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PubMed2002

Unique features of fowl adenovirus 9 gene transcription.

We examined the transcriptional organization of fowl adenovirus 9 (FAdV-9) and analyzed temporal transcription profiles of its early and late mRNAs. At least six early and six late transcriptional regions were identified for FAdV-9. Extensive splicing was observed in all FAdV-9 early transcripts examined. Sequence analysis of the cDNAs representing the early proteins identified untranslated leader sequences, precise locations of splice donor and acceptor sites, as well as polyadenylation signals and polyadenylation sites. A unique characteristic, compared to other adenoviruses, was the detection by RT-PCR of multiple transcripts specific for each of five late genes (protein III, pVII, pX, 100K, and fiber), suggesting that FAdV-9 late transcripts undergo more extensive splicing than reported for other adenoviruses.

Adenovirus Early Proteins↗
PubMed2006

Analysis of genetic elements regulating the methionine adenosyltransferase gene in Leishmania infantum.

Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, inasmuch as its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Our prior studies have shown that the Leishmania infantum genome contains two identical copies of the gene encoding MAT (MAT2 gene), arranged in head-to-tail configuration and alternating with another gene, called LORIEN that contains a zinc-finger motif. Both genes are constitutively expressed throughout the promastigote stage of the parasite cell cycle, and their flanking regions were detected by RT-PCR. Luciferase (luc) reporter assays indicated the presence of regulatory elements within the MAT2 3'UTR and intergenic region, and fragments responsible for such regulation were identified by deletional analysis. By site-directed mutagenesis of the wild-type -42 AG recognized in the trans-splicing of the MAT2 gene, the AG slightly downstream (position -36) was observed to be able to generate the same levels of luc expression, thus suggesting that potentially this gene has alternative spliced leader acceptor sites. The stability of MAT2 and LORIEN transcripts was very similar in both logarithmic and stationary phases. However, cycloheximide (CHX) inhibition of protein synthesis increased MAT2 and LORIEN mRNA levels in the logarithmic phase only, an indication that these genes are regulated in promastigotes at the post-transcriptional level by protein factors that targets both transcripts for degradation. However, during the stationary phase, another CHX-independent factor also led to MAT2 and LORIEN mRNAs degradation, indicating the existence of different mechanisms operating on the post-transcriptional regulation of these two genes.

Animals↗
PubMed2006

Expression of mouse Abcg2 mRNA during hematopoiesis is regulated by alternative use of multiple leader exons and promoters.

ABCG2 encodes a transmembrane transporter associated with multidrug resistance in various cancer cells. ABCG2 is also highly expressed in hematopoietic stem cells (HSCs) and is down-regulated in most committed progenitors, whereas expression is sharply up-regulated during erythroid differentiation. The mechanisms for regulation of ABCG2 expression in hematopoietic cells are poorly understood. We have recently identified three novel leader exons (termed E1A, E1B, and E1C) located in the 5'-untranslated region of mouse Abcg2 mRNA by data base searches and reverse transcription-PCR. In a mouse erythroid cell line, reverse transcription-PCR analysis showed that the transcript containing E1B exon was the only isoform detected. Consistently, the E1B-containing transcript was the predominant isoform of Abcg2 mRNA in primary Ter119+ erythroid cells from mouse bone marrow as well as in mouse fetal liver cells. In contrast, the E1A-containing transcript was highly expressed in c-Kit+, Sca-1+, Lin- (KSL) bone marrow cells, especially in CD34- KSL fraction, which is highly enriched for repopulating HSCs. The differential expression pattern of Abcg2 mRNA isoforms in mouse HSCs and erythroid cells was confirmed by 5'-rapid amplification of cDNA ends, indicating that at least two different promoters control mouse Abcg2 transcription during hematopoiesis. Promoter functional assays using EGFP as reporter gene demonstrated that the E1A 5'-flanking region had promoter activity, which contains multiple putative hematopoietic transcription factor binding sites. In summary, our data show that the expression of Abcg2 during hematopoiesis is transcriptionally regulated by alternative use of multiple leader exons and promoters in a developmental stage-specific manner.

5' Untranslated Regions↗
PubMed1991

Identification of a novel transcription unit in the human insulin-like growth factor-II gene.

The human insulin-like growth factor-II (hIGF-II) gene has until now been thought to be composed of eight exons, including three independent leader exons. In the present study two additional exons, one leader exon and one alternatively used ordinate exon, have been newly identified. They were abundantly expressed in human histiocytoma tissue, generating mRNA species of about 5.0 kb in length. The new leader exon shows significant sequence similarity with the rE1 exon, previously reported to be transcribed only in the rat, and is mapped at nearly the same genomic location as in the rat. On the other hand, sequence similarity with another exon in the corresponding region of the rat genome was also found. It was, however, obvious that the rat sequence would not work as an active exon, since both splice acceptor and donor sites were deviated considerably from the consensus sequences. It has thus become apparent that the complex transcription unit of a single-copy hIGF-II gene comprises at least 10 exons, including four leader exons, one alternative exon and three common protein-coding exons.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells.

We have recently described a signal transducing molecule, 2B4, expressed on all NK and T cells that mediate non-MHC-restricted killing. The gene encoding this molecule was cloned and its nucleotide sequence determined. The encoded protein of 398 amino acids has a leader peptide of 18 amino acids and a transmembrane region of 24 amino acids. The predicted protein has eight N-linked glycosylation sites, suggesting that it is highly glycosylated. Comparison of 2B4 with sequences in the databanks indicates that 2B4 is a member of Ig supergene family, and it shows homology to murine and rat CD48 and human LFA-3. Northern blot analysis has shown at least three transcripts for 2B4 in adherent lymphokine-activated killer cells of several mouse strains and TCR-gamma/delta dendritic epidermal T cell lines but not in allospecific T cell clones. These three mRNA are the products of differential splicing of heterogeneous nuclear RNA. Southern blot analysis of genomic DNA from several mouse strains revealed that 2B4 belongs to a family of closely related genes. The 2B4 gene has been mapped to mouse chromosome 1 by analysis of 2B4 expression in recombinant inbred mouse strains.

Amino Acid Sequence↗
PubMed2003

Genetic content and evolution of adenoviruses.

This review provides an update of the genetic content, phylogeny and evolution of the family Adenoviridae. An appraisal of the condition of adenovirus genomics highlights the need to ensure that public sequence information is interpreted accurately. To this end, all complete genome sequences available have been reannotated. Adenoviruses fall into four recognized genera, plus possibly a fifth, which have apparently evolved with their vertebrate hosts, but have also engaged in a number of interspecies transmission events. Genes inherited by all modern adenoviruses from their common ancestor are located centrally in the genome and are involved in replication and packaging of viral DNA and formation and structure of the virion. Additional niche-specific genes have accumulated in each lineage, mostly near the genome termini. Capture and duplication of genes in the setting of a 'leader-exon structure', which results from widespread use of splicing, appear to have been central to adenovirus evolution. The antiquity of the pre-vertebrate lineages that ultimately gave rise to the Adenoviridae is illustrated by morphological similarities between adenoviruses and bacteriophages, and by use of a protein-primed DNA replication strategy by adenoviruses, certain bacteria and bacteriophages, and linear plasmids of fungi and plants.

Adenoviridae↗
PubMed1993

Translation of equine infectious anemia virus bicistronic tat-rev mRNA requires leaky ribosome scanning of the tat CTG initiation codon.

We have examined the translational regulation of the equine infectious anemia virus (EIAV) bicistronic tat-rev mRNA. Site-directed mutagenesis of the tat leader region followed by expression of the tat-rev cDNA both in vitro and in transiently transfected cells established that tat translation is initiated exclusively at a CTG codon. Increasing the efficiency of tat translation by altering the CTG initiator to ATG resulted in a dramatic decrease in translation of the downstream (rev) cistron, indicating that leaky scanning of the tat CTG initiation codon permitted translation of the downstream rev cistron. Since the tat leader sequences precede the major EIAV splice donor and are therefore present at the 5' termini of both spliced and unspliced viral mRNAs, the expression of all EIAV structural and regulatory proteins is dependent on leaky scanning of the tat initiator.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and developmental expression of the alpha-2 tubulin gene of Caenorhabditis elegans.

Alpha tubulin isotypes are encoded by at least four genes designated alpha-1 to alpha-4 in the nematode Caenorhabditis elegans. We describe here, molecular cloning of the alpha-2 tubulin gene, located on chromosome I, that encodes a protein of 449 amino acids that has high homology to human, mouse and Drosophila alpha tubulins, but relatively lower homology to the yeast alpha tubulins. The alpha-2 tubulin gene is trans-spliced to the SL1 leader sequence. Northern analysis shows that the gene is increasingly transcribed during the early (L1-L3) larval stages but has a lower level of transcription in L4 L4 larvae, adults, and embryos. Using an alpha-2-lacZ fusion gene expression in transgenic animals, we show that the gene is expressed in a tissue-specific manner in the intestine, pharyngeal muscle cells, and a subset of neurons which include a class of DB and VB motor neurons in the ventral nerve cord, posterior touch receptor neurons, PLML, PLMR, in the lumbar ganglia; PVT in the pre-anal ganglion, and ALA in the dorsal ganglion in the head. Our results support the notion that tubulin structure may contribute to the functional specialization of microtubules.

Amino Acid Sequence↗
PubMed1984

mRNAs from human adenovirus 2 early region 4.

The molecular structure of the mRNAs from early region 4 of human adenovirus 2 has been studied by Northern blot analysis, S1 nuclease analysis, and sequence analysis of cDNA clones. The results make it possible to identify four different splice donor sites and six different splice acceptor sites. The structure of 12 different mRNAs can be deduced from the analysis. The mRNAs have identical 5' and 3' ends and are thus likely to be processed from a common mRNA precursor by differential splicing. The different mRNA species are formed by the removal of one to three introns, and they all carry a short 5' leader segment. The introns appear to serve two functions; they either place a 5' leader segment in juxtaposition with an open reading frame or fuse two open translational reading frames. The early region 4 mRNAs can encode at least seven unique polypeptides.

Adenoviruses, Human↗
PubMed1997

Mouse IL-1 receptor antagonist isoforms: complementary DNA cloning and protein expression of intracellular isoform and tissue distribution of secreted and intracellular IL-1 receptor antagonist in vivo.

IL-1R antagonist (IL-1Ra) is a competitive inhibitor of the binding of IL-1 to IL-1R. IL-1Ra refers to two different proteins derived from the same gene by alternate splicing of two different first exons. One protein contains a leader sequence and is secreted (sIL-1Ra), whereas the other remains intracellular (icIL-1Ra). We describe the cloning of mouse icIL-1Ra cDNA, the expression of the recombinant mouse icIL-1Ra protein, and the tissue distribution of sIL-1Ra and icIL-1Ra mRNA and of icIL-1Ra protein in control and LPS-injected mice. As described in the human and the rabbit, mouse icIL-1Ra protein differs from mature mouse sIL-1Ra protein by seven amino acids at the amino terminus. In addition, human and mouse icIL-1Ra are 77% identical. Regulation of IL-1Ra isoforms was examined in normal mice and after LPS injection. Circulating levels were undetectable in control mice, but were strongly increased 4 h after LPS injection. Using a ribonuclease protection assay (RPA), we found that icIL-1Ra mRNA was expressed constitutively in skin and in LPS-stimulated RAW 264.7 murine macrophages. Consistent with the RNA studies, Western blot analysis showed that murine icIL-1Ra protein was constitutively expressed in skin and in LPS-stimulated RAW 264.7 cells. In contrast, sIL-1Ra mRNA was not detected by RPA in tissues of control mice, but was strongly up-regulated in the lung, spleen, and liver after LPS injection. Using RPA, primer extension assay and 5' rapid amplification of cDNA ends, we were able to demonstrate the presence of different transcription start sites for murine sIL-1Ra mRNA.

Alternative Splicing↗
PubMed1992

Genomic organization of the mouse granzyme A gene. Two mRNAs encode the same mature granzyme A with different leader peptides.

Granzyme A is a serine protease that, together with the other granular components of cytotoxic T lymphocyte (CTL) cells, has been implicated in the cytolysis process. We report here two different messages and the genomic organization of the mouse granzyme A gene. The granzyme A gene is composed of six exons spanning 7 kilobases. Alternative splicing of the second exon results in the two transcripts. The two mRNA species encode the same mature granzyme A protein but with different leader sequences. The first (HF1) encodes a typical leader signal sequence similar to other granzymes, but the second (HF2) putative leader sequence is different and less hydrophobic. Both messages are present in cultured CTL cell lines and in normal lymphoid tissues. They are both induced when CTL cells are activated in vitro or in vivo. Both messages can be translated in vitro, although the HF1 message appears to be much more efficient as a template. The putative 5' promoter region of the HF gene sequenced (500 base pairs of upstream sequences) contains no well defined promoter sequences aside from the TATA box. The results suggest that (a) granzyme A may be produced with putative different leader sequences from two different mRNAs; (b) this may provide a model system for studying alternate splicing and the evolution of a complex enzymatic system in an organelle; and (c) the genomic DNA reported will be useful for studying transcription regulations involved in controlling the specific expression pattern of this gene.

Amino Acid Sequence↗
PubMed1990

Tandemly duplicated Caenorhabditis elegans collagen genes differ in their modes of splicing.

Caenorhabditis elegans contains 50 to 150 collagen genes dispersed throughout its genome. We have determined the complete nucleotide sequences of two collagen genes, col-12 and col-13, that are separated by only 1800 bases and are transcribed in the same direction. The 951 nucleotides of their coding regions differ by only five nucleotides (99.5% identity). The amino acid sequences are identical except for two conservative amino acid changes within the putative secretory signal sequences, so the mature forms of the col-12 and col-13 collagens would be identical. The position and sequence of the intron (52 base-pairs) within the coding region of each gene are perfectly conserved. In contrast to the coding regions and the introns, the 5' and 3' flanking regions show little sequence similarity, col-12 and col-13 are expressed at similar levels at the same developmental stages, and appear to utilize conserved TATA boxes and transcription start sites. The major differences between the genes is that, preceding the initiator ATG, col-12 has a cis-spliced intron, while col-13 is transspliced. Thus, col-12 and col-13 are essentially identical in all aspects except that the col-12 mRNA has a 26-nucleotide cis-spliced leader at the same place where the col-13 mRNA has a 22-nucleotide trans-spliced leader. These results suggest that col-12 and col-13 are derived from a gene duplication and that sequence homology in the coding regions, but not in the flanking regions, has been maintained by gene conversion. The fact that the only significant difference between the two genes is in their modes of splicing suggests that cis and trans-splicing can be interchanged during gene evolution.

Amino Acid Sequence↗
PubMed1992

Exon sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae.

A survey of exon sequence and structure requirements for splicing was undertaken using labeled pre-tRNA substrates prepared by in vitro transcription of bacterial promoter-yeast tRNA(Tyr) gene fusions. Transcription templates were assembled from oligonucleotide cassettes allowing analysis of 22 derivatives affecting each of the potential secondary and certain tertiary interactions in the pre-tRNA. Effects on both excision of the intervening sequence by yeast endonuclease and joining of exons by ligase were examined. Replacements within the D- and T-stems and anticodon stems revealed that while the primary sequences of these segments were not essential for splicing, formation of base-paired structures was required. Replacements which altered the primary sequence while retaining the secondary structure of the aminoacyl stem allowed efficient excision by endonuclease but reduced joining by ligase. Potentially, the effects of changes within these stems may be indirect through effects on adjacent or overall structure. The presence of either structured or unstructured 5' leader and/or 3' trailer sequences had no effect on either splicing step. Alterations in the conserved Levitt tertiary pair (G15/C48), previously implicated in splicing of pre-tRNA(Phe), did not alter splicing of pre-tRNA(Tyr). A precursor in which the small (type I) extra arm in pre-tRNA(Tyr) was replaced with the large (type II) extra arm sequence from tRNA(Ser) was efficiently spliced. These and previous results suggest that only limited features of exon sequence or structure are recognized by the splicing enzymes.

Base Sequence↗
PubMed1993

A nuclear lamin of the nematode Caenorhabditis elegans with unusual structural features; cDNA cloning and gene organization.

This report describes the characterization of the nuclear lamin CeLam-1 of the nematode Caenorhabditis elegans by molecular analysis of the corresponding complete cDNA and gene sequences. The primary structure of CeLam-1, representing only the third non-vertebrate lamin sequence currently known, follows essentially the features displayed by the B-type lamins of vertebrates and Drosophila. The nematode lamin shows, however, some exceptional properties. First, it lacks the SPTR sequence in front of the coil 1a domain which constitutes the major mitotic cdc2 kinase phosphorylation site. Second, two prominent deletions occur in the CeLam-1 sequence. One eliminates 14 amino acid residues from the coil 2 domain. A larger deletion of approximately 25 residues results in the shortest lamin tail domain documented so far. The latter corresponds to a region which varies considerably in sequence from highly acidic in vertebrate B-type lamins to rather basic in Drosophila lamin Dmo. CeLam-1 is encoded by a single 2.3 kb mRNA which is abundantly expressed in mixed-stage worm populations. The 5'-end of the mRNA is generated by trans-splicing to the SL1 leader sequence. The CeLam-1 gene extending over 2.7 kb is located on chromosome I. The gene is composed of 6 exons and 5 short introns, which all interrupt the coding sequence. Surprisingly, none of the intron positions has a counterpart in either the Drosophila lamin Dmo or the vertebrate lamin genes.

Amino Acid Sequence↗
PubMed1992

Late promoter of human papillomavirus type 8 and its regulation.

Human papillomavirus type 8 (HPV8) belongs to the HPV types associated with skin carcinomas of patients with epidermodysplasia verruciformis (EV). Its noncoding regulatory sequences (NCR) were shown to drive the expression of the reporter gene chloramphenicol acetyltransferase (cat) in transient assays with human epithelial cells (HT3 cells). This constitutive activity could be enhanced by coexpression of the HPV8 transactivator protein E2. The analysis of 5' deletions of the NCR showed that the EV-specific sequence motif M33 and the neighboring AP1 site are essential for the promoter activity, whereas 44 nucleotides located immediately upstream of M33 are strongly inhibitory. The same effects were observed in simian virus 40-immortalized fetal keratinocytes (SV61 cells) and spontaneously immortalized skin keratinocytes (HaCaT cells). By using primer extension and RNase protection analyses two promoters could be identified within the HPV8 NCR. A nested set of weak signals, corresponding to start sites between positions 175 to 179, represented the previously described E6 promoter. The vast majority of transcripts was initiated at position 7535 and shown to undergo processing at an NCR-internal splice donor (positions 1 to 8). The promoter P7535 is similar to late promoters of other skin-associated papillomaviruses as far as localization, transcript structure, and sequence characteristics are concerned. To confirm that P7535-initiated transcripts proceed indeed to the L1 gene for the major capsid protein, viral mRNAs from an HPV8-induced lesion of a patient with EV were characterized by RNase protection and sequence analysis of polymerase chain reaction-amplified cDNAs. The NCR leader (positions 7535 to 4) appeared in two messages with three exons each. The third exon started with the second ATG codon of L1 in both cases; the short central exons from the 3' part of the early coding region were defined by a common splice acceptor site (position 3303) and different splice donor sites (positions 3443 and 3704).

Adult↗
PubMed1996

Characterization of the spliced pol transcript of feline foamy virus: the splice acceptor site of the pol transcript is located in gag of foamy viruses.

Foamy viruses, or spumaviruses, are distinct members of the Retroviridae. Here we have characterized the long terminal repeat of the feline, or cat, foamy virus by determining the locations of the transcriptional start site and the poly(A) addition site. The splice donor and splice acceptor sites of the subgenomic mRNA responsible for Pro-Pol protein expression were identified by nucleotide sequencing of the corresponding cDNAs. The leader exon of the feline foamy virus is 57 nucleotides long. The splice acceptor of the subgenomic pol mRNA was found to be located in gag. The location of the splice acceptor of the human foamy virus pol mRNA was confirmed to map in gag. The pol splice acceptor site in gag of the cat foamy virus is located further downstream than that of human foamy virus.

Animals↗
PubMed1997

Differentially expressed, abundant trans-spliced cDNAs from larval Brugia malayi.

Isolation and cloning of abundant reverse transcriptase-polymerase chain reaction (RT-PCR) products from the filarial nematode Brugia malayi using the conserved nematode spliced leader sequence and poly A as amplification targets has allowed us to identify abundant, stage specific transcripts from infective and post-infective larvae. The predicted protein products of the most prominent full-length transcripts from mosquito-derived L3 parasites are: (i) Bm-ALT-1, a homologue of a Dirofilaria immitis abundant larval protein: (ii) Bm-CPI-1, a cystatin-type cysteine protease inhibitor; (iii) Bm-ALT-3, a novel predicted 6 kDa glycine/tyrosine-rich protein; and (iv) Bm-TPH-1, a homologue of a mammalian translationally-controlled tumour protein. Some transcripts were not full-length but had mis-primed at A-rich stretches of coding sequence: the most abundant of these was Bm-col-3, a which encodes a collagen homologous to Bp-COL-1 of Brugia pahangi. Similar analysis of abundant spliced leader (SL)/oligo-dT products from fourth-stage larvae 9 days post-infection yielded two dominant transcripts: (i) Bm-cdd-1, which encodes a protein with homology to cytidine deaminase, differing at only one amino acid position from its homologue described in Brugia pahangi; and (ii) the same truncated form of Bm-col-3 found in L3 preparations. Expression of the major transcripts was assessed by PCR amplification of cDNA libraries derived from each stage of the life cycle. alt1, alt-3 and cpi-1 were all found to be specific to the L3 stage, while cdd-1 was found only in the L4 cDNA library. Expression of these larval-specific transcripts was not detected in either microfilarial or adult libraries.

Aedes↗
PubMed1998

Characterization and expression of the phytochrome gene family in the moss Ceratodon purpureus.

In the moss Ceratodon purpureus, phytochrome is encoded by two different genes, CpPHY1 and CpPHY2. CpPHY2 represents a conventional type phytochrome characterized by a C-terminus homologous to the catalytic domain of bacterial sensor histidine kinases, whereas CpPHY1 represents an unique phytochrome, which carries a C-terminus homologous to the catalytic domain of eukaryotic serine/threonine/tyrosine kinases. Southern blot analysis revealed that CpPHY1 is present in different Ceratodon cultivars which were collected in Germany and in Finland, implying that CpPHY1 represents a functional and active gene in Ceratodon, but CpPHY1 homologous genes could not be detected in another moss, Physcomitrella patens, or in Arabidopsis thaliana. cDNA analysis of CpPHY1 revealed the presence of a hitherto unnoticed intron within the 3' region. This results in a change of the sequence of the 11 C-terminal amino acids from KLSSHSYLTSK to FSSYQDSYPSTEELS. CpPHY1 and CpPHY2 mRNAs appear to accumulate in a light-independent manner, with CpPHY2 being much more strongly expressed than CpPHY1. Accordingly, in crude protein extracts, CpPHY2 is clearly detectable by Western blot analysis, whereas CpPHY1 is not. Light-dependent expression of CpPHY2 can be detected at the post-transcriptional level; during a 7-day period of dark adaptation, pronounced CpPHY2 accumulation occurs. Upon transfer to white light, dark-accumulated CpPHY2 is depleted within 24 h. That depletion can be completely inhibited by the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), implying that photosynthesis is strongly involved in the adjustment of phytochrome steady-state concentrations in Ceratodon. The presence of an ORF within the 5' UTR region of CpPHY2 (uORF) encoding peptide MKEFSSTSRSLMIVGIY suggests regulation at the translational level. The uORF resides on a short intron which is excised from the 5' leader in a light-dependent manner, resulting in the formation of an alternative uORF encoding peptide MEEEEDCVP.

Amino Acid Sequence↗