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PubMed1987

Different transcript abundance of two divergent ATP synthase subunit 9 genes in the mitochondrial genome of Petunia hybrida.

The mitochondrial genome of Petunia hybrida line 3704 has been found to contain two transcribed genes for the proteolipid subunit of the ATP synthase complex (atp 9). The 5' and 3' flanking sequences of the second atp 9 gene differ from those of another atp 9 gene previously sequenced by Young et al. (1986), while the coding region exhibits only one silent base change. The transcript termini of the two divergent atp 9 genes map to different locations, most of which are surrounded by sequences homologous to putative mitochondrial transcription signals (Hiesel and Brennicke 1985; Schuster et al. 1986; Young et al. (1986). Because transcripts from the atp 9 genes differ in abundance, divergence in the flanking regions of these two genes must affect regulatory elements which control either transcription rate or transcript stability.

Amino Acid Sequence↗
PubMed1988

Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. An abundant transcript induced by transformation of fibroblasts.

Transfection of an activated rat oncogene into NIH3T3 fibroblasts leads to transformation and induction of a metastatic phenotype. To identify genes whose activation might mediate these processes, we used a differential screening strategy. A 1.5-kb transcript is induced fiftyfold, constitutes 1% of ras transformed cell messenger RNA (mRNA) and is the most abundantly induced message in these cells. Our sequence data shows that it encodes murine cathepsin L, a potent collagenolytic and elastinolytic lysosomal enzyme. The murine clone was used to isolate human cathepsin L complementary DNA (cDNA) clones. The complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L are presented and compared to other papain family cysteine proteinases. Northern analysis shows that both human and murine cathepsin L probes hybridize to a 1.5-kb transcript in several tissues, but also to a 4-kb transcript in human kidney. These clones will facilitate studies of the structure, expression, and function of cathepsin L, including its unexpected upregulation in transformation.

Amino Acid Sequence↗
PubMed1990

Measurement of histone mRNA transcript abundance in Xenopus oocytes by a quantitative primer extension assay.

A quantitative primer extension method was used to measure the mass of histone gene transcripts in mature oocytes of the amphibian Xenopus laevis. The procedure, using a large excess of gene-specific oligonucleotide primer and continuous incorporation of a radiolabeled deoxynucleoside triphosphate precursor, is more sensitive and quantitative than primer extension assays employing end-labeled primers. It was determined that there are stoichiometric amounts, approximately 2 X 10(8) copies, of mRNA for each of the five major histone gene classes in mature Xenopus oocytes. These observations are consistent with a model whereby transcription of these genes is coordinately regulated in a cell cycle-independent manner during amphibian oogenesis.

Animals↗
PubMed2006

Abundant transcripts from retrotransposons are unstable in fully grown mouse oocytes.

One physiological function proposed for RNA interference (RNAi) is to constrain expression of repetitive elements and thereby reduce the incidence of retrotransposition. Consistent with this model is that inhibiting the RNAi pathway results in an increase in expression of repetitive elements in preimplantation mouse embryos. Mouse oocytes are essentially transcriptionally quiescent providing a unique opportunity to assess the stability of repetitive element-derived transcripts in these cells. We compared the transcriptome of freshly isolated fully grown germinal vesicle (GV)-intact oocytes to that of oocytes in which meiotic maturation in vitro was inhibited for 48 h by milrinone. Consistent with the aforementioned function for RNAi is that the abundance of only a relatively small number of transcripts decreased in the cultured oocytes, when compared to changes that occur during maturation or following fertilization, and of those, several belonged to mobile elements.

Animals↗
PubMedDate not supplied

Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley.

Responses to drought and salinity in barley (Hordeum vulgare L. cv. Tokak) were monitored by microarray hybridization of 1463 DNA elements derived from cDNA libraries of 6 and 10 h drought-stressed plants. Functional identities indicated that many cDNAs in these libraries were associated with drought stress. About 38% of the transcripts were novel and functionally unknown. Hybridization experiments were analyzed for drought- and salinity-regulated sequences, with significant changes defined as a deviation from the control exceeding 2.5-fold. Responses of transcripts showed stress-dependent expression patterns and time courses. Nearly 15% of all transcripts were either up- or down-regulated under drought stress, while NaCl led to a change in 5% of the transcripts (24 h, 150 mM NaCl). Transcripts that showed significant up-regulation under drought stress are exemplified by jasmonate-responsive, metallothionein-like, late-embryogenesis-abundant (LEA) and ABA-responsive proteins. Most drastic down-regulation in a category was observed for photosynthesis-related functions. Up-regulation under both drought and salt stress was restricted to ESTs for metallothionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress. A number of functionally unknown transcripts from cDNA libraries of drought-stressed plants showed up-regulation by drought but down-regulation by salt stress, documenting how precisely transcript profiles report different growth conditions and environments.

Disasters↗
PubMed2006

Regional variation in mRNA transcript abundance within the ventricular wall.

Previous studies have shown that regional variation in ion channel gene expression contributes to electrical heterogeneity within the walls of the cardiac ventricles. To map the extent of regional variation in gene expression in the ventricular walls and to begin to understand its genesis we have performed a microarray analysis of gene expression in the epicardial and endocardial tissues of the rat adult left ventricle. While the vast majority of the genes are expressed at uniform levels across the ventricular wall, a total of 36 transcripts (representing less than 0.1% of the genes expressed in the ventricle) are expressed more abundantly in either epicardium or endocardium. One of these differentially expressed genes is the sodium channel gene Scn5a, which is expressed at higher levels in the endocardium than in the epicardium of rat heart. The transcription factor genes Irx3, Irx5 and Etv1 were found to be expressed in transmural gradients across the ventricular wall of rat heart and also of canine heart. The Irx3 and Irx5 genes were expressed in an inverse pattern to that of the Kcnd2 (Kv4.2) gene in rat heart, suggesting that these transcription factors may act as negative regulators of Kcnd2 expression in vivo.

Animals↗
PubMed1991

Isolation and characterization of cDNA clones for NADP-malic enzyme from leaves of Flaveria: transcript abundance distinguishes C3, C3-C4 and C4 photosynthetic types.

To study the control of enhanced synthesis of enzymes associated with C4 photosynthesis relative to non-C4 plants, we investigated the expression of NADP-malic enzyme (NADP-ME) in different photosynthetic types of Flaveria. Complementary DNA clones encoding NADP-ME were constructed using poly(A)+ RNA from leaves of Flaveria trinervia (C4) and F. linearis (C3-C4) and identified by homology to a cDNA clone (500 bp) encoding NADP-ME from maize (Zea mays L. [39]). The sequence of one clone from each species was determined. The Flaveria clones were 90% homologous over a 564 nucleotide region encoding the carboxy terminal end of the derived polypeptide; sequence similarity to the maize transcript in this region was 71%. Both Flaveria clones detected a 2/3 kb transcript by hybridization to poly(A)+ RNA from expanding leaves of F. trinervia, F. linearis and F. pringlei (C3). The level of transcripts paralleled previously observed NADP-ME activity and abundance differences determined in these species, suggesting that control of the expression of NADP-ME in different photosynthetic types is predominantly at the transcriptional/post-transcriptional level. Southern analysis of genomic DNAs from F. trinervia, F. linearis and F. pringlei indicated a low copy number for this gene in all three species.

Amino Acid Sequence↗
PubMed1991

Glycerol 3-phosphate dehydrogenase gene expression in cultured 3T3-L1 adipocytes: regulation by insulin, dexamethasone and dibutyryl cAMP at the level of mRNA abundance, transcription and mRNA stability.

In fully differentiated 3T3-L1 adipocytes, glycerol 3-phosphate dehydrogenase (G3PDH:Sn-glycerol 3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) is subject to regulation by hormones and dibutyryl cAMP. An increase by insulin (4-fold) and decrease by dexamethasone (by 50%) and dibutyryl cAMP (by 70%) was observed for G3PDH mRNA abundance as analyzed by Northern blot hybridization. In addition, incubation of adipocytes with dibutyryl cAMP resulted in 3-fold increase in G3PDH gene transcription as measured by nuclear transcript elongation assay. The effects of these modulators on the control of G3PDH mRNA stability were also investigated. The G3PDH mRNA has a half-life of about 125 min. Dibutyryl cAMP caused an increase in G3PDH mRNA degradation by greater than 2-fold (t1/2 = 55 min) whereas insulin had an opposite effect (t1/2 = 240 min) and dexamethasone was without any effect on G3PDH mRNA stability. Taken together, our results directly demonstrate that in fully differentiated 3T3-L1 adipocytes the regulation of G3PDH gene expression by dibutyryl cAMP and insulin is exerted by alterations in transcription as well as mRNA stability.

3T3 Cells↗
PubMed2005

Profiling of diurnal patterns of metabolite and transcript abundance in potato (Solanum tuberosum) leaves.

Diurnal changes in carbohydrates and a broad range of primary metabolites were analysed through a diurnal period in potato leaves (Solanum tuberosum cv. Desiree) using an established gas chromatography-mass spectrometry based metabolic profiling protocol alongside conventional spectrophotometric technologies. In tandem, we profiled transcript levels using both a custom array containing approximately 2,500 cDNA clones predominantly representing transcripts involved in primary metabolism and commercially available arrays containing approximately 12,000 cDNA clones that gave coverage of transcript levels over a broader functional range. The levels of many metabolites and transcripts varied during the diurnal period with 56 significant differences observed in the metabolite contents and 832 significant differences recorded in transcript levels. Whilst a large number of the differences would be expected from what has been known previously, several novel changes were observed in these experiments. Notably, qualitative comparison of the combined data sets obtained from the parallel analysis of transcripts and metabolites suggests relatively few changes in gene expression strongly correlate with changes in metabolite levels during a diurnal cycle. Furthermore, these changes appear to be confined to the central metabolic pathways. However, principal component analysis of the metabolic profiles obtained here revealed that metabolite patterns change progressively through a diurnal period suggesting the operation of mechanisms for tight temporal regulation of metabolite composition.

Amino Acids↗
PubMed1997

Expression cloning of a rat testicular transcript abundant in germ cells, which contains two leucine zipper motifs.

The aim of the present study was to identify specific, novel germ cell markers that could be used to monitor normal and abnormal spermatogenesis. Of several cloned cDNAs isolated from an adult rat testis cDNA library using an expression screening strategy, clone 813B4 (700 base pairs) hybridized exclusively to three mRNA transcripts in samples isolated from rat testes on and after Day 21 of life and to epididymides from some, but not all, adult rats. After further screening, two identical clones encoding a 2.2-kilobase cDNA (KTT4) were isolated and found to contain an open reading frame of 578 amino acids including two leucine zipper motifs. On Northern blots, KTT4 mRNA was abundant in samples from round spermatids, and homologous mRNAs were present in testes from mice and marmosets. A zoo blot revealed that the KTT4 gene is conserved in humans, monkeys, mice, dogs, and cattle. On sections of rat testes, KTT4 mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter was abundant in round and elongating spermatids until step 15. Expression of KTT4 was not altered by ethane dimethane sulphonate-induced androgen withdrawal, but in rats treated 14 days previously with methoxyacetic acid, a marked reduction in KTT4 was noted associated with the depletion of round spermatids. In conclusion, the present study identified a conserved gene expressed in meiotic and post-meiotic germ cells; database searches have shown it to be homologous to recently published sequences for an outer dense fiber protein of the sperm tail (Odf2/Odf84).

Amino Acid Sequence↗
PubMed2004

Transcript abundance of rml1, encoding a putative GT1-like factor in rice, is up-regulated by Magnaporthe grisea and down-regulated by light.

We isolated and sequenced both genomic DNA and cDNA clones, which encoded a putative GT1-like protein with 385 amino acids, from cultivated rice (Oryza sativa ssp. indica). This protein shows significant amino acid sequence similarities with trihelix DNA-binding GT-1a/B2F and GT-1 factors that were identified in dicot plants. Northern blotting analysis indicated that the transcript of the rice GT-1 factor in seedling was up-regulated by the rice blast fungus Magnaporthe grisea, down-regulated by various continuous light conditions and expressed rhythmically in light/dark cycles. This GT1-like factor gene was therefore designated as rml1 (rice gene regulated by M. grisea and light). The putative RML1 protein, encoded by this single copy gene, is thus identified as a new member of the plant-specific GT family of transcription factors in rice.

Amino Acid Sequence↗
PubMed2006

Cloning and characterization of a cDNA encoding cholesterol side-chain cleavage cytochrome P450 (CYP11A1): tissue-distribution and changes in the transcript abundance in ovarian tissue of Japanese eel, Anguilla japonica, during artificially induced sexual development.

Cholesterol side-chain cleavage cytochrome P450 (CYP11A1: P450scc) is a crucial steroidogenic enzyme that catalyzes an initial step in the production of all classes of steroids. A cDNA encoding Japanese eel P450scc was cloned and characterized. The cDNA putatively encoded 521 amino acid residues with high homology to those of other vertebrate forms. The recombinant P450scc produced in COS-7 cells efficiently catalyzed the conversion of 25-hydroxycholesterol into pregnenolone. By northern blot, a single P450scc transcript of approximately 3.3 kb was detected in both ovary and head kidney. Transcript levels of this enzyme significantly increased throughout ovarian development artificially induced by salmon pituitary homogenate, which suggests that gonadotropic stimuli can induce ovarian expression of the P450scc gene in teleosts, as has been reported in mammals. Furthermore, RT-PCR analysis revealed that gene expression of three steroidogenic enzymes, P450scc, P450c17 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) show distinctly different tissue-specific patterns of expression in the Japanese eel. The P450scc gene was expressed in ovary and head kidney while the sole source of the P450c17 transcript was ovary. In contrast, 3beta-HSD transcript was detected in all tissues examined, brain, liver, spleen and trunk kidney, etc. These suggest that some steroidogenic enzymes are also expressed in non-endocrine tissues and could potentially regulate the local and/or circulating steroid levels in teleosts, as they do in mammals.

3-Hydroxysteroid Dehydrogenases↗
PubMed2004

Variation in transcript abundance during somatic embryogenesis in gymnosperms.

Somatic embryogenesis of Norway spruce (Picea abies L.) is a versatile model system to study molecular mechanisms regulating embryo development because it proceeds through defined developmental stages corresponding to specific culture treatments. Normal embryonic development involves early differentiation of proembryogenic masses (PEMs) into somatic embryos, followed by early and late embryogeny leading to the formation of mature cotyledonary embryos. In some cell lines there is a developmental arrest at the PEM-somatic embryo transition. To learn more about the molecular mechanisms regulating embryogenesis, we compared the transcript profiles of two normal lines and one developmentally arrested line. Ribonucleic acid, extracted from these cell lines at successive developmental stages, was analyzed on DNA microarrays containing 2178 expressed sequence tags (ESTs) (corresponding to 2110 unique cDNAs) from loblolly pine (Pinus taeda L.). Hybridization between spruce and pine species on microarrays has been shown to be effective (van Zyl et al. 2002, Stasolla et al. 2003). In contrast to the developmentally arrested line, the early phases of normal embryo development are characterized by a precise pattern of gene expression, i.e., repression followed by induction. Comparison of transcript levels between successive stages of embryogenesis allowed us to identify several genes that showed unique expression responses during normal development. Several of these genes encode proteins involved in detoxification processes, methionine synthesis and utilization, and carbohydrate metabolism. The potential role of these genes in embryo development is discussed.

Cycadopsida↗
PubMed2004

The nuclear factor HCF145 affects chloroplast psaA-psaB-rps14 transcript abundance in Arabidopsis thaliana.

The high chlorophyll fluorescence (hcf)145 mutant of Arabidopsis thaliana is specifically affected in photosystem (PS)I function as judged from spectroscopic analysis of PSII and PSI activity. The defect is because of a severe deficiency of PSI core subunits, whereas levels of the four outer antenna subunits of PSI were less reduced in hcf145. Pulse labelling of chloroplast proteins indicated that synthesis of the two largest PSI reaction-centre polypeptides, Psa (photosystem I subunit) A and PsaB, is significantly affected by the mutation. A comparison of stationary transcript levels with rates of transcription demonstrates that hcf145 induces a decreased stability and, probably, transcription of the tricistronic psaA-psaB-rps (small-subunit ribosomal protein)14 mRNA, which is generated by the plastid-encoded RNA polymerase. Translation inhibition experiments excluded translational defects as primary cause of impaired mRNA stability. Larger primary transcripts, which also contain sequences of the ycf3 (hypothetical chloroplast reading frame) gene located upstream of the psaA-psaB-rps14 operon and generated by the action of the nuclear-encoded RNA polymerase, are not targeted by the mutation. Real-time reverse transcription (RT)-PCR analysis has successfully been applied to quantify defined intervals of the tricistronic transcript and it was established that the psaA region is less stable than the rps14 region in hcf145. The hcf145 gene has been mapped on the upper part of chromosome 5.

Arabidopsis↗
PubMed2000

Analysis of expressed sequence tags of retinal pigment epithelium: cystatin C is an abundant transcript.

In order to identify genes that are expressed in the retinal pigment epithelium (RPE), randomly chosen clones of a cDNA library of cultured human foetal RPE cells were analyzed by sequencing. Of 164 informative expressed sequence tags (ESTs), 88 matched the sequences of 74 genes for proteins of known or presumed function. Approximately a third of these represented genes with involvement in gene/protein expression, with a major subcategory concerned with protein turnover. In particular, the gene coding for precursor cystatin C was represented by 3 independent ESTs, and plaque hybridization estimated the frequency of cystatin C clones in the library to be 1.3%. Cystatin C mRNA in cultured RPE cells was confirmed by Northern blotting and by reverse transcription polymerase chain reaction (RT-PCR) with identification of the cystatin C sequence as the product of the reaction. The survey also revealed 25 novel human sequences representing genes that are active in RPE. One of these was localized near a recently identified, new autosomal recessive retinitis pigmentosa locus. In conclusion, the findings specifically demonstrate the unexpected presence of cystatin C mRNA at fairly high abundance in cultured human RPE cells, and, more generally, serve as a model study establishing the usefulness of the EST approach for further characterizing the molecular basis of the activities of the RPE.

Cells, Cultured↗
PubMed2006

Two serine proteases from Anopheles dirus haemocytes exhibit changes in transcript abundance after infection of an incompatible rodent malaria parasite, Plasmodium yoelii.

Serine proteases are involved in regulation of innate immune responses, such as haemolymph coagulation, melanization reaction and antimicrobial peptide synthesis. Although several serine proteases have been characterized in Anopheles gambiae (A. gambiae), few were cloned from other malaria vectors. In this study, we identified three cDNA fragments of serine proteases (AdSp1, AdSp2 and AdSp3) from haemocytes of an oriental malaria vector, Anopheles dirus (A. dirus), by cloning of fragments amplified with degenerate primers into the T-vector. RT-PCR analysis demonstrated that both AdSp1 and AdSp3 genes were also expressed in salivary gland. Basic local alignment search tool (BLAST) search found that both AdSp1 and AdSp3 were highly similar in sequence to A. gambiae Sp14A and Sp14D2, insects prophenoloxidase activating enzyme (PPAE) and Drosophila protease easter. Semi-quantitative RT-PCR indicated the transcription level of both AdSp1 and AdSp3 in haemocytes of A. dirus infected with Plasmodium yoelii (P. yoelii) was significant higher than that fed on 5% glucose or normal mouse blood at 7 days after the infectious meal (p<0.05), when P. yoelii oocysts began to be melanized by A. dirus. Our results indicated that both AdSp1 and AdSp3 might play an important role during melanotic encapsulation of P. yoelii by A. dirus.

Animals↗
PubMed2001

Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental development, promoter sequences of genes and biological activity of protein products.

Multiple interferon (IFN)-tau genes exist in cattle, but it has remained unclear how many are expressed, the extent of their variation, and whether different genes exhibit similar patterns of expression and code for proteins with similar biological activities. A total of 118 complementary DNA (cDNA) were bi-directionally sequenced from reverse-transcribed bovine (bo) conceptus RNA over the period from blastocyst formation until day 25 of pregnancy. Fourteen different cDNAs, encoding eight different IFN-tau, were confirmed unique. All showed high sequence conservation (>98% nucleotide identity; >96% amino acid identity). The cDNA fell into three, recently evolved, phylogenetic groups (tau1, 2, and 3). Mean concentrations of IFN-tau messenger RNA were greater at day 17 and day 19 than at day 14 and day 25, with different genes showing comparable expression patterns, although there appeared to be a major bias in expression of two genes (for boIFN-tau1c and tau3a) in blastocysts. Genes representing members of the three boIFN-tau groups were cloned. Their promoter regions were conserved over regions considered important for transcriptional activation. Recombinant protein generated in Escherichia coli from representative genes in the three groups had similar but not identical antiviral activities. In summary, many IFN-tau genes, which are probably under similar transcriptional control, are expressed in bovine trophoblast during the peri-implantation period of development.

Amino Acid Sequence↗
PubMed2005

The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promotes high-level expression by increasing transcript abundance and translation efficiency.

The involvement of regions located upstream of the translation start site in the expression of two Arabidopsis thaliana nuclear COX5c genes encoding subunit 5c of mitochondrial cytochrome c oxidase has been analysed. It was observed that these regions, which include a leader intron, direct the tissue-specific expression of the gus reporter gene, mainly in root and shoot meristems, actively growing tissues and vascular strands. Expression was also observed in flowers, specifically localized in anthers, stigma, and the receptacle, and in developing seeds. GUS activity measurements in protein extracts from transformed plants indicated that expression levels are higher than those observed with the constitutive CaMV 35S promoter. Removal of the leader intron produced a significant decrease in expression to values only slightly higher than those observed with a promoterless gus gene. Histochemical staining of plants transformed with the intronless construct revealed expression only in pollen, suggesting that regulatory elements capable of directing pollen-specific expression are present upstream of the intron. The COX5c-2 intron also increased GUS expression levels when fused in the correct orientation with the promoter of the unrelated COX5b-1 gene. Comparison of GUS activity values with the transcript levels suggests that the intron also increases translation efficiency of the corresponding mRNA. The results obtained point to an essential role of the intron present in the 5'-non-coding region of all known COX5c genes in directing the expression of these genes in plants.

5' Untranslated Regions↗