Yeast artificial chromosomes with 200- to 800-kilobase inserts of human DNA containing HLA, V kappa, 5S, and Xq24-Xq28 sequences

Abstract

Sequences hybridizing to several human gene probes have been recovered as cloned inserts in yeast artificial chromosomes (YACs). Among 2300 YACs made from human leukocyte DNA (totaling about 0.1 genomic equivalent of human DNA) we have found two, 200 and 780 kilobases (kb), containing sequences of V kappa I immunoglobulin (V = variable); one, 240 kb, with class I HLA; and 11, 200-800 kb, with 5S rRNA-encoding DNA (rDNA). Fifty human YACs from a hamster-human cell hybrid with only the Xq24-Xq28 portion of the X chromosome include one that contains two anonymous probe sequences, DX13 and St14, previously inferred by indirect means to lie within about 70 kb of one another in Xq28. The YACs specific for human DNA arise at a frequency equivalent to the fraction of cellular DNA that is human-specific. Furthermore, the human YACs, formed in a 280-fold excess of hamster DNA, do not hybridize to a hamster DNA probe, indicating that individual YACs do not contain a combination of human and hamster DNA. To confirm that sequences are not scrambled, the YACs containing V kappa I or DX13 and St14 sequences were shown to produce restriction fragments identical in mobility to fragments detected by the same probes in total human DNA digested with the same enzymes. YACs may therefore provide large clones to bridge gene mapping at the chromosome level to molecular analyses of small fragments of genomic DNA.