Time of suppression of human rRNA genes in mouse-human hybrid cells.
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Animals↗
Biomedical subjects
M Rechsteiner
Publications and source records attributed to M Rechsteiner.
Degradation of proteins microinjected into cultured mammalian cells.
Iodinated proteins were degraded after injection into HeLa cells at first-order rates with half-lives varying from three hours for the trout monhistone chromosomal protein, HMG-T, -to 60 hours for whale myoglobin. Fluoresceinated-bovine serum albumin (fl-BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I-BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS-acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrasting, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl-BSA, which exhibited perinuclear accumulation in approximately one-half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.
Animals↗
Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells.
The nonhistone chromosomal protein HMG1 associated rapidly with the nuclei of HeLa cells and bovine fibroblasts following its introduction into the cytoplasm by red cell-mediated microinjection. A number of non-nuclear proteins, on the other hand, failed to concentrate in HeLa or bovine fibroblast nuclei. Autoradiography of thin sections showed that 125I-labeled HMG1 localized within nuclei, and further established that it remained associated with metaphase chromosomes at mitosis. When uninjected HeLa cells were fused with 125I-HMG1-injected HeLa cells, the labeled molecules equilibrated between nuclei within 12 hr. Similar results were obtained with bovine fibroblasts, indicating that a dynamic equilibrium exists between HMG1 and chromatin within living cells. Electrophoresis of 125I-HMG1 retrieved from HeLa cells or bovine fibroblasts up to 48 hr after injection showed that more than 80% of the molecules were intact. Autoradiographic analysis of cells fixed over a period of several days after injection produced apparent half-lives for 125I-HMG1 of 80 hr in HeLa cells and 100 hr in bovine fibroblasts.
Cell Fusion↗
Kinetics of human chromosome loss from 3T3-human hybrid cells.
Human chromosomes were lost from populations of 3T3-HeLa and 3T3-diploid human fibroblast (HF) hybrids with similar first-order kinetics. Whereas loss began immediately in 3T3-HF hybrids, there was a lag of 5-10 cell divisions before chromosome loss began in 3T3-HeLa hybrids. Human chromosome loss was not affected by aminopterin selection, the use of polyethylene glycol rather than Sendai virus as fusagen, or by the presence of one or two 3T3 genomes. However, when cell division was retarded by growing 3T3-HF hybrids in low serum or at low temperatures, fewer human chromosomes were lost. This suggests that cell cycle traverse is important in chromosome loss. The distribution of human chromosomes among hybrid metaphases indicated that gradual chromosome loss occurred in all hybrids rather than extensive loss from a portion of the hybrids. During the period of chromosome loss, increased numbers of individual asynchronously condensed human chromosomes were randomly distributed among hybrid metaphases.
Animals↗
The turnover of tRNAs microinjected into animal cells.
Red cell-mediated microinjection has been used to study tRNA turnover in SV3T3 mouse cells and TC7 cells, an African green monkey kidney line. The turnover of endogenous tRNA, measured by labeling with 3H-methionine, was first-order with half-lives of approximately one day in SV3T3 and two days in TC7 cells. 32PtRNA isolated from E. coli or TC7 cells turned over at the same rate as endogenous tRNA when injected into either SV3T3 or TC7 cells. This demonstrates that cellular processes, not properties inherent to tRNAs, are responsible for the difference in tRNA turnover observed between SV3T3 and TC7 cells. These results further indicate that the mechanism of tRNA turnover in mammaliam cells does not distinguish prokaryotic from eukaryotic tRNAs. In contrast to unmodified tRNA, glyoxalated tRNA was rapidly degraded upon injection. Thus altered tRNA's, like altered proteins, are turned over more rapidly in animal cells.
Anticodon↗
Red cell-mediated microinjection.
I have reviewed the current status of microinjection based on fusion of red blood cells and tissue culture cells. Macromolecules are introduced into red blood cells during hypotonic hemolysis, and the resealed red cells are then fused to tissue culture cells with Sendai virus. The procedure has been used to inject ferritin, thymidine kinase, bovine serum albumin, and transfer RNA molecules into large numbers of tissue culture cells. Physiologically significant amounts of various macromolecules can be transferred, and preliminary studies show that [125I]bovine serum albumin and transfer RNA are stable within recipient culture cells. Tissue culture cells remain viable following microinjection. Red cell-mediated microinjection should facilitate the study of various processes, such as macromolecular turnover and genetic regulation, that are not easily studied with conventional biochemical techniques.
Animals↗
Magnitude and significance of NAD turnover in human cell line D98/AH2.
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Cell Line↗
Studies on the intranuclear distribution of human and mouse genomes and formation of human-mouse hybrid cells.
Autoradiographic examination of early hybrid cells formed by the fusion of 3H-thymidine labeled D98/AH2 cells and 3T3-4E cells revealed that human and mouse chromosomes are often separated within metaphase and interphase nuclei. Although the marked separation of human and mouse chromosomes progressively disappeared with succeeding cell division, the occurrence of sectored nuclei in 16-cell hybrid colonies and the labeling pattern of human chromosomes within separated metaphases indicate that separation of human and mouse chromosomes may persist through several mitoses. Fusion of 3H-thymidine labeled D98/AH2 cells and 3T3-4E cells was coupled with aminopterin selection to study other aspects of hybrid cell formation. Hybrid cells arose from heterokaryons by cell division. Nuclear fusion may have occurred in two out of several thousand heterokaryons examined. However, these could reflect the close apposition of adjacent nuclei. A large fraction (greater than 0.5) of D98/3T3 heterokaryons underwent at least one cell division. However, the number of hybrid colonies containing more than eight cells at ten days following fusion was about 0.03 of the total number of heterokaryons. Many hybrid colonies arrested growth before the 8-cell stage, and the cells in such colonies exhibited nuclear abnormalities.
Animals↗
Turnover at nicotinamide adenine dinucleotide in cultures of human cells.
The rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C-adenine. In one set of experiments, cells were labeled by growth in medium containing 14C-adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C-adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C-adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C-adenine and the rate of entry of 14C-adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C-adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse-chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half-life of an intact NAD molecule is 60 +/- 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.
Adenine↗
Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells.
Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3-4F cells are unable to utilize nicotinic acid. When 3T3-4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the four-day period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.
Aminopterin↗
Pyridine nucleotide metabolism in mitotic cells.
The biosynthesis and turnover of nicotinamide adenine dinucleotide (NAD) have been examined in mitotic cells of the human culture line, D98/AH2. No significant difference in the incorporation of nicotinic acid or nicotinamide could be detected between mitotic and interphase cells. The distribution of newly-incorporated nicotinic acid among the various pyridine nucleotides was also identical in mitotic and interphase cells. Whereas previous results have shown that the nucleus is necessary for NAD biosynthesis, the present results show that an intact nucleus is not required. In contrast to the equivalent rates of biosynthesis in mitotic and interphase cells, the pyridine ring of NAD was lost twice as fast from mitotic as from interphase cells. Loss of the pyridine ring to the medium is not necessarily an accurate measure of turnover, and the difference between mitotic and interphase cells may reflect differential reutilization of the pyridine ring within the cell. However, it is clear that NAD turnover is substantial in mitotic cells and possibly greater in mitotic cells than interphase cells.
Cell Line↗