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PubMed1991

Eukaryotic initiation factor (eIF)-4F. Implications for a role in internal initiation of translation.

In order to study the eukaryotic translation initiation mechanisms of "internal initiation," "re-initiation," and/or "coupled internal initiation," a series of model mRNAs have been constructed which contain two non-overlapping open reading frames (ORFs) that encode different lengths of rabbit alpha globin. These mRNAs, along with the bicistronic constructs TK/CAT and TK/P2CAT developed by Pelletier and Sonenberg (Pelletier, J., and Sonenberg, N. (1988) Nature 334, 320-325, 1988), were used to program an in vitro rabbit reticulocyte lysate translation system. Cap-dependent and cap-independent translation were distinguished by monitoring translation in the presence or absence of exogenously added cap analog (m7GTP). Messenger RNAs which translate both ORF1 and ORF2 by a cap-dependent mechanism, as well as mRNAs that translate ORF2 by a cap-independent mechanism while still translating ORF1 in a cap-dependent fashion have been obtained. These same alpha globin mRNAs differ by no more than 45 nucleotides in intercistronic length. Initiation factor addition studies were performed in this same in vitro translation system. Both eukaryotic initiation factor (eIF)-4F and, to a lesser extent, eIF-4B can stimulate translation of an internally located ORF independent of upstream ORF translation and in a manner not dependent on mRNA cap recognition. This indicates that the cap-recognition initiation factor, eIF-4F, and eIF-4B facilitate cap-independent and internal initiation of an open reading frame.

Animals↗
PubMed1997

Maturation and translation mechanisms involved in the expression of a myb gene of rice.

We have isolated two overlapping cDNAs coding for a MYB-related protein expressed in aerobic and anaerobic rice (Oryza sativa) roots and coleoptiles. Analysis of their sequences reveals some peculiar features, suggesting the presence of post-transcriptional regulation events: an upstream ORF, two unspliced introns and a putative leucine zipper in the ORF coded by the unspliced RNA. Transient expression in protoplasts indicates that the upstream ORF inhibits expression of a downstream coding sequence. Finally, we demonstrated that anoxia, in roots, increases the ratio between the spliced and the unspliced mRNA and affects the expression of other myb-related genes.

Gene Expression Regulation, Developmental↗
PubMed2000

Infectious hypodermal and hematopoietic necrosis virus of shrimp is related to mosquito brevidensoviruses.

We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus (PstDNV).

Amino Acid Sequence↗
PubMed1997

Comprehensive physical map of the Cydia pomonella granulovirus genome and sequence analysis of the granulin gene region.

A cloned strain of Cydia pomonella granulovirus, CpGV-M1, was obtained using successive rounds of an in vivo limiting dilution method. A detailed physical map of the genome was constructed using 11 restriction enzymes. The region containing the granulin gene and an open reading frame immediately upstream of the granulin gene was sequenced. This region showed a high degree of homology to the equivalent region from Cryptophlebia leucotreta granulovirus with 98% amino acid identity for the granulins and 68% identity for the putative polypeptides encoded by the upstream ORFs. These latter polypeptides contained two zinc finger-like motifs and showed a low degree of homology to ME53 from Autographa californica nucleopolyhedrovirus (AcMNPV). Evidence is presented for a similar upstream ORF in Artogeia rapae GV also. Hybridization studies showed that the CpGV genome had a similar overall organization to the Artogeia rapae GV genome. Hybridization between CpGV and AcMNPV was limited to fragments spanning about 15% of each genome suggesting that very few genes are highly conserved between GVs and NPVs.

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↗
PubMed1992

Sequence encoding ribosomal protein L33 of Lactococcus lactis.

A cloned fragment from Lactococcus lactis chromosome encoding the L33 ribosomal protein was sequenced. Two incomplete open reading frames (ORFs) were also found: the upstream ORF shows similarity to the tetracycline-resistance protein (Tet) of Bacillus stearothermophilus, and the downstream ORF shows homology to a protein of Bacillus subtilis participating in sporulation (SpoVE), and to proteins of Escherichia coli involved in cell division (FtsW) and the maintenance of cell shape (RodA).

Amino Acid Sequence↗
PubMed2002

Cloning and sequencing of the genes encoding cyclic tetrasaccharide-synthesizing enzymes from Bacillus globisporus C11.

The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from alpha-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to alpha-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.

Amino Acid Sequence↗
PubMed1996

A cis-acting element in the BCL-2 gene controls expression through translational mechanisms.

The bcl-2 gene becomes activated in many types of human cancers and contributes to neoplastic cell expansion, as well as to resistance to radiation and chemotherapy, by blocking programmed cell death or apoptosis. The expression of this proto-oncogene is regulated at both the transcriptional and post-transcriptional levels. DNA sequence comparisons of human, mouse, rat and chicken bcl-2 cDNAs revealed the presence of an open reading frame (ORF) [correction of (OFR)] located upstream of the normal coding region. Because upstream ORFs (uORFs) have been associated with translational repression, we analysed the functional significance of the 11 amino-acid uORF in the human BCL-2 gene (-119 to -84 bp). Deletion of this uORF from chloramphenicol acetyltransferase (CAT) reporter gene constructs that contained the bcl-2 promoter and entire 5'-untranslated region (5'-UTR), as well as introduction of an A-->T mutation at position -119 bp that destroyed the AUG-initiation codon, significantly increased CAT activity in HeLa, CEM, and other cell lines, without producing a corresponding elevation in CAT mRNA levels. Positioning this uORF, together with its accompanying Kozak sequences, between a heterologous promoter from SV40 and a CAT reporter gene resulted in marked inhibition of CAT protein production without a decrease in CAT mRNA. Mutation of the start codon (ATG-->TTG) of this uORF completely abolished its inhibitory activity, consistent with a translational mechanism. Taken together, these findings suggest that the uORF located within the 5'UTR of the bcl-2 gene is necessary and sufficient for translational regulation of bcl-2 gene expression.

Animals↗
PubMedDate not supplied

A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti.

During free-living reproductive growth, Sinorhizobium meliloti accumulates poly-beta-hydroxybutyrate (PHB) and glycogen, and produces and excretes exopolysaccharides and beta-1,2-glucan. In previous investigations, PHB-minus mutants of S. meliloti 41 were obtained and studied; and the genes for PHB biosynthesis, phaAB and phaC, were described. In this work, the role of an open reading frame (orf) upstream of phaAB is studied. This orf is designated aniA because the gene was found to be expressed during anaerobic growth. Under low oxygen conditions, glycogen decreases and the production of extracellular polymeric substances (EPS) is partially repressed. When the aniA mutant is incubated under oxygen-limiting conditions, the only significant change observed is an overproduction of EPS. Subsequent in planta tests showed that although the mutant strain produced abundant nodules, only very low acetylene-reduction activity was detected, indicating that nitrogen fixation was not adequately supported by endogenous substrates.

Alcohol Oxidoreductases↗
PubMed1997

Chicken repeat 1 (CR1) elements, which define an ancient family of vertebrate non-LTR retrotransposons, contain two closely spaced open reading frames.

Chicken repeat 1 (CR1) elements comprise a family of non-long terminal repeat (LTR) retrotransposons that have several noteworthy features. For example, whereas most other non-LTR elements have poly(A) tracts or other simple A-rich repeats at their 3' ends, the 3' ends of CR1 elements conform to the consensus [(CATTCTRT)(GATTCTRT)1-3]. CR1 elements also display an unusual bias for severe 5' truncations: only approx. 30 (out of a total of approx. 30 000) CR1 elements in the chicken genome include significant portions of the pol-like open reading frame (ORF) that we previously identified and partially sequenced [Burch et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8199-8203]. In the present study we derived a consensus sequence for this entire ORF (ORF2) as well as an upstream ORF (ORF1) and part of a 5' untranslated region (UTR). The conceptual translation product of ORF2 is predicted to contain an endonuclease domain in addition to a reverse transcriptase domain. These results suggest that CR1 elements retrotranspose using a "nick and prime" mechanism similar (but not identical) to other families of non-LTR elements.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of the groESL operon from Bacillus subtilis.

The sequence of the 10 N-terminal amino acids of a Bacillus subtilis protein that cross-reacts with antibody to Escherichia coli GroEL was used to design a set of degenerate oligonucleotide probes. These probes identified a clone which carries almost the entire groESL operon from a B. subtilis subgenomic library. By chromosomal walking, an additional fragment carrying the 3' end of groESL and its flanking sequence was isolated. Sequence analysis revealed two open reading frames (ORFs) in the cloned DNA. The upstream ORF encodes a 10-kDa protein which has 47% amino acid identity with E. coli GroES. The downstream ORF encodes a 58-kDa protein which is 62% identical to E. coli GroEL. A 2.1-kb groESL mRNA from B. subtilis was detected independently by Northern (RNA) blot analyses with a groES- and a groEL-specific probe. This demonstrated that groES and groEL are in an operon. The groESL promoter was located by using a promoter-probing plasmid, and the apparent transcription start site was mapped by primer extension analysis. The same promoter is utilized under normal and heat shock conditions. This promoter has the same features as a typical sigma A promoter. A strain in which the groESL operon was under the control of the sucrose-inducible sacB promoter was created. With this strain, it was possible to show that both groES and groEL are essential genes under both normal and heat shock conditions.

Amino Acid Sequence↗
PubMed2025

The transcriptional and translational landscape of HCoV-OC43 infection.

The coronavirus HCoV-OC43 circulates continuously in the human population and is a frequent cause of the common cold. Here, we generated a high-resolution atlas of the transcriptional and translational landscape of OC43 during a time course following infection of human lung fibroblasts. Using ribosome profiling, we quantified the relative expression of the canonical open reading frames (ORFs) and identified previously unannotated ORFs. These included several potential short upstream ORFs and a putative ORF nested inside the M gene. In parallel, we analyzed the cellular response to infection. Endoplasmic reticulum (ER) stress response genes were transcriptionally and translationally induced beginning 12 and 18 hours post infection, respectively. By contrast, conventional antiviral genes mostly remained quiescent. At the same time points, we observed accumulation and increased translation of noncoding transcripts normally targeted by nonsense mediated decay (NMD), suggesting NMD is suppressed during the course of infection. This work provides resources for deeper understanding of OC43 gene expression and the cellular responses during infection.

Humans↗
PubMed1998

Mutational analysis of Bax and Bcl-2 in childhood acute lymphoblastic leukaemia.

In childhood acute lymphoblastic leukaemia there are large interpatient variations in levels of the apoptosis-regulating proteins Bax and Bcl-2, but the molecular basis for this variation is unknown. Point-mutations in bax have been reported in cell lines derived from haematological malignancies. Frameshift mutations, which result in reduced Bax levels, have also been found in colon cancer of the microsatellite mutator phenotype. Bcl-2 overexpression, or gain of function mutations in the open reading frame (ORF) or in the translational repressor, the upstream ORF(uORF) of bcl-2, might also be important in deregulating its function or expression. We have therefore analyzed 21 bone marrow aspirates from untreated childhood acute lymphoblastic leukaemia and 2 from myeloid leukaemia for mutations in box and bcl-2. DNA sequence analysis of the ORFs of bax and bcl-2 and of the uORF of bcl-2 revealed no mutations, despite the large range in expression levels. Thus, mutations within the (u)ORFs of bax and bcl-2 that (in)activate or deregulate Bax and Bcl-2 are infrequent in primary childhood acute leukaemia and do not play a major role in regulation of the encoded proteins in this disease.

Adolescent↗
PubMed1990

Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases.

A detailed analysis of the 8130-bp open reading frame (ORF) of gene toxA and of an upstream ORF designated utxA, indicates the presence of a transcription terminator stem-loop for toxA, promoter sequences, and Shine-Dalgarno boxes for toxA and utxA. No transcription terminator between toxA and utxA is suggested by the sequence. ToxA contains two domains, one-third (C-terminal) with a repetitive structure and the residual two-thirds with no repetitions. The 2499-bp sequence encoding the repetitive structure is composed of nine groups of different short repetitive oligodeoxyribonucleotides (SRONs). A combination of these SRONs codes for five groups of combined repetitive oligopeptides (CROPs). Seven 50-amino acid (aa) CROPs and 23 CROPs of 21 aa in length are noticed. The CROPs are generally highly conserved, but four exhibit variability and possibly represent 'hot spots' of the repetitive structure. The reactivity of the C-terminal repeat with monoclonal antibody 1337C8 indicates that this part contains the carbohydrate-binding domain of ToxA. In this region homology exists between the ToxA repeats and the glucosyltransferases of Streptococci. We propose that binding of ToxA to cells occurs via the C-terminal repeat domain, with the N-terminal domain being responsible for toxic function.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and characterization of comC, a late competence gene of Bacillus subtilis.

comC is a Bacillus subtilis gene required for the development of genetic competence. We have cloned a fragment from the B. subtilis chromosome that carries comC and contains all the information required to complement a Tn917lac insertion in comC. Genetic tests further localized comC to a 2.0-kilobase HindIII fragment. Northern (RNA) blotting experiments revealed that an 800-base-pair comC-specific transcript appeared at the time of transition from exponential to stationary phase during growth through the competence regimen. The DNA sequence of the comC region revealed two open reading frames (ORFs), transcribed in the same direction. The upstream ORF encoded a protein with apparent sequence similarity to the folC gene of Escherichia coli. Insertion of a chloramphenicol resistance determinant into this ORF and integration of the disrupted construct into the bacterial chromosome by replacement did not result in competence deficiency. The downstream ORF, which contained the Tn917lac insertion that resulted in a lack of competence, is therefore the comC gene. The predicted protein product of comC consisted of 248 amino acid residues and was quite hydrophobic. The comC gene product was not required for the expression of any other com genes tested, and this fact, together with the marked hydrophobicity of ComC, suggests that it may be a component of the DNA-processing apparatus of competent cells.

Amino Acid Sequence↗
PubMed2005

New lnu(C) gene conferring resistance to lincomycin by nucleotidylation in Streptococcus agalactiae UCN36.

Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 microg/ml) but susceptible to clindamycin (MIC = 0.12 microg/ml) and erythromycin (MIC = 0.06 microg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA(N2), LnuA, and LnuA' lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

Anti-Bacterial Agents↗
PubMed2000

Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region.

A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized. This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids). An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene. Sequence analysis revealed three ORFs upstream of the gene. The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A. tumefaciens. In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions. These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B. henselae and other gram-negative bacteria such as A. tumefaciens.

Agrobacterium tumefaciens↗
PubMed1993

Molecular genetics in Saccharomyces kluyveri: the HIS3 homolog and its use as a selectable marker gene in S. kluyveri and Saccharomyces cerevisiae.

We cloned the Saccharomyces kluyveri HIS3 homolog, k-HIS3, and made a partial deletion of the gene. The k-HIS3 gene complemented a HIS3 deletion in S. cerevisiae. The DNA sequences of the open reading frames (ORFs) of the HIS3 homologs are 70% identical at the DNA level and 83% identical at the deduced amino acid level. The ORF upstream of the k-HIS3 gene is related to the PET56 gene of S. cerevisiae found upstream of the HIS3 gene of S. cerevisiae. The ORF downstream from the k-HIS3 gene is not related to the DED1 gene found downstream of the HIS3 gene in S. cerevisiae.

Amino Acid Sequence↗