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Biomedical subjects

B J McCarthy

Publications and source records attributed to B J McCarthy.

At least 19 recordsLinked to original sources
PubMed1992

Synthesis and secretion of apolipoprotein E occur independently of synthesis and secretion of apolipoprotein B-containing lipoproteins in HepG2 cells.

The synthesis and secretion of apolipoprotein (apo) E, a major protein component of very low density lipoproteins, were examined in the human hepatoma cell line HepG2 under metabolic conditions known to stimulate lipogenesis and the production of apoB-containing lipoproteins. When HepG2 cells were incubated in the presence of fetal bovine serum (5 or 10%) or canine chylomicron remnants (5 or 10 micrograms of protein), the secretion of triglycerides and cholesteryl esters of lipoproteins of d less than 1.063 g/ml increased, as determined by the incorporation of [14C]acetate. Determination of the distribution of apoE among media lipoproteins by agarose column chromatography showed that the majority of secreted apoE was associated with large lipoproteins when cells were incubated with fetal bovine serum. However, immunoblot analysis of total media apoE revealed that incubating cells with or without the lipogenic factors had no effect on the amount of apoE secreted. Pulse-chase and continuous labeling experiments demonstrated that the synthesis and secretion of apoE did not vary under the different metabolic conditions, even though there was a 5-fold increase in apoB secretion in response to increased lipogenesis. Neither apoE nor apoB mRNA levels responded to the lipogenic stimuli. We conclude that the synthesis and secretion of apoE are independent of the production of apoB-containing lipoproteins in HepG2 cells.

Acetates↗
PubMed1979

Rapid quantitation of individual RNA species in a complex population.

Many investigations require quantitation of one or more individual RNA species in complex populations. Existing methods are tedious when multiple samples are to be assayed. A method is presented which allows rapid and accurate quantitation of many species of RNA simultaneously. Recombinant plasmids containing cDNA inserts are electrophoresed in agarose and blotted to nitrocellulose. After hybridization with labeled RNA and autoradiography, bands are quantitated by scanning. The results were calibrated by solution hybridization. The approach has been validated through the use of plasmids containing inserts of Drosophila cDNA and RNA of cultured cells.

Animals↗
PubMed1979

The E. coli gene encoding heat stable toxin is a bacterial transposon flanked by inverted repeats of IS1.

Restriction endonuclease subclones of the Escherichia coli gene encoding the heat stable (ST) toxin exhibit a stem and loop structure similar to those seen in many procaryotic transposons. An EcoRI DNA fragment encoding tetracycline (Tc) resistance but no transposition functions was spliced into the ST gene in one of these subclones. By monitoring Tcr, we were able to show that the ST gene transposes. Restriction and DNA sequence data strongly suggest that the ST transposon, Tn 1681, is flanked by inverted repeats of IS1.

Bacterial Toxins↗
PubMed1979

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.

Animals↗
PubMed1979

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

Base Sequence↗
PubMed1979

Translation of messenger RNA from fetal and adult mouse kidney.

Polyadenylated mRNA was prepared from fetal, adult, and regenerating adult mouse kidneys. The polyadenylated portion is 2% of total RNA in the fetus, falls abruptly to 1% after birth, and returns to about 2% in the adult. In vitro translation of these mRNA's showed great similarities between 17 and 20 day fetal samples and between the normal and regenerating adult samples, but few similarities between the fetal and adult samples. The data do not support the hypothesis that renal growth following contralateral nephrectomy may entail the de-repression of genes active in the fetus but not active in the normal adult.

Aging↗
PubMed1978

Induction of the Drosophila heat shock response in isolated polytene nuclei.

When Drosophila salivary glands are disrupted in cytoplasm from heat-shocked Drosophila Kc cells in culture, puffs are induced in the polytene nuclei at the heat shock loci. The in vitro response increases with time of incubation, reaching in vivo levels after 2 hr, and is sensitive to alpha-amanitin. Cytoplasm from control Kc cells induces poorly, but prolonged exposure to air or the addition of hydrogen peroxide stimulates induction to the levels observed with cytoplasm from heat-shocked Kc cells. Autoradiography confirms that the heat shock loci induced to puff in vitro are the most actively transcribing sites in the genome.

Amanitins↗
PubMed1978

In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites.

A method for mutagenizing circular DNA molecules has been developed that uses synthetic oligodeoxynucleotide restriction sites as mutagens. A single synthetic restriction site is introduced at random by cleaving circular DNA with a nonspecific double-strand endonuclease. The restriction site is then ligated to the ends and the molecule is subsequently recircularized. These small additions to the genome are mapped by digestion with the appropriate restriction enzyme. Rearrangements such as duplications and deletions can be engineered at will by using the added restriction sites. This technique has been used to produce a fine-structure map of RSF1050, a ColE1 derivative, 60% of which is a transposable DNA sequence encoding the TEM beta-lactamase (Tn3). A subset of the mutations, mapping within a narrow region of Tn3, result in an increased frequency of Tn3 transposition; mutations in other regions abolish transposition entirely.

Bacteriocin Plasmids↗
PubMed1978

In vitro transcription of heat-shock-specific RNA from chromatin of Drosophila melanogaster cells.

Polyadenylylated RNA synthesized after heat shock was isolated from polysomes of cultured cells of Drosophila melanogaster and used as template to prepare cDNA. An excess of poly(A)-RNA from heat-shocked cells hybridized to 80% of the cDNA, whereas cytoplasmic poly(A)-RNA from cells grown at 25 degrees could drive only half of the cDNA probe into hybrid. These sequences were removed from the cDNA population by annealing to poly(A)-RNA from cells grown at 25 degrees. The unreacted material represented only heat-shock-induced mRNA sequences, as shown by a second cycle of hybridization. Isolated chromatin was transcribed in vitro at 25 degrees with Escherichia coli RNA polymerase, with mercurated UTP as precursor. RNA transcribed from chromatin that was prepared from cells 1 hr after the temperature was shifted to 37 degrees hybridized with 100-fold faster kinetics to the heat-shock-specific cDNA probe than did RNA transcribed from chromatin of cells grown at 25 degrees. Therefore, heat shock results in a change in chromatin structure recognizable by E. coli RNA polymerase.

Cells, Cultured↗
PubMed1977

Acetylation and phosphorylation of Drosophila histones. Distribution of acetate and phosphate groups in fractionated chromatin.

The phosphorylation and acetylation patterns of Drosophila histones were studied after whole cell incubation with ortho [32P] phosphate or [3H] acetate. Radioactive phosphate associated mainly with H1, H3, and H4 and radioactive acetate was associated mainly with H3, H4, and H2B. H3 showed the highest specific activity of both labels. Two chromatin fractionation methods were employed to investigate the distribution of acetate and phosphate groups in histones form template-active and template-inactive regions. The levels of both acetate and phosphate groups were higher in histones from template-active regions.

Acetates↗
PubMed1977

Limited accessibility of chromatin satellite DNA to RNA polymerase from Escherichia coli.

An attempt was made to elucidate some of the factors influencing the fidelity with which isolated chromatin from mouse L-cells is transcribed by RNA polymerase from Escherichia coli by analyzing the in vitro transcript for the presence of satellite sequences. These sequences are absent from cellular RNA and therefore reflect aberrant transcription. The results indicate that satellite sequences are underrepresented in chromatin transcripts relative to those of DNA. This selectivity is insensitive to many variables in procedures for the isolation and transcription of chromatin. However, lowering the ratio of enzyme to template further reduced the proportion of satellite sequences in the transcript. We conclude that a primary factor influencing the extent of aberrant transcription is the level of enzyme used. Under limiting enzyme conditions, an efficient selection against satellite sequences is observed. However, under conditions of enzyme excess, the enzyme initiates chains at weaker secondary promoters localized in regions of the chromatin containing satellite DNA.

Animals↗
PubMed1976

Fidelity of chromatin transcription in vitro.

Chromatin and DNA from Schneider's Drosophila melanogaster cell line 2 were transcribed in vitro with Escherichia coli RNA polymerase. Using mercurated UTP as precursor, the newly synthesized RNA could be separated from DNA and endogenous RNA by affinity chromatography on sulfhydryl-Sepharose 6B. Characterization of the transcription products with complementary DNA (cDNA) made from polyadenylated nuclear RNA and with fractionated cDNA probe demonstrated a fair quantitative fidelity in the in vitro transcript from chromatin which was not evident when DNA was transcribed. However, as shown by hybridization to total nuclear RNA, E. coli RNA polymerase transcribed both DNA strands from chromatin in vitro. We conclude that E. coli polymerase is able to distinguish sections of chromatin at which rapid synthesis of RNA occurs in the cell.

Cell Line↗
PubMed1976

Histone-histone interactions within chromatin. Preliminary characterization of presumptive H2B-H2A and H2B-H4 binding.

Cyanogen bromide peptide analyses of cross-linked histones show that histone 2B possesses separate binding sites for histones 2A and 4. Various polymorphic forms of H2A and H4 in chromatin are bound to H2B through these binding sites. These interactions are thus a fundamental structural feature of chromatin. Additional data strongly suggest that H2B is bound to H2A and H4 simultaneously in vivo. Mouse L cells were treated with either tetranitromethane (TNM) or ultraviolet (uv) light at 280 nm in order to induce the cross-linking of histone 2B in their chromatin to either histone 4 or 2A. The characteristics of these cross-linking agents and the reactions they induce suggest that the cross-links define loci within the actual binding sites between these histones. Characterization of the cyanogen bromide peptide fragments of the H2B-H4 and H2B-H2A dimers showed that the C terminal half of H2B contains the link to H4 (also near its C terminus), whereas the N-terminal half contains the link to H2A. Treatment of the cells with both uv light and TNM results in the appearance of a single new trimer which we presume to be H2A-H2B-H4. We conclude form this that H2B interacts simultaneously with H2A and H4 in chromatin through separate binding sites. Further analysis shows that several acetylated subspecies of H4 are represented in the H2B-H4 dimer and that at least two of the polymorphic forms of H2A are present in the H2B-H2A dimer. These data reveal that the H2A-H2B-H4 association is an important underlying structural feature of chromatin.

Binding Sites↗