The retinoblastoma gene product, p110
RB1
, appears to regulate cell growth by modulating the activities of nuclear transcription factors. The elements that specify the transport of p110
RB1
into the ...nucleus have not yet been explored. We now report the identification of a basic region, KRSAEGGNPPKPLKKLR, in the C terminus of p110
RB1
, which has sequence similarity to known bipartite nuclear localization signals (NLSs). A two-amino-acid mutation introduced into this putative NLS to give mutant NLS(NQ) or deletion of the entire NLS (δNLS) abrogated exclusive nuclear localization, yielding proteins which were distributed either equally throughout the cell or predominantly in the cytoplasm. A mutant protein NLS(NQ)/δ22 containing both the mutated NLS and a deletion of exon 22, previously shown to disrupt the interaction of p110
RB1
with several cellular transcription factors and oncoproteins, accumulated only in the cytoplasm. When fused to the C terminus of Escherichia coli β-galactosidase, the RB1 NLS directed this protein to the nucleus, indicating that the motif is not only necessary but also sufficient for nuclear transport. Neither NLS(NQ) nor δNLS was hyperphosphorylated in vivo, but both retained their abilities to interact, in vitro, with simian virus 40 large T antigen, adenovirus E1a, and the cellular transcription factor E2F. When transfected at multiple copy number, the NLS mutant alleles displayed reduced biological activity, measured by inhibition of growth of the osteogenic sarcoma cell line Saos-2, which has no wild-type RB1. Naturally occurring mutations and deletions in exon 25 of RB1 which disrupt the NLS may lead to partial or complete inactivation of p110
RB1
and may be responsible for some retinoblastoma and other tumors.
The temperature-sensitive (ts) mouse L-cell mutation, ts A1S9, has previously been shown to affect nuclear DNA replication and to be complemented by active and inactive human X chromosomes. Herein, ...the molecular cloning and characterization of a human gene, denoted A1S9, that complements the ts A1S9 lesion is described. A DNA-mediated gene transfer approach was used to correct the ts A1S9 cell defect with genomic human DNA. Primary, secondary and tertiary temperature-resistant transformants were isolated. These retained a human DNA region encompassing the complementing human A1S9 genomic locus. The transferred human DNA was recovered from a genomic library of a secondary transformant and from a human X chromosome-specific DNA library. A single-copy fragment derived from the A1S9 locus was found, by Northern blot analysis, to contain coding DNA sequences. This DNA segment was used as a probe to identify human cDNA clones of 2.7 kbp, which complemented the ts A1S9 lesion as assessed by transfection and temperature selection. Nucleotide sequence analysis revealed that the A1S9 cDNA encompassed a single open reading frame of 2409 bp which could encode a protein of 90 kilodaltons. Computer-aided search in a protein/DNA data bank did not reveal any sequence with strong homology to the A1S9 gene or its predicted polypeptide. Interspecies Southern blot hybridization detected DNA sequences homologous to the A1S9 gene in various vertebrates, but not in yeast. The A1S9 gene expression was found to be low in quiescent cells. Once the cells were induced to proliferate, the expression increased and was maintained at a constant level throughout the cell cycle. Southern blot analysis of human-rodent hybrids, and in situ hybridization to human metaphase chromosomes, allowed the regional assignment of the A1S9 gene to Xp11.2-p11.4. Northern blot analysis revealed that human cell lines harbouring increasing numbers of inactive X chromosomes (e.g. 47,XXX, 49,XXXXX) express the A1S9 gene at the same level as control cells (45,X), suggesting that A1S9 does not escape X inactivation.
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.
The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts ...AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.
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