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T J Koerner

Publications and source records attributed to T J Koerner.

At least 19 recordsLinked to original sources
PubMed1995

Cloning and characterization of COX14, whose product is required for assembly of yeast cytochrome oxidase.

Nuclear respiration-deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G93 lack cytochromes a and a3 and detectable cytochrome oxidase activity. Other respiratory chain carriers and the ATPase complex are present at near wild-type levels, indicating that the mutations specifically affect cytochrome oxidase. Since synthesis of the mitochondrially derived subunits 1, 2, and 3 of cytochrome oxidase is normal, the defect cannot be related to transcription of the endogenous genes or processing and translation of the corresponding RNAs. The results of Western analysis of the cytochrome oxidase subunits encoded in nuclear DNA also argues against an effect of the mutations on expression of these constituents. The G93 mutants are complemented by a nuclear gene, designated COX14. The product of this gene is a low molecular mass protein of 7,960 Da. A gene fusion expressing a biotinylated form of Cox14p complements cox14 mutants, indicating partial functional equivalence. The biotinylated derivative has been helpful in localizing Cox14p to the mitochondrial membrane and demonstrating that it is not a hitherto unrecognized subunit of cytochrome oxidase, although it does appear to be associated with a high molecular weight complex. This evidence, combined with the assembly-arrested phenotype of cox14 mutants, indicates that Cox14p, like several other recently described mitochondrial constituents, provides an important function at some late stage of the cytochrome oxidase assembly pathway.

Amino Acid Sequence↗
PubMed1991

Effects of TNF alpha on the expression of class II MHC molecules in macrophages induced by IFN gamma: evidence for suppression at the level of transcription.

Tumor necrosis factor-alpha (TNF-alpha) induces surface expression of class II major histocompatibility (MHC) molecules (la molecules) in many cells, including macrophage-like cell lines. When we tested the effects of this cytokine on murine peritoneal macrophages, TNF alpha had little effect on surface expression of la. The strong expression of such molecules induced by interferon-gamma (IFN gamma) was, however, suppressed moderately by TNF alpha. These effects were reflected at the level of specific messenger RNA (mRNA) as detected by Northern blot analysis. Furthermore, the locus of control appears to be transcriptional; in nuclear run-on assays, TNF alpha suppressed the IFN gamma-induced enhancement of transcription for the murine beta-chain of I-A (I-A beta.). Taken together the data suggest that TNF alpha has little effect on class II MHC genes and surface expression in murine peritoneal macrophages, that TNF alpha is a modest suppressant of such molecules when their levels are raised by IFN gamma, and that these suppressive effects are mediated at the level of transcription.

Animals↗
PubMed1990

Gene regulation in macrophage activation: differential regulation of genes encoding for tumor necrosis factor, interleukin-1, JE, and KC by interferon-gamma and lipopolysaccharide.

Although macrophage activation is induced in a complex manner by signals such as interferon-gamma (IFN gamma) and bacterial lipopolysaccharide (LPS) and depends on alterations in levels of specific proteins due to differences in gene expression, the complexity of gene regulation during macrophage activation in regard to multiple signals is not fully appreciated. To probe this question, we selected four model genes encoding for tumor necrosis factor (TNF), interleukin-1 (IL-1), and the immediate early genes JE and KC. After analyses of Northern blots for specific mRNA, LPS was found to enhance levels of mRNA for IL-1, TNF, JE, and KC. IFN gamma initiated heightened mRNA levels for JE but did not alter specific mRNA for IL-1, TNF, or KC. When IFN gamma and LPS were combined, additive effects on levels of specific mRNA for JE, enhancement of mRNA for TNF, suppressed mRNA for KC, and no effect on mRNA for IL-1 were observed. When transcription of these genes was assessed by nuclear "run on" experiments, LPS increased transcription of KC and TNF but not of IL-1 or JE, implying that the increased levels of mRNA for JE and IL-1 were attributable to increased stability of mRNA. Likewise, IFN gamma did not initiate transcription of JE. When IFN gamma and LPS were given together, IFN gamma enhanced the LPS-induced transcription of TNF and KC, suggesting decreased stability of mRNA for KC. A distinct pattern of regulation for each of the four genes was thus observed. Taken together, the data suggest that gene regulation in macrophage activation represents a complex response of enhanced and suppressed transcription and mRNA stability, the precise pattern of which depends on the stimuli given to the macrophages and the gene examined.

Animals↗
PubMed1989

Molecular mechanisms regulating the expression of class II histocompatibility molecules on macrophages. Effects of inductive and suppressive signals on gene transcription.

The surface expression of class II MHC molecules (immune associated or Ia Ag) is an acquired and important property of macrophages. Recent evidence from several laboratories demonstrates that mRNA levels for class II genes reflect levels of surface expression. We have investigated the effects of agents that regulate Ia expression, either positively as IFN-gamma or negatively as bacterial LPS or maleylated proteins, on transcription of these genes. By using probes for I-A beta and I-E beta as representative class II MHC genes, we found that IFN-gamma induces transcription in murine peritoneal macrophages of I-A beta and of I-E beta as determined in nuclear run-on assays. The measured transcription peaked 6 to 10 h after administration of IFN-gamma to the macrophages. This augmented expression of transcription was markedly suppressed by administration of either LPS or maleylated protein to the macrophages. Collectively, the data indicate that regulation of transcription by either positive or negative stimuli, acting through surface receptors and binding sites, is a major mechanism for controlling the expression of class II MHC molecules in macrophages.

Albumins↗
PubMed1989

Expression of macrophage p120 depends on early protein synthesis.

We have previously identified a group of early proteins preceding the expression of a 120-kDa protein (p120) which coincides with tumoricidal activation in peritoneal macrophages. In the present report, we have asked whether the in vitro induction of new or enhanced expression of p120 depends on early protein synthesis and RNA synthesis during the treatment period. Expression of p120 was sensitive to pretreatment of the macrophages with either actinomycin D or cycloheximide, indicating that both active protein synthesis and RNA synthesis were required. When poly-adenylated RNA isolated from various macrophage populations was translated in a rabbit reticulocyte in vitro translation system, only mRNA isolated from cells which express p120 was able to direct synthesis of a 120-kDa polypeptide. This product showed identical mobility to p120 induced in intact activated macrophages radiolabeled with [35S]methionine. The presence of translatable p120 mRNA was dependent upon treatment of thioglycollate-elicited macrophages with both IFN-gamma plus LPS at low doses, as is expression of p120 in intact cells. Accumulation of translatable p120 mRNA was blocked by treatment with cycloheximide, indicating that active protein synthesis was required during the induction period. These results suggest that the presence of specific translatable mRNA encoding the p120 polypeptide is dependent upon the expression of early macrophage gene products.

Animals↗
PubMed1988

Characterization of lipopolysaccharide-induced macrophage gene expression.

A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages. Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization. When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen. In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures. All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment. The time course of expression differed among the individual genes. Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation. In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h. Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA. The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC. Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes. Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS.

Animals↗
PubMed1987

The early competence genes JE and KC are differentially regulated in murine peritoneal macrophages in response to lipopolysaccharide.

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide (LPS) has been previously documented to induce accumulation of mRNA for the early or competence genes JE and KC; the data further suggested that multiple pathways existed for the transduction of the LPS signal, since induction of mRNA for JE was related to breakdown of polyphosphoinositides while induction of KC was not (Introna et al. 1987 J. Immunol. 138, 3891). This study provides analysis of the regulation of the expression of these genes by using the nuclear transcription assay. We present evidence that LPS enhanced transcriptional activity of the KC gene, but not of the JE gene. By contrast, serum stimulation of quiescent BALB/c-3T3 fibroblasts induced transcription of the JE and KC genes. The data imply that expression of the KC gene in LPS-treated macrophages is regulated transcriptionally, while that of the JE gene is regulated post-transcriptionally. Furthermore, there appear to be two mechanistic pathways for the induction of JE mRNA depending upon the stimulus and upon the cell type: one involving transcriptional and one post-transcriptional control.

Animals↗
PubMed1987

Characterization of the yeast HEM2 gene and transcriptional regulation of COX5 and COR1 by heme.

The respiratory deficiency of two noncomplementing mutants of Saccharomyces cerevisiae (C41 and N28) has been shown to be due to mutations in HEM2, the structural gene for delta-aminolevulinate dehydratase. The mutants are unable to convert delta-aminolevulinic acid to porphobilinogen and are not complemented by the hem2 mutant GL4 (Gollub, E. G., Liu, K.-P., Dagan, J., Adlersberg, M., and Sprinson, D. B. (1977) J. Biol. Chem. 252, 2846-2854). A gene capable of complementing the respiratory deficiency of C41 and N28 has been cloned by transformation of a hem2 mutant with a recombinant plasmid library of wild type yeast nuclear DNA. The sequence of the protein encoded by the cloned gene exhibits extensive homology to the recently reported sequence of human delta-aminolevulinate dehydratase (Wetmur, J. G., Bishop, D. F., Cantelmo, C., and Desnick, R. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7703-7707). Several approaches were taken to study the effect of heme on transcription of PET genes known to code for subunit components of respiratory enzymes and of mitochondrial ATPase. The first involved measurements of the steady state levels of mRNAs for subunit 5 of cytochrome oxidase and the beta subunit of F1 ATPase in wild type and in a hem2 mutant. Secondly, transcription of the genes coding for the cytochrome oxidase and ATPase subunits as well as of the COR1 gene coding for the 44-kDa core 1 subunit of coenzyme QH2-cytochrome c reductase was quantitated by fusing the 5'-flanking and part of the coding region of each gene to the lacZ gene of Escherichia coli in vectors capable of integrating into yeast chromosomal DNA. The different lacZ fusions were integrated into nuclear DNA of a wild type strain and of hem2 mutants allowing expression of beta-galactosidase to be studied as a function of intracellular heme. These experiments indicate that the promoters of the genes for subunits of the respiratory complexes are regulated by heme. In contrast, the expression of the ATPase subunit appears to be heme-independent. Because neither subunit 5 of cytochrome oxidase nor the core 1 subunit of coenzyme QH2-cytochrome c reductase are hemoproteins, transcriptional regulation by heme may be a general mechanism for controlling the synthesis of mitochondrial proteins involved in respiration.

Alleles↗
PubMed1987

Regulation of tumor necrosis factor (TNF) expression: interferon-gamma enhances the accumulation of mRNA for TNF induced by lipopolysaccharide in murine peritoneal macrophages.

The secretion of tumor necrosis factor (TNF) by macrophages is initiated by lipopolysaccharide (LPS); considerable evidence indicates that such secretion can be potentiated by interferon-gamma (IFN-gamma). The present studies show that accumulation of mRNA for tumor necrosis factor, which represents an important regulatory focus for controlling secretion of TNF, is enhanced by physiologic doses of IFN-gamma (20 units/ml of purified recombinant IFN-gamma). mRNA for TNF induced by LPS, which was maximal 2 hr after LPS was applied to the cells, was enhanced 5- to 8-fold by IFN-gamma as determined by Northern blot analysis. Interferon did not change the kinetics of accumulation but did change the dose effects of LPS in that increasing amounts of LPS led to increasing amounts of TNF mRNA in IFN-gamma-treated macrophages. IFN-gamma itself, however, did not induce expression of TNF mRNA. These studies document that IFN-gamma potentiates the cytoplasmic accumulation of mRNA for TNF induced in murine peritoneal macrophages by LPS.

Animals↗
PubMed1987

Suppressed expression of surface Ia on macrophages by lipopolysaccharide: evidence for regulation at the level of accumulation of mRNA.

The surface expression of class II major histocompatibility molecules (immune associated or Ia antigens) is an acquired property of macrophages, essential to their ability to interact effectively with T lymphocytes. Surface expression of Ia is induced by stimulants such as interferon-gamma and is suppressed by agents such as lipopolysaccharide (LPS). Recent studies on several cultured cell lines indicate that interferon-gamma can heighten cellular levels of mRNA encoding Ia, and the level of such mRNA may represent an important regulatory focus for controlling expression of surface Ia. Murine peritoneal macrophages were treated with interferon-gamma and/or LPS and expression of Ia mRNA determined by Northern blot analysis with a probe specific for the murine beta-chain of I-A. mRNA specific for I-A beta was not detectable in explanted macrophages obtained from sites of sterile inflammation but was induced by treatment of purified recombinant interferon-gamma. This effect was dose dependent and was optimal by 24 hr after stimulation. Ia-specific mRNA preceded the surface expression of Ia as monitored by a radioimmunoassay using a monoclonal antibody specific for I-A beta. When a physiologic dose of LPS was added concomitantly with the interferon-gamma, the time course of induction if Ia-specific mRNA was not altered, but the amount of such mRNA detected was suppressed 40 to 80%. This effect was dependent on the dose of LPS, and the levels of mRNA correlated closely with subsequent surface expression of Ia. The ability of LPS to suppress both mRNA and cell surface Ia expression required that the suppressive agent be added within 12 hr of the inducing stimulus. This is the time frame during which accumulation of mRNA occurs. Thus the data demonstrates that accumulation of specific mRNA is a major regulatory focus governing expression of Ia both by interferon-gamma and LPS.

Animals↗
PubMed1987

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase.

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1986

Yeast/E. coli shuttle vectors with multiple unique restriction sites.

Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously in Saccharomyces cerevisiae and in E. coli; (2) they contain the beta-lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of beta-galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these auxotrophic markers in yeast and E. coli; (7) both plasmids are retained with high frequency in yeast grown under non-selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for construction of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for autonomous replication in yeast. The derivative plasmids YIp351 and YIp352 can therefore be used to integrate specific sequences into yeast chromosomal DNA.

Cloning, Molecular↗
PubMed1985

Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase.

The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino-acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene.

Acylation↗
PubMed1985

Cloning and characterization of the yeast nuclear gene for subunit 5 of cytochrome oxidase.

The nuclear gene COX5 coding for subunit 5 of cytochrome oxidase has been cloned by transformation of the cox5-1 mutant aE4-238/AL1 with a library of yeast genomic DNA. The recombinant plasmid pG46/ST2 bearing a nuclear DNA insert of 1.17 kilobase pairs restores the ability of cox5 mutants to respire and to synthesize a wild type subunit 5. The COX5 gene has been sequenced and determined to code for a 153-amino acid long protein with a molecular weight of 17,121. The amino-terminal 20 residues comprise the signal peptide. The sequence starting from residue 21 matches the partial sequence reported for the mature subunit 5. The sequence of the subunit 5 gene indicates that the mature protein has a molecular weight of 14,858 which agrees with previous size estimates based on electrophoretic migration. The primary sequence and polarity profile of yeast subunit 5 establishes that it is homologous to subunit 4 of bovine cytochrome oxidase.

Amino Acid Sequence↗
PubMed1985

Nuclear mutants of Saccharomyces cerevisiae with altered subunits 4, 5, and 6 of cytochrome oxidase.

A collection of pet mutants of Saccharomyces cerevisiae has been screened for lesions in cytochrome oxidase. Three different complementation groups have been identified to consist of strains with altered forms of subunits 4, 5, or 6 that are known to be encoded by nuclear genes. The mutant proteins cross-react with antiserum to the holoenzyme or to the individual subunits but exhibit either an increase or decrease in size. In each instance the mutation imparts a respiratory deficient phenotype which is due to reduced levels of cytochrome oxidase activity in the mitochondria. These results indicate that each of the three proteins is required either for the catalytic activity or for the assembly of functional cytochrome oxidase.

Electron Transport Complex IV↗
PubMed1984

Assembly of the mitochondrial membrane system. CBP1, a yeast nuclear gene involved in 5' end processing of cytochrome b pre-mRNA.

Noncomplementing mutations in a nuclear gene (CBP1) of Saccharomyces cerevisiae D273-10B specifically affect the synthesis of cytochrome b, a mitochondrially encoded carrier of the respiratory chain. The nuclear mutants have been shown to have lowered levels of cytochrome b-specific transcripts. This phenotype is attributed to the inability of the mutant strains to process the 5' end of the cytochrome b pre-mRNA. Impairment of the processing function encoded by the CBP1 gene introduces an instability in the transcripts and promotes nucleolytic degradation. Mutations in CBP1 can be suppressed by a p- genome in which the 5' untranslated leader of the oli1 gene (subunit 9 of the ATPase) is fused near the 5' side of the cytochrome b coding sequence. The rearranged genome allows the cytochrome b gene to be transcribed from the oli1 promoter and results in novel cytochrome b transcripts with the 5' leader sequence of the oli1 mRNA. The presence of the oli1 leader sequence confers stability to the RNA and circumvents the CBP1 processing function.

Base Sequence↗