Somatic Cell and Molecular Genetics最新文献

Gene targeting can be used for genetic studies of human cell lines and has significant potential for somatic cell gene therapy. These applications are however restricted by the low frequency of homologous recombination in higher eukaryotes compared to the relatively efficient nonhomologous integration of transfected DNA into the genome. As part of our attempts to overcome this problem, we compared two widely used transfection methods for their efficiency in gene targeting. To our surprise we found that, for conditions that render similar frequencies of nonhomologous integrants, lipofection is much less efficient than electroporation in generating targeted clones. This suggests that nonhomologous and homologous recombination have different requirements for DNA delivery in human cells.

Tc1/mariner transposons are widespread in the eukaryotes. In vitro transposition test indicated that the transposase is the only protein that is needed in transpositions. It was shown later that the reconstructed Tc1-like transposon, "sleeping beauty" in fish, and the Tc1 transposon in C. elegans jumps in human cells. This discovery indicates that the Tc1/mariner transposon may be engineered as a somatic gene therapy vector if coupled with an efficient gene delivery system. We introduced the Tc1 transposon from C. elegans into different mammalian cell lines and detected the transposition events, indicating that Tc1 transposon functions in different mammalian cells. Interestingly, a high inversion frequency of the transposon was also detected, suggesting that this type of transposon may add variations to host genome when it is horizontally transferred into a new species.

Glutathione synthesis, a vital cellular process, depends on L-cystine uptake by the amino acid transporter, System x-C. Here we show that a second transporter, System X-AG, is required for normal System x-C activity and glutathione maintenance by employing somatic cell mutants of CHO-K1. Uptake by System x-C in two X-AG-null mutants is significantly lower than that of CHO-K1, either under control conditions or after prolonged treatment with an electrophile. In addition, levels of glutathione in control and treated mutant cells are less than half those of wild-type CHO-K1 or of a pseudorevertant. The significance of this reduction was tested by chemical challenge: mutants are twofold more sensitive than wild type to reactive oxygen species generated by phenylbenzoquinone and to damage produced by the anticancer drug, cisplatin. These results suggest that System X-AG provides a significant portion of the glutamate used to energize the uptake of cystine required for the synthesis of glutathione.

Extracellular phosphate concentrations are maintained by coordinated regulation of specific homeostatic mechanisms. A novel gene, the type IIb sodium-phosphate cotransporter (Npt2b), was recently cloned and is expressed within intestinal tissues, indicating that the transporter may be an important regulator of phosphate reabsorption. Another gene, human stanniocalcin-2 (STC2), was previously shown to decrease phosphate uptake into kidney cells in vitro. Because of the important role that STC2 may play in phosphate homeostasis, we considered the peptide hormone a candidate for the phosphate wasting disease autosomal dominant hypophosphatemic rickets (ADHR), previously localized to chromosome 12p13. The purpose of our study was to determine the chromosomal localization of human NPT2b and STC2. In the present work, NPT2b was localized to human chromosome 4p15-p16, and STC2 to 5q33-tel. Because STC2 did not map to 12p13, the hormone was excluded as the ADHR gene, however it should be considered a candidate for other diseases involving phosphate homeostasis.

The chromo multigene superfamily encodes numerous proteins involved in chromatin structure or organization. The prototypical member of this superfamily is HP1 of Drosophila melanogaster. We now present the sequence of a human HP1Hs gamma pseudogene and assign it to chromosome 11p14 by radiation hybrid mapping. The coding regions of at least three other human genes for HP1 orthologues and another pseudogene are very similar in sequence. These results demonstrate that HP1-type sequences have been duplicated multiple times in the mammalian genome.

Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.

We have isolated and characterized the immediate 5'-flanking region (886 bp) of the gene encoding human HOXA-7. When the total sequence was compared with those of mice, 93% of the 3' 518 bp (nt 370-886) sequences were identical, in which the 245 bases just preceding the AUG initiator codon (nt 614) was as highly conserved as in the coding region (nt 614-886). Sequences further upstream (nt 1-370) by comparison were highly diverged. In the 245 bp region, 8 stop and 3 initiation (including the initiator) codons were located, and a 50-aa long presumptive polypeptide was encoded. Nucleotide sequence analysis revealed three Sp1 and one AP2 binding sites, as well as one CAAT box. However, there was no consensus sequence for a TATA box in the 5' flanking region. One RARE repeat, one krox20 and three Hox-PBC binding sites were detected. Since many of the factor recognition sites were located in the immediate 5' flanking sequences of a highly-conserved region, it might be speculated that a regulatory mechanism for Hox gene expression is conserved throughout the evolution and one possible mechanism could be at the post-translational level.

Rat embryo fibroblasts (REFs) are inefficiently transformed by RAS-oncogenes. Induction of p16INK4A expression by RAS has been suggested to contribute to this resistance. Glucocorticoid hormones, (DEX), enhance REF transformation by RAS and facilitates the isolation of transformed and immortal cell lines. We show that DEX induced cell proliferation is paralleled by a decrease in Cdkn2a gene transcripts, suggesting a mechanism for hormone promotion. The mechanisms of progression into hormone independent cell lines were examined. Twenty-two of 30 clones which reached a population size of approximately 10(6) cells could be established as cell lines. All lines studied showed homozygous deletions of the Cdkn2 loci (Cdkn2a and Cdkn2b) on RNO5. LOH was found for all RNO5 genetic markers examined in 7 of 19 cell lines, suggesting non-disjunction events. In the remaining 12 cell lines, both copies of Cdkn2 appeared to be lost by deletions/rearrangements, some of which could by demonstrated by karyotype analysis. We conclude that (i) clonal expansion of RAS-transfected REF by DEX is paralleled by down-regulation of Cdkn2a expression; (ii) homozygous deletion of Cdkn2 were estimated to occur at a frequency of 2 x 10(-8)/cell/generation or higher, and (iii) deletion/rearrangements and nondisjunction appear to be the main mechanisms leading to deletion of Cdkn2.

The hASNA-I is a novel human arsenite-stimulated ATPase identified as the human paralogue of the ATPase component of the arsenite efflux system in E. coli. The hASNA-I has distinct biochemical properties and a dual nuclear and cytoplasmic distribution. Immunohistochemical staining showed a distinct pattern of hASNA-I expression in cells within normal tissues, and its overexpression in breast cancer. Recently, the yeast two-hybrid system has identified hASNA-I as a cellular partner of metallothionein II suggesting an additional role in Zn homeostasis and cellular detoxification. This report describes the assignment of hASNA-I to human chromosome 19 by somatic-cell hybrid PCR mapping, the isolation of a chromosome 19-specific cosmid clone, and the genomic structure and exon-intron boundaries of hASNA-I. Our results indicate that the coding region of hASNA-I consists of 4 exons spanning 6 kb on band 19q13.3. These data will facilitate molecular analysis of the role of hASNA-I in human disease.

We purified to near homogeneity a previously identified 100 kDa mammalian homologous DNA pairing protein. The purified 100 kDa protein also catalyzed high levels of cell-free homologous DNA recombination activity. This ATP-dependent activity was capable of forming conservative recombinant products between two circular, double-stranded DNA molecules. We were unable to detect any DNA polymerase, DNA ligase, or 5' or 3' exonuclease activity associated with this purified material. The purified 100 kDa protein bound silver nitrate as well as a monoclonal antibody specific for nucleolin. A recombinant protein comprised of the Escherichia coli maltos-ebinding protein fused to the carboxyl-terminal two-thirds of human nucleolin possessed homologous DNA pairing activity. These data indicate that the 100 kDa homologous DNA pairing protein is nucleolin. The observation that nucleolin can carry out homologous DNA strand pairing in vitro raises the prospect that it may function similarly in vivo.