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Lecture 15

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Biological Sciences
Mark Fitzpatrick

th BIOA01H3 – Lecture 15 Week of October 1 : DNA, the stuff of heredity Chapter 9 Genetic Recombination  Ultimate source of genetic diversity is mutation of DNA sequence  Since mutations are usually very rare, diversity amplified through various mechanisms that shuffle existing mutations into novel combinations  Cutting and pasting of DNA backbones into new combinations called genetic recombination  Allows “jumping genes” to move, inserts some viruses into chromosome of their hosts, underlies spread of antibiotic resistance among bacteria and Achaea, and is at the heart of meiosis in eukaryotic organisms 9.1 Mechanism of Genetic Recombination In its most general sense, genetic recombination requires the following: o Two DNA molecules that differ from one another o Mechanism for bringing DNA molecules into close proximities o Collection of enzymes to “cut”, “exchange”, and “paste” DNA back together  Two similar double helixes lie close together  Most recombination that occurs in this chapter are between regions of DNA that are very similar, but not identical  Such regions are called homologous  Homology allows different DNA molecules to line up and recombine precisely  Once homologous regions of DNA paired, enzymes break covalent bond in each of four sugar-phosphate backbones  Free ends of each backbone then exchanged and reattached to those of other DNA molecules  Result is two recombined molecules  Cutting and pasting of four DNA backbones results in one recombination event 9.2 Genetic Recombination in Bacteria  Historically first associated w/ meiosis in sexually reproducing eukaryotes 9.2a Genetic Recombination Occurs in E. coli  E. coli can be grown in minimal medium containing water, an organic carbon source such as glucose, and a selection of inorganic salts, including one, such as ammonium chloride, that provides nitrogen  Growth medium can be in liquid form or gel form by adding agar to the liquid medium  In order to detect genetic recombination, need some sort of detectable diff. that could be shown to occur in changing combinations  The diff that proved most useful was related to nutrition  Cells require various amino acids for synthesis of proteins  Strains that are able to synthesize necessary amino acids called prototrophs  Mutant strains that are unable to synthesize amino acids called auxotrophs; only grow if amino acid provided in growth medium  Strain that cannot manufacture own arginine represented by genetic shorthand argA-  argA refers to one of the genes that govern a cell’s ability to synthesize arginine from simple inorganic molecules  given strain of bacteria might carry this gene in normal form, argA+, or its mutant form, argA-  alternative forms of gene called alleles  prokaryotic cells have one circular chromosome that carries one particular allele for each gene  using mutagens such as x-rays or UV light, Lederberg and Tatum isolated two different strains of E. coli carrying distinctive combinations of alleles for various metabolic genes 1  one particular gene could grown only if vitamin biotin and amino acid methionine were added to culture medium  Second mutant strain didn’t need biotin or methionine but could grown only if amino acids leucine and threonine were added along w/ vitamin thiamine  Two multiple-mutant strains of E. coli represented in genetic notation as followed: Strain 1 bio-met-leu+thr+thi+ Strain 2 bio+met+leu-thr-thi-  Lederberg and Tatum mixed about 100million cells of two mutant strains together and placed them on a minimal medium  Several colonies grew, though none of original cells carried all of normal alleles needed for growth  Might be thinking, “They are mutants. Maybe some of originally mutated alleles went back to normal.”  Possibly easily discounted by plating large numbers of cells from original strains onto minimal medium separately  If mutation responsible for initial results w/ mixed cultures, then colonies should have also appeared when strains plated separately. There were none. 9.2b Bacterial Conjugation Brings DNA of Two Cells into Close Proximity  In eukaryotes, genetic recombination occurs in diploid cells by exchange of segments between pairs of chromosomes  In bacteria, that are haploid organisms, each cell typically has own, circular chromosome  Transfer of genetic information is unidirectional, from one donor cell to a recipient cell  Bacteria cells conjugate  cells contact each other by long tubular structure called sex pilus then forms cytoplasmic bridge  During conjugation, copy of part of DNA of one cell moves through cytoplasmic bridge into other cell  Once DNA from one cell enters other, genetic recombo occur  Conjugation facilitates a kind of sexual reproducing in prokaryotic organisms The F Factor and Conjugation  Conjugation initiated by bacterial cell containing small circle of DNA in addition to main circular chromosomal DNA  Small circles are called plasmids but this particular one is called fertility plasmid or F factor  F factor carries several genes as well as replication origin that permits copy to be passed on to each daughter cell during division  example of vertical inheritance  During conjugation, F Factor also has ability to be copied and passed directly from donor cell to recipient  example of horizontal inheritance  Donor cells called F+ cells b/c contain F factor, able to mate w/ recipient cells but not w/ other donor cells  Recipient cells lack F factor and are called F- cells  F factor carries about 20 genes, several encode proteins of sex pilus (F pilus)  During conjugation, F plasmid replicates using special type of DNA replication called rolling circle  Recipient cell becomes F+, but no chromosomal DNA transferred between cells in this process therefore, no genetic recombination occurs between DNA of two different cells in such a mating  So why are we including F factor conjugation if it doesn’t recombine DNA of different cells? Answer lies with Hfr cells  Hfr Cells and Genetic Recombination  In some F+ cells, F factor comes into close proximity with main chromosome & undergoes recombination  When two circular DNA molecules recombine, simply fuse together into one larger circle  F factor becomes part of main bacterial chromosome  Special donor cells called Hfr cells (Hfr = high frequency recombination)  Although recombination event integrated F factor into host chromosome, it occurred in one cell, not between two different cells  Hfr cells called high-frequency recombination b/c can promote recombination between DNA of diff. cells by “exporting” copies of chromosomal genes to another cell as explained as followed: 2  When F factor integrated into bacterial chromosome, genes still available for expression  Therefore, Hfr cells make sex pili & can conjugate w/ F- cell  Segment of F factor moves through conjugation bridge into recipient, bringing single-stranded chromosomal DNA behind it  Again is rolling circle replication, both donor and recipient cells restore DNA to double strandedness  In this situation, circle that rolls is entire Hfr donor chromosome  Recall that when F factor transfer by itself, recipient cells often become F+  In Hfr cells, origin of transfer near middle of integrated F factor  only half of F factor DNA transferred at front of chromosomal DNA  Other half of F factor can follow only after rest of entire chromosome  very unusual for recipient cell to obtain entire F factor & become Hfr as well  Most likely, recipient cell become partial diploid; have two copies of only genes that came through conjugation ridge on donor chromosomal DNA segment ~ Look at textbook page 187 ~ for rest of info Mapping Genes by Conjugation  Discovered by Francis Jacob (proposed operon model for regulation of gene expression in bacteria) and Elie L. Wollman  Began experiments by mating Hfr and F cells that differed in number of alleles  At reg. intervals after conjugation commenced, removed some cells and agitated them in blender to break apart mating pairs  Then cultured separated cells and analyzed them for recombinants  Found that longer allowed cells to conjugate before separation, greater number of donor genes entered recipient and produced recombinants  By noting order & time at which genes transferred, able to map and assign relative positions of several genes in E. coli chromosome 9.2c Transformation and Transduction Provide Additional Sources of DNA for Recombination  DNA can transfer from one bacterial cell to another by two additional mechanisms: transformation and transduction  Like conjugation, transfer DNA in one direction and create partial diploids in which recombination can occur between alleles in homologous DNA regions  Unlike conjugation, transformation and transduction enable recipient cells to recombine w/ DNA obtained from dead donors Transformation  Bacteria simply take up pieces of DNA released into environment as other cells disintegrate  Phenomenon discovered while understanding how bacteria causes pneumonia in mice  Cells of virulent strains of Streptococcus pneumonia surrounded by polysaccharide capsule, whereas nonvirulent cells were not  Griffith found that mixture of heat-killed virulent cells + living nonvirulent cells still caused pneumonia  The substance capable of transforming nonvirulent bacteria to virulent form was DNA  Geneticist established that in transformation of Streptococcus, linear DNA fragments taken up from disrupted virulent cells recombine w/ chromosomal DNA of nonvirulent cells in same as conjugation Transduction  DNA transferred from donor to recipient cells inside head of infecting bacterial virus  Infection cycles of viruses infect bacteria, called bacteriophages  In general, transduction begins when new phages assemble within infected bacterial cell; sometimes incorporate fragments of host cell DNA along with, or instead of, viral DNA  After host cell killed, new phages released may then attach to another cell and inject bacterial DNA into recipient cell  Intro of this DNA makes recipient cell partial diploid and allows recombination to take place 3  Recipients not killed b/c received bacterial DNA rather than infective viral DNA 2 types of transduction, arising from different infection cycles of phage involved: 1) Generalized 2) Specialized 1) Generalized Transduction o All donor genes equally likely to be transferred, associated w/ some virulent bacteriophages, which kill host cells during each cycle of infection (lytic cycle) o During infection by virulent phage, host bacterial chromosome degraded to provide raw material for synthesis of new phage chromosomes o Sometimes fragment of host chromosome avoids degradation & packed into head of new phage by mistake o Particular phage now contains small random sample of bacterial genes instead of phage genes o When host cell burst to release new phage, transducing phage can mechanically infect recipient cell o It will deliver a liner piece of DNA from donor cells rather than infectious phage chromosome o Newly infected recipient cell will survive; incoming DNA may then pair & recombine, with homologous regions on recipient chromosome 2) Specialized Transduction o One of most studied bacteriophages is phage lambda, which infects E. coli o Again, a mistake in infection cycle result in transfer of bacterial genes from donor to recipient cell o In this case, diff type of mistake, in a diff. infection cycles, gives rise to a diff type of transduction (specialized) o Lambda is temperate bacteriophages  when lambda infects new host, it determines whether this cell likely to be robust & long-lived host o Is it starving? Is it suffering from DNA damage? o If host cell passes this molecular health checkup, then lambda chromosome lines up with small region of homology on bacterial chromosome and phage-coded enzyme catalyzes single recombination event o Phage thus integrated into host chromosomal DNA and is called prophage o Prophage then replicated & passed to daughter cells along w/ rest of bacterial chromosome as long as conditions remains favorable (lysogenic cycle) o If host cell becomes inhospitable, prophage activates several genes, releases itself from chromosome by recombination event, and proceeds to manufacture new phages, which are released as cell bursts result of lytic growth o The “mistake” occurs when prophage excised from chromosome o Sometimes this recombination event is imprecise; bacterial DNA removed from host chromosome, some prophage DNA left behind o Result  bacterial DNA packaged into new phages & carried to recipient cells o Since transducing phage defective, having left some of its genes behind in host, does not kill its new host o Only bacterial genes that are close to integration site of phage likely be incorporated into phage chromosome by recombination mistake o Typically, only genes coding for galactose & biotin metabolism transferred at high frequency by phage lambda  Conjugation, transformation, and transduction all ways in which DNA from two diff. bacterial cells bought into close proximity  Homologous regions may then pair & recombine to give rise to recipient cell that carries diff. collection of alleles than previously  These processes create more diversity in DNA sequence among members of pop. than mutation & binary fission alone 9.3 Genetic Recombination in Eukaryotes: Meiosis Sexual reproduction  production of offspring through the union of male and female gametes – i.e. eggs and sperm cells in animals 4  Depends on meiosis, specialized process of cell division that recombines DNA sequences & produces cells w/ half number of chromosomes present in somatic cells  At fertilization, nuclei of egg and sperm cell fuse, producing cell called zygote, in which chromosome # typical of species is restored  W/o halving chromosome number by meiotic divisions, fertilization would double number of chromosomes in each subsequent generation  Both meiosis and fertilization mix genetic info into new combos 9.3a Meiosis Occurs in Different Places in Different Organismal Life Cycles  Gametes are made by meiosis only in animals  Haploid products of meiosis are spores in plants and some fungi Animals  Follows a pattern in which diploid phase dominates life cycles, haploid phase reduced, and meiosis followed direction by gamete formation  In males, each of four nuclei produced by meiosis enclosed in separate cell by cytoplasmic divisions, and each of four cells differentiates into functional sperm cell  In females, only one of four nuclei becomes functional as egg cell nucleus  Fertilization restores diploid phase of life cycle  Thus animals are haploids only as sperm or eggs, and no mitotic divisions occur during haploid phase of life cycle Most Plants and Some Fungi  Organisms alternate between haploid and diploid generations, either generation may dominate & mitotic divisions occur in both phases  Fertilization produces diploid generation  individuals called sporophyt
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