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H D Royer

Publications and source records attributed to H D Royer.

49 records · Page 3Linked to original sources
PubMed1984

Genes encoding the Ti beta subunit of the antigen/MHC receptor undergo rearrangement during intrathymic ontogeny prior to surface T3-Ti expression.

To obtain further information about the ontogeny of the T-cell antigen/MHC receptor, a Ti beta subunit cDNA probe and heteroantisera specific for the Ti alpha and Ti beta subunits were utilized to characterize human T-lineage cells. Analysis of thymic tumors and normal thymocytes at both the DNA and protein levels demonstrates that Ti beta gene rearrangement is evident in stage II (T11+T6+T3-) and stage III (T11+T6-T3+), but not stage I (T11+T6-T3-) thymocytes. In contrast, surface expression of Ti alpha and Ti beta molecules is exclusively restricted to stage III thymocytes. Thus human T-lineage ontogeny is characterized by an orderly series of differentiation steps wherein Ti beta gene rearrangement precedes surface expression of the T3-Ti molecular complex.

Cloning, Molecular↗
PubMed1984

Purification and NH2-terminal amino acid sequencing of the beta subunit of a human T-cell antigen receptor.

To obtain information about the structural basis for T-cell antigen recognition, a T3-associated Ti receptor molecule was isolated from crude membranes of the REX human thymic tumor line and purified by affinity chromatography with an anti- clonotypic monoclonal antibody in conjunction with preparative gel electrophoresis. NH2-terminal amino acid sequencing of the beta subunit unambiguously identified the amino acids in positions 2-12. Comparative protein sequence analysis by computer search demonstrated that this Ti beta sequence bore weak, but definite, homology to the first framework of the variable region of human lambda light chain. Anti-sera to a synthetic peptide corresponding to positions 2-11 precipitated the denatured Ti beta subunit from REX, thus confirming the above sequence. This information suggests that the Ti beta subunit is distantly related to human immunoglobulin lambda light chain and, moreover, should be of use in the molecular cloning of the Ti beta gene.

Amino Acid Sequence↗
PubMed1984

Clonotypic surface structure on human T lymphocytes: functional and biochemical analysis of the antigen receptor complex.

Recent studies using cloned antigen-specific T lymphocytes and monoclonal antibodies directed at their various surface glycoprotein components have led to identification of the human T cell antigen receptor as a surface complex comprised of a clonotypic 90KD Ti heterodimer and the monomorphic 20/25KD T3 molecules. Approximately 30,000-40,000 Ti and T3 molecules exist on the surface of human T lymphocytes. These glycoproteins are acquired and fully expressed during late thymic ontogeny, thus providing the structural basis for immunologic competence. The alpha and beta subunits of Ti bear no precursor-product relationship to one another and are encoded by separate genes. The presence of unique peptides following proteolysis of different Ti molecules isolated by noncrossreactive anticlonotypic monoclonal antibodies supports the notion that variable regions exist within both the alpha and beta subunits. Moreover, N-terminal amino acid sequencing of the Ti beta subunit shows that it bears homology to the first V-region framework of immunoglobulin light chains and represents the product of a gene that rearranges specifically in T lymphocytes. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand (antigen/MHC), enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor crosslinking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.

Antibodies, Monoclonal↗
PubMed1980

Identification of 5-methylcytosine in DNA fragments immobilized on nitrocellulose paper.

A method to identify 5-methylcytosine (m5Cyt) in DNA immobilized on nitrocellulose paper by using antibody against m5Cyt raised in rabbits is described. Immobilized restriction fragments of DNA are incubated first with purified antibody against m5Cyt and then with goat anti-rabbit IgG labeled with 125I. Restriction fragments containing m5Cyt are visualized by autoradiography. By using this method, a heavily methylated fragment of about 1700 base pairs was identified in nuclear DNA fom Chinese hamster cells, the methylation pattern of calf thymus satellite I DNA was examined, and chloroplast DNAs that were extracted from various stage of the Chlamydomonas life cycle were compared. Little if any methylation was detected in chloroplast DNA from vegetative cells or from male gametes, whereas homologous DNAs from female gametes and from zygotes were heavily methylated. The sensitivity of the method was examined with calf thymus satellite I DNA (which contains approximately 40 m5Cyt residues per repeat unit of 1400 base pairs) and with phi X174 virion DNA (which contains a single m5Cyt per molecule). The presence of m5Cyt was detected with as little as 40 ng of phi X174 DNA containing 0.02 pmol of m5Cyt and with 100 ng of satellite DNA containing about 0.5 pmol of m5Cyt. Thus, the method makes possible the identification of individual methylated sites in purified DNAs in the size range of single genes.

5-Methylcytosine↗
PubMed1979

The organization of the ribosomal RNA genes of Chironomus tentans and some closely related species.

Southern gel analysis of total DNA from Chironomus tentans showed that the rRNA genes (rDNA) are homogeneous in structure. After cloning in Escherichia coli plasmid pBR313, the rDNA organisation was further studied by restriction fragment analysis and R-loop mapping. No heterogeneity could be detected by heteroduplex analysis of six different cloned rRNA cistrons. R-loop sizes of 1.69 and 3.63 kilobases (kb) were measured for the 18S and 28S rRNA coding sequences. The two spacers are 0.75 and 1.77 kb long. Southern gel analysis showed also a homogeneous rDNA structure for a Canadian population of C. tentans and C. pallidivittatus. The same technique indicated, however, that the rDNA of two other closely related species of C. thummi and C. melanotus is heterogeneous in structure. A possible correlation between this heterogeneity and the presence of heterochromatin in these species is discussed.

Animals↗
PubMed1979

Methylation of chloroplast DNAs in the life cycle of Chlamydomonas.

Methylation patterns of Chlamydomonas chloroplast DNAs (chlDNAs) were examined in the vegetative, gametic, and zygotic stages of the life cycle. Restriction endo-nuclease fragment patterns produced by EcoRI, BamHI, Hpa II, and Msp I were compared; the last two cleave DNA at the sequence C-C-G-G, but Hpa II is blocked by prior methylation of the internal cytidine whereas Msp I is not. chlDNAs from vegetative cells of both mating types showed no evidence of methylation at C-C-G-G. Gametic mt+ chlDNA was heavily methylated at C-C-G-G, whereas the homologous chlDNA from mt- gametes showed very slight methylation at C-C-G-G. Methylation of additional sites in chlDNA from mt+ gametes but not from mt- gametes was shown by blockage of some EcoRI and BamHI sites that were cleaved in the chlDNA from vegetative cells. chlDNA from 6-hr zygotes was much more methylated than gametic mt+ DNA, as shown by its almost total resistance to cleavage by all four restriction enzymes. These findings support and extend previous evidence that chlDNA of mt+ cells is methylated during gametogenesis and that further methylation occurs after gametic fusion in the young zygotes.

Journal Article↗
PubMed1979

Cloning of the structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12.

The gene (ompA) for the major outer membrane protein II* from Escherichia coli K-12 has been cloned on a 5-megadalton EcoRI fragment by using phage lambda as vector. The gene is expressed during the lytic cycle of the recombinant phage and the insoluble membrane-bound protein was detected in phage plaques with a simple radioimmunoassay. Transfer of the EcoRI fragment into plasmid pSC101 and expression in a host lacking protein II* led to overproduction of protein II* and decreased production of two other major outer membrane proteins. Expression of the plasmid pSC101-ompA+ in minicells derived from an ompA minicell-producing strain led to synthesis, at high rates, of this protein and massive accumulation of a second cell envelope protein most likely representing the biosynthetic precursor of protein II*.

Bacterial Proteins↗
PubMed1977

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

The non-tandem inverted duplication in the 2-mum DNA of Saccharomyces cerevisiae has a length of 0.19 mum and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and self-annealing of the 2-mum monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67mum+/-0.06 mum (S-loop) and 0.86 mum+/-0.05 mum (L-loop) length. Two additional size classes which comprised 5-10% of the measured molecules had contour lengths of around 1.7 mum and 2.1 mum. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-mum and 6-mum circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-mum monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.

Chromatography, Gel↗
PubMed1976

Non Xenopus-like DNA sequence organization in the Chironomus tentans genome.

We have examined the sequence organization of Chironomus tentans DNA by means of optical and hydroxyapatite renaturation kinetics of total DNA fragment sizes of 0.36, 2.6 and 13.5 kilobases (kb) as well as isolated middle repeat DNA at sizes of 0.36 and 13.5 kb. 90% of the DNA renatured as unique sequences of a genome of 0.20 pg with the balance of DNA renaturing as middle repetitive sequences present on averge 90 times per haploid genome. At a DNA fragment length of 13.5 kb, 35% of the DNA was trapped on the hydroxyapatite as middle repetiitive fraction. We concluded C. tentans DNA to have a mean repeat length of about 4.3 kb distributed throuth out at least 35% of the genome with an inter repeat spacing of at least 13.5 kb but possibly being distributed throughout the whole genome with an inter repeat spacing of 36 kb. This shows C. tentans DNA organization not to follow the most ubiquitous Xenopus model but to be similar to the organization of Drosophila melanogaster DNA.

Animals↗
PubMed1976

Synthesis of high molecular weight polypeptides in Escherichia coli minicells directed by cloned Saccharomyces cerevisiae 2-micron DNA.

The minicell-producing Escherichia coli strain P 678-54 was transformed with a series of defined PTY chimeric plasmids consisting of yeast 2-micron DNA and E. coli plasmid pCR1. In minicells the integrated 2-micron DNA from yeast directed specifically the synthesis of six polypeptides with apparent molecular weights of 15,000, 17,500, 20,000, 22,000, 37,000, AND 48,000. The specificity of five other polypeptides, which cover a molecular weight range of 19,000 to 28,000, has not yet been established with certainty. Neither the orientation of the integrated DNA, nor the inversion which distinguishes the two structural forms of 2-micron DNA affected the polypeptides synthesized. However, integration at a given EcoRI site appeared to be correlated with the absence of one particular polypeptide band; this suggests that at least one of these sites is located in an expressed region of the DNA.

DNA, Recombinant↗
PubMed1976

Characterization of 2-mum DNA of Saccharomyces cerevisiae by restriction fragment analysis and integration in an Escherichia coli plasmid.

Electrophoretic analysis of EcoRI and HindIII restriction fragments of 2-mum supercoiled DNA of Saccharomyces cerevisiae indicated that this class of DNA is heterogeneous and probably consists of two types of molecules. Integration of the 2-mum yeast DNA in E. coli plasmid pCR1 directly showed that existence of two types of molecules as each of these could be individually inserted into separate bacterial plasmids. The difference between the two types of 2-mum circles is due to an inversion of about 1.6 X 10(6) daltons. The inversion is flanked by a reversed duplicated sequence of 0.45 X 10(6) daltons. Possible implications of this structure are dicussed.

Chromosome Mapping↗
PubMedDate not supplied

Isolation of single stranded DNA from purified hepatitis A-virus.

Hepatitis A-virus was purified from human stools by three purification steps. Virus was identified by radioimmunoassay and purity monitored with immune electron microscopy. Virus particles, serologically and morphologically identical, banded in CsCl in two density ranges at 1.31-1.34 g/cm3 and at 1.41-1.43 g/cm3. Virions of density 1.31-1.34 g/cm3 were shown to contain single-stranded DNA of different size classes. Class I 1.33 kb, class II 4.61 kb in addition a small amount of molecules was detected with lengths up to 15 kb.

DNA, Single-Stranded↗