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Biomedical subjects

L M Staudt

Publications and source records attributed to L M Staudt.

At least 55 records · Page 3Linked to original sources
PubMed1993

Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes.

The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.

Amino Acid Sequence↗
PubMed1992

Oct-3 and mammalian development: correction of discussion.

The title of the 5 June report on page 1445 by R. C. deL. Milton et al. should have been "Total chemical synthesis of a D-enzyme: The enantiomers of HIV-1 protease show reciprocal chiral substrate specificity." Figure 3 in the same report (p. 1447) was inadvertently printed upside down. The labels "L-HIV protease" and "D-HIV protease" were therefore under the wrong illustrations. The correct figure is printed below. [See figure in the PDF file]

Animals↗
PubMed1992

Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen.

Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes. All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2. To address the role of Oct-2 in the T-cell lineage, we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells. Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells. In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek, Oct-2 was induced by antigen stimulation, with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h. Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A, as well as by protein synthesis inhibitors. These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation. The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes.

Animals↗
PubMed1991

Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin.

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.

Amino Acid Sequence↗
PubMed1991

Oct-3 is a maternal factor required for the first mouse embryonic division.

Oct-3 is a POU domain transcription factor that binds the octamer DNA motif and is present in mouse oocytes before and after fertilization. When fertilized oocytes were injected with antisense Oct-3 oligonucleotides or double-stranded DNA containing the octamer motif, embryonic DNA synthesis was inhibited and the embryos were arrested at the one-cell stage. In vitro synthesized Oct-3 mRNA rescued the developmental block induced by antisense Oct-3 oligonucleotide. We conclude that maternally inherited Oct-3 is required for DNA replication and division of the one-cell embryo.

Animals↗
PubMed1991

A B cell population that dominates the primary response to influenza virus hemagglutinin does not participate in the memory response.

The early primary B cell response of BALB/c mice to the influenza virus A/PR/8/34 (PR8) hemagglutinin (HA) is dominated by B cells that utilize a single V kappa gene in association with one of two closely related VH genes. We have used an anti-idiotypic reagent that recognizes a light chain-associated idiotope (23-1 Id) on these antibodies to follow their presence during the anti-HA response. Quantitation of 23-1Id+ antibodies at different time points during the anti-HA response indicates that the 23-1Id+ B cell response peaks early after primary immunization and is not re-induced by secondary challenge. Furthermore, 23-1Id+ titers in serum decay rapidly between the first and second week after immunization, and the HA-specific 23-1Id+ precursor B cell population does not significantly expand in the months following immunization. These results indicate that despite their predominance during the primary response, 23-Id+ B cells abruptly disappear from the response and do not mature into memory B cells.

Animals↗
PubMed1991

Chromosomal location of the octamer transcription factors, Otf-1, Otf-2, and Otf-3, defines multiple Otf-3-related sequences dispersed in the mouse genome.

Chromosomal locations have been assigned for the octamer transcription factor, Otf, gene family (previously named the octamer-binding protein, Oct, gene family) using an interspecific backcross of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice and the BXH recombinant inbred strains. Molecular probes for Otf-1 and Otf-2 recognized single loci on mouse chromosomes 1 and 7, respectively, whereas probes for Otf-3 recognized a minimum of eight independently segregating loci (designated Otf-3a through Otf-3h). Members of the Otf-3 family mapped to mouse chromosomes 1, 2, 3, 6, 14, 17, and the X chromosome, indicating that the Otf family has become widely dispersed during evolution. Several Otf loci mapped near developmental mutations, raising the possibility that these mutations result from defects in Otf family members.

Animals↗
PubMed1991

Immunoglobulin gene transcription.

Transcriptional regulation of the immunoglobulin (Ig) genes is one of the best-studied systems for understanding tissue-restricted gene control. Dissection of the Ig genes reveals that each contains multiple regulatory elements that are preferentially active in B lymphocytes. These include a promoter and one or more enhancer elements. Biochemical analysis and cloning of the genes for transactivator proteins has uncovered a variety of distinct proteins that are often members of gene families. The transactivators themselves are under complex regulation at the transcriptional level as well as by interactions with other proteins in the cell. It appears that the coordinate action of a select constellation of these transactivators conveys the information for tissue-specific expression of the Ig genes.

Animals↗
PubMed1990

A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR/8/34 influenza virus hemagglutinin.

Approximately 50% of the primary antibody response of BALB/c mice to the A/PR/8/34 influenza virus hemagglutinin is directed to the Cb site, one of the four major antigenic regions of the molecule. To determine the structural basis of the anti-Cb site response, we have examined the paratypic and genetic diversity exhibited by a panel of 24 primary and 4 secondary response mAb specific for this antigenic region. Reactivity pattern analysis demonstrated 20 distinct fine specificities among these antibodies, and V region gene sequence analysis showed that they are encoded by 17 different VH gene segments from 6 VH gene families and 14 different VK gene segments from 6 VK gene groups. Despite this overall diversity, many of the antibodies can be placed in a limited number of sets based on the shared expression of VH and/or VK genes. One set contains antibodies encoded by a single gene of the VK4/5 group in combination with one of two closely related genes from the J558 VH family. This set accounts for half of the Cb site-specific primary response hybridomas, indicating that the representation of the various anti-Cb site B cell specificities during the primary response to A/PR/8/34 influenza virus is not uniform. The preferential participation of B cells expressing this VH/VK combination is largely responsible for the dominance of anti-Cb site antibodies in the primary anti-hemagglutinin response.

Amino Acid Sequence↗
PubMed1990

A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo.

The murine oct-3 gene encodes a transcription factor containing a POU-specific domain and a homeodomain. In marked contrast to other homeodomain-encoding genes, oct-3 is expressed in the totipotent and pluripotent stem cells of the pregastrulation embryo and is down-regulated during differentiation to endoderm and mesoderm, suggesting that it has a role in early development. The oct-3 gene is also expressed in primordial germ cells and in the female germ line.

Amino Acid Sequence↗
PubMed1990

V region gene usage and somatic mutation in the primary and secondary responses to influenza virus hemagglutinin.

Most (80 to 90%) primary antibodies specific for the Sb site of influenza virus (A/PR/8/34) hemagglutinin share an Id (designated C4). Secondary antihemagglutinin(Sb) antibodies also exhibit the C4 Id although less frequently (10 to 15%). We have analyzed the V region nucleotide sequences of primary and secondary antibodies with the C4 Id. Primary C4 antibodies are encoded by the same Vk gene, belonging to the Vk8 group, usually rearranged to Jk5. The H chains are diverse, encoded by VH genes belonging to at least four different VH families, a variety of DH genes, and either JH2, JH3, or JH4. There is only one somatic mutation among seven Vk and two VH genes encoding primary C4 antibodies. Secondary C4 antibodies are also encoded by the same Vk8-Jk5 gene segment and by diverse VH genes. Additional heterogeneity in the secondary response is caused by somatic mutation.

Amino Acid Sequence↗
PubMed1990

Characterization of chicken octamer-binding proteins demonstrates that POU domain-containing homeobox transcription factors have been highly conserved during vertebrate evolution.

The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarily conserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, we identified two homologous octamer-binding proteins in chicken B cells. When the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, we sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, we were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. Low-stringency screening of a chicken embryonic cDNA library allowed us to clone one of these genes. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. The predicted protein sequence of the chicken Oct-1 gene demonstrated that the gene for Oct-1 has been highly conserved during vertebrate evolution with an overall 96% amino acid sequence identity between the chicken and human proteins. The previously described POU domain (termed POU for its presence in the Pit-1, Oct-1/Oct-2, and Unc-86 genes) and homeobox domain are 100% conserved between the two protein products. Together, our data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.

Amino Acid Sequence↗
PubMed1989

fos/jun and octamer-binding protein interact with a common site in a negative element of the human c-myc gene.

A negative element has previously been localized to a 57-base pair segment approximately 300 base pairs upstream of the human c-myc promoter P1. Within this element, a 26-base pair region was protected in vitro from DNase I digestion with a HeLa cell nuclear factor(s). Two specific DNA-protein complexes were identified in gel retardation assays using HeLa cell nuclear extracts and an oligonucleotide probe spanning the footprinted region. Exonuclease and chemical footprint analyses suggested that the binding sites for both complexes are almost entirely overlapping. One of the complexes was eliminated by oligonucleotide competitors possessing known AP-1 binding sites. This same complex reacted strongly with anti-fos immunoglobulin suggesting a role for c-fos in governing c-myc expression. Precipitation of fos protein bound to c-myc DNA that was immobilized on beads confirmed the involvement of c-fos in a specific complex with the c-myc upstream sequence. In contrast, the other complex seen by the c-myc probe could not be competitively inhibited by AP-1 binding sites and was not affected by anti-fos antibody. Instead, this complex was efficiently eliminated by unlabeled oligonucleotides containing the octamer DNA motif found in immunoglobulin gene promoters. Purified octamer-binding proteins formed stable complexes with the 26-base pair c-myc sequences. These results demonstrate that degeneracy in the consensus recognition sequences of these distinct factors allows each of them to bind the c-myc negative element. The interaction of known transcriptional activators with a negative element suggests that the same factors can mediate both transcriptional activation and repression.

Base Sequence↗
PubMed1988

A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain.

The homeobox domain is shared by Drosophila homeotic proteins, yeast mating type proteins, and some functionally uncharacterized mammalian proteins. A lymphoid-restricted human protein that binds to the immunoglobulin octamer regulatory motif was shown to contain an amino acid sequence that has 33% amino acid identity with the consensus sequence of the previously cloned homebox domains. This homeobox gene was localized to chromosome 19, thus mapping separately from other human homebox genes. A mutant protein containing amino acid substitutions within a putative helix-turn-helix motif in the homeobox domain did not bind DNA detectably. This human homeobox protein was shown to bind the same DNA sequence as the homeobox domains of the yeast mating type proteins and Drosophila homeotic protein, suggesting that homeobox proteins may have closely related DNA binding characteristics.

Amino Acid Sequence↗
PubMed1988

Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.

An octamer DNA sequence plays a critical role in directing transcription of immunoglobulin genes in B lymphocytes. A new technique of direct binding of radioactive DNA was used to screen a complementary DNA expression library from the BJAB cell line in lambda gt11 phage to derive molecular cDNA clones representing a putative B lymphocyte-specific octamer binding protein. The plaques were screened with DNA containing four copies of the octamer sequence and positive phage recombinants were identified. The fusion protein produced on inducing a lysogen of one phage bound to a monomeric octamer probe. The cDNA insert from this phage hybridized to messenger RNA found in B lymphocytes, but not in most other cells. Thus, this cDNA derives from a gene (oct-2) that specifies an octamer binding protein expressed preferentially in B lymphocytes, proving that, for at least one gene, a cell-specific transcription factor exists and its amount is controlled through messenger RNA availability.

Cloning, Molecular↗
PubMed1986

Structural and functional implications of a restricted antibody response to a defined antigenic region on the influenza virus hemagglutinin.

A group of hybridoma antibodies that recognize structurally overlapping epitopes on the influenza virus hemagglutinin have been analyzed for the sequence of their immunoglobulin heavy and light chain variable regions. All VH regions derive from the same gene family, and only two Vk genes, from different families, are involved. The repetitive and restricted use of these variable region genes indicates that considerable structural requirements influence the generation of antibodies specific for this region of the hemagglutinin. The degree of amino acid variability which is permissive for interaction with this region suggests that two thirds of the possible replacement mutations may abolish either antibody function or specificity. Analysis of the somatic mutation which occurred in the individual antibodies indicates that the light chains acquired replacement mutations at the rate predicted for random mutation. The heavy chains, however, accumulated a 3-fold excess of replacement mutations over that predicted for random accumulation, correlating with the dominant role they apparently play in determining fine differences in the specificity of these antibodies. The effect of somatic mutation on the clonal amplification and diversification of these B cell lineages is discussed.

Animals↗