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M C Weiss

Publications and source records attributed to M C Weiss.

At least 91 records · Page 5Linked to original sources
PubMed1981

Short-lived cytoplasmic regulators of gene expression in cell cybrids.

Somatic cell hybridization is a valuable tool for investigation the control of gene expression in eukaryotic cells. Studies of hybrid cells, heterokaryons, reconstructed cells and cybrids (cytoplasmic hybrids) have suggested that cytoplasmic factors may be involved in this regulatory process. Unfortunately, studies of this kind usually require that hybrid or modified cells be maintained for some time in a selective environment during which chromosomal losses or other changes may modify the genetic functions of the cells and thus vitiate conclusions about the mechanism of gene regulation. We report here the preparation of cybrids between enucleated mouse fibroblasts (Cl-1-D) and differentiated rat hepatoma cells (Fao) and the use of a combination of histological techniques to identify these modified cells early after fusion without the use of selective media. We found that albumin production in most cybrids was suppressed (extinguished) at 12-20 h after fusion but was restored by 48 h. These results suggest that there is a cytoplasmic factor in the fibroblast which exerts negative control over expression of the albumin gene, but which in the absence of the fibroblast nucleus, is not renewed and therefore short-lived.

Albumins↗
PubMed1981

Transfer of heritable properties by cell hybridization: specificity and the role of selective pressure.

Enucleated chloramphenicol (CAP) resistant mouse L-cells (LEA-2A) were fused with the mouse hepatoma cells (BW1J). The resultant cybrids expressed CAP resistance (the property used in selection of the cybrids), and also expressed the hepatic-specific functions of the BW1J parent. Hybrids between these same cells, on the other hand, exhibited chloramphenicol resistance and extinction of the hepatoma-specific properties. Cybrids were also prepared between enucleated rat hepatoma cells (FT-2) and mouse erythroleukemia cells (C19TK). The resultant cybrids selected in tyrosine-free medium expressed phenylalanine hydroxylase, an enzyme normally appeared to be the result of activation of the previously silent gene of the C19TK cells. These cybrids, however, did not express any other liver-specific functions present in the FT-2 cytoplast donor. These experiments suggest that the transfer of heritable properties by cell cybridization is selection specific and that activation or extinction observed in hybrids may not occur in cybrids of the same cells.

Animals↗
PubMed1981

Expression of the albumin gene in rat hepatoma cells and their dedifferentiated variants.

Rat hepatoma clones whose cells do and do not produce albumin, as well as somatic hybrid between the two types of cells, have been examined for albumin mRNA. A direct proportionality between the rate of albumin production and the concentration of albumin mRNA sequences was found for all albumin-producing hepatoma and hybrid clones, indicating that rate of synthesis of the protein is determined by the concentration of its mRNA. Albumin-negative dedifferentiated variant and somatic hybrid cells contain fewer than one to five molecules of albumin mRNA per cell; the block in expression of the gene appears to be at the same (probably transcriptional) level in variants and their somatic hybrids.

Albumins↗
PubMed1981

Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids.

We have used a combination of a sensitive immunocytochemical stain for intracellular albumin, and Hoechst 33258 dye for identification of parental nuclei to investigate the time-course of extinction, reexpression, and activation of albumin production in fusion products of 1s (hyperdiploid) or 2s (hypertetradiploid) rat hepatoma cells with mouse fibroblasts (L cells or embryonic cells). In all combinations, the initial event is extinction of albumin production. Extinction occurs immediately after fusion when the mouse fibroblast is a normal embryonic (senescent?) cell. In the case of an L cell, rat albumin is synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurs. Thereafter, albumin production ceases. 8-12 d after fusion, young hybrid colonies are found to resume the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin begins (activation). The patterns of reexpression and activation indicate (a) that chromosome loss is not necessary for either event to occur and (b) that the cells active in the synthesis of mouse albumin are a subpopulation of those cells already engaged in the production of rat albumin. We conclude that (a) extinction is mediated by diffusible factor(s) from the L-cell parent that act in the hepatoma nucleus to prevent the formation of new albumin messenger RNA; (b) reexpression and activation are gene dosage-dependent but extinction is not; and (c) previously active genes are more rapidly expressed than previously silent ones.

Albumins↗
PubMed1980

Coordinate secretion of mouse alphafetoprotein, mouse albumin and rat albumin by mouse hepatoma-rat hepatoma hybrid cells.

Mouse heptoma cells that secrete large amounts of alpha-fetoprotein (AFP) and albumin have been crossed with rat hepatoma cells that secret only albumin, and in relatively small amounts, to investigate the influence of each parental genome upon the expression of serum proteins. All of the ten independent hybrid clones examined produce mouse AFP and both mouse and rat albumin; none produces rat AFP. The absence of production of rat AFP by the hybrids suggests that different mechanisms are involved in the initiation and in the maintenance of expression of this function. The secretion of the three proteins by the hybrid cells is coordinate: Whatever the growth phase (exponential or stationary) and irrespective of the amounts produced over a wide range, the ratio secreted of mouse AFP to mouse albumin is near to one, and that of mouse albumin to rat albumin is near to five. In addition, even though the pattern of protein secretion during the growth cycle of hybrid cells is different from those of both parents, the products of both parental genomes conform to the new hybrid pattern. Finally, some hybrids secrete less of the proteins with increasing numbers of cell generations, yet all three continue to be secreted in coordinate fashion. Since the rates of secretion of serum proteins probably reflect their rates of synthesis, we conclude that coordinate secretion indicates coordinate synthesis, and may reflect coordinate transcription of the relevant genes.

Albumins↗
PubMed1980

Chromatin repeat length in somatic hybrids.

In order to study the mechanisms by which a characteristic repeat length is inherited in somatic cells, it was necessary to develop a method for determining repeat length with a precision of 1 to 2 base pairs. Hybrid clones between parental cell lines differing in repeat length by 6 base pairs were isolated. The four independent hybrid clones characterized had repeat lengths intermediate between those of the parental lines; however, it could be demonstrated that these repeat lengths are unique values and do not arise from a double distribution of the parental repeat lengths. It therefore is concluded that repeat length in somatic cells is determined by a common pool of diffusible substances.

Animals↗
PubMed1980

Chromatin repeat length correlates with phenotypic expression in hepatoma cells, their dedifferentiated variants, and somatic hybrids.

Chromatin repeat length is known to differ among the tissues of organisms. We have examined the repeat length of somatic cells in culture and have found an unexpected correlation between this parameter and the state of differentiation of the cells. Three sequentially derived subclones of a rat hepatoma, similar in phenotype, present a constant repeat length of 189 (+/- 1) base pairs. Three independent variant clones do not express hepatic functions and each has a different repeat length: 184 base pairs, 188 base pairs, and 192 base pairs. Somatic hybrids between the two types of cells in which liver functions are extinguished have a repeat length close or identical to that of the nonexpressing parent. In the case of one hybrid clone that reexpresses the liver phenotype and has undergone some chromosome loss, the 189-base-pair repeat length characteristic of the well-differentiated cells is reestablished. Finally, an amelanotic variant clone derived from a pigmented hamster melanoma clone also shows a signficantly altered repeat length. No correlations have been observed between repeat length and either generation time or chromosome number. Maintenance of the expression of differentiation therefore appears to be associated with a constant repeat length, and loss of the former with a modification in repeat length, suggesting that chromatin packaging is intimately involved in the regulation of gene expression.

Animals↗
PubMed1979

Dedifferentiated variants of a rat hepatoma: analysis by cell hybridization.

Two independent dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma, produce trans-acting diffusible substances(s) that extinguish the expression of liver-specific proteins when hybridized with a well-differentiated cell line of the same origin (Fao and Fu5-5, respectively). H5 x Fao hybrids show total and stable extinction of four liver functions and clonal variability in the expression of three others. FaoflC2 x Fu5-5 hybrids are initially flat (like FaoflC2 cells), and die in glucose-free medium where survival requires expression of hepatic gluconeogenic enzymes, but then evolve to hepatoma-like and finally round morphology; these latter cells express all liver functions analyzed including the gluconeogenic enzymes. Two exceptional clones that remained flat long enough for complete analysis showed extinction of all hepatic functions not expressed by FaoflC2 cells. We conclude that this transitory extinction reflects the action and then loss of extinguishing factor(s) contributed by FaoflC2. When crossed with BW1-J mouse hepatoma cells. FaoflC2 causes stable extinction of mouse aldolase B. We propose that production of extinguishing factor(s) is the rule for dedifferentiated variants.

Animals↗
PubMed1979

Expression of fetal and neonatal hepatic functions by mouse hepatoma-rat hepatoma hybrids.

In order to analyze the mechanisms implicated in the expression of differentiated functions during development, we have studied ten hybrid clones arising from fusion of cells of a mouse hepatoma characterized by the expression of only fetal hepatic functions with those of a rat hepatoma which express, like adult hepatocytes, a set of neonatal as well as fetal hepatic functions. The cells of most hybrid clones contain one set of chromosomes of each parent and coexpress the hepatic functions common to both parents. Among the hepatic proteins characteristic of only one parental line, some continue to be expressed while others are extinguished. The three functions out of the eight examined which are subject to extinction are expressed uniquely by the rat parental cells and appear only near or at birth during normal liver development. These results suggest that regulatory mechanisms (whose final effect is negative) operate in fetal cells to inhibit the expression of differentiated functions limited to a later stage of development.

Animals↗
PubMed1978

Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously.

Hybridization of cells of defined and different histotypes has been carried out to investigate whether the expression (or reexpression) of parental functions is mutually exclusive, as is expected if the generally assumed rule of discreteness of differentiation applies to hybrid cells. A cross of pigmented mouse melanoma cells and albumin-producing rat hepatoma cells gave rise to hybrids containing essentially one set of chromosomes from each parent and producing neither melanin nor albumin. Cells of one hybrid clone are shown to retain the potential to reexpress both parental differentiations. Successive subclonings of this hybrid have shown that cells which reexpress one function may retain the potential to reexpress the other, and that freshly isolated, morphologically homogeneous subclones may produce pigment or albumin, but not both; there successive and exclusive shifts of phenotype are documented, and in these cases, chromosome loss is very slight. The use of immunoadsorbed antisera has revealed that most (if not all) of the albumin produced by the hybrid cells is of the mouse type. We conclude that both parental determinations are retained by the hybrid cells, and that the parental differentiations are reexpressed only in a mutually exclusive fashion.

Albumins↗
PubMed1975

Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells.

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin . The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.

Alanine Transaminase↗
PubMed1975

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.

Alanine Transaminase↗
PubMed1974

Expression of differentiated functions in hepatoma cell hybrids: high frequency of induction of mouse albumin production in rat hepatoma-mouse lymphoblast hybrids.

We have studied the production of serum albumin by somatic hybrids between well-differentiated 2s and 1s rat hepatoma cells (Faza), which produce serum albumin, and sub-diploid mouse leukemic lymphoblasts (Lc), which do not produce albumin. We determined the rat or mouse origin of the albumin by double immunodiffusion, using immuno-adsorbed noncrossreacting antisera. Each of 12 karyologically identified 2s hybrid clones (Lc2F) produces both rat and mouse albumin. Moreover, unlike 1s hybrids reported previously, eight of nine 1s hybrids (LcF) also produce mouse albumin; six of them produce rat albumin as well. One clone from the 1s cross produces only rat albumin.

Albumins↗
PubMed1973

Expression of differentiated functions in hepatoma cell hybrids: alanine aminotransferase.

The expression of alanine aminotransferase (EC 2.6.1.2), an enzyme that is inducible in the liver, has been examined in somatic hybrid cells formed by crossing well-differentiated rat hepatoma cells with rat diploid epithelial cells, the former characterized by high activity and inducibility of the enzyme, and the latter by the absence of detectable activity. The hybrid cells that contain essentially complete chromosomal sets of the two parents show only very low activity and little inducibility. Among numerous "segregated" hybrid subclones, which have lost up to 40% of the chromosomes initially present, several show expression of intermediate levels of enzyme activity and very little inducibility, and two independent subclones are characterized by full re-expression of both baseline and inducible enzyme activity. The electrophoretic mobility of the enzyme from the latter hybrids, from the hepatoma parental cells, and from rat liver is identical. The absence of a correlation between total chromosome number of the hybrid cells and re-expression of alanine aminotransferase suggests that the loss of specific chromosomes is required for re-expression. In these hybrid cells, the re-expression of alanine aminotransferase baseline and inducibility is independent of that of tyrosine aminotransferase inducibility.

Alanine Transaminase↗