Molecular cloning of human A1S9 locus: An X-linked gene essential for progression through S phase of the cell cycle

Abstract

The temperature-sensitive (ts) A1S9 mouse L-cell mutant is defective in an X-linked gene essential for the progression of cells through the S phase of the cell duplication cycle. We recently reported the complementation of the ts A1S9 cell defect with total human DNA and the isolation of independent temperature-resistant transformants that retained a common set of human specific Alu-containing fragments. Here we describe the molecular cloning of these human DNA sequences from one of the secondary transformants. ST-1-0. A genomic library prepared from ST-1-0 was screened with a total human DNA probe, and two recombinant bacteriophages carrying overlapping segments were isolated. The cloned region was extended in both directions using a human X-chromosome specific library. In total, a human region spanning 42 kb in length, and containing all the Alu-specific DNA sequences found in ST-1-0, was isolated in five overlapping recombinant phages. The A1S9 gene appeared to be larger than the DNA recovered in individual phage isolates, as was assessed by transfection experiments. A single-copy probe derived from the phage DNA was shown to be conserved in independent primary, secondary, and tertiary transformants of ts A1S9 cells and mapped to the X chromosome by molecular hybridization. Northern blot hybridization of this probe with poly(A)+ mRNA derived from ST-1-0 cells identified a transcript of about 3.6 kb.

A gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme.

“…At the amino acid level, the translated D8 gene shows a 45.3% identity to the human UBA1 gene, which encodes the ubiquitin-activating enzyme El (13). The UBAI gene appears to be identical to the AIS9 gene, a human gene that corrects a defect in DNA replication in the tsAlS9 mouse cell cycle mutant (11,12). An independently isolated mouse cell mutant with a similar phenotype has been shown to have a thermolabile El ubiquitin-activating enzyme (17,18).…”

“…This kb (12); it is therefore incomplete, presumably at its 5' end. screening revealed two entries with an z45% identity in the The other cDNA sequence, which also identifies a 3.5-kb parts overlapping with the D8 gene.…”

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.

“…One of these clones, denoted pcAlS9-H2, and p6-1 were aligned with respect to the genomic A1S9 locus as shown in Figure 1. This was accomplished by a combination of genomic Southern blots, dot blots and hybridization with five recombinant phages containing DNA inserts that span the entire A1S9 locus (Zacksenhaus and Sheinin, 1989). In this way it was demonstrated that the 3' end of the cDNA hybridized with the E2.2 genomic fragment and contained an EcoRI site found in the latter fragment as indicated in Figure 1.…”

“…The entire locus was recovered from a genomic library prepared from a secondary transformant. A single copy fragment, designated E2.2, derived from the A1S9 locus, was found to be conserved in independent transformants and to identify a transcript of an estimated 3.5 kb in length on Northern blots (Zacksenhaus and Sheinin, 1989). Using the E2.2 probe, the A1S9 gene has been localized to the centromeric region on the short arm of the X chromosome at Xpl 1.2-p1 1.4, and shown not to share homology with the Y chromosome (Zacksenhaus et al, 1990).…”

Sex chromosome evolution: platypus gene mapping suggests that part of the human X chromosome was originally autosomal.

“…escape inactivation (32)(33)(34)(35)(36) suggest that the process is still Ipials was initially exincomplete in some regions of the human X chromosome, am the ancestral maiperhaps because these genes lack sequences that respond to aller marsupial X chroa signal spreading from an X inactivation center. The obserriabsence also from the vation that some genes that escape inactivation in humans are ficult to explain on this inactivated in mouse suggests that the inactivation system verged earlier from the may be more completely evolved in mouse (31 …”