Biomedical subjects

S T Brentano

Publications and source records attributed to S T Brentano.

10 recordsLinked to original sources

PubMed1992

cAMP post-transcriptionally diminishes the abundance of adrenodoxin reductase mRNA.

Adrenodoxin reductase (AR; ferridoxin: NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein that mediates electron transport from NADPH to all known mitochondrial forms of cytochrome P450. AR mRNA was found in all human adult and fetal tissues examined; however, it was vastly more abundant in tissues that synthesize steroid hormones. The ratio of the 18- form of mRNA lacking 18 alternately spliced bases to the 18+ form was approximately 100:1 and remained constant irrespective of the tissue or hormonal manipulation, indicating that the alternate splicing is a passive nonregulated event. AR protein was unchanged by forskolin treatment of human JEG-3 cytotrophoblast cells for 24 h, but the mRNA diminished. Phorbol 12-myristate 13-acetate and cycloheximide had no effect, even though these agents had the expected effects on P450scc and adrenodoxin mRNAs. cAMP decreased the abundance of AR mRNA expressed from both transfected plasmids and the endogenous gene, indicating the effect was post-transcriptional. AR gene transcription in JEG-3 cells and promoter-chloramphenicol acetyltransferase constructs transfected into JEG-3 cells were unresponsive to forskolin. Powerful basal transcription elements were identified between -46 and -214 bases from the principal transcriptional initiation site, a region containing six elements closely resembling the binding site for transcription factor SP1.

Cells, Cultured↗

PubMed1992

Regulation of human cytochrome P450scc and adrenodoxin messenger ribonucleic acids in JEG-3 cytotrophoblast cells.

Cycloheximide generally inhibits steroidogenesis, but has different effects on the accumulation of the mRNAs for various steroidogenic enzymes in different species, tissues, and cell lines. In bovine adrenocortical cells, cycloheximide prevents ACTH- or cAMP-induced accumulation of the mRNAs for cytochrome P450scc and adrenodoxin, but in human cells, cycloheximide induces the accumulation of adrenodoxin mRNA. To study the potential role of the 3'-untranslated regions, and especially the AU-rich regions, of adrenodoxin and P450scc mRNAs in cycloheximide-sensitive regulation of mRNA accumulation, we constructed a series of vectors expressing P450scc or adrenodoxin mRNA with its own or each other's 3'-untranslated sequences and transfected them into human JEG-3 cytotrophoblast cells. Removal of the AU-rich 3'-untranslated sequences of adrenodoxin mRNA and replacing them with the 3'-untranslated region of P450scc did not alter the abundance or apparent stability of this mRNA, or its inducibility by cycloheximide or cAMP. Substituting the AU-rich 3'-untranslated region of adrenodoxin mRNA (which contains three copies of the AUUUA sequence) for the 3'-untranslated region of P450scc did not alter the inducibility of P450scc mRNA with forskolin. Inhibition of transcription with actinomycin-D elicited no difference in the adrenodoxin mRNA half-life in JEG-3 cells treated with forskolin, cycloheximide, or both. RNA polymerase run-on assays show little effect of forskolin on adrenodoxin gene transcription, while P450scc gene transcription was induced. These data suggest that the principal means for regulating P450scc mRNA is transcriptional, while the principal regulation of adrenodoxin is posttranscriptional. This posttranscriptional regulation of adrenodoxin mRNA is not mediated by the AUUUA sequences or other segments of the 3'-untranslated region.

Adrenodoxin↗

PubMed1990

Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements.

Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450scc. Adrenal steroidogenesis is regulated primarily by two hormones: adrenocorticotropin, which works via cyclic AMP (cAMP) and protein kinase A, and angiotensin II, which works via Ca2+ and protein kinase C. Forskolin and 8-bromo-cAMP stimulated, while prolonged treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) additively suppressed accumulation of endogenous P450scc mRNA in transformed murine adrenal Y1 cells. In Y1 cells transfected with 2,327 base pairs of the human P450scc promoter fused to the bacterial gene for chloramphenicol acetyltransferase (CAT), forskolin increased CAT activity 900% while combined TPA plus A23187 reduced CAT activity to 15% of the control level. Forskolin induced the P450scc promoter as rapidly as a promoter containing two cAMP-responsive elements fused to a simian virus 40 promoter, a system known to respond directly to cAMP. Basal expression was increased by sequences between -89 and -152 and was increased further by sequences between -605 and -2327. This upstream region also conferred inducibility by cAMP. TPA plus A23187 transiently increased CAT activity before repressing it, reflecting the complex actions of angiotensin II in vivo. Repression by prolonged treatment with TPA plus A23187 was mediated by multiple elements between -89 and -343. Induction of CAT activity by forskolin was not diminished by treatment with TPA plus A23187, nor were the regions of the promoter responsible for regulation by the two pathways coisolated. Thus, the human gene for P450scc is repressed by TPA plus A23187 by mechanisms and sequences independent of those that mediate induction by cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Tissue-specific, cyclic adenosine 3',5'-monophosphate-induced, and phorbol ester-repressed transcription from the human P450c17 promoter in mouse cells.

Cytochrome P450c17 is the single microsomal enzyme catalyzing steroid 17 alpha-hydroxylase and 17-20-lyase activities. It is expressed and regulated by tropic hormones in the human adrenal and gonads, but is not expressed in the placenta. To study the transcriptional regulation of the human P450c17 gene, we constructed 11 plasmids containing serial deletions of its 5' nontranslated region driving expression of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into mouse adrenal Y1 and testis MA-10 cells and incubated with forskolin, 8-bromo-cAMP, or 12-O-tetradecanoyl-phorbol-13 acetate (TPA) for 12 h. Interpretation of results from standard constructions was difficult, apparently because some transcription was incorrectly initiated by DNA sequences in the vector. Therefore, we built a modified CAT reporter vector that eliminated detectable read-through transcription. In Y1 cells, the basal activity of constructs containing from -82 to -184 basepairs (bp) of 5' flanking DNA was between 80-150% of the promoterless control. Constructs containing at least -235 bp of this DNA expressed CAT at 540% of the control value, but addition of sequences to -774 had no further effect. Forskolin increased the expression of CAT activity to 300% above basal with constructions containing DNA from -184 to -774 bp. Constructs containing between -184 and -310 bp expressed CAT at 50% of the forskolin-induced levels in cells treated with TPA. Both basal and cAMP-induced expression were much lower in MA-10 cells than in Y1 cells and increased with increasing promoter length to -774.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1986

Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA.

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.

Animals↗

PubMed1986

The 35-nucleotide spliced leader sequence is common to all trypanosome messenger RNA's.

In Trypanosomatidae the messenger RNA's (mRNA's) that code for the variant surface glycoproteins (VSG's), tubulins, calmodulin, and at least a subset of other proteins contain a common 35-nucleotide leader sequence at their 5' ends. Hybrid-arrested in vitro translation has been used to show that all mRNA's in both African and South American trypanosomes contain this 35-nucleotide sequence. Oligonucleotides complementary to this sequence blocked translation of all trypanosome mRNA's in a rabbit reticulocyte lysate system, but did not inhibit translation of mRNA's from other organisms lacking this sequence. An oligonucleotide complementary to the VSG mRNA downstream from the spliced leader sequence arrested only VSG synthesis. Thus, the 35-nucleotide leader sequence is a general feature of all trypanosome mRNA's. The high specificity of oligonucleotides complementary to the spliced leader for their target sequence suggests that analogues permeable to the cell membrane may be useful in the treatment of trypanosomal infections.

Base Sequence↗

PubMed1984

Expression of antigenic regions of a trypanosome variable surface glycoprotein in E. coli using Bal-31 nuclease digestion.

We have used the expression of a trypanosome variable surface glycoprotein (VSG) in E. coli to produce VSG serotype-specific antisera which have none of the cross-reacting specificities characteristic of antisera prepared against purified VSGs. This was accomplished by treating restriction fragments of VSG cDNAs with Bal-31 nuclease to facilitate expression of their open reading frames in the E. coli expression vector, pMR100 (4). The resultant VSG-beta-galactosidase fusion proteins possess various antigenic regions of the original VSG. This provides a rapid means for producing VSG-specific antisera for reagent use and has the capability of large scale production of antigen for immunological investigation.

Animals↗

PubMed1984

A DNA sequence analysis package for the IBM personal computer.

We present here a collection of DNA sequence analysis programs, called "PC Sequence" (PCS), which are designed to run on the IBM Personal Computer (PC). These programs are written in IBM PC compiled BASIC and take full advantage of the IBM PC's speed, error handling, and graphics capabilities. For a modest initial expense in hardware any laboratory can use these programs to quickly perform computer analysis on DNA sequences. They are written with the novice user in mind and require very little training or previous experience with computers. Also provided are a text editing program for creating and modifying DNA sequence files and a communications program which enables the PC to communicate with and collect information from mainframe computers and DNA sequence databases.

Amino Acid Sequence↗

PubMed1984

DNA rearrangements of the variable surface antigen genes of the trypanosomes.

The trypanosome genome contains several hundred (and perhaps several thousand) genes for the trypanosome variable surface glycoproteins (VSGs). In an individual trypanosome only one of these genes is expressed at a given instant; the others are transcriptionally silent. This differential gene expression is responsible for the sequential antigenic variation displayed by trypanosomes. It is mediated by two types of genomic rearrangements of these VSG genes. The best understood rearrangement type is the formation of a transcriptionally-active expression-linked extra copy (ELC) of a transcriptionally-silent basic copy (BC) gene. This duplication and translocation event places the ELC near a chromosomal end (a telomere) where it is apparently located downstream from a strong promotor. Some VSG genes are not expressed via this ELC mechanism. These genes, which seem to already be near telomeres, are activated by a different non-duplication associated ( NDA ) type of mechanism. We have used recombinant DNA techniques to clone and determine the sequences of genes expressed by both the ELC and NDA mechanisms. Comparison of these sequences reveals that sequences flanking the VSG coding regions are similar. This indicates that there is a sequence correlation between the two mechanisms of expression. We have also shown that when bloodstream trypanosomes expressing a specific VSG via the ELC mechanism are established in culture, the resultant procyclic trypanosomes rapidly stop synthesizing the VSG mRNA (and the VSG) but retain the ELC of the VSG gene. This demonstrates that transcription of an ELC can cease without the loss of that ELC and may indicate the presence of other factors regulating VSG gene transcription.

Animals↗

PubMed1983

Comparison of the expression-linked extra copy (ELC) and basic copy (BC) genes of a trypanosome surface antigen.

A recombinant clone of an expression-linked extra copy (ELC) gene of a trypanosome-variable surface glycoprotein was sequenced. In addition the sequences of the corresponding cDNA and portions of the two basic copy genes were determined. Comparison of these sequences reveals that the 5' boundary of the ELC-transposed segment (2.2 kb) occurs within a repetitive sequence about 700 bp upstream from the start codon of the coding sequence. This sequence does not contain internal symmetries and is not homologous with the repetitive sequence at the 3' boundary. The first 35 nucleotides of the cDNA are different than the corresponding ELC sequence and presumably were transcribed from another genomic location. A restriction fragment containing predominantly sequences outside of the 5' boundary hybridizes to a Pst I fragment whose length is variable in different trypanosome clones. This hybridization pattern is similar to that observed using probes for surface glycoprotein genes that are expressed via the nonduplication-associated (NDA) mechanism rather than the ELC mechanism. This indicates that there is a sequence correlation between these two DNA rearrangement mechanism.

Amino Acid Sequence↗