Biomedical subjects

C E Bauer

Publications and source records attributed to C E Bauer.

64 records · Page 4Linked to original sources

PubMed1988

Transcription of the Rhodobacter capsulatus nifHDK operon is modulated by the nitrogen source. Construction of plasmid expression vectors based on the nifHDK promoter.

We characterized the Rhodobacter capsulatus nifHDK promoter by nucleotide sequencing and nuclease S1 analysis of mRNA-protected DNA probes. Comparison of this promoter to nifP and ntrP promoters from other species reveals extensive homology to the canonical nifP consensus sequence. Using lac fusions we have demonstrated that transcription of the nifHDK operon is totally repressed when the growth medium is supplemented with ammonia, becomes fully derepressed in ammonia-free medium, and proceeds at intermediate levels when other nitrogen sources are used. Based on this information, we constructed plasmid expression vectors in which the rates of transcription from cloned DNA fragments are determined by the nitrogen source used in the growth medium.

Base Sequence↗

PubMed1988

Analysis of the Rhodobacter capsulatus puf operon. Location of the oxygen-regulated promoter region and the identification of an additional puf-encoded gene.

In an attempt to identify features of an oxygen-regulated promoter, we have determined the location of transcription initiation for the puf operon. The position for the oxygen-regulated promoter was demonstrated by several independent means to be located 699 base pairs (bp) upstream from the pufB structural gene. DNA sequence analysis of the promoter region demonstrates the presence of a 26-base pair region of dyad symmetry followed by a sequence containing homology to promoters which use the RNA polymerase sigma 60 subunit (ntrA) for recognition of DNA. In addition to the oxygen-regulated promoter, a region responsible for low-level constitutive expression of the puf operon was shown to initiate transcription 511 bp upstream from the pufB gene. In contrast to the oxygen-regulated promoter, this second promoter contains no obvious secondary structure nor sequence homology to ntrA-dependent promoters. DNA sequence analysis demonstrates the existence of an additional open reading frame (designated as pufQ) that is located between the promoters and the pufB structural gene. A translational fusion of pufQ to lacZ was used to demonstrate that pufQ is efficiently translated and regulated in a manner analogous to a translational fusion of pufM to lacZ. Finally, we also demonstrate that puf operon transcription initiation and regulation does not involve any puf-encoded gene products.

Amino Acid Sequence↗

PubMed1988

Rhodobacter capsulatus puf operon encodes a regulatory protein (PufQ) for bacteriochlorophyll biosynthesis.

Biosynthesis of the photochemical apparatus by purple nonsulfur photosynthetic bacteria is known to be inhibited by molecular oxygen and high light intensity. Polypeptides that bind bacteriochlorophyll (BChl) to form the light-harvesting I (LH-I) and reaction-center (RC) complexes are encoded by a single transcriptional unit termed the puf operon. In this investigation we demonstrate that the first structural gene in the puf operon (pufQ) of Rhodobacter capsulatus encodes a protein that is required for BChl biosynthesis and that there exists a linear relationship between the amount of pufQ expression and the level of BChl synthesis. Protein sequence similarity exists between PufQ and the region of RC polypeptides that are known to bind BChl and quinone. These observations suggest that pufQ may regulate BChl biosynthesis by a "carrier polypeptide" mechanism as originally proposed by Lascelles.

Amino Acid Sequence↗

PubMed1987

Role of homology in site-specific recombination of bacteriophage lambda: evidence against joining of cohesive ends.

Bacteriophage lambda integration and excision take place at specific loci called attachment sites. Earlier work has shown that efficient recombination requires the identical sequence to be present in both attachment sites throughout the seven-base-pair region between the points of strand exchange. A plausible model for the role of homology postulates that Int, the site-specific recombinase, makes double-strand breaks at attachment sites such that each broken end has a short single-strand protrusion. Recombination would then depend upon the capacity of these protrusions to form Watson-Crick helices--i.e., to anneal--a process that might require perfect complementarity between the cohesive ends. To test this model, we have studied Int-promoted crosses in which one attachment site is a heteroduplex. Specifically, we constructed sites in which the seven-base-pair region between the points of strand exchange contains one or more noncomplementary pairs. The double-strand break and annealing mechanism predicts that crosses with these heteroduplex sites should yield one completed recombinant and one broken site. We find that such nonreciprocal recombination is uncommon and that the typical outcome of crosses involving a heteroduplex site is a reciprocal recombinant in which both products are resealed. Moreover, the occasional appearance of nonreciprocal products can be explained by our finding that Int can cleave heteroduplex attachment sites after recombination is completed. Taken together, our data strongly indicate that bacteriophage lambda recombination does not proceed by the homology-dependent annealing of cohesive ends; acceptable alternatives for the role of homology are discussed.

Bacteriophage lambda↗

PubMed1986

Mutational analysis of integrase arm-type binding sites of bacteriophage lambda. Integration and excision involve distinct interactions of integrase with arm-type sites.

Integrative recombination between specific attachment (att) regions of the bacteriophage lambda genome (attP) and the Escherichia coli genome (attB) results in a prophage flanked by the hybrid recombinant sites attL and attR. Each att site contains sequences to which proteins involved in recombination bind. Using site-directed mutagenesis, we have constructed a related set of point mutations within each of the five Int "arm-type" binding sites located within attP, attL and attR. Footprint analyses of binding demonstrate that mutating the arm-type sites significantly disrupts the binding of Int. Recombination analyses of mutant att sites in vivo and in vitro demonstrate that only three wild-type arm-type sites within attP are required for efficient integrative recombination. Similar analyses demonstrate that efficient excision can occur with two other different sets of wild-type arm-type sites in attL and attR. These results demonstrate that integrative and excisive recombination may involve interactions of Int with distinct and different subsets of arm-type sites.

Attachment Sites, Microbiological↗

PubMed1985

Identification and characterization of mutants affecting transcription termination at the threonine operon attenuator.

Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized. These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes. Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol). Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain. The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis. In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants. Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator. The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem. The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination. Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.

Base Sequence↗

PubMed1985

Extent of sequence homology required for bacteriophage lambda site-specific recombination.

Bacteriophage lambda integration and excision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position -3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position -2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the -2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.

Attachment Sites, Microbiological↗

PubMed1985

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis.

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.

Coliphages↗

PubMed1984

DNA interactions during bacteriophage lambda site-specific recombination.

Extensive research on site-specific recombination has provided many details, particularly with respect to the protein-DNA interactions. However, very little is known about the molecular mechanism of recombination during synapsis and strand exchange. Presumably, these steps of recombination involve various forms of DNA-DNA, DNA-protein, and protein-protein interactions. One stage at which DNA-DNA interactions may be occurring is at the level of synapsis where the recombining DNAs are juxtaposed. In this paper we have presented evidence that homology-dependent DNA interactions do occur within the overlap region before strand exchange. This interaction is presumably at the synaptic stage of recombination. Furthermore, we have demonstrated that the homology-dependent interactions require that only one strand of attB have homology to attP. Another stage in recombination at which DNA-DNA interactions could occur is during strand exchange where complementary strands from the recombining parents are paired and resealed. We have also presented evidence that homology-dependent DNA interactions occur during strand exchange prior to the resealing of the strands and that disruption of this interaction results in nonreciprocal recombination. Taken together, these results suggest that DNA-DNA interactions during reciprocal site-specific recombination occur during at least two stages in the reaction.

Bacteriophage lambda↗

PubMed1977

Qualitative evaluation of macroscopic flocculation tests for syphilis.

Five thousand two hundred and two sera were tested in parallel using the Rapid Plasma Reagin (RPR) Card Test, the Reagin Screen Test (RST), and the Venereal Disease Research Laboratory (VDRL) Slide Test. The fluorescent treponemal antibody absorption (FTA-ABS) test was then used to confirm the reactive specimens. This comparison study was to evaluate the RPR and the RST when used in a screening program. Of the total 5,202 samples, 199 (3.8%) were VDRL reactive, 187 (3.6%) were RPR reactive, and 183 (3.5%) were RST reactive. Of the 199 VDRL reactive, 116 (58.2%) were confirmed by the FTA-ABS, 137 (73.3%) of the 187 RPR reactives were confirmed by the FTA-ABS and of the 183 RST reactives, 115 (62.8%) were confirmed by the FTA-ABS. Included in this discussion are some testing and procedural problems encountered during the study.

Evaluation Studies as Topic↗