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Biomedical subjects

M L Gefter

Publications and source records attributed to M L Gefter.

At least 91 records · Page 5Linked to original sources
PubMed1978

Control of immunoglobulin secretion in the murine plasmacytoma line MOPC 315.

Cells of the 315LV-1 (derived from NP1) variant line of MOPC 315 contain approximately 1% the normal intracellular level of the heavy (alpha) chain of IgA and no detectable light (lambda2) chain. The synthesis rate of alpha-chain in the variant, however, is similar to that in cells of the parent line. Moreover the relative amount of translatable alpha-chain mRNA that can be extracted from 315LV-1 cells is about the same as for parental cells. No light-chain synthesis can be detected either in vivo or in vitro in a wheat germ cell-free system. The 315LV-1 heavy chain synthesized in vivo or in vitro has slightly greater electrophoretic mobility than normal H chain and turns over rapidly intracellularly. The variant fails to secrete any of its heavy chain, despite the fact that its H chain mRNA is bound to membranes, as one would expect for a secretory protein message. Fusion of 315LV-1 cells with cells of a kappa-producing MPC 11 variant line leads to stabilization of the intracellular H chain and also to full recovery of secretion of the H chain as an H2L2 molecule.

Animals↗
PubMed1978

Regulation of immunoglobulin biosynthesis in the murine plasmacytoma MOPC 315.

We have examined certain aspects of IgG biosynthesis by constructing hybrids between MPC11 (gamma2b, kappa) and MOPC 315 (alpha,lambda2) that have lost the ability to synthesize one or the other heavy chain. Cells express the three chains in a stable fashion, and both autologous (parental) and heterologous (nonparental) H and L chain pairs form and are secreted. The alpha H chain was found in polymeric form when associated with the heterologous kappa L chain. The lambda2 L chain covalently assembled to the heterologous gamma2b H chain. Surprisingly, autologous pairing was always favored over heterologous pairing in vivo by 5 to 10:1 in terms of rate of assembly. Similar ratios were maintained in the secreted protein. These results suggest that co-expression of particular H and L chain pairs is predetermined. Evolution presumably operates to improve antigen recognition as well as rate of assembly of active molecules.

Animals↗
PubMed1977

Synthesis of messenger RNA-like molecules in isolated myeloma nuclei.

Nuclei isolated from mouse myeloma cells grown in tissue culture are capable of synthesizing RNA for prolonged periods of time. Addition of cytoplasmic extracts to the system stimulates slightly the rate and prolongs the time of synthesis. As judges by sedimentation in SDS and in formamide gradients, the size of the RNA synthesized is heterogeneous from smaller than 10S to larger than 45S, thus resembling in vivo made RNA. The characteristics of some of the RNA are in keeping with those expected to be for mRNA. Fifty percent of the RNA synthesis is sensitive to alpha-amanitin. After an incubation of two hours in the absence of alpha-amanitin about 10 percent of the newly synthesized RNA is found outside of the nuclei; it sediments with a broad distribution at 18S. A considerable fraction of the RNA that is released from nuclei in vitro can promote the formation of polyribosomes, and contains molecules that are polyadenylated and "capped".

Amanitins↗
PubMed1975

In vitro synthesis of transfer RNA. I. Purification of required components.

We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.

Chromatography, Gel↗
PubMed1975

In vitro synthesis of transfer RNA. II. Identification of required enzymatic activities.

We have shown that the synthesis of active su+III tRNATyr from a phi80psu+III DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-dependent RNA polymerase, catalyzes the formation of a large molecular weight transcript, initiating synthesis at a specific site 41 nucleotides proximal to the 5' end of the su+III tRNATyr structural gene and continuing at least 100 nucleotides beyond the 3' terminus of the su+III tRNATyr sequence. The second required component, designated Fraction V, allows purified DNA-DEPENDENT RNA polymerase to function in tRNA synthesis. We have shown that this fraction contains an endonuclease that together with DNA-dependent RNA polymerase is responsible for the synthesis of su+III tRNATyr "precursor". Thus, su+III tRNATyr precursor is not itself the primary transcription product of the su+III tRNATyr gene, but rather, it arises as a result of post-transcriptional cleavage of a much larger transcript by the action of the nuclease present in Fraction V. The third enzymatic activity required for synthesis of active su+III tRNATyr is a ribonuclease (RNase P III) that specifically catalyzes the removal of the 3' extra nucleotides from the su+III tRNATyr precursor. The fourth activity required for synthesis of tRNA is a previously identified endonuclease, RNase P, that specifically catalyzes the removal of the 5' extra nucleotides from tRNA precursors. The properties of RNase P purified according to the procedure developed in this laboratory have been compared with those of the enzyme purified from ribosomes according to the procedure described by Robertson et al. (Robertson, H.D., Altman, S., and Smith, F.D. (1972) J.Biol. Chem. 247, 5243-5251.).

Coliphages↗
PubMed1975

Physical studies of the interaction between the Escherichia coli DNA binding protein and nucleic acids.

The interaction of nucleic acid with the Escherichia coli DNA-binding protein has been studied by fluorescence emission spectroscopy and sedimentation velocity analysis. The protein binds to single-strand DNA with an apparent equilibrium dissociation constant of 2 X 10(-9). It binds to the homopolymers poly (dA) and poly (dT) slightly more tightly, but has a larger apparent equilibrium dissociation constant to poly (dC). The protein also binds tightly to ribohomopolymers and to tRNA, but not to duplex DNA. By the use of defined-length oligonucleotides, it has been shown that the protein binds to DNA in a highly cooperative manner. The extent of cooperativity is seen as the difference in binding between an isolated monomeric protein molecule bound to DNA and two or more molecules binding to contiguous sites.

Bacterial Proteins↗
PubMed1975

DNA replication.

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Bacteriophages↗
PubMed1974

Properties of the Escherichia coli in DNA binding (unwinding) protein: interaction with DNA polymerase and DNA.

The E. coli DNA binding protein reduces the activity of the single-strand-specific nucleases associated with all three DNA polymerases known in E. coli. A slight excess of binding protein over that required to saturate the DNA template leads to total inhibition of activity of the 3' --> 5' nucleases associated with DNA polymerases I and III, but restores maximum activity of the DNA polymerase II-associated nuclease. The binding protein forms a specific complex with DNA polymerase II in the absence of DNA, and it is this complex that degrades a DNA.binding protein complex. Binding protein also facilitates the binding of DNA polymerase II to single-stranded DNA, whereas the binding to DNA of DNA polymerase I is inhibited. These data may explain the specificity with which the binding protein enhances the synthetic ability of DNA polymerase II.

Animals↗