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PubMed1988

Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter.

The antigen receptor of the T lymphocyte consists of two variable T-cell receptor chains (either TCR-alpha, TCR-beta or TCR-gamma, TCR-delta) noncovalently linked to four different invariant membrane proteins (CD3-gamma, CD3-delta, CD3-epsilon, and the CD3-zeta homodimer). The CD3 genes are expressed early in thymocyte development, preceding the rearrangement and expression of the T-cell receptor genes. Here we report the isolation and structural analysis of the human CD3-epsilon gene. The gene consisted of nine exons. Three exons, encoding the junction of leader peptide and mature protein, were extremely small (21, 15, and 18 base pairs, respectively). The murine gene contained only two such miniexons, the sequences of which were not homologous to those of the three human miniexons. But from comparisons of intron sequences the regions surrounding the human miniexons III and IV appeared to be closely related to those surrounding the murine miniexons III and IV. The most-3' miniexon in the human gene (IVa) had no murine counterpart and appeared not to duplicate any of the other miniexons. Sequence analysis of CD3-epsilon cDNA clones isolated from four independent libraries gave no evidence for alternative use of these miniexons. Like CD3-delta, the CD3-epsilon gene was transcribed from a weak, nontissue-specific, TATA-less promoter. Pulsed-field electrophoresis showed that the human CD3-epsilon gene was separated from the CD3-gamma, CD3-delta gene pair by at least 30 kilobases, but by no more than 300 kilobases.

Amino Acid Sequence↗
PubMed1997

Gene linkage and steady state RNAs suggest trans-splicing may be associated with a polycistronic transcript in Schistosoma mansoni.

Spliced leader (SL) trans-splicing generates the 5' end of mature mRNAs through the addition of a small exon to pre-mRNAs in some flagellates (kinetoplastida and euglenoids) and metazoans (nematodes and flatworms). Although SL addition in the kinetoplastida and a subset of nematode genes serves to resolve multicistronic mRNAs into monocistronic, capped mRNAs, information regarding the functional significance of trans-splicing in flatworms is limited. We describe here the identification and characterization of a closely linked gene upstream from the trans-spliced enolase gene in the flatworm Schistosoma mansoni. This gene produces a non-trans-spliced mRNA encoding a ubiquinol binding protein, UbCRBP, that is a component of the ubiquinol-cytochrome C reductase complex. The distance between the UbCRBP polyadenylation site and the enolase trans-splice acceptor site is exceptionally short, only 54 nucleotides. Primer extension (5' RACE), RT-PCR, and RNase mapping have identified steady state, cis-spliced RNAs which significantly overlap both the UbCRBP and enolase genes. These transcripts contain the 5' ends of mature UbCRBP mRNAs; extend through UbCRBP, across the intergenic region, and a significant distance 3' into the enolase gene. Interestingly, the close linkage between the UbCRBP and enolase genes is conserved in a second flatworm, Fasciola hepatica, which also trans-splices the downstream enolase gene. Taken together, the role of SL addition in resolving multicistronic transcripts in both C. elegans and the kinetoplastida, the conservation of UbCRBP/enolase gene linkage in two divergent trematodes, and the multicistronic organization of schistosome UbCRBP/enolase RNAs are consistent with the suggestion that these two genes are likely to be cotranscribed and that trans-splicing in flatworms may be associated with polycistronic transcripts.

Amino Acid Sequence↗
PubMed1988

A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear viral genome.

Epstein-Barr virus (EBV) immortalizes B-lymphocytes efficiently in vitro and in most or all of the cells a latent infection is established. Only a few viral genes are expressed during latency and these apparently maintain cell immortalization by EBV. A 1.9 kb cDNA clone of an EBV mRNA has been isolated from a B95-8 cDNA library and this cDNA corresponds to a new member of the group of EBV latent cycle genes. On Northern blots two polyadenylated transcripts 1.7 and 2.0 kb in length have been detected by the cDNA clone in B95-8 cells as well as in latently infected cell lines and in various cell lines derived from Burkitt's lymphomas. The cDNA sequence showed that this gene consists of nine exons and showed that this gene consists of nine exons and that the transcription unit crosses the terminal repeats. Thus, the intact gene is created upon infection by the circularization of the linear viral DNA molecule at the terminal repeats. One major open reading frame is present in the cDNA apparently coding for a 53 kd protein. A strongly hydrophilic part of the protein is followed by a very hydrophobic C-terminus, perhaps suggesting a membrane protein. The gene is unrelated to the other latent cycle genes, the EBV nuclear antigens (EBNA 1-4), the leader protein (LP), and the latent membrane protein (LMP) genes. This is the first example of a viral gene whose coding sequence is only created by joining of the ends of a linear virus.

Amino Acid Sequence↗
PubMed2002

Regulation of hepatic insulin-like growth factor I leader exonusage in lambs: effect of immunization against growth hormone-releasing factor and subsequent growth hormone treatment.

The establishment of a GH-responsive endocrine IGF-I network is essential for the regulation of postnatal growth. Transcripts of exons 1 and 2 of the mammalian IGF-I gene are alternately spliced onto exon 3, generating class 1 and class 2 mRNA, respectively, each encoding individual signal peptides. The liver is largely responsible for the synthesis of circulating IGF-I and is the main site of expression for class 2 mRNA. The aim of this study was to examine the regulation of hepatic class 1 and 2 mRNA levels in response to changed GH status. Lambs were actively immunized against GRF to suppress GH secretion; hepatic IGF-I mRNA leader exon usage was examined in the presence and absence of GH replacement and in control-immunized lambs. Lambs immunized against GRF exhibited a 17% (P < 0.001) decrease in growth rate as assessed by whole body weight gain, accompanied by decreased circulating IGF-I concentrations (P < 0.001), which were increased by subsequent GH treatment (P < 0.001). Hepatic class 1 and 2 IGF-I mRNA levels decreased in GRF-immunized lambs, although only class 2 transcripts decreased significantly (P < 0.001). Subsequent GH treatment induced increases in class 1 and 2 mRNA levels (P < 0.001) but the increase in class 2 message exceeded that for class 1 (P < 0.001). Thus, the percentage of total IGF-I mRNA accounted for by class 2 mRNA was 45% in control lambs, decreased to less than 20% in GRF-immunized lambs, but increased to 72% in the GRF-immunized lambs treated with GH and correlated with circulating IGF-I concentrations. These data suggest physiological significance for class 1 and 2 IGF-I mRNA species in GH action. Possible functions for such alternative splicing mechanisms are discussed.

Age Factors↗
PubMed2001

An E-box motif residing in the exon/intron 1 junction regulates both transcriptional activation and splicing of the human norepinephrine transporter gene.

The norepinephrine transporter (NET) is responsible for the rapid NaCl-dependent uptake of norepinephrine into presynaptic noradrenergic nerve endings. Recently, we have characterized the structural organization of the 5' upstream promoter region of the human NET (hNET) gene. A new intron of 476 base pairs was found in the middle of the 5'-untranslated leader sequence and was shown to robustly enhance the promoter activity. Here, we show that the first hNET intron enhances both the homologous hNET and the heterologous thymidine kinase promoter activities in an orientation- and position-dependent manner. The first hNET intron exhibited a similar promoter-enhancing effect in both SK-N-BE(2)C (NET-positive) and HeLa (NET-negative) cell lines, showing that its function is not cell-specific. Transient transfection assays of a series of deletional constructs show that the first hNET intron contains subdomains with either positive or negative regulatory functions. Furthermore, DNase I footprinting analysis demonstrated that the 5' side of the intron, encompassing the splice donor site, is prominently protected by nuclear proteins isolated from both SK-N-BE(2)C and HeLa cells. The protected nucleotide sequence contains a consensus E-box motif, known to regulate diverse eukaryotic genes, which overlaps with the splice donor site of the first intron. We demonstrate that two basic helix-loop-helix proteins, upstream stimulatory factors 1 and 2, are major proteins interacting at this site and that the E-box is at least in part responsible for the promoter-enhancing activity of the first intron. Furthermore, site-directed mutagenesis of the splice donor site of the first intron affects both correct splicing and transcriptional activity. Taken together, our results indicate that a cis-element residing at the first exon/intron junction, encompassing an E-box motif, has a unique dual role in basal transcriptional activation and splicing of hNET mRNA.

Base Sequence↗
PubMed1999

A novel form of human neuropsin, a brain-related serine protease, is generated by alternative splicing and is expressed preferentially in human adult brain.

We have cloned cDNAs encoding two isoforms of a human novel serine protease. They encoded sequences of 260 and 305 amino acids, and both showed significant homology to mouse neuropsin. Mouse neuropsin has been reported to be involved in hippocampal plasticity, therefore we designated the proteins as type 1 and type 2 neuropsin, respectively. The amino acid sequences of the two types of human neuropsin were identical, except that type 2 carried an insert of 45 amino acids at the C-terminus of the leader sequence. The essential three amino acids in the active site triad, His, Asp, and Ser, and the single putative N-glycosylation site were conserved in human and mouse neuropsin. Sequence analysis of the 946 bp genomic DNA spanning the region encoding the insertion sequence revealed that two isoforms were generated in human brain by alternative splicing. However, the mouse genomic sequence did not conserve the 3' acceptor consensus sequence at the corresponding position, suggesting that type 2 neuropsin was a species-specific splice variant. When the open reading frames of human neuropsin were expressed in insect cells, both types of neuropsin were detected in the conditioned media by western blot analysis using anti-human neuropsin serum. Northern blot hybridization and reverse transcription-polymerase chain reaction showed predominant expression of type 1 neuropsin in pancreas. Type 2 neuropsin was preferentially expressed in human adult brain and hippocampus, although both types were expressed in fetal brain and placenta in comparable amounts. Dot blot hybridization showed that neuropsin was expressed in various regions of adult brain, including the hippocampus and cerebral cortex, and also in various fetal tissues. These results suggest that human type 2 neuropsin may be important to the adult brain plasticity, although both types may be necessary for the development of the nervous system.

Adult↗
PubMed1990

High level expression of the envelope glycoproteins of the human immunodeficiency virus type I in presence of rev gene using helper-independent adenovirus type 7 recombinants.

The effect of rev (art/trs) gene on the level of HIV-1 envelope (env) expression using recombinant adenovirus was investigated. Recombinant adenoviruses expressing either the envelope or the rev gene of the human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the gene into an expression cassette. The expression cassette contained the adenovirus type 7 major late promoter, followed by leader 1 of the adenovirus tripartite leader and a portion of intron between leaders 1 and 2, leaders 2 and 3, and a hexon polyadenylation signal. The cassette was then inserted at the terminal region between the E4 and ITR regions of the adenovirus 7 genome with a concomitant E3 region deletion (80-87 m.u.). A549 cells infected with the recombinant virus containing the env gene produced the envelope glycoproteins gp160, gp120, and gp41. HIV-1 envelope gene expression was greatly enhanced (20- to 50-fold) in the cells that were simultaneously infected with the recombinant adenovirus containing the rev gene as measured by ELISA and Western blotting. Interestingly, this effect was observed despite the lack of the 5' down splice site for rev and seems to be post-transcriptional. Another recombinant adenovirus which contains both the rev and the env genes was constructed by inserting the rev gene in the deleted E3 region and the env gene in the terminal cassette. This double recombinant virus expressed high levels of env antigen in A549 cells similar to those attained upon co-infection with two separate recombinant viruses containing the rev or env gene. Furthermore, the rev gene nucleotide sequence could be altered without altering the amino acid sequence and its sequences truncated by 17 amino acids from the C-terminus had no effect of rev function.

Adenoviridae↗
PubMed1994

Synergistic antiviral activities of oligonucleoside methylphosphonates complementary to herpes simplex virus type 1 immediate-early mRNAs 4, 5, and 1.

An oligonucleoside methylphosphonate (ONMP) complementary to the splice acceptor site of immediate-early (IE) pre-mRNAs 4 and 5 (IE4,5SA) inhibits herpes simplex virus type 1 (HSV-1) growth in vitro and in infected animals. The antiviral effect appears to be due to inhibition of IE pre-mRNA 4 and 5 splicing and/or IE4 gene expression (M. Kulka, M. Wachsman, S. Miura, R. Fishelevich, P. S. Miller, P. O. P. Ts'o, and L. Aurelian, Antiviral Res. 20:115-130, 1993). We describe the potentiation of antiviral activity when we targeted two IE genes with different ONMPs. A psoralen derivative of an ONMP complementary to the IE mRNA 1 (IE1) translation initiation site (IE1TI) covalently bound a 2.8-kb transcript that hybridized with a 20-base oligonucleotide complementary to the 5' leader sequence of IE1 but not a 20-base oligonucleotide complementary to the first intron of IE1. IE1TI inhibited IE1 gene expression and virus replication in cells infected with HSV-1 in vitro. Inhibition was specific because it was not observed with oligomers mutated in two (IE1TImu1) or four (IE1TImu2) central residues or in cells infected with an IE1 deletion mutant (HSV-1 dl1403). IE1TI potentiated the antiviral activity of IE4,5SA (synergistic effect), while potentiation was not observed when IE4,5SA was mixed with IE1TImu1. A similar synergistic effect was seen when IE1TI was mixed with an ONMP complementary to the translation initiation site of IE mRNA 4 but not with an ONMP complementary to the translation initiation site of IE mRNA 5. These findings suggest that synergistic antiviral activity is mediated by targeting at least two IE genes (IE1 and IE4).

Animals↗
PubMedDate not supplied

The soluble CTLA-4 receptor: a new marker in autoimmune diseases.

A soluble form of cytotoxic T lymphocyte-associated antigen-4 (sCTLA-4) was recently found and shown to possess B7 binding activity. sCTLA-4 is generated by alternatively spliced mRNA. The mRNA encoding sCTLA-4 consists of 3 exons: exon 1 encodes a leader peptide, exon 2 the ligand binding domain, and exon 4 the cytoplasmic tail, but it lacks the transmembrane domain encoded by exon 3. The altered transcript is detected in resting CD4 and CD8 T cells and its expression is inhibited after 24--48 h of activation and returns to the prestimulation level after 72--120 h of activation. Low levels of sCTLA-4 have been detected in normal human serum and increased serum levels have been observed in several autoimmune diseases (e.g. Graves' disease, myasthenia gravis, systemic lupus erythematosus, and systemic sclerosis). The biological significance of increased sCTLA-4 serum level has not been clarified. On one hand, sCTLA-4 may bind B7 expressed on antigen-presenting cells and is thus able to interfere with the B7:CD28-mediated costimulation of T cell responses. On the other hand, sCTLA-4 may also be capable of interfering with B7:CTLA-4 interactions, thereby blocking the negative signal imparted via the full-length form of CTLA-4. This double-edged nature of B7 blocking by sCTLA-4 may result in different outcomes of the clinical course of disease.

Alternative Splicing↗
PubMed1990

Linkage of two human pregnancy-specific beta 1-glycoprotein genes: one is associated with hydatidiform mole.

A genomic clone containing two linked human pregnancy-specific beta 1-glycoprotein (PS beta G) genes has been isolated and characterized. The two genes are arranged in the same 5'----3' orientation; the 3' region (including the A2 and B-C exons) of the upstream gene, PSGGA, is linked to the 5' region (including the 5'/L and L/N exons) of PSGGB, the downstream gene. Depending upon the domains compared, PSGGA and PSGGB share 92-98% nucleotide and 86-95% amino acid sequence identity with PSG93, the most abundant PS beta G transcript. The 3' exon (B-C) of PSGGA contains four alternative splice sites and three polyadenylylation sites, which account for the 3' heterogeneity previously reported in the PS beta G family. Each of the predicted PSGGA-encoded proteins would have a different carboxyl terminus. PSGGB corresponds to the previously identified cDNA PSG6, which encodes proteins containing a 34-amino acid leader peptide and a 108-amino acid N domain, which is one amino acid shorter than the majority of PS beta G N domains. Additionally, the PSGGB-encoded proteins contain the cell-surface recognition tripeptide Arg-Gly-Asp, shared by several previously reported PS beta Gs as deduced from cDNA sequences. Northern blot hybridization performed with a PSGGB-specific oligonucleotide probe to the N domain revealed that PSGGB or a PSGGB-like gene encodes a major 1.7-kilobase mRNA in hydatidiform mole tissues and a major 2.0-kilobase mRNA in term placenta tissues. Moreover, the PSGGB-specific probe hybridized most strongly with mRNA from molar trophoblastic tissue, suggesting that the PSGGB-like species may be the gene preferentially expressed in gestational trophoblastic disease. Additionally, the sequence of a 2315-base-pair PS beta G cDNA (PSG95) that contains an N-A1-A2-B2-C domain arrangement is reported. The coding region of PSG95 is identical to the previously reported cDNA clones PSG1d and FL-NCA, but PSG95 contains an additional 518 and 523 base pairs in the 3' end as compared with PSG1d and FL-NCA, respectively.

Amino Acid Sequence↗
PubMed1984

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

Animals↗
PubMed1998

Localization and processing from a polycistronic precursor of novel snoRNAs in maize.

We have shown previously that groups of U14 snoRNA genes are clustered with other, novel snoRNAs in maize. These genes are transcribed polycistronically from an upstream promoter to give a precursor snoRNA, which is processed by a splicing-independent mechanism. The clusters contain both box C/D snoRNAs, thought to guide rRNA O-ribose methylations, and the first plant box H/ACA snoRNA so far identified, thought to guide an rRNA pseudo-uridylation. Here we show that four novel snoRNAs identified as members of U14-containing gene clusters each show distinct sub-nucleolar localizations. Two of the snoRNAs (snoR2, a box H/ACA snoRNA, and snoR3, a box C/D snoRNA) colocalise closely with nucleolar rDNA transcription sites. A third box C/D snoRNA, U49, is localised to a more extended region which includes the transcription sites. On the other hand snoR1, another box C/D snoRNA, is located in a quite different region of the nucleolus, and shows a similar distribution to that of 7-2/MRP, a snoRNA involved in the later pre-rRNA cleavage reactions. This may indicate that this snoRNA is involved at later stages of processing, whereas the other snoRNAs are involved early or cotranscriptionally. Probes to intergenic spacer regions of the precursor snoRNA have been used to determine the location of the precursor. This shows a clear labelling of both the dense fibrillar component of the nucleolus, and of coiled bodies. This distribution implies that the polycistronic precursor is imported into the nucleolus for processing to the mature snoRNAs, and that the import or processing pathway involves coiled bodies.

Cell Nucleolus↗
PubMed1994

Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control.

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.

Base Sequence↗