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Biomedical subjects

C F Beck

Publications and source records attributed to C F Beck.

At least 55 records · Page 3Linked to original sources
PubMed1989

Three light-inducible heat shock genes of Chlamydomonas reinhardtii.

Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light.

Chlamydia↗
PubMed1988

Point mutations that affect translation initiation of the transposon Tn10 tetA gene.

Single base-pair changes affecting the 5' untranslated leader of the tetracycline-resistance gene (tetA) mRNA resulted in increased or decreased levels in expression of tetA-lacZ protein fusions. The base changes affected the rate of initiation of translation of tetA mRNA because operon fusions revealed that the mutations had little or no effect on transcription. The translational efficiency of wild-type and mutant tetA mRNAs varied by more than a factor of 2000. The observed variations could be correlated with stabilization and destabilization of RNA secondary structures. These structures, located 5' from the translation start codon, sequester the Shine-Dalgarno sequence.

Base Sequence↗
PubMed1988

Monoclonal antibodies as tools in membrane biochemistry. Identification and partial characterization of the dicarboxylate transporter from pea leaf mitochondria.

Monoclonal antibodies specific for the dicarboxylate transporter of pea mitochondria were prepared and used to identify this substrate carrier. The hybridoma library was derived from mice that had been immunized with total mitochondrial membranes. The monoclonal antibodies specific for the dicarboxylate transporter were selected by screening hybridoma supernatants for their ability to inhibit malate- and succinate-dependent oxalacetate reduction by osmotically shocked pea leaf mitochondria. Three monoclonal antibodies were shown to be specific for the dicarboxylate transporter by their ability to 1) inhibit malate and succinate metabolism without affecting alpha-ketoglutarate, citrate, pyruvate, glycine, glutamate, or aspartate metabolism by mitochondria and 2) inhibit malate uptake by a partially purified transporter fraction reconstituted into asolectin vesicles. The dicarboxylate transporter was identified by Western blotting and immunoprecipitation and had an apparent molecular mass of 26,000 Da. The techniques described should prove useful for identifying a number of membrane proteins that can be assayed in situ but are difficult to assay following dissolution of the membrane.

Aminomethyltransferase↗
PubMed1987

Functional in vivo verification in E. coli of promoter activities from the rDNA/tDNA(Val)(GAC) leader region of Zea mays chloroplasts.

Restriction fragments containing upstream sequences of the rRNA operon from Zea mays chloroplasts were tested for promoter activity in vivo by insertion into an E. coli promoter-probe vector. The expression of this vector's reporter gene, which codes for alkaline phosphatase, was stimulated more than 1,500-fold upon linkage with the chloroplast rRNA promoter. Site specific mutagenesis of the invariant T of the -10 sequence of this promoter reduced the expression of the reporter gene to 2% of the wild type. This indicates that the chloroplast rRNA promoter, which directs transcriptional initiation 117 bp upstream of the 16S rRNA gene, is also active in the bacterial system. A restriction fragment further upstream containing the gene for tRNA(Val) (GAC) also showed strong promoter activity (29% as compared with the rRNA promoter). This promoter activity probably reflects the chloroplast promoter directing the synthesis of the tRNA(Val) (GAC) primary transcript. Surprisingly, this restriction fragment also displayed promoter activity (13% compared with the rRNA promoter) in reverse orientation.

Base Sequence↗
PubMed1986

Sequence conservation and region shuffling in an endoglucanase and an exoglucanase from Cellulomonas fimi.

Cellulomonas fimi produces an endoglucanase and an exoglucanase which bind strongly to cellulose. Each enzyme contains three distinct regions: a short sequence of about 20 amino acids containing only proline and threonine (the Pro-Thr box); an irregular region, rich in hydroxyamino acids, of low charge density, and which is predicted to have little secondary structure; and an ordered region of higher charge density which contains a potential active site, and which is predicted to have secondary structure. The Pro-Thr box is conserved almost perfectly in the two enzymes. The irregular regions are 50% conserved, and the conserved sequences include four Asn-Xaa-Ser/Thr sites. The ordered regions appear not to be conserved, but the potential active sites both have the sequence Glu-Xaa7-Asn-Xaa6-Thr; they occur at widely separated sites in the two regions. The order of the regions is reversed in the two enzymes: irregular-Pro-Thr box-ordered in the endoglucanase; ordered-Pro-Thr box-irregular in the exoglucanase. The genes for the two enzymes appear to have arisen by shuffling of two conserved sequences and either one or two other sequences.

Actinomycetales↗
PubMed1986

Promoter-probe vectors for the analysis of divergently arranged promoters.

A series of plasmid-based promoter-probe vectors has been constructed which are particularly useful for the analysis of divergent control regions. Each vector contains a pair of divergently oriented indicator genes whose expression can be monitored over a wide range by simple assay methods. These genes are separated by different polylinkers. Specifically, the beta-galactosidase gene (lacZ) was employed in combination with either the galactokinase gene (galK) or the alkaline phosphatase gene (phoA). In all cases translational stop codons are present in all three reading frames upstream from the initiation codon. The vectors permit direct detection of promoters--independent of insert orientation--on indicator plates after transformation. Using this vector system, we further characterized the divergent tet control regions of transposon Tn10 and plasmid pBR322.

Alkaline Phosphatase↗
PubMed1986

Monoclonal antibodies specific for Corynebacterium sepedonicum, the causative agent of potato ring rot.

BALB/c mice were immunized with Corynebacterium sepedonicum, and spleen cells from the immunized animals were fused with cells of the mouse myeloma line P3-X63-Ag8.653. Several hybridoma cell cultures were selected for further study. Monoclonal CS-B-5 was specific for C. sepedonicum and did not react significantly with other closely related phytopathogenic corynebacteria in an enzyme-linked immunosorbent assay (ELISA). As few as 10(3) organisms could be detected. This approach should prove useful for developing improved diagnostic procedures for a number of bacterial plant pathogens.

Animals↗
PubMed1985

Tetracycline uptake by susceptible Escherichia coli cells.

Experiments measuring the initial uptake of commercial (3H) tetracycline exhibit two distinct kinetic phases: a rapid phase followed by a slow phase. (3H) tetracycline purified by chromatography on a Dowex 50WX2 column exhibited only monophasic rapid uptake when tested with susceptible Escherichia coli cells. Cyanide inhibited the uptake of purified (3H) tetracycline only partially while transport of proline and maltose was entirely abolished. Energy independent accumulation of tetracycline may be accounted for by binding to cellular constituents. Uptake of tetracycline--as measured by inhibition of beta-galactosidase synthesis--was strongly affected by a shift in temperature from 37 degrees C to 21 degrees C while carrier-mediated transport systems revealed only minor reductions. Taken together with the non-saturability of tetracycline uptake and the evidence for diffusion of tetracycline through phospholipid bilayers [Argast and Beck (1984) Antimicrob Agents Chemother 26:263-265] these data support the hypothesis that tetracycline enters the cytoplasm by diffusion.

Cyanides↗
PubMed1985

Resistance to various tetracyclines mediated by transposon Tn10 in Escherichia coli K-12.

Levels of resistance to tetracycline, chlortetracycline, demethylchlortetracycline, doxycycline, oxytetracycline, methacycline, pyrrolidinotetracycline, minocycline, and beta-chelocardin of Escherichia coli K-12 carrying transposon Tn10 or defined DNA segments of Tn10 were determined. In all cases, tetA was the only gene required for resistance. Doxycycline was the most effective inducer of tetA gene expression.

DNA Transposable Elements↗
PubMed1984

Tetracycline diffusion through phospholipid bilayers and binding to phospholipids.

The ability of tetracycline to pass through phospholipid bilayers by diffusion was investigated. Liposomes did not retain enclosed tetracycline. Accumulation of tetracycline was observed with liposomes containing entrapped Tet repressor protein. These results indicate that the drug can pass through lipid bilayers. The antibiotic was also shown to bind to liposomes and isolated phospholipids.

Chemical Phenomena↗
PubMed1984

Identification of additional genes on transposon Tn10: tetC and tetD.

Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R. Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively. The TetD protein was found to be membrane associated. Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested. The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K. Schollmeier and W. Hillen, J. Bacteriol. 160:499-503, 1984).

Bacterial Proteins↗
PubMed1982

A multifunctional gene (tetR) controls Tn10-encoded tetracycline resistance.

The tetracycline resistance regulatory gene (tetR) of transposon Tn10 was analyzed by a combination of methods involving gene fusion and cloning. This gene is located on a 695-base pair HincII DNA fragment near the center of Tn10. The direction of transcription is opposite to that of neighboring gene tetA, which encodes the TetA protein. The gene product of the tetR gene (the TetR protein) has a molecular weight of 23,000. tet-R-lacZ gene fusions encode fusion beta-galactosidases that are membrane bound, indicating that the TetR protein itself is membrane associated. Mutants defective in tetR result in constitutive tetracycline resistance, but the level of resistance is reduced. Expression of the tetR gene is induced by tetracycline; in the absence of tetracycline, the TetR protein turns off its own synthesis.

Bacterial Proteins↗
PubMed1981

Periplasmic phosphatases in Salmonella typhimurium LT2. A biochemical, physiological, and partial genetic analysis of three nucleoside monophosphate dephosphorylating enzymes.

Three periplasmic nucleoside monophosphate-splitting phosphatases from Salmonella typhimurium, 2':3'-cyclic nucleotide 2'-phosphodiesterase, nonspecific acid phosphatase I, and nonspecific acid phosphatase II, were separated by column chromatography. They are characterized with respect to their substrate specificities, Km values, pH optima, molecular weights, and sensitivity to inhibition by Pi and EDTA. Nonspecific acid phosphatase II has not been reported previously. The physiological roles of the various phosphatases are assessed by studies on mutant strains. Selection procedures were developed for the isolation of mutants defective in the synthesis of one or more of the phosphatase activities. Analysis of these mutant strains revealed that 2':3'-cyclic nucleotide 2'-phosphodiesterase is the major 3'-nucleotide dephosphorylating activity, and nonspecific acid phosphatase II is most active against 5'-nucleotides at concentrations below 1 mM. Triple mutants, lacking all periplasmic nucleotide-splitting activities, are viable. Regulatory mutants with elevated levels of nonspecific acid phosphatase II activity were isolated. The gene for 2':3'-cyclic nucleotide 2'-phosphodiesterase (pde) was located between purA and argI at 135 min on the S. typhimurium linkage map.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1980

The tetracycline-resistance transposon Tn10 inhibits translocation of Tn10.

Using a set of overlapping deletion mutants in the tetracycline-resistance transposon Tn10, it has been established that certain regions of the Tn10 genome exert a powerful inhibition on translocation of an intact Tn10 element into the bacterial genome. Such inhibition is strongly temperature dependent: at 37 degrees C translocation is inhibited by at least a factor of 100; no inhibition of translocation is detected at 30 degrees C.

Chromosome Deletion↗
PubMed1979

A genetic approach to analysis of transposons.

Integration of the tetracycline resistance transposon Tn10 into lacI of a lacI-lacZ gene fusion permits the isolation of deletions that excise DNA from one end of Tn10 and fuse Tn10 genes with lacZ in such a manner that chimeric proteins with beta-galactosidase activity are produced. The synthesis of the chimeric proteins is under the same control as the transposon genes. Thus, regulation of expression of Tn10 genes can be investigated by measuring beta-galactosidase activity. Analysis of Tn10-lacZ fusions revealed different deletion endpoints within Tn10; lacZ has been fused to at least three different Tn10 genes or operons. Two of these genes are under the control of a tetracycline repressor.

Chromosome Deletion↗