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Endothelial cells preparing to die by apoptosis initiate a program of transcriptome and glycome regulation.

The protein-based changes that underlie the cell biology of apoptosis have been extensively studied. In contrast, mRNA- and polysaccharide-based changes have received relatively little attention. We have combined transcriptome and glycome analyses to show that apoptotic endothelial cell cultures undergo programmed changes to RNA transcript abundance and cell surface polysaccharide profiles. Although a few of the transcriptome changes were protective, most appeared to prepare cells for apoptosis by decreasing the reception and transduction of pro-survival signals, increasing pro-death signals, increasing abundance of apoptotic machinery, inhibiting cellular proliferation, recruiting phagocytes to regions of cell death, and promoting phagocytosis. Additional transcriptomal changes appeared to alter the synthesis and modification of cell surface glycosaminoglycans. The resultant reduced abundance of sulphated cell surface glycosaminoglycans may further promote cell death by inhibiting the presentation of extracellular matrix-tethered survival factors to their receptors on dying cells. We propose that the transcriptome and glycome regulation presented here synergize with previously described protein-based changes to guide the apoptotic program.

Apoptosis↗

Connexin43 and the brain transcriptome of newborn mice.

Our previously reported cDNA array datasets from neonatal wild-type and Cx43-/- (approved gene symbol Gja1) mouse brains were further analyzed to identify underlying interlinkages in the brain transcriptome. The analysis revealed that no gene cohort sharing either primary function or chromosomal location was significantly altered (up-and down-regulation were roughly balanced) in Cx43-/- brains, but each cohort exhibited significant perturbation of transcript abundance proportions and reduced expression variability and coordination. By comparing pairwise expression correlations of all genes with one another in wild-type brains, we found genes exhibiting remarkable similarity or opposition to the coordination profile (set of synergistically, antagonistically, and independently expressed partners) of Cx43, one of the most similar being pannexin1, a vertebrate homolog of invertebrate gap junction proteins. This study indicates striking redundancy of expression controls over functional pathways and suggests that certain genes may play roles similar to or opposite that of Cx43 in organizing the brain transcriptome.

Animals↗

Cloning and characterization of murine carnitine acetyltransferase: evidence for a requirement during cell cycle progression.

We have employed a newly developed differential screening technique (reverse strand priming) to identify murine carnitine acetyltransferase (CARAT) as a growth- and cell cycle-regulated gene in S3T3 mouse fibroblasts. Sequence analysis of the full-length cDNA clone and homology comparisons have revealed 87% homology to the human CARAT gene. On Northern blots we were able to measure a 2-3-fold induction 18 h after a mitogenic stimulus following serum deprivation as well as after release from a sodium butyrate block. The cell cycle induction pattern of the CARAT gene was analysed in mouse fibroblasts at different stages of the unperturbed cell cycle. Fractions obtained by elutriation of an exponentially growing culture showed a biphasic maximum of transcript abundance in the G1 and G2 phases of the cell cycle. CARAT expression was investigated in several organs of the adult mouse. Among those measured, CARAT expression was highest, relative to liver, in heart muscle (56-fold) and testis (21-fold). Using both conventional antisense oligodeoxynucleotides and novel single-stranded antisense phagemid DNA, we obtained evidence that the CARAT enzyme function is necessary for progression through G1 and into the S-phase of the cell cycle.

Amino Acid Sequence↗

The transcribed genome and the heritable basis of essential hypertension.

Gene expression can be now assessed quantitatively and comprehensively. In addition to reflecting the specialized differentiation of the cell or tissue type from which gene expression is sampled, it also manifests patterns determined by inheritance. Thus gene expression is a phenotypic trait, at least when assessed comprehensively. This trait shows familial aggregation and segregation patterns indicative of an inherited contribution. The molecular evolution of genes includes mutations affecting regulatory sequences in the genome that influence gene expression in cis and in trans. Such mutations may increase in frequency in a population either by genetic drift or by selection. Traits of gene expression, acting alone or in concert with other gene expression traits, may generate phenotypes that extend beyond transcript abundance. Indeed, the divergence of species and the traits that distinguish related species appear to rely importantly on inherited divergence in the control of gene expression. Variation in gene expression may contribute to the pathogenesis of a prevalent human disease trait that shows heritability--essential hypertension. Along with other common heritable diseases, hypertension susceptibility arises from the actions of multiple genome sequence variations. The identity of such variation has proven elusive when sought by methods that have been successfully applied to Mendelian diseases. This review explores the potential to uncover hypertension genes by exploiting quantitative variation in the heritable control of gene expression.

Animals↗

Floral induction in tissue culture: a system for the analysis of LEAFY-dependent gene regulation.

We have developed a versatile floral induction system that is based on ectopic overexpression of the transcription factor LEAFY (LFY) in callus. During shoot regeneration, flowers or floral organs are formed directly from root explants without prior formation of rosette leaves. Morphological and reporter gene analyses show that leaf-like structures are converted to floral organs in response to LFY activity. Thus, increased levels of LFY activity are sufficient to bypass normal vegetative development and to direct formation of flowers in tissue culture. We found that about half of the cultured cells respond to inducible LFY activity with a rapid upregulation of the known direct target gene of LFY, APETALA1 (AP1). This dramatic increase in the number of LFY-responsive cells compared to whole plants suggested that the tissue culture system could greatly facilitate the analysis of LFY-dependent gene regulation by genomic approaches. To test this, we monitored the gene expression changes that occur in tissue culture after activation of LFY using a flower-specific cDNA microarray. Induction of known LFY target genes was readily detected in these experiments. In addition, several other genes were identified that had not been implicated in signaling downstream of LFY before. Thus, the floral induction system is suitable for the detection of low abundance transcripts whose expression is controlled in an LFY-dependent manner.

Arabidopsis↗

A small subpopulation of blastospores in candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and beta-1,6-glucan pathway genes.

The resistance of Candida albicans biofilms to a broad spectrum of antimicrobial agents has been well documented. Biofilms are known to be heterogeneous, consisting of microenvironments that may induce formation of resistant subpopulations. In this study we characterized one such subpopulation. C. albicans biofilms were cultured in a tubular flow cell (TF) for 36 h. The relatively large shear forces imposed by draining the TF removed most of the biofilm, which consisted of a tangled mass of filamentous forms with associated clusters of yeast forms. This portion of the biofilm exhibited the classic architecture and morphological heterogeneity of a C. albicans biofilm and was only slightly more resistant than either exponential- or stationary-phase planktonic cells. A submonolayer fraction of blastospores that remained on the substratum was resistant to 10 times the amphotericin B dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes coding for enzymes in the ergosterol (ERG1, -3, -5, -6, -9, -11, and -25) and beta-1,6-glucan (SKN and KRE1, -5, -6, and -9) pathways was performed by quantitative RT-PCR. The results indicate a possible association between the high level of resistance exhibited by the blastospore subpopulation and differential regulation of ERG1, ERG25, SKN1, and KRE1. We hypothesize that the resistance originates from a synergistic effect involving changes in both the cell membrane and the cell wall.

Amphotericin B↗

Analysis of the yeast transcriptome with structural and functional categories: characterizing highly expressed proteins.

We analyzed 10 genome expression data sets by large-scale cross-referencing against broad structural and functional categories. The data sets, generated by different techniques (e.g. SAGE and gene chips), provide various representations of the yeast transcriptome (the set of all yeast genes, weighted by transcript abundance). Our analysis enabled us to determine features more prevalent in the transcriptome than the genome: i.e. those that are common to highly expressed proteins. Starting with simplest categories, we find that, relative to the genome, the transcriptome is enriched in Ala and Gly and depleted in Asn and very long proteins. We find, furthermore, that protein length and maximum expression level have a roughly inverse relationship. To relate expression level and protein structure, we assigned transmembrane helices and known folds (using PSI-blast) to each protein in the genome; this allowed us to determine that the transcriptome is enriched in mixed alpha-beta structures and depleted in membrane proteins relative to the genome. In particular, some enzymatic folds, such as the TIM barrel and the G3P dehydrogenase fold, are much more prevalent in the transcriptome than the genome, whereas others, such as the protein-kinase and leucine-zipper folds, are depleted. The TIM barrel, in fact, is overwhelmingly the 'top fold' in the transcriptome, while it only ranks fifth in the genome. The most highly enriched functional categories in the transcriptome (based on the MIPS system) are energy production and protein synthesis, while categories such as transcription, transport and signaling are depleted. Furthermore, for a given functional category, transcriptome enrichment varies quite substantially between the different expression data sets, with a variation an order of magnitude larger than for the other categories cross-referenced (e.g. amino acids). One can readily see how the enrichment and depletion of the various functional categories relates directly to that of particular folds.

Amino Acids↗

Mapping herpes simplex virus type 1 latency-associated transcript sequences that protect from apoptosis mediated by a plasmid expressing caspase-8.

LAT (latency-associated transcript) is the only herpes simplex virus type 1 (HSV-1) transcript abundantly expressed during neuronal latency. LAT expression is required for the high reactivation phenotype of HSV-1 and this phenotype correlates with LAT's anti-apoptosis properties. LAT nucleotides 1 to 1499 inhibit caspase-8 (death receptor apoptotic pathway), but not caspase-9 (mitochondrial apoptotic pathway), -induced apoptosis as efficiently as larger LAT fragments. LAT sequences important for inhibiting caspase-8-induced apoptosis were also localized. The ability of LAT nucleotides 1 to 1499 to efficiently inhibit caspase-8-induced apoptosis correlates with the high reactivation phenotype of a mutant virus expressing just the first 1.5 kb of LAT (nucleotides 1 to 1499).

Animals↗

Variation in transcriptional regulation of cyclin dependent kinase inhibitor p21waf1/cip1 among human bronchogenic carcinomas.

BACKGROUND: Cell proliferation control depends in part on the carefully ordered regulation of transcription factors. The p53 homolog p73, contributes to this control by directly upregulating the cyclin dependent kinase inhibitor, p21waf1/cip1. E2F1, an inducer of cell proliferation, directly upregulates p73 and in some systems upregulates p21 directly. Because of its central role in controlling cell proliferation, upregulation of p21 has been explored as a modality for treating bronchogenic carcinoma (BC). Improved understanding of p21 transcriptional regulation will facilitate identification of BC tissues that are responsive to p21-directed therapies. Toward this goal, we investigated the role that E2F1 and p73 each play in the transcriptional regulation of p21. RESULTS: Among BC samples (N = 21) p21 transcript abundance (TA) levels varied over two orders of magnitude with values ranging from 400 to 120,000 (in units of molecules/106 molecules beta-actin). The p21 values in many BC were high compared to those observed in normal bronchial epithelial cells (BEC) (N = 18). Among all BC samples, there was no correlation between E2F1 and p21 TA but there was positive correlation between E2F1 and p73alpha (p < 0.001) TA. Among BC cell lines with inactivated p53 and wild type p73 (N = 7) there was positive correlation between p73alpha and p21 TA (p < 0.05). Additionally, in a BC cell line in which both p53 and p73 were inactivated (H1155), E2F1 TA level was high (50,000), but p21 TA level was low (470). Transiently expressed exogenous p73alpha in the BC cell line Calu-1, was associated with a significant (p < 0.05) 90% increase in p21 TA and a 20% reduction in E2F1 TA. siRNA mediated reduction of p73 TA in the N417 BC cell line was associated with a significant reduction in p21 TA level (p < 0.01). CONCLUSION: p21 TA levels vary considerably among BC patients which may be attributable to 1) genetic alterations in Rb and p53 and 2) variation in TA levels of upstream transcription factors E2F1 and p73. Here we provide evidence that p73 upregulates p21 TA in BC tissues and upregulated p21 TA may result from E2F1 upregulation of p73 but not from E2F1 directly.

Carcinoma, Bronchogenic↗

Gene discovery using computational and microarray analysis of transcription in the Drosophila melanogaster testis.

Identification and annotation of all the genes in the sequenced Drosophila genome is a work in progress. Wild-type testis function requires many genes and is thus of potentially high value for the identification of transcription units. We therefore undertook a survey of the repertoire of genes expressed in the Drosophila testis by computational and microarray analysis. We generated 3141 high-quality testis expressed sequence tags (ESTs). Testis ESTs computationally collapsed into 1560 cDNA set used for further analysis. Of those, 11% correspond to named genes, and 33% provide biological evidence for a predicted gene. A surprising 47% fail to align with existing ESTs and 16% with predicted genes in the current genome release. EST frequency and microarray expression profiles indicate that the testis mRNA population is highly complex and shows an extended range of transcript abundance. Furthermore, >80% of the genes expressed in the testis showed onefold overexpression relative to ovaries, or gonadectomized flies. Additionally, >3% showed more than threefold overexpression at p <0.05. Surprisingly, 22% of the genes most highly overexpressed in testis match Drosophila genomic sequence, but not predicted genes. These data strongly support the idea that sequencing additional cDNA libraries from defined tissues, such as testis, will be important tools for refined annotation of the Drosophila genome. Additionally, these data suggest that the number of genes in Drosophila will significantly exceed the conservative estimate of 13,601.

Animals↗

Evidence for distinguishable transcripts of the putative testis determining gene (ZFY) and mapping of homologous cDNA sequences to chromosomes X,Y and 9.

Oligonucleotide sequences based on the amino acid sequence of the putative testis determining gene ZFY have been used to isolate a 1.3 Kb Hind III Y genomic DNA fragment CMPXY1 and three human testis cDNA sequences (CMPXY2, CMPXY3 and CMPXY4). These sequences detect at least four potential exons on the Y (Y1, Y3, Y4 and Y5), three on the X (X1, X2 and X3) and three of autosomal origin (A1, A2 and A3) as determined by comparing the fragments detected by different clones. Analysis with subfragments of CMPXY4 shows that Y3 is unique to the Y and that Y4 and X1 are homologous. Y5 and X3 are detected by the same subfragment of CMPXY4. This is also the case for Y1, X2, A1, A2 and A3. Thus these exons may contain further regions of homology between the X, Y and an autosomal locus. The X-linked sequences all lie in Xp21.2-Xp22.1 and studies with XX males have placed the Y-linked sequences in distal Yp adjacent to the Y-autosomal homologous sequence GMGY3. We have confirmed these localizations by in situ hybridization with CMPXY4 and have shown additionally that the autosomal sequences of both the CMPXY4 sequence and GMGY3 map to 9p22-9pter. Restriction analysis demonstrates that CMPXY1/XY2/XY3 differ in sequence from CMPXY4 at three restriction enzyme sites, thus suggesting that they are transcribed from different but closely related genes and that CMPXY4 must be either X-linked or autosomal in origin. This indicates that more than one of the loci containing ZFY-related sequences are transcribed and potentially fulfil functionally distinct roles in the human sex determining pathway. Northern blot analysis of human foetal testis RNA has shown that three low abundance transcripts of 5, 6 and 8 Kb can be detected by ZFY-related DNA sequences.

Base Sequence↗

Transcription profile of Helicobacter pylori in the human stomach reflects its physiology in vivo.

BACKGROUND: Little is known about levels of expression of Helicobacter pylori genes in the human host. We therefore developed a quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay to measure transcript profiles of H. pylori in the human stomach. METHODS: In vivo expression of 16 genes on the cag pathogenicity island and of 18 putative virulence genes was quantitated by isolation of total RNA directly from infected human gastric mucosa. The results were compared with in vitro expression determined from H. pylori cells grown in culture. RESULTS: The highest levels of expression were found for cag1 and cag25 and for genes, such as urease and catalase, that may be important for bacterial homeostasis in the relatively hostile environment of the gastric mucosa. Transcript abundance, relative to 16S rRNA, was lower in vivo than in vitro, which suggests that H. pylori cells are in stationary phase in the gastric environment. This was particularly apparent for cagA. Since CagA is arguably of unique importance, in terms of interaction with the host, tight control of its in vivo expression might be particularly important. CONCLUSIONS: qRT-PCR is a powerful tool to measure gene expression in human or animal tissue that contains minute amounts of microbial mRNA, and the results reflect on the physiology of the pathogen in its natural host.

Adult↗

Cloning of 17beta-hydroxysteroid dehydrogenase-I cDNAs from Japanese eel ovary.

17beta-hydroxysteroid dehydrogenase-I (17beta-HSD-I) is a key steroidogenic enzyme for estradiol-17beta (E(2)) production. cDNAs encoding 17beta-HSD-I were cloned for the first time in lower vertebrates from the ovary of a teleost, the Japanese eel. The deduced amino acid sequence from these cDNAs was approximately 50% identical to mammalian 17beta-HSD-Is. 17beta-HSD-I mRNA was not detected in previtellogenic ovaries by Northern blotting. However, transcript abundance increased in early vitellogenic ovaries obtained from fish artificially matured by gonadotropic treatment, but thereafter did not appear to change further. Recombinant 17beta-HSD-I expressed in human kidney 293 cells selectively converted estrone to E(2), but androstenedione, testosterone, or E(2) were not converted to any other steroids. Although it is widely accepted that E(2) is produced from testosterone in other species of teleosts, the substrate specificity of eel 17beta-HSD-I suggests that a steroidogenic pathway for production of E(2) from androstenedione via estrone exists in the Japanese eel ovary.

17-Hydroxysteroid Dehydrogenases↗

Gene expression during formation of earlywood and latewood in loblolly pine: expression profiles of 350 genes.

The natural variability of wood formation in trees affords opportunities to correlate transcript profiles with the resulting wood properties. We have used cDNA microarrays to study transcript abundance in developing secondary xylem of loblolly pine (Pinus taeda) over a growing season. The cDNAs were selected from a collection of 75 000 ESTs that have been sequenced and annotated (http://web.ahc.umn.edu/biodata/nsfpine/). Cell wall thickness and climatic data were related to earlywood and latewood formation at different time points during the growing season. Seventy-one ESTs showed preferential expression in earlywood or latewood, including 23 genes with no significant similarity to genes in GenBank. Seven genes involved in lignin synthesis were preferentially expressed in latewood. The studies have provided initial insights into the variation of expression patterns of some of the genes related to the wood formation process.

Expressed Sequence Tags↗

The control of herpes simplex virus type-1 late gene transcription: a 'TATA-box'/cap site region is sufficient for fully efficient regulated activity.

The transcriptional programme of herpes simplex virus type 1 (HSV-1) is organised into three principle phases; immediate-early (IE), early (E) and late. The appearance of IE gene products provides the switch for E transcription. Abundant expression of late genes requires viral DNA replication. There is some overlap between E and late genes according to their degree of dependence on DNA replication. The pattern of expression of gene US11 is regulated with 'true-late' kinetics (Johnson et al., 1986). In a transient assay system, regulation of a plasmid-borne US11 promoter mimics its viral counterpart, and has a similar dependence on DNA replication for abundant expression. Using plasmids which contain a functional HSV-1 origin of replication (ORIS), we have identified the sequence requirements for the expression of late genes. All DNA sequence elements necessary for fully efficient regulated expression of US11 lie within 31 bp of the RNA cap sites; therefore it appears that a late gene promoter consists only of a proximal 'TATA-box' and cap-site region. We tested this hypothesis by removing the distal upstream region of the gD promoter (which is required for its normal regulation as an early promoter) and linking this truncated promoter to ORIS. This resulted in the conversion of gD promoter regulation to late gene kinetics during virus superinfection. The implications of these results for the mechanisms of HSV gene regulation are discussed.

Base Sequence↗

Transcription profiling of signal transduction-related genes in sugarcane tissues.

A collection of 237,954 sugarcane ESTs was examined in search of signal transduction genes. Over 3500 components involved in several aspects of signal transduction, transcription, development, cell cycle, stress responses and pathogen interaction were compiled into the Sugarcane Signal Transduction (SUCAST) Catalogue. Sequence comparisons and protein domain analysis revealed 477 receptors, 510 protein kinases, 107 protein phosphatases, 75 small GTPases, 17 G-proteins, 114 calcium and inositol metabolism proteins, and over 600 transcription factors. The elements were distributed into 29 main categories subdivided into 409 sub-categories. Genes with no matches in the public databases and of unknown function were also catalogued. A cDNA microarray was constructed to profile individual variation of plants cultivated in the field and transcript abundance in six plant organs (flowers, roots, leaves, lateral buds, and 1st and 4th internodes). From 1280 distinct elements analyzed, 217 (17%) presented differential expression in two biological samples of at least one of the tissues tested. A total of 153 genes (12%) presented highly similar expression levels in all tissues. A virtual profile matrix was constructed and the expression profiles were validated by real-time PCR. The expression data presented can aid in assigning function for the sugarcane genes and be useful for promoter characterization of this and other economically important grasses.

Computational Biology↗

Relative transcript quantification by quantitative PCR: roughly right or precisely wrong?

BACKGROUND: When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches 12 may lead to opposing biological conclusions. CONCLUSION: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results.

Animals↗

Microarray analysis of uterine epithelial gene expression during the implantation window in the mouse.

In mice, the uterus becomes transiently receptive to the hatched blastocyst on the day of implantation to allow its attachment to the luminal epithelium and subsequent invasion into the uterus. This uterine preparation for implantation is regulated by estradiol-17beta and progesterone, acting through their transcription factor receptors. Using ovariectomized mice treated with physiological regimens of these hormones, combined with methods to isolate RNA specifically from the uterine epithelium followed by transcriptome analysis on cDNA microarrays, 222 genes whose transcript abundance was specifically increased by estradiol-17beta and progesterone treatment were identified. Gene ontology analysis revealed an emphasis on genes involved with immune responses, extracellular matrix metabolism, and cell-to-cell communication. In situ hybridization to uterine sections isolated through the first 6 d of pregnancy identified novel sets of genes such as Bach, Myd88, Cd14, Isg20, and Lrp2 whose expression was restricted to the uterine epithelium during the implantation window. Particularly notable was the expression of the mRNA for members of the signaling pathway from the Toll-like receptors to its downstream targets such as Irg-1. The identification of these genes showing a cell type hormonally regulated pattern of expression in the uterus suggests novel functions for them during implantation.

Animals↗