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M L Gefter

Publications and source records attributed to M L Gefter.

At least 73 records · Page 4Linked to original sources
PubMed1981

Dual expression of lambda genes in the MOPC-315 plasmacytoma.

The expression of two kappa light chain immunoglobulins in the MPC-11 mouse myeloma is well established, the two protein products being apparently from RNA transcripts derived from separate, rearranged kappa alleles in the MPC-11 genome. Recently, the characterization of kappa-related RNAs and protein products in several lambda-producing myelomas has indicated that multiple expression of light chain RNAs is a common event in myelomas and other cells of the B-lymphocyte lineage. These studies suggest that, although many light chain alleles may function to make RNA and protein in a given B-lymphocytic cell, only one complete, functional light chain is generally translated from the RNAs present in a single cell. The myeloma, MOPC-315, synthesizes and secretes an antibody which has an alpha heavy chain and a lambda II light chain. The DNA of MOPC-315 either has no kappa genes or has only a fragment of one, but it certainly has no kappa genes in the embryonic configuration. Rearrangement of its lambda genes has been observed but the exact nature of the rearrangement is not known. Because initial observations suggested that an immunoglobulin-related protein other than alpha and lambda II was present in MOPC-315 cells, we undertook to derive molecular cDNA clones from the MRNA in MOPC-315 tumour cells. Analysis of the clones has now identified two lambda chain mRNA species: a normal lambda II chain mRNA and another which directs the synthesis of a deleted form of a lambda I protein. The nucleotide sequence of the deleted lambda I mRNA shows that it resulted from a joining of the sequence encoding amino acid 31 of the variable region directly to the constant region coding sequence.

Amino Acid Sequence↗
PubMed1981

Two structurally distinct and independently regulated idiotypic families associated with the A/J response to azophenylarsonate.

At least two distinct subpopulations of A/J anti-azophenylarsonate (Ars) antibodies can be defined on the basis of serological cross-reactivity and sequence homology of heavy and light chains. The most frequently occurring idiotype family, IdCR, was initially identified by a rabbit antiserum and is present only in A/J mice. Examination of several randomly selected hybridoma proteins which failed to express IdCR-associated determinants revealed a second set of closely related molecules. The second idiotype family, Id36-60, is recognized by a rabbit antiserum prepared against the hybridoma protein 36-60. It is present in practically all A/J and BALB/c, but not B10. A Ars immune sera. On average, A/J immune sera contain approximately one-fifth as much Id36-60 as IdCR. A/J mice can be suppressed for subsequent expression of either idiotype by neonatal injection of anti-IdCR or anti-Id36-60 antisera. Suppression of IdCR does not affect the production of Id36-60 and vice versa. Together, these idiotype systems provide two independent markers for monitoring network regulation of the anti-Ars antibody response in A/J mice.

Animals↗
PubMed1981

Base substitution in an intervening sequence of a beta+-thalassemic human globin gene.

beta globin gene fragments from a patient with homozygous beta+-thalassemia have been cloned and subjected to restriction endonuclease, nucleotide sequence, and in vitro trancription analyses. Restriction endonuclease mapping of the cloned gene fragments revealed no deletions or other rearrangements, and transcription of the thalassemic gene appeared to be normal in vitro. However, nucleotide sequence analysis of the beta+-thalassemic gene fragments permitted identification of a single base change in the body of the small intervening sequence. This nucleotide change creates a sequence much like that of the 3' splice site of the small intervening sequence. The presence of a potential anomalous splicing site as a result of this base change suggests a mechanism for defective posttranscriptional processing of beta globin mRNA precursor molecules in beta+-thalassemia.

Base Sequence↗
PubMed1981

Multiple expression of Ig lambda-chain encoding RNA species in murine plasmacytoma cells.

The murine plasmacytoma MOPC 315, which synthesizes a complete IgA molecule containing lambda 2 light chains, is shown to synthesize a polypeptide related to the lambda 1 light chain. The abnormal lambda 1 polypeptide (m.w. approximately equal to 18,000) is encoded by a shorter polyA+ mRNA than the lambda 1 mRNAs of lambda 1-producing plasmacytomas. Polypeptides containing lambda 1-specific antigenic determinants have been found to be synthesized both in vitro by translations of RNA and in intact cells. HOPC-1, a lambda 1-producing plasmacytoma, was found to synthesize, in addition to the normal lambda 1-encoding mRNA, a lambda 2 RNA of normal length. A lambda 2-producing hybridoma does not appear to synthesize RNA encoding lambda 1 sequences. The results presented here show that more than 1 light chain gene may be transcribed within a single plasmacytoma cell. Although more than 1 light chain gene may be transcribed, the product of only 1 gene appears to be utilized for the synthesis of complete immunoglobulin molecules. This suggests novel mechanisms for the control of immunoglobulin expression.

Animals↗
PubMed1980

T lymphocyte-mediated suppression of myeloma function in vitro. III. Regulation of antibody production in hybrid myeloma cells by T lymphocytes.

To investigate the mechanisms by which T lymphocytes regulate myeloma function in vitro, the effects of regulatory T cells on antibody secretion by a hybrid myeloma cell line were examined. Suppressor T cells (Ts) specific for idiotypic determinants on M315 (IgA, lambda 2 anti-2,4-dinitrophenol and anti-2,4,6-trinitrophenol [TNP]) and MPC 11 (IgG2b, kappa) myeloma proteins inhibit antibody secretion by the appropriate parental myeloma cells. When cocultured with a hybrid cell line derived by fusion of MOPC 315 and MPC 11 myelomas, the idiotype-reactive Ts inhibit secretion of only the immunoglobulin (Ig) bearing the relevant idiotype. In contrast, syngeneic TNP-reactive cytolytic T lymphocytes (CTL) inhibit antibody secretion by TNP-binding MOPC 315 cells but not by MPC 11 cells in the presence of soluble TNP-keyhole limpet hemocyanin (KLH), and this inhibition probably represents a prelytic effect of the CTL. Such TNP-reactive CTL, in the presence of TNP-KLH, inhibit both IgA and IgG secretion by the MOPC 315-MPC 11 hybrid, which is consistent with a prelytic effect. Thus, myeloma hybrids are a useful tool for investigating the effector function of regulatory T cells. These results are discussed with reference to the mechanisms of action of regulatory T cells and their relevance to modulation of physiologic humoral immune responses.

Animals↗
PubMed1980

Structure and in vitro transcription of human globin genes.

The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

Base Sequence↗
PubMed1980

Enzyme-catalyzed DNA unwinding. The role of ATP in helicase III activity.

The enzyme helicase III catalyzes ATP-dependent unwinding of double-stranded DNA (Yarranto, G. T., Das, R. H., and Gefter, M. L. (1979) J. Biol. Chem. 254, 11997-12001). The free enzyme is able to bind to double- and single-stranded DNA. In the presence of ATP the enzyme can bind single- but not double-stranded DNA. The enzyme catalyzes an ADP-ATP exchange reaction in the absence of DNA. It is suggested that there is an enzyme.phosphate complex that discriminates between the two forms of DNA. These results are discussed in relation to a model that accounts for catalytic unwinding of DNA coupled to ATP hydrolysis.

Adenosine Triphosphatases↗
PubMed1980

Hybridoma proteins expressing the predominant idiotype of the antiazophenylarsonate response of A/J mice.

Hybridoma cell lines that secrete monoclonal antiazophenylarsonate antibodies were isolated from the fusion of A/J splenic lymphocytes with a myeloma cell line. A small percentage of these hybridoma proteins were recognized by rabbit antisera that detect the crossreactive idiotype characteristics of the antiazophenylarsonate response of A/J mice. The isotype, pI value, and amino-terminal sequences of four independently derived idiotype-positive hybridoma proteins were determined. These proteins were either of the IgG1 or IgG2a heavy chain class. For two mice tested, the majority of the idiotype in the immune serum was shown to be of the same isotype as the fusion-derived monoclonal antibodies. The pI values of the hybridoma proteins differed from one another and ranged from 6.9 to 7.6. Amino acid sequences of the heavy chains showed a significant degree of homology with each other, but each chain was unique in the framework or the first complementarity determining region (or both). A comparable pattern of sequence variation was evident for the light chains. The azophenylarsonate idiotype, therefore, appears to consist of a family of nonidentical but closely related molecules that are the product of more than one germline gene or the result of somatic mutation of a single germline gene.

Amino Acid Sequence↗
PubMed1980

DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.

We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.

Adenoviruses, Human↗
PubMed1980

Unique determinants associated with hybridoma proteins expressing a cross-reactive idiotype: frequency among individual immune sera.

It is possible to prepare antisera that recognize individual members of a family of hybridoma proteins in which all members of the family express the cross-reactive idiotype (IdCR) characteristic of the A/J anti-Ars response. These hybridoma-specific reagents also recognize a small percentage of the IdCR+ anti-Ars antibodies, which constitute the normal A/J anti-Ars response. Thus, the previously reported sequence heterogeneity of our IdCR+ hybridoma proteins actually represents components of the normal A/J anti-Ars response. Determinants associated with one particular IdCR+ hybridoma protein, 36-71 (Id36-71), were found on approximately 0.01 to 5.0% of the IdCR+ antibodies in all of the A/J hyperimmune sera that were examined. Determinants associated with 2 other IdCR+ hybridoma proteins, 16-46-4-8 (Id16-46) and 44-10 (Id44-10) were also found in almost all A/J immune sera, again at various concentrations. These results indicate that the heritable trait (IdCR) is expressed as a large number of antibody sequences that probably arise from the somatic diversification of a limited number of germ line genes. The pattern of diversification is qualitatively similar among individual mice but varies with regard to the relative proportions of the individual antibody components.

Animals↗
PubMed1979

Enzyme-catalyzed DNA unwinding. A DNA-dependent ATPase from E. coli.

We have isolated a new DNA-dependent ATPase from E. coli. The enzyme has been purified to greater than 90% purity. It appears to be composed of two identical polypeptide chains of molecular weight 20,000. The enzyme catalyzed the hydrolysis of ATP in the presence, but not in the absence, of single-stranded DNA. Double-stranded DNA is not a cofactor. The products of hydrolysis are ADP and Pi. The enzyme also catalyzed strand separation of duplex DNA in the presence of ATP and E. coli DNA binding protein. Two E. coli proteins capable of promoting strand separation have been reported previously and have been termed helicase I and II (Abdel-Monem, M., and Hoffmann-Berling, H. (1977) Eur. J. Biochem. 79, 33-38). Accordingly, this protein is named helicase III.

Adenosine Triphosphatases↗
PubMed1979

RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor.

We have analyzed the RNA produced in vitro by incubating nuclei from HeLa cells infected with adenovirus serotype 2. Our results show that adenovirus-specific RNA is produced at a linear rate for up to 2.5 hr. Hybridization analysis of RNA produced in nuclei isolated 18 hr after infection indicated that transcription begins at the "late promoter" at map position 16.5. Sequence analysis of the 5' termini of the in vitro transcripts showed that this system initiates RNA chains de novo at the correct promoter and that the 5' terminus is capped.

Amanitins↗
PubMed1979

Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein.

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.

Adenosine Triphosphatases↗
PubMed1979

Properties and applications of monoclonal antibodies directed against determinants of the Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.

Animals↗