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T Manser

Publications and source records attributed to T Manser.

67 records · Page 4Linked to original sources
PubMed1985

Shared idiotopes among antibodies encoded by heavy-chain variable region (VH) gene members of the J558 VH family as basis for cross-reactive regulation of clones with different antigen specificity.

A wide idiotype cross-reactivity was observed among six groups of monoclonal antibodies specific for arsonate and nitrophenyl haptens, hemagglutinin of PR8 and X31 influenza viruses, dextran, A48-idiotype, and a set of six monoclonal antibodies with unknown antigenic specificity. All of these antibodies are encoded by heavy-chain variable region (VH) genes belonging to the J558 VH family. This idiotypic cross-reactivity was determined by studying the binding of these antibodies to a panel of six monoclonal anti-idiotype antibodies, each one raised against a member of the six groups of monoclonal antibodies. The administration at birth of two such monoclonal anti-idiotype antibodies induced a long-lasting suppression not only of the corresponding idiotype but also of VH-related idiotypes with different antigenic specificities. These results suggest that the idiotypes encoded by VH genes that belong to the same VH gene family are interactive one with another. The possible physiological consequences of this immunochemical cross-reactivity are discussed.

Animals↗
PubMed1985

Human U1 small nuclear RNA genes: extensive conservation of flanking sequences suggests cycles of gene amplification and transposition.

The DNA immediately flanking the 164-base-pair U1 RNA coding region is highly conserved among the approximately 30 human U1 genes. The U1 multigene family also contains many U1 pseudogenes (designated class I) with striking although imperfect flanking homology to the true U1 genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb) regions of the human genome spanning U1 homologies. Two clones contain one true U1 gene each, and the third bears two class I pseudogenes 9 kb apart in the opposite orientation. We show by genomic blotting and by direct DNA sequence determination that the conserved sequences surrounding U1 genes are much more extensive than previously estimated: nearly perfect sequence homology between many true U1 genes extends for at least 24 kb upstream and at least 20 kb downstream from the U1 coding region. In addition, the sequences of the two new pseudogenes provide evidence that class I U1 pseudogenes are more closely related to each other than to true genes. Finally, it is demonstrated elsewhere (Lindgren et al., Mol. Cell. Biol. 5:2190-2196, 1985) that both true U1 genes and class I U1 pseudogenes map to chromosome 1, but in separate clusters located far apart on opposite sides of the centromere. Taken together, these results suggest a model for the evolution of the U1 multigene family. We speculate that the contemporary family of true U1 genes was derived from a more ancient family of U1 genes (now class I U1 pseudogenes) by gene amplification and transposition. Gene amplification provides the simplest explanation for the clustering of both U1 genes and class I pseudogenes and for the conservation of at least 44 kb of DNA flanking the U1 coding region in a large fraction of the 30 true U1 genes.

Base Sequence↗
PubMed1985

The generation of major and minor idiotype-bearing families of anti-p-azophenylarsonate antibodies; stochastic utilization of VH gene segments.

An average of 50% of anti-p-azophenylarsonate (Ars) antibodies bear a cross-reactive idiotype, IdCR, and an average of 15% bear a relatively minor idiotype, Id, in A/J mice. To begin to investigate the processes that influence the expressed levels of these idiotype-bearing antibodies in serum, we have examined the frequency among preimmune B cells of cells that utilize the heavy chain variable region gene segment (VH) needed for IdCR and that which is needed for Id anti-Ars antibody expression. Our results indicate these VH gene segments are functionally rearranged at frequencies one would expect for random usage. The frequency of VH gene segment utilization is similar to, if not higher than, that of VHCR, arguing that the predominance of IdCR-over Id-bearing antibodies is not due to preferential usage of the VHCR gene segment. In addition to the analysis of Ars-immune sera pooled from several mice, we have examined 20 individual A/J mice to determine whether the relative serum levels of IdCR- and Id-bearing antibodies are strictly regulated relative to each other. Among individuals, we find that IdCR and Id antibody levels fluctuate over a 28-fold and a 120-fold range, respectively. The ratio of IdCR to Id antibody levels was found not to be strictly regulated, varying over a 300-fold range. Linear regression analysis of IdCR relative to Id concentrations shows a correlation coefficient of only 0.093. Indeed, rare mice can be found that generate greater levels of Id-bearing antibodies than those bearing IdCR. These results are indicative of a stochastic process involved during the generation of these IdCR-and Id-bearing antibody families. Models accounting for the generation of this highly variable serologic response derived from a preimmune repertoire in which VH gene segments are equivalently utilized are discussed.

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PubMed1984

Influence of clonal selection on the expression of immunoglobulin variable region genes.

The humoral immune response of the mouse to certain antigens is characterized by the dominant expression of a single or limited number of related, immunoglobulin variable region (V) structures by antibody-secreting lymphocytes. Such dominance could be due to preferred expression of these V regions in the B cell population prior to the immune response or could result from the action of selective or regulatory mechanisms during the immune response. Expression of a heavy chain variable region (VH) gene segment that partially encodes a V region structure that dominates the immune response to para-azophenylarsonate (Ars) in strain A mice was examined in the B cell population of Ars nonimmune mice. This VH gene segment participates in encoding several hundred thousand different V region structures expressed in this B cell population. The immune system is therefore capable of recurrently selecting a single V region structure from such a repertoire for dominant expression by antibody-secreting lymphocytes during an immune response.

Animals↗
PubMed1984

Localization of human U1 small nuclear RNA genes to band p36.3 of chromosome 1 by in situ hybridization.

U1 small nuclear RNA (U1 snRNA) is encoded by a large family of genes (30-125 copies/haploid genome) which are transcribed by RNA polymerase II. U1 snRNA is thought to function in gene splicing. Since the U1 genes were found to be greater than 20 kb apart by analyzing genomic phage clones, the chromosomal location of U1 genes in the human genome was determined using Southern filter analysis of DNA isolated from human-rodent somatic cell hybrids and by in situ hybridization. Human DNA digested with PvuII and probed with a U1-specific probe, pD2, show several major hybridizing fragments. Of these, two human PvuII fragments of 1.4 kb and 2.4 kb had unique mobilities compared to mouse fragments. In a study of 19 cell hybrids, the human-specific U1 fragments segregated with the chromosome 1 markers peptidase C and adenylate kinase 2. All other chromosomes showed greater than or equal to 19% discordancy . An additional 13 karyotyped cell hybrids, analyzed by Southern filter analysis, confirmed the assignment of this class of U1 genes to chromosome 1. Additional digests with MspI and PstI indicated that most U1 genes are located on chromosome 1. To determine if the U1 RNAs are located predominantly at one site or dispersed over chromosome 1, a tritium-labeled U1 probe was hybridized in situ to metaphase chromosomes. The majority of the grains were at band 1p36 .3, suggesting that most of the U1 genes are located in this region.

Animals↗
PubMed1984

Isolation of hybridomas expressing a specific heavy chain variable region gene segment by using a screening technique that detects mRNA sequences in whole cell lysates.

A technique is described that allows single hybridoma cell colonies to be assayed for the productive rearrangement of a single immunoglobulin variable region (V) gene segment by utilizing expression of V mRNA for analysis. Hybridomas growing in microwell tissue culture plates are lysed in situ, cellular RNA is directly transferred to nitrocellulose by filtration, and specific immunoglobulin mRNA is detected by hybridization of the filter with a DNA probe. The method is simple and sensitive. A single species of mRNA can be detected in a lysate of 1000 cells; 5000 hybridoma colonies can be easily screened per day. The technique has been successfully used to isolate cell lines from nonimmune mice expressing a particular heavy chain variable region (VH) gene segment.

Animals↗
PubMed1982

Human U1 loci: genes for human U1 RNA have dramatically similar genomic environments.

We have cloned and sequenced several distinct loci from the human genome whose sequences agree exactly and are colinear with the sequence of the small nuclear RNA U1. There appear to be 100-150 such U1 loci in the human genome. Although these loci are not in close clusters or in small tandem repeats in the genome, the majority of them have remarkably similar genomic environments. Several potential RNA polymerase termination sites exist in the immediate 3' flanking regions of the loci studied; however, no TATA box is found in the immediate 5' flanking sequences. These observations, with the findings of others concerning the transcription of U1, suggest that U1 RNA is synthesized from a family of transcription units. These data are consistent with the proposal that RNA polymerase II may synthesize primary transcripts from these transcription units, which are processed at both the 5' and 3' ends to yield mature U1 RNA and possibly other RNA species of unknown function.

Base Sequence↗
PubMed1981

Direct repeats flank three small nuclear RNA pseudogenes in the human genome.

We previously demonstrated that pseudogenes complementary to the small nuclear RNAs U1, U2 and U3 are dispersed and abundant in the human genome. Here we report that three pseudogenes (U1.101, U2.13 and U3.5) are flanked by perfect short direct repeats of 16 to 19 base pairs. In all three pseudogenes. the upstream direct repeat abuts a DNA sequence corresponding to the 5' end of the mature snRNA; in U2.13 and U3.5, the downstream direct repeat immediately follows the truncated 3' end of the snRNA sequence, whereas in U1.101, the downstream direct repeat is separated from the 3, end of the full-length snRNA sequence by a short A-rich region. We consider the direct repeats to be an indication that these three pseudogenes were created by insertion of snRNA information into a new chromosomal locus. To explain why the upstream repeat abuts a DNA sequence complementary to the 5' end of the mature snRNA, we propose a model for insertion that uses a reverse transcript of the snRNA as an intermediate. Furthermore, we note similarities between the structure of all three pseudogene loci and the Alu family of middle repetitive DNA sequences. These similarities suggest that some Alu family sequences are mobile genetic elements that can transpose to new chromosomal loci using as an intermediate a cDNA copy of an RNA transcribed from the Alu family element by RNA polymerase III.

Animals↗
PubMed1980

NAD turnover in microplasmodia of physarum polycephalum.

The rate of NAD turnover in microplasmodia of Physarum polycephalum was investigated using a double labeling technique with (14C)-adenine or adenosine and (3H)-nicotinamide. The half-life of an NAD molecule in Physarum was estimated to be 25 min, which is shorter than in either E. coli or human cell lines. The half-life of NAD in the presence of an inhibitor of NADase and poly ADPR synthase, 5-methylnicotinamide, was also investigated, but found to be indistinguishable from controls. The possible reasons for this and for the rapid turnover is discussed in the light of the known functions for NAD in prokaryotes and eukaryotes.

NAD↗
PubMed1980

Arginine-rich histones do not exchange between human and mouse chromosomes in hybrid cells.

Following division of HeLa-3T3 heterokaryons, human and mouse chromosomes occupy distinct regions within the resulting hybrid nuclei. This favorable orientation of genomes has allowed us to determine whether histones exchange between chromosomes in vivo. Acrylamide gel electrophoresis of the proteins from HeLa cells labeled with 3H-arginine during S phase showed that the core histones were labeled preferentially, constituting 30% of the total cellular tritium and 50% of the label in a crude nuclear fraction. Autoradiographic analysis of cells formed by fusion of 3H-arginine-labeled HeLa cells and 3T3-4E cells showed that 3H-arginine-labeled proteins did not migrate between nuclei in heterokaryons; hybrid cells formed from such heterokaryons contained nuclei in which 3H proteins occupied a sector within the nucleus; "sectored nuclei" could persist for at least 4 days; and the unequal distribution of 3H proteins did not change during DNA synthesis. Electron microscopic examination of hybrid nuclei failed to reveal a physical partition between human and mouse chromosome sets. Sectored nuclei were also observed in synkaryons derived from 3H-arginine-labeled HeLa and unlabeled HeLa cells, indicating that the unequal distribution of 3H-arginine-labeled proteins in HeLa-3T3 hybrid cells did not result from species-specific binding of proteins and DNA. The persistent unequal distribution of 3H-arginine-labeled proteins within hybrid nuclei in the apparent absence of a barrier between mouse and human chromosomes indicates that histones, the principal 3H-arginine-labeled proteins do not dissociate from DNA in vivo.

Animals↗
PubMedDate not supplied

Effect of IFN-gamma on the immune response in vivo and on gene expression in vitro.

T lymphocytes produce a variety of immunoregulatory molecules including gamma interferon (IFN-gamma) and antigen-specific suppressor and enhancer factors. During our studies of active substances obtained from cloned T-cell lines, we observed that certain fractions administered to mice resulted in enhancement of immune responses. Preliminary characterization of the substance suggested that it could be IFN-gamma and we therefore undertook a study of the action of IFN-gamma produced by recombinant DNA methodology on immune responses. We found that for several antigens, administration of IFN-gamma to mice leads to two- to five-fold enhancement of antibody formation provided that the IFN-gamma and antigen are administered together. The effect was dose dependent, giving a maximal response at 500-600 anti-viral units per mouse. Preliminary studies suggest that the macrophage may be the target of IFN-gamma action. Addition of IFN-gamma to cultures of a macrophage cell line leads to a greater than 10-fold increase in the level of RNA coding for I-region-encoded cell surface molecules.

Animals↗
PubMedDate not supplied

Expression of a VHC kappa chimaeric protein in mouse myeloma cells.

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.

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