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PubMed1990

A novel oncofetal gene is expressed in a stage-specific manner in murine embryonic development.

A novel cDNA clone obtained from a murine T-lymphoma library hybridizes to transcripts expressed in placenta and embryos (Pem) in a stage-specific manner. The Pem cDNA sequence predicts an intracellular hydrophilic protein with no significant sequence similarity to other DNA or protein sequences. Pem transcripts are abundant in 7- and 8-day mouse embryos, but decrease precipitously thereafter. On Day 9 they become abundant in placenta and yolk sac, persisting there until parturition. Although Pem transcripts are present in immortalized and tumorigenic cell lines from several different cell lineages, they are not detectable in any of 15 adult tissues tested. The expression of Pem during fetal development and its presence in immortalized and neoplastic cell lines is consistent with the properties expected of an "oncofetal" gene.

Amino Acid Sequence↗
PubMed2005

Structural organization of the human complexin 2 gene (CPLX2) and aspects of its functional activity.

We report here on the in vitro and in silico characterization of the organization of the human complexin 2 (CPLX2) gene. This encodes for a protein of 134 amino acid residues, contains five exons, is localized on human chromosome 5q35.3, and spans more than 87 kb. We performed in silico analysis of the CPLX2 5' untranslated region (UTR) and propose an alternative variant of the gene transcript. Compared to the mRNA reported earlier [McMahon, H.T., Missler, M., Li, C., Sudhof, T.C., 1995. Complexins: cytosolic proteins that regulate SNAP receptor function. Cell 83, 111-119.], this transcript bears a partly altered 5'-UTR associated with the same open reading frame. Both CPLX2 transcripts share exons III-V; the alternative transcript is devoid of exons I and II, and includes exon A instead. Exon A is localized within CPLX2 intron 2 about 7 kb upstream to exon III. Using reverse transcription polymerase chain reaction (RT-PCR) we detected both types of transcripts in human brain mRNA. In silico data suggest that two putative alternative TATA-less promoter regions separated by 74 kb govern the expression of two CPLX2 transcripts. Several potential transcription start sites were detected by primer extension for each of two alternative CPLX2 transcripts. The relative abundance of the alternative transcripts was investigated in human and rat forebrain, cerebellum, and hippocampus. Whereas both transcripts were detected in human and rat brain, their expression levels were found to vary significantly among the regions investigated. The organization of CPLX2 transcripts is conserved in humans and rodents.

3' Untranslated Regions↗
PubMed1997

Identification of two estrogen receptor transcripts with novel 5' exons isolated from a MCF7 cDNA library.

Two novel transcripts of human estrogen receptor (ER) have been identified that differ in the 5' untranslated sequence. It has previously been determined that an alternate ER transcript is generated from transcription initiated upstream of the main ER cap site (P1), and utilizes a splice acceptor site at +163. Here we report the isolation of 21 ER clones from a MCF7 cDNA library. Eleven of these clones correspond to transcripts that initiate at the P1 cap site, whereas the remaining 10 clones are derived from two previously unidentified ER transcripts (designated E and H) that both utilize the +163 splice acceptor site. A panel of breast and endometrial carcinoma cell lines were screened by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of the E and H transcripts. It was found that all ER-positive cell lines expressed both of the novel transcripts. In addition, 10 primary human breast cancers were analyzed, of which six expressed the E transcript and five abundantly expressed the H transcript. These data indicate that expression of ER in human breast cancers can be dependent upon an alternate promoter at least 20 kb upstream of the primary cap site for ER.

Base Sequence↗
PubMed1998

Transcripts of maize RbcS genes accumulate differentially in C3 and C4 tissues.

RbcS genes exist as multigene families in most plant species examined. In this paper, we report an investigation into the expression patterns of two maize RbcS genes, designated in this report as RbcS1 and RbcS2. We present the sequence of RbcS2 and show that the structure of the gene has several features in common with other monocot RbcS genes. To determine whether RbcS1 and RbcS2 fulfil different functional roles with respect to the C3 and C4 carbon fixation pathways, we have investigated the expression patterns of the two genes in different maize tissue types. Transcripts of both genes are found at high levels specifically in bundle-sheath cells of maize seedling leaves, indicating that both genes are expressed in the C4-type pattern. However, we show that RbcS1 transcripts are relatively more abundant than RbcS2 transcripts in C3 tissues such as husk leaves. These results are discussed with respect to the evolution of C4 carbon fixation and the mechanisms required for the cell-specific expression of RbcS genes.

Amino Acid Sequence↗
PubMed2001

Stability of barley aleurone transcripts: Dependence on protein synthesis, influence of the starchy endosperm and destabilization by GA3.

We have studied the stability of Barley aleurone and embryo expressed (Balem) transcripts in aleurone layers. The Per1, Ole1 and Ole2 transcripts are abundant during desiccation and in dry resting seeds, while B12D and B22E transcripts are expressed mainly during seed maturation and germination. From 21 to 40 days post anthesis (DPA) incubation of aleurone layers resulted in a substantial, but differential reduction in the levels of these transcripts. In contrast, Balem transcript levels in aleurone layers of incubated embryoless grains were (except for B22E) similar to those of freshly dissected layers. Cycloheximide lowered transcript levels significantly. This indicates that a protein-synthesis-dependent mRNA-stabilizing mechanism is active in the aleurone cells when attached to the starchy endosperm. At the onset of seed desiccation (40 DPA), half-lives of transcripts to be stored in the dry seed were up to several days longer than the half-life of B22E, which decreases during seed maturation. While the Per1, Ole1 and Ole2 transcript levels decline rapidly in the aleurone layers of mature, germinating seeds, the genes are actively transcribed and their transcripts highly stable in the aleurone of incubated embryoless seeds. The expression of Ole1 and Ole2, as well as Per1, can be repressed 100-1 000-fold by gibberellic acid (GA3) in a dose-dependent manner. Abscisic acid can counteract the GA3 repression. Incubations with transcriptional and translational inhibitors indicate that GA3 inhibits the transcription of these genes and at the same time induces a protein-synthesis-dependent mechanism destabilizing their mRNA molecules present.

Journal Article↗
PubMed1989

Identification of new human repetitive sequences: characterization of the corresponding cDNAs and their expression in embryonal carcinoma cells.

We have identified new repeated interspersed DNA sequences by analysis of homologous RNA transcripts from a human teratocarcinoma cell line (NTERA-2 clone D1). The abundance of transcripts varies upon retinoic acid induced differentiation of NTERA-2/D1 cells, and it is highest when the cells display the embryonal carcinoma phenotype. The expression of these novel repeated sequences appears to be tissue specific as no detectable expression was found in various cell lines of different embryological derivation. Characterization of the RNA transcripts by analysis of recombinant cDNA clones indicated that transcripts of different genomic units are present in undifferentiated embryonal teratocarcinoma cells. Nucleotide sequencing of the cloned cDNAs reveals a complex structure composed by unique and tandemly repeated sub-elements.

Base Sequence↗
PubMed2000

BodyMap: a human and mouse gene expression database.

BodyMap is a human and mouse gene expression database that has been maintained since 1993. It is based on site-directed 3'-ESTs collected from non-biased cDNA libraries constructed at Osaka University and contains >270 000 sequences from 60 human and 38 mouse tissues. The site-directed nature of the sequence tags allows unequivocal grouping of tags representing the same transcript and provides abundance information for each transcript in different parts of the body. Our collection of ESTs was compared periodically with other public databases for cross referencing. The histological resolution of source tissues and unique cloning strategy that minimized cloning bias enabled BodyMap to support three unique mRNA based experiments in silico. First, the recurrence information for clones in each library provides a rough estimate of the mRNA composition of each source tissue. Second, a user can search the entire data set with nucleotide sequences or keywords to assess expression patterns of particular genes. Third, and most important, BodyMap allows a user to select genes that have a desired expression pattern in humans and mice. BodyMap is accessible through the WWW at http://bodymap.ims.u-tokyo.ac.jp

Animals↗
PubMed1995

Tumor necrosis factor-alpha modulates monocyte/macrophage apoprotein E gene expression.

apo E has been shown to modulate cholesterol balance in arterial wall cells. Production of apo E by macrophages in atherosclerotic plaques could thereby influence the development of the plaque lesion. Cytokines, including TNF alpha, have been identified in human lesions, therefore, we undertook a series of studies to evaluate the effect of TNF alpha on monocyte/macrophage apo E production. The addition of TNF alpha to freshly isolated human monocytes led to a four- to fivefold increase of apo E mRNA abundance. The addition of TNF alpha to fully differentiated macrophages either had no effect or modestly inhibited apo E mRNA expression. THP1 human monocytic cells also responded to TNF alpha in a phenotype-specific manner. Treatment of these cells with TNF alpha produced a dose- and time-dependent increase in apo E mRNA. This increase was reflected in apo E synthesis and was associated with inhibition of DNA synthesis, and with induction of c-fos and ICAM-1 gene expression. Cell-permanent analogues of ceramide did not reproduce TNF alpha effect on apo E, but antagonists of protein kinase C did inhibit its effect. TNF alpha induction of apo E mRNA abundance was associated with stimulation of apo E promoter-dependent gene transcription. In summary, TNF alpha stimulates apo E gene transcription, mRNA abundance, and protein synthesis in the monocyte/macrophage in a phenotype-specific manner. Such regulation could significantly modify the amount of apo E present in vessel wall lesions.

Apolipoproteins E↗
PubMed2004

Differential expression of three plastidial sigma factors, OsSIG1, OsSIG2A, and OsSIG2B, during leaf development in rice.

We isolated and characterized two rice nuclear genes, OsSIG2A and OsSIG2B, encoding the putative sigma-factor of the plastid RNA polymerase. Deduced protein sequences predicted a plastid-localizing signal in the N-terminus and subsequent polypeptides similar to known SIG2 proteins. Gene expression analysis revealed that the OsSIG2A transcript is more abundant than the OsSIG2B transcript in all tissues tested and that both rice SIG2s are expressed from earlier stages of leaf development than that in the case of OsSIG1. These results indicate differential expression of SIG genes in leaf morphogenesis, suggesting the existence of tissue- and stage-specific functions of SIG proteins for transcriptional regulation of chloroplast genes in plant development.

Amino Acid Sequence↗
PubMed1992

Identification and characterization of the regulated pattern of expression of a novel mouse gene, meg1, during the meiotic cell cycle.

The gene designated meg1 (meiosis expressed gene) is a new mouse gene identified during a search for mammalian genes potentially involved in meiotic processes. Two classes of complementary DNAs were isolated from an adult mouse testis complementary DNA library, which shared the same 3' end including the entire putative coding region but differed in their 5' ends. Only one of these complementary DNA classes appeared to correspond to the very abundant 0.75-kilobase testicular transcript of meg1. Sequence analysis predicts a 10.8-kilodalton protein which is highly charged and lysine rich. It is also relatively rich in potential phosphoacceptor amino acids (approximately 17%), several of which are located in phosphorylation consensus sequences. The pattern of expression of meg1 was studied utilizing a combined Northern blot and in situ hybridization analysis. Of the adult tissues examined, meg1 transcripts were detected exclusively in testis. Analysis of mRNA from testes of two germ cell deficient mutant strains did not reveal significant levels of meg1 transcripts. Analysis of RNA from enriched populations of spermatogenic cells from adult testes and localization by in situ hybridization revealed that meg1 transcripts are most abundant in pachytene spermatocytes. These results suggest a role for meg1 during germ cell differentiation, possibly during meiotic prophase.

Amino Acid Sequence↗
PubMed2006

[Screening and detecting of proteins interacting with mPem].

mPem, a homeobox gene, is expressed in a time and stage specific manner during murine ontogeny. Pem transcripts are abundant in 7- and 8-day mouse embryos, but decrease precipitously thereafter. On Day 9 they become abundant in placenta and yolk sac, persisting there until parturition. Although Pem transcripts are not detectable in most of adult tissues, they are present in reproductive system such as testis, epididymis and ovary. This indicates a important role for Pem during embryogenesis and reproductive development. To study the function of mPem protein, we used a GAL4 based yeast two-hybrid assay to screen a 7-day mouse embryo library with full-length of mPem. 3 proteins were found interacting with mPem protein. One of theses is Mdfic. We confirmed the interaction between mPem and Mdfic in yeast and in vitro. Mdfic, MyoD family inhibitor domain containing, encodes the myoD family inhibitor domain (I-mfa domain). The interaction between mPem and Mdfic suggested they maybe form the transcriptional regulator complex to regulate embryo differentiation.

Animals↗
PubMed1990

Increased expression of a gene coding for NAD:glyceraldehyde-3-phosphate dehydrogenase during the transition from C3 photosynthesis to crassulacean acid metabolism in Mesembryanthemum crystallinum.

We utilized differential plaque hybridization to identify three cDNA clones for transcripts which increase in abundance during the salinity-induced transition from C3 photosynthesis to crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum. Although there are differences in the abundance of these transcripts in unstressed tissue, steady-state levels of all three increased within 30 h following irrigation with 0.5 M NaCl. One cDNA encodes the cytosolic form of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) (NAD-GAPDH], an enzyme involved in the production of phosphoenolpyruvate for CO2 fixation at night and the conversion of pyruvate to storage carbohydrate during the day. Coding region and 3'-noncoding sequence probes were used to examine the expression of NAD-GAPDH transcripts in leaf and root tissue. We show that the gene encoding the NAD-GAPDH cDNA is expressed in both leaf and root tissue during C3 photosynthesis and CAM. NAD-GAPDH transcript levels increase rapidly in leaf (but not in root) tissue during the transition to CAM. Our data indicate that the predominant NAD-GAPDH transcript expressed during C3 photosynthesis and CAM is encoded by a single gene in M crystallinum. These results imply that the transition to CAM in some cases involves an upward readjustment in the level of a gene product expressed during C3 photosynthesis, rather than the expression of a CAM-specific isoform with unique regulatory or kinetic properties.

Amino Acid Sequence↗
PubMed1995

Analysis of the human CD10/neutral endopeptidase 24.11 promoter region: two separate regulatory elements.

The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes, and a variety of epithelial cells. To further define the tissue-specific and developmentally related expression of CD10/NEP, we have characterized two separate regulatory regions that control the transcription of 5' alternatively spliced CD10/NEP transcripts. These type 1 and 2 CD10/NEP regulatory regions are both characterized by the presence of multiple transcription initiation sites and the absence of classic TATA boxes and consensus initiator elements. The purine-rich type 1 regulatory region, which includes 5' UTR exon 1 sequence, is characterized by multiple putative PU.1 binding sites and consensus ets-binding motifs. In marked contrast, the GC-rich type 2 regulatory region contains multiple putative Sp1 binding sites, a potential consensus retinoblastoma control element (RCE), and an inverted CCAAT box. In the majority of tissues examined to date, type 2 CD10/NEP transcripts were more abundant; the abundance of type 1 transcripts was more variable, with the highest type 1 levels in fetal thymus and certain lymphoblastic leukemia cell lines.

Base Sequence↗
PubMed1995

Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.

Animals↗
PubMed2003

Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis.

PURPOSE: To characterize the gene expression pattern in the human trabecular meshwork (TM) and identify candidate genes for glaucoma by expressed sequence tag (EST) analysis as part of the NEIBank project. METHODS: RNA was extracted from dissected human TM and used to construct unamplified, un-normalized cDNA libraries in the pSPORT1 vector. More than 4000 clones were sequenced from the 5' end. Clones were clustered and identified using GRIST software. In addition, the expression patterns of genes encoding olfactomedin-domain proteins were analyzed by RT-PCR. RESULTS: After non-mRNA contaminants were removed, 3459 independent TM-expressed clones were obtained. These were grouped in 1888 clusters, potentially representing individual expressed genes. Transcripts for the myocilin gene, a locus for inherited glaucoma, formed the third most abundant cluster in the TM collection, and several other genes implicated in glaucoma (PITX2, CYP1B1, and optineurin) were also represented. One abundant TM transcript was from the gene for the angiopoietin-like factor CTD6, which is located at on the long arm of chromosome 1, area 36.2-36.1 in the region of the glaucoma locus GLC3B, whereas other transcripts were from genes close to known glaucoma loci. The TM collection contains cDNAs for genes that are preferentially expressed in the lymphatic endothelium (matrix Gla protein, apolipoprotein D precursor, and selenoprotein P precursor). In addition to EST profiling, RT- PCR was used to detect transcripts of the olfactomedin-domain proteins latrotoxin receptor Lec3 and optimedin in the TM. CONCLUSIONS: The TM libraries are a good source of molecular markers for TM and candidate genes for glaucoma. The abundance of myocilin cDNAs corresponds to the critical role of this gene in glaucoma and contrasts with libraries derived from cultured tissue. The expression profile raises the possibility that cells of the TM and Schlemm's canal may be more similar to lymphatic, rather than blood vascular endothelium.

Aged↗
PubMed2004

Severe subacute GM2 gangliosidosis caused by an apparently silent HEXA mutation (V324V) that results in aberrant splicing and reduced HEXA mRNA.

We have characterized the molecular basis of beta-hexosaminidase A (HEX A) deficiency in a patient ascertained through an ophthalmologic examination that revealed cherry red spots on his retina. The absence of neurological deficit in this child until 3 3/4 years of age indicated residual HEX A must be present. Three HEXA mutations, 10T > C (S4P) and 972T > A (V324V) on the maternal allele, and 1A > T (M1L) on the paternal allele were identified. The effects of the amino acid substitutions on HEX A expressed in COS-7 cells were analyzed; as expected, no HEX A activity was associated with the M1L mutation but surprisingly, the S4P mutation resulted in 59% of the HEX A activity expressed by the wild type cDNA. The effect of the S4P change was much less than that of another HEXA mutation, G269S, associated with an adult onset form of G(M2) gangliosidosis. This indicated that the S4P change was not the cause of disease and suggested that one of the mutations on the maternal allele, 10T > C or 972T > A, had its effect at the mRNA level. This was confirmed by Northern blot analysis that showed only 7% of the normal level of HEXA mRNA in proband fibroblasts. Analysis of the residual mRNA by RT/PCR and sequencing revealed normal transcripts from both the maternal and paternal allele, as well as a low abundance aberrant transcript from the maternal allele. Sequencing of this aberrant transcript revealed a new exon 8 donor site created by the 972T > A mutation that resulted in a 17 bp deletion and destabilization of the resulting abnormal transcript. The remaining normal mRNA produced from the 972T > A allele must account for the delayed onset of clinical symptoms in this child.

Alternative Splicing↗
PubMed2004

Expression patterns and phylogenetic analysis of two xylanase genes (htxyl1 and htxyl2) from Helminthosporium turcicum, the cause of northern leaf blight of maize.

A xylanase gene (htxyl2) was cloned from Helminthosporium turcicum, the cause of northern leaf blight of maize by screening the genomic library from the fungus using a approximately 500 bp PCR fragment of the gene as a probe. The gene is a second xylanase gene cloned from the fungus and it is different from the previously cloned xylanase gene (htxyl1). The two genes are grouped into separate clades in a phylogenetic tree based on DNA sequences. The patterns of htxyl1 and htxyl2 transcript accumulation were studied in vitro with different carbon and nitrogen sources and during infection of maize by the pathogen. In culture high htxyl1 transcript was detected in mycelium obtained from medium containing birchwood xylan and birchwood xylan plus xylose as sole carbon sources. On the other hand htxyl2 transcript was detected in a medium containing birchwood xylan plus xylose but not in the medium where only birchwood xylan was used as the sole carbon source. Addition of glucose to the basal inducing media had inhibitory effect to the expression of both genes. The htxyl1 transcript accumulation declined sharply with an increase in concentration of ammonium sulphate and glutamic acid, while increased htxyl2 transcript was observed with an increase in concentrations of the two nitrogen sources. Abundant htxyl2 transcript was detected at early and late stages of infection of maize by Helminthosporium turcicum. On the other hand no detectable htxyl1 transcript was found in northern blot under the test conditions. This study demonstrated that in H. turcicum xylanolytic system htxyl1 and htxyl2 are differentially expressed and might play important roles in the saprophytic and pathogenic phases of the fungus, respectively.

Amino Acid Sequence↗
PubMed2004

Cloning and developmental expression of mouse pygopus 2, a putative Wnt signaling component.

Recent studies in Drosophila identified pygopus, which encodes a PHD finger protein, as an additional nuclear component of the canonical Wingless(Wg)/Wnt signaling pathway. In this study, we describe the molecular cloning and expression analysis of a mouse pygopus gene, mpygo2. mpygo2 transcripts were detected in almost all adult mouse tissues examined, whereas transcripts of another mouse pygopus gene, mpygo1, were detected only in heart tissue. Abundant mpygo2 transcripts were observed during embryogenesis in multiple developmental sites. Consistent with the demonstrated role of the Wnt-beta-catenin-LEF/TCF signaling pathway in mammalian skin development, mpygo2 expression was detected in the developing epidermis and hair follicles, which suggests that mpygo2 might mediate the effect of this signaling pathway in mouse skin.

Amino Acid Sequence↗