HUMAN DISTAL EQ DNA--ANALYSIS WITH YACS

对人类细胞遗传学部分DNA的系统分析

Systematic analysis of DNA from portions of the human cytogenetic bands Xq24-Xq28 is proposed, based on the use of yeast artificial chromosomes (YACs). Some genes are included in which lesions cause monogenic disease. Those genes could be isolable in intact form, providing long-term potential for medical applications. The study could also be a pilot project for the complete analysis of a chromosomal region. To initiate the work a test library of total human DNA in YACs has been developed to be screened for clones of interest. A second library is being organized from a hamster-human somatic cell hybrid in which the Xq24-Xq28 region is the only known human DNA; that library now contains 10 Mb of human-specific DNA in 60 clones, and will be expanded to l-hit (30 Mb) by the time the project tenure would begin. In the library from the hybrid, clones containing human DNA occur with a frequency equal to their fraction of the cellular DNA (1/250), and have already yielded two YACs which hybridize to a single Xq28 probe; those two clones overlap in their restriction patterns, suggesting that the YACs contain unrearranged DNA segments. A number of available probes will be used to screen the two libraries, and subsidiary mapping techniques will be used to search for overlaps. Contigs will be particularly sought for the MN12-st14 and glucose-6-phosphate dehydrogenase (G6PD)-Factory VIII-color blindness regions of Xq28. Selected regions will be analyzed for the locations of transcribed and of certain repetitive sequences, by a combination of restriction site mapping and screening of cDNA libraries. Finally, an intact form of the G6PD gene will be sought in order to assess the requirements for expression and mRNA processing in transfected cells.