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Y Bergman

Publications and source records attributed to Y Bergman.

At least 37 records · Page 2Linked to original sources
PubMed1990

Extinction of Ig genes expression in myeloma x fibroblast somatic cell hybrids is accompanied by repression of the oct-2 gene encoding a B-cell specific transcription factor.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of differentiation-specific traits of the nonfibroblast parental cell. To explore the genetic basis of this phenomenon, we have used a series of somatic cell hybrids between myeloma cells and fibroblasts. Previous findings show that in these hybrids expression of the immunoglobulin (Ig) genes was extinguished at the transcriptional level. Our present results show that NF-kappa B transcription factor, known to be critical for kappa-chain enhancer activity, is present although in a lower amount, in the nucleus and in the cytosolic fraction of most of these hybrids (probably attached to the previously postulated I-kappa B inhibitor). In contrast, the expression of the NF-A2/OTF-2 transcription factor encoded by the oct-2 gene, which binds to the octameric motif located in the Ig promoters and heavy chain gene enhancer, is extinguished at the transcriptional level. Our data thus suggest that extinction of Ig genes expression occurs via an indirect mechanism in which a fibroblast factor suppresses transcription factor(s) which are critical for Ig transcription.

Animals↗
PubMed1989

Adenovirus E1A products activate the Ig k-chain enhancer in fibroblasts. A possible involvement of the NF-kB binding site.

Proteins encoded by the early region 1A (E1A) regulate transcription of viral and cellular genes. The mechanism of this trans-regulation remains unclear but it is of considerable interest as an example of transcription regulation through cellular intermediates. We have therefore studied the effect of E1A products on the activity of the k-L chain gene enhancer. In our previous studies we have found that the k-chain enhancer is activated by E1A products in fibroblasts. Using in vivo competition experiments we found that the E1A regulatory effect involves trans-acting factors that can be titrated out by the k-chain enhancer sequences. Our transfection experiments suggest that the k3 enhancer fragment, encompassing the NF-kB binding site, is one of the sites through which the adenovirus E1A products exert their transactivation effect. The level of the nuclear factors that bind to the NF-kB binding site increases in fibroblasts that constitutively express E1A products. Understanding the mechanisms through which E1A regulates transcription should help to illuminate cellular mechanisms of gene control in normal and transformed cells.

Adenovirus Early Proteins↗
PubMed1989

Extinction of expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells.

Most murine plasma-cell tumors show a t(12;15) reciprocal chromosomal translocation which truncates the first exon of one of the myc gene alleles and fuses it to one of the switch regions of the immunoglobulin (Ig) heavy-chain locus. This results in constitutive activation of the translocated myc gene and the production of smaller-sized mRNA molecules, which are initiated at new sites in the first myc intron. The normal myc allele is not expressed in these myeloma cells. We have studied the expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells. Our previous findings show that Ig gene expression is extinguished in such hybrids. In the present work we found that the hybrids contain the normal and translocated myc genes. In contrast to the myeloma parental cells which express the translocated myc gene, the hybrids are similar to the L-cells in expressing only the normal myc allele. Our results suggest that the L-cell, fibroblast-like phenotype, is dominant in these hybrids, and show that the translocated myc gene is expressed in a tissue-specific manner in the context of the myeloma cell, and is not expressed when subjected to a fibroblast-like cellular environment.

Alleles↗
PubMed1989

Mapping of murine IgE epitopes involved in IgE-Fc epsilon receptor interactions.

The generation of anti-IgE monoclonal antibodies has permitted the identification of various serological epitopes on the IgE molecule. The relationship of the sites on IgE recognized by such antibodies to the Fc epsilon receptor (Fc epsilon R) interaction site has been determined using cross-inhibition studies. However, interpretation of this type of experiment is limited by problems of steric hindrance. Thus, to accomplish precise mapping on the IgE molecule of the Fc epsilon R interaction site and the binding sites of various anti-IgE mAb, we employed site-directed mutagenesis of the IgE heavy chain gene. To this end we have constructed and expressed a recombinant murine constant epsilon heavy chain (C epsilon) gene bearing a (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH region. Several site-specific mutants in the C epsilon 3 and C epsilon 4 domains of this recombinant C epsilon gene were prepared and expressed by transfection into the light chain-producing J558L myeloma cell line. The resulting IgE antibodies were tested for binding to mast cells and to various anti-IgE mAb. The mutants produced include a proline to histidine point mutant at amino acid residue 404 in the C epsilon 3 domain, a mutant with a truncated C epsilon 4 domain, a mutant with a 45 amino acid deletion in the carboxy end of C epsilon 3, and a chimeric human C epsilon in which the human C epsilon 3 was replaced by the homologous mouse C epsilon 3 domain. These mutants have permitted the localization, to the C epsilon 3 domain, of the epitopes recognized by the 84.1C and 95.3 anti-IgE mAb. The 84.1C mAb recognizes a site on IgE which is identical or very close to the Fc epsilon R binding site, and 95.3 recognizes a site on IgE which is related, but not identical to the Fc epsilon R binding site. The antigenic determinant recognized by the 51.3 mAb, which is inefficient at blocking the IgE-Fc epsilon R interaction, has been mapped to the C epsilon 4 domain. When tested for binding to the Fc epsilon R on RBL-2H3 cells, the point mutant bound to the Fc epsilon R with twofold reduced affinity, while the C epsilon 3 deletion mutant and the mutant truncated in C epsilon 4 lost all receptor binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1988

Regulation of the Ig kappa-chain enhancer by the adenovirus E1A gene products. Repression in lymphoid cells, activation in fibroblasts.

Proteins encoded by early region 1A (E1A) regulate transcription of viral and cellular genes. The mechanism of this trans-activation is not understood, but is of considerable interest as an example of transcription regulation through a cellular intermediate. We have therefore studied the effect of E1A products on the activity of the kappa L chain gene promoter and enhancer. By using transient and stable transfections into lymphoid and nonlymphoid cells, we found that the E1A proteins have a pleiotropic effect on the regulation of the mouse kappa-chain gene enhancer. In lymphoid cells the E1A products repress kappa-chain enhancer, whereas in fibroblasts, the kappa-chain enhancer is activated by the E1A products whether the E1A gene is in an extrachromosomal location or stably integrated in the genome. Furthermore, a functional kappa-chain promoter, containing the octanucleotide and "TATA" sequences is needed in order to be transcribed in E1A-producing cells. This ability of E1A products to negatively and positively regulate kappa-chain transcription may reflect a more general phenomenon in which a given cellular protein could participate in a variety of different cellular controls.

Adenoviridae↗
PubMed1988

The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro.

In this paper we characterize the chromatin structure and nuclear proteins associated with different transcriptional states of the mouse serum albumin gene. We found the albumin gene to be transcribed in liver at rates 1000-fold or greater than in other tissues tested. We discovered seven DNase I hypersensitive sites encompassing the albumin gene only in liver chromatin, with strong hypersensitivity at the promoter and the enhancer, which is over 10 kb upstream. Using a gel retardation assay, we found a liver nuclear protein, or set of proteins, which binds specifically to DNA of a liver-specific hypersensitive site that maps 3.5 kb upstream, between the promoter and enhancer. Footprinting, heat insensitivity, and binding competition experiments indicate that the protein(s) have characteristics similar to a heat-stable, liver-abundant protein that binds to the albumin promoter and other enhancer and promoter sequences. Finally, we asked whether the liver-specific factors that cause DNase I hypersensitivity in vivo are present concurrently at the various sites in chromatin. We devised a simple new method to reveal that in liver, individual albumin genes are hypersensitive simultaneously at the promoter, the enhancer, and the -3.5-kb site. Thus, transcriptionally active albumin genes appear to contain tissue-abundant factors that are present at three widely spaced points in chromatin, yet at the same point in time. Similar factors binding simultaneously to at least two of these sites could create a specific structure in chromatin required for high-level albumin gene transcription.

Animals↗
PubMed1987

Expression in transgenic mice of two genes of different tissue specificity integrated into a single chromosomal site.

Transgenic mice were used to study the expression of pairs of genes with distinctly different tissue specificities, covalently linked and integrated into the same chromosomal site. A transgenic strain carrying, in close proximity and in the same orientation, the rat fast skeletal muscle myosin light-chain 2 (MLC2) gene and the mouse rearranged immunoglobulin kappa light-chain gene expressed the immunoglobulin gene specifically in the lymphoid tissues, whereas rat MLC2 transcripts were found in skeletal muscle but not in the spleen or the other tissues that were tested. In another transgenic strain, carrying the rat MLC2 gene and a modified rat skeletal muscle actin gene (actin-globin chimeric gene), transcripts of the rat MLC2 gene were detected in skeletal muscle only, whereas the actin-globin transcripts were detected in skeletal muscle as well as in the heart. Moreover, the expression of the chimeric gene was also developmentally regulated. Expression was higher in cardiac muscle than in the skeletal muscle of neonatal mice, whereas expression was higher in skeletal muscle in adult mice. This pattern is consistent with the regulation of the expression of the endogenous skeletal muscle actin gene. Thus, in those transgenic strains that expressed both genes, each gene retained its tissue specificity, in spite of their close proximity. These results indicate a high degree of autonomy of the control elements included in the cloned genomic DNA fragment and demonstrate that a single chromosomal site can be permissive for the proper expression of two genes with different tissue specificities.

Actins↗
PubMed1984

Two regulatory elements for immunoglobulin kappa light chain gene expression.

By using internal deletions within a rearranged immunoglobulin kappa light chain gene, the presence of an intron regulatory sequence (enhancer) has been confirmed. Its presence is required for high-level transcription from a plasmid after transfection into myeloma cells. Transfection efficiency was monitored by the activity of a deleted H4 histone gene included in the plasmid. The intron element could be moved upstream of the gene in both orientations, fulfilling the definition of an enhancer. By using 5' deletions, a second regulatory element was located upstream of the "TATA" box, between positions -69 and -104. These two elements both are required for efficient kappa chain gene expression.

Animals↗
PubMed1983

Biosynthesis, glycosylation, and in vitro translation of the human T cell antigen Leu-4.

Two families of polypeptides were found in immunoprecipitates formed from human T cell lines by using the monoclonal antibody anti-Leu-4. When analyzed by one- and two-dimensional SDS-PAGE, the first family consisted of members with m.w. of 26,000 and isoelectric points that varied from very basic to acidic. The second family consisted of members with m.w. of 22,000 to 23,000 and isolectric points that varied from neutral to very acidic. The biosynthetic processing and intracellular glycosylation of several of these polypeptides were examined. Some, but not all, intracellular forms of the Leu-4 antigen contained N-linked oligosaccharides of the high mannose type. By using messenger RNA from human T cell lines, intracellular forms of Leu-4 were shown to be accurately translated and properly processed by Xenopus oocytes in vitro.

Animals↗
PubMed1982

Biosynthesis and processing of a human T lymphocyte antigen.

The biosynthesis and processing of Leu-1, a human T lymphocyte antigen, has been studied with the use of a monoclonal antibody. This molecule exists on the cell surface as a 67,000 m.w. glycoprotein. Through a series of pulse-labeling studies, in conjunction with the use of the antibiotic tunicamycin and the enzyme Endo-H, the details of glycosylation, processing, and deposition at the cell membrane were examined. The protein backbone of the molecule is 58,000 m.w. High-mannose sugars are added to asparagine residues during synthesis. Within 20 min, these high mannose sugars are converted to complex type carbohydrates, including fucose. The fully processed glycoprotein appears at the cell surface within 30 min after synthesis. This sequence of events is similar to that for other cell surface glycoproteins, including HLA and vesicular stomatitus virus glycoprotein.

Acetylglucosaminidase↗
PubMed1979

Induction of secretion of IgM from cells of the B cell line 38c-13 by somatic cell hybridization.

Cells of the murine B cell line 38C-13 possess immunoglobulins of the IgM class on their surface but do not secrete them. Upon hybridization of 38C-13 cells with murine myeloma cells, hybridoma clones were obtained that secreted both pentameric IgM of 38C-13 origin and the myeloma protein. All hybridoma clones synthesized and secreted large amounts of homogeneous IgM with a half disappearance time of about 2 hr, typical of mature plasma cells. Concomitantly with the induction of IgM secretion, the hybridoma cells lost their surface IgM. The possibility of separate pathways for the synthesis of membrane and secreted IgM is discussed.

Animals↗
PubMed1978

B lymphocytes contain three species of mu chains.

The murine B cell line cloned from a single cell, 38C-13, synthesizes three species of mu chains, that of cell surface membrane IgM (m-mu), that of secreted IgM (s-mu) and that of intracellular IgM (i-mu). They differ in their mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Sequence analysis of the different mu chains suggests that they are identical in the N-terminal as well as in their C-terminal positions. The ratio between incorporated radioactive monosaccharides to radioactive amino acids into the three different mu chains was higher in s-mu than in m-mu, but nevertheless m-mu migrated more slowly than s-mu on sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, since this ratio may also be influenced by the rate of synthesis, it may not represent a real molar ratio of carbohydrate to protein. Studies with normal spleen cells clearly indicated the presence of the same three types of mu chains.

Amino Acid Sequence↗
PubMed1978

A quantitative fluorometric assay for detection and characterization of Fc receptors.

A new quantitative fluorometric binding assay that uses fluoresceinated aggregated IgG is proposed for the study of Fc receptors. The method was compared with a radiolabeling binding assay on three well characterized murine cell lines (38C-13, EL4, and BW). The apparent association constant of the binding and the amount of aggregated IgG bound per cell at saturation were calculated. The fluorometric assay enables the detection of 5 X 10(-10) M bound aggregated IgG. Inhibition studies with monomeric IgG, reduced and alkylated aggregated IgG, and aggregated F(ab')2 fragments of IgG confirmed the specificity of the assay. Staphylococcal protein A inhibited the binding of the aggregated IgG to Fc receptors.

Antibody Specificity↗
PubMed1977

Cell surface components of carcinogen-induced lymphoid tumors in SJL/J mice.

Preleukemic cells could be detected in the bone marrow cell population of SJL/J mice within several days after induction of leukemia by repeated feedings with a chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Bone marrow cells collected 7, 30 or 60 days following carcinogenic treatment, developed lymphoid tumors upon transplantation into syngeneic irradiated recipients. The incidence of these tumors varied between 40--45% when the bone marrow cells were collected and transferred 7--30 days after feeding with DMBA, and raised to an incidence of 80% when transferred 60 days after carcinogen administration (compared to 50% incidence in the DMBA-treated bone marrow donors). A survey of several cell surface components on the lymphoid tumor cells, obtained after transplantation of preleukemic cells, indicated that most of the tumor lines bore both the Thy-1.2 antigen (weak) and the Fc receptor, whereas the rest were positive only for the Fc receptor. None of these tumor cell lines would yield a significant amount of cell-bound immunoglobulin.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1977

Characterization of a carcinogen-induced murine B lymphocyte cell line of C3H/eB origin.

A carcinogen-induced lymphoid tumor, denoted 38C-13, obtained in a T cell-depleted mouse of C3H/eB strain, was adapted to continuous culture in vitro and characterized with respect to its cell surface components. The cells possess IgM class immunoglobulins on their surface but do not secrete it. This membrane IgM is composed of mu and L-chains that are similar in apparent molecular weight to those of an IgM myeloma protein. It is also homogeneous as revealed by isoelectric focusing. The cells possess Fc receptors but lack complement receptors as well as Thy-1 and Ia alloantigens. These characteristics indicate that 38C-13 cells are transformed counterparts of small B lymphocytes at an early stage of differentiation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1977

An IgM-producing tumor with biochemical characteristics of a small B lymphocyte.

A lymphocytic tumor, 38C-13, induced by the chemical carcinogen 7, 12-dimethylbenz(a)anthracene in C3H/eB mice and adapted to tissue culture, produces 7-8 S IgM with "core" carbohydrates (N-acetylglucosamines, mannoses), but not "branch" carbohydrates (neuraminic acids, fucoses, galactoses) attached to the mu heavy, but not to the light chains. Turnover of the 7-8 S 38C-13 IgM is slow (half disappearance time = 10-15 h). The IgM is released from the cells as 7-8 S IgM. The ratio of IgM synthesis to the synthesis of all cellular glycoproteins is 0.005-0.01. After comparison of these data with data obtained with normal B lymphocytes before and after mitogenic stimulation, we conclude that 38C-13 tumor cells are transformed counterparts very near or within the population of small, mitogen-sensitive, resting B lymphocytes.

9,10-Dimethyl-1,2-benzanthracene↗