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1.
Somatic cell hybridization techniques have allowed the preparation of interspecies hybrids that express the features of both parental cell lines. We have studied hybrids made with human myeloma cells fused to a continuous mouse myeloma cell line. In the present study we analyzed the kinetics of leucine influx and efflux in Ig producer and nonproducer hybrids. We found no statistical difference in amino acid influx; however, the rates of efflux were markedly increased in nonproducer hybrids as compared to the producers. The producer cells were tested further in puromycin known to inhibit protein synthesis. Under these conditions amino acid influx was not altered, but efflux was markedly increased resembling the findings in nonproducers. We conclude that hybrids that synthesize human immunoglobulins show decreased efflux of labeled leucine and this effect can be abolished by inhibition of protein synthesis. This difference in the efflux rate appears to be a consequence of immunoglobulin synthesis, rather than a component of a control mechanism of Ig synthesis. 相似文献
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Differences between the centromeric regions of mouse and human chromosomes have been revealed with a staining technique which is based on the quenching by 5-bromodeoxyuridine of the fluorescence of the dye 33258 Hoechst. The differences can be used to distinguish between human and mouse chromosomes in human-mouse hybrids. 相似文献
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The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes. 相似文献
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The mouse embryonal carcinoma (EC) line, PCC4, was used to construct a series of somatic cell hybrids which contain a single or a few human chromosomes. The hybrids all retained the EC phenotype as determined by morphology, expression of SSEA-1, lack of cell surface H-2 antigen and cytokeratin filaments, high alkaline phosphatase levels, the ability to form EC tumors ectopically in nude mice, and the ability to differentiate in response to retinoic acid. Constitutively differentiated cloned lines were derived from retinoic acid-treated hybrid cultures. Several derived lines had a phenotype indistinguishable from that of parietal endoderm cells, which includes synthesis of large amounts of laminin, type IV procollagen, and plasminogen activator. One differentiated line showed a fibroblast-like morphology. The differentiated lines derived from two of the hybrids, MCP6 and GEOC4, stably maintained the sole human chromosomal component present in the EC progenitors. These EC hybrids therefore provide a system to study developmental regulation of the introduced and stably maintained human genetic material derived from a variety of cell types. 相似文献
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The terminal phase of DNA replication was studied by autoradiography in hybrids between human lymphocytes and mouse fibroblasts. The hybrids contained on the average only 11 human chromosomes. It was found that the sequence of terminal DNA replication for the human chromosomes in the hybrids was the same as the sequence of terminal replication for the corresponding chromosomes in the human lymphocytes. Furthermore, it was shown that the maintenance of the normal terminal replication sequence of the human chromosomes in the hybrids was not dependent on the presence of any specific human chromosome. The results suggest that the timing of terminal DNA replication is determined independently in each human chromosome. 相似文献
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Fragile X expression was studied in human-mouse cell hybrids prepared from lymphocytes and fibroblasts obtained from a mentally retarded male. The patient showed a fragile X in 29-35.5% of his lymphocytes in medium 199 (M199) and in M199 plus fluorodeoxyuridine (FdU). One lymphocyte hybrid clone showed no expression in M199 and low expression in M199 + FdU. The other lymphocyte hybrid clone showed significantly increased expression in both media, comparable to levels in the parental cells. Fibroblast cultures from the patient showed no fragile X expression in M199 and 17% expression in M199 + FdU. Fragile X expression was also found in fibroblast hybrid clones in M199 and was significantly enhanced by the addition of FdU. Fragile X expression in one clone was consistently lower than in the other two clones and in the parental fibroblasts. Our results indicate that the level of fragile X expression varies in the hybrid clones, since frequencies similar to those of parental cells and suppressed frequencies were found. The presence or absence of a specific human chromosome did not correlate with the level of fragile X expression. 相似文献
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The recombination of alkylated H and L chains of a human myeloma protein (Jo) was studied by means of circular dichroism (CD). Marked CD changes were observed at 295 and 235 nm when H and L chains recombined. The change in the CD maximum at 235 nm was followed with time after mixing preparations of H and L chains in the pH range between 4 and 6. The recombination reaction was slow and followed second order kinetics. The observed rate constants were markedly dependent on pH. The pH dependence of the rate constant was analyzed assuming that there are two forms of H chain which are in a pH-dependent equilibrium with each other. 相似文献
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Activity of multiple light chain genes in murine myeloma cells producing a single, functional light chain 总被引:42,自引:0,他引:42
Two cloned lambda 1-producing myelomas (HOPC-1, MOPC-104E) contain rearranged kappa genes and levels of mature-sized kappa RNA comparable to those found in kappa-producing myeloma cells. Another lambda 1-producing myeloma tumor line (HOPC-2020) and a lambda 1-containing B cell leukemia line (BCL1) also contain significant levels of kappa RNA. One lambda 11-producing line (MOPC-315) contains no detectable kappa RNA, but it also has no kappa genes in the embryonic configuration. kappa-related proteins are not detectable in the lambda 1-producing lines by standard procedures, but by sensitive methods at least two lines contain kappa protein fragments. The MOPC-104E line produces both a 14.5K kappa fragment that is not readily detectable because of its low rate of synthesis and short half-life (T 1/2 less than 5 min), and a major 16.5K protein that lacks kappa cross reactivity but is demonstrable by translation of purified MOPC-104E kappa RNA. The HOPC-1 kappa RNA also encodes a short-lived 14K kappa fragment. The MPC-11 line, which produces a mature kappa RNA and protein as well as an 800 base kappa fragment RNA and kappa protein fragment, has both kappa alleles rearranged, one apparently aberrantly between J and C kappa. Two different kappa RNA species, one the same size as the MPC-11 kappa fragment RNA, frequently are present in kappa RNA-containing Abelson murine leukemia virus-transformed lymphoid cells as well as in 18 and 19 day murine fetal liver. For light chains, neither allelic nor isotype exclusion is generally evident in myeloma and lymphoma cells; rather both produce only a single functional light chain. Models of light chain activation must explain restriction by considering the functional properties of the light chain rather than light chain gene expression. 相似文献
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W. Michael Kuehl Barry A. Kaplan Matthew D. Scharff Marion Nau Tasuku Honjo Philip Leder 《Cell》1975,5(2):139-147
Cultured MPC 11 mouse myeloma cells synthesize not only γ2b heavy and κ light chains but also a carboxyl terminal (constant region) fragment of κ light chain. In vitro translational analysis of total cytoplasmic and microsomal RNA indicates that these cells contain RNA which directs synthesis of both a light chain precursor and a light chain fragment precursor. Variant clones which do not synthesize either heavy or light chains continue to synthesize the light chain fragment. One such “nonproducing” variant was studied in detail. It does not contain translatable mRNA for the intact light chain but does contain RNA which is translated into the light chain fragment precursor. Nucleic acid hybridization analysis with a cDNA probe specific for the constant region of κ light chains revealed that microsomal RNA from the wild-type cell contains both a 14S and a 10S species of κ specific RNA, whereas the variant contains only the 10S species. Translational analysis of these same RNAs indicates that the 14S species codes for the light chain precursor, while the 10S RNA codes for the light chain fragment precursor. 相似文献
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Thomas B. Shows 《Biochemical genetics》1972,7(3-4):193-204
Based on somatic cell genetic analysis, autosomal gene linkage is reported for the supernatant enzymes of human isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) in human-mouse cell hybrids. The IDH, MDH linkage was not linked to the X and E17 chromosomes or to 12 additional human enzyme markers.This work was supported in part by grants from the U.S. Public Health Service (Child Health and Human Development) and the United Health Foundation of Western New York. 相似文献
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Electrophoretically homogeneous myeloma light chain mRNA and its translation in vitro 总被引:12,自引:0,他引:12
mRNA coding for the light chain of a myeloma protein has been purified to give one band in acrylamide gel electrophoresis. This pure RNA (S~13.5) could be translated into the light chain in a heterologous cell-free translation system. The light chain synthesized is apparently slightly larger than the light chain secreted by the tumor. 相似文献
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Gasparian ME Ostapchenko VG Dolgikh DA Kirpichnikov MP 《Biochemistry. Biokhimii?a》2006,71(2):113-119
The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents. 相似文献
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Ala U Piro RM Grassi E Damasco C Silengo L Oti M Provero P Di Cunto F 《PLoS computational biology》2008,4(3):e1000043
Background
Even in the post-genomic era, the identification of candidate genes within loci associated with human genetic diseases is a very demanding task, because the critical region may typically contain hundreds of positional candidates. Since genes implicated in similar phenotypes tend to share very similar expression profiles, high throughput gene expression data may represent a very important resource to identify the best candidates for sequencing. However, so far, gene coexpression has not been used very successfully to prioritize positional candidates.Methodology/Principal Findings
We show that it is possible to reliably identify disease-relevant relationships among genes from massive microarray datasets by concentrating only on genes sharing similar expression profiles in both human and mouse. Moreover, we show systematically that the integration of human-mouse conserved coexpression with a phenotype similarity map allows the efficient identification of disease genes in large genomic regions. Finally, using this approach on 850 OMIM loci characterized by an unknown molecular basis, we propose high-probability candidates for 81 genetic diseases.Conclusion
Our results demonstrate that conserved coexpression, even at the human-mouse phylogenetic distance, represents a very strong criterion to predict disease-relevant relationships among human genes. 相似文献19.
G H Jones 《Biochemical and biophysical research communications》1973,51(1):88-93
A protein (denoted L′) which is similar in structure to immunoglobulin light chain has been isolated from the mouse plasma cell tumor, RPC-20. L′ has a molecular weight which is about 6000 daltons less than light chain. The exact nature of the relationship between L′ and light chain has not been established. 相似文献
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