Background: Prostate cancer gene 3 (PCA3) encodes a prostate-specific mRNA that has shown promise as a prostate cancer diagnostic tool. This report describes the characterization of a prototype quantitative PCA3-based test for whole urine. Methods: Whole-urine specimens were collected after digital rectal examination from 3 groups: men scheduled for prostate biopsy (n ‫؍‬ 70), healthy men (<45 years of age with no known prostate cancer risk factors; n ‫؍‬ 52), and men who had undergone radical prostatectomy (n ‫؍‬ 21). PCA3 and prostate-specific antigen (PSA) mRNAs were isolated, amplified, and quantified by use of Gen-Probe DTS400 ® Systems. Prostate biopsy results were correlated with the PCA3/PSA mRNA ratio, and PSA mRNA concentrations were used to normalize PCA3 signals and confirm the yield of prostate-specific RNA. Assay precision, specimen stability, and mRNA yield were also evaluated. Results: The specimen informative rate (fraction of specimens yielding sufficient RNA for analysis) was 98.2%. In this clinical research study, ROC curve analysis of prebiopsy specimens yielded an area under the curve of 0.746; sensitivity was 69% and specificity 79%. Serum PSA assay specificity was 28% for this same group. PCA3 and PSA mRNAs were undetectable in postprostatectomy specimens except for one man with recurrent prostate cancer. Assay interrun CVs were

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.

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)

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 -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.

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.