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Biomedical subjects

G H Rosenberg

Publications and source records attributed to G H Rosenberg.

14 recordsLinked to original sources

Marsupial light chains: IGK with four V families in the opossum Monodelphis domestica.

A full-length and several partial cDNAs encoding IGK light chains from the marsupial South American opossum, Monodelphis domestica, were isolated and characterized. Using these clones as a starting point, the expressed IGKV repertoire was sampled by anchored polymerase chain reaction using an IGKC-specific primer. Based on nucleotide sequences of twenty unique, expressed IGKV-J combinations, there are at least four IGKV families and two J segments. Southern blot analysis revealed each IGK-V family contains multiple gene segments totaling at least thirty-five IGKV in the opossum genome. No evidence for particular, recurrent IGKV-J combinations in the opossum IGK repertoire was seen, rather the V-J combinations appeared random and diverse. Each of the four IGKV families appear more closely related to V segments from placental mammals than to each other, suggesting the duplication of the IGKV families prior to the separation of marsupials and placental mammals more than one-hundred-million years ago. Overall, the complexity of opossum light chain V segments appears greater than that found in the heavy chain, and light chains are likely to contribute significantly to Ig diversity in this species. With this report, the homologues encoding all three classes of eutherian Ig chains, IGH, IGL, and IGK, have been described in a non-placental mammal.

Amino Acid Sequence↗

Marsupial light chains: complexity and conservation of lambda in the opossum Monodelphis domestica.

The Ig lambda chains in the South American opossum, Monodelphis domestica, were analyzed at the expressed cDNA and genomic organization level, the first described for a nonplacental mammal. The V lambda segment repertoire in the opossum was found to be comprised of at least three diverse V lambda families. Each of these families appears to be related to distinct V lambda families present in placental mammals, suggesting the divergence of these genes before the separation of metatherians and eutherians more than 100 million years ago. Based on framework and constant region sequences from full-length cDNAs and intron sequences from genomic clones, it appears that there are multiple functional J lambda-C lambda pairs in the opossum locus. The opossum J lambda-C lambda sequences are phylogenetically clustered, suggesting that these gene duplications are more recent and species specific. Sequence analysis of a large set of functional, expressed V lambda-J lambda recombinations is consistent with an unbiased, highly diverse lambda light chain repertoire in the adult opossum. Overall, the complexity of the Ig lambda locus appears to be greater than that found in the Ig heavy chain locus in the opossum, and light chains are therefore likely to contribute significantly to Ig diversity in this species.

Amino Acid Sequence↗

V(H) repertoire of a marsupial (Monodelphis domestica).

When contrasted with information available for placental mammals, very little is known about the development of immunocompetence in marsupials. Marsupials, however, provide interesting immunology problems, since most appear to be born at a stage of development much less mature than that of placental mammals. To further understand the marsupial immune system, the Ig repertoire of the short-tailed opossum, Monodelphis domestica, was characterized. The majority of the V(H) clones were isolated in an unbiased manner by screening a spleen cDNA phage library, using C region probes, or anchored PCR, using C region-specific primers paired with vector specific primers. Analysis of 54 unique V(H) sequences from this marsupial revealed the presence of two V(H) families in the expressed Ig repertoire. The larger family, which contributed the majority of the clones identified, appears to be derived from 10 to 12 germline V(H) segments. The second family of clones is derived from a single germline V(H). Both V(H) families are related to the group III sequences described in other vertebrates. Unusual codon bias differences between the two families may result in very different patterns of somatic mutation within the opossum Ig repertoire.

Amino Acid Sequence↗

Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe.

The architectural features of 73 introns found in 36 genes of the fission yeast Schizosaccharomyces pombe have been compiled and tabulated. The introns from S. pombe can be grouped into two size classes. Intron features are discussed in comparison to intron features of Saccharomyces cerevisiae and other eukaryotes. The results indicate that S. pombe displays quite different architectural features than the budding yeast S. cerevisiae. However, particularly in the 3' region, S. pombe introns also appear to differ from mammalian introns.

Base Sequence↗

prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns.

We have generated a bank of temperature-sensitive (ts) Schizosaccharomyces pombe mutant strains. About 150 of these mutants were transformed with a ura4 gene containing an artificial intron. We screened these ts mutants for mutants deficient in splicing of the ura4 intron. With this approach three mutants were isolated which have a general defect in the splicing process. Two of these mutants fall into the prp1 complementation group and one defines a new complementation group, prp4.

Genes, Fungal↗

Frequencies of Band 3 variants of human red cell membranes in some different populations.

A variant of Band 3, the major protein of the erythrocyte membrane, was observed by Mueller and Morrison in 1977 in 6-7% of healthy blood donors on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes treated with pronase. Pronase treated red cells containing this first recognized variant [here designated 'Band 3-Memphis (m)'] section had two bands of about 63,000 and 60,000 Mr while pronase treated normal cells had only the lighter Mr band. The present study includes data on the frequency of variants resembling Band 3-Memphis in patients of different ethnic groups and on random donors obtained earlier in Memphis. These variants were detected by the original method of Mueller and Morrison and were not associated with recognized clinical or haematological abnormalities. Significantly higher gene frequencies for the variants of the (m) type were observed in American Indians, African Americans and Filipinos than in Caucasians; putative heterozygotes and homozygotes were identified among each of these groups. The frequency of silent Band 3 polymorphisms in different populations should be considered in the interpretation of clinical findings associated with the presence of Band 3 variants.

Anion Exchange Protein 1, Erythrocyte↗

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product. This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene. Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly. A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site. No functional product is produced from this transcript. An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product. Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns. We discuss the possibility that S. pombe introns represent a simple type of eucaryotic intron.

Base Sequence↗

Quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels onto positively charged nylon membranes.

A method for the reproducible and quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a single sheet of either Zetabind or Gene Screen Plus membranes is presented. This procedure uses commercially available equipment and includes three crucial parameters: the omission of methanol from the transfer buffer, the use of thin (0.75-mm) resolving gels, and a newly developed protocol for pretreatment of the polyacrylamide gel after electrophoresis and before electroblotting. This combination of parameters yields a blot that both qualitatively and quantitatively reflects the proteins in the original polyacrylamide gel.

Electrochemistry↗

Further characterization of T cell receptor chains of marsupials.

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.

Amino Acid Sequence↗

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials.

cDNAs encoding IgM heavy chain constant region (Cmu) were isolated from two metatherians (marsupials)--the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cmu in both species. The domain size and structure of the marsupial Cmu sequences were compared with other Cmu sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.

Amino Acid Sequence↗