Biomedical subjects

J C Crowley

Publications and source records attributed to J C Crowley.

14 recordsLinked to original sources

PubMed1995

Differential regulation of TNF-alpha production by listeriolysin-producing versus nonproducing strains of Listeria monocytogenes.

Only Listeria monocytogenes that produce listeriolysin O (LLO) elicit protective immunity. Given the importance of tumor necrosis factor alpha (TNF-alpha) in anti-Listeria immunity, we have investigated TNF-alpha production by macrophages after they ingested live LLO-producing compared to LLO-non-producing bacteria. We used two genetically engineered strains of Listeria that differed only in their ability (Ly+) or inability (Ly-) to produce LLO. Ly+ and Ly- caused the same kinetics of increased mRNA abundance for TNF-alpha during the first 90 min after phagocytosis. However, only Ly+ caused sustained transcription of TNF-alpha mRNA, and this may account for the increased release of TNF-alpha. The transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) prevented the sustained abundance of cytokine mRNA 20 h after ingestion of Ly+. In addition, nuclear run-on assays indicated sustained transcription of TNF-alpha genes only after ingestion of Ly+. LLO itself was not responsible for the ability of Ly+ to stimulate the sustained transcription of the TNF-alpha genes. Instead, LLO may allow Listeria to survive within macrophages so that other bacterial products cause sustained TNF-alpha gene transcription. Both Ly+ and Ly- produced molecules, isolated by 50% ammonium sulfate, that induced cytokine production. In conclusion, we now report that Ly+ causes sustained transcription of the TNF-alpha gene and production of TNF-alpha by macrophages in vitro. We speculate that the TNF-alpha may activate endothelium and thus allow the recruitment of T cells to sites of infection. This may contribute to the ability of only LLO-producing Listeria to induce protective immunity.

Animals↗

PubMed1995

Characterization of recombinant polioviruses expressing regions of rotavirus VP4, hepatitis B surface antigen, and herpes simplex virus type 2 glycoprotein D.

Recombinant polioviruses expressing antigens from rotavirus, herpes simplex virus type 2, and hepatitis B virus were generated. Fusion of the heterologous polypeptides to the amino terminus of the poliovirus polyprotein did not prevent myristylation of VP0, suggesting a novel mechanism of myristylation for these recombinant viruses. The effects of the parental genetic background, different foreign sequences, and different insert sizes on growth characteristics were compared. Both the size and the nature of the heterologous sequence appeared to be factors influencing the growth and stability of recombinant polioviruses. All of the recombinants showed a temperature-sensitive phenotype, regardless of the genetic background (attenuated or wild type) from which they were derived. Preliminary studies with transgenic mice carrying the poliovirus receptor gene are discussed.

Animals↗

PubMed1990

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation, characterization and regulation of the SPO7 sporulation gene.

SPO7 is one of several previously identified genes from the yeast Saccharomyces cerevisiae that is required for sporulation but not for vegetative growth. The SPO7 gene has been cloned by functional complementation and physically mapped 15-16 kb to the left of CEN1. Gene-disruption experiments confirmed that the cloned gene was the bona fide SPO7 gene. SPO7 codes for a 0.95-kb transcript that is expressed at approximately the same level in both vegetative and sporulating cells. The gene was sequenced and has the capacity to encode a 259-amino acid protein that does not appear to be related to other known proteins.

Amino Acid Sequence↗

PubMed1990

Oxygenator failure.

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Cardiopulmonary Bypass↗

PubMed1989

Cloning of chromosome I DNA from Saccharomyces cerevisiae: mutational analysis of the FUN2 transcribed region.

To increase the number of mutationally defined genes on chromosome I from Saccharomyces cerevisiae, most of the FUN2 (Function Unknown Now) transcribed region was deleted by gene replacement. Strains containing the deletion were viable, but grew with a 20% longer generation time. The mutation was recessive. Mutant haploids were able to mate, and homozygous mutant diploids were able to sporulate, giving asci containing four viable ascospores. These results indicate FUN2 is dispensable for the life cycle of S. cerevisiae, but required for an optimal growth rate.

Chromosome Deletion↗

PubMed1988

Measles virus L protein evidences elements of ancestral RNA polymerase.

We have determined the nucleotide sequence of the measles virus (MV) L gene using a cDNA library encompassing the entire MV genome (J. Crowley et al. (1987) Intervirology, 28, 65-77). The L gene is 6639 nucleotides in length, and contains a single long open reading frame that could code for a protein of 247,611 kDa. Both the L gene and in particular the predicted L protein of MV bear substantial homology to their counterparts in Sendai virus and Newcastle disease virus, suggesting that the multifunctional nature of paramyxovirus L proteins imposes strong evolutionary constraints. The predicted MV L protein also contains distinct elements of a postulated ancestral RNA polymerase.

Amino Acid Sequence↗

PubMed1988

Sequence variability and function of measles virus 3' and 5' ends and intercistronic regions.

Sequences critical for the activity of the measles virus (MV) RNA polymerase in transcription and replication were analyzed using a MV genomic cDNA library containing overlapping clones encompassing the entire MV genome. Clones corresponding to the 3' and 5' ends of the MV genome were identified and sequenced, and these sequences were confirmed by primer extension experiments. Neither (+) nor (-) strand leader RNAs were detected in MV-infected cell extracts, using high specific activity riboprobes made form these clones. Clones representing each of the MV gene boundaries were also sequenced, and variations including point mutations, insertions, and deletions were noted. Together with the sequence of the MV L gene region, this report completes the sequence determination of the MV genome.

Base Sequence↗

PubMed1987

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures.

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16-1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M1 replication defects of mak16-1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade 1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1.2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C. The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential).

Base Sequence↗

PubMed1987

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region.

To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized. Starting with the previously cloned ADE1 gene (J. C. Crowley and D. B. Kaback, J. Bacteriol. 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization. The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1). The CDC15 gene was located by isolating plasmids from a YCp50 S. cerevisiae genomic library that complemented the cdc15-1 mutation. S. cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization. The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene. This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1. CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S. cerevisiae plasmid vectors. Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15. In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element. Notably, a higher than average proportion of the DNA examined was transcribed into RNA.

Centromere↗

PubMed1986

Transcriptional map of the measles virus genome.

We have constructed a recombinant DNA library of the measles virus genome and identified gene-specific clones containing sequences coding for portions of each of the six viral structural proteins, as well as clones coding for intercistronic sequences. By Northern blotting, we could order the clones according to the pattern of individual gene-specific and readthrough mRNAs. Clones corresponding to the N, P/C and M genes were identified by correlation of the mRNAs with their in vitro translation products; clones corresponding to the H, F and L genes were identified by indirect evidence. The results indicated that the gene order in measles virus is that of a typical paramyxovirus (3'-N,P/C,M,F,H,L-5'), but that the M and F transcripts each contain 1.5 times the coding capacity needed for synthesis of these proteins in vivo.

Cloning, Molecular↗

PubMed1984

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene.

The ADE1 gene of Saccharomyces cerevisiae was isolated by complementation in S. cerevisiae from a yeast genomic DNA library carried on plasmid YEp13. Electron microscopy of R-loop-containing DNA indicated the location of the ADE1 gene on the plasmid insert. Gene disruption and gene replacement were used to demonstrate that the ade1-complementing sequence was the actual ADE1 gene that maps on chromosome I. ade1 strains which normally form red colonies form white ones when transformed with the cloned ADE1 gene. This property should be very useful, since it enables detection of plasmids carrying this gene under nonselective conditions.

Chromosomes↗

PubMed1983

Cell saver.

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Blood Cells↗

PubMed1981

Instrumentation needs of research universities.

This article assesses the status of scientific instruments in major research universities and identifies factors that facilitate or impede their development, acquisition, use, and maintenance. Sixteen universities, six national and government laboratories, and nine commercial laboratories were visited; over 700 individuals were interviewed. Data on instrument acquisition and age were collected. Instrumentation was examined in physics, chemistry, biological sciences, earth sciences, and electrical engineering. The study found that the quality of university instrumentation has seriously deteriorated, due principally to a relative decrease in instrumentation funding, inflexibility within the project grant system, and insufficient support for maintenance.

Costs and Cost Analysis↗

PubMed1976

Distribution and removal of human serum albumin-technetium 99m instilled intranasally.

The efficacy of antiviral drugs and vaccines administered intranasally may depend upon the technique of application. The distribution and time-course of removal of human serum albumin-technetium 99m (HSA-Tc 99m)-instilled intranasally were studied in eleven healthy volunteers using a gamma camera and an anterior sodium iodide scintillation detector. In 100 randomized studies material was delivered as drops in the supine position or as a spray to seated subjects. A significantly higher proportion of 'good' distributions (62 in 73 tests) was obtained with drops compared with spray (1 in 27). The volume administered was varied between 0.10 ml and 0.75 ml and the concentration of HSA was changed from 3 to 30% with no significant effect upon the distribution of time-course of removal; pertechnetate in isotonic saline was distributed and removed in a manner comparable to HSA-Tc 99m. Activity recorded by the detector showed an initial rapid fall associated with removal of most of the material from the nasal cavity, followed by a slower decline associated with the removal of material mainly from the anterior region of the nose. A multidose study confirmed that frequent administration by drops is required to maintain a high level of activity in the nasal cavity. Using this technique it should be possible to correlate measurements of antiviral efficacy and vaccines take-rates with certain characteristics of intranasal applicators; such studies may lead to the design of better devices.

Administration, Intranasal↗