UC Irvine

To investigate the molecular basis of dosage compensation in Drosophila, a recombinant lambda phage containing the Drosophila melanogaster glucose-6-phosphatase dehydrogenase (G6PD) gene was isolated by differential screening of a Drosophila genomic lambda library with poly(A)+RNA obtained from polyribosomes enriched for or depleted of G6PD mRNA sequences. Of 44 000 plaques screened, a single phage, lambda DmG21, showed hybridization with the enriched poly(A)+RNA but not the depleted one. Confirmation that the Drosophila DNA fragment cloned in lambda DmG21 contains the G6PD gene sequence is based on the following observations. lambda DmG21 DNA shows hybridization only to the 18D region of the salivary gland X-chromosome, which is the known cytological locus for the G6PD gene. In vitro translation of the poly(A)+mRNA selected by hybridization to lambda DmG21 DNA sequences shows a polypeptide product of apparent Mr 55 000, identical to that of the monomeric unit of G6PD. When the putative coding sequence of G6PD is cloned into the expression vector lambda gt11, recombinant plaques are recognized by anti-G6PD immunoglobulin. A transcriptional map of the G6PD gene shows that it is divided into two exons, 0.9 kb (exon I) and 1.8 kb (exon II) long, which are separated by a 2.4-kb intron. The G6PD mRNA is 2.0 kb in length and the steady-state level of the mRNA is similar in both sexes. Measurement of the copy number of the G6PD gene in males and females shows the gene to be present once per X-chromosome in both sexes. No amplification of the gene sequence was observed in males. These results are, therefore, in agreement with the previous suggestion that dosage compensation is the result of enhanced transcription of X-linked genes in males.