertion sequences – inverted repeat sequence and coding for transposase Transposons- inverted repeat and central genes, including host genes- most notably antibiotic resistance

Transposable Elements Transposable elements (TEs) Segments of DNA that move around cell genome Transposition is movement of TEs, jumping gene Target site of TE is not homologous with TE No crossing over TEs can move in two ways Cut-and-paste, original TE leaves Copy-and-paste, original TE stays in place

Why is it important Proteins for recombination, excision and insertion, replication and packaging provide a “molecular toolkit” for genetic engineering