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Expression of Sry, the mouse sex determining gene.

In the mouse, Sry is expressed by germ cells in the adult testis and by somatic cells in the genital ridge. Transcripts in the former exist as circular RNA molecules of 1.23 kb, which are unlikely to be efficiently translated. We have used RNase protection to map the extent of the less abundant Sry transcript in the developing gonad. We demonstrate that it is a linear mRNA derived from a single exon. This begins in the unique region 5' of the protein coding region and extends several kilobases into the 3' arm of the large inverted repeat which bounds the Sry genomic locus. Knowledge of this transcript, which is very different from that of the human SRY gene, allows us to predict its protein product and reveals several features which may be involved in translational control. Our data is also consistent with there being two promoters for the Sry gene, a proximal one that gives functional transcripts in the genital ridge and a distal promoter used in germ cells in the adult testis. As RNase protection is a quantitative technique, a detailed timecourse of Sry expression was carried out using accurately staged samples. Sry transcripts are first detectable just after 10.5 days post coitum, they reach a peak at 11.5 days and then decline sharply so that none are detected 24 hours later. This was compared with anti-Müllerian hormone gene expression, an early marker of Sertoli cells and the first known downstream gene of Sry. Amh expression begins 20 hours after the onset of Sry expression at a time when Sry transcripts are at their peak. While this result does not prove a direct interaction between the two genes, it defines the critical period during which Sry must act to initiate Sertoli cell differentiation.

Amino Acid Sequence↗

Prostatic growth and development are regulated by FGF10.

We have examined the role of Fibroblast Growth Factor 10 (FGF10) during the growth and development of the rat ventral prostate (VP) and seminal vesicle (SV). FGF10 transcripts were abundant at the earliest stages of organ formation and during neonatal organ growth, but were low or absent in growth-quiescent adult organs. In both the VP and SV, FGF10 transcripts were expressed only in a subset of mesenchymal cells and in a pattern consistent with a role as a paracrine epithelial regulator. In the neonatal VP, FGF10 mRNA was expressed initially in mesenchymal cells peripheral to the peri-urethral mesenchyme and distal to the elongating prostatic epithelial buds. At later stages, mesenchymal cells surrounding the epithelial buds also expressed FGF10 transcripts. During induction of the SV, FGF10 mRNA was present in mesenchyme surrounding the lower Wolffian ducts and, at later stages, FGF10 transcripts became restricted to mesenchymal cells subadjacent to the serosa. We investigated whether the FGF10 gene might be regulated by androgens by analysing the levels of FGF10 transcripts in SV and VP organs grown in serum-free organ culture. While FGF10 transcript levels increased after treatment with testosterone in the SV (but not VP), these changes were not sensitive to anti-androgen treatment, and thus it is likely that FGF10 mRNA was not directly regulated by testosterone. Also, FGF10 mRNA was observed in the embryonic female reproductive tract in a position analogous to that of the ventral prostate in males suggesting that FGF10 is not regulated by androgens in vivo. Recombinant FGF10 protein specifically stimulated growth of Dunning epithelial and BPH1 prostatic epithelial cell lines, but had no effect on growth of Dunning stromal cells or primary SV mesenchyme. Furthermore, FGF10 protein stimulated the development of ventral prostate and seminal vesicle organ rudiments in serum-free organ culture. When both FGF10 and testosterone were added to organs in vitro, there was no synergistic induction of development. Additionally, development induced by FGF10 was not inhibited by the addition of the anti-androgen Cyproterone Acetate demonstrating that the effects of FGF10 were not mediated by the androgen receptor. Taken together, our experiments suggest that FGF10 functions as a mesenchymal paracrine regulator of epithelial growth in the prostate and seminal vesicle and that the FGF10 gene is not regulated by androgens

Aging↗

Enhanced expression of the tie receptor tyrosine kinase mesenger RNA in the vascular endothelium of metastatic melanomas.

Angiogenesis of human melanomas has been the focus of intense interest since it was shown that the spread and prognosis of primary tumors is correlated with their vascularization (N. Weidner, J. P. Semple, W. R. Welch, and J. Folkman, N. Engl. J. Med., 324: 1-8, 1991). Basic fibroblast growth factor (bFGF) and its high-affinity receptor FGFR-1 have been implicated in melanoma growth and angiogenesis (R. Halaban, Y. Funasaka, J. Lee, J. Rubin, D. Ron, and D. Birnbaum, Fibroblast Growth Factors in Normal and Malignant Melanocytes, pp. 232-243. New York: The New York Academy of Sciences, 1991). We have studied the expression of the Tie endothelial cell receptor tyrosine kinase mRNA in skin and primary cutaneous melanomas as well as in their skin and brain metastases by in situ hybridization. The Tie probe hybridized very weakly with the vascular endothelium of capillaries of normal skin, while it was detected in larger arteries and veins as well as in capillaries around sweat glands. However, capillaries and medium-sized vessels within cutaneous and brain metastases of melanoma were strongly positive for Tie mRNA. In contrast, endothelial cells contained very little or no FGFR-1 transcripts, whereas abundant FGFR-1 mRNA was present in melanoma tumor cells and in fibrovascular stroma. In agreement with these findings, a Tie-specific amplified cDNA band was obtained by reverse transcription-polymerase chain reaction from melanoma metastases but not from normal skin. These results suggest a role for the Tie receptor in the angiogenesis associated with melanoma metastases.

Base Sequence↗

Expression of transforming growth factor beta in the embryonic avian lens coincides with the presence of mitochondria.

During their maturation, lens cells lose all membrane bound organelles, including mitochondria. In chicken embryos this process begins in the central lens fibers beginning around embryonic day 12 (E12). Transforming growth factor beta (TGF beta) is a multipotent growth modulator thought to play a role in numerous developmental processes. TGF beta 1 has been localized to mitochondria in rat liver cells and muscle cells. In the present study, we examined the expression of TGF beta isoform mRNAs and proteins during chicken embryonic lens development. PCR analysis demonstrated TGF beta 2 and TGF beta 3 transcripts in the lens epithelium and fibers throughout pre- and post-hatching development. TGF beta isoforms were detected throughout the lens epithelium and fibers early in development (E6). However by E19, the distribution of TGF beta 2 and TGF beta 3 transcripts and proteins coincided with regions of the lens that contained mitochondria. In addition, intense TGF beta staining was observed in the basal portions of the equatorial epithelial cells, a region with abundant mitochondria. Transcripts for TGF beta 1 and TGF beta 4 were not detected in any tissue or time frame examined. Similarly, no immunostaining for TGF beta 1 was observed.

Animals↗

Transcriptional analysis of the virion-sense genes of the geminivirus beet curly top virus.

The genome of the geminivirus beet curly top virus (BCTV) consists of a single circular DNA containing overlapping open reading frames (ORFs) located on both the virion-sense and complementary-sense DNA strands. To investigate the expression of these ORFs, RNA extracted from infected Nicotiana benthamiana and Beta vulgaris has been examined for the presence of viral transcripts. An abundant 1.1-kb virion-sense polyadenylated RNA and four complementary-sense polyadenylated RNAs of 1.7, 1.5, 1.3, and 0.7 kb have been identified by northern blot hybridization, confirming the bidirectional transcription strategy implied by the arrangement of ORFs. We previously demonstrated that two overlapping virion-sense ORFs are involved in coat protein synthesis (ORF V1) and viral single-stranded DNA accumulation (ORF V2). Mutants of a third virion-sense ORF (ORF V3), located upstream and overlapping ORFs V1 and V2, retain the ability to replicate efficiently in N. benthamiana leaf discs but produce an asymptomatic infection in N. benthamiana and B. vulgaris at low frequency, associated with reduced levels of viral DNA compared to wild-type infection. Our data support the recent suggestion that ORF V3 participates in virus movement. The 1.1 kb virion-sense RNA comprises a population of overlapping transcripts with 5' termini suitably positioned for the expression of ORFs V1, V2, and V3. The overlapping arrangement of the transcripts and juxtaposition of putative regulatory elements could provide a means for the temporal control of virion-sense gene expression.

Amino Acid Sequence↗

Molecular cloning of two exo-beta-glucanases and their in vivo substrates in the cell walls of lily pollen tubes.

Full-length cDNA sequences of two exo-beta-glucanases, LP-ExoI and LP-ExoII, secreted into cell walls of lily (Lilium longiflorum) pollen tube, were determined by RT-PCR. LP-ExoI exhibited over 80% similarity to LP-ExoII at both DNA and amino acid levels. RT-PCR showed that LP-ExoI transcripts were abundant in pollen grains and tubes, but could not be detected in leaf, stem, stigma, style, ovary, petal, filament, young root, young bud, and scale leaf of bulb. However, LP-ExoII transcripts ubiquitously existed in all the tissues tested. To determine the potential substrates of exo-beta-glucanases, cell wall components of lily tissues were analyzed. Linkage analysis revealed that pollen tubes contained high levels of 3-Glc in hemicellulose (44.3%), while pollen grains had no detectable 3-Glc. The hemicellulose fraction of pollen tubes was treated with lichenase and the product was analyzed by HPLC-PAD to determine the origin of 3-Glc. Specific tetra-saccharide was liberated from hemicellulose of pollen tubes, suggesting the presence of 1,3 : 1,4-beta-glucan in lily pollen tube hemicellulose. The structure of this 1,3 : 1,4-beta-glucan may be different from cereal plant 1,3 : 1,4-beta-glucan, since tri-saccharide was not detected in hemicellulose fraction after lichenase treatment. LP-ExoI and LP-ExoII, expressed in pollen grains and tubes, may be involved in the regulation of pollen tube elongation by hydrolyzing callose and 1,3 : 1,4-beta-glucan within pollen tube walls.

Amino Acid Sequence↗

Is human T-cell lymphotropic virus type I really silent?

The role of the cellular immune response to human T-cell lymphotropic virus type I (HTLV-I) is not fully understood. The low level of HTLV-I protein expression in peripheral blood lymphocytes has led to the widely held belief that HTLV-I is transcriptionally silent in vivo. However, most HTLV-I-infected individuals mount a strong and persistently activated cytotoxic T-lymphocyte (CTL) response to the virus; this observation implies that there is abundant chronic transcription of HTLV-I genes. Here we show that HTLV-I Tax protein expression rises quickly in freshly isolated peripheral blood lymphocytes, but that expressing cells are rapidly killed by CTLs. Mathematical analysis of these results indicates that the CTL response is extremely efficient and that the half-life of a Tax-expressing cell is less than a day. We propose that HTLV-I protein expression in circulating lymphocytes is undetectable by current techniques because of the efficiency of the CTL-mediated immune surveillance in vivo.

HTLV-I Infections↗

Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis.

Public databases now include vast amounts of recently acquired DNA sequences that are only partially annotated and, furthermore, are often annotated by automated methods that are subject to errors. Maximum information value of these databases can be derived only by further detailed analyses that frequently require careful examination of records in the context of biological functions. In this study we present an example of such an analysis focused on plant glycerolipid synthesis. Public databases were searched for sequences corresponding to 65 plant polypeptides involved in lipid metabolism. Comprehensive search results and analysis of genes, cDNAs and expressed sequence tags (ESTs) are available online (http://www.canr.msu.edu/lgc). Multiple alignments provided a method to estimate the number of genes in gene families. Further analysis of sequences allowed us to tentatively identify several previously undescribed genes in Arabidopsis. For example, two genomic sequences were identified as candidates for the palmitate-specific monogalactosyldiacylglycerol desaturase (FAD5). A candidate genomic sequence for 3-ketoacyl-acyl-carrier protein (ACP) synthase involved in mitochondrial fatty acid biosynthesis was also identified. Biotin carboxyl carrier protein (BCCP) in Arabidopsis is encoded by at least two genes, but the most abundant BCCP transcript so far has not been characterized. The large number (>165,000) of plant ESTs also provides an opportunity to perform "digital northern" comparisons of gene expression levels across many genes. EST abundance in general correlated with biochemical and flux characteristics of the enzymes in Arabidopsis leaf tissue. In a few cases, statistically significant differences in EST abundance levels were observed for enzymes that catalyze similar reactions in fatty acid metabolism. For example, ESTs for the FatB acyl-ACP thioesterase occur 21 times compared with 7 times for FatA acyl-ACP thioesterase, although flux through the FatA reaction is several times higher than through FatB. Such comparisons may provide initial clues toward previously undescribed regulatory phenomena. The abundance of ESTs for ACP compared with that of stearoyl-ACP desaturase and FatB acyl-ACP thioesterase suggests that concentrations of some enzymes of fatty acid synthesis may be higher than their acyl-ACP substrates.

Databases, Factual↗

Regulation of pathways determining cholesterol availability in the baboon placenta with advancing gestation.

Low density lipoprotein (LDL) is accepted as the primary source of cholesterol for progesterone biosynthesis in the primate placental syncytiotrophoblast. We hypothesized that the syncytiotrophoblast may, however, derive significant amounts of cholesterol from sources in addition to the LDL pathway, especially during early pregnancy or when faced with a paucity of lipoprotein-cholesterol. To test this, alternate cholesterol-providing pathways were assessed in placentae at early (Days 60-61), mid (Days 98-102), and late (Days 160-167) gestation in the baboon (Papio sp., term approximately 184 days). Expression of LDL receptor mRNA transcripts in an enriched fraction of syncytiotrophoblast cells was approximately 13-fold greater (P < 0.05) in mid and late gestation than in early pregnancy, although no differences were observed in whole villous tissue. The abundance of transcripts for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme responsible for de novo cholesterol synthesis, remained unchanged in syncytiotrophoblast cells; however, HMG-CoA reductase activity declined approximately 2-fold from early to late pregnancy (P < 0.01), with a commensurate decline in immunoreactive HMG-CoA reductase protein. Activities for acyl-coenzyme A:cholesterol acyl transferase (ACAT), a rate-limiting enzyme for cholesterol esterification, were greater (P < 0.05) at early and mid pregnancy in placental homogenates than in those from late pregnancy, while ACAT-1 mRNA concentrations and cholesterol ester hydrolase activity remained unchanged. These results, taken together, suggest that although de novo synthesis has the potential to provide a measure of the cholesterol used for placental progesterone production during early baboon pregnancy, its contribution declines with advancing gestational age as LDL receptor-derived cholesterol becomes the major source of substrate. Changes in LDL receptor mRNA abundance suggest differences in mechanisms regulating cholesterol homeostasis in steroidogenically active syncytiotrophoblasts vs. proliferative nonendocrine cell types in the placenta.

Animals↗

Adenovirus E1A-regulated transcription factor p120E4F inhibits cell growth and induces the stabilization of the cdk inhibitor p21WAF1.

Adenovirus E1A proteins influence cell growth and phenotype through physical interactions with cellular proteins that regulate basic processes such as cell cycle progression, DNA synthesis, and differentiation. p120E4F is a low-abundance cellular transcription factor that represses the adenovirus E4 promoter and is regulated by E1A, through a phosphorylation-induced reduction of its DNA binding activity, to permit activation of the E4 promoter during early infection. To determine the normal biological role of p120E4F, we assessed its ability to influence fibroblast cell growth and transformation. p120E4F suppressed NIH 3T3 fibroblast colony formation but had little effect when coexpressed with E1A and/or activated ras. Cells that overexpressed p120E4F were inhibited in their ability to enter S phase, had elevated levels of the cdk inhibitor p21WAF1, and reduced cyclin D-cdk4/6 kinase activity. The increase of p21WAF1 levels occurred through a p53-independent posttranscriptional mechanism that included a three- to fourfold increase in the half-life of p21WAF1 protein. Coexpression of activated ras with p120E4F stimulated cyclin D1 expression, elevated cyclin D-cdk4/6 kinase activity, and accelerated cell growth. These data suggest an important role for p120E4F in normal cell division and demonstrate that p21WAF1 can be regulated by protein turnover.

Adenoviridae↗

Expression of connexin31 and connexin43 genes in early rat embryos.

Gap junctions have been reported to play a pivotal role in coordinating embryonic development. Here we report the temporal and spatial pattern of connexin31 that has been found to be coexpressed with connexin43 in preimplantation rat embryos. Connexin31 and connexin43 transcripts are abundant in the zygote and degraded in the two- and four-cell stage to low levels for connexin31 and to undetectable ones for connexin43. The uncompacted eight-cell stage lacks the transcripts of both connexins. Reexpression of connexin43 and connexin31 mRNA is found from the compacted eight-cell stage onward. The connexin31 antigen, however, is already detected intracellularly at the uncompacted eight-cell stage. At the blastocyst stage, both connexins are coexpressed in the trophectoderm as well as in the inner cell mass. After implantation, compartmentalization of both connexins is observed. Connexin31 is now expressed exclusively by the cells of the ectoplacental cone and extraembryonic ectoderm, whereas connexin43 is restricted to the cells of the embryo proper. This compartmentalization in connexin expression between the derivatives of the inner cell mass and the trophectoderm may maintain the different developmental programs. THus, connexin31 seems not to be related to the first step in trophoblast lineage development and could serve as a compensatory channel during preimplantation development.

Animals↗

Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA.

Two novel members of the human cAMP-dependent protein kinase inhibitor (PKI) gene family, PKIB and PKIG, were cloned. The deduced proteins showed 70% and 90% identity with mouse PKIbeta and PKIgamma respectively. Both the already identified pseudosubstrate site and leucine-rich nuclear export signal motifs were defined from the 11 PKIs of different species. The PKIB and PKIG genes were mapped respectively to chromosome 6q21-22.1, using a radiation hybrid GB4 panel, and to chromosome 20q13.12-13.13, using a Stanford G3 panel. Northern-blot analysis of three PKI isoforms, including the PKIA identified previously, revealed significant differences in their expression patterns. PKIB had two transcripts of 1.9 kb and 1.4 kb. The former transcript was abundant in both placenta and brain and the latter was expressed most abundantly in placenta, highly in brain, heart, liver, pancreas, moderately in kidney, skeletal muscle and colon, and very little in the other eight tissues tested. PKIG was widely expressed as a 1.5-kb transcript with the highest level in heart, hardly detectable in thymus and peripheral blood leucocytes and was moderately expressed in the other tissues, with slightly different levels. However, PKIA was specifically expressed as two transcripts of 3.3 kb and 1.5 kb in heart and skeletal muscle. The distinct expression patterns of the three PKIs suggest that their roles in various tissues are probably different.

Adaptor Proteins, Signal Transducing↗

Highly identical cassettes of gene regulatory elements, genomically repetitive and present in RNA.

A region in the first intron of a metallothionein-encoding gene of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) regulates its 5' promoter activity. Within this region is a 290-bp cassette of six sequence motifs that are present in other genes in this species and posited to operate as regulatory elements. The cassette, present at high multiplicity in the genome, was used to screen genomic DNA clones. Of these, six diverse individuals were partially sequenced and found to have segments 94% identical to the 290-bp cassette in the SpMTA gene. Their next 80 bp diverged from the SpMTA sequence but were highly identical among the six non-SpMTA clones and contained an additional regulatory motif. These diverse clones thus contained 370-bp cassettes of an overall 94% sequence identity and an apparent content of seven regulatory elements. The regulatory cassettes were transposon-like, insofar as the termini of the highly identical regions consisted of 24- to 25-bp inverted repeats, bracketed by 6- to 9-bp direct repeats in the divergent regions. In addition to being in transcripts of the SpMTA intron, the cassette was found in other sea urchin embryo poly(A)+ RNAs, in eggs and embryos, and enriched in pluteus ectoderm. The cassette sequence was present in moderate abundance in transcripts in both sense and antisense orientation. We report the presence of a transposon-like cassette of regulatory elements that is also represented in RNA, which potentially could function differently from previously described transposons.

Animals↗

Comparative genomics of gene expression in the parasitic and free-living nematodes Strongyloides stercoralis and Caenorhabditis elegans.

Although developmental timing of gene expression is used to infer potential gene function, studies have yet to correlate this information between species. We analyzed 10,921 ESTs in 3311 clusters from first- and infective third-stage larva (L1, L3i) of the parasitic nematode Strongyloides stercoralis and compared the results to Caenorhabditis elegans, a species that has an L3i-like dauer stage. In the comparison of S. stercoralis clusters with stage-specific expression to C. elegans homologs expressed in either dauer or nondauer stages, matches between S. stercoralis L1 and C. elegans nondauer-expressed genes dominated, suggesting conservation in the repertoire of genes expressed during growth in nutrient-rich conditions. For example, S. stercoralis collagen transcripts were abundant in L1 but not L3i, a pattern consistent with C. elegans collagens. Although a greater proportion of S. stercoralis L3i than L1 genes have homologs among the C. elegans dauer-specific transcripts, we did not uncover evidence of a robust conserved L3i/dauer 'expression signature.' Strikingly, in comparisons of S. stercoralis clusters to C. elegans homologs with RNAi knockouts, those with significant L1-specific expression were more than twice as likely as L3i-specific clusters to match genes with phenotypes. We also provide functional classifications of S. stercoralis clusters.

Animals↗

Pediococcus acidilactici ldhD gene: cloning, nucleotide sequence, and transcriptional analysis.

The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb insert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase and pyruvate-formate lyase double mutant. The nucleotide sequence of ldhD encodes a protein of 331 amino acids (predicted molecular mass of 37,210 Da) which shows similarity to the family of D-2-hydroxyacid dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme has both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rpsB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of ldhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a 1-kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential growth but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either cotranscription of ldhD and rpsB or transcription of rpsB alone.

Amino Acid Sequence↗

The beta3 integrin gene is expressed at high levels in the major haematopoietic and lymphoid organs, vascular system, and skeleton during mouse embryo development.

Integrins are a family of cell surface molecules that mediate the attachment of cells to the extracellular matrix (ECM). These alphabeta heterodimers are involved in many biological processes. We used northern blotting and in situ hybridization to study the pattern of beta3 integrin gene expression during mouse embryogenesis. Northern blotting detected two species of beta3 mRNA from 7 to 17 days post coitum (dpc). These transcripts were abundant in the adult testis, kidney, liver, spleen, and heart. In situ hybridization experiments detected high levels of beta3 in the major haematopoietic and lymphoid organs: yolk sac, liver, and thymus. Moreover, beta3 transcripts were also detected in the vascular system, where beta3 integrin probably plays a key role in angiogenesis and vasculogenesis. We also detected a hybridization signal in the gut, the bronchioles of the lungs, and the bladder wall. beta3 transcripts were also present in the medullary regions of the adrenal glands and in the developing skeleton. Our study shows that beta3 gene expression is not restricted to the liver and gut during mouse development. We also detected beta3 integrin mRNA in the yolk sac, vessels, lung, bladder, and developing bones. Our data suggest that beta3 integrin plays a key role in many important physiological processes like haematopoiesis, angiogenesis, phagocytosis, and bone resorption.

Animals↗

Mixed messages: transcription patterns in failing and recovering human myocardium.

In previous studies, mechanical support of medically refractory hearts with a left ventricular assist device (LVAD) has induced regression of many morphological and functional abnormalities characteristic of failing human hearts. To identify transcriptional adaptations in failing and LVAD-supported hearts, we performed a comprehensive transcription analysis using the Affymetrix microarray platform and 199 human myocardial samples from nonfailing, failing, and LVAD-supported human hearts. We also used a novel analytical strategy that defines patterns of interest based on multiple intergroup comparisons. Although over 3088 transcripts exhibited significantly altered abundance in heart failure, most of these did not exhibit a consistent response to LVAD support based on our analysis. Of those 238 with a consistent response to LVAD support, more than 75% exhibited persistence or exacerbation of HF-associated transcriptional abnormalities whereas only 11%, 5%, and 2% exhibited partial recovery, normalization, and overcorrection responses, respectively. Even among genes implicated by previous reports of LVAD-associated myocardial improvements, partial or complete normalization of transcription did not predominate. The magnitude of differences in transcript abundance between nonfailing and failing hearts, and between failing an LVAD-supported hearts, tended to be low with changes greater than or equal to 2-fold infrequently observed. Our results indicate that morphological or functional myocardial improvements may occur without widespread normalization of pathological transcriptional patterns. These observations also suggest that many failure-associated transcriptional changes have only a limited role in regulating cardiac structure and function and may represent epiphenomena and/or nonspecific myocardial plasticity responses. Differences in mRNA localization, translation efficiency, and posttranslational protein modifications or interactions may be more pivotal in regulating myocardial structure and function.

3-Phosphoinositide-Dependent Protein Kinases↗

Transcriptional analysis of the region of the herpes simplex virus type 1 genome containing the UL8, UL9, and UL10 genes and identification of a novel delayed-early gene product, OBPC.

The region of the UL component of the herpes simplex virus type 1 genome between nucleotides 17,793 and 25,150 includes three open reading frames that code for the protein products of the UL8, UL9, and UL10 genes (D.J. McGeogh, M.A. Dalrymple, A.J. Davison, A. Dolan, M.C. Frame, D. McNab, L.J. Perry, J.E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). We have mapped and characterized the overlapping transcripts in this region and have found that, in addition to the low-abundance UL8 and UL9 transcripts and the abundant UL10 transcript, at least two additional transcription units, designated UL8.5 and UL9.5, are specified by this region of the genome. The 5' ends of the UL8, UL8.5, and UL9 transcripts were mapped to nucleotides 20,682, 22,351, and 23,381, respectively. The 5' terminus of the UL9.5 transcript has not yet been mapped. The 3' ends of the UL8, UL8.5, UL9, and UL9.5 transcripts are coterminal at nucleotide 18,197. The 5' end of the UL10 mRNA, which is transcribed from the strand opposite that specifying the UL8, UL8.5, UL9, and UL9.5 transcripts, lies within the UL9 open reading frame at nucleotide 22,944, while the 3' terminus was mapped to nucleotide 24,666. Time course studies demonstrated that the UL8 and UL9 transcripts are members of the early kinetic class, the UL8.5 mRNA is a delayed-early transcript, and the UL9.5 and UL10 transcripts belong to the true-late kinetic class. Examination of the nucleotide sequence of the UL8.5 transcript revealed a potential open reading frame that overlaps and is in frame with the C-terminal half of the open reading frame encoding the origin-binding protein (OBP), the product of the UL9 gene. In vitro translation of the UL8.5 transcript demonstrated that it encodes a protein with an apparent molecular mass of 53 kDa. This protein was recognized by antibody directed against the C-terminal region of OBP and has thus been designated OBPC. A protein with an identical apparent molecular mass was also recognized by this antibody in infected-cell lysates, indicating that OBPC is synthesized during viral infection.

Animals↗