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Biomedical subjects

R Grassmann

Publications and source records attributed to R Grassmann.

26 records · Page 2Linked to original sources
PubMed1991

A pair of selectable herpesvirus vectors for simultaneous gene expression in human lymphoid cells.

The genome of Herpesvirus saimiri, a lymphotropic virus of non-human primates, was used to develop a vector system for transducing foreign genes into primary human T-cells and T-lymphoid cell lines. Recombinant viruses were obtained by homologous recombination of the viral genome with linearized plasmid DNA. The plasmid used contained a fragment of virion DNA, a hygromycin-B-resistance marker (HyR), and a multiple cloning site for the insertion of additional expression cassettes. The resulting recombinants were efficiently enriched and were plaque-purified. The virus mediating HyR and a H. saimiri strain carrying the Geneticin-resistance marker were used to infect the human T-lymphoid cell line Jurkat. Lymphocytes with a double-resistant phenotype were shown to contain the two different H. saimiri recombinants persisting as episomes at high multiplicity. The H. saimiri vector system will be suitable to study cooperating regulatory genes in T-lymphocytes.

Base Sequence↗
PubMed1991

Persistence of selectable herpesvirus saimiri in various human haematopoietic and epithelial cell lines.

Herpesvirus (h.) saimiri, an infectious agent of squirrel monkeys, is capable of persisting in T lymphocytes of various primate species. It has been used as a vector for the functional analysis of regulatory genes in primary human T lymphocytes. As it is not yet known whether other cell types are capable of supporting viral persistence, various human cell lines were investigated using selectable h. saimiri recombinants. The lines chosen represent cells from the epithelium and connective tissue as well as from all haematopoietic lineages, i.e. cells of B and T lymphoid origin as well as myeloid-, fibroblast- and carcinoma-derived cultures converted to Geneticin or hygromycin B resistance, and harbouring episomal DNA of the selectable recombinants. The Burkitt's lymphoma-derived cell line Raji also contained simultaneously persisting episomes of the Epstein-Barr virus. Most of the cell cultures except a pancreatic carcinoma line and foreskin fibroblasts did not produce infectious virus. These observations show that a herpesvirus genome can persist episomally in a broad range of cultured cell types. The variety of infectable cell types and species suggests the presence of a widely distributed and well conserved virus receptor for h. saimiri. Thus the h. saimiri genome could be applied more generally as a vector.

Blotting, Northern↗
PubMed1991

In vitro binding of human T-cell leukemia virus rex proteins to the rex-response element of viral transcripts.

Human T-cell leukemia virus (HTLV-I, HTLV-II) rex protein function is required for the cytoplasmic expression of incompletely spliced viral transcripts encoding structural proteins. The effect is mediated by a cis-acting rex-response element (RRX) which is located near the 3' end of all viral mRNAs. We show that rex polypeptides of HTLV-I and HTLV-II expressed in Escherichia coli are capable of specifically binding RRX-containing transcripts of both viruses in cell-free assays. Binding analyses with deletion variants of rex proteins revealed a domain with RNA-binding activity in the first 77 N-terminal amino acids. Removal of a basic peptide of 19 amino acids from the N terminus abrogated RNA binding, whereas a beta-galactosidase fusion protein containing this peptide bound to the RRX. These results suggest that direct binding of rex protein to the RRX is important for rex-mediated regulation of viral gene expression and that a short stretch of positively charged amino acids contributes to the specific binding of rex to its target RNA.

Carrier Proteins↗
PubMed1989

Transformation to continuous growth of primary human T lymphocytes by human T-cell leukemia virus type I X-region genes transduced by a Herpesvirus saimiri vector.

The role of the X region of the genome of the human T-cell leukemia virus type I (HTLV-I) in the immortalization of lymphocytes has been difficult to distinguish from its role in viral replication as this region encodes at least two genes, tax and rex, required for replication and the expression of viral proteins. To determine whether the X region does encode immortalizing functions, a fragment of the HTLV-I provirus capable of expressing known X-region proteins was inserted into the genome of a transformation-defective, replication-competent Herpesvirus saimiri. Infection of fresh mitogen-activated human cord blood and thymocytes yielded immortal T-cell lines that had the same phenotype (CD4+, CD5+, HLA class II+, interleukin 2 receptor alpha-chain +) as lymphocytes transformed by cocultivation with HTLV-I. These experiments demonstrate that the X region encodes the functions of HTLV-I that immortalize a distinct subpopulation of human T cells. The experiments also demonstrate the utility of the H. saimiri vector for the transduction of heterologous genes into human T cells.

Cell Transformation, Neoplastic↗
PubMed1989

Selectable recombinant herpesvirus saimiri is capable of persisting in a human T-cell line.

Strategies were developed to insert the neo resistance marker into the junction between L DNA and the right terminal repetitive H DNA of herpesvirus saimiri. Recombinant viruses were selectable in permissive epithelioid cultures. The human T-cell line Jurkat could be infected persistently with the Neor virus; the cells contained episomal viral DNA in high copy number. This selectable vector should be generally applicable for gene expression in human T cells.

Cell Line↗
PubMed1988

A gene for dihydrofolate reductase in a herpesvirus.

The enzyme dihydrofolate reductase (DHFR) is found ubiquitously in both prokaryotes and eukaryotes. It is essential for de novo synthesis of purines and of deoxythymidine monophosphate for DNA synthesis. Among viruses, however, only the T-even and T5 bacteriophage have been found to encode their own DHFR. In this study a gene for DHFR was found in a specific subgroup of the gamma or lymphotropic class of herpesviruses. DNA sequences for DHFR were found in herpesvirus saimiri and herpesvirus ateles but not in Epstein-Barr virus, Marek's disease virus, herpes simplex virus, varicella-zoster virus, herpesvirus tamarinus, or human cytomegalovirus. The predicted sequence of herpesvirus saimiri DHFR is 186 amino acids in length, the same length as human, murine, and bovine DHFR. The human and herpesvirus saimiri DHFRs share 83 percent positional identity in amino acid sequence. The herpesvirus saimiri DHFR gene is devoid of intron sequences, suggesting that it was acquired by some process involving reverse transcription. This is to our knowledge the first example of a mammalian virus with a gene for DHFR.

Amino Acid Sequence↗