The goal of this proposal is to utilize homologous recombination strategies to generate defined interstitial and terminal deletions of individual human chromosomes. A nested series of interstitial and terminal deletions would aid in generating deletions in the human X-chromosome and using them to map several markers on this chromosome. Interstitial deletions will be generated first in YACs containing human DNA. A recombination vector containing a fragment of human clotting factor IX (F9) at one end and a human repetitive element (LINE 1) at the other with selectable markers in between (HIS3 for yeast and neo for mammalian cells) will be introduced into yeast containing a 650 kb YAC which has the complete F9 gene. Homologous recombination (HR) between the vector and its target sequences in the YAC would result in deletions extending from the F9 to each of several L1 sequences located on the chromosome. Similar experiments will be conducted in rodent-human somatic cell hybrids containing an intact human X-chromosome or part of it translocated onto the tip of a rodent chromosome. HR events will be isolated by selection for G418-resistant colonies followed by screening by the polymerase chain reaction (PCR). Terminal deletions in the human X-chromosome will be generated by targeting the L1 repeat elements with a vector containing a dominant selectable marker and human telomeric sequences. Homologous recombination of the introduced DNA with each of its homologous sequences on the X-chromosome will yield a nested series of terminal deletions. The deletions that are generated will be characterized at the molecular as well as cytological level. An ordered set of deletions of increasing size will be used to map markers on the X-chromosome. The map that is generated will be compared to existing maps. If successful, this strategy could be employed to generate similar deletions in any human chromosome.
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