Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication.

Abstract

The temperature‐sensitive ts A1S9 mutation of mouse L cells was previously shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes in human‐ts A1S9 somatic cell hybrids. We report the isolation of cDNA clones which correct the ts A1S9 lesion, using as a probe a genomic fragment derived from the human A1S9 locus. The nucleotide sequence of the A1S9 cDNA encompasses a single open reading frame of 2409 bp which could encode a heretofore unreported protein of 90 393 daltons. Southern blot hybridization of the A1S9 cDNA probe with DNA from various species revealed homologous sequences in vertebrates but not in yeast. Northern blot analysis of serum‐starved, synchronized cells demonstrated that the A1S9 gene was expressed at a relatively low level in quiescent cells and at a higher and constant level throughout the cell cycle. Human cell lines harbouring increasing numbers of inactive X chromosomes (47, XXX, 49, XXXXX) were found to express the A1S9 gene at the same level as control cells (45, X), suggesting that the gene does not escape X chromosome inactivation.

Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme

“…It has been reported that the expression of E1 is higher during the active cell cycle and lower in quiescent cells and in terminally differentiated tissues like muscle (22). This corresponds well to the reduced NER activity that we and others have observed in terminally differentiated tissues (reviewed in ref.…”

Application of microarrays to the analysis of the inactivation status of human X-linked genes expressed in lymphocytes

“…PCTAIRE-1, cdc2-related protein kinase (PCTK1) and ubiquitin-activating enzyme (UBE1) were represented and have previously been reported as escaping inactivation 5,6 although for the latter there have been conflicting reports concerning its inactivation status. 4,44 Our data indicate that UBE1 is expressed at significant levels in lymphocytes and that there appears to be an excess of female transcripts (see Table 3), which may reflect partial escape from inactivation. PCTK1 also shows a slight excess of female transcripts; however, as for UBE1, the standard deviation intervals overlap and the interpretation concerning their inactivation status remains tentative.…”

Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.

“…Thus, either the requisite enzymes are available at these varied subcellular sites or ubiquitin-protein conjugates generated within the cytoplasm are subsequently targeted to their ultimate site or both. Interestingly, native El is -220 kDa (subunit equals 110 kDa) and contains a highly charged putative nuclear localization sequence near its amino terminus (6,7,9). Eukaryotic cells harboring temperaturesensitive mutations in El display a cell-cycle arrest phenotype, suggesting putative roles for El within the nucleus (see refs.…”

“…Recently, we (6) and others (7)(8)(9) have cloned and sequenced the cDNA for El from human, yeast, and wheat. A single ubiquitous El mRNA of 3.5 kilobases (kb) predicts a mature protein of =110 kDa.…”