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PubMed1999

Organisation of the mouse and human 5T4 oncofoetal leucine-rich glycoprotein genes and expression in foetal and adult murine tissues.

The human 5T4 oncotrophoblast leucine-rich glycoprotein may contribute to the process of placentation or metastasis by modulating cell adhesion, shape and motility. To understand better the role of 5T4 in development and cancer, the gene structure has been elucidated from both human and mouse genomic clones and mRNA expression has been studied in foetal and adult mouse tissues. The protein coding region is located within the second of two exons, the first exon comprising solely of 5'-untranslated region. Upstream there are no TATA or CAAT boxes, but there are a number of potential Sp1 binding sites. The murine and human proteins show a homologous domain organisation of the leucine rich repeats (LRR) and associated N- and C-terminal flanking regions, although the hydrophilic sequence which intervenes between the two LRR domains contains six additional amino acids in the mouse. The signal peptide, transmembrane region and cytoplasmic tail sequences are identical as are 6 out of the 7 potential N-linked glycosylation sites. Mouse 5T4 transcripts are abundant in placenta and also highly expressed in embryos while in adult tissues transcripts are restricted to brain and ovary. These patterns of expression and the genomic organisation are discussed in relation to possible function and other recently described LRR containing proteins.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

3' Untranslated Regions↗
PubMed1998

Molecular cloning of cDNAs and expression of mRNAs encoding alpha and beta subunits of soluble guanylyl cyclase from medaka fish Oryzias latipes.

Two cDNA clones encoding alpha and beta subunits of soluble guanylyl cyclase (GC) were isolated from medaka fish (Oryzias latipes) cDNA libraries and designated as OlGCS-alpha1 and OlGCS-beta1, respectively. The ORF of OlGCS-alpha1 encodes 678 amino acids and that of OlGCS-beta1 encodes 614 amino acids. Both subunits have a putative catalytic domain conserved with membrane GC and adenylyl cyclases. The amino acid sequences of OlGCS-beta1 and mammalian soluble GC subunits are highly conserved throughout the entire protein, suggesting that amino acid sequences outside the catalytic domain are important for the functioning of the beta subunit. On the contrary, amino acid sequences of alpha subunits outside the catalytic domain are fairly divergent between fish and mammals. Reverse transcription/PCR (RT/PCR) analysis showed that OlGCS-alpha1 and OlGCS-beta1 transcripts were abundant in the brain, eye, spleen and testis. RT/PCR analysis demonstrated that both transcripts were present in unfertilized eggs, were reduced immediately after fertilization, then increased again. In addition, our results suggest that expression patterns of OlGCS-alpha1 and OlGCS-beta1 genes are different from each other both during development and in adults.

Amino Acid Sequence↗
PubMed1989

Unusual behavior of the cytoplasmic transcript of hsr omega: an abundant, stress-inducible RNA that is translated but yields no detectable protein product.

Although a major site of transcription in heat shock, the Drosophila hsr omega gene does not encode any known heat shock proteins. Instead, studies of the hsr omega transcripts suggest that the RNA molecules, rather than encoded proteins, are the active products of this gene. The cytoplasmic RNA, omega 3, is spliced and polyadenylated and yet has only very small open reading frames (ORFs), and these are poorly conserved in different Drosophila species. Surprisingly, the work reported here leads us to conclude that one of the tiny ORFs in this RNA is translated. This ORF, designated ORF-omega, is notable in being the only ORF that shows sequence conservation in the three Drosophila species examined. However, translation of this ORF does not lead to detectable accumulation of the protein product. We suggest that ORF-omega may be an example of an unusual type of translated ORF. The act of translation itself may be important rather than the generation of a functional protein product. This nonproductive translation may play a role in regulation of cellular activities.

Animals↗
PubMed1995

Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.

Journal Article↗
PubMed1996

Differential display protocol with selected primers that preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.

A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.

Animals↗
PubMed1987

Sequences controlling histone H4 mRNA abundance.

The post-transcriptional regulation of histone mRNA abundance is manifest both by accumulation of histone mRNA during the S phase, and by the rapid degradation of mature histone mRNA following the inhibition of DNA synthesis. We have constructed a comprehensive series of substitution mutants within a human H4 histone gene, introduced them into the mouse L cell genome, and analyzed their effects on the post-transcriptional control of the H4 mRNA. Our results demonstrate that most of the H4 mRNA is dispensable for proper regulation of histone mRNA abundance. However, recognition of the 3' terminus of the mature H4 mRNA is critically important for regulating its cytoplasmic half-life. Thus, this region of the mRNA functions both in the nucleus as a signal for proper processing of the mRNA terminus, and in the cytoplasm as an essential element in the control of mRNA stability.

Animals↗
PubMed1985

Regulation of T-cell receptor gene expression in human T-cell development.

A cDNA clone encoding the alpha chain of the human T-cell antigen receptor was isolated by screening a library from the human T-cell line Jurkat with a mouse alpha-chain cDNA clone. This human alpha-chain clone, together with a human antigen receptor beta-chain cDNA clone, was used to determine the stage of T-cell development at which antigen receptor mRNAs first appear. Blot-hybridization analysis of mRNA isolated from a panel of human thymic tumor lines clearly demonstrated that beta-chain transcripts could be detected in all T-lineage cells. However, alpha-chain transcripts were only found in the most phenotypically mature lines, which express the antigen receptor-associated molecule T3. Furthermore, beta-chain transcripts were abundant in RNA prepared from purified T3-negative thymocytes, whereas alpha-chain transcripts were virtually absent. From these results we conclude that alpha-chain expression occurs later in thymic ontogeny than that of the beta chain and propose that it controls surface expression of the antigen receptor-T3 complex.

Amino Acid Sequence↗
PubMed1991

polo encodes a protein kinase homolog required for mitosis in Drosophila.

We show that mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed; normal bipolar spindles with polyploid complements of chromosomes; bipolar spindles in which one pole can be unusually broad; and monopolar spindles. We have cloned the polo gene from a mutant allele carrying a P-element transposon and sequenced cDNAs corresponding to transcripts of the wild-type locus. The sequence shows that polo encodes a 577-amino-acid protein with an amino-terminal domain homologous to a serine-threonine protein kinase. polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos but are specifically concentrated in dividing cells in larval discs and brains. In the cell cycles of both syncytial and cellularized embryos, the polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic.

Amino Acid Sequence↗
PubMed1996

Cellular distribution of CD44 gene transcripts in colorectal carcinomas and in normal colonic mucosa.

AIM: To study the cellular distribution of CD44 mRNA transcripts in tissue sections of colorectal cancer and corresponding normal colonic mucosa in order to correlate the findings with information from immunohistochemical methods and previous data from analysis by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: In situ hybridisation (ISH) analysis of CD44 standard (CD44s) and variant (CD44v) mRNA in cryostat sections of normal and neoplastic colonic mucosa with 35S-labelled riboprobes. Immunohistochemistry was performed on cryostat sections from the same patients using monoclonal antibodies directed against epitopes encoded by CD44 exon 1 (F.10.44.2), exon 5, (Hermes 3), exon 7 (23.6.1), and exon 11 (2F10). RESULTS: CD44s and CD44v transcripts were both strikingly increased in carcinomas compared with corresponding normal mucosa and the abundant CD44v transcripts in tumour tissues were localised exclusively in the cancer cells. CD44s transcripts were present in cancer, inflammatory and resident stromal cells, but the relative amount in carcinoma cells was greater. Immunohistochemical staining broadly paralleled these results, but some clumps of tumour cells showed clear heterogeneity with regard to CD44 protein content. There were also some scattered focal discrepancies in the quantity and distribution of mRNA transcripts and proteins, respectively. CONCLUSIONS: The ISH technique provides powerful independent corroboration of elevated CD44 gene expression and disproportionately high transcription of CD44v isoforms in carcinoma cells observed in earlier immunohistochemical and RT-PCR studies. It unequivocally localises the abnormally elevated gene transcription within the cancer cells and not in the surrounding inflammatory cells.

Antigens, Neoplasm↗
PubMed1996

Expression of retinoic acid receptor beta in human renal cell carcinomas correlates with sensitivity to the antiproliferative effects of 13-cis-retinoic acid.

The differentiation and growth suppressive effects of retinoic acid are mediated through retinoic acid nuclear receptors (RARs and RXRs), which are ligand-activated transcription factors. Recent data suggest that both altered and regulated expression of RARs are linked to retinoic acid response in a cell context-dependent manner. This study examined the antiproliferative effects of 13-cis-retinoic acid (cRA) on 12 renal cancer cell lines and correlated these findings with the basal and induced expression of RAR-alpha, -beta and -gamma. Eleven of 12 renal cancers that were either resistant to or only minimally inhibited by cRA did not basally express RAR-beta as determined by Northern blot analysis. In these cells, cRA treatment did not induce RAR-beta expression. In contrast, 1 of 12 cell lines (SK-RC-06) was >90% inhibited by cRA and basally expressed RARbeta. Furthermore, RAR-beta mRNA in SK-RC-06 cells was up-regulated by cRA treatment. Amplification of cDNA using PCR and RAR-beta isoform-specific primer pairs revealed that only SK-RC-06 cells expressed the RAR-beta1 isoform. Expression of RAR-alpha transcripts was abundant in all 12 cell lines examined, whereas low levels of RAR-gamma transcripts were detectable in 6 of 10 renal cancers. Expression of RAR-alpha and RAR-gamma was not affected by cRA. These data showing that the majority of renal cancer cell lines are resistant to cRA suggest that: (a) resistance to the antiproliferative action of cRA correlates with repressed RAR-beta mRNA expression; and (b) the antiproliferative effects of cRA in renal cancer cells are mediated through RAR-beta1.

Antineoplastic Agents↗
PubMed2003

Quantification and glucocorticoid regulation of glucocorticoid receptor transcripts in two human leukemic cell lines.

We have quantified the basal and glucocorticoid-regulated levels of different transcripts from the human glucocorticoid receptor (GR) gene in the T-cell acute lymphoblastic leukemia cell line, CEM-C7, and in the B lymphoblastoid cell line, IM-9. Highly specific quantitative, reverse transcription-polymerase chain reaction assays measured total GR transcripts, transcripts encoding the isoforms glucocorticoid receptor alpha (GRalpha) and glucocorticoid receptor beta (GRbeta), and transcripts containing different forms of exon 1: 1A1, 1A2, 1A3, 1B, and 1C. GRalpha and GRbeta transcripts are coordinately upregulated in CEM-C7 cells and coordinately downregulated in IM-9 cells by dexamethasone. The concentration of GRalpha mRNA is more than a 1000-fold higher than that for GRbeta mRNA. Transcripts with different exon 1 forms are all upregulated in CEM-C7 cells and all downregulated in IM-9 cells by dexamethasone, but transcripts containing exons 1A1, 1A2, or 1A3 are regulated to a higher degree than transcripts containing exon 1B or exon 1C. However, exon 1B- and exon 1C-containing transcripts are substantially more abundant than exon 1A-containing transcripts, with exon 1A3-containing transcripts more abundant than exon 1A1- or exon 1A2-containing transcripts. Analysis using models for glucocorticoid receptor autoregulation kinetics suggests that the minor 1A3-containing transcript component could be important for GR protein upregulation, and hence apoptosis, in CEM-C7 cells. These studies suggest that GRalpha transcripts containing exons 1A3, 1B, and 1C contribute most to the intracellular level of GR mRNA and may be the most relevant for steroid-mediated apoptosis in T-lymphoblasts.

Blotting, Western↗
PubMed1997

Regulation of luteinizing hormone-receptor and follicle-stimulating hormone-receptor messenger ribonucleic acid levels during development in the neonatal mouse ovary.

Normal gonadal development is dependent upon stimulation by the gonadotropic hormones, and it is likely that control of ovarian development is regulated by expression of gonadotropin receptors. In this study, quantitative changes in LH- and FSH-receptor mRNA levels were measured in the ovary during development in normal mice and in hypogonadal (hpg/hpg) mice, which lack circulating gonadotropins. The relative abundance of alternate transcripts encoding LH and FSH receptors was also determined. Results show that shortened transcripts of the receptors were abundant at all ages. Full-length transcripts of the LH receptor were not detectable until postnatal Day 5 although shortened transcripts encoding the extracellular domain of the receptor were present from birth. Between Days 5 and 7, LH-receptor transcript levels showed a marked increase in normal animals but no change in hpg/hpg animals. FSH-receptor transcripts encoding all domains of the receptor were detectable at low levels at birth, increased in concentration between Days 3 and 5, and peaked at Day 10. In hpg/hpg animals, FSH-receptor mRNA levels were normal up to Day 7 but failed to increase thereafter. These results show that early development of both LH- and FSH-receptor mRNA levels in the ovary is gonadotropin-independent. This coincides with early folliculogenesis to the primary stage. Further development of LH-receptor mRNA levels is gonadotropin-dependent although FSH-receptor mRNA levels continue to increase independently until early secondary follicles are present.

Animals↗
PubMedDate not supplied

A comparison of selected mRNA and protein abundances in human liver.

In order to obtain an estimate of the overall level of correlation between mRNA and protein abundances for a well-characterized pharmaceutically relevant biological system, we have analyzed human liver by quantitative two-dimensional electrophoresis (for protein abundances) and by Transcript Image methodology (for mRNA abundances). Incyte's LifeSeq database was searched for expressed sequence tag (EST) sequences corresponding to a series of 23 proteins identified on 2-D maps in the Large Scale Biology (LSB) Molecular Anatomy database, resulting in estimated abundances for 19 messages (4 were undetected) among 7926 liver clones sequenced. A correlation coefficient of 0.48 was obtained between the mRNA and protein abundances determined by the two approaches, suggesting that post-transcriptional regulation of gene expression is a frequent phenomenon in higher organisms. A comparison with published data (Kawamoto, S., et al., Gene 1996, 174, 151-158) on the abundances of liver mRNAs for plasma proteins (secreted by the liver) suggests that higher abundance messages are strongly enriched in secreted sequences. Our data confirms this: of the 50 most abundant liver mRNAs, 29 coded for secreted proteins, while none of the 50 most abundant proteins appeared to be secreted products (although four plasma and red blood cell proteins were present in this group as contaminants from tissue blood).

DNA, Complementary↗
PubMed1997

Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation.

The cDNA for the monocarboxylate transporter MCT2 from rat testis has been cloned and sequenced. The derived protein sequence shows 82% identity with that from hamster. Rat MCT2 has a relative insertion of five amino acids in the N-terminal sequence preceding the first predicted transmembrane segment. MCT2 appears to be less highly conserved between species than MCT1. Using Northern blotting of RNA from rat and mouse tissues, MCT2 message was demonstrated to be abundant in the testis where a smaller, less abundant MCT2 transcript was also present. Low levels of a slightly different-sized transcript were found in rat and mouse liver, and mouse kidney. In hamster, only one-size transcript was detected at relatively high abundance in all the tissues examined. Antibodies were raised against a peptide derived from the extreme C-terminus of rat MCT2, and Western blotting with these detected MCT2 in membrane fractions prepared from rat testis, liver and brain but not those from heart or skeletal muscle. In hamster, MCT2 was detected in liver, heart and testis but not in brain [Garcia, Brown, Pathak, and Goldstein (1995) J. Biol. Chem. 270, 1843-1849]. For both rat MCT1 and MCT2 there were marked differences between the relative abundance of their respective messages and the amount of protein in membrane fractions from different tissues. This suggests that expression of both of these transporters in different tissues may be species-specific and regulated post-transcriptionally. The different-sized MCT2 transcripts may arise from alternative splicing. Starvation of rats for up to 48 h did not lead to any change in MCT1 or MCT2 expression in the liver, as determined by either Northern or Western blotting.

Amino Acid Sequence↗
PubMed1998

Disruption of the 5' and 3' splice sites flanking the major latency-associated transcripts of herpes simplex virus type 1: evidence for alternate splicing in lytic and latent infections.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are the only viral gene products expressed within latently infected neurones. The most abundant (major) LATs consist of two collinear nuclear polyA- RNAs of 2 kb and 1.5 kb which it has been suggested represent stable introns derived from a less abundant primary transcript (minor LAT). Consistent with this proposition is the identification of consensus splice donor and acceptor sites flanking major LATs which are conserved between HSV types 1 and 2. Here we test the functionality of the predicted splice sites within the context of the virus genome during productive infection in vitro and latent infection in vivo. To this end viruses in which the LAT splicing signals were disrupted by site-directed mutagenesis were constructed. We report that mutation of the splice acceptor site abrogates 2 kb major LAT generation during productive infection but does not significantly influence major LAT synthesis during neuronal latency. Similarly, mutation of the splice donor site significantly reduces levels of 2 kb major LAT during productive infection but has no detectable effect on the generation of 2 kb major LAT during neuronal latency as assessed by Northern and in situ hybridization analyses of latently infected neuronal tissue. From these data it can be concluded that the proposed splice sites flanking the major LAT region are dispensable for 2 kb major LAT production in neurones latently infected with HSV-1 but constitute functional splicing signals in productively infected non-neuronal cells.

Alternative Splicing↗
PubMed1994

Chalcone synthase from Camellia sinensis: isolation of the cDNAs and the organ-specific and sugar-responsive expression of the genes.

Three full-length cDNAs (CHS1, CHS2 and CHS3) encoding chalcone synthase (CHS; EC 2.3.1.74) were isolated from young leaves of Camellia sinensis. Each cDNA encoded 389 amino acid residues, which showed 93-96% identity to one another. Oligonucleotides were synthesized on the basis of the 5'-untranslated sequences of the cDNAs and their corresponding transcripts could be distinguished. The CHS1, CHS2 and CHS3 transcripts were abundant in the leaves and stems. After an initial treatment with water in darkness, the transcripts fell to very low levels in the young leaves. These levels were increased by a subsequent treatment with fructose, sucrose or maltose in darkness, and they increased still further upon a treatment with glucose, sucrose or maltose under continuous light. These results indicate that the CHS1, CHS2 and CHS3 transcripts are expressed in various organs and respond to sugars in young leaves in a similar manner. The effect of continuous light is also discussed.

Acyltransferases↗
PubMed1998

Human papillomavirus type 33 DNA and E6-E7 transcripts in late passages of the UT-DEC-1 vaginal keratinocyte cell line.

Transcription of human papillomavirus (HPV) type 33 early region was analysed in the UT-DEC-1 keratinocyte cell line, which has been derived from a HPV-33-containing mild vaginal dysplasia. Fifteen cDNA clones from transcripts from the E6-E7 open reading frames were constructed and analysed. Most clones represented viral transcripts spliced within the E6 open reading frame, probably encoding the E7 protein. Interestingly, a less abundant unspliced transcript species with coding capacity for the full length E6 protein was found, reported here for the first time for the malignancy-associated HPV type 33.

Base Sequence↗