LECTURE 7 RECOMBINATION MECHANISMS Reading Ch 5 p 121 122 p 137 138 Ch 6 p 178 185 Problems Ch 6 6 21 6 22 6 24 First we ll cover ordered octads from last lecture s notes Then we ll review data from breeding experiments that established the properties of recombination and cover one molecular model for recombination Breeding experiments demonstrate that recombination is caused by a physical exchange between homologous chromosomes In 1931 Creighton and McClintock working with maize and Stern working with Drosophila demonstrated that reciprocal exchanges between homologous chromosomes are the physical basis of recombination Both groups followed chromosomes that were physically marked with cytologically visible abnormalities so that a maternally derived homolog could be easily distinguished from the paternal one The marked chromosomes also carried mutations in order to monitor which progeny were the result of recombination We ll cover Creighton and McClintock s experiment in lecture Creighton and McClintock used two different forms of Chromosome 9 in their experiment One form was normal and the other had two cytological abnormalities a heterochromatic knob at one end and a piece of chromosome 8 fused at the other end a translocation which you ll hear more about later in the course In addition to these physical differences these two chromosomes were genetically marked to detect recombination events One marker gene controlled kernel color C colored c colorless and the other controlled kernal starch metabolism Wx starchy wx waxy They set up the following cross knob translocation C Wx c wx x c wx c wx All the parental types resulting from this cross retained the parental type chromosomal cytology The chromosomes of the recombinant progeny C wx and c Wx were then examined Creighton and McClintock showed that each recombinant carried only one of the cytological abnormalities C wx and c Wx indicating that physical exchange between the cytologically marked and normal chromosome 9 homologs had occurred in the previous generation Creighton and McClintock s paper ended with the statement The foregoing evidence points to the fact that cytological crossing over occurs and is accompanied by the expected types of genetic cross over DNA molecules break and rejoin during recombination When viewed through a high powered microscope recombinant chromosomes with cytologically visible markers appear to result from two homologous chromosomes breaking and exchanging parts during recombination To test this hypothesis Meselson and Weigle infected bacteria with two different viruses that were genetically marked They had grown the viruses in heavy 13C and 15N and light 12C and 14N isotopes of carbon and nitrogen in order to differentially label the viral DNA They infected a bacterial cell with both viruses under conditions that prevented viral replication After allowing time for recombination and repackaging of viral DNA into particles they collected the viral particles and analyzed them on a density gradient Non recombinant viruses were found at the extreme ends of the gradient representing all heavy and all light compositions Recombinant viruses were found at intermediate parts of the density gradient The density of the recombinant virus depended upon the position of the recombination event if a recombinant derives most of its alleles from the heavy chromosome it was found closer to the heavy region of the gradient vice versa for the light chromosome A B D HEAVY a b d LIGHT Non recombinants ABD heavy and abd light Recombinants abD ABd aBD Abd AbD aBd etc were found at intermediate regions in the density gradient depending upon the fraction of each of the parental chromosome it contains Gene conversion is the unidirectional transfer of genetic information Gene conversion is any deviation from the expected 2 2 segregation of parental alleles Let s consider an example in the yeast S cerevisiae A diploid yeast cell is heterozygous at the gene B B b When induced to sporulate the resulting tetrads usually segregate 2 2 for the parental alleles Occasionally a tetrad results that doesn t share this segregation pattern but instead segregate 3 1 These tetrads cannot be classified as PD NPD or T The deviations from the expected are the result of gene conversion at the B locus In Neurospora 6 2 2 6 5 3 3 5 and 3 1 1 3 aberrant 4 4 gene conversion octads can be observed The latter half chromatid conversion octads can only be explained if the two strands of the DNA double helix carry information for two different alleles heteroduplex DNA at the end of meiosis Consider a 5 3 octad m m m The box indicates a pair of spores that arose by a mitotic division Remember that an octad contains four pairs of cells and that each pair of cells should be genetically identical Thus two strands of double helical DNA on the chromatid of the haploid cell that gives rise to these nonidentical daughters must have contained information for both alleles of the gene one for the allele and one for the m allele after meiosis In 6 2 and 2 6 octads the mismatch in heteroduplex DNA is repaired prior to mitosis Gene conversion is not explained by mutation since the allele that is converted always changes to the other allele segregating in the cross not to a new allele Gene conversion is associated with recombination of flanking markers What else happens when gene conversion happens on a chromosome Consider that gene B above is tightly linked to genes A and C If a diploid cell ABC abc is induced to sporulate then the majority of the progeny are PDs ABC ABC abc abc However gene conversion does occur at low levels giving rise to the following combinations of alleles when the tetrads are examined 1 2 3 4 ABC ABC ABC ABC ABC ABc AbC abC aBc aBC abc Abc abc abc abc abc Half of the tetrads that exhibit gene conversion at B also show recombination of flanking markers A and C and the other half do not show recombination It is only among the subset of tetrads that experienced gene conversion at B that the recombination frequency of the tightly linked A and C genes is that high Any model that was proposed for recombination must satisfy the following points 1 Chromosomes physically break exchange parts and rejoin 2 Reciprocal products are created 3 Recombination can occur anywhere along the DNA 4 Recombination is precise mutations are not induced during the process 5 Gene conversion can give rise to unequal segregation of two different alleles About 50 of gene
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