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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↗
PubMed1995

Characterization of SFP2, a putative sulfate permease gene of Saccharomyces cerevisiae.

The SFP2 gene of Saccharomyces cerevisiae has been characterized. The deduced amino acid sequence contained twelve highly hydrophobic domains and showed 50, 47, 44 and 48% homologies to Neurospora crassa sulfate permease II (CYS14), soybean GMAK170 nodulin, human colon mucosa protein (DRA) and a putative open reading frame (ORF) downstream of Escherichia coli prs (phosphoribosyl pyrophosphatate synthetase) gene, respectively, in the aligned regions. Cells lacking SFP2 were viable and displayed no obvious decrease in their growth rate. Southern blot analysis revealed that SFP2 exists as a single copy in haploid genome. Northern blot analysis showed that SFP2 produced a 2.8-kb transcript which was highly expressed under sulfur derepressing condition. SFP2 mRNA was found to turn over with a half-life of approximately 15 min, which may contribute to the regulation of sulfate permease function, and reached its maximal level in about 22 h after depression.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome.

The citrus tristeza closterovirus (CTV) RNA genome was cloned as cDNA generated from both CTV-specific double-stranded RNA and genomic RNA, and the sequence of the 3' 7292 nucleotides was determined. The sequenced portion contained eight open reading frames potentially encoding, in the 5' to 3' direction, proteins with the apparent molecular weights of 65, 61, 27, 25 (capsid protein, CP), 18, 13, 20, and 23 kDa, and a potential noncoding region of 277 nucleotides. The 65-kDa protein is a viral homolog of cellular hsp70 heat shock proteins (hsp), the 61-kDa protein is distantly related to the hsp90 proteins, and the 27-kDa protein is a diverged copy of the CP. Database searches did not identify any protein sequences of significant similarity to the remaining four ORFs downstream of the CP. A specific four-gene module consisting of the hsp70 protein, the hsp90-related protein, the diverged copy of the CP, and the CP itself was found to be common in organization between CTV and beet yellows closterovirus. All four proteins in this module were highly conserved, indicating that these viruses probably have evolved from a common ancestor.

Amino Acid Sequence↗
PubMed1987

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins.

The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.

Amino Acid Sequence↗
PubMed1996

Nucleotide sequence of the genome of a citrus isolate of olive latent virus 1.

The 3699 nt genome of olive latent virus 1 (OLV-1), described years ago from Southern Italy as a putative sobemovirus, was completely sequenced. OLV-1 genomic RNA was not polyadenylated and had a structure virtually identical to that of species of the Necrovirus rather than the Sobemovirus genus. Five open reading frames (ORFs) were identified, of which the 5'-proximal encoded a 23K protein and ended with an amber codon whose readthrough could yield a putative 82K product. This polypeptide had extensive sequence similarity with polymerases of serotypes A and D of tobacco necrosis necrovirus (TNV-A and TNV-D) and species of the family Tombusviridae and related genera (Dianthovirus and Machlomovirus). Two small ORFs followed, which encoded polypeptides of 8K and 6K, respectively. The 6K product had extensive homology with the comparable 6K protein of TNV-A and was also related to the 11K protein of shallot latent carlavirus, one of the "triple block" polypeptides involved in cell-to-cell virus movement. The 3'-proximal ORF was in the same position as the coat protein (CP) cistron of necroviruses and encoded a 30K product related to CP of both TNV-A and -D. Computer-assisted comparative analysis of structural and non-structural proteins of OLV-1, TNV-A and TNV-D disclosed on overall distant relationship between OLV-1 and TNV-D. OLV-1 genome appeared homologous to that of TNV-A, but differences from TNV-A were the absence of the small ORF downstream of the CP cistron and in the low degree of sequence identity in CP (39% aa identity). OLV-1 is serologically distantly related to TNV-A and even more distantly related to TNV-D. We propose that OLV-1 is a necrovirus species in its own right.

Amino Acid Sequence↗
PubMed2006

Cloning and molecular characterization of three novel LMW-i glutenin subunit genes from cultivated einkorn (Triticum monococcum L.).

Three novel low molecular weight (LMW) glutenin subunits from cultivated einkorn (Triticum monococcum L., A(m)A(m), 2n = 2x = 14) were characterized by SDS-PAGE and molecular weights determined by MALDI-TOF-MS. Their coding genes were amplified and cloned with designed AS-PCR primers, revealing three complete gene sequences. All comprised upstream, open reading frame (ORF), downstream and no introns were present. The deduced amino acid sequences showed that all three genes, named as LMW-M1, LMW-M3 and LMW-M5, respectively, belonged to the LMW-i type subunits with the predicted molecular weight between 38.5206 and 38.7028 kDa. They showed high similarity with other LMW-i type genes from hexaploid bread wheats, but also displayed unique features. Particularly, LMW-M5 subunit contained an extra cysteine residue in the C-terminus except for eight conserved cysteines, which resulted from a single-nucleotide polymorphism (SNP) of the T-C transition, namely arginine --> cysteine substitution at position 242 from the N-terminal end. This is the first report that the LMW-i subunit contained nine cysteines residues that could result in a more highly cross-linked and more elastic glutenin suggesting that LMW-M5 gene may associates with good quality properties. In addition, a total of 25 SNPs and one insertions/deletions (InDels) were detected among three LMW-i genes, which could result in significant functional changes in polymer formation of gluten. It is anticipated that these SNPs could be used as reliable genetic markers during wheat quality improvement. The phylogenetic analysis indicated that LMW-i type genes apparently differed from LMW-m and LMW-s type genes and diverged early from the primitive LMW-GS gene family, at about 12.92 million years ago (MYA) while the differentiation of A(m) and A genomes was estimated at 3.98 MYA.

Amino Acid Sequence↗
PubMed1991

Two bacteriophage T4 base plate genes (25 and 26) and the DNA repair gene uvsY belong to spatially and temporally overlapping transcription units.

The bacteriophage T4 DNA recombination-repair gene uvsY located at or near an origin of DNA replication and adjacent to the late base plate genes 25 and 26. Our present results reveal a complex transcription pattern in the region encompassing these genes. Most significantly, uvsY and two ORFs, downstream of it, all of which are transcribed from a middle promoter before the onset of DNA replication, are also part of a larger late transcription unit which includes the base plate genes 25 and 26. The late genes 25 and 26 are transcribed not only late, but also early from one or several early promoters further upstream. Translation, however, is inhibited by secondary structures which sequester the ribosome binding site in the early transcript. We discuss possible advantages of these transcriptional patterns for T4 DNA recombination, replication, and repair. The predicted and in vivo-expressed 23.9-kDa product of gene 26 is smaller than the reported size of gene 26 protein isolated from base plates, suggesting that nascent gp26 might be processed to a larger protein during assembly.

Base Sequence↗
PubMed1995

Sequence and transcriptional analysis of a DNA region involved in the production of capsular polysaccharide in Streptococcus pneumoniae type 3.

The nucleotide (nt) sequence of a 9704-bp EcoRI fragment of Streptococcus pneumoniae (Sp) type-3 DNA has been determined and found to contain one partial and five complete open reading frames (ORFs). One of these ORFs corresponds to the cap3 A gene coding for the UDP-glucose (UDPGlc) dehydrogenase which is directly responsible for the transformation of some unencapsulated serotype-3 Sp mutants to the encapsulated phenotype [Arrecubieta et al., J. Bacteriol. 176 (1994) 6375-6383]. The two ORFs downstream from this gene (cap3B and cap3C) encode proteins with molecular masses of 49 and 34 kDa. Analysis of the deduced amino acid (aa) sequences of Cap3B and Cap3C shows homology to polysaccharide synthases and UDPG1c pyrophosphorylases, respectively. Furthermore, genetic complementation analysis showed that cap3C restored the galU defect of an Escherichia coli mutant. Northern blots have shown that cap3A, cap3B and cap3C constitute a single transcriptional unit, and primer extension analysis has revealed that the transcription start point is preceded by a nt sequence identical to the sigma 70 consensus promoter sequence of E. coli. The sequence upstream from this cluster also has a high degree of similarity with genes postulated to be essential for capsular production in several Gram+ bacteria. However, Northern blot analysis and insertion-duplication mutagenesis indicated that genes located in this region are not necessary for type-3 capsule production in the Sp strain 406.

Amino Acid Sequence↗
PubMedDate not supplied

Analysis of genes encoding the cell division protein FtsZ and a glutathione synthetase homologue in the cyanobacterium Anabaena sp. PCC 7120.

Heterocysts, cells specialized in nitrogen fixation in Anabaena sp. PCC 7120, lose the potential for cell division once fully differentiated. This suggests that cell division activity is differentially regulated in heterocysts and vegetative cells. FtsZ has been shown to play a crucial role in bacterial cell division. Two degenerate oligonucleotide primers were designed to detect, by polymerase chain reaction (PCR), an ftsZ homologue from the heterocystous cyanobacterium Anabaena sp. PCC 7120. A PCR-amplified DNA fragment was cloned and used as a probe to isolate the entire ftsZ gene of Anabaena sp. PCC 7120. The deduced amino acid sequence shares strong similarities with other FtsZ proteins, suggesting remarkable conservation of the FtsZ protein during evolution. An ORF downstream of ftsZ, which would be transcribed in the opposite direction compared to ftsZ, could encode a polypeptide with significant sequence similarity to the glutathione synthetase from Escherichia coli. Inactivation experiments in vivo for both ftsZ and the glutathione synthetase gene did not yield any double recombinants either in the presence or in the absence of combined nitrogen, suggesting that both genes are essential for cell growth under these conditions.

Amino Acid Sequence↗
PubMed2004

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11.

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.

Amino Acid Sequence↗
PubMed2005

Nucleotide sequence and transcriptional analysis of the pif gene of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV).

Defective viruses, not transmissible alone, increase the transmissibility of complete genotypes in natural populations of Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV). The defective phenotype is associated with a 15 kb deletion, which includes the pif (per os infectivity factor) gene. The sequence of a 2.4 kb fragment that includes pif was determined. Multiple transcripts encompassing pif were detected by Northern blot analysis. RT-PCR and nuclease protection analysis demonstrated the presence of run-through transcripts starting upstream of pif. A 2.0 kb messenger started from a CTAAG promoter motif located 11 nt upstream of the pif start codon, and ended 450 nt downstream from the pif stop codon. This pif mRNA included a small downstream ORF (homologous to Se37). A transcript of 0.8 kb was detected that may correspond to a specific transcript from this small ORF. This transcript would start at a late consensus motif internal to pif coding sequences, ending at the same polyadenylation signal as the pif transcript. These transcription features resemble those of pif transcription in Spodoptera littoralis NPV, although the genomic location of pif is not equivalent in the two viruses. SfMNPV pif can encode a protein of 529 amino acids, closely related to Spodoptera exigua MNPV PIF.

Amino Acid Sequence↗
PubMed2026

A-to-I RNA editing remodels 5'-UTR initiation codons to tune translational output.

A-to-I RNA editing is a prevalent post-transcriptional modification in higher eukaryotes that converts adenosine to inosine within RNA molecules. Because inosine is interpreted as guanosine during translation, editing can alter codon identity and potentially influence translation initiation signals. Here, we examined whether A-to-I editing within the 5' untranslated region (5'-UTR) can remodel upstream initiation codons and thereby tune downstream translation. Using luciferase-based reporter systems, we show that AUA-to-AUI editing generates an initiation-competent inosine-containing codon, whereas AUG-to-IUG editing markedly attenuates initiation and can relieve uORF-mediated repression. Quantitative in vitro and cellular assays establish the initiation hierarchy AUA&#x2009;<&#x2009;AUI&#x2009;<&#x2009;AUG, with IUG exhibiting strongly reduced initiation efficiency. Importantly, AUI-mediated upstream initiation did not behave like a canonical AUG-initiated uORF in the tested contexts; its effect on downstream ORF translation was modest and context-dependent. Transcriptome-wide bioinformatic analysis identified endogenous human transcripts whose 5'-UTRs harbor editing sites compatible with initiation-codon gain or attenuation. Reporter validation using native 5'-UTR sequences supports the possibility that editing-dependent initiation-codon remodeling can tune translational output in living cells, particularly through AUG-to-IUG-mediated derepression. Together, these findings establish a reporter-based framework in which A-to-I editing can remodel 5'-UTR initiation codons, while highlighting the need for endogenous protein-level and native-locus validation to determine physiological relevance.

RNA Editing↗
PubMed2001

Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication.

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5' leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

Animals↗
PubMed1988

Regulation of two nested proteins from gene 49 (recombination endonuclease VII) and of a lambda RexA-like protein of bacteriophage T4.

Phage T4 gene 49, encoding recombination endonuclease VII, specifies, by initiation from an AUG and an internal GUG codon, two in-frame overlapping peptides (of 18 and 12 kD). The gene is transcribed early and late, albeit from different promoters. The sequence predicts that in long early transcripts, initiated far upstream of the coding sequence, the Shine-Dalgarno sequence of the first ribosome binding site can be sequestered in a hairpin and/or cleaved. These processes might reduce initiation from the first AUG and facilitate initiation of the 12-kD peptide from the internal GUG. The potential of this hairpin to participate in Y structures or cruciforms suggests possible autoregulation. Shorter, more stable late transcripts initiated from a late promoter immediately upstream of the first ribosome binding site cannot form this hairpin. More efficient translation of the longer 18-kD gene 49 peptide from these late transcripts accounts for the strong dependence of endonuclease VII activity on late gene expression. An ORF downstream from gene 49 can be translated from a motA-dependent transcript that starts inside gene 49 as well as from the gene 49 transcripts. Its initiation codon overlaps the stop codon of gene 49, suggesting some coupling of translation. The deduced protein resembles, among others, the RexA protein of phage lambda. Possible implications for T4 recombination and for the interference of lambda lysogens with T4 gene 49 and rII mutants are discussed.

Amino Acid Sequence↗
PubMed2001

The regulation of ornithine decarboxylase gene expression by sucrose and small upstream open reading frame in tomato (Lycopersicon esculentum Mill).

We identified a near-full-length cDNA clone encoding ornithine decarboxylase (ODC) from tomato (Lycopersicon esculentum Mill). It contained a small upstream open reading frame (uORF) within its 5' untranslated region. An in vitro translation assay demonstrated that the uORF repressed expression of downstream ORF. Neither nucleotide nor predicted peptide sequence of the uORF was responsible for the repression. The presence of upstream AUG codon was shown to be responsible. ODC expression appeared to be organ specific. The ODC gene was expressed in roots, hypocotyls and sink leaves but not in source leaves. ODC transcripts were observed in apical meristem of primary roots, and were distributed in cells of cortex layer preferentially. ODC expression responded immediately to sucrose availability via the sucrose-specific pathway independent of hexokinase. Sucrose induction of ODC gene was seen in roots, hypocotyls and flowers but not in mature leaves. Moreover, only the root apical meristem responded to sucrose availability. These observations indicate that the spatial pattern of ODC expression is closely associated with cell proliferation and that sucrose sensing plays a major role in the spatial pattern of ODC expression. Also, the differential regulation of ODC and arginine decarboxylase gene expression by factors modulating plant growth suggests that they would have different physiological roles in plant development.

5' Untranslated Regions↗
PubMed1990

Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions.

The genome organization of porcine respiratory coronavirus (PRCV), a newly recognized agent which has a close antigenic relationship to the enteropathogenic transmissible gastroenteritis virus (TGEV), was studied. Genomic RNA from cell-cultured PRCV (French isolate RM4) was used to produce cDNA clones covering the genomic 3' end to the start of the spike (S) glycoprotein gene (7519 nucleotides). Six open reading frames (ORFs) were identified that allowed the translation of three coronavirus structural proteins and three putative non-structural (NS) polypeptides, homologous to TGEV ORFs designated NS3-1, NS4 and NS7. Pairwise alignment of PRCV nucleotide and amino acid sequences with sequence data available for three TGEV strains revealed a 96% overall homology. However, the genome of PRCV exhibited two important distinctive features. The first was that the S gene lacked 672 nucleotides in the 5' region and encoded a truncated form of the S polypeptide, and secondly, the first NS ORF downstream of the S gene was predicted to be non-functional as a consequence of a double deletion. The significance of genomic deletions with respect to tissue tropism and evolution of coronaviruses is discussed.

Amino Acid Sequence↗
PubMed1993

Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis.

A 5.4 kb HindIII fragment of Lactococcus lactis subsp. lactis was identified using a homologous dnaK probe generated by PCR and cloned in Escherichia coli. Upstream sequences were generated by inverse PCR. The two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orf1-grpE-dnaK-orf4. orf1 encodes a 39 kDa protein of unknown function which shows considerable sequence homology with the Orf39 and Orfa proteins of Bacillus subtilis and Clostridium acetobutylicum, respectively. The downstream ORFs showed high homology to the grpE and dnaK genes of other prokaryotes. The DnaK protein has a characteristic 24-amino-acid deletion exhibited by all the known DnaK proteins of Gram-positive species. In many bacteria the dnaK and dnaJ genes are found as part of the same operon. The L. lactis dnaK operon is unusual in that the dnaK gene is followed by a putative transcription terminator and a fourth large ORF which shares no homology with the dnaJ genes of other bacteria but has a small degree of homology with various membrane proteins. Vegetative promoter sequences are found upstream of both orf1 and orf4. A 12 bp inverted repeat is found upstream of the putative promoter of orf1 and an 8 bp inverted repeat is found between this promoter and the orf1 initiation codon. These repeats are thought to be involved in regulation of the heat-shock genes. The DnaK homologue is induced approximately 3-fold on heat shock at 42 degrees C.

Amino Acid Sequence↗
PubMed2004

A motif co-occurrence approach for genome-wide prediction of transcription-factor-binding sites in Escherichia coli.

Various computational approaches have been developed for predicting cis-regulatory DNA elements in prokaryotic genomes. We describe a novel method for predicting transcription-factor-binding sites in Escherichia coli. Our method takes advantage of the principle that transcription factors frequently coregulate gene expression, but without requiring prior knowledge of which groups of genes are coregulated. Using position weight matrices for 49 known transcription factors, we examined spacings between pairs of matrix hits. These pairs were assigned probabilities according to the overrepresentation of their separation distance. The functions of many open reading frames (ORFs) downstream from predicted binding sites are unknown, and may correspond to novel regulon members. For five predictions, knockouts with mutated replacements of the predicted binding sites were created in E. coli MG1655. Quantitative real-time PCR (RT-PCR) indicates that for each of the knockouts, at least one gene immediately downstream exhibits a statistically significant change in mRNA expression. This approach may be useful in analyzing binding sites in a variety of organisms.

Base Composition↗