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PubMed1992

A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts.

Leishmania DNA 1 (LD1) is a 27.5-kb sequence that occurs as an inverted repeat in a 55-kb multicopy, circular DNA in Leishmania infantum ITMAP263. The sequence is also found with a different genomic organization, possibly a tandem array, within a 1.5-Mb chromosome in all Leishmania isolates. About 26 stable transcripts of LD1 sequence, ranging from 0.6 to 15 kb, are found in ITMAP263. Transcripts were detected from both strands of the entire LD1 sequence, but the inverted repeat nature of the circular molecule prevented determination of whether transcription proceeded in one or both directions. Nine abundant transcripts (0.6-8.4 kb) from adjacent regions on the same strand of the repeat unit may represent mature mRNAs. One of these transcripts was shown to contain the 39-nucleotide spliced leader sequence characteristic of the 5' termini of trypanosomatid mRNAs. Several transcripts from the other strand of the repeat unit are also abundant and contain sequence complementary to some of the putative mRNAs. Less abundant, larger transcripts that span sequences encoding abundant mRNAs are also present, suggesting that transcription of LD1 is polycistronic.

Animals↗
PubMed1988

trans splicing in Leishmania enriettii and identification of ribonucleoprotein complexes containing the spliced leader and U2 equivalent RNAs.

The 5' ends of Leishmania mRNAs contain an identical 35-nucleotide sequence termed the spliced leader (SL) or 5' mini-exon. The SL sequence is at the 5' end of an 85-nucleotide primary transcript that contains a consensus eucaryotic 5' intron-exon splice junction immediately 3' to the SL. The SL is added to protein-coding genes immediately 3' to a consensus eucaryotic 3' intron-exon splice junction. Our previous work demonstrated possible intermediates in discontinuous mRNA processing that contain the 50 nucleotides of the SL primary transcript 3' to the SL, the SL intron sequence (SLIS). These RNAs have a 5' terminus at the splice junction of the SL and the SLIS. We examined a Leishmania nuclear extract for these RNAs in ribonucleoprotein (RNP) particles. Density centrifugation analysis showed that the SL RNA is predominantly in RNP complexes at 60S, while the SLIS-containing RNAs are in complexes at 40S. We also demonstrated that the SLIS can be released from polyadenylated RNA by incubation with a HeLa cell extract containing debranching enzymatic activity. These data suggested that Leishmania enriettii mRNAs are assembled by bimolecular or trans splicing as has been recently demonstrated for Trypanosoma brucei. Furthermore, we determined the partial sequence of the Leishmania U2 equivalent RNA and demonstrated that it cosediments with the SL RNA at 60S in a nuclear extract. These RNP particles may be analogous to so-called spliceosomes that have been demonstrated in other systems.

Animals↗
PubMed1994

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

Amino Acid Sequence↗
PubMed1995

The Euglena gracilis intron-encoded mat2 locus is interrupted by three additional group II introns.

The 4,144 nt Euglena gracilis chloroplast psbC intron 2 has been characterized as a single, cis-spliced 593 nt group II intron interrupted by an open reading frame of 758 codons in the loop region of domain IV. The 2,277 nt coding region of orf 758 is interrupted by two additional group II introns of 369 nt and 352 nt. Another 553 nt group II intron is located in the 5' untranslated leader region of orf 758. Because the psbC intron 2 orf encodes a maturase-like protein that has reverse transcriptase domains and a domain X characteristic of group II intron-encoded proteins, the locus has been designated mat2. The psbC intron 2 is the first member of a new category of twintron, characterized by introns within a gene within another intron. A potential role of psbC intron 2 as a "founder" intron involved in the spread of introns to new sites in the plastid genome of the Euglenophycae is discussed.

Amino Acid Sequence↗
PubMed1988

Functional analysis of alternatively spliced tyrosinase gene transcripts.

Three different cDNA clones (pmcTyr1, pmcTyr2 and pmcTyr3) representing mRNAs originating by alternative splicing of the primary transcript of mouse tyrosinase gene, were identified and characterized by sequence analysis and by a functional assay. These cDNAs were subcloned into the newly constructed expression vector pHD. After electroporation of these hybrid clones into tyrosinase negative cells, protein extracts were prepared and tested for tyrosinase enzyme activity. Only the cDNA insert of pmcTyr1 was able to confer tyrosinase enzyme activity. This cDNA encodes a protein 533 amino acid residues in length containing a putative leader peptide of 18 amino acids and six putative glycosylation sites. Comparisons of the deduced amino acid sequence of the cDNA clone pmcTyr1 with the protein sequence of tyrosinases from man, Streptomyces, Neurospora and with haemocyanin subunits from a spider showed two regions of sequence conservation. One of these regions is known to be involved in copper binding. Since this gene with the coding capacity for tyrosinase is absent in all studied c-locus lethal deletion mutant mice, we have evidence that albinism in mice is caused by mutations of the tyrosinase gene.

Amino Acid Sequence↗
PubMed1988

Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.

Capsid↗
PubMed1981

Expression of SV40 T antigen under control of adenovirus promoters.

We have obtained novel adenovirus-SV40 recombinant viruses that express wild-type SV40 large T and small t antigens under the control of different adenovirus promoters. Hybrids were constructed in vitro with SV40 DNA that contains the entire early coding region but lacks the transcriptional promoter. Recombinants were isolated by a strong biological selection for viruses that express SV40 T antigen. Analysis of several recombinant genomes indicates that they contain the SV40 A gene inserted in a variety of positions and orientations in the adenoviral genome. Moreover, the set of hybrid transcripts reveals an unexpected variety of splicing patterns. Some hybrid mRNAs transcribed from the adenovirus late promoter appear to contain the adenovirus tripartite leader sequence. Other hybrid mRNAs were transcribed from adenovirus early promoters. All recombinant mRNAs contain intact SV40 early sequences that have normal splice patterns and produce wild-type T antigens. Biochemical characterization of SV40 T antigens overproduced by the hybrid viruses indicates that they are structurally indistinguishable from wild-type SV40 large T antigen and are functionally equivalent to the D2 protein.

Adenoviruses, Human↗
PubMed1999

Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperature-sensitive and controlled by a single-nucleotide polymorphism.

Granule-bound starch synthase (GBSS), a product of the waxy gene in rice (Oryza sativa L.), is necessary for the synthesis of amylose in the endosperm. In an extended pedigree of 89 rice cultivars, we have previously shown that all cultivars with more than 18% amylose had the sequence AGGTATA at the leader intron 5' splice site, while all cultivars with a lower proportion of amylose had the sequence AGTTATA. This single-nucleotide polymorphism reduces the efficiency of GBSS pre-mRNA processing. It also results in alternate splicing at multiple sites, some of which have non-consensus sequences. Here we demonstrate that this same G-to-T polymorphism is also associated with differential sensitivity to temperature during the period of grain development. Cultivars with the sequence AGTTATA have a substantial increase in accumulation of mature GBSS transcripts at 18 degrees C compared to 25 or 32 degrees C. The selection of leader intron 5' splice sites is also affected by temperature in these cultivars. A 5' splice site -93 upstream from that used in high-amylose varieties predominates at 18 degrees C. At higher temperatures there is increased utilization of a 5' splice site at -I and a non-consensus site at +1. Potential implications of differential 5' splice site selection and associated differences in 3' splice site selection on transcript stability and translational efficiency are discussed.

Alternative Splicing↗
PubMed1993

Ribosomal protein L32 of Saccharomyces cerevisiae regulates both splicing and translation of its own transcript.

Ribosomal protein L32 of Saccharomyces cerevisiae regulates the splicing of its own transcript (1, 2) apparently by interacting with a structure composed largely of the 5' exon. However, even in strains overproducing L32 mRNA, e.g. from a cDNA copy of the gene, little accumulation of L32 is observed after a brief pulse label. When the 5' leader of the RPL32 mRNA is replaced by an exogenous leader, the amount of pulse-labeled L32 increases severalfold, suggesting that L32 regulates the translation of its own mRNA, acting through sequences in the 5' region. This conclusion was confirmed by the observation that in cells carrying a chimeric gene in which the L32 leader is fused to LacZ coding sequences, the presence of a second gene that overexpresses L32 itself reduces the level of beta-galactosidase by 50%, in spite of a doubling of L32-lacZ fusion mRNA, presumably due to stabilization of the message. Mutations within the 5' leader that abolish the regulation of splicing also abolish the regulation of translation, suggesting that the regulation of translation by L32 involves a structure similar to that proposed for the regulation of splicing. In cells overproducing L32-mRNA about half the excess mRNA was found in ribonucleoproteins of < 25 S, unassociated with ribosomal particles. Much of the rest was found in ribonucleoproteins of 80-120 S.

Base Composition↗
PubMed1990

Identification and initial characterization of a fourth leader exon and promoter of the human IGF-II gene.

Transcription of the human insulin-like growth factor II (IGF-II) gene is regulated by activation of multiple promoters which act in a tissue-specific and development-dependent manner. Previously, we have identified three different promoters, one of which is active in adult liver tissue only, whereas the other two are active in many fetal tissues and adult non-hepatic tissues. Here we report the identification of a fourth leader exon of the human IGF-II gene indicating the presence of a fourth promoter. Transcription from this promoter yields a 5.0 kb IGF-II mRNA species, which is detected in fetal liver and leiomyosarcoma tissue.

Animals↗
PubMed1995

The concerted evolution of 5S ribosomal genes linked to the repeat units of other multigene families.

We review all instances in which the nuclear 5S rRNA genes of fungi, protist, nematode, and arthropod species have been reported to be linked to the tandemly repeated units of the rDNA, trans-spliced leader, and histone multigene families. The evolution of these gene arrangements is analyzed by mapping them to independently derived phylogenies. These analyses show that 5S rRNA genes have repeatedly become linked to diverse tandemly repeated gene families and that such linkages have also been subsequently inverted or lost in some species. These variable gene linkages are probably the result of stochastic gains and losses of variant repeat units, where functional 5S rRNA had transposed, by the mechanisms which are responsible for the concerted evolution of tandemly repeated multigene families. We discuss the possible mechanisms of 5S rRNA gene transposition and suggest that the characteristics of their promoter elements, transcription, and termination signals may allow functional copies of these genes to be fortuitously transposed through an RNA intermediate. We also review the evidence which shows that the linked 5S rRNA gene copies are transcribed. We conclude that the observed patterns of 5S rRNA gene linkages to the repeat units of other tandemly repeated multigene families have likely arisen due to fortuitous recombination events and are unlikely to represent the remnants of an eubacterial-like arrangement of rDNA operons or to have been established due to selective pressures.

Biological Evolution↗
PubMed1989

An alternate promoter in the glucokinase gene is active in the pancreatic beta cell.

An alternate promoter in the glucokinase gene is active in the beta cell and produces a glucokinase mRNA which is longer and that has a different leader sequence and translation start site than the hepatic glucokinase mRNA. The glucokinase beta cell promoter is located at least 12 kilobases upstream from the glucokinase hepatic promoter. Transcription from the glucokinase beta cell promoter initiates over a region of 62 bases. The absence of a TATA box homology in the proximal promoter region may account for the diffuse transcriptional initiation. Translation of the beta cell glucokinase mRNA predicts a glucokinase isozyme that is different from the hepatic isozyme by 15 amino acids at the N terminus. The use of alternative promoters apparently enables the glucokinase gene to be regulated by insulin in the liver and by glucose in the beta cell, thus possibly constituting an important feedback control loop for maintaining glucose homeostasis. Alternate RNA splicing of the beta cell glucokinase mRNA predicts at least two beta cell glucokinase isoforms. An alternate splice acceptor site in the 4th exon of the glucokinase gene was identified in two glucokinase cDNAs from rat insulinoma tissue. Use of the alternate splice acceptor site results in a 51-nucleotide in frame deletion in the beta cell glucokinase mRNA and removal of 17 amino acids from a region of the protein situated between the putative glucose and ATP binding domains. Analysis of the pattern of RNA splicing in tissues containing beta cells indicates that the splice acceptor site utilized in producing hepatic glucokinase mRNA is also utilized in the beta cell.

Adenoma, Islet Cell↗
PubMed2004

Characterization of Trypanosoma cruzi TcRjl locus and analysis of its transcript.

RJLs represent a recently described family of the Ras-related GTP-binding proteins. The Trypanosoma cruzi orthologue, TeRjl, was isolated and its locus was characterized in a region of almost 5 kb. Its 660 bp orf, predicting a protein of 24.13 kDa, is present as a single copy gene in T. cruzi I lineage, and from 1-2 copies in T. cruzi II lineage. TcRjl shares 73% aa sequence similarity with its closest identified orthologue, T. brucei TbRjl. RT-PCR experiments revealed that TcRjl is transcribed in mRNA in the 3 main life forms of the parasite, while Northern hybridization demonstrated that TcRjl is transcribed in T. cruzi epimastigotes as at least 2 transcripts, one of around 950 nt and the other of 1500 nt. Splice-leader addition was mapped to a single site at -69 bp upstream of TcRjl orf indicating that the two mRNA types may derive in differences at the 3' of TcRjl mRNA. TcRjl locus presents considerable synteny with Rjl loci from Trypanosoma brucei and Leishmania major as available from their respective genome projects.

Amino Acid Sequence↗
PubMed1985

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 7. Continuous gene for apocytochrome b in strain EF1 (CBS 356) and sequence variation in the region of intron insertion in strain ade 7-50h.

The third BamHI fragment, containing most of gene for apocytochrome b, has been cloned and sequenced in the Schizosaccharomyces pombe strain EF1 (CBS 356). In contrast to strain ade 7-50h- (50) from the Leupold collection, in which the gene is interrupted by an intron of group II (Lang et al. 1984), the homologous gene in strain EF1 is continuous. This demonstrates that the intron in the gene for apocytochrome b is optional. Aligning the EF1 sequence with the homologous regions in strain 50, 2 base pair changes were found in the leader and 14 in the coding region. These changes led to 12 altered triplets, but 9 of them specify the same amino acid. Seven base changes were clustered within a stretch of 30 base pairs in the region in which the intron is inserted in strain 50. Five out of the resulting six triplet changes were also silent. These sequence variations around the highly conserved splice point region may be linked to the insertion or excision of the intron.

Ascomycota↗
PubMed1983

Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product.

The amino-terminal amino acid sequences of gp85 and gp37, the envelope glycoproteins of Rous sarcoma virus (RSV), were determined. Alignment of these sequences with the amino acid sequence predicted from the complete nucleotide sequence of the Prague strain of RSV, subgroup C (PR-C), has allowed us to delineate the env gene-coding region of this virus. The coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5045 to nucleotide 6862 and predict sizes of 341 amino acids (36,962 molecular weight) for gp85 and 198 amino acids (21,566 molecular weight) for gp37. Carbohydrate makes a significant contribution to the observed molecular weights of these polypeptides--the amino acid sequence contains 14 potential glycosylation sites (Asn-X-Ser/Thr) in gp85 and two in gp37. Experiments aimed at estimating the number of carbohydrate side chains yielded results consistent with most or all of these sites being occupied. Although an initiation codon is located early (codon 4) in the open reading frame, it is likely that splicing yields an mRNA on which translation initiates at the same AUG as that of the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62-amino-acid-long leader peptide. This leader contains the hydrophobic sequence (signal sequence) necessary for translocation across the endoplasmic reticulum and is completely removed from the env gene product during translation. The polyprotein precursor, Pr95env, is cleaved to gp85 and gp37 at the carboxyl side of the basic sequence:-Arg-Arg-Lys-Arg-. gp85 is attached through a disulphide linkage to gp37, and although the positions of the cysteines involved in this linkage are not known, the presence of a 27-amino-acid-long hydrophobic region at the carboxy-terminus of gp37 is consistent with its role as a membrane anchor for the viral glycoprotein complex. The location of host range variable regions with respect to the possible tertiary structure of the complex is discussed.

Amino Acid Sequence↗
PubMed1991

The R-U5-5' leader sequence of neurovirulent wild mouse retrovirus contains an element controlling the incubation period of neurodegenerative disease.

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.

Amino Acid Sequence↗
PubMed1981

Gene and mRNA for precursor polypeptide VI from adenovirus type 2.

We present a 1,040-base-pair-long sequences of adenoviruses type 2 DNA which encodes the complete gene for precursor polypeptide VI (pVI). pVI consists of 250 amino acids amounting to a molecular weight of 26,990. The proteolytic cleavage maturing pVI to virion polypeptide VI removes 33 amino acids from the amino-terminal end of the polypeptide, thus giving the mature polypeptide VI a molecular weight of 23,400. The UAA stop codon terminating pVI translation is separated by 84 nucleotides from the initiator triplet for the hexon gene. Both polypeptides are encoded by the same translational reading frame, suggesting the evolution of pVI and hexon as separate proteins by the introduction of a termination codon and selection of a new splice acceptor site in an ancestral fused polypeptide chain. The splice site where the common tripartite leader is attached to the pVI mRNA precedes the initiator codon for pVI translation by one nucleotide and forms, together with other late splice acceptor sites, a late adenovirus consensus acceptor site. We also demonstrate that the 3' end of the mRNA's belonging to the L2 3'-cotermination family is located only 31 nucleotides upstream from the splice junction of the pVI mRNA. Furthermore, we show that four novel polypeptides of molecular weights 80,000, 39,000, 36,000, and 10,500 are encoded by region L2.

Adenoviruses, Human↗
PubMed1991

Transcription initiation in the two leader exons of the rat IGF-I gene occurs from disperse versus localized sites.

Rat IGF-I mRNAs contain two different 5'-UTR sequences as a result of alternate splicing of leader exons. Using a combination of solution hybridization/RNase protection and primer extension assays, we have mapped the transcriptional start sites in these leader exons. There appear to be three putative transcription start sites in exon 1 spread over a 140-bp region, the most upstream of which defines a 381 bp-long exon 1. There appear to be three distinct start sites in exon 2, the most upstream of which defines a greater than 770 bp-long exon 2. The two downstream start sites in exon 1 together account for approximately 70% of IGF-I gene expression in adult rat liver. Essentially all of the remaining IGF-I gene expression comes from the second start site in exon 2. Rat IGF-I gene transcription may therefore be regulated by two distinct promoter regions, a disperse promoter for exon 1, with several transcription initiation sites, and a more typical promoter region for exon 2, which controls transcription initiation from a discrete region.

Animals↗