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Biomedical subjects

S Carleton

Publications and source records attributed to S Carleton.

12 recordsLinked to original sources
PubMed1996

Direct identification and typing of Mycobacterium tuberculosis by PCR.

We have developed a rapid PCR assay that types strains of Mycobacterium tuberculosis by generating distinct DNA fingerprints directly from primary cultures. This assay allows strain identification analogous to that achieved by the standard restriction fragment length polymorphism method, and fingerprints are obtained in less than 8 h. This assay does not require subculturing, DNA purification, restriction digestion, Southern blotting, or nucleic acid hybridization. Rapid and precise identification of M. tuberculosis strains permits immediate molecular epidemiologic studies. The assay can be converted to a computer-automated system by employing fluorescently labeled PCR primers and the Perkin-Elmer DNA sequencer so that unknown-specimen fingerprints are identified by computer comparison to a database of M. tuberculosis strain fingerprints.

Bacterial Typing Techniques↗
PubMed1995

Adaptational response in transcription factors during development of myocardial hypertrophy.

Cardiac hypertrophy is characterized, among others, by the molecular events which selectively activate the expression of genes for contractile proteins within individual myocardial cells. As such, myosin light chain 2 (MLC-2), which is upregulated in the hypertrophic state in both rat and human, serves as a marker for hypertrophy. In an attempt to investigate the gene regulatory mechanisms of this phenomenon, we tested the hypothesis that certain transcription factors are directly involved in the development of cardiac hypertrophy by demonstrating the presence of cardiac tissue-specific regulatory elements in the 5'-flanking region of the MLC-2 promoter and testing them in the gel mobility shift assay for their binding activity to nuclear proteins from hypertrophied and normal cardiac tissue. In nuclear extracts from the ventricular tissues of the spontaneously hypertensive rat (SHR), distinctive changes in two families of activator proteins, the A/T-rich DNA-binding transcription factors, myocyte enhancer factor (MEF-2) and CArG-binding factor, manifested in a developmentally dictated manner paralleling the evolution of cardiac hypertrophy in these animals. Extracts isolated from brains and skeletal muscle tissues from the same animals did not exhibit the changes in binding activity. Also, the changes were not apparent when a distal negative regulatory element (CSS), which confers cardiac-specific expression, was tested in gel mobility shift assays. The ubiquitous TATA-binding proteins remained unchanged in comparing SHR with the control strain WKY in the same assay. These data support the notion that the expression of specific transcription factors is modulated in response to hypertrophy related signals which execute changes at the gene level effecting the enrichment of certain contractile proteins in an effort discrete and estranged from the basal transcription machinery.

Adaptation, Physiological↗
PubMed1994

Identification of alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit.

Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the alpha-cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the alpha-cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3'-end of rabbit alpha-cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of alpha-cardiac myosin heavy chain expression in extraocular muscle.

Amino Acid Sequence↗
PubMed1994

Differential expression of the myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis.

In the present study, we have used single chicken blastoderms of defined early developmental stages, beginning with the prestreak stage, stage 1 (V. Hamburger and H. L. Hamilton, J. Morphol. 88:49-92, 1951), to analyze the onset of cardiac myogenesis by monitoring the appearance of selected cardiac muscle tissue-specific gene transcripts and the functional expression of the myocyte enhancer factor 2 (MEF-2) proteins. Using gene-specific oligonucleotide primers in reverse transcriptase PCR assay, we have demonstrated that the cardiac myosin light-chain 2 (MLC2) and alpha-actin gene transcripts appear as early as stage 5, i.e., immediately after the cardiogenic fate assignment at stage 4. Consistent with this observation is the developmental expression pattern of DNA-binding activity of BBF-1, a cardiac muscle-specific member of the MEF-2 protein family, which also begins at stage 5 prior to MEF-2. Differential expression of DNA-binding complexes is also observed with another AT-rich DNA sequence (CArG box) as probe, but the binding pattern with the ubiquitous TATA-binding proteins remains unchanged during the same developmental period. Thus, the cardiogenic commitment and differentiation of the precardiac mesoderm, as exemplified by the appearance of cardiac MEF-2, MLC2, and alpha-actin gene products, occur earlier than previously thought and appear to be closely linked. The onset of skeletal myogenic program follows that of the cardiogenic program with the appearance of skeletal MLC2 at stage 8. We also observed that mRNA for the MEF-2 family of proteins appears as early as stage 2 and that for CMD-1, the chicken counterpart of MyoD, appears at stage 5. The temporal separation of activation of cardiac and skeletal MLC2 genes, which appears immediately after the respective fate assignments, and those of cardiac MEF-2 and CMD-1, which occur before, are consistent with the established appearance of the myogenic programs and with the acquisition pattern of the two tissue-specific morphological characteristics in the early embryo. The preferential appearance of BBF-1 activity in precardiac moesderm, relative to that of MEF-2, indicates that these two protein factors are distinct members of the MEF-2 family and provides a compelling argument in support of the potential role of BBF-1 as a regulator of the cardiogenic cell lineage determination, while cardiac MEF-2 might be involved in maintenance of the cardiac differentiative state.

Age Factors↗
PubMed1988

Isolation and characterization of an autonomously replicating sequence from Ustilago maydis.

DNA fragments that function as autonomously replicating sequences (ARSs) have been isolated from Ustilago maydis. When inserted into an integrative transforming vector, the fragments increased the frequency of U. maydis transformation several-thousandfold. ARS-containing plasmids were transmitted in U. maydis as extrachromosomal elements through replication. They were maintained at a level of about 25 copies per cell but were mitotically unstable. One ARS characterized in detail, which we called UARS1, was localized to a 1.7-kilobase fragment. UARS1 contained a cluster of active sequences. This element could be reduced further into three separate subfragments, each of which retained ARS activity. The smallest one was 383 base pairs (bp) long. Although not active itself in yeast, this small fragment contained seven 8-bp direct repeats, two contiguous 30-bp direct repeats, and five 11-bp units in both orientations with sequences similar but not identical to the consensus sequence found to be crucial for ARS activity in Saccharomyces cerevisiae.

Base Sequence↗
PubMed1988

Anemia and erythropoiesis in patients with the acquired immunodeficiency syndrome (AIDS) and Kaposi sarcoma treated with zidovudine.

We evaluated the development of anemia as part of a trial of zidovudine therapy in patients with the acquired immunodeficiency syndrome (AIDS) with Kaposi sarcoma. Patients were randomized to one of three treatment groups or a placebo group. Transfusion-requiring anemia (hemoglobin less than 100 g/L) developed in 6 of 15 patients on zidovudine therapy at a mean of 6 weeks after starting treatment. A fall in the reticulocyte count was the earliest peripheral blood indicator of toxicity. Mean corpuscular volume increased markedly in patients on zidovudine therapy not developing anemia yet remained stable or only slightly increased in those who became anemic. Bone marrow examination showed pure red cell aplasia in 2 patients, erythroid maturation arrest in 1, and erythroid hypoplasia in 3. Megaloblastic erythropoiesis was present in 2 of the 6 anemic patients. Erythropoietin levels measured at the time of first transfusion were increased. Thus, the anemia associated with zidovudine therapy is due to red cell hypoplasia or aplasia.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Replication control for pT181, an indirectly regulated plasmid.

PT181 is a fully sequenced Staphylococcus aureus plasmid whose size is 4,437 bp. It specifies tetracycline resistance and has a copy number of about 22 per cell in exponentially growing cultures. The functional organization of the pT181 replicon is centered around the coding sequence for a 35-kd protein, RepC, that is absolutely required for replication of the plasmid. The replication origin is contained within the repC coding sequence and the region immediately 5' to the RepC start is involved in control of the plasmid replication rate. PT181 replication is controlled at the level of RepC synthesis by a negative regulatory system that is functionally similar to that of the Co1E1 and IncFII plasmids of Escherichia coli. The pT181 control circuit involves 2 short transcripts, RNA I and RNA II, that are transcribed from the region specifying the 5' end of the untranslated repC mRNA leader and in the opposite direction. These are referred to as countertranscripts. The countertranscripts regulate RepC synthesis by a mechanism that probably involves interaction with the repC mRNA leader in a manner that interferes with translation. Both of the countertranscripts seem to be necessary for normal replication control; their separate roles remain unclear. Unlike plasmids of the Co1E1 and IncFII groups, plasmids such as Co1E1 are considered to have direct regulation of replication because the inhibitory element of the copy control circuit directly inhibits the initiation of replication. Plasmids such as pT181 are considered to have indirect regulation of replication because the product of the regulated step, RepC, is trans-active. Plasmids of the IncFII type are considered to have direct regulation of replication because the product of the regulated step, RepA is cis-active The analysis of pT181 replication physiology has illustrated 2 important differences between directly and indirectly regulated plasmids: a) for directly regulated plasmids, copy mutants specifying a normal inhibitor substance but an inactive target site exclude the wild-type or recessive mutants by directly interfering with their replication. Analogous mutants of indirectly regulated plasmids coexist readily with the wild-type and all mutants (although they do manifest segregational incompatibility) because the Rep protein is always shared by all plasmids in the cell, regardless of its source. b) Mutations of directly regulated plasmids in the region where target transcript and countertranscript overlap may give rise to totally new incompatibility groups because they engender independently self-correcting copy pools.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1984

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein.

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.

Bacterial Proteins↗
PubMed1984

Control of pT181 replication II. Mutational analysis.

We describe the isolation and analysis of mutations affecting the regulation of Staphylococcus aureus plasmid pT181 replication. Previous results suggested that regulation is achieved by control of the synthesis of RepC, a plasmid-coded replication protein and that the primary negative control element is CopA RNA, which consists of two transcripts that are complementary to the 5' region of the repC mRNA leader. CopA inhibition probably involves a base pairing interaction with the complementary region of the RepC mRNA leader which would facilitate the formation of a downstream stem-loop in the leader that occludes the repC ribosome binding site. RepC is freely diffusible so that regulation of pT181 replication is indirect. Both CopA RNA-sensitive (recessive) and -insensitive (dominant) mutants were isolated. The recessives have defects in CopA RNA structure or activity, the dominants have defects in the site of action (target) of the inhibitor. Some dominants were located within the copA coding sequence. These therefore affect the structure of CopA RNA as well as that of its target. Other dominant mutations mapped outside of the copA gene and therefore produced wild-type CopA RNA. In contrast to directly regulated plasmids, pT181 copy mutants producing wild-type inhibitor could be co-maintained with the wild-type plasmid and mutational changes in inhibitor-target specificity did not change incompatibility specificity.

Bacillus subtilis↗
PubMed1983

Determination of plasmid copy number by fluorescence densitometry.

A simple and reliable method for the determination of plasmid copy numbers by direct fluorescence densitometry of ethidium bromide-stained electrophoretic gels was developed. In developing the method, the following parameters were evaluated and controlled: plasmid DNA trapping in the linear chromosomal DNA, staining-destaining kinetics for ethidium bromide, linearity of the fluorescence response, and the effect of the molecular topology of DNA on ethidium bromide binding to DNA in agarose.

DNA, Bacterial↗
PubMed1982

Coding sequence for the pT181 repC product: a plasmid-coded protein uniquely required for replication.

pT181 is a 4.4-kilobase plasmid from Staphylococcus aureus specifying tetracycline resistance and present in about 20 copies per cell. The existence of a diffusible pT181 product required for plasmid replication has been proposed on the basis of trans-complementable thermosensitive mutants defective in plasmid maintenance (phenotype Tsr). In this report, the Tsr mutants are shown to have primary replication defects, and the genetic complementation data are confirmed biochemically. All of five mutations are in a single cistron, the repC cistron; interruption of the plasmid DNA molecule at any of three neighboring restriction sites inactivates repC function. Analysis of the DNA sequence in this region reveals an open reading frame of 939 base pairs which encodes the repC product, a 313-amino acid protein. pT181 replication has been demonstrated in cell-free extracts to require specifically a pT181-coded protein of approximately the same size, and it is proposed that this protein is, indeed, the repC product. Preliminary evidence is discussed suggesting that the pT181 replication rate is controlled at the level of synthesis of the repC protein.

Bacterial Proteins↗