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York University
BIOL 3110
Michael Gadsden

Cloning a Gene and Selection by Complementation of a Mutation Binary Fission Animation: http://highered.mcgraw- animation_-_cell_division.html E .coli and strains There is not one type of E.coli genetically. It has been altered from its wildtype form significantly and often. K12 is considered to be one of the wild type strains and is lysogenic for Lambda phage and has the F’ plasmid. The F factor or episome is an extrachromosomal DNA that is found in some cells and can be passed on by conjugation to other cells. The plasmids we use in the lab are not like this and have been created by scientists to perform various functions. Genotypes There are many different strains of E. coli – mostly lab generated. Below is the genotype for one highly used strain called JM109: + endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB Δ(lac-proAB) e14- [F' traD36 proAB lacI lacZΔM15] hsdR17(r m ) K- K+ Notes on this genotype • endA1 = endonuclease A1 deficient • Δ (delta) means that this genetic location/mutation has a delection • F’ refers to the natural fertility plasmid • tra genes are involved in DNA transfer during conjugation • hsd genes are involved in the restriction/methylation system • lacZ you should be familiar with Another strain LE392: LE392: glnV44 supF58 (lacY1 or ΔlacZY) galK2 galT22 metB1 trpR55 hsdR514(r m ) - + K K See the “E. coli Genotype Description” document on the course site as well. K-12 Genome This is the Chromosome of the E. coli strain K-12. It is closest to the originally isolated strain (in 1922) compared with the spectrum of strains in labs today. They were mostly derived from this strain or the B strain. Some Features of the E. coli Chromosome Many genes encoding enzymes of a single biochemical pathway are clustered into operons Operons equally distributed on both strands ~ 5 Mbp in size ~ 40% of predicted proteins are of unknown function Average protein contains ~ 300 amino acids Insertion sequences (IS elements) present => mobile genetic sequences The Formation of Hfr Strains and Chromosome Mobilization • Plasmids are extrachromosomal circular DNA that is much smaller than the Chromosome. They are NOT necessary to cell survival and thus most cells lack plasmids UNLESS thay carry a gene that gives them better fitness (ie an ampicillin resistance gene (bla =β-lactamase) in the presence of ampicillin) • Only found in SOME bacteria and Some yeast naturally. • F plasmid is an episome; can integrate into host chromosome • Cells possessing a non-integrated F plasmid are called F+ • Cells possessing an integrated F plasmid are called Hfr (high frequency of recombination) – High rates of genetic recombination between genes on the donor chromosome and those of the recipient Presence of the F plasmid results in alterations in cellproperties •Ability to synthesize F pilus •Mobilization of DNA for transfer to another cell •Alteration of surface receptors so that cell can no longer act as a recipient in conjugation The Formation of Hfr Strains and Chromosome Mobilization • Insertion sequences (mobile elements) are present in both the F plasmid and E. coli chromosome – Facilitate homologous recombination – requires HOMOLOGOUS ends (yellow) – same DNA sequence on donor and target. F plasmid is integrated into the genome by this mechanism. It can also be excised by the reverse process. Genetic Map of the F (Fertility) Plasmid of E. coli IS = Insertion sequence (see next slide). Tra region are a cluster of tra genes that allow transfer of the plasmids to other cells – makes a pilus and transfers DNA through it. F′ plasmids Previously integrated (in the genome) F plasmids that have excised and captured some chromosomal genes (thus F plasmid plus some chromosomal sequences). Figure 11.19 Transposable elements • In Bacteria: – Insertion sequences (IS) – Transposons – Certain bacteriophages • Contain transposase gene – required to cut and ligate DNA from a donor DNA to a target. Most also have inverted terminal DNA sequence repeats at ends of DNA. ie 5’ GATCA_____________________TGATC 3’ 3’ CTAGT_____________________ACTAG 5’ Insertion sequences –Simplest transposable element. –Carry only genes necessary for mobility (tnp = transposase). Transposons carry additional genes (e.g. resistance). NOTE: Some Mobile elements have inverted repeats at each end while others have Direct repeats. LINE’s lack LTR’s but encode a Reverse Transcriptase that is highly specific for LINE mRNA. SINE’s (~500bp) lack LTR’s and do not encode any genes – they are transcribed and then reverse transcribed by LINE Rtase. Table 5-3 Molecular Biology of the Cell (© Garland Science 2008) Note – the Insertion Sequence (element) doesn’t have “extra” genes other than those necessary to move the DNA (transposase). The Transposons have other genes – in this example genes for antibiotic resistance. This is one important method used by bacteria to pass antibiotic resistance throughout a population. Figure 5-68 Molecular Biology of the Cell (© Garland Science 2008) Basic Methods of Tra
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