Biomedical subjects

D Levens

Publications and source records attributed to D Levens.

46 records · Page 3Linked to original sources

PubMed1986

Improved survival of island flaps after prolonged ischemia by perfusion with superoxide dismutase.

Perfusion of rat groin flaps after 10 and 11 hours of complete ischemia with superoxide dismutase, an oxygen free-radical scavenger, significantly improved the survival of these flaps. This finding provides further evidence for the important role that oxygen-derived free radicals play in ischemic injury. The study also demonstrates that while restoration of blood supply alone is not enough to ensure tissue survival after prolonged ischemia, chemical agents can be utilized to achieve viable flaps beyond what was believed to be "a point of no return".

Animals↗

PubMed1985

Myelopathy following intrathecal chemotherapy in a patient with extensive Burkitt's lymphoma and altered immune status.

A 30-year-old homosexual man presented with widespread Burkitt's lymphoma. On the basis of immunologic and viral studies, he was suspected of having the acquired immune deficiency syndrome. Following chemotherapy that included intrathecal cytosine arabinoside and methotrexate, brain stem edema, paraplegia, and an elevated cerebrospinal fluid level of myelin basic protein developed. Autopsy revealed vacuolar demyelination of spinal cord, brain stem, and cerebellum. The pathologic findings were similar to those reported to occur in myelopathy associated with intrathecal chemotherapy, but far more extensive. The contribution of the suspected acquired immune deficiency syndrome is unknown.

Acquired Immunodeficiency Syndrome↗

PubMed1985

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.

Base Sequence↗

PubMed1982

Transcriptional initiation and processing of the small ribosomal RNA of yeast mitochondria.

We have identified the nucleotide at which transcription initiates on the yeast mitochondrial small (14 S) rRNA gene by sequencing of RNA labeled at the 5' initiating triphosphate with vaccinia virus guanylyltransferase [alpha-32P]GTP (in vitro capping reaction). Initiation occurs within the stem of a 12-base palindromic repeat. The initiation sequence has homology with the large (21 S) ribosomal RNA initiation sequence that has been previously determined. We have also sequenced the 5' and 3' ends of the mature 14 S rRNA after labeling with T4 polynucleotide kinase and RNA ligase, respectively. These sequences demonstrate that about 80 nucleotides are cleaved from the 5' end of a precursor to produce the mature 14 S rRNA. This cleavage is imprecise in that the processing occurs at one of five adjacent nucleotides 77 to 81 nucleotides downstream from the 5' initiation site. The 3' ends of this precursor and the mature 14 S rRNA are unique and identical.

Base Sequence↗

PubMed1982

The biogenesis and regulation of yeast mitochondria RNA polymerase.

Yeast mitochondrial RNA polymerase is a nuclear-coded protein of approximately 90,000 daltons comprised of two 45,000-dalton subunits of pI 6.9 to 7.0. To investigate the nature of the initial translation product of the RNA polymerase, we have analyzed those products of a cell-free translation system directed by yeast RNA that are immunoreactive with antibodies to the 45,000-dalton peptide of polymerase. A precursor of one or more of the subunits of the polymerase, 2,000 daltons later than the mature product, has been characterized using immunoreaction, immunocompetition, and peptide digestion. The role of transcription of the polymerase gene in catabolite repression of mitochondrial development has been investigated by analyzing the changes in cell-free synthesis of the RNA polymerase precursor during glucose and raffinose growth. The results indicate an increase in precursor synthesis and probably in the corresponding transcript abundance during glucose derepression. In contrast, the precursor is present at high levels until stationary phase during raffinose growth. These data indicate the involvement of increased transcription of the polymerase gene in the process of derepression.

Cell Nucleus↗

PubMed1982

Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system.

We have developed an in vitro transcription system for yeast mitochondrial rRNA genes. Using highly purified yeast mitochondrial RNA polymerase and bacterial plasmids carrying DNA segments containing the mitochondrial rRNA sites of transcriptional initiation, we have been able to demonstrate correct initiation of transcription in vitro. By directly sequencing the transcription products, we show that transcription in vitro of both the 14S and 21S rRNAs is initiated at precisely the same site as it is in vivo. Transcription of the rRNA genes is highly sensitive to ionic strength and RNA polymerase concentration. Additional factors or modified conditions may be necessary to permit accurate transcription of mitochondrial protein genes.

Base Sequence↗

PubMed1982

Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [alpha-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above--i.e., 20-40 nucleotides upstream of the tRNAPhe gene--had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.

Chromosome Mapping↗

PubMed1981

Transcriptional initiation and 5' termini of yeast mitochondrial RNA.

We have used vaccinia virus guanylyltransferase to label polyphosphate-terminated yeast mitochondrial RNAs in vitro with [alpha-32P]GTP. Hybridization of RNA labeled in vitro indicates the presence of multiple transcriptional initiation sites in both grande and petite mitochondrial genomes. Agarose/urea gel electrophoresis of capped RNA suggests the existence of a precursor to the small (14 S) rRNA. In contrast, direct examination of the large (21 S) rRNA by partial ribonuclease T1 digestion reveals a complete lack of processing of the 5' end of the primary transcript of this RNA.

DNA Restriction Enzymes↗

PubMed1981

Mitochondrial transcription complex from Saccharomyces cerevisiae.

A DNA protein complex has been isolated from the mitochondria of Saccharomyces cerevisiae. The complex transcribes RNA complementary to mtDNA in a nonrandom manner. The RNA polymerase activity contained in the transcription complex is not dependent on the addition of exogenous template. The activity is rendered template-dependent by autolysis and can be further purified by heparin-Sepharose 4B chromatography. The activity is inhibited by heparin, Mn2+, and increasing ionic strength. The activity requires Mg2+ and ribonucleotides. The preferred template for the template dependent activity is poly[d(AT)]. The majority of the RNA synthesized by the transcription complex from endogenous DNA is complementary to the DNA strands directing the synthesis of the large and small ribosomal RNA. In yeast the 21 S and 14S rRNA genes are widely separated, therefore the transcription of these two regions but not of the intervening regions by the transcription complex suggests the existence of at least two transcriptional promoters on the yeast mitochondrial genome.

DNA, Fungal↗

PubMed1981

Purification of mitochondrial RNA polymerase from Saccharomyces cerevisiae.

The RNA polymerase from the mitochondria of Saccharomyces cerevisiae has been extensively purified by Sepharose 4B, heparin Sepharose 4B phosphocellulose, and DEAE-Sephadex A-50 chromatography. The activity co-sediments with a 45,000-dalton polypeptide at 6.3 S in glycerol gradients. The activity is inhibited by antibodies to the 45,000-dalton polypeptide. The activity is not inhibited by rifampicin or alpha-amanitin. It requires Mg2+ and is inhibited by elevated ionic strength and Mn2+. The most efficient template for the RNA polymerase is poly[d(AT)], with mtDNA being the preferred natural template. The RNA polymerase transcribes mtDNA from the petite strain F11 in a nonrandom manner.

DNA-Directed RNA Polymerases↗