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PubMed2002

Characterization of the p10 gene region of Anticarsia gemmatalis nucleopolyhedrovirus.

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) p10 gene region was cloned, sequenced and the putative p10 gene expression characterized by Northern-blot analysis. Sequence analysis of the p10 gene region indicated the presence of two complete open reading frames (ORFs) of 713 and 281 nucleotides, which codes for polypeptides of 273 and 93 amino acids, with homology to the P26 and P10 proteins of baculoviruses, respectively. Two additional partial ORFs, coding for partial polypeptides of 110 and 146 amino acids, showed homology to the p22.2 gene of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) and p74 genes of different baculoviruses, respectively. A small ORF of 224 nucleotides coding for a protein of 74 amino acids showed homology to the 3'-end of the early p94 gene of AcMNPV. A putative baculovirus very late promoter motif TAAG was identified in the 5'-non-translated region (5'-UTR) at position-54 upstream of the start codon. The consensus polyadenylation sequence AATAAA is present 146nt downstream of the termination codon and the p10 ORF is flanked by the p26 and p74 ORFs. Homology comparisons showed that the P10 protein of AgMNPV is most closely related (82% amino acid sequence identity) to the P10 from the Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV). Transcriptional analysis of the AgMNPV p10 gene showed that p10-specific transcripts could be detected late in infection.

Amino Acid Sequence↗
PubMed1990

Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J.

Lactobacillus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in lambda gt11 and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and HJ4 lysogens produced beta-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in lambda EMBL3 with the insert DNA from phage HJ4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A Bg/II fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into Lactobacillus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an acitivity spectrum that was the same as that of helveticin J.

Amino Acid Sequence↗
PubMed1995

Determination of a 17,484 bp nucleotide sequence around the 39 degrees region of the Bacillus subtilis chromosome and similarity analysis of the products of putative ORFs.

We have determined a 17,484 bp nucleotide sequence around the 39 degrees region, located about 480 kb downstream from the zero position of the Bacillus subtilis chromosome physical map. Among the 17 putative ORFs identified, orf1 and orf2 seem to correspond to mtlA and mtlB, encoding mannitol-specific phosphotransferase enzyme II and mannitol-1-phosphate dehydrogenase, respectively. orf4 seems to be another signal peptidase I gene (sipS) of B. subtilis. The putative products of six ORFs were similar to known proteins in data banks, namely a hypothetical 29.7 kDa protein of Escherichia coli (Orf7), a lactam utilization protein (Orf8), the urea amidolyase of yeast (Orf12), the IcIR regulatory protein for aceAB of Salmonella typhimurium (Orf13), penicillin-binding protein 2 (Orf16) and aryl-alcohol dehydrogenase (Orf17). The amino acid sequence of Orf3 showed 34% identity with that of LeuC of B. subtilis, though they seem to be functionally different. The remaining seven ORFs did not show similarity to any known proteins.

Amino Acid Sequence↗
PubMed1994

The prediction of human exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames.

Discriminant analysis is applied to the problem of recognition 5'-, internal and 3'-exons in human DNA sequences. Specific recognition functions were developed for revealing exons of particular types. The method based on a splice site prediction algorithm that uses the linear Fisher discriminant to combine the information about significant triplet frequencies of various functional parts of splice site regions and preferences of oligonucleotides in protein coding and intron regions (Solovyev, Lawrence, 1994). The accuracy of our splice site recognition function is about 97%. A discriminant function for 5'-exon prediction includes hexanucleotide composition of upstream region, triplet composition around the ATG codon, ORF coding potential, donor splice site potential and composition of downstream intron region. For internal exon prediction, we combine in a discriminant function the characteristics describing the 5'-intron region, donor splice site, coding region, acceptor splice site and 3'-intron region for each open reading frame flanked by GT and AG base pairs. The accuracy of precise internal exon recognition on a test set of 451 exon and 246693 pseudoexon sequences is 77% with a specificity of 79% and a level of pseudoexon ORF prediction of 99.96%. The recognition quality computed at the level of individual nucleotides is 89% for exon sequences and 98% for intron sequences. A discriminant function for 3'-exon prediction includes octanucleotide composition of upstream intron region, triplet composition around the stop codon, ORF coding potential, acceptor splice site potential and hexanucleotide composition of downstream region.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Simulation↗
PubMed2004

Reinitiation and recycling are distinct processes occurring downstream of translation termination in yeast.

The circularisation model of the polysome suggests that ribosome recycling is facilitated by 5'-3' interactions mediated by the cap-binding complex eIF4F and the poly(A)-binding protein, Pab1. Alternatively, downstream of a short upstream open reading frame (uORF) in the 5' untranslated region of a gene, posttermination ribosomes can maintain the competence to (re)initiate translation. Our data show that recycling and reinitiation must be distinct processes in Saccharomyces cerevisiae. The role of the 3'UTR in recycling was assessed by restricting ribosome movement along the mRNA using a poly(G) stretch or the mammalian iron regulatory protein bound to the iron responsive element. We find that although 3'UTR structure can influence translation, the main pathway of ribosome recycling does not depend on scanning-like movement through the 3'UTR. Changes in termination kinetics or disruption of the Pab1-eIF4F interaction do not affect recycling, yet the maintenance of normal in vivo mRNP structure is important to this process. Using bicistronic ACT1-LUC constructs, elongating yeast ribosomes were found to maintain the competence to (re)initiate over only short distances. Thus, as the first ORF to be translated is progressively truncated, reinitiation downstream of an uORF of 105nt is found to be just detectable, and increases markedly in efficiency as uORF length is reduced to 15nt. Experiments using a strain mutated in the Cca1 nucleotidyltransferase suggest that the uORF length-dependence of changes in reinitiation competence is affected by peptide elongation kinetics, but that ORF length per se may also be relevant.

Base Sequence↗
PubMed2004

A novel outer-membrane anion channel (porin) as part of a putatively two-component transport system for 4-toluenesulphonate in Comamonas testosteroni T-2.

Inducible mineralization of TSA (4-toluenesulphonate) by Comamonas testosteroni T-2 is initiated by a secondary transport system, followed by oxygenation and oxidation by TsaMBCD to 4-sulphobenzoate under the regulation of TsaR and TsaQ. Evidence is presented for a novel, presumably two-component transport system (TsaST). It is proposed that TsaT, an outer-membrane porin, formed an anion-selective channel that works in co-operation with the putative secondary transporter, TsaS, located in the inner membrane. tsaT was identified as a 1017-bp ORF (open reading frame) on plasmid pTSA upstream of the TSA-catabolic genes in the tsa operon. Expression of tsaT was regulated by TsaR, the transcriptional activator of the tsa regulon. The presence of tsaT was concomitant with the presence of the tsa operon in different TSA-degrading isolates. tsaT was expressed in Escherichia coli and was detected in the outer membrane. A 22-amino-acid leader peptide was identified. Purified protein reconstituted in lipid bilayer membranes formed anion-selective channels with a single-channel conductance of 3.5 nS in 1 M KCl. Downstream of tsaT, a constitutively expressed 720-bp ORF (tsaS) was identified. tsaS coded for a hydrophobic protein predicted to have six transmembrane helices and which is most likely localized in the cytoplasmic membrane. tsaS is adjacent to tsaT, but showed a different transcriptional profile.

Benzenesulfonates↗
PubMed1997

Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family.

The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence. Only one homologous region was detected. This region was subsequently cloned and partially sequenced. Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected. Sequencing of the cloned S. typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S. enteritidis. These nucleotide sequences mapped upstream of the S. typhi chromosomal region homologous to the S. enteritidis virulence plasmid. The general organization of the cloned S. typhi chromosomal fragment was similar to the fimbriae-encoding region of the S. typhimurium virulence plasmid. The deduced product of orf8 in the S. typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S. typhi chromosome and in the S. enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol: disulphide oxidoreductases. The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli. The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes. The products of both genes could be detected using the T7 RNA polymerase promoter expression system. The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller. The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference. The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein.

Amino Acid Sequence↗
PubMed2004

Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs.

To find novel short coding sequences from accumulated full-length cDNA sequences, proteomic analysis of small proteins expressed in human leukemia K562 cells was performed using high-resolution nanoflow liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Our analysis led to the identification of 54 proteins not more than 100 amino acids in length, including four novel ones. These novel short coding sequences were all located upstream of the longest open reading frame (ORF) of the corresponding cDNA. Our findings indicate that the translation of short ORFs occurs in vivo whether or not there exists a longer coding region in the downstream of the mRNA. This investigation provides the first direct evidence of translation of upstream ORFs in human cells, which could greatly change the current outline of the human proteome.

Amino Acid Sequence↗
PubMed1997

Bovine coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA.

The mRNA encoding the 49-kDa nucleocapsid protein (N) of the bovine coronavirus is bicistronic. A 23-kDa protein, termed the I protein for the 'internal' open reading frame (ORF), is also synthetized but in the +1 reading frame beginning 61 nt downstream of the N start codon. Sequences flanking the N and I start codons suggest that the I ORF might be accessed by scanning ribosomes passing over the N start codon. Here we test this idea and demonstrate with translation studies both in vitro and in vivo that the I protein is synthesized according to the leaky scanning model for initiation of translation on the subgenomic N mRNA molecule.

Animals↗
PubMed1997

The effect of a nested set of C-terminal substituted deletions on the function of the alpha subunit of Escherichia coli RNA polymerase.

A genetic screen was devised to obtain plasmid-borne rpoA alleles exhibiting partial or no complementation of the chromosomal Escherichia coli rpoA341 allele responsible for a pleiotropic phenotype. Nine of the ten mutants obtained carried single base pair deletions within the 3' end of rpoA resulting in frameshifting into a 72 codon +1 orf extending from within codon L262 and terminating 16 bp downstream of the rpoA reading frame. These frameshifts give rise to a set of substituted alpha deletions that are all of the same size (334 aa) and carry segments of the Orf sequence replacing the alpha region from the C-terminus (residue 329) to various points between 272 > 319. The in vivo properties of this nested set of nine C-terminal-substituted derivatives of the alpha subunit of RNA polymerase have been assessed in terms of their assembly and transcriptional proficiency. The results indicate: (i) replacement of as much as 42 C-terminal residues of the alpha subunit does not prevent formation of a transcriptionally proficient holoenzyme form of RNA polymerase capable of complementing rpoA112(Ts); (ii) the extreme C-terminal Orf region, like that of alpha itself, is exposed in holoenzyme; (iii) these substituted deletions are not commonly functional at class I activated promoters.

Alleles↗
PubMed1988

The bovine papillomavirus P2443 promoter is E2 trans-responsive: evidence for E2 autoregulation.

The bovine papillomavirus type 1 (BPV-1) P2443 promoter is located just upstream of the E2, E3, E4 and E5 open reading frames (ORFs) and is active in both transformed rodent cells and in productively infected warts. Analysis of viral RNA structures suggests that transcripts from this promoter encode the E2 transactivator as well as the E5 oncoprotein. To study expression of P2443, the chloramphenicol acetyltransferase (CAT) reporter gene was placed downstream of this promoter, deleting the E2 and E5 ORFs, in a plasmid which contained all BPV-1 upstream sequences including the long control region (LCR). By itself, this plasmid demonstrated a low level of activity in transient assays and could be transactivated to a high level by the full-length E2 product. Transactivation of P2443 expression by E2 required the LCR in cis in an orientation- and position-independent manner, suggesting that this transactivation was mediated through the E2 responsive enhancer elements (E2RE) located within the LCR. Primer extension analysis of the 5' ends of the viral transcripts from pooled cells expressing these P2443/CAT plasmids confirmed that E2 transactivation results in an increase in the steady-state levels of RNA initiated from the P2443 promoter. Furthermore, E2 transactivation of the P2443 promoter could be inhibited by the trans-repressor encoded by the 3' portion of the E2 ORF. Thus, expression of the E2 transactivator and the E5 oncoprotein is directly regulated by transcriptional factors encoded by the E2 ORF.

Animals↗
PubMed1999

A novel internal open reading frame product expressed from a polycistronic mRNA of porcine epidemic diarrhoea virus may not contribute to virus attenuation.

Cell-culture-adapted (ca) porcine epidemic diarrhoea virus (PEDV) contains three internal open reading frames (I ORF) within the nucleocapsid protein gene and lacks the downstream counterpart of porcine transmissible gastroenteritis virus ORF7 or feline infectious peritonitis virus ORF6a. To confirm whether such features also exist in wild-type (wt) PEDV, the 3' 1800 nucleotides of its genome were sequenced and were found to be identical to those of ca virus. The coding potential of I-1 ORF was ascertained by transient expression in Vero cells followed by immunofluorescence using antipeptide sera. The I-1 protein was synthesized as a 12 kDa non-phosphorylated PEDV-specific protein that was not present in detectable amounts in virions. However, a low copy number of I-1 in the virion would suggest it is a structural component. Nevertheless, identical nucleotide sequences and gene expression strategies of attenuated ca virus and its virulent parent, wt PEDV, demonstrate that the 3' 1800 nucleotides or the genes and gene products encoded therein may not contribute to virus attenuation.

Amino Acid Sequence↗
PubMed1994

Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site: a dual role for polypyrimidine tracts in trypanosomes?

The procyclin-associated genes (PAGs) of Trypanosoma brucei are located downstream of tandemly repeated procyclin genes and belong to the same alpha-amanitin-resistant polycistronic transcription units. In procyclic form trypanosomes the PAG 1 pre-mRNA is alternatively spliced to give rise to three transcripts of 2.7 kb, 1.8 kb and 1.3 kb. The two larger transcripts contain additional short open reading frames (ORFs) upstream of the major ORF. Trans-splicing to generate these transcripts occurs downstream of three different polypyrimidine tracts. A minor population of procyclin mRNAs is also generated by alternative splicing at a polypyrimidine tract that begins 524 bp upstream of the major splice acceptor site of the procyclin beta-gene. The same polypyrimidine tract is also required for accurate polyadenylation of mRNAs from the upstream procyclin alpha-gene (1). Alternatively polyadenylated forms of PAG 1 mRNAs can also be detected. All polyadenylation sites are found at a similar distance upstream of splice-acceptor sites, in each case with a polypyrimidine tract between them. Our results point to a dual role for polypyrimidine tracts in the maturation of trypanosome mRNAs.

Alternative Splicing↗
PubMed2000

Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme.

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5' end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

Animals↗
PubMed1991

Translation of a polycistronic mRNA in the presence of the cauliflower mosaic virus transactivator protein.

Polycistronic mRNAs containing an upstream beta-glucuronidase (GUS) and a downstream chloramphenicol acetyltransferase (CAT) reporter open reading frame (ORF) were expressed in transfected plant protoplasts. CAT expression could be strongly induced by coexpression of the cauliflower mosaic virus encoded translation transactivator. Transactivation was abolished when an upstream ORF overlapped the CAT ORF for a long distance. No specific sequence elements were required for transactivation but the presence of a short ORF upstream of the GUS ORF strongly enhanced the process. The inhibitory effect of additional presumed stem structures inserted into various regions of the reporter mRNAs indicates that both ORFs are translated by ribosomes that associate with the RNA at the 5' end and reach the ORFs by a linear migration mechanism.

Base Sequence↗
PubMed2001

Functional analysis of six ORFs from Saccharomyces cerevisiae chromosome IV: two-spored asci produced by disruptant of YDR027c and strain-dependent DNA heterogeneity around YDR036c.

Six S. cerevisiae FY1679 heterozygous deletion mutants were made by replacing six open reading frames (ORFs) of the chromosome IV right arm with kanMX4 selection marker. Haploid and homozygous diploid deletion mutants were obtained from sporulation, dissection and mating experiments. No essential genes were found. The basic phenotypic analysis showed that the haploid and homozygous deletants for the ORF YDR027c (LUV1, VSP54 or RKI1) grew slowly. The diploid homozygous deletants for this ORF had a low frequency of sporulation. They produced asci with no more than one or two haploid spores and the majority of these spores formed were not viable. The deletion of the other ORFs, YDR022c (CIS1), YDR030c (RAD28), YDR032c (PST2), YDR033w (MRH1) and YDR036c, did not change the phenotypes tested in strain FY1679 or the first four ORFs in strain CEN.PK2. This work showed some differences in the DNA sequences between FY1679 and CEN.PK2: the regions immediately 1 kb upstream from YDR036c in these two strains are too different to hybridize properly, preventing deletion of YDR036c in the CEN.PK2 background by recombination with a disruption cassette designed for FY1679. In addition, there are different sets of transposable elements on the other side of the ORF, the differences starting at about 3.5 kb downstream from YDR036c.

Chromosomes, Fungal↗
PubMed2005

Collateral damage: spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa.

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1-0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.

Base Sequence↗
PubMed1994

Characterization of a Cu/Zn superoxide dismutase-encoding gene region in Drosophila willistoni.

A Cu/Zn superoxide dismutase-encoding gene (Sod) from Drosophila willistoni was cloned and sequenced. The gene shows a typical structure for a fruit-fly Sod gene, with a coding region of 462 bp in two exons separated by a 417-bp intron. Comparison of the Sod sequences from D. willistoni and D. melanogaster suggests that these species are only remotely related. Downstream from the Sod gene, there is an ORF on the opposite strand that putatively encodes the last exon of an unidentified gene. The polyadenylation signals of the two genes are separated by only 61 bp in D. willistoni, conforming to the common picture of compact dipteran genomes.

Amino Acid Sequence↗