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Biomedical subjects

E R Olson

Publications and source records attributed to E R Olson.

34 records · Page 2Linked to original sources
PubMed1991

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein.

We describe the isolation and analysis of an Escherichia coli gene, dppA, and its role in dipeptide transport. dppA maps near min 79 and encodes a protein (DppA) that has regions of amino acid similarity with a peptide-binding protein from Salmonella typhimurium (OppA). Like OppA, DppA is found in the periplasmic space and thus is most likely a dipeptide-binding protein. Insertional inactivation of dppA results in the inability of a proline auxotroph to utilize Pro-Gly as a proline source. dppA-dependent Pro-Gly utilization does not require any of the three major proline transport systems, demonstrating that DppA is not simply a dipeptidase. An in vivo competition assay was used to show that DppA is probably involved in the transport of dipeptides other than Pro-Gly. Transcription of dppA is repressed by the presence of casamino acids, suggesting that the cell alters its dipeptide transport capabilities in response to an environmental signal.

Amino Acid Sequence↗
PubMed1990

Point mutations in a pBR322-based expression plasmid resulting in increased synthesis of bovine growth hormone in Escherichia coli.

The bGH cDNA coding for bovine growth hormone (bGH) is expressed poorly in Escherichia coli using a pBR322-based expression plasmid. Random mutagenesis of the plasmid gave rise to two types of plasmid mutants which increased the expression of bGH. One class had single base changes in the first four codons of the bGH sequence. The second class had single base changes in regions of the plasmid involved in controlling plasmid replication but had little effect on plasmid copy number.

Amino Acid Sequence↗
PubMed1990

Transcription-dependent competition for a host factor: the function and optimal sequence of the phage lambda boxA transcription antitermination signal.

Ordered development of lambdoid phages relies on systems of transcription termination and antitermination. The phage-encoded N early regulatory proteins, acting with the Nus proteins of Escherichia coli, modify RNA polymerase to a form that overrides many transcription termination signals. These modifications require cis-acting sites, nut, located downstream of the early phage promoters. The nut sites in phages lambda, 21, and P22, which share similarities but are not identical, contain two signals, boxA and boxB. We demonstrate that although a consensus sequence for the boxA signal (boxAcon), 5'CGCTCTTTA, is found only in P22, changes to consensus in the nutR sites of lambda and 21 create more effective antitermination signals than the wild-type signals. An in vivo competition assay demonstrates that a lambda nut region with boxAcon outcompetes nut regions with wild-type, as well as other variations of the boxA sequence, for the host NusB protein. This suggests that boxA influences NusB activity in N-mediated antitermination. Successful competition by boxAcon requires transcription of the nut site as well as N activation. Nucleotide replacement further demonstrates that bases at both ends of boxA are important for antitermination.

Bacterial Proteins↗
PubMed1989

Effect of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin in E. coli.

We have studied the expression of bovine somatotropin (BSt) to gain more understanding of various factors affecting translation in E. coli. The unmodified cDNA coding for mature bovine somatotropin does not produce significant amounts of BSt in E. coli using a pBR322-derived vector. However, a translation fusion with 16 codons from trpLE in front of BSt cDNA results in greater than 20% of total cell protein as the fusion product. Analysis of transcription by measuring the rate and integrity of the mRNA confirms that a post-transcriptional event is responsible for the poor expression of the BSt cDNA. There are two potential stem-loop structures in the 5' region of the mRNA which may interfere with translation. To study their effect on translation, lacZ fusions and oligonucleotide mutagenesis were carried out. The results demonstrate that the secondary structure involving the initiation codon blocks translation initiation. Removal of this stem-loop results in a 100-fold increase in BSt expression. However, the expression level is still low, amounting to only 0.5-1% of total cell protein. High level expression can be obtained by replacement of the beginning sequence of BSt cDNA with trpLE codons. These results suggest that in addition to the secondary structure, the nucleotide sequence or amino acid context within the beginning of BSt is incompatible with one of the steps in translation initiation.

Cloning, Molecular↗
PubMed1989

Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA.

Southern blot analysis of 15 proU transposon insertions in Salmonella typhimurium indicated that this operon is at least 3 kilobase pairs in length. The nucleotide sequence of 1.5-kilobase-pair fragment that contains the transcriptional control region of the proU operon and the coding sequences specifying 290 amino acids of the first structural gene of the operon was determined. The predicted amino acid sequence of the product of this gene shows extensive similarity to the HisP, MalK, and other proteins that are inner membrane-associated components of binding protein-dependent transport systems. S1 mapping and primer extension analysis of the proU mRNAs revealed several species with different 5' ends. Two of these endpoints are sufficiently close to sequences that have weak similarities to the consensus -35 and -10 promoter sequences that they are likely to define two transcription start sites. However, we cannot rule out the possibility that some or all of the 5' endpoints detected arose as a result of the degradation of a longer mRNA. The expression of proU-lacZ operon fusions located on plasmids was normal in S. typhimurium regardless of the plasmid copy number. The sequences mediating normal, osmoregulated expression of the proU operon were shown by subcloning to be contained on an 815-base-pair fragment. A 350-base-pair subclone of this fragment placed onto a lacZ expression vector directed a high-level constitutive expression of beta-galactosidase, suggesting that there is a site for negative regulation in the proU transcriptional control region which has been deleted in the construction of this plasmid.

Amino Acid Sequence↗
PubMed1989

Genetic analysis of the N transcription antitermination system of phage lambda.

Regulation of coliphage lambda gene expression occurs, in part, by a process of termination and antitermination of transcription. The N gene product, one of two lambda encoded antitermination proteins, acts with host encoded proteins, Nus, at sites, nut, located downstream of the early promoters to render the transcribing RNA polymerase resistant to many downstream termination signals. We discuss the nature of some of the proteins and sites involved in this process.

Amino Acid Sequence↗
PubMed1988

Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound.

Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction. The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence. No similarity was found with the promoter areas of Rhizobium trifolii, R. meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.

Base Sequence↗
PubMed1984

The nusA recognition site. Alteration in its sequence or position relative to upstream translation interferes with the action of the N antitermination function of phage lambda.

The phage lambda transcription antitermination protein, pN, acts with host factors, Nus, at sites on the phage genome, nut, to render RNA polymerase resistant to subsequent downstream termination signals. The NusA protein appears to recognize a seven to eight base-pair consensus sequence (5'Py-G-C-T-C-T-T(T)3') called boxA that is found in the promoter-proximal part of the nut region. Two types of change within or near the boxA sequence in the nutR region are shown to interfere with pN-mediated antitermination of transcription that has initiated at the upstream pR promoter. (1) A change of one base-pair (from G to T at the second position) in the boxA sequence significantly reduces pN action. (2) We prove that a frameshift mutation, cro delta 62, at the end of the gene promoter-proximal to the lambda nutR region, interferes with the pN antitermination reaction by allowing translation to proceed beyond cro into the nutR region. Using a series of plasmid constructions, we now show that the inhibition of antitermination caused by the cro delta 62 mutation can be suppressed when translation is terminated upstream from this mutation.

Bacterial Proteins↗
PubMed1983

Evidence that a nucleotide sequence, "boxA," is involved in the action of the NusA protein.

We report the isolation of a mutation, boxA1, in the nutR region of the phage lambda genome. The nutR region, located downstream of the pR promoter, includes the site nutR where the lambda N protein is thought to act to render subsequent transcription termination-resistant. We have previously suggested that the boxA sequence, 5'CGCTCTTA3' (or its RNA analog), located 8 bp promoter-proximal to nutR, might be the recognition site for the E. coli host factor, NusA, which has been shown to be necessary for N action. The boxA1 mutation, an A:T to T:A transversion, results in a changed boxA sequence upstream of nutR, CGCTCTTT. This change is necessary for lambda to effectively use the NusA of Salmonella typhimurium, a NusA function not normally active with the N product of lambda. Other lambdoid phages with unique N functions and nut sites that are normally active with the NusA of Salmonella have boxA sequences with the terminal three Ts. Moreover, sequences closely resembling boxA have been found near transcription termination sequences in E. coli operons where NusA has been shown to be involved in termination. These findings identify boxA as an important recognition signal for the NusA protein.

Bacterial Proteins↗
PubMed1982

Analysis of nutR: a region of phage lambda required for antitermination of transcription.

The N gene product of coliphage lambda acts with host factors (Nus) through sites (nut) to render subsequent downstream transcription resistant to a variety of termination signals. These sites, nutR and nutL, are downstream, respectively, from the early promoters PR and PL. Thus a complicated set of molecular interactions are likely to occur at the nut sites. We have selected mutations in the nutR region that reduce the effectiveness of pN in altering transcription initiating at the PR promoter. DNA sequence analysis of three independently selected mutations revealed, in each case, a deletion of a single base pair in the cro gene. Consideration of the effect of such mutations on the extension of translation of cro message into the adjacent downstream nut region led to the identification of a consensus sequence CGCTCT(T)TAA that appears to play a role in the recognition of a host factor, possibly the NusA protein.

Bacteriophage lambda↗
PubMed1981

Stellate block for trigeminal zoster.

Stellate ganglion block for relief of pain and prevention of ophthalmic complications in trigeminal herpes zoster has been advised for many years. In a series of 27 patients, control of pain was dramatic after local anesthetic block of the stellate ganglion. This report is presented to stimulate renewed interest in this old form of therapy. Because of the retrospective nature of this investigation, the lack of adequate numbers and the absence of controls, no conclusions on efficacy of treatment can be made. However, enough hope of success is presented to justify a controlled series in a large metropolitan area where adequate numbers of patients can be accumulated for a double-blind protocol.

Bupivacaine↗