Telomerase activity in prostate sextant needle cores from radical prostatectomy specimens.

Human telomerase acts to maintain functioning telomeres, which are required for cellular immortality and very likely for cancer progression. Telomerase activity is present in about 85% of human cancers tested, but it has not been found in most human somatic cells and tissues. We used the Telomeric Repeat Amplification Protocol to perform telomerase activity assays on sextant needle core samples obtained from 35 freshly excised radical retropubic prostatectomy specimens. Similar assays were done on prostatic tissues obtained by means of other urologic procedures from 8 patients without prostate cancer. Telomerase activity was found in one or more specimens from 32 of 35 prostate cancer patients (91%), but was not detectable in all biopsy specimens from 7 of 8 cancer-free patients (88%). Further analysis showed that cancers more poorly differentiated, with higher Gleason scores, were always associated with a higher rate of telomerase detection and stronger telomerase activity. Moreover, comparison of telomerase activity in needle core samples with the volume of cancer in surrounding tissue as observed on corresponding histologic slides showed that stronger activity was positively correlated with a higher cancer volume. Prognostic indicators of prostate cancer and the expression of telomerase appear to be linked. The presence of telomerase activity in prostate tissue may aid in the detection of prostate cancer and produce additional prognostic information.

Multiple negative and positive cis-acting elements control the expression of the murine CD4 gene.

The cis-acting elements located within 15 kb 5' of the murine CD4 gene transcriptional start site and the first intron of the CD4 gene have been investigated using deletion constructs. Our transient transfection data indicate that the expression of the murine CD4 gene is controlled by multiple positive and negative regulatory cis-acting elements. There are at least two cis-acting elements that have a positive effect on the expression of the CD4 gene and at least four regions of DNA that have a negative effect. The positive control elements are located about 13.5 kb 5' of the promoter and within the flanking sequences of the first intron. The DNA between the 5' enhancer and the promoter contains at least two regions that exert a negative effect on CD4 expression. In addition to the positive effect that the first intron has on CD4 expression, there are two regions within the first intron that have a negative effect. These two negative regulatory elements correspond to two T-cell-specific DNase I hypersensitive sites found in the first intron.

Characterization and expression of a gene encoding serine tRNA5 from Escherichia coli.

The genes for translational components frequently are located together on the Escherichia coli genome. We have reported previously that the gene for a serine tRNA lies directly downstream from infA, the gene encoding initiation factor IF1. Here we characterize this tRNA gene, named serW. The serW gene expresses a minor form of serine tRNA(GGA) which recognizes the most frequently used serine codons, UCC and UCU. Two promoters were identified by S1 nuclease mapping: P1, which lies about 72 bp upstream from the structural gene; and P2, which lies about 35 bp upstream. Expression from P1 and P2 is comparable under conditions of rapid growth. The P2 promoter is followed by a GC-rich element characteristic of promoters regulated by ppGpp. A putative hairpin structure followed by a stretch of U residues about 25 nucleotides following the mature tRNA sequence resembles a rho-independent termination signal. The upstream gene, infA, is followed by a transcriptional terminator, but S1 mapping shows considerable readthrough. This serW expression appears to rely both on its own promoters and on promoters further upstream. The downstream gene, encoding an unidentified protein of about 100 kDa, is expressed in the opposite orientation and also is followed by a termination signal. Therefore serW is expressed both as a monocistronic gene and in combination with infA.

Transcription of retinoic acid receptor genes in transgenic mice increases CD8 T-cell subset.

Retinoic acid (RA), a vitamin A derivative is known to have a number of effects on the immune system such as inducing cytotoxic T-lymphocytes in vivo and inducing the rejection of skin grafts. However, the molecular mechanisms of these actions are unclear. The retinoic acid receptors which belong to the steroid/thyroid receptor superfamily, are known to bind to regulatory elements of certain genes thereby regulating gene transcription. To determine if expression of retinoic acid receptors in vivo under normal physiological conditions is also regulating genes involved in immunological function, we assayed the human retinoic acid receptor gamma gene driven by a T-cell specific lck-promoter in transgenic mice. Using FACS analysis, we showed that mice expressing the RAR gamma-transgene had significantly increased numbers of CD4-/CD8+ cells compared to controls.

T cell-specific protein-DNA interactions occurring at the CD4 locus: identification of possible transcriptional control elements of the murine CD4 gene.

The cis-acting transcriptional control elements of the murine CD4 gene were investigated within 75 kb of chromatin associated with the CD4 locus. DNase I hypersensitive (DH) sites were identified in several T and non-T cell lines, and in freshly isolated thymocytes. A total of 22 DH sites were found, seven of which are present only in T cells expressing CD4 or CD8. The T cell-specific DH sites are located in four regions: (i) 5' of the first exon of CD4, (ii) in the first intron, (iii) near the second and third exons, and (iv) 3' of the CD4 gene. Some of these sites inversely correlate to the CD4 expression at defined stages of T cell development, suggesting a role for these sites in repression of this gene. The T cell-specific DH sites were subcloned and analyzed for protein-DNA interactions using the electrophoretic mobility shift assay. All T cell-specific DH sites analyzed appear to be a consequence of T cell-specific protein-DNA interactions. We have also identified the nuclear matrix attachment regions (MARs) and repetitive elements associated with the CD4 gene. Two nuclear MARs, separated by a region of highly repetitive DNA, are located 5' of the gene. Another region of highly repetitive DNA exists within the third intron. We discuss the implications of our results for the developmental regulation of CD4 expression.

Structure and expression of the infA operon encoding translational initiation factor IF1. Transcriptional control by growth rate.

The cellular levels of the three translational initiation factors, IF1, IF2, and IF3, increase as a function of growth rate in parallel with those of ribosomes. Therefore both ribosomal and initiation factor gene expression is under metabolic control. To address how expression of the Escherichia coli gene for IF1, infA, is regulated, a 3-kilobase region of the genome surrounding infA was sequenced. The 5' and 3' termini of in vivo infA transcripts were defined by S1 nuclease mapping, and mRNA size was measured by Northern blot hybridization. The infA gene is transcribed by two promoters, P1 and P2, which generate transcripts of 525 and 330 nucleotides, apparently ending at the same rho-independent terminator. Analyses of operon and protein fusions to lacZ demonstrate that neither infA transcription nor translation is affected by high cellular levels of IF1. However, P2, but not P1, increases in activity as a function of the growth rate of the cell and is the dominant promoter in rich medium. Therefore, metabolic control of infA expression occurs exclusively at the level of transcription by the P2 promoter.

The existence of two genes between infB and rpsO in the Escherichia coli genome: DNA sequencing and S1 nuclease mapping.

A number of genes encoding proteins involved in transcription and translation are clustered between 68 and 69 minutes on the Escherichia coli genome map and are transcribed clockwise as two operons: the metY operon, containing metY, P15A, nusA, infB; and about a kilobase further downstream, the rpsO and pnp operon. The DNA sequence between infB and rpsO was determined and two open reading frames were detected which code for proteins of 15,200 (P15B) and 35,091 (P35) daltons. Maxicell analysis showed a relatively strong expression of P15B whereas P35 was synthesized more weakly. An overlap of the termination codon of P15B and the initiator codon for P35 suggests that translation of P15B and P35 may be coupled. S1 nuclease mapping of in vivo transcripts between infB and rpsO provided no evidence for major promoters but detected a moderately efficient rho-independent terminator between infB and P15B. The results indicate that P15B and P35 are expressed as part of the metY operon, but that some transcriptional read through into the rpsO operon also occurs, thereby, functionally linking the expression of these two complex systems.

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.