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Biomedical subjects

P Leder

Publications and source records attributed to P Leder.

At least 271 records · Page 15Linked to original sources
PubMed1976

Purification and translation of an immunoglobulin lambda chain messenger RNA from mouse myeloma.

Here we describe the 500-fold purification of an mRNA encoding an immunoglobulin lambda light chain derived from the mouse myeloma tumor, RPC-20. Purification involves the isolation of membrane-bound polysomes, oligo(dT)-cellulose chromatography, and sucrose gradient centrifugation under conditions favoring denaturation of polynucleotide complexes. The mRNA purified in this way directs the cell-free synthesis of a polypeptide which is five or six amino acids longer than the mature form of RPC-20 light chain. In addition to directing the synthesis of a precursor-like polypeptide, the mRNA migrates on electrophoresis as a band containing approximately 1150 nucleotides, about 500 more than required to encode the mature form of the light chain.

Cell Line↗
PubMed1975

Differential expression of alpha- and beta-globin genes during differentiation of cultured erythroleukemic cells.

Murine erythroleukemic cells induced to differentiate in vitro with dimethylsulfoxide provide a model for events involved in the regulated expression of the globin genes. Here we examine alpha- and beta-globin gene expression in such cells which contain no detectable globin RNA prior to induction. To quantitate alpha- and beta-globin RNAs in cellular RNA samples by molecular hybridization techniques, highly radioactive complementary DNAs were synthesized using mouse alpha- and beta-globin RNAs purified by formamide gel electrophoresis. Maximally induced erythroleukemic cells and mouse reticulocytes contain nearly equal relative amounts of alpha- and beta-globin RNA. During the period in which globin RNA accumulates in differentiating erythroleukemic cells, however, alpha- and beta-globin RNAs are not present in equivalent amounts. alphaRNA is present in substantial excess (alpha/beta ratio 3.7) early in induction, and the alpha/beta RNA ratio progressively approaches 1 as differentiation proceeds further. These observations directly suggest that the alpha- and beta-globin genes are differentially expressed during cellular differentiation and raise questions as to how relative expression of globin genes is controlled during normal development.

Animals↗
PubMed1975

Turnover of protein synthetic elongation and initiation factors in Escherichia coli.

In an effort to understand the regulation of expression of the factors required for protein biosynthesis, we have measured the turnover of elongation factor G (EF-G) and initiation factor 2 (IF-2). Using a quantitative assay which involves immunoprecipitation and gel electrophoresis, it was found that both of these factors are very stable cellular proteins, but that their rates of accumulation are quite different, indicating a differential rate of synthesis. The effects of amino acid starvation and the relaxed response were also examined. The results showed that under these conditions EF-G and IF-2 are neither synthesized nor degraded.

Bacterial Proteins↗
PubMed1975

Regulation of initiation and elongation factor levels in Escherichia coli as assessed by a quantitative immunoassay.

These studies are directed toward determining whether the structural genes for protein biosynthetic factors comprise an operon subject to coordinate regulation in Escherichia coli. To assess coordinate expression of these genes, an immunoassay was devided to enable accurate quantitation of initiation and elongation factors in crude bacterial extracts. The antibodies made against highly purified initiation factor 2 (IF-2A and IF-2B) and elongation factor G (EF-G) are shown to inhibit the appropriate in vitro reactions and precipitate proteins co-migrating with appropriate factors on polyacrylamide gels. Immunoprecipitation in combination with gel electrophoresis was employed to make quantitative measurements of the amounts of IF-2 (A and B) relative to EF-G present in cells at different growth rates. The results show that the ratio of EF-G to IF-2 varies in a consistent way with the generation time of the cell. IF-2 levels remain constant as cells double more rapidly, WHEREAS THE EF-G content increases with more rapid cell growth.

Animals↗
PubMed1975

Butyric acid, a potent inducer of erythroid differentiation in cultured erythroleukemic cells.

Butyric acid is an unusually potent inducer of erythroid differentiation in cultured erythroleukemic cells. It is effective at one hundredth the concentration required of dimethylsulfoxide, a most effective inducing agent. Studies using a variety of analogues and metabolites suggest that the structural features of butyric acid are rather stringently required for induction. This effect is considered in view of the fact that butyric acid is a naturally occurring fatty acid, is effective in relatively low concentrations, and is widely used to form derivatives of cAMP.

Butanols↗
PubMed1975

Differentiation in erythroleukemic cells and their somatic hybrids.

Clones of erythroleukemic cells differ in the extent to which they (1) undergo differentiation spontaneously and (2) can be induced to differentiate in the presence of dimethylsulfoxide. Here we demonstrate that relative differences in globin gene expression within and between clones largely reflect differences in the proportion of cells participating in differentiation rather than uniform differences in the extent to which all cells in these clones undergo differentiation. We call this phenotype of a clone its characteristic probability of differentiation, a property that reflects the likelihood that a cell of this clone will undergo erythrodifferentiation under given conditions. We have examined somatic hybrid cells formed between similar erythroleukemic lines, between phenotypically different erythroleukemic lines, between phenotypically different erythroleukemic lines, and between erythroleukemic cells and mouse fibroblasts. Results of these experiments demonstrate that the spontaneous and induced probabilities of differentiation may be altered in an uncoupled fashion, suggesting that each is determined at different steps leading to a common pathway of globin gene expression.

Animals↗
PubMed1975

Induction of globin mRNA in Friend leukemia virus-infected cells and its presence in viral 60S RNA.

The Friend leukemia virus (FLV)-infected cell line, T-3-Cl-2, undergoes a form of erythroid differentiation in culture when treated with an appropriate inducer, such as dimethylsulfoxide ((CH3)2SO). Thus, whereas untreated cells contain no detectable hemoglobin, treated cells accumulate hemoglobin in quantities comparable to those in the mature mouse red blood cell. We have investigated the mechanism of hemoglobin induction by quantitating the number of globin genes and the amount of globin mRNA in cells before and during the period of hemoglobin accumulation. The results indicate the number of globin genes does not change as the cells accumulate hemogtobin: There are less than 5 globin genes per haploid genome. On the other hand, whereas cells lacking hemoglobin contain little, if any, globin mRNA, hemoglobin-containing cells accumulate, on the average, 8,000 molecules of globin mRNA per cell. The most direct, although, by no means, the only interpretation of these results is that the induction of hemoglobin synthesis involves transcriptional activation of the globin genes. Using this same cell line, we show that mouse globin mRNA sequences are also present in viral particles purified from the culture medium of globin-producing cells. These globin mRNA sequences are absent from viral particles derived from T-3-Cl-2 cells which are not producing globin mRNA. Virus-associated globin mRNA sequences sediment in association with 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60 S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin mRNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin mRNA does not become associated with the 60S viral RNA complex; and that globin mRNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin mRNA. The presence of globin mRNA sequences in FLV particles has important implications in terms of our ability to distinguish between host and viral RNAs in viral particles and in terms of the possible role RNA tumor viruses might play in transduction of genetic information.

Animals↗
PubMed1974

Enzymatic synthesis of solid phase-bound DNA sequences corresponding to specific mammalian genes.

With oligo(dT)-cellulose as primer, RNA-dependent DNA polymerase catalyzes the synthesis of cellulose-bound DNA that is complementary to mouse globin mRNA. The resulting cellulose-bound or solid phase complementary DNA hybridizes specifically with globin mRNA and permits the recovery of intact globin mRNA. This simple technique for the synthesis of solid phase-bound complementary DNA provides an additional and convenient method for the purification of specific genetic sequences.

Animals↗
PubMed1974

The organization and diversity of immunoglobulin genes.

We have used purified mouse immunoglobulin light chain mRNA and synthetic DNA which is complementary to it to assess the reiteration frequency of gene sequences corresponding to the kappa constant region of the mouse immunoglobulin light chain. These studies indicate that the constant region sequence is represented only two to three times per haploid mouse genome, a finding that rules out a simple stringent germ line mechanism which would require the constant region sequence to be represented hundreds if not thousands of times. Hybridization studies involving (125)I-labeled myeloma light chain mRNA yield interesting results which may eventually permit us to distinguish between the remaining somatic mutation and recombinational germ line hypotheses. These results reveal a major component of relatively unique frequency and a minor component with a reiteration frequency of approximately 30 to 50 copies per haploid genome. As discussed, these results do not permit us to distinguish unambiguously between a germ line model and a type of somatic mutation model that permits germ line genes corresponding to each kappa subgroup. The results do, however, clearly rule out the existence of thousands of variable region sequences so closely related to the MOPC-41 V-region as to permit extensive stable cross-hybridization.

Alleles↗
PubMed1974

An association between globin messenger RNA and 60S RNA derived from Friend leukemia virus.

The association between certain cellular RNAs and purified RNA tumor viruses prompted us to examine the possibility that specific host messenger RNAs might also be incorporated into RNA tumor viruses. Using a mouse cell line infected with Friend leukemia virus, T-3-Cl-2, which can be induced to accumulate mouse-globin messenger RNA, we show that mouse-globin messenger RNA sequences are present in viral particles purified from the culture medium of globin-producing cells. These globin messenger RNA sequences are absent from viral particles derived from T-3-Cl-2 cells that are not producing globin messenger RNA. Virus-associated globin messenger RNA sequences sediment in association with the 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin messenger RNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin messenger RNA does not become associated with the 60S viral RNA complex; and that globin messenger RNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin messenger RNA.

Animals↗