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PubMed1996

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Amino Acid Sequence↗
PubMed1993

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins.

The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium. Strains carrying mutations located downstream of nifB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations. DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB. The gene products of modB and modC are homologous to ChlJ and ChlD of Escherichia coli and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively. ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein. The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other. Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum. Compared with that for the parental nifHDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants. No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants. The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase. Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake.

Amino Acid Sequence↗
PubMed2000

Transcriptional organization and regulation of a polycistronic cold shock operon in Sinorhizobium meliloti RM1021 encoding homologs of the Escherichia coli major cold shock gene cspA and ribosomal protein gene rpsU.

A homolog of the major eubacterial cold shock gene cspA was identified in Sinorhizobium meliloti RM1021 by luxAB reporter transposon mutagenesis. Here we further characterize the organization and regulation of this locus. DNA sequence analysis indicated that the locus includes three open reading frames (ORFs) encoding homologs corresponding to CspA, a novel 10.6-kDa polypeptide designated ORF2, and a homolog of the Escherichia coli ribosomal protein S21. Transcription analysis indicated that this locus produced two different-sized cspA-hybridizing transcripts upon cold shock, a 400-nucleotide (nt) RNA encoding cspA alone and a 1, 000-nt transcript encoding cspA-ORF2-rpsU. The sizes of the transcripts agreed with the location of the transcription start site determined by primer extension and the locations of two putative transcriptional terminators. The promoter of the cspA-ORF2-rpsU locus had -10 and -35 elements similar to the E. coli sigma(70) consensus promoter and, like the cspA locus of E. coli, included an AT-rich region upstream of the -35 hexamer. The promoter of the S. meliloti cspA locus was found to impart cold shock-induced mRNA accumulation. In addition, the 5'-untranslated region (5' UTR) was found to increase the fold induction of cspA transcripts after cold shock and depressed the level of luxAB mRNA prior to cold shock, another feature similar to cspA regulation in E. coli. No "cold box" was identified upstream of the S. meliloti cspA gene, however, and there was no other obvious sequence identity between the S. meliloti 5' UTR and that of E. coli. DNA hybridization analysis indicated that outside the cspA-ORF2-rpsU cold shock locus there are several additional cspA-like genes and a second rpsU homolog.

Amino Acid Sequence↗
PubMed2005

Genetic variations in Lycoris radiata var. radiata in Japan.

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.

Base Sequence↗
PubMed2005

Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence.

Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.

Animals↗
PubMed2000

A satellite DNA containing CENP-B box-like motifs is present in the antarctic scallop Adamussium colbecki.

The DNA of the Antarctic scallop Adamussium colbecki was found to contain a highly repeated sequence identifiable upon restriction with endonuclease BglII. The monomeric unit - denominated pACS (about 170bp long) - was cloned. Southern blot hybridization yielded a ladder-like banding pattern, indicating that the repeated elements are tandemly arranged in the genome and therefore represent a sequence of satellite DNA. Sequence analysis of five different clones revealed the presence of various subfamilies, some of which showed a high degree of divergence. In each clone, regions homologous to the mammalian CENP-B box were observed. A region homologous to the CDEIII centromeric sequence of yeast was also found in one of the clones. These observations suggest a relationship of the pACS family to the centromeric area in A. colbecki.

Animals↗
PubMed2001

Chromosome mapping and phylogenetic analysis of the cytosolic acetyl-CoA carboxylase loci in wheat.

The cytosolic isoform of plant acetyl-CoA carboxylase is a multidomain enzyme involved in the synthesis of very-long-chain fatty acids and in secondary metabolism. Chromosome mapping of wheat identified one locus containing cytosolic acetyl-CoA carboxylase genes (Acc-2) and a related partially processed pseudogene (Psi-Acc-2) in the distal region of the long arm of wheat homoeologous group 3 chromosomes. Multiple copies of the Acc-2 genes, whose presence was suggested by sequence analysis, are likely to be arranged in tandem repeats. At least three out of five genes cloned from hexaploid wheat map to this locus. Another locus containing Acc-2--related sequences is present in the distal region of the long arm of chromosome 5D. The identity of the hybridizing DNA present at this locus remains unknown. A system based on PCR-cloning and DNA sequence analysis of acetyl-CoA carboxylase genes was developed to address various phylogenetic and systematics questions in grasses. It was applied to reconstruct the phylogeny of the Acc-2 genes from D- and S-genome Aegilops and A-genome Triticum diploid species, AABB- and AAGG-genome tetraploid wheat, and AABBDD-genome hexaploid wheat, as well as from rye and barley. The combined cytogenetic and molecular evolution approach allowed assignment of gene sequences included in phylogenetic analysis to specific loci on homoeologous chromosomes. Recurring gene duplication followed by chromosome translocation and/or possible loss of some gene copies, as well as loss of introns, occurred in the gene family in different plant lineages. Two major Acc-2 clades appeared before the divergence of barley and rye. Nucleotide substitution rates in different parts of the Acc-2 gene were assessed. This analysis of the Acc-2 loci provides detailed information regarding evolutionary events at a low--copy-number locus containing important functional genes. These events are likely to be common and to play a significant role in shaping grass genomes.

Acetyl-CoA Carboxylase↗
PubMed1997

Sequence and characterization of an Ehrlichia chaffeensis gene encoding 314 amino acids highly homologous to the NAD A enzyme.

DNA sequence analysis of the nadA gene of Ehrlichia chaffeensis revealed a 942 bp open reading frame with the capacity to encode 314 amino acids. The amino acid sequence of the E. chaffeensis quinolinate synthetase A (NAD/A) has 53.6% identity and 82% similarity to the NAD A of the cyanelle of Cyanophora paradoxa. Portions of the homologous genes of E. canis and E. muris were also sequenced. The amino acid sequences of the NAD A of E. canis and E. muris have 89.2% and 93.2% homology, respectively, to the NAD A of E. chaffeensis. We propose that the nadA gene may be an excellent candidate for a genetic tool for the phylogenetic study of ehrlichiae.

Bacterial Proteins↗
PubMed1996

Cloning and characterization of murine endothelial constitutive nitric oxide synthase.

Complementary DNA clones encoding mouse endothelial constitutive nitric oxide synthase (ecNOS) were isolated by plaque hybridization from a murine fetal cardiac lambda ZAP II cDNA expression library using a full-length human ecNOS cDNA as the hybridization probe. DNA sequence analysis indicates a 1202 amino acid protein showing significant sequence identity with human as well as bovine ecNOS.

Amino Acid Sequence↗
PubMed1993

Cloning and sequence analysis of cDNAs encoding human hippocampus N-methyl-D-aspartate receptor subunits: evidence for alternative RNA splicing.

Several cDNA clones encoding human N-methyl-D-aspartate receptor (hNR1) subunit polypeptides were isolated from a human hippocampus library. Degenerate oligodeoxyribonucleotide (oligo) primers based on the published rat NR1 (rNR1) amino acid (aa) sequence [K. Moriyoshi et al. Nature 354 (1991) 31-37] amplified a 0.7-kb fragment from a human hippocampus cDNA library, via the polymerase chain reaction (PCR). This fragment was used as a probe for subsequent hybridization screening. DNA sequence analysis of 28 plaque-purified clones indicated three distinct classes, designated hNR1-1, hNR1-2 and hNR1-3, presumably generated by alternative RNA splicing. One of these clones, hNR1-1(5A), was isolated as a full-length cDNA. The hNR1-2 and hNR1-3 cDNAs represented 66.8 and 98.9%, respectively, of the total aa coding information predicted for the polypeptides. The hNR1 cDNAs demonstrated an 84-90.8% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequences of hNR1-1, hNR1-2 and hNR1-3 would encode 885-, 901- and 938-aa proteins, respectively, that have 99.1-99.8% identity with the corresponding rodent NR1 (roNR1) subunits. The changes between the predicted aa sequences of hNR1 and the corresponding roNR1 subunits are confined to the extracellular N-terminal regions. We have also identified two possible allelic variations of the hNR1-3 cDNA that result in aa substitutions in the extracellular N- and C-terminal regions. One of these naturally occurring aa variations is situated within a potential glutamate-binding site.

Alternative Splicing↗
PubMed1997

Characterization of the genes encoding integrative and excisive functions of Lactobacillus phage øg1e: cloning, sequence analysis, and expression in Escherichia coli.

øg1e is a temperate phage of the Lactobacillus strain G1e. The phage-host junctions attR and attL cloned from the lysogen have a 24-bp common (core) sequence implicated in recombination. DNA sequencing analysis of a 5.2-kbp SacI fragment of the øg1e phage genome (42.5 kbp) revealed two possible open reading frames (ORF), xis and int, and the phage attachment (recombination) site (attP), whose 24-bp sequence is identical to the core sequence detected in attR and attL. The deduced int product (Int) is a basic protein of 391 amino acids with an estimated pI of 9.70, and significantly resembles other presumed integrases encoded by the Lactobacillus and Lactococcus phages including øadh and øLC3, as well as the Escherichia coli phages such as lambda. The predicted øg1e xis protein (Xis) is small and very acidic (66 amino acids; pI 4.55), and shows a resemblance (32% overall identity) with a putative excisionase encoded by the Staphylococcus phage ø11. The øg1e Int with a deduced molecular mass of 45.5 kDa was overproduced in E. coli cells, and electrophoretically analyzed.

Amino Acid Sequence↗
PubMed1997

Regulation of ornithine utilization in Pseudomonas aeruginosa (PAO1) is mediated by a transcriptional regulator, OruR.

We have used transpositional mutagenesis of a proline auxotroph (PAO951) to isolate an ornithine utilization (oru) mutant of Pseudomonas aeruginosa (PAO951-4) that was unable to use ornithine efficiently as the sole carbon and nitrogen source. DNA sequence analysis of the inactivated locus confirmed that the transposon had inserted into a locus whose product demonstrated significant primary sequence homology to members of the AraC family of transcriptional activators. DNA mobility shift assays affirmed this potential regulatory function and indicated that the inactivated gene encodes a transcriptional regulator, which has been designated OruR. In trying to define the ornithine utilization phenotype further, a similar inactivation was engineered in the wild-type strain, PAO1. The resulting isolate (PAO1R4) was totally unable to use ornithine as the sole carbon source. Despite the intensified phenotype, this isolate failed to demonstrate significant changes in any of the catabolic or anabolic enzymes that are known to be subject to regulation by the presence of either ornithine or arginine. It did, however, show modified levels of an enzyme, ornithine acetyltransferase (OAcT), that was previously thought to have merely an anaplerotic activity. Definition of this oruR locus and its effects upon OAcT activity provide evidence that control of ornithine levels in P. aeruginosa may have a significant impact upon how the cell is able to monitor and regulate the use of arginine and glutamate as sources of either carbon or nitrogen.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence and analysis of the centromeric region of yeast chromosome IX.

We have determined the nucleotide sequence of a cosmid (pIX338) containing the centromere region of yeast (Saccharomyces cerevisiae) chromosome IX. The complete nucleotide sequence of 33.8 kb was obtained by using an efficient directed sequencing strategy in combination with automated DNA sequencing on the A.L.F. DNA sequencer. Sequence analysis revealed the presence of 17 open reading frames (ORFs), four of them previously known yeast genes (sly12, pan1, sts1 and prl1), a tRNA gene and the centromere motif. Exhaustive database searches detected sequence homologues of known function for as many as 14 of the 17 ORFs. These include a mammalian tyrosine kinase substrate; the Escherichia coli cell cycle protein MinD; the human inositol polyphosphate-5-phosphatase (gene OCRL) involved in Lowe's syndrome, a developmental disorder; and helicases, for which the new yeast member defines a distinct DEAD/H-box subfamily. A surprisingly large fraction of the ORFs (at least six out of 17) in the centromeric region are apparently involved in RNA or DNA binding.

Adenosine Triphosphatases↗
PubMed1998

Mucin biosynthesis: molecular cloning and expression of bovine lung mucin core 2 N-acetylglucosaminyltransferase cDNA.

A cDNA clone containing a 2,150-bp insert was isolated from a bovine lung lambdagt10 cDNA library by cross-species hybridization using a DNA probe generated by polymerase chain reaction (PCR) employing a human cDNA that encodes mucin core 2 beta6-N-acetylglucosaminyltransferase (hC2TF) as the template. The bovine cDNA (bcDNA) insert was devoid of 220 bp of the 5' portion of the C2TF open reading frame (ORF), as predicted from the human counterpart. Southern blotting analysis suggested that the coding region of this C2TF gene is in one exon. To construct a full-length bovine C2TF (bC2TF) cDNA, a genomic DNA fragment containing the 5' portion of the ORF of the bC2TF gene was cloned from a lambdaEMBL bovine genomic DNA library and ligated to the 5' end of the cloned cDNA insert. DNA sequence analysis showed that the complete ORF of bC2TF gene was 1,281 bp in length, which corresponds to a polypeptide of 427 amino acids. Catalytically active bC2TF was expressed in sf21 insect cells infected with recombinant baculovirus containing the ORF of the bC2TF gene. The recombinant bC2TF catalyzed the synthesis of core 2, but not core 4 and blood group I structures. Western blotting analysis showed that the recombinant bC2TF migrated with the same mobility (approximately 55 kD) as the native bovine tracheal C2TF. Immunohistochemical analysis showed that in bovine trachea, the bC2TF was present at the surface epithelium and in the submucosal glands, with the latter being the major site of distribution.

Amino Acid Sequence↗
PubMed2006

Molecular characterization of a new begomovirus infecting Sida cordifolia and its associated satellite DNA molecules.

Two virus isolates Hn57 and Hn60 were obtained from Sida cordifolia showing mild upward leaf-curling symptoms in Hainan province of China. Comparison of partial sequences of DNA-A like molecule confirmed the existence of a single type of begomovirus. The complete nucleotide sequence of DNA-A of Hn57 was determined to be 2757 nucleotides, with a genomic organization typical of begomoviruses. Complete sequence comparison with other reported begomoviruses revealed that Hn57 DNA-A has the highest sequence identity (71.0%) with that of Tobacco leaf curl Yunnan virus. Consequently, Hn57 was considered to be a new begomovirus species, for which the name Sida leaf curl virus (SiLCV) is proposed. In addition to DNA-A molecule, two additional circular single-stranded satellite DNA molecules corresponding to DNAbeta and DNA1 were found to be associated with SiLCV isolates. Both DNAbeta and DNA1 were approximately half the size of their cognate genomic DNA. Sequence analysis shows that DNAbeta of Hn57 and Hn60 share 93.8% nucleotide sequence identity, and they have the highest sequence identity (58.5%) with DNAbeta associated with Ageratum leaf curl disease (AJ316027). The nucleotide sequence identity between DNA1 of Hn57 and that of Hn60 was 83.8%, they share 58.2-79.3% nucleotide sequence identities in comparison with other previously reported DNAl.

DNA, Satellite↗
PubMed1994

Isolation of a cytochrome P450 homologue preferentially expressed in developing inflorescences of Zea mays.

Four cDNA clones exhibiting preferential hybridization to transcripts present in developing maize tassels were isolated by differential screening. One of these cDNA clones hybridizes to transcripts detectable only in the shoot apex. The abundance of this transcript is significantly higher in developing inflorescence apices than in vegetative apices. DNA sequence analysis of a 2107 nucleotide cDNA clone corresponding to this transcript revealed that the transcript encodes a polypeptide of 547 amino acids, with a molecular mass of 58.4 kDa. This polypeptide shares significant sequence similarity with members of the cytochrome P450 monooxygenase gene superfamily, including the conserved C-terminal domains typical of the cytochrome P450 monooxygenases.

Amino Acid Sequence↗
PubMed1994

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of the murine homologue of the rat/human MRC OX-2 gene.

A 350 bp amplicon, obtained by PCR-select subtractive hybridization from RNA derived from mesenteric lymph nodes (MLN) cells from mice pre-immunized with allogeneic lymphocytes 36hrs prior to receiving donor-specific skin grafts, and showing > 98% homology with a published sequence for the rat MRC OX-2 gene, was used as a hybridization probe to screen a cDNA library constructed from adult mouse MLN treated in the same fashion. Several clones were identified which, on DNA sequence analysis, predicted a 218 amino acid protein showing significant homology with the rat and human MRC OX-2 gene product.

Adoptive Transfer↗