Biomedical subjects

D L Court

Publications and source records attributed to D L Court.

At least 55 records · Page 3Linked to original sources

PubMed1991

Functional importance of sequence in the stem-loop of a transcription terminator.

Intrinsic transcription terminators of prokaryotes are distinguished by a common RNA motif: a stem-loop structure high in guanine and cytosine content, followed by multiple uridine residues. Models explaining intrinsic terminators postulate that the stem-loop sequence is necessary only to form structure. In the tR2 terminator of coliphage lambda, single-nucleotide changes reducing potential RNA stem stability eliminated tR2 activity, and a compensatory change that restored the stem structure restored terminator activity. However, multiple changes in the stem sequence that should have either maintained or increased stability reduced terminator activity. These results suggest that the ability of the stem-loop structure to signal transcription termination depends on sequence specificity and secondary structure.

Bacteriophage lambda↗

PubMed1991

RNaselll activation of bacteriophage lambda N synthesis.

The bacteriophage lambda N gene product is one of the first genes expressed during phage development. N protein allows the expression of other phage genes by altering the transcription elongation process so as to prevent transcription termination. We have found that N levels may be modulated soon after induction or infection. Using N-lacZ fusions, we determined that cells containing RNaselll have at least a fourfold greater expression than cells defective for RNaselll. This effect is exerted at the post-transcriptional level. RNaselll processes an RNA stem structure in the N-leader RNA. Removal of the stem structure by deletion increases N expression and prevents further stimulation by RNaselll. The base of this stable stem is adjacent to the N ribosome binding site. We present a model for control of N synthesis in which this stable stem inhibits ribosome access to the N mRNA.

Bacteriophage lambda↗

PubMed1991

High expression of era gene.

For high production of Era protein, lambda Ea8.5 gene has been chosen from Gene Bank because the Ea8.5 protein contains a sequence of several amino acids at its N-terminus which is identical to that of Era and also because it can be highly expressed in E. coli. The 5'-end sequence of era gene was substituted by a synthetic oligonucleotide which was identical to that of the 5'-end of Ea8.5 gene, so that the transcripts of era gene was provided with a strong translational initiation signal without changing the amino acid sequence of its translational product. Plasmid pCE31, which harbored the recombined era gene under the control of PL promoter, could express very high levels of Era protein, while synthesis of other cellular proteins was nearly shut off during the period of induction. As a result, Era constituted over 80% of the total cellular protein. Electrophoretic pure Era protein with specific guanine nucleotide-binding activity was obtained by a simple procedure including lyzing the cells and washing the pellet of the lysate.

Bacterial Proteins↗

PubMed1990

Expression and characterization of RNase III and Era proteins. Products of the rnc operon of Escherichia coli.

The synthesis rates of ribonuclease III (RNase III) and Era proteins are relatively low, and expression of the era gene is translationally coupled with expression of the rnc gene. Expression of both genes is negatively controlled by RNase III itself. We have constructed plasmids that overproduce RNase III and/or Era proteins under the control of the lambda PL promoter. A plasmid with the rnc gene under PL control expresses RNase III at levels greater than 40% of total cellular protein. Another plasmid with the era gene under PL control and a modified translation-initiation signal produces up to 80% of total cell protein as Era. Each protein has been purified using simple and rapid procedures. Purified RNase III protein specifically processes mRNA transcripts containing known RNase III sites. The purified Era protein binds GDP and GTP and has GTPase activity. Kinetic analysis shows that one molecule of GTP or GDP is bound/Era peptide with a Kd of 5.5 microM for GTP binding and 1.0 microM for GDP binding. The Km of the Era GTPase is 9.0 microM, and the maximum catalyzed rate of GTP hydrolyzed/min/mol of Era protein at 37 degrees C is 9.8 mmol.

Bacterial Proteins↗

PubMed1990

Expression of HIV-1 integrase in E. coli: immunological analysis of the recombinant protein.

Sequences encoding the human immunodeficiency virus type 1 (HIV-1) integrase gene have been cloned and expressed in Escherichia coli. The expressed protein is a lambda cII fusion protein of 37 kD containing the carboxyl-terminal 23 [corrected] amino acids of reverse transcriptase fused to the entire integrase sequence and is insoluble, a feature which allows partial purification away from soluble bacterial proteins. As judged by its reactivity with HIV positive sera in Western blot and in enzyme-linked immunosorbent assay (ELISA), the recombinant integrase retains antigenicity similar to native protein. Additionally, ELISA data obtained with the cloned protein indicate that patients infected with HIV-1 who are at different stages of progression to AIDS have antibodies reactive with the cloned integrase. HIV-2 positive human sera are also reactive with the cloned integrase. Rabbit antibodies produced against the recombinant protein react both by ELISA and Western blot with the homologous bacterially expressed protein, recognize both virion HIV-1 integrase and reverse transcriptase in Western blots, and immunoprecipitate an HIV-1 virion protein of 34 kD. Unlike human antisera from patients infected with HIV-1 or HIV-2 which are frequently reactive with both HIV-1 and HIV-2 integrase, the rabbit antibodies are type specific, reacting with HIV-1, but not with HIV-2 integrase by Western blot.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli.

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.

Alleles↗

PubMed1989

Murine monoclonal antibodies which recognize active sites of Escherichia coli NusA protein and epitope mapping by gene fusion.

Seven hybridomas producing murine monoclonal antibodies reactive against NusA protein of Escherichia coli were prepared. Antigenic determinants of these monoclonal antibodies have been mapped by immunoblotting analyses using fusion proteins containing parts of NusA. The epitope of the N14 antibody maps in a hydrophobic amino acid (aa) cluster and consists of at least Ala-181 and Ser-183 residues. nusA1 and nusA11 mutations, which cause aa changes of these residues, abolish the antigenic reactivity to the N14 antibody. These antibodies react with intact NusA protein, indicating that the epitopes are exposed on the surface of NusA. Most of these epitopes cluster around the nusA1 and nusA11 mutation loci.

Amino Acid Sequence↗

PubMed1989

Autoregulation of RNase III operon by mRNA processing.

RNase III has been implicated in the control of gene expression by the processing of mRNA. We have found that the rnc operon is autoregulated; rnc- mutant strains oversynthesize the operon's mRNA and protein products. A site in the 5'-noncoding region of the operon's message is cleaved by RNase III. This site-specific cleavage appears to be the initial step in the functional inactivation of the message, since the half-life of the cut message is dramatically shorter than that of the uncut message.

Base Sequence↗

PubMed1989

Conditionally lethal amber mutations in the leader peptidase gene of Escherichia coli.

The lep gene of Escherichia coli encodes the leader peptidase which cleaves amino-terminal leader sequences of secreted proteins. To facilitate the study of structure-function relationships of the leader peptidase, 22 amber mutations in lep were isolated by localized mutagenesis. These amber mutants grew at 32 degrees C but not at 42 degrees C in the presence of a temperature-sensitive amber suppressor. Most of them were lethal under sup0 conditions. However, one amber mutant, the lep-9 mutant, exhibited temperature-sensitive growth in the sup0 strain, indicating that the amber fragment is active at 32 degrees C but not at 42 degrees C. Protein precursors of the maltose-binding protein and OmpA accumulate strikingly in the lep-9 mutant.

Chromosome Mapping↗

PubMed1989

Genetic analysis of the rnc operon of Escherichia coli.

RNase III, an Escherichia coli double-stranded endoribonuclease, is known to be involved in maturation of rRNA and regulation of several bacteriophage and Escherichia coli genes. Clones of the region of the E. coli chromosome containing the gene for RNase III (rnc) were obtained by screening genomic libraries in lambda with DNA known to map near rnc. A phage clone with the rnc region was randomly mutagenized with a delta Tn10 element, and the insertions were recombined onto the chromosome, generating a series of strains with delta Tn10 insertions in the rnc region. Two insertions that had Rnc- phenotypes were located. One of them lay in the rnc gene, and one was in the rnc leader sequence. Polarity studies showed that rnc is in an operon with two other genes, era and recO. The sequence of the recO gene beyond era indicated it could encode a protein of approximately 26 kilodaltons and, like rnc and era, had codon usage consistent with a low level of expression. Experiments using antibiotic cassettes to disrupt the genes rnc, era, and recO showed that era is essential for E. coli growth but that rnc and recO are dispensable.

Bacterial Proteins↗

PubMed1989

Temperature-sensitive lethal mutant of era, a G protein in Escherichia coli.

The era gene of Escherichia coli encodes a GTP-binding protein which has similarities to elongation factor Tu and the Saccharomyces cerevisiae RAS protein. To investigate its function, mutations affecting era were isolated. A mini-Tn10 insertion, which truncated 22 amino acids from the COOH end of Era, did not affect cell growth. By using this mini-Tn10 insert as a coselectable marker, a temperature-sensitive lethal era mutant was isolated by localized mutagenesis using P1 phage transduction. A single-base G to A change was found at position 23, causing a tyrosine residue to be substituted for the cysteine residue at position 8 (era-770), in addition to the COOH-terminal mini-Tn10 disruption. Both alterations were necessary for the temperature-sensitive phenotype. Purified Era-770 mutant protein exhibited reduced binding to GTP compared with that of the wild-type Era protein.

Amino Acid Sequence↗

PubMed1988

nusA amber mutation that causes temperature-sensitive growth of Escherichia coli.

The nusA134 mutation was isolated from a sup0 strain as a temperature-sensitive mutant which grew at 32 degrees C but not at 42 degrees C. Immunoblot analysis showed that this mutant produced a 31,000-dalton nusA-encoded protein instead of the full-size 54,500-dalton product. Sequence and genetic analyses of the mutant nusA gene revealed a substitution of T for C at the PstI site (i.e., CTGCAG to CTGTAG), thereby creating a nonsense UAG codon. These results indicate that nusA134 is an amber mutation and that the 31,000-dalton amber fragment is active for Escherichia coli growth at 32 degrees C but not at 42 degrees C. Most lambda bacteriophage variants tested grew normally on the nusA134 mutant both at permissive and at nonpermissive temperatures. However, lambda r32, which carries an IS2 insertion beyond the tR1 terminator, was restricted at 42 degrees C. Defects in the transcriptional antitermination process, but not in transcription termination, were observed. A comparative study of nusA134 protein and a PstI-truncated protein suggests that truncation of the peptide chain at the PstI site by the amber mutation, rather than the loss of the glutamine residue, is primarily responsible for the defect in antitermination. The mode of the involvement of mutant nusA proteins in the N-mediated antitermination reaction is discussed.

Bacterial Proteins↗

PubMed1988

Functional organization of the murine leukemia virus reverse transcriptase: characterization of a bacterially expressed AKR DNA polymerase deficient in RNase H activity.

The functional organization of the murine leukemia virus reverse transcriptase was investigated by expressing a molecular clone containing AKR MuLV reverse transcriptase-coding sequences in Escherichia coli. A purified preparation of the expressed enzyme (pRT250 reverse transcriptase) consisted primarily of a 69-kilodalton protein that has normal levels of murine leukemia virus polymerase activity but 10-fold-reduced levels of RNase H compared with the viral enzyme. The deficit in RNase H activity was correlated with the absence of 60 to 65 amino acids normally present at the carboxyl end of murine leukemia virus reverse transcriptase. The results provide additional experimental evidence for the localization of polymerase and RNase H domains to the N- and C-terminal regions of reverse transcriptase, respectively.

AKR murine leukemia virus↗

PubMed1987

Detection of heterologous fusion proteins in Escherichia coli with a monoclonal antibody.

Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli. We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays. However, there are some CII-hybrid protein species with which the antibody does not react. Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.

Animals↗

PubMed1987

Analysis of nutR, a site required for transcription antitermination in phage lambda.

Deletions extending from the cro gene into boxA and nutR of the Rho-dependent tR1 terminator of bacteriophage lambda have been generated and cloned between promoters and the galK gene of Escherichia coli on a multicopy plasmid. Terminators placed between the promoters and galK restrict transcription and expression of galK on these plasmids. However, when lambda N protein is provided, and if a functional N interaction site, nutR, is intact, transcription antitermination occurs and galK expression increases. Deletions into the nutR region affect the ability to antiterminate. From the results obtained we conclude that: boxA, a site believed to bind host factors (Nus), is not required for transcription antitermination in this system; the host NusA function is required even in the absence of boxA; nutR is required for N antitermination; translation across the nutR sequence prevents N-dependent antitermination.

Bacteriophage lambda↗

PubMed1986

Mutations of bacteriophage lambda that define independent but overlapping RNA processing and transcription termination sites.

Bacteriophage lambda int gene expression is regulated differentially from transcripts originated at the pL and pI promoters. Transcripts initiated at pI terminate at the site tI and express int gene product efficiently. Polymerases starting at pL do not terminate at tI, due to the antiterminating activity of lambda N protein. The pL transcripts are unable to express Int protein efficiently because sib, a control site overlapping tI in the unterminated RNA, is processed by host RNase III. We have isolated lambda sib- mutants by their inability to inhibit int expression from pL transcripts. sib mutations were genetically mapped to the left of the lambda attachment site, and do not structurally alter this site for recombination. Several sib mutations do alter the nucleotide sequence of the overlapping sib and tI sites. The lambda sib- mutants tested prevent RNA processing but do not affect transcription termination in vivo.

Attachment Sites, Microbiological↗

PubMed1986

Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo.

Previous studies have attributed two activities to the NusA protein of Escherichia coli; namely, termination and antitermination of transcription. To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene (nusAts11). The mutant cells produce a thermolabile NusA protein and grow at 32 degrees C, but not at 42 degrees C. At 42 degrees C, nusAts11 is recessive to nusA+ and nusA1, indicating the absence of its active gene product at that temperature. In the mutant, the efficiency of termination at the lambda tR1 terminator decreases, resulting in an increased expression of distal gene(s). On the other hand, the synthesis of the beta-galactosidase and beta beta' subunits of RNA polymerase is reduced in the mutant. This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system. These results suggest that the NusA protein plays both negative and positive modulator roles in vivo. The mutation nusAts11, unlike nusA1, does not block lambda phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant. Besides, the nusAts11 allows lambda Nam7Nam53byp phage growth under sup0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant.

Bacterial Proteins↗

PubMed1986

A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment.

We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.

Antibodies, Neoplasm↗