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Distinct post-transcriptional modifications result into seven alternative transcripts of the CC-NBS-LRR gene JA1tr of Phaseolus vulgaris.

The generation of splice variants has been reported for various plant resistance (R) genes, suggesting that these variants play an important role in disease resistance. Most of the time these R genes belong to the Toll and mammalian IL-1 receptor-nucleotide-binding site-leucine-rich repeat (TIR-NBS-LRR) class of R genes. In Phaseolus vulgaris, a resistance gene cluster (referred to as the B4 R-gene cluster) has been identified at the end of linkage group B4. At this complex resistance cluster, three R specificities (Co-9, Co-y and Co-z) and two R QTLs effective against the fungal pathogen Colletotrichum lindemuthianum, the causal agent of anthracnose, have been identified. At the molecular level, four resistance gene candidates encoding putative full-length, coiled-coil (CC)-NBS-LRR R-like proteins, with LRR numbers ranging from 18 to 20, have been previously characterized. In the present study, seven cDNA corresponding to truncated R-like transcripts, belonging to the CC-NBS-LRR class of plant disease R genes, have been identified. These seven transcripts correspond to a single gene named JA1tr, which encodes, at most, only five LRRs. The seven JA1tr transcript variants result from distinct post-transcriptional modifications of JA1tr, corresponding to alternative splicing events of two introns, exon skipping and multiple 'aberrant splicing' events in the open reading frame (ORF). JA1tr was mapped at the B4 R-gene cluster identified in common bean. These post-transcriptional modifications of the single gene JA1tr could constitute an efficient source of diversity. The present results provide one of the few reports of transcript variants with truncated ORFs resulting from a CC-NBS-LRR gene.

Alternative Splicing↗

Chloroplast ATPase genes in the diatom Odontella sinensis reflect cyanobacterial characters in structure and arrangement.

We have cloned and sequenced a 5200 base restriction fragment and an overlapping 3100 base fragment of the large single copy region of the chloroplast genome of the diatom Odontella sinensis, which hybridized to several ATPase gene probes. These fragments contain six closely linked reading frames that were identified as atpI, atpH, atpG, atpF, atpD, and atpA, coding for subunits IV, III, II, I, delta, and alpha, respectively. Remarkably, the genes atpG and atpD, which are nucleus-encoded in chlorophyll a + b plants, are present in the Odontella chloroplast gene cluster. They map at the same positions as in cyanobacteria. The genes atpD and atpF overlap by four base-pairs as in certain photosynthetic and heterotrophic eubacteria. Upstream from the atpA gene cluster an open reading frame coding for 251 amino acid residues was found, which shows sequence similarity to ATP-binding subunits of periplasmic prokaryotic and eukaryotic transport systems. No similar reading frame is present in the land plant chloroplast genomes analysed so far. Sequences and arrangement of the genes are discussed with respect to the peculiar evolution of the chlorophyll a + c-containing chromophytic plastids.

Adenosine Triphosphatases↗

Nucleotide sequence of a gene essential for viral DNA replication in the baculovirus Autographa californica nuclear polyhedrosis virus.

The nucleotide sequence of the 60.1- to 65.5-m.u. region of Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined. Seven large open reading frames were identified. Two open reading frames potentially encoding gene products of 143 and 38 kDa were found in the counterclockwise direction upstream of the p6.9 gene. Four additional open reading frames were found in the opposite direction. Analysis of the predicted amino acid sequence of the 143-kDa gene revealed a potential leucine zipper motif, a putative nuclear localization signal, and seven amino acid motifs previously identified in a number of proteins involved in NTP binding and DNA/RNA unwinding. The mutation in a DNA replication defective temperature-sensitive mutant was fine mapped to the carboxy terminus of the ORF1(p143) gene. Sequence analysis of the mutation site identified a single base change of a guanine to an adenine, resulting in the substitution of a methionine for valine. This mutation resides seven amino acids downstream of the putative NTP-binding motif of the ORF1(p143) gene product and results in a DNA negative mutant. Together these data strongly suggest that the ORF1(p143) gene product is a baculovirus helicase.

Amino Acid Sequence↗

The 2p21 deletion syndrome: characterization of the transcription content.

The vast majority of small-deletion syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We have previously identified a homozygous deletion of 179,311 bp on chromosome 2p21 as the cause of a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria. We now present the transcription content of this region: Multiple splicing variants of the genes protein phosphatase 1B (formerly 2C) magnesium-dependent, beta isoform (PPM1B), SLC3A1, and KIAA0436 (approved gene symbol PREPL) were identified and their patterns of expression analyzed. The spliced variants are predicted to have additional functions compared to the known variants and their patterns of expression fit the tissues affected by the syndrome. The first exon of an additional gene (C2orf34) is encoded in the deleted region and the gene is not expressed in the patients. In addition several transcripts with very short open reading frames are also encoded in the deletion. The identification of all transcripts encoded in the region deleted in the patients is the first step in the study of the genotype-phenotype correlation of the 2p21 patients.

Alternative Splicing↗

The sequence of a Drosophila Cyp4e2 cytochrome P450-encoding cDNA.

A composite 1458-bp cDNA that encodes cytochrome P450 (P450) Cyp4e2 has been constructed from clones isolated from two Drosophila embryonic cDNA libraries. The Drosophila cDNA open reading frame encodes a protein of 486 amino acids that is 40% identical and 61% similar to Cyp4d1 from Drosophila. The predicted protein is unusual in that it appears to lack the hydrophobic N-terminus typical of microsomal P450s and also contains a small insertion at its C-terminus.

Amino Acid Sequence↗

Compilation of all genes encoding two-component phosphotransfer signal transducers in the genome of Escherichia coli.

Bacteria have devised sophisticated His-Asp phosphorelay signaling systems for eliciting a variety of adaptive responses to their environment, which are generally referred to as the "two-component regulatory system." The widespread occurrence of the His-Asp phosphorelay signaling in both prokaryotes and eukaryotes implies that it is a powerful device for a wide variety of adaptive responses of cells to their environment. The two-component signal transducers contain one or more of three common and characteristic phosphotransfer signaling domains, named the "transmitter, receiver, and histidine-containing phosphotransfer (HPt) domains." The recently determined entire genomic sequence of Escherichia coli allowed us to compile systematically a complete list of genes encoding such two-component signal transduction proteins. The results of such an effort, made in this study, revealed that at least 62 open reading frames (ORFs) were identified as putative members of the two-component signal transducers in this single species. Among them, 32 were identified as response regulator and 23 were identified as orthodox sensory kinases. In addition, E. coli has five hybrid sensory kinases. The precise location of each ORF was mapped on a physical map of the entire E. coli genome. All of these ORFs were then compiled and annotated extensively.

Amino Acid Sequence↗

Expression of genes from Rahnella aquatilis that are necessary for mineral phosphate solubilization in Escherichia coli.

Rahnella aquatilis is a Gram-negative bacterium that can fix atmospheric nitrogen and also has the ability to solubilize mineral phosphate. We have cloned the genes that confer the mineral phosphate solubilizing (Mps) trait from this organism by mobilizing a cosmid library of R. aquatilis into Escherichia coli HB101. A 7.0-kb EcoRI fragment from a cosmid, when transferred into E. coli strains HB101 and DH5 alpha, conferred the ability to solubilize hydroxyapatite and the production of gluconic acid to E. coli. The relative amounts of soluble phosphate and gluconic acid produced by the cloned 7.0-kb EcoRI fragment in E. coli were significantly higher than those of R. aquatilis. Nucleotide sequence analysis revealed two complete open reading frames (ORF1 and ORF2) and a partial ORF, ORF1 and ORF2 encoded proteins of molecular mass 10 kDa and 44 kDa. The 44-kDa protein showed extensive sequence similarity to pqqE of Erwinia herbicola, Klebsiella pneumoniae and A. Acinetobacter calcoaceticus. The 10-kDa protein revealed strong similarity to the pqqD of K. pneumoniae and A. calcoaceticus.

Chemotactic Factors↗

Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a.

The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail. In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6-phospho-3-hexuloisomerase (PHI) (rmpB). The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI. The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects.

Aldehyde-Lyases↗

Complementation analysis of the Dichelobacter nodosus fimN, fimO, and fimP genes in Pseudomonas aeruginosa and transcriptional analysis of the fimNOP gene region.

The causative agent of ovine footrot, the gram-negative anaerobe Dichelobacter nodosus, produces polar type IV fimbriae, which are the major protective antigens. The D. nodosus genes fimN, fimO, and fimP are homologs of the Pseudomonas aeruginosa fimbrial assembly genes, pilB, pilC, and pilD, respectively. Both the pilD and fimP genes encode prepilin peptidases that are responsible for cleavage of the leader sequence from the immature fimbrial subunit. To investigate the functional similarity of the fimbrial biogenesis systems from these organisms, the D. nodosus genes were introduced into P. aeruginosa strains carrying mutations in the homologous genes. Analysis of the resultant derivatives showed that the fimP gene complemented a pilD mutant of P. aeruginosa for both fimbrial assembly and protein secretion. However, the fimN and fimO genes did not complement pilB or pilC mutants, respectively. These results suggest that although the PilD prepilin peptidase can be functionally replaced by the heterologous FimP protein, the function of the PilB and PilC proteins may require binding or catalytic domains specific for the P. aeruginosa fimbrial assembly system. The transcriptional organization and regulation of the fimNOP gene region were also examined. The results of reverse transcriptase PCR and primer extension analysis suggested that these genes form an operon transcribed from two sigma70-type promoters located upstream of ORFM, an open reading frame proximal to fimN. Transcription of the D. nodosus fimbrial subunit was found to increase in cells grown on solid media, and it was postulated that this regulatory effect may be of significance in the infected footrot lesion.

Amino Acid Sequence↗

Transcriptional mapping of the 3' end of the bovine syncytial virus genome.

The bovine syncytial virus, a member of the retroviral subfamily Spumavirinae, causes a persistent, asymptomatic infection in cattle. Nucleotide sequence analysis of the viral genome revealed two overlapping reading frames in the 3' region, traditionally occupied by accessory-function genes in other complex retroviruses. In order to analyze the transcripts from the accessory-gene region, we designed oligonucleotide primers complementary to sequences within the 5' and 3' long terminal repeats (LTRs) for use with the PCR. Southern blot analysis of amplification products revealed eight major cDNA bands. Eleven distinct cDNA clones were subsequently isolated and characterized. The initial splice donor in each clone is located 49 bp downstream from the mRNA cap site in the 5' LTR. The primary splice acceptor site was located 17 bp upstream from the proximal 3' open reading frame known as BF-ORF1. A second major splice acceptor was localized to a region upstream of the second open reading frame, BF-ORF2. Clones were identified which spliced directly to each of these sites. Additional splice donor and acceptor sites within BF-ORF1 and BF-ORF2 and the 3' LTR were variously used to generate a complex array of multiply spliced transcripts. Each of these transcripts remained in frame and coded for a potential protein product.

Amino Acid Sequence↗

Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains.

Nucleotide sequence comparisons were performed on a highly heterogeneous region of three human cytomegalovirus strains, Toledo, Towne, and AD169. The low-passage, virulent Toledo genome contained a DNA segment of approximately 13 kbp that was not found in the Towne genome and a segment of approximately 15 kbp that was not found in the AD169 genome. The Towne strain contained approximately 4.7 kbp of DNA that was absent from the AD169 genome, and only about half of this segment was present, arranged in an inverted orientation, in the Toledo genome. These additional sequences were located at the unique long (UL)/b' (IRL) boundary within the L component of the viral genome. A region representing nucleotides 175082 to 178221 of the AD169 genome was conserved in all three strains; however, substantial reduction in the size of the adjacent b' sequence was found. The additional DNA segment within the Toledo genome contained 19 open reading frames not present in the AD169 genome. The additional DNA segment within the Towne genome contained four new open reading frames, only one of which shared homology with the Toledo genome. This comparison was extended to five additional clinical isolates, and the additional Toledo sequence was conserved in all. These findings reveal a dramatic level of genome sequence complexity that may explain the differences that these strains exhibit in virulence and tissue tropism. Although the additional sequences have not altered the predicted size of the viral genome (230 to 235 kbp), a total of 22 new open reading frames (denoted UL133 to UL154), many of which have sequence characteristics of glycoproteins, are now defined as cytomegalovirus specific. Our work suggests that wild-type virus carries more than 220 genes, some of which are lost by large-scale deletion and rearrangement of the UL/b' region during laboratory passage.

Base Sequence↗

[cDNA cloning and sequence analysis of the seventh segment of maize rough dwarf virus genome].

The double strand RNA of maize rough dwarf virus (MRDV) was prepared from the maize samples showing symptoms which was from the Luanchen county of Heibei province of China. The primers were designed according to the known sequence of MRDV, the cDNA sequence of the seventh segment of MRDV was obtained by RT-PCR, the S7 sequence was analyzed by computer after sequencing. The results showed: the full length of the S7 cDNA is 1936 bp and equal to that of the S7 cDNA from abroad, the two open reading frame(ORF1 and ORF2) contained in the S7 segment are also unchanged. In comparison with the S7 segment from Italy, the homology of S7 nucleotide is 87.7% and the homology of ORF1 amino acid sequence is 91.6%. However, the MRDV S7 segment and the rice black strike dwarf virus S8 segment showed 95.5% nucleotide identities and 93.5% ORF1 amino acid identities.

Amino Acid Sequence↗

[Molecular cloning and sequence analyzing of cytoplasmic ribosomal protein gene OsRPS7 from rice (Oryza sativa)].

Using the cDNA of rye cytoplasmic ribosomal protein ScRPS7 as a query probe, a highly homologous rice genomic contig was obtained from Huada rice genome database. The full-length cDNA sequence of rice cytoplasmic ribosomal protein S7 was assembled by informatics based on the contig. Furthermore, with the two primers designed according to this assembled cDNA, the full-length cDNA of rice ribosomal protein was cloned by RT-PCR and named as OsRPS7. The cDNA was 919bp in length and contained a complete Open Reading Frame (ORF) of 576bp, encoding a protein of 192 amino acid residues. The deduced amino acids of OsRPS7 showed 88%,72% and 72% identity with those from Secale cereale, Arabidopsis thaliana and Brassica oleracea, respectively. The genome structure of OsRPS7 was analyzed, and its function was predicted in this paper.

Amino Acid Sequence↗

Conditional rather than absolute requirements of the capsid coding sequence for initiation of methionine-independent translation in Plautia stali intestine virus.

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5' region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5' part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5' part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3' part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.

Base Sequence↗

The sequence of a 12 kb fragment on the left arm of yeast chromosome XI reveals five new open reading frames, including a zinc finger protein and a homolog of the UDP-glucose pyrophosphorylase from potato.

We report the sequence of a 12,399 bp DNA fragment from the left arm of Saccharomyces cerevisiae chromosome XI. This fragment is located between the genetic loci mif2 and mak11. We have detected five new open reading frames (ORFs) longer than 300 bp provisionally called YKL248 to YKL252. One of them, YKL248 encodes a homolog of the UDP-glucose pyrophosphorylase from potato. The product of YKL251 contains the consensus for zinc binding proteins, similar to those of a number of fungal transcriptional activators. The three other ORFs do not show significant homology to known protein.

Amino Acid Sequence↗

Sequencing of chromosome I of Saccharomyces cerevisiae: analysis of the 42 kbp SPO7-CENI-CDC15 region.

Determination of the DNA sequence and preliminary functional analysis of a 42 kbp centromeric section of chromosome I have been completed. The section spans the SPO7-CEN1-CDC15 loci and contains 19 open reading frames (ORFs). They include an apparently inactive Ty1 retrotransposon and eight new ORFs with no known homologs or function. The remaining ten genes have been previously characterized since this part of the yeast genome has been studied in an unusually intensive manner. Our directed sequencing allows a complete ordering of the region.

Chromosome Mapping↗

Sequencing of an 18.8 kb fragment from Saccharomyces cerevisiae chromosome VI.

The nucleotide sequence of lambda phage clone 4121, which contains the 18.8 kb fragment of Saccharomyces cerevisiae chromosome VI left arm, was determined. This sequence had seven open reading frames (ORFs), four of which were identical to known genes (ACT1, YPT1, TUB2 and RPO41). Another three ORFs (4121orfR003, 4121orfR004 and 4121orfRN001) were highly homologous to FET3 multi-copper oxidase, glucose transport protein, and hypothetical protein of YIL106w on chromosome IX, respectively. 4121orfRN01 is suggested to contain an intron.

Amino Acid Sequence↗

Molecular cloning of a new erythropoiesis-stimulating activity (ESA36) from Streptomyces thermoviolaceus and expression in Escherichia coli.

Previous studies have shown that an Actinomycetes strain produces a novel protein with a molecular weight of 87,000 which stimulated the growth of murine erythroid progenitors in vitro, and we purified the protein. In this study, we have isolated and sequenced a clone encoding a polypeptide which had the equivalent activity. The clone contained an open reading frame of 334 amino acids. The activity was confirmed by the expression in E. coli as a fusion protein with glutathion S-transferase (GST). Based on the differences of molecular weight and N-terminal amino acid sequences, this protein was considered to be different from that previously reported and was named "ESA36".

Amino Acid Sequence↗