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PubMed1995

The distinct and developmentally regulated patterns of expression of members of the mouse Cdc25 gene family suggest differential functions during gametogenesis.

Cdc25 genes encode protein threonine/tyrosine phosphatases that activate cyclin-dependent kinases. A cDNA encoding the murine homologue of the human Cdc25C gene was isolated in studies designed to identify those genes involved in regulating the mitotic and meiotic cell cycles of developing mouse germ cells. Northern blot and in situ hybridization along with immunoblot analyses were performed to determine the patterns of expression of Cdc25C compared to those of a second Cdc25 family member, Cdc25B. Murine Cdc25C transcripts of 2.1 kb were detected in midgestation embryos and in several adult tissues, including testis and ovary. The highest levels of Cdc25C transcripts were detected in the testis, with low levels of the 2.1-kb transcript and abundant levels of a 1.9-kb transcript that was not detected in the other tissues examined. In the testis, Cdc25C expression was localized in germ cells, specifically in late pachytene-diplotene spermatocytes and round spermatids, whereas Cdc25B expression was most readily detected in the somatic cells. In the ovary, Cdc25C expression was apparent in cumulus granulosa cells, whereas the expression of Cdc25B was detected in both growing oocytes and somatic cells, including the granulosa cells. These results not only indicate that the expression of the murine Cdc25 genes is lineage- and developmental stage-specific in the mouse testis and ovary but also suggest that the Cdc25 genes may have different functions in the germinal and somatic compartments of the ovary and the testis, only some of which are involved in cellular proliferation.

Animals↗
PubMed2004

Analysis of mouse germ-cell transcriptome at different stages of spermatogenesis by SAGE: biological significance.

The transcriptomes of mouse type A spermatogonia (Spga), pachytene spermatocytes (Spcy), and round spermatids (Sptd) were determined by sequencing the respective SAGE (Serial Analysis of Gene Expression) libraries. A total of 444,015 tags derived from one Spga, two Spcy, and one Sptd library were analyzed, and 34,619 different species of transcripts were identified, 5279 of which were novel. Results indicated the germ-cell transcriptome comprises of more than 30,000 transcripts. Virtual subtraction showed that cell-specific transcripts constitute 12-19.5% of the transcriptome. Components of the protein biosynthetic machinery are highly expressed in Spga. In Spcy transcription factors are abundantly expressed while transcripts encoding proteins involved in chromosome remodeling and testis-specific transcripts are prominent in Sptd. The databases generated by this work provide very useful resources for cellular localization of genes in silico. They are also extremely useful as sources for identification of splice variants of genes in germ cells.

Animals↗
PubMed1999

The mat-r open reading frame is transcribed from a non-canonical promoter and contains an internal promoter to co-transcribe exons nad1e and nad5III in wheat mitochondria.

The expression of the mat-r locus (mat-r-nad1e-nad5III) was studied in wheat mitochondria. Transcription initiation sites were mapped by S1 protection, primer extension and capping experiments. Two different transcription initiation sites were found. One, non-canonical promoter of low expression level generates a transcript containing the complete mat-r open reading frame (orf), suggesting that this form is the maturase-reverse transcriptase mRNA. A second transcription initiation site, found within the coding region of the mat-r orf, directs the transcription of an abundant co-transcript containing the carboxy-terminal region of the mat-r orf, exon e of the nad1 gene, exon III of the nad5 gene and their respective trains-introns. The co-transcript promoter carries the consensus motif of plant mitochondrial promoters. Analysis of transcript sequences reveals the presence of editing sites in analogous positions in both nad1e and nad5III trans-introns, suggesting that RNA editing is necessary for the trans-splicing process.

Base Sequence↗
PubMed2005

Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages.

Clk/STY is a LAMMER protein kinase capable to phosphorylate serine/arginine-rich (SR) proteins that modulate pre-mRNA splicing. Clk/STY alternative splicing generates transcripts encoding a full-length kinase and a truncated catalytically inactive protein. Here we showed that clk/STY, as well as other members of the family (e.g. clk2, clk3 and clk4), are up-regulated during HMBA-induced erythroleukemia cell differentiation. mRNAs coding for the full-length and the truncated forms were responsible for the overall increased expression. In clk/STY, however, a switch was observed for the ratio of the two alternative spliced products. In undifferentiated cells the full-length transcript was more abundant whereas the transcript encoding for the truncated form predominated at latter stages of differentiation. Surprisingly, overexpression of clk/STY did not alter the splicing switch upon differentiation in MEL cells. These results suggest that clk/STY might contribute to control erythroid differentiation by a mechanism that implicates a balance between these two isoforms.

Acetamides↗
PubMed2000

An adipocyte-derived plasma protein, adiponectin, adheres to injured vascular walls.

Adipose tissue secretes a variety of proteins into the bloodstream. We have previously reported a novel cDNA, apM1 (adipose most abundant gene transcript 1), which is specifically and abundantly expressed in adipose tissue [1]. Primary structure analysis predicted that the apM1 gene product possesses significant homology to collagens VIII, X and complement factor C1q, and we named it adiponectin. In the current study, we analyzed characteristics of adiponectin in vitro and in vivo. Adiponectin protein was proved to be secreted into the medium when the cDNA was transfected to COS cells. Anti-adiponectin cross-reactivities were abundantly detected in the human plasma. In solid-phase binding assays, adiponectin specifically bound to collagen types I, III and V, which are present in vascular intima. Immunohistochemical analysis revealed that adiponectin was detected in the walls of the catheter-injured vessels but not in the intact vascular walls. These data suggest that adiponectin is a plasma protein produced by adipose tissue and accumulates in vascular walls when the endothelial barrier is injured.

Adipocytes↗
PubMed1997

Differential expression of CuZn- and Fe-superoxide dismutase genes of tobacco during development, oxidative stress, and hormonal treatments.

Chloroplasts of Nicotiana tabacum have two superoxide dismutases: a Fe- and a CuZn-containing enzyme, encoded by the nuclear genes sodB and sodCp, respectively. As a first step in studying the physiological function of these two enzymes, we compared the expression of sodB and sodCp in different plant organs, in response to hormonal treatments, and upon treatment with paraquat and Norflurazon. The sodCp transcript and active enzyme were detected only in young leaves of mature plants. The sodB transcript was more abundant in young compared to old leaves, but the enzymatic activity was higher in mature and senescent leaves. sodCp and sodB exhibited a different expression pattern upon treatment with abscisic acid, indole-3-acetic acid, kinetin, gibberellin, and 1-aminocyclopropane-1-carboxylate. Paraquat treatment caused a decrease in abundance of both transcripts, although the dose dependency of this decrease differed. Norflurazon-induced photooxidation resulted in a 10-fold increase of sodCp mRNA whereas the sodB transcript level was 25% higher than the control. These differences in expression might explain why both plastid-located superoxide dismutase enzymes are needed, particularly under stress conditions.

Amino Acid Sequence↗
PubMed1999

BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize.

The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript (rbcL) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2-conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize.

Amino Acid Sequence↗
PubMed2004

The NtcA-dependent P1 promoter is utilized for glnA expression in N2-fixing heterocysts of Anabaena sp. strain PCC 7120.

Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N(2)-fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNA(I) originates from a canonical NtcA-dependent promoter (P(1)) and RNA(II) originates from a sigma(70)-type promoter (P(2)), RNA(IV) is influenced by NtcA but the corresponding promoter (P(3)) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNA(V), which has previously been detected only in in vitro transcription assays and should originate from P(4). However, in heterocysts, which are differentiated cells specialized in N(2) fixation, RNA(I) was the almost exclusive glnA transcript. Analysis of P(glnA)::lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P(1), P(2), or P(3) and P(4) have the ability to promote transcription. Mutation of the NtcA-binding site in P(1) eliminated P(1)-directed transcription and allowed increased use of P(2). The NtcA-binding site in the P(1) promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

Ammonia↗
PubMed2006

Analysis of gene expression in operons of Streptomyces coelicolor.

BACKGROUND: Recent studies have shown that microarray-derived gene-expression data are useful for operon prediction. However, it is apparent that genes within an operon do not conform to the simple notion that they have equal levels of expression. RESULTS: To investigate the relative transcript levels of intra-operonic genes, we have used a Z-score approach to normalize the expression levels of all genes within an operon to expression of the first gene of that operon. Here we demonstrate that there is a general downward trend in expression from the first to the last gene in Streptomyces coelicolor operons, in contrast to what we observe in Escherichia coli. Combining transcription-factor binding-site prediction with the identification of operonic genes that exhibited higher transcript levels than the first gene of the same operon enabled the discovery of putative internal promoters. The presence of transcription terminators and abundance of putative transcriptional control sequences in S. coelicolor operons are also described. CONCLUSION: Here we have demonstrated a polarity of expression in operons of S. coelicolor not seen in E. coli, bringing caution to those that apply operon prediction strategies based on E. coli 'equal-expression' to divergent species. We speculate that this general difference in transcription behavior could reflect the contrasting lifestyles of the two organisms and, in the case of Streptomyces, might also be influenced by its high G+C content genome. Identification of putative internal promoters, previously thought to cause problems in operon prediction strategies, has also been enabled.

Bacterial Proteins↗
PubMed1991

Differential expression of natriuretic peptide genes in cardiac and extracardiac tissues.

Cardiac myocytes secrete a family of natriuretic and diuretic peptides, including atrial natriuretic factor (ANF) and brain natriuretic peptide or the rat hormone isoANF. These peptides share structural and functional similarities, but their respective physiological roles have yet to be elucidated. Differential expression of natriuretic peptide genes may reflect distinct physiological or pathophysiological functions for these peptides. To test this hypothesis, we have determined the sites of expression of the ANF and isoANF genes in rat tissues using polymerase chain reaction amplification of ANF and isoANF transcripts. Like ANF mRNA, isoANF mRNA was detected in all heart compartments, and the transcription initiation sites of the isoANF gene, determined from primer extension experiments, were identical in atria and ventricles. In the adult heart, the ventricular isoANF mRNA concentration is only 3 times lower than in atria, and in sharp contrast to ANF, the isoANF gene is constitutively expressed in ventricles during postnatal development. Since ventricles are at least 20 times larger than atria, this implies that isoANF is mostly a ventricular hormone, whereas ANF is essentially an atrial hormone in normal adult hearts. Low levels of isoANF mRNA were found in few extracardiac tissues, including hypothalamus, brain, lung, and aorta. IsoANF transcripts were more abundant than ANF transcripts in aorta, whereas ANF and isoANF mRNA levels were similar in lung. In brain and hypothalamus, ANF transcripts were only 7- and 2-fold greater than isoANF transcripts. The presence of isoANF transcripts in brain and hypothalamus suggests that isoANF is the rat homolog of the human hormone brain natriuretic peptide. Thus, the expression of these two natriuretic peptide genes is not coordinated, suggesting that ANF and isoANF may play different physiological roles in cardiac and extracardiac tissues.

Animals↗
PubMed1991

Macrophage colony-stimulating factor (CSF-1) gene expression in human T-lymphocyte clones.

Macrophage colony stimulating factor (CSF-1) is one of several cytokines that control the differentiation, survival, and proliferation of monocytes and macrophages. A set of 11 human T-cell clones, chosen for their phenotypic diversity, were tested for their ability to express CSF-1 mRNA. After 5 hours of stimulation with phorbol myristate acetate (PMA) + calcium ionophore (Cal), all T-cell clones expressed a major 4-kb transcript, a less abundant 2-kb transcript, and several other minor species. This pattern of expression is typical for CSF-1 mRNAs. Furthermore, of the two alloreactive T-cell clones analyzed, only one showed a definitive message for CSF-1 on specific antigenic stimulation, but with delayed kinetics and less efficiency. Both conditions of stimulation induced the release of CSF-1 protein by T cells in the culture medium. Together, these findings demonstrate for the first time that normal T cells are able to produce CSF-1, previous reports being limited to two cases of tumoral cells of the T-cell lineage.

Antigens↗
PubMed1993

Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.

Amino Acid Sequence↗
PubMed2007

Temporal expression of transcripts related to embryo quality in bovine embryos cultured from the two-cell to blastocyst stage in vitro or in vivo.

The post-fertilization embryo culture environment can have a dramatic effect on the pattern of gene expression in the embryo and it is widely acknowledged that bovine embryos derived from in vitro culture are of inferior quality to those derived in vivo. The objective of this study was to examine temporal variation in the mRNA abundance of several transcription and translation factors known to differ between blastocysts produced following culture in vitro and in vivo. Embryos were recovered from two in vitro culture systems SOF1 or SOF2 at five developmental stages: 2- to 4-cell, 8-cell, 16-cell, morula, and blastocyst. In vivo embryos were produced from superovulated and artificially inseminated heifers and recovered at approximately 40 hr or 3, 4, 5, and 7 days postinsemination. Blastocysts were also produced following in vitro maturation, in vitro fertilization and culture in the ewe oviduct. Analysis of relative transcript abundance for FOXO3A, EEF1G, HMG2, and REA was performed using quantitative real-time PCR. Irrespective of culture environment each transcript followed, approximately the same general pattern of expression where relative abundance decreased dramatically from the 2- to 4-cell stage to 8-cell stage and increased from the morula to blastocyst stage (P < 0.05). Transcripts for GNBL2 were not observed between the 2- and 16-cell stage of development. Relatively high expression at the 2- to 4-cell indicated that these transcripts are most likely of maternal origin produced in the oocyte during growth and final maturation. A culture-induced change in mRNA abundance of transcription and translation factors was evident in embryos that were produced not only between in vivo and in vitro culture environments but also between different in vitro culture systems.

Animals↗
PubMed2007

Localization and comparative analysis of acid-sensing ion channel (ASIC1, 2, and 3) mRNA expression in mouse colonic sensory neurons within thoracolumbar dorsal root ganglia.

Reducing colonic mechanosensitivity is an important potential strategy for reducing visceral pain. Mice lacking acid-sensing ion channels (ASIC) 1, 2, and 3 show altered colonic mechanosensory function, implicating ASICs in the mechanotransduction process. Deletion of ASICs affects mechanotransduction in visceral and cutaneous afferents differently, suggesting differential expression. We determined relative expression of ASIC1, 2, and 3 in mouse thoracolumbar dorsal root ganglia (DRG) by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis (QPCR) and specifically in retrogradely traced colonic neurons isolated via laser capture microdissection. Localization of ASIC expression in DRG was determined with fluorescence in situ hybridization (FISH) and retrograde tracing. QPCR of whole thoracolumbar DRG revealed and abundance of ASIC2 > ASIC1 > ASIC3. Similarly, FISH of all neurons in thoracolumbar DRG demonstrated that ASIC2 was expressed in the most (40 +/- 1%) neurons, followed by ASIC3 (24 +/- 1%), then ASIC1 (18 +/- 1%). Retrograde tracing from the distal colon labeled 4 +/- 1% of neurons in T10-L1 DRG. In contrast to whole DRG, FISH of colonic neurons showed ASIC3 expression in 73 +/- 2%, ASIC2 in 47 +/- 0.5%, and ASIC1 in 30 +/- 2%. QPCR of laser captured colonic neurons revealed that ASIC3 was the most abundant ASIC transcript, followed by ASIC1, then ASIC2. We conclude that ASIC1, 2, and 3 are expressed preferentially in colonic neurons within thoracolumbar DRG. In particular ASIC3, the least abundant in the general population, is the most abundant ASIC transcript in colonic neurons. The prevalence of ASIC3 in neurons innervating the colon supports electrophysiological data showing that it makes a major contribution to colonic mechanotransduction and therefore may be a target for the treatment of visceral pain.

Acid Sensing Ion Channels↗
PubMed2005

Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plants.

Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species.

ATP Phosphoribosyltransferase↗
PubMed1993

Coexpression of multiple acetylcholine receptor genes in neurons: quantification of transcripts during development.

A large family of genes encoding subunits of nicotinic ACh receptors (AChRs) has been identified in vertebrates and shown to be expressed in the nervous system. The multiplicity of genes raises questions about which gene products coassemble to produce native receptor subtypes and how the expression of receptor genes is regulated in neurons. We report here that five neuronal AChR genes are expressed in the chick ciliary ganglion at both early and late times in development. Quantitative RNase protection experiments demonstrated that at embryonic day 18 (E18) the ganglion contains about 1800 copies of alpha 7 transcript per neuron, 900 copies of alpha 3 transcript per neuron, and 200-300 copies each of alpha 5, beta 2, and beta 4 transcripts per neuron. The same five genes are expressed at significantly lower levels at E8 but show the same rank order of abundance in transcripts per neuron. Few, if any, transcripts were found for the alpha 2, alpha 4, alpha 8, and beta 3 AChR genes in ciliary ganglion RNA at either E8 or E18. The 6- and 13-fold increases previously reported for two classes of AChRs on the neurons between E8 and E18 approximate the 4-14-fold increases observed here in AChR gene mRNA levels per neuron over the same time period. The alpha 3, alpha 5, alpha 7, and beta 4 genes have previously been correlated with subunits of ciliary ganglion AChRs, but the beta 2 gene has not. The abundance of beta 2 transcripts raises the possibility either that the known AChRs in the ganglion have a more complex subunit composition than previously described or that additional receptor subtypes remain to be discovered. Northern blot analysis revealed no changes in transcript pattern for the alpha 3, alpha 5, and beta 4 genes between E8 and E18; a small change may occur in the transcript pattern for the alpha 7 gene. In situ hybridizations demonstrated that alpha 5 and beta 4 transcripts are expressed in essentially all ciliary ganglion neurons as has been shown previously for the more abundant alpha 3 transcript and inferred for the alpha 7 transcript. The results indicate that neurons can stably coexpress multiple AChR genes, including three of the alpha type, and that transcript levels may be rate limiting for accumulation of AChRs during development.

Animals↗
PubMed2002

A 3'-UTR variant of the inducible porcine hsp70.2 gene affects mRNA stability.

Large individual differences were observed in the abundance of transcripts from the hsp70.2 gene in primary fibroblast cultures sampled from 15 different pigs. While previously described functional promoter variants of this gene can partly account for the high variability of heat-induced increased abundance of transcripts, they are unrelated to the observed highly variable absolute amounts of hsp70.2 transcripts. Comparative sequence analysis revealed an alteration of the 3'-untranslated region (3'-UTR) sequences in these samples. The variant 3'-UTR allele proved to increase the half life of the hsp70.2 mRNA in reporter gene assays. It is suggested that the cellular stress response is significantly affected by the action and interaction of both promoter and 3'-UTR variants of the hsp70.2 gene.

3' Untranslated Regions↗