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1.
Wayne K. Hoffman Peter Lalley Jean Deb Butler Sheldon Orloff Joseph D. Schulman Anil B. Mukherjee 《In vitro cellular & developmental biology. Plant》1981,17(8):735-740
Summary Using lipochromosomes (phospholipid-entrapped chromosomes) we have transferred the human HGPRT gene into HGPRT deficient mouse
cells (A9) with a frequency of approximately 1×10−5 (Mukherjee et al., Proc. Natl. Acad. Sci. USA 75: 1361–1365; 1978). Two other genes located on the long arm of the human
X-chromosome were also expressed in two independently derived populations of transferents (A9/GT3 and A9/GT4). We report here
the chromosomal and enzymatic composition of human HGPRT-positive clones from each subpopulation analyzed in detail with alkaline
Giemsa-11 staining.
All the clones expressed human PGK and HGPRT, but one (A9/GT4C6) lacked human G6PD. In each of four clones examined microscopically,
a small piece of presumptive human chromatin was visible in the karyotypes of most cells. The chromatin fragment was free
or attached in each cell of an individual clone. When integrated, the human chromosomal fragment in each clone appeared associated
with the centromere of the same telocentric A9 chromosome (No. 6 by Q-banding).
These data suggest that: (a) substantial human chromosomal fragments can be transferred into recipient cells using the lipochromosome
technique; (b) clones from human HGPRT positive A9 transferent subpopulations may or may not possess other human X-linked
markers; (c) the stability of lipochromosomally transferred genes varied from clone to clone and stability is generally poor
in the absence of continuous selection pressure (e.g., HAT); (d) when multiple X-linked human genes were transferred to mouse
cells a cytologically detectable human chromosomal fragment was identified free or attached to a host chromosome; and (e)
integration of transferred human chromosomal material into mouse chromosomes may occur at preferential site(s) in the recipient
genome.
This research was sponsored in part by the Office of Health and Environmental Research U.S. Department of Energy under Contract
W-7405-eng-26 with the Union Carbide Corporation. 相似文献
2.
Cystinotic and normal fibroblasts: Differential susceptibility to cysteine toxicity in vitro 总被引:2,自引:0,他引:2
Sheldon Orloff Anil B. Mukherjee Jean DeB Butler Barbara Foley Joseph D. Schulman 《In vitro cellular & developmental biology. Plant》1980,16(8):655-660
Summary Extracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagle's minimal essential medium containing supplemental fetal
bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion.
In cocultivation experiments of [3H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective
survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally
contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular
cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration
of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells. 相似文献
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Rat cytomegalovirus gene expression in cardiac allograft recipients is tissue specific and does not parallel the profiles detected in vitro 下载免费PDF全文
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We present a new method for fine-mapping a disease susceptibility locus using a case-control design. The new method, termed the weighted average (WA) statistic, averages the Cochran-Armitage (CA) trend test statistic and the difference between the Hardy-Weinberg disequilibrium test statistic for cases and controls (the HWD trend). The main characteristics of the WA statistic are that it improves on the weaknesses, and maintains the strengths, of both the CA trend test and the HWD trend test. Data from three different populations in the Genetic Analysis Workshop 14 (GAW14) simulated dataset (Aipotu, Karangar, and Danacaa) were first subjected to model-free linkage analysis to find regions exhibiting linkage. Then, for fine-scale mapping, 140 SNPs within the significant linkage regions were analyzed with the WA test statistic on replicates of the three populations, both separately and combined. The regions that were significant in the multipoint linkage analysis were also significant in this fine-scale mapping. The most significant regions that were obtained using the WA statistic were regions in chromosome 3 (B03T3056-B03T3058, p-value < 1 x 10(-10)) and chromosome 9 (B09T8332-B09T8334, p-value 1 x 10(-6)). Based on the results of the simulated GAW14 data, the WA test statistic showed good performance and could narrow down the region containing the susceptibility locus. However, the strength of the signal depends on both the strength of the linkage disequilibrium and the heterozygosity of the linked marker. 相似文献
8.
ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT 总被引:13,自引:6,他引:7 下载免费PDF全文
Charles E. Ganote Jared J. Grantham Harold L. Moses Maurice B. Burg Jack Orloff 《The Journal of cell biology》1968,36(2):355-367
Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow. 相似文献
9.
The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms. 相似文献
10.