Differences In Transcription Rates Research Articles

Microheterogeneity in the Kinetics and Sex-Specific Response to Type I IFN.

The response to type I IFNs involves the rapid induction of prototypical IFN signature genes (ISGs). It is not known whether the tightly controlled ISG expression observed at the cell population level correctly represents the coherent responses of individual cells or whether it masks some heterogeneity in gene modules and/or responding cells. We performed a time-resolved single-cell analysis of the first 3 h after invivo IFN stimulation in macrophages and CD4+ T and B lymphocytes from mice. All ISGs were generally induced in concert, with no clear cluster of faster- or slower-responding ISGs. Response kinetics differed between cell types: mostly homogeneous for macrophages, but with far more kinetic diversity among B and T lymphocytes, which included a distinct subset of nonresponsive cells. Velocity analysis confirmed the differences between macrophages in which the response progressed throughout the full 3 h, versus B and T lymphocytes in which it was rapidly curtailed by negative feedback and revealed differences in transcription rates between the lineages. In all cell types, female cells responded faster than their male counterparts. The ISG response thus seems to proceed as a homogeneous gene block, but with kinetics that vary between immune cell types and with sex differences that might underlie differential outcomes of viral infections.

Interpretable and tractable models of transcriptional noise for the rational design of single-molecule quantification experiments.

The question of how cell-to-cell differences in transcription rate affect RNA count distributions is fundamental for understanding biological processes underlying transcription. Answering this question requires quantitative models that are both interpretable (describing concrete biophysical phenomena) and tractable (amenable to mathematical analysis). This enables the identification of experiments which best discriminate between competing hypotheses. As a proof of principle, we introduce a simple but flexible class of models involving a continuous stochastic transcription rate driving a discrete RNA transcription and splicing process, and compare and contrast two biologically plausible hypotheses about transcription rate variation. One assumes variation is due to DNA experiencing mechanical strain, while the other assumes it is due to regulator number fluctuations. We introduce a framework for numerically and analytically studying such models, and apply Bayesian model selection to identify candidate genes that show signatures of each model in single-cell transcriptomic data from mouse glutamatergic neurons.

Age-associated bimodal transcriptional drift reduces intergenic disparities in transcription.

This study addressed the question of how well the quantitative transcriptome structure established in early life is maintained and how consistently it appears with increasing age, and if there is age-associated alteration of gene expression (A3GE), how much influence the Huntington's disease (HD) genotype exerts on it. We examined 285 exonic sequences of 175 genes using targeted PCR sequencing in skeletal muscle, brain, and splenic CD4+ T cells of wild-type and HD mice. In contrast to the muscle and brain, T cells exhibited large A3GE, suggesting a strong contribution to functional decline of the organism. This A3GE was markedly intensified in age-matched HD T cells, which exhibited accelerated aging as determined by reduced telomere length. Regression analysis suggested that gene expression levels change at a rate of approximately 3% per month with age. We found a bimodal relationship in A3GE in T cells in that weakly expressed genes in young mice were increasingly transcribed in older animals whereas highly expressed genes in the young were decreasingly expressed with age. This bimodal transcriptional drift in the T cell transcriptome data causes the differences in transcription rate between genes to progressively reduce with age.

Comparing the Expression of Olfaction-Related Genes in Gypsy Moth (Lymantria dispar) Adult Females and Larvae from One Flightless and Two Flight-Capable Populations

In insects, flight and sophisticated olfactory systems go hand in hand and are essential to survival and evolutionary success. Females of many Lepidopteran species have secondarily lost their flight ability, which may lead to changes in the olfactory capabilities of both larval and adult stages. The gypsy moth, Lymantria dispar, an important forest pest worldwide, is currently undergoing a diversification process with three recognized subspecies: the Asian gypsy moth (AGM) L. dispar asiatica, the Japanese gypsy moth L. dispar japonica (JGM) and the European gypsy moth (EGM) L. dispar dispar. Females of EGM populations from North America have lost their flight capacity whereas the JGM and AGM females are flight capable, making this an ideal system to investigate the relationship between flight and olfaction. We used next-generation sequencing to obtain female antennal and larval head capsule transcriptomes in order to (i) investigate the differences in expression of olfaction-related genes among populations; (ii) identify the most similar protein sequences reported for other organisms through a BLAST search, and (iii) establish the phylogenetic relationships of these sequences with respect to other insect species. Using this approach, we identified 115 putative chemosensory genes belonging to five families of olfaction-related genes. A principal component analysis revealed that the gene expression patterns of female antennal transcriptomes from different subspecies were more similar among them than to the larval head capsules of their respective subspecies supporting strong chemosensory differences between the two developmental stages. An analysis of the shared and exclusively expressed genes for three populations shows no evidence that loss of flight affects the number or type of genes being expressed. These results indicate either that (a) loss of flight does not impact the olfactory gene repertoire or that (b) the secondary loss of flight in the American EGM populations may be too recent to have caused major changes in the genes being expressed. However, we found higher expression values for IRs, OBPs GRs and ORs in EGM females, suggesting that differences in transcription rates could be an adaptation of flightless females to their chemical environment. Differences in olfactory genes and their expression in the

Shaping the murine macrophage phenotype: IL-4 and cyclic AMP synergistically activate the arginase I promoter.

Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but was correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; that is, differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.

Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

BackgroundExpression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells.MethodsTo determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA.ResultsFPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage.ConclusionWe demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.

Expression of mitochondrial genes and transcription estimation in different brain areas in Alzheimer's disease patients

Accumulation of mitochondrial defects is hypothesised to play a role in the complex pathophysiology of the sporadic form of Alzheimer's disease (SAD). Changes in expression of mtDNA encoded genes have been reported in SAD. However no conclusive results were obtained by using different methodologies and analysing distinct transcripts in a variety of brain areas. Here, we measured the expression of five mitochondrial-encoded genes in three brain areas and in lymphocytes from patients and controls by performing reverse transcription followed by quantitative real-time PCR. The analysis of gene expression was also performed by carrying out classic dot-blot experiments to evaluate the two techniques. SAD and control samples showed similar gene expression levels. Estimation of the transcription rate was also measured. No differences were observed when comparing patients and controls. Selective differences in transcription rates were observed only when samples were pooled by tissue, lymphocytes being the tissue with the highest rates.

Regulation of the packaging of Bacillus thuringiensis delta-endotoxins into inclusions.

During sporulation, many Bacillus thuringiensis subspecies synthesize several related delta-endotoxins which are packaged into bipyramidal intracellular inclusions. These inclusions are solubilized in the alkaline, reducing conditions of the midguts of susceptible insect larvae and are converted by proteolysis to active toxins. The toxins insert into the membranes of cells lining the midgut and form cation-selective channels, which results in lethality. There are three delta-endotoxins, Cry1Ab3, Cry1Ca1, and Cry1Da1, present in the inclusions produced by a B. thuringiensis subsp. aizawai cell. While the ratio of the steady-state mRNAs for these three protoxins has been shown to differ (cry1Ab3/cry1Ca1/cry1Da1 mRNA ratio, 4:2:1), the half-lives of the cry1Da1 and cry1Ab3 mRNAs were found to be similar, indicating that there were differences in the transcription rates. The relative contents of these delta-endotoxins in purified inclusions from B. thuringiensis subsp. aizawai have been measured previously, and an even greater relative deficiency of the Cry1Da1 protoxin (ratio, 20:12:1) was found. In order to account for this deficiency, other steps which could be involved in inclusion formation, such as translation and packaging, were examined. The three cry genes have the same dual overlapping promoters, but the ribosome binding sequence for the cry1Da1 gene was not the consensus sequence. Translation was enhanced about fourfold by changing to the consensus sequence. In addition, the relative amount of Cry1Da1 protoxin in inclusions was twofold lower when cells were sporulated in Luria-Bertani (LB) medium than when cells were sporulated in a glucose-yeast extract medium. This difference was attributable to packaging since the relative amounts of Cry1Da1 antigen in cells sporulating in the two media were the same. Some factor(s) required for packaging of the Cry1Da1 protoxin in inclusions is apparently limiting in LB medium. Differences in the initial transcription rates, translation efficiencies, and packaging all contribute to the delta-endotoxin composition of an inclusion.

Platelet count and interleukin 6 gene polymorphism in healthy subjects.

BackgroundInterleukin 6 (IL-6) is thought to play important roles in the development of reactive thrombocytosis caused by inflammation by its stimulatory effect on megakaryocytopoiesis. A G/C polymorphism of the IL-6 gene at position -174 has been found to be associated to different transcription rates. Specifically, subjects with the CC genotype showed lower plasma IL-6 levels compared with GC or GG subjects. Given this difference in transcription rates of IL-6 we speculated on different platelet count according to this IL-6 polymorphism.MethodsThe G/C polymorphism of the IL-6 gene at position -174, serum IL-6 concentration and platelet count were prospectively analyzed in 59 (25 women) consecutive healthy subjects.ResultsSubjects who were homozygotes for the C allele at position -174 of the IL-6 gene (Sfa NI genotype) showed significantly lower platelet count than carriers of the G allele, despite similar age, sex, body mass index and proportion of smokers (205400 ± 44088 vs 239818 ± 60194, p = 0.047). This was in parallel to differences in peripheral white blood cell count (5807 ± 1671 vs 6867 ± 1192 × 109/ml, p = 0.01).ConclusionThis is the first description, to our knowledge, of a genetical influence on basal platelet counts, which appears to be partially dependent on a polymorphism of the IL-6 gene, even in the absence of inflammation.

Disruption of plastid-encoded RNA polymerase genes in tobacco: expression of only a distinct set of genes is not based on selective transcription of the plastid chromosome.

Plastids of higher plants operate with at least two distinct DNA-dependent RNA polymerases, which are encoded in the organelle (PEP) and in the nucleus (NEP), respectively. Plastid run-on assays and Northern analyses were employed to analyse gene expression in tobacco mutant plastids lacking the PEP genes rpoA, rpoB or rpoC1. Hybridisation of run-on transcripts to restriction fragments representing the entire tobacco plastid chromosome, as well as to selected plastid gene-specific probes, shows that all parts of the plastid DNA are transcribed in rpo-deficient plastids. In comparison to wild-type chloroplasts, which are characterized by preferential transcription of photosynthesis-related genes in the light, mutant plastids exhibit a different transcription pattern with less pronounced differences in the hybridisation intensities between the individual genes. The analysis of steady-state transcript patterns and transcription rates of selected genes in both types of plastids demonstrates that differences in transcription rates are not necessarily paralleled by corresponding changes in transcript levels. The accumulation of large transcripts in the mutant plastids indicates that processing of primary transcripts may be impaired in the absence of PEP. These data suggest that, contrary to the prevailing view, much of the regulation of NEP-driven plastid gene expression in the rpo-deficient mutants is not based on differential promoter usage but is exerted at post-transcriptional levels.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million-year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, DNase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation.

Evolutionary adaptations of gene structure and expression in natural populations in relation to a changing environment: A multidisciplinary approach to address the million‐year saga of a small fish

We have used an experimentally based strategy to address molecular mechanisms underlying adaptation in Fundulus heteroclitus. In an attempt to falsify the hypothesis that selection is a major driving force in the maintenance of genetic diversity, we employed a multidisciplinary approach including allelic isozyme and mtDNA phylogeography, kinetic analyses of allelic isozymes, analysis of variation in coding and regulatory DNA sequences, metabolic biochemistry, organismal physiology, and selection experiments. Observed differences in gene structure and expression led us to make testable predictions about differences in metabolic flux, whole organism performance, and differential survival between allotypes. We have shown that variation in the lactate dehydrogenase-B (Ldh-B) protein results in differences in physiological function and is correlated with differences in survival at high temperatures. Recent work has investigated the role of variation in Ldh-B expression. There are differences in the levels of Ldh-B protein, mRNA, and transcription rate. We have addressed the mechanisms responsible for differences in transcription rate by a combination of sequence comparison, Dnase I footprinting, and functional analyses both in vitro and in vivo. We have shown that variation in the regulatory sequence of Ldh-B is responsible for the differences in transcription rate between populations and that the patterns of variation are inconsistent with a neutral model of molecular evolution. This functional differentiation, coupled with departures from neutral expectations, suggests that natural selection has acted on the regulation of Ldh-B. This article illustrates the value of a multidisciplinary approach in addressing problems in gene structure, expression, and evolutionary adaptation. J. Exp. Zool. 282:71–94, 1998. © 1998 Wiley-Liss, Inc.

HuD, a neuronal-specific RNA-binding protein, is a potential regulator of MYCN expression in human neuroblastoma cells.

HuD is one of a family of neural antigens recognised by the sera of patients with antibody-associated paraneoplastic encephalomyelitis. Localised exclusively to neurons, these proteins are among the earliest markers of the developing nervous system. Sequence analysis suggests that HuD is an RNA-binding protein. Hu protein levels were determined for the three cell types characterising human neuroblastoma cell lines: sympathoadrenal neuroblasts (N), substrate-adherent Schwann/glial/melanoblastic precursors (S) and stem cells (I) which can give rise to both N and S cells. Western blot analysis showed similar levels of protein in three N-type cell lines; S cells have no detectable Hu protein. Northern blot analysis indicated that N cells express all three Hu genes, HuD, HuC and Hel-N1. N cells, mostly from MYCN-amplified cell lines, have consistently higher steady-state levels of MYCN mRNA than S cell counterparts. Nuclear run-on and mRNA half-life experiments revealed no differences in transcription rate or mRNA stability between N and S cells from the LA-N-1 cell line, implicating differences in post-transcriptional regulation. HuD is postulated to be instrumental in splicing/processing and/or stabilisation of mRNAs involved in cell growth and neuronal differentiation. As determined by gel-mobility shift assays, HuD fusion protein binds to the 3'UTR of human MYCN mRNA. Analysis of HuD deletion mutants has demonstrated that the first and second RNA-recognition motifs (RRMs) are required for binding. Whether HuD regulates MYCN expression and thereby influences tumour aggressiveness is of major interest.

The ts13 mutation in the TAF(II)250 subunit (CCG1) of TFIID directly affects transcription of D-type cyclin genes in cells arrested in G1 at the nonpermissive temperature.

The general transcription initiation factor TFIID contains the TATA-binding protein (TBP) and TBP-associated factors (TAFs) implicated in the function of gene-specific activators. Previous studies have indicated that a hamster cell line (ts13) with a point mutation in the TAF(II)250/CCG1 (TAF(II)250) gene shows temperature-sensitive expression of a subset of genes and arrests in late G1 at 39.5 degrees C. Here, we report the identification of cell cycle-specific (G1-specific) genes that appear to be regulated directly through TAF(II)250 both in vivo and in vitro. Transcription rates of several cell cycle-regulatory genes were determined by run-on assays in nuclei from ts13 cells grown at permissive (33 degrees C) and nonpermissive (39.5 degrees C) temperatures. Temperature-dependent differences in transcription rates were observed for cyclin A, D1, and D3 genes. In transient-transfection assays, the human cyclin D1 promoter fused to a luciferase reporter showed a temperature-dependent reduction in activity in ts13 cells but not in parental BHK cells. In in vitro assays, upstream sequence-dependent transcription from the human cyclin D1 promoter was significantly reduced in ts13 nuclear extracts preincubated at 30 degrees C but not in similarly treated BHK nuclear extracts, and transcription in the ts13 extract was restored by addition of an affinity-purified human TFIID. Preincubation of the ts13 nuclear extracts did not affect the function of several GAL4-activation domain fusion proteins (GAL4-VP16, GAL4-p65, and GAL4-p53) on either the adenovirus major late or cyclin D1 core promoter bearing GAL4 sites, further indicating that the effect of the TAF(II)250 mutation is both core promoter and activator specific.

Regulation of Interleukin-10 Gene Expression: Possible Mechanisms Accounting for Its Upregulation and for Maturational Differences in Its Expression by Blood Mononuclear Cells

AbstractInterleukin-10 (IL-10) downmodulates phagocytic immune responses and accentuates humoral responses. Human neonates exhibit broad immune deficits that parallel actions of IL-10. We postulated that IL-10 production would be diminished in neonatal blood cells. We found that IL-10 production by lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMNCs) in vitro was greater by adult cells than by term cells and preterm cells. Additional studies were undertaken to identify mechanisms responsible for the developmental differences in IL-10 gene expression. IL-10 transcription was present in freshly isolated adult and neonatal cells in the absence of detectable levels of transcript. Transcription rates were not different between adult and neonatal cells. IL-10 transcripts were approximately 40% more abundant in adult cells than in term cells and were consistent with differences in secreted protein; however, no differences were noted in mRNA stability. IL-10 half-life was 60 minutes for both adult and term PBMNCs. We conclude that up-regulation of IL-10 gene expression in PBMNCs is modulated at the post-transcriptional level, that IL-10 protein production and mRNA content are greater in activated cells from adults compared with those from neonates, and that maturational differences in IL-10 expression are not due to differences in transcription rate or mRNA stability. Maturational differences in IL-10 expression might be due to differences in subpopulations of cytokine-producing cells or differences in nucleo-cytoplasmic transport.

Descriptive and functional characterization of variation in theFundulus heteroclitus Ldh-B proximal promoter

Variation in enzyme expression may be an important mechanism for physiological and evolutionary adaptation. The Ldh-B locus in the teleost fish Fundulus heteroclitus is one of a very few loci for which an evolutionary difference in transcription rate between populations has been demonstrated. To begin to understand the molecular modifications that are responsible for altering transcription, we have characterized the Ldh-B proximal promoter using a combination of sequence analysis, transient transfection, and in vivo footprinting. The Ldh-B gene has several transcription start sites and a TATA-less, Inr (initiator of transcription motif) containing promoter with multiple Sp1-like motifs. Transfection experiments reveal that Sp1 sites, TCC repeats, and Inrs are functional components of the proximal promoter. We find substantial sequence variation between populations within the proximal promoter (250 bp from the transcription start sites) and footprinting analysis indicates that some of this sequence variation is associated with differential protein binding to the apparent TFIID binding site and Sp1 sites. Together, these data suggest that variation in the Ldh-B proximal promoter may play a role in the observed difference in transcription rates between northern and southern populations of F. heteroclitus.

Transcriptional activation of the urokinase receptor gene in invasive colon cancer

The plasminogen activator urokinase promotes tumor invasion by converting plasminogen into plasmin, which degrades several extracellular matrix components. Urokinase can bind to a specific cell surface receptor, which leads to accelerated plasmin production. While there is good evidence indicating a role for this binding site in tumor invasion/metastasis, there is little information concerning the regulation of urokinase receptor expression in invasive cancer. To address this question a series of colon cancer cell lines, which demonstrate either a high or low ability to invade an extracellular matrix-coated porous filter, was characterized for receptor expression at the transcriptional and post-transcriptional levels. The invasive cell lines possessed 10-fold more receptors than their non-invasive counterparts as shown by cross-linking experiments and by Western blotting. Northern blotting indicated that this disparity in receptor number could be largely accounted for by a different amount of steady-state mRNA encoding the binding site. However, neither gene amplification nor enhanced mRNA stability could account for the augmented receptor protein observed for the invasive colon cancer cell types. In contrast, nuclear run-on experiments with representative cell lines revealed that the 10-fold difference in receptor display between the invasive-competent and invasive-deficient cells could be largely accounted for by differences in transcription rates. Transcription of the u-PAR gene in the receptor-deficient GEO cells, but not in the receptor-rich RKO cells, could be augmented by protein kinase C stimulation. These findings provide a clear rationale for studies to determine if the urokinase receptor promoter in invasive colon cancer is activated in cis or in trans.

Inappropriate transcription from the 5' end of the murine dihydrofolate reductase gene masks transcriptional regulation.

Using the nuclear run-on assay we found that in proliferating cells the transcription rate in the 5' end of the murine dihydrofolate reductase (dhfr) gene was approximately ten-fold higher than in the 3' end of the gene, suggesting transcriptional attenuation within the dhfr gene. However, when the transcription rate was measured by pulse-labeling, the rate was uniform throughout the gene, and the 5' dhfr signal was approximately ten-fold lower relative to a control gene signal than in the run-on assay. Previously, the activity of a dhfr promoter linked to a luciferase reporter gene was shown to increase about ten-fold at the G1/S-phase boundary following stimulation of serum-starved cells. To determine if the run-on procedure would detect growth regulation of the endogenous dhfr gene, serum-starved and -stimulated NIH 3T3 cells were analyzed. Using a dhfr 5' end probe no difference in transcription rate between these growth states was detected and the dhfr 3' end probe did not detect signal above background. In a cell line that was amplified at the dhfr locus, the transcription rate in the 5' end of the gene increased less than two-fold in stimulated cells, but the rate in the 3' end of the gene increased five- to seven-fold. Therefore, the dhfr gene is growth regulated at the level of transcription, but the nuclear run-on assay was only able to detect a difference in transcription rate in the 3' end of the gene in amplified cells. We suggest that isolation of nuclei may activate dhfr transcription complexes that normally are activated only at the G1/S-phase boundary.

Evolutionary adaptation to different thermal environments via transcriptional regulation.

Populations of the teleost fish Fundulus heteroclitus are subjected to the clinical variation in environmental temperatures that occurs along the eastern seacoast of North America. In concordance with this change in temperature is the clinal variation in the enzyme concentration of the heart-type lactate dehydrogenase (LDH-B; E.C.1.1.1.27). Previously we have shown that the compensating change in the LDH-B enzyme concentration is due to a change in the amount of LDH-B mRNA, but we did not define whether this was due to differences in mRNA stability or to differences in rate of transcription. The results presented here help clarify the molecular mechanism responsible for the variation in Ldh-B gene expression: the rate of transcription from the Ldh-B locus is significantly different between populations, and this difference is responsible for the compensatory change in LDH-B enzyme concentration.

Splicing of the platelet-derived-growth-factor A-chain mRNA in human malignant mesothelioma cell lines and regulation of its expression.

Platelet-derived-growth-factor (PDGF) A-chain transcripts differing in the presence or absence of an alternative exon-derived sequence have been described. In some publications, the presence of PDGF A-chain transcripts with this exon-6-derived sequence was suggested to be tumour specific. However, in this paper it was shown by reverse-transcription polymerase-chain-reaction (PCR) analysis that both normal mesothelial cells and malignant mesothelioma cell lines predominantly express the PDGF A-chain transcript without the exon-6-derived sequence. This sequence encodes a cell-retention signal, which means that the PDGF A-chain protein is most likely to be secreted by both cell types. In cultured normal mesothelial cells, the secreted PDGF A-chain protein might be involved in autocrine growth stimulation via PDGF alpha receptors. However, human malignant mesothelioma cell lines only possess PDGF beta receptors. If this also holds true in vivo, the PDGF A-chain protein produced and secreted by malignant mesothelial cells might have a paracrine function. In a previous paper, we described elevated expression of the PDGF A-chain transcript in human malignant mesothelioma cell lines, compared to normal mesothelial cells. In this paper, the possible reason for this elevation was studied. First, alterations at the genomic level were considered, but cytogenetic and Southern-blot analysis revealed neither consistent chromosomal aberrations, amplification nor structural rearrangement of the PDGF A-chain gene in the malignant cells. Possible differences in transcription rate of the PDGF A-chain gene, and stability of the transcript between normal and malignant cells, were therefore studied. The presence of a protein-synthesis inhibitor, cycloheximide, in the culture medium did not significantly influence the PDGF A-chain mRNA level in normal mesothelial and malignant mesothelioma cell lines. Furthermore, nuclear run-off analysis showed that nuclear PDGF A-chain mRNA levels varied in both cell types to the same extent as the levels observed in Northern blots. Taken together, this suggests that increased transcription is the most probable mechanism for the elevated mRNA level of the PDGF A-chain gene in human malignant mesothelioma cell lines.