Mapping Minimally Reiterated Genes on Diploid Chromosomes by in Situ Hybridization

150 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.A general method for the localization of minimally reiterated genes on diploid chromosomes by in situ hybridization was developed. To obtain a detectable autoradiographic signal, carrier-free ('125)I-CTP prepared by the method of Ward (unpublished), and was incorporated into complementary RNA (cRNA) probes by in vitro transcription of recombinant plasmids containing eucaryotic gene sequences. ('125)I-cRNA probes were labeled to high specific radioactivity (3.5 x 10('9) dpm/ug); sufficient to map single gene copies.To test the specificity and sensitivity of hybridization in situ, ('125)I-cRNA, prepared by transcription of a recombinant plasmid containing the Drosophila 5S RNA genes, was hybridized to polytene and diploid chromosomes. This model system was chosen because the chromosomal location of the 5S RNA genes was known and the kinetics of hybridization in situ had been characterized. The ('125)I-cRNA probe was specific for the 5S RNA gene loci. No other sites were labeled on either polytene or diploid chromosomes. The rate of appearance of silver-grains over the known genetic loci indicated that the sensitivity of the ('125)I-cRNA probe.To test the resolution of in situ gene localization, the chromosomal sites of integration of SV40 DNA sequences were mapped in two virus-transformed human-mouse hybrid somatic cell lines. These hybrids contain the entire mouse chromosome complement but only human chromosome 7, to which the integrated viral genes had been assigned. The SV40 DNA sequences integrated into the DNA of these two cell lines was characterized by filter hybridization which demonstrated that the two clones were derived from independently-transformed human cells in which th viral DNA had become integrated at two different sites on chromosome 7. ('125)I-cRNA, prepared using purified SV40 DNA as template, was hybridized to chromosomes of both cell lines to distinguish these two sites cytologically. The viral integration sites mapped to the same position on chromosome 7 (q31) in each hybrid line. It was concluded from these data that minimally reiterated gene sequences could be localized on diploid chromosomes with a precision of one silver-grain diameter (the equivalent of about 6.2 x 10('7) nucleotide pairs in the human genome). The significance of this work is that it permits the localization of essentially any gene which can be cloned in a recombinant DNA vector.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD