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J Swindle

Publications and source records attributed to J Swindle.

17 recordsLinked to original sources
PubMed1996

The calmodulin-ubiquitin (CUB) genes of Trypanosoma cruzi are essential for parasite viability.

The CUB genes represent single copy genes in the diploid Trypanosoma cruzi genome. In this report data are presented which demonstrate that a single expressed CUB gene is necessary for parasite viability. Although either CUB gene could be deleted individually, repeated attempts to simultaneously delete both genes were unsuccessful. The essential nature of the CUB genes was further supported by studies which demonstrated positive selection for CUB gene expression. Positive selection was demonstrated by carrying out dual gene replacements which showed that both native CUB genes could be efficiently deleted provided the CalB1 calmodulin gene was simultaneously replaced by a CUB gene protein coding sequence. Although the function of the CUB gene product remains unknown the experiments presented here indicate the product is likely to play an important role in the parasites' life cycle.

Animals↗
PubMed1996

Adenovirus-mediated transfer of a gene encoding acyloxyacyl hydrolase (AOAH) into mice increases tissue and plasma AOAH activity.

Although the host response to gram-negative bacterial infection follows largely from the interactions of bacterial lipopolysaccharides (LPS or endotoxin) with host cells, little information is available concerning the mechanisms by which the host eliminates or detoxifies LPS. Acyloxyacyl hydrolase (AOAH) is an enzyme, found in phagocytic cells, that catalyzes the enzymatic deacylation of the lipid A moiety of LPS. Enzymatically deacylated LPS is much less potent than LPS at inducing responses in human cells, and it can antagonize the ability of LPS to activate human macrophages, neutrophils, and endothelial cells. Despite these observations, the physiologic role of LPS deacylation remains undefined. To investigate the ability of AOAH to carry out LPS deacylation in vivo, we produced a recombinant adenovirus carrying a gene encoding (AOAH) (Ad.CMV-AOAH) and employed this vector to elicit transient overexpression of AOAH in mice. Mice infected with Ad.CMV-AOAH expressed high levels of the enzyme in plasma, liver, spleen, and kidney. Although adenovirus-induced hepatitis reduced hepatic uptake of intravenously injected [3H]LPS, animals expressing the transgene deacylated a larger fraction of the [3H]LPS taken up by their livers than did mice infected with a control adenovirus. These studies indicate that AOAH can catalyze the deacylation of LPS in vivo, and they provide evidence that the rates of hepatic LPS uptake and deacylation are not closely linked.

Acylation↗
PubMed1995

Expression of mammalian cytokines by Trypanosoma cruzi indicates unique signal sequence requirements and processing.

A vector based upon the calmodulin-ubiquitin 2.65 locus of Trypanosoma cruzi has enabled the expression and secretion of the murine cytokines interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) by transfected T. cruzi. The T. cruzi-derived cytokines were bioactive and produced by both epimastigotes and mammalian forms. The native coding sequence of IL-2 was sufficient to cause secretion of the protein, but the gamma-IFN signal sequence had to be replaced by the IL-2 signal sequence (IL-2/gamma-IFN) to allow efficient secretion of gamma-IFN. The amino acid sequences at the N-termini of the secreted T. cruzi-derived cytokines were different from the expected murine secreted protein. The secreted IL-2 was cleaved six amino acids downstream from the murine signal sequence cleavage site, and the hybrid IL-2/gamma-IFN molecule was cleaved three amino acids downstream from the predicted signal cleavage site in the IL-2/gamma-IFN molecule. These apparent differences in signal peptide sequence requirements and cleavage sites most likely indicate that the signal sequence processing in trypanosomes is distinct from that of higher eukaryotes.

Amino Acid Sequence↗
PubMed1994

Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements.

We describe here a strategy for introducing simultaneous, independent gene replacements into the Trypanosoma cruzi chromosome. The goal of this study was to use two linear DNA fragments to simultaneously replace the CalA2 calmodulin and FUS1 ubiquitin-fusion genes with the neomycin resistance (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. One clone (D6), of thirty G418-resistant clones analyzed, carried the desired dual gene replacement. CDNA sequence analysis indicated that the CAT mRNA was accurately trans-spliced using the previously identified FUS1 mini-exon addition site. However, DNA sequence analysis of the intergenic sequence immediately upstream of the neo(r) gene in clone D6 identified a mutation which altered the pattern of trans-splicing of the neo(r) mRNA. Possible effects of this mutation on 3' splice acceptor site selection are discussed.

Animals↗
PubMed1993

Using simultaneous, tandem gene replacements to study expression of the multicopy ubiquitin-fusion (FUS) gene family of Trypanosoma cruzi.

Many genes in trypanosomes exist as members of multicopy gene families. Due to this fact it is frequently difficult to determine if specific members of a gene family are expressed. We describe here a strategy for simultaneous tandem gene replacement in T. cruzi which leads to the replacement of the gene of interest by a silent reporter gene, the expression of which can be assayed in stable transformants. To determine if the FUS1 gene (one of 5 copies of the ubiquitin-fusion, FUS, gene family) was expressed, stable G418-resistant transformants were isolated in which the tandemly arrayed CUB2.65 and FUS1 genes were precisely replaced by the neomycin phosphotransferase (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. All stable clones carrying the tandem gene replacements were shown to express the CAT activity indicating that FUS1 is expressed in mid-log epimastigotes. Northern blot analysis of parasites carrying the tandem gene replacements indicated that at least one other member of the FUS gene family is expressed and that there were no apparent polar effects on the expression of genes downstream of the replacement events. These experiments have demonstrated the utility of tandem gene replacements as a means of inserting a nonselected reporter gene into the chromosome, facilitating the molecular genetic analysis of the expression of multicopy gene families.

Animals↗
PubMed1993

Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption.

Analysis of gene expression in Trypanosoma cruzi has been impeded by the lack of efficient, stable, DNA-mediated transfection systems. We describe here the establishment of such a system for T. cruzi. Stable transformants were isolated following integration of the circular transforming plasmid into the chromosome by homologous recombination. Mutants with a disrupted PUB12.5 polyubiquitin gene, resulting from targeted integration of the plasmid vector, have been isolated. A mutant harboring the disrupted PUB12.5 gene lacks the intact PUB12.5 mRNA as well as transcripts corresponding to the truncated gene. Genomic Southern-blot analysis indicates that the inserted plasmid is tandemly repeated in each of the clones analyzed. A secondary recombination event in one clone resulted in a deletion within the 2.65 calmodulin-ubiquitin locus, encompassing the sequence from the CalA2 calmodulin gene to the PUB12.5 polyubiquitin gene.

Animals↗
PubMed1993

The calmodulin-ubiquitin associated genes of Trypanosoma cruzi: their identification and transcription.

We describe here the identification of the calmodulin-ubiquitin associated (CUB) genes of Trypanosoma cruzi. A single CUB gene resides in a 1.5-kb DNA sequence linking the calmodulin and ubiquitin genes in the 2.65 and 2.8 loci (CUB2.65 and CUB2.8 respectively). The CUB genes also share the same coding strand as the flanking calmodulin and ubiquitin genes. DNA sequence analysis reveals that each CUB gene contains an open reading frame which would encode a protein of 208 amino acids. The CUB protein shares homology with the recently identified calcium binding EFH5 protein of T. brucei. Transcription of the CUB genes results in the generation of a mRNA of approximately 1.0 kb. CUB cDNA sequence analysis following PCR amplification of the CUB mRNA population indicates that both genes are expressed and trans-spliced, but utilize different 3' acceptor sites for the trans-splicing reaction.

Amino Acid Sequence↗
PubMed1990

Linkage of the calmodulin and ubiquitin loci in Trypanosoma cruzi.

We describe here the organization of the calmodulin genes of Trypanosoma cruzi and their linkage to the ubiquitin gene family. The nucleotide sequence of the CalA2 gene has been determined and is 85% homologous to the protein coding sequence of the calmodulin genes of the African trypanosome, Trypanosoma brucei. The proteins encoded by CalA2 and the T. brucei genes contain a single mismatch out of a total of 149 amino acids. The genome of T. cruzi contains eight calmodulin genes present at two distinct loci (2.8 and 2.65) each of which is linked to downstream ubiquitin genes. Within each locus two calmodulin gene families have been defined, CalA and CalB. Each calmodulin locus consist of alternating tandem arrays of the CalA and CalB genes. Both 2.8 and 2.65 calmodulin loci and their respective downstream ubiquitin genes share the same DNA coding strand. Transcription of the calmodulin genes in the epimastigote stage of T. cruzi results in the generation of two stable mRNAs of 1.6Kb and 1.1Kb.

Amino Acid Sequence↗
PubMed1988

Purification and properties of the NusB protein of Escherichia coli.

Mutations in the nusB gene of Escherichia coli block transcriptional antitermination mediated by the N gene protein of bacteriophage lambda. We describe here two methods of overproducing the NusB protein in E. coli and a method of purifying NusB to apparent homogeneity on a large scale. Purified NusB directly stimulates transcriptional antitermination by the lambda N protein in vitro. It behaves as a monomer (Mr = 15,689) during gel permeation chromatography and gradient sedimentation. The number of NusB molecules in a wild type E. coli K12 cell ranges from about 3,000 to about 6,000 molecules/cell, depending on the growth medium, and is about 50-80% of the number of molecules of the core component of RNA polymerase in the cell. This implies that NusB has a major role in regulating chain elongation during the transcription of E. coli genes. Many E. coli strains with nusB mutations cannot grow at low temperature. However, a sup+ strain with the suppressible amber mutation nusBam115 can grow at 42 degrees C. Since such a strain does not produce NusB protein detectable by immunoprecipitation with anti-NusB, normal amounts of NusB are not essential for the survival of E. coli at 42 degrees C.

Bacterial Proteins↗
PubMed1988

The genomic organization and transcription of the ubiquitin genes of Trypanosoma cruzi.

We describe here the organization of the ubiquitin genes of the parasitic protozoan Trypanosoma cruzi. T. cruzi contains greater than 100 ubiquitin coding sequences all of which are clustered into a 27 kb segment of the genome. Two types of ubiquitin coding sequences were found. There are five fusion genes (FUS1-5) consisting of a ubiquitin coding sequence fused to a basic non-ubiquitin sequence. The T. cruzi ubiquitin fusion protein is 84% homologous to the product of the UBI gene of Saccharomyces cerevisiae. The non-ubiquitin domains of the two proteins are 67% homologous. There are five polyubiquitin coding genes (PUB) each consisting of varying lengths of polyubiquitin coding sequence and terminating with a single copy of the larger fusion gene. Transcription of the ubiquitin genes results in the generation of six major poly(A)+ mRNAs. The pattern of transcription accurately reflects the genomic organization, in that the transcripts consist of either a single copy of the ubiquitin fusion coding sequence or varying lengths of polyubiquitin (up to 52 copies of the ubiquitin coding unit) each ending with a single copy of the ubiquitin fusion sequence. Finally, there are heat shock elements 5' to the PUB genes and transcription patterns are altered under conditions of stress.

Amino Acid Sequence↗
PubMed1984

The nucleotide sequence of the Escherichia coli K12 nusB (groNB) gene.

The nusB (groNB) gene product of Escherichia coli plays a pivotal role in allowing bacteriophage lambda N protein to function as an antiterminator of mRNA transcription and in modulating host gene expression. In addition it is essential for bacterial viability since mutations in it result in a cold-sensitivity phenotype for growth. We have previously cloned the nusB gene and shown it to code for a 14,500-Mr protein. Here we present the primary DNA sequence of the nusB gene. From the sequence we deduce that it codes for a slightly basic protein (21 basic as opposed to 20 acidic amino acids) composed of 139 amino acids with a cumulative 15,689-Mr. The predicted N-terminal amino acid sequence as well as the overall amino acid composition agrees well with that of the purified protein.

Amino Acid Sequence↗
PubMed1981

Identification of the E. coli groNB(nusB) gene product.

The E. coli groNB(nusB) gene product has been previously shown to be necessary for bacteriophage lambda N protein function. The product of the groNB gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells with various lambda groNB+ transducing phage derivatives. It is a polypeptide with an apparent molecular weight of 14,000 daltons. Transducing phage carrying either a deletion or an amber mutation in the groNB gene fail to synthesize the 14,000-Mr polypeptide chain upon infection of a sup+ host. However, am+ revertants of the lambda groNBam phage do induce the synthesis of the polypeptide.

Bacterial Proteins↗
PubMed1980

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function.

Escherichia coli mutants, called groNB, which block the growth of bacteriophage lambda at the level of action of the gene N product, have been isolated as survivors at 42 degrees C of bacteria carrying a) the defective prophage lambda bio11 i lambda cI857 delta H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage lambda cI857. In addition, groNB bacterial mutants have been isolated at 37 degrees C, as large colony formers in the presence of lambda i lambda cI h434, lambda i lambda cI h lambda, and lambda i lambda cI h80 phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42 degrees C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of lambda growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro+ for lambda growth. Lambda transducing phages carrying the groNB+ bacterial gene have been isolated. GroNB mutant bacteria lysogenized by the transducing phage acquire the Gro+ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.

Bacteriophage lambda↗