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J A Prendergast

Publications and source records attributed to J A Prendergast.

8 recordsLinked to original sources
PubMed1992

A comparison of the flanking regions of the mouse cytotoxic cell proteinase genes.

T lymphocyte activation correlates with the transcriptional induction of a variety of genes that encode proteins that are believed to play a role in specific effector functions of the mature cells. Transcripts corresponding to members of the cytotoxic cell proteinase (CCP) family of genes accumulate with different kinetics depending upon the nature of the T cell stimulus. The profile of expression for each family member is unique. Sequences corresponding to the 5' and 3' flanking regions of each of the CCP genes were isolated and sequenced. A comparison of these sequences reveal regions of conservation that are consistent with the differential expression observed and indicate potential regulatory elements.

Animals↗
PubMed1992

Quantitative polymerase chain reaction analysis of cytotoxic cell proteinase gene transcripts in T cells. Pattern of expression is dependent on the nature of the stimulus.

A quantitative polymerase chain reaction assay was developed that allowed us to monitor transcript levels corresponding to individual members of the cytotoxic cell proteinase (CCP) gene family during T cell activation. Selective expression was observed and shown to depend upon the mode of T cell antigen receptor stimulation. Mitogen or allogeneic stimulation of cells resulted in the appearance of transcripts corresponding to all the genes measured, whereas alpha CD3 antibody produced a response restricted to just two family members. This differential gene activation represents a heterogeneity in cytotoxic T lymphocytes that has not been recognized previously. It may indicate that the T cell branch of the immune system can distinguish between different forms of stimulation and respond by synthesizing a specific set of effector and ancillary molecules that is most appropriate for lysis of cells bearing that type of antigen. Only CCP1 transcripts correlated with cytotoxicity for all modes of stimulation. The patterns for the others are suggestive of distinct and ancillary, rather than direct effector, roles in the lytic mechanism.

Animals↗
PubMed1992

Peritoneal exudate lymphocyte and mixed lymphocyte culture hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin.

We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.

Animals↗
PubMed1991

A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

Alleles↗
PubMed1991

Structure and evolution of the cytotoxic cell proteinase genes CCP3, CCP4 and CCP5.

A family of serine proteinases is believed to be important in cell-mediated cytotoxicity. Presented here are the genomic sequences for three murine members of this cytotoxic cell proteinase (CCP) family: the CCP3, CCP4 and CCP5 genes. All three of these genes have introns inserted at the same codon sites and the same exon distribution of the active site residues. These characteristics are also shared with the CCP1 and CCP2 genes, the charter members of the CCP gene family. Phylogenetic analysis using intron and exon sequences suggests that all five genes arose by various duplication events. This analysis also indicates that the recently described HuCCPX and CCP2 genes originated from recombination events between genes of different lineages. A phylogenetic and Southern analysis of the recombinant HuCCPX gene suggests that the human genome contains an additional CCP gene that has yet to be described. Finally, evidence is presented suggesting that the cDNA clone originally describing the CCP5 gene was derived from an alternately spliced transcript.

Amino Acid Sequence↗
PubMed1991

The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles.

After the initiation of bud formation, cells of the yeast Saccharomyces cerevisiae direct new growth to the developing bud. We show here that this vectorial growth is facilitated by activity of the MYO2 gene. The wild-type MYO2 gene encodes an essential form of myosin composed of an NH2-terminal domain typical of the globular, actin-binding domain of other myosins. This NH2-terminal domain is linked by what appears to be a short alpha-helical domain to a novel COOH-terminal region. At the restrictive temperature the myo2-66 mutation does not impair DNA, RNA, or protein biosynthetic activity, but produces unbudded, enlarged cells. This phenotype suggests a defect in localization of cell growth. Measurements of cell size demonstrated that the continued development of initiated buds, as well as bud initiation itself, is inhibited. Bulk secretion continues in mutant cells, although secretory vesicles accumulate. The MYO2 myosin thus may function as the molecular motor to transport secretory vesicles along actin cables to the site of bud development.

Actins↗
PubMed1990

Size selection identifies new genes that regulate Saccharomyces cerevisiae cell proliferation.

A centrifugation procedure to enrich for enlarged cells has been used to isolate temperature-sensitive cdc mutants of the yeast Saccharomyces cerevisiae. Among these mutants are strains containing mutations that arrest proliferation at the regulatory step start. These new start mutations define two previously unidentified genes, CDC67 and CDC68, and reveal that a previously identified gene, DNA33 (here termed CDC65), can harbour start mutations. Each new start mutation permits significant biosynthetic activity after transfer of mutant cells to the non-permissive temperature. The cdc68-1 start mutation causes arrest of cell proliferation without inhibition of mating ability, while the cdc65-1 and cdc67-1 mutations inhibit zygote formation and successful conjugation. The identification of new start genes by a novel selection procedure suggests that the catalog of genes that influence start is large.

Cell Division↗