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PubMed1983

Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region.

We have constructed two simian virus 40 early replacement recombinants that have the coding sequences for hepatitis B virus surface antigen (HBsAg). One construction, LSV-HBsAg, has the coding region for HBsAg but not the portion encoding the putative pre-surface antigen leader. Transformed monkey kidney cells (COS) infected with this recombinant express large quantities of the characteristic partially glycosylated HBsAg molecule, which are assembled into 22-nm particles that appear similar to those produced by human liver cells infected with hepatitis B virus. This result indicates that the pre-surface antigen sequences are not required for the synthesis of HBsAg or its assembly into particulate structures. The second recombinant, LSV-HBpresAg, has the entire surface antigen gene, including the putative promoter and pre-surface antigen region. COS cells infected with this recombinant plasmid produce 40- to 50-fold less HBsAg than those infected with the LSV-HBsAg recombinant plasmid. RNA mapping studies suggest that the transcription of the HBsAg gene is initiated at more than one site, or alternatively, that RNA splicing of transcripts occurs in the pre-surface antigen region.

Animals↗
PubMed1998

Consequences of maternal undernutrition for fetal and postnatal hepatic insulin-like growth factor-I, growth hormone receptor and growth hormone binding protein gene regulation in the rat.

The mechanisms that contribute to postnatal growth failure following intrauterine growth retardation (IUGR) are poorly understood. We demonstrated previously that nutritional deprivation in the pregnant rat leads to IUGR in offspring, postnatal growth failure and to changes in endocrine parameters of the somatotrophic axis. The present study examines the effects of maternal undernutrition (30% of the ad libitum available diet; IUGR group) throughout pregnancy on hepatic insulin-like growth factor-I (IGF-I), growth hormone receptor (GHR) and GH-binding protein (GHBP) gene expression using solution hybridisation/RNase protection assays (RPAs). Animals were killed at fetal (E22, term=23 days) and postnatal (birth, days 5, 9, 15, 21) ages, livers were collected and RNA extracted for RPAs. Results demonstrate the presence of all IGF-I mRNAs resulting from transcription start sites (ss) in exon 1 (ss1/2, ss3, ss2 spliced), exon 2, the two IGF-I E-domain variants (Ea and Eb) as well as GHR and GHBP mRNAs in hepatic tissue at E22 in both the ad libitum fed and IUGR offspring. In the postnatal liver, IGF-I ss1/2, ss3, ss2 spliced, Ea and Eb IGF-I variants as well as GHR and GHBP mRNA transcripts increased in abundance from birth to day 21. IGF-I exon 2 transcripts were relatively constant from E22 until postnatal day 15, then increased at postnatal day 21 in both the ad libitum fed and IUGR offspring. The expressions of all hepatic IGF-I leader exon ss and Ea domain variants were significantly reduced in IUGR offspring (P<0.05) from E22 to postnatal day 9. In contrast, relative abundance of hepatic IGF-I Eb variants, GHR and GHBP mRNAs were unaltered in IUGR offspring compared with the ad libitum fed animals. Whether these postnatal effects of undernutrition are a direct consequence of IUGR or whether they are related, in part, to differences in postnatal food intake remains to be investigated. In summary, we have demonstrated that hepatic IGF-I ss within exon 1 and exon 2 are coordinately regulated. Use of exon 1 ss increased during normal development and decreased with IUGR without changes in GHR or GHBP gene expression. Eb transcripts, thought to represent GH-dependent endocrine regulation of IGF-I, were unchanged in IUGR. These results suggest a possible postreceptor defect in GH action as a consequence of IUGR.

Aging↗
PubMed1998

Translational efficiency is up-regulated by alternative exon in murine IL-15 mRNA.

IL-15 promotes the growth of T cells and shares properties of IL-2. IL-2 is produced exclusively by T cells, while IL-15 message is expressed by a variety of tissues. However, it has been difficult to demonstrate IL-15 in the supernatants of many cells that express message for this cytokine. This suggests that IL-15 production is regulated by post-transcriptional controls. In this study, we cloned three types of murine IL-15 cDNA isoforms generated by alternative splicing and compared the translational efficiency among these isoforms. The translational efficiency of isoforms with alternative exon 5 containing another 3' splice site was significantly higher than that of IL-15 cDNA with originally described exon 5, which is generated by internal splicing of alternative exon 5. The translation product of the isoform containing alternative exon 5 has a shorter open reading frame due to stop codons in additional sequence, followed by a new AUG codon, and displays a shorter leader sequence. The shorter isoform of the IL-15 was detected in peritoneal macrophages stimulated with IFN-gamma and LPS, which expressed an abundant level of alternative exon 5. These results suggest that normal IL-15 production in stimulated macrophages is regulated by splicing of alternative exon 5.

Alternative Splicing↗
PubMed1991

Structure and expression of human germline VH transcripts.

The human VH5 family consists of two functional genes and one pseudogene. We have found a novel 1.2-kb VH5 gene transcript in normal fetal liver and cord blood and in transformed B lineage cells. VH5-positive cDNA clones were isolated from precursor B acute lymphoblastic leukemia, B chronic lymphoblastic leukemia, Epstein-Barr Virus-transformed B cell lines, and cord blood, and were identified as transcripts of unrearranged VH5 genes (germline transcripts). The cDNA clones were derived from both functional and pseudo-VH5 genes. Most germline transcripts appear to initiate at the normal VH promoter and are cleaved and polyadenylated at sites several hundred bases downstream of the VH5 coding region. Correct splicing of the leader intron was observed in all clones. In functional and pseudo-VH5 cDNAs, an open translational reading frame extends from the leader to a termination codon in the nonamer. Only limited polymorphisms were observed in the coding as well as flanking regions of the VH5 transcripts. Functional and pseudo-VH5 transcripts and previously identified murine germline VHJ558 transcripts are discussed.

Animals↗
PubMed1992

Minigene rescues acetylcholinesterase lethal mutations in Drosophila melanogaster.

The gene encoding acetylcholinesterase in Drosophila melanogaster is over 34,000 base-pairs long. We have constructed a 5800 base-pair minigene containing 1500 base-pairs of genomic sequence upstream from the transcription start spliced to the coding sequence, but lacking the nine introns. After germline genetic transformation, this minigene rescues acetylcholinesterase lethal mutants. Tissue-specific distribution appears normal. This allows us to test site-directed mutations of acetylcholinesterase. In a first effort, deletion of most of the unusual 1000 bases leader and its intriguing short open reading frames showed no effect on gene expression. The way is open to study in vivo the structure-function relationships of acetylcholinesterase and insecticide resistance.

Acetylcholinesterase↗
PubMed1989

Transcripts from the co-transposed segment of variant surface glycoprotein genes are in Trypanosoma brucei polyribosomes.

In Trypanosoma brucei the 5' proximal flanking sequences of a variant surface glycoprotein (VSG) gene, the co-transposed segment, are transcribed in a variant antigenic type- and stage-specific fashion along with the VSG gene. The precursor transcripts are subsequently processed to yield smaller transcripts from the co-transposed segment as well as the VSG mRNA. These co-transposed segment transcripts are quite abundant, polyadenylated and contain the spliced leader sequence, all characteristics of trypanosome mRNAs. We have found that all of the co-transposed segment transcripts from two VSG genes are present in polyribosomes. The nucleotide sequence of much of the co-transposed segment of one of these VSG genes, however, has no open reading frames coding for proteins longer than 49 amino acids. These results suggest that co-transposed segment transcripts do not encode essential proteins even though they are present in polyribosomes and may be translated.

Animals↗
PubMed1994

Structure of the L-histidine decarboxylase gene.

Two species of L-histidine decarboxylase (HDC) mRNA were found in the KU-812-F basophilic cell line, but only the 2.4-kilobase (kb) one encodes the functional HDC (Mamune-Sato, R., Yamauchi, K., Tanno, Y., Ohkawara, Y., Ohtsu, H., Katayose, D., Maeyama, K., Watanabe, T., Shibahara, S., and Takishima, T. (1992) Eur. J. Biochem. 209, 533-539). The 3.4-kb one encodes a truncated HDC protein and is also found in human leukemia-derived cell lines HEL and KCL-22. To clarify the mechanisms that regulate transcription of the HDC gene and generate the two species of mRNA, we have isolated genomic DNA clones coding for the HDC from human genomic libraries. Structural analysis of the isolated clones revealed that the human HDC gene is composed of 12 exons spanning approximately 24 kb. Genomic DNA blot analysis suggested that HDC is encoded by a single copy gene. The structural analysis also demonstrated that the heterogeneity of the HDC mRNA is caused by an insertion of the seventh intron sequence and alternative use of the splicing acceptor site at the 12th exon. The transcription start site of the HDC gene and the nucleotide sequences of the promoter and first exon regions were determined. We found a TATA-like sequence, a GC box, four CACC boxes, four GATA consensus sequences, and six leader-binding protein-1 binding motifs in the promoter region of the HDC gene.

Base Sequence↗
PubMed1988

Variant specific transcripts from the co-transposed segments of variant surface glycoprotein genes in Trypanosoma brucei.

We examined steady-state transcripts from the sequences immediately upstream of the 1 and 11 variant surface glycoprotein (VSG) genes in Trypanosoma brucei of the IsTaR 1 serodeme. These sequences, the co-transposed segment, differ in size and sequence, and produce a variety of variant-specific polyadenylated transcripts. Most, if not all, of these transcripts, some of which may be precursors, contain the spliced leader (SL). Variant specific transcription extends from 5' of the co-transposed segment to 100-400 nucleotides downstream of the major VSG mRNA polyadenylation site. The 1 VSG mRNA has at least 2, and possibly 3, SL addition sites; the major site is 120 nucleotides downstream of the polyadenylation site identified by sequencing a cDNA from the co-transposed segment. Two general models for the production of the co-transposed segment transcripts are discussed.

Animals↗
PubMed2005

SL trans-splicing: easy come or easy go?

Is spliced-leader (SL) trans-splicing an ancestral eukaryotic characteristic that has been lost in multiple lineages, or did it arise independently in the various phyla in which it occurs? Recent studies have discovered SL trans-splicing in new metazoan phyla, including the chordates. Its discovery in chordates identifies, for the first time, a phylum that clearly contains both trans-splicing and non-trans-splicing major groups, and defines a limited and well-understood field in which to study the evolutionary dynamics of SL trans-splicing. In this article, I summarize the evolutionarily relevant aspects of SL trans-splicing and consider the interplay among SL trans-splicing, pre-mRNA splice-signal syntax and evolutionary genomics.

Animals↗
PubMed1992

Cytoplasmic accumulation of a normally mitochondrial malonyl-CoA decarboxylase by the use of an alternate transcription start site.

Malonyl-CoA decarboxylase, a normally mitochondrial enzyme, accumulates in the cytoplasm of specialized glands to cause production of multiple methyl-branched fatty acids. Evidence was presented that a single copy of the decarboxylase gene present in the goose genome codes for both the mitochondrial form found in extremely low amounts in the liver and the cytosolic form found in large amounts in uropygial glands. To elucidate how a single gene encodes both forms, the malonyl-CoA decarboxylase gene and the cDNAs for both the mitochondrial (liver) and the cytoplasmic (gland) species were cloned and sequenced. The decarboxylase gene, found in a 21-kb segment of cloned genomic DNA, is composed of five exons of 0.521, 0.118, 0.156, 0.145, and 1.93 kb interrupted by 6.9, 1.5, 0.45, and 9.3-kb introns. Exon 1 revealed two ATGs in frame 150 bp apart. cDNA for the cytoplasmic form and mitochondrial form showed identical nucleotide sequence, except that the latter was longer than the former. The longest cDNA for the cytoplasmic form of the enzyme extended only 44 bp 5' to the second ATG and the position corresponded to the transcription initiation site of the cytoplasmic form revealed by primer extension and RNase protection. The cDNA for the mitochondrial form isolated from the library extended 19 bp further upstream. Primer extension and RNase protection indicated that transcripts for the mitochondrial form initiated upstream from the first ATG. The N-terminal segment of the open reading frame initiated at the first ATG showed an amphipathic signal sequence appropriate for mitochondrial import. A putative full length mRNA for the mitochondrial form of the enzyme when translated in vitro yielded a 55-kDa primary translation product which was processed by removal of about 5 kDa during uptake into goose liver mitochondria. These results strongly suggest that in most tissues transcription initiates 5'- to the first ATG, generating a transcript that would generate a protein with an N-terminal leader for transport into mitochondria. In the uropygial gland the use of an alternate promoter generates transcripts initiated between the two ATGs and the translation product accumulates in the cytoplasm since it lacks a mitochondrial targeting sequence.

Alternative Splicing↗
PubMed1996

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

Animals↗
PubMed1990

Developmentally regulated transporter in Leishmania is encoded by a family of clustered genes.

We have previously cloned a gene for a developmentally regulated transport protein from the trypanosomatid protozoan Leishmania enriettii. We demonstrate here that this transporter is encoded by a single family of tandemly clustered genes containing approximately 8 copies of the 3.6 kilobase repeat unit. Transcriptional mapping defines a contiguous 3.3 kilobase region of the repeat unit that encodes the mRNA. The 5' end of the mature mRNA contains the spliced leader or mini-exon previously identified in kinetoplastid protozoa, while the 3' ends of the mRNA are heterogeneous in sequence and in location of the polyadenylation site. We have identified genomic restriction fragments that flank the tandem repeat on the 5' and 3' sides and which may be linked to sequences required for expression of the gene family. Other species of Leishmania also contain sequences that hybridize to the cloned L. enriettii gene at high stringency.

Animals↗
PubMed1981

The nucleotide sequence of the gene for protein IVa2 and of the 5' leader segment of the major late mRNAs of adenovirus type 5.

We present here the primary structure of the region of human adenovirus 5 (Ad5) DNA from nucleotide 4001 through the HindIII site at nucleotide 6246 (map positions 0.11 to 0.17). The corresponding region in the closely related adenovirus type 2 (Ad2) encodes the spliced mRNA for viral protein IVa2 (Chow et al., 1979; Persson et al., 1979). Reverse transcription of the Ad5 pIVa2 mRNA localized the 5' terminus of the mRNA to approximately position 5840, and its splice coordinates to positions 5706 and 5427. From the data of Aleström et al. (1980) for Ad2, the 3' end of this mRNA was inferred to be specified by Ad5 nucleotide 4060. The nucleic acid data allow us to predict an Mr of 50873 for the IVa2 protein of Ad5, which is close to the experimentally determined value of 50000 (Persson et al., 1979). The DNA sequence described here also includes the information for the 5'-terminal leader segment of the major late mRNAs of Ad5.

Adenoviruses, Human↗
PubMed1994

Retinoic acid and granulocyte colony-stimulating factor synergistically induce leukocyte alkaline phosphatase in acute promyelocytic leukemia cells.

In this report we show a strong synergistic interaction between granulocyte colony-stimulating factor (G-CSF) and all-trans retinoic acid (ATRA) on the expression of leukocyte alkaline phosphatase (LAP) in freshly isolated acute promyelocytic leukemia (APL) blasts as well as in NB40 and HL-60 cell lines. The strong synergism observed in these cell types was not evident in two acute leukemia cell lines (K562 and GF-D8), in normal granulocytes, and in monocytes. In freshly isolated leukocytes derived from chronic myelogenous leukemia (CML), in the stable phase of the disease, a weaker interaction between ATRA and G-CSF was documented. The cross-talk between the cytokine and the retinoid was studied in detail in NB4, an immortalized APL leukemia cell line, retaining the 15-17 chromosomal translocation involving the retinoic acid receptor type alpha. The treatment of NB4 cells with G-CSF alone or ATRA alone leads to no increase and to minor induction in LAP activity, respectively. If the cells are treated with the two compounds simultaneously, a dramatic elevation of LAP is observed after 4 days. The synergism between G-CSF and ATRA is evident at concentrations of the retinoid between 10(-7) and 10(-5) mol/L and at concentrations of the cytokine between 1 and 10 ng/mL. The simultaneous presence of the two compounds is necessary to obtain maximal increase of LAP activity and the effect is cell density-dependent. Synergism is specific for G-CSF, and it is not observed with other cytokines and functional inducers of the granulocyte. The augmentation of LAP activity is the consequence of an increased transcriptional rate of the liver/bone/kidney-type (L/B/K-type) alkaline phosphatase gene, as determined by Northern blotting and nuclear run-on analysis using specific cDNA probes. Only one of the two possible alternatively spliced forms of L/B/K-type alkaline phosphatase transcript is detected in NB4 cells after stimulation with G-CSF and ATRA. This mRNA form, which is the one observed in normal polymorphonuclear leukocytes, contains the most upstream leader exon. In NB4 cells, ATRA induces G-CSF, alpha, and beta retinoic acid receptor transcripts, whereas G-CSF has minor effects on the expression of these mRNAs.

Alkaline Phosphatase↗
PubMed1997

Additional organizational features of the murine folylpolyglutamate synthetase gene. Two remotely situated exons encoding an alternate 5' end and proximal open reading frame under the control of a second promoter.

Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5' end. These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a encodes most of the 5'-untranslated region. Exon A1b encodes a downstream segment of the 5'-untranslated region, two ATG start codons, and a unique mitochondrial leader peptide as well as 15 additional amino acids of cytosolic FPGS not encoded by all previously identified (Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol. Chem., 271, 23820-23827) splice variants. It was also found by direct sequencing of genomic fragments that although exon A1b is spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to exon 1 are found approximately 9.5 kilobases upstream from exons B1a, B1b, and B1c. Exons A1a and A1b are separated from each other by a 124-nucleotide intron. Sequencing of the region 5' to exon A1a revealed a nucleotide sequence that was promoter-like and different from the downstream promoter region in the content of putative cis-acting elements. Primer extension analysis identified a number of potential transcription start sites within the more 3' end of this region. FPGS RNA transcripts incorporating exons A1a and A1b were detected in both normal mouse tissues, particularly, liver and kidney, and also to a varying extent in tumors; FPGS RNA transcripts incorporating exons B1a, B1b, and B1c were detected mainly in tumors. Thus, transcription of the FPGS gene in this tissue-specific manner appears to reflect the different usage of alternates to exon 1 under the control of different promoters. An unusual splice variant identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of the downstream promoter region along with exons B1c and B1b and exons 2-15.

Alternative Splicing↗
PubMed1982

An adenovirus agnogene.

The nucleotide sequence of a 550 base pairs long segment, located between map positions 21 and 22.5 in the adenovirus type 2 genome has been determined. S1 nuclease mapping and sequence analysis of cDNA copies of adenovirus mRNA demonstrated that the established sequence includes the i-leader which is spliced to the 5'-end of certain adenovirus mRNAs (1). The i-leader which is 440 nucleotides long contains an open translational reading frame which is preceded by an AUG triplet and which terminates in the third segment of the tripartite leader. A polypeptide with the same molecular weight as predicted from the DNA sequence was identified by in vitro translation of mRNA which had been selected by hybridization to DNA fragments, containing sequences from the i-leader. The results thus suggest that the i-leader, unlike other adenovirus leader segments, is used for translation.

Adenoviruses, Human↗
PubMed1997

The transcription promoter of the spliced leader gene from Trypanosoma cruzi.

A putative promoter element responsible for transcription of the spliced leader (SL) gene of Trypanosoma cruzi was identified by overlapping deletion and linker scanning analyses of the upstream flanking sequences using the bacterial chloramphenicol acetyltransferase (CAT) gene as a reporter in transient transfections of cultured epimastigotes. Deletion or substitution of a proximal sequence element (PSE) between positions -53 and -40 relative to the transcription start point eliminated CAT gene expression. Comparison of SL genes from several strains of T. cruzi revealed two alternative sequence patterns for the putative SL PSE, both composed of a short run of purines followed by a run of pyrimidines. Moreover, an examination of these sequences supports the subdivision of T. cruzi isolates into two divergent groups. Double-stranded oligonucleotides containing the sequence of the PSE exhibited specific gel mobility shifts after incubation with T. cruzi nuclear extracts, suggesting that a transcription factor binds this site. Finally, experiments designed to increase the level of CAT expression from the SL promoter suggest that it is not a strong promoter in cultured T. cruzi epimastigotes.

Animals↗
PubMed2001

Regulation of laminin beta2 chain gene expression in human cancer cell lines.

The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain mRNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.

Alternative Splicing↗