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PubMed2004

Production of wheat-Leymus racemosus chromosome addition lines.

We produced ten wheat-Leymus racemosus chromosome addition lines. Eight chromosomes (A, C, F, H, I, J, k, and l) were recovered as disomic additions and two (E and n) as monosomic. Screening of the addition lines was done by fluorescence in situ hybridization using several repetitive sequences as probes, which allowed us to identify different L. racemosus chromosomes and find many aberrant L. racemosus chromosomes. RFLP analysis revealed partial conservation of homology between L. racemosus and wheat chromosomes, depending on the homologous groups. Chromosomes A and l belonged to group 2, chromosomes C and I to group 5, and chromosome k to group 6. Chromosomes H and J were a mixture of groups 1, 3, and 7, chromosome n of groups 3 and 7, and chromosomes E and F were of group 4 and others. Comparison of our addition lines with other addition lines showed large cytological differences.

Blotting, Southern↗
PubMed2002

Characterization of the ysa pathogenicity locus in the chromosome of Yersinia enterocolitica and phylogeny analysis of type III secretion systems.

Several Gram negative bacteria use a complex system called "type III secretion system" (TTSS) to engage their host. The archetype of TTSS is the plasmid-encoded "Yop virulon" shared by the three species of pathogenic Yersinia (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica). A second TTSS, called Ysa (for Yersinia secretion apparatus) was recently described in Y. enterocolitica 8081, a strain from serotype O:8. In this study, we describe the ysa locus from A127/90, another strain of serotype O:8, and we extend the sequence to several new genes encoding Ysp proteins which are the substrates of this secretion system, and a putative chaperone SycB. According to the deduced protein sequences, the ysa system from A127/90 is identical to that of 8081. It is different from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. We further demonstrated that the ysa locus is only present in biotype IB strains of Y. enterocolitica. Including this new Ysa system, a phylogenetic analysis of the 26 known TTSSs was carried out, based on the sequence analysis of three conserved proteins. All the TTSSs fall into five different clusters. The phylogenetic tree of these TTSSs is completely different from the evolutionary tree based on 16S RNA, indicating that TTSSs have been distributed by horizontal transfer.

Base Sequence↗
PubMed2004

A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, Paralichthys olivaceus.

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.

Amino Acid Sequence↗
PubMed2004

Characterization of the dog Agouti gene and a nonagoutimutation in German Shepherd Dogs.

The interaction between two genes, Agouti and Melanocortin-1 receptor ( Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs ( Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.

Agouti Signaling Protein↗
PubMed2003

Site-directed mutational analysis of the novel catalytic domains of alpha-aminoadipate reductase (Lys2p) from Candida albicans.

The alpha-aminoadipate reductase, a novel enzyme in the alpha-aminoadipic acid pathway for the biosynthesis of lysine in fungi, catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, NADPH and MgCl(2). This reaction requires two distinct gene products, Lys2p and Lys5p. In the presence of CoA, Lys5p posttranslationally activates Lys2p for the alpha-aminoadipate reductase activity. Sequence alignments indicate the presence of all functional domains required for the activation, adenylation, dehydrogenation and alpha-aminoadipic acid binding in the Lys2p. In this report we present the results of site-directed mutational analysis of the conserved amino acid residues in the catalytic domains of Lys2p from the pathogenic yeast Candida albicans. Mutants were generated in the LYS2 sequence of pCaLYS2SEI by PCR mutagenesis and expressed in E. coli BL21 cells. Recombinant mutants and the wild-type Lys2p were analyzed for their alpha-aminoadipate reductase activity. Substitution of threonine 416, glycine 418, serine 419, and lysine 424 of the adenylation domain (TXGSXXXXK, residues 416-424) resulted in a significant reduction in alpha-aminoadipate reductase activity compared to the unmutagenized Lys2p control. Similarly replacement of glycine 978, threonine 980, glycine 981, phenylalanine 982, leucine 983 and glycine 984 of the NADPH binding domain (GXTGFLG, residues 978-984) caused a drastic decrease in alpha-aminoadipate reductase activity. Finally, substitution of histidine 460, aspartic acid 461, proline 462, isoleucine 463, glutamine 464, arginine 465, and aspartic acid 466 of the putative alpha-aminoadipic acid binding domain (HDPIQRD, residues 460-466) resulted in a highly reduced alpha-aminoadipate reductase activity. These results confirm the hypothesis that specific amino acid residues in highly conserved catalytic domains of Lys2p are essential for the alpha-aminoadipate reductase activity.

Adenosine Triphosphate↗
PubMed2006

Results of complete hemivertebra excision followed by circumferential fusion and anterior or posterior instrumentation in patients with type-IA formation defect.

To evaluate the results of surgical treatment in patients with unlocked full-segmented hemivertebra treated by excision. Twenty-six patients with a mean age of 12.4+/-1.7 years were included in the study. The mean duration of follow-up was 47.8+/-21.9 months. Diagnosis of type-IA hemivertebra was established by clinical, radiological, CT, and MRI evaluation. Preoperatively, patients were randomly allocated into two groups. In the first group, patients underwent anterior hemivertebrectomy initially; this was followed by posterior excision of the hemivertebra, posterior instrumentation, and fusion. In the second group, posterior components of the hemivertebra were excised at first, then the hemivertebra body was excised anteriorly, and this was followed by anterior instrumentation and fusion. For both groups, compression was applied to the convex side while distraction was applied to the concave side. Frontal and sagittal plane analysis of radiograms obtained preoperatively, postoperatively, and after a minimum period of 2 years was performed. The balance was analyzed clinically and radiologically by the measurement of the lateral trunk shift (LT) and shift of head (SH). The mean preoperative and postoperative Cobb angles were 45.5 degrees -/+11.4 degrees and 16.8 degrees -/+7.9 degrees, respectively, and postoperatively, a mean correction rate of 64.4+/-13.9% was obtained (P=0.00). The mean correction rate was 61.2+/-13.3% (19.2 degrees -/+7.6 degrees) for the last follow-up visit. Sagittal plane analysis demonstrated either conservation of physiological sagittal contours or a normalizing effect following excision of hemivertebra combined with anterior or posterior instrumentation. When postoperative balance values were compared, a statistically significant correction was found in terms of LT and SH values. Although none of the patients had complete balance (SH: 0 mm) or balanced curves (0 mm<SH<15 mm) preoperatively, 20 (76.9%) of the patients had a balanced trunk after surgical intervention. Circumferential fusion could be achieved in all cases. No neurological complication developed, the only complication was delayed wound healing. In view of these data, it is concluded that these techniques can be safely used for this patient group at low thoracic, thoracolumbar, and lumbar levels of vertebral column with high correction rates.

Adolescent↗
PubMed2006

Phylogenetic analysis of partial S1 and N gene sequences of infectious bronchitis virus isolates from Italy revealed genetic diversity and recombination.

A total of ten infectious bronchitis virus (IBV) isolates collected from commercial chickens in Italy in 1999 were characterized by RT-PCR and sequencing of the S1 and N genes. Phylogenetic analysis based on partial S1 gene sequences showed that five field viruses clustered together with 793/B-type strains, having 91.3-98.5% nucleotide identity within the group, and one isolate had very close sequence relationship (94.6% identity) with 624/I strain. These two IBV types have been identified in Italy previously. The other three variant isolates formed novel genotype detected recently in many countries of Western Europe. For one of these variant viruses, Italy-02, which afterwards became the prototype strain, the entire S1 gene was sequenced to confirm its originality. In contrast, phylogenetic analysis of more conserved partial N gene sequences, comprising 1-300 nucleotides, revealed different clustering. Thus, three variant IBVs of novel Italy-02 genotype, which had 96.7-99.2% S1 gene nucleotide identity with each other, belonged to three separate subgroups based on N gene sequences. 624/I-type isolate Italy-06 together with Italy-03, which was undetectable using S1 gene primers, shared 97.7% and 99.3% identity, respectively, in N gene region with vaccine strain H120. Only one of the 793/B-type isolates, Italy-10, clustered with the 793/B strain sharing 99.3% partial N gene identity, whereas the other four isolates were genetically distant from them (only 87.7-89.7% identity) and formed separate homogenous subgroup. The results demonstrated that both mutations and recombination events could contribute to the genetic diversity of the Italian isolates.

Animals↗
PubMed2005

Chromosomal organization, evolutionary relationship, and expression of zebrafish GnRH family members.

Multiple forms of gonadotropin-releasing hormone (GnRH) are found in different vertebrates. In this study, we have cloned cDNA encoding the full-length gnrh3 and gnrh2 from zebrafish brain and characterized their structure and expression patterns. We performed phylogenetic analysis and compared conserved syntenies in the region surrounding the GnRH genes from human, chicken, pufferfish, and zebrafish genores. The gnrh3 and gnrh2 genes were mapped to LG17 and LG21, respectively. The zebrafish genome appears to lack an ortholog to human GNRH1, and the human genome appears to lack an ortholog of gnrh3. Expression of gnrh3 began in the olfactory pit at 24-26 h postfertilization and expanded to the olfactory bulb during early larval stage. Expression of gnrh2 is always in the midbrain. In addition, GnRH is also expressed in boundary cells surrounding seminiferous cysts of the testis. Thus, this detailed phylogenetic, chromosomal comparison, and expression study defines the identity and the evolutionary relationship of two zebrafish gnrh genes. We propose a model describing the evolution of gnrh genes involving ancestral duplication of the genes followed by selective loss of one gene in some teleosts.

Animals↗
PubMed2006

Cloning, expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1, a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers. The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene. In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves. The peak expression signal was observed in mesophyll cells under low phosphorus (P) induction. A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris. At the meantime, the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters deficient. Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.

Cloning, Molecular↗
PubMed1989

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F1F0-ATPase disrupt ATP-driven proton translocation.

Three missense mutants in subunit a of the Escherichia coli F1F0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp119----His, Ser152----Phe, or Gly197----Arg substitutions in subunit a. Function was not completely abolished by any of the mutations. The F0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F1F0-ATPase. The H+ translocation capacity of F0 was reduced in all three mutants. ATP-driven H+-translocation was also reduced, with the response in the Gly197----Arg mutant being almost nil and that in the Asp119----His and Ser152----Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly197----Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp119----His and Ser152----Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp231----stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB+ plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp231----stop) background strain was shown to bind F1-ATPase nearly normally despite lacking subunit a in its membrane.

Adenosine Triphosphate↗
PubMed1990

The structure and expression of a distantly related member of the beta-gamma crystallin super gene family from Xenopus.

In an attempt to understand amphibian crystallin gene regulation, we have isolated and partially characterized several genomic clones which hybridized to the gamma 1 cDNA probe from Rana temporaria. A complete sequence of one of these clones showed slight homology to the mammalian beta and gamma crystallins. The deduced amino acid sequence of the coding region and its alignment as a folding unit indicated that all the topologically equivalent residues involved in maintaining the protein folding pattern are highly conserved. Northern blot analysis of total RNAs derived from several adult tissues, including eye lens, suggested that an RNA of approximately 700 nucleotides long is present in lens, heart, spleen and embryos of later stages of development but not in retina, oocytes and embryos of early developmental stages.

Amino Acid Sequence↗
PubMed1996

ER beta: identification and characterization of a novel human estrogen receptor.

A novel estrogen receptor (hereinafter referred to as ER beta) was cloned using degenerate PCR primers. A comparison of the amino acid sequence of ER beta with the "classical' ER (ER alpha) shows a high degree of conservation of the DNA-binding domain (96%), and of the ligand-binding domain (58%). In contrast, the A/B domain, the hinge region and the F-domain are not conserved. Northern blot analysis revealed that ER beta is expressed in human thymus, spleen, ovary and testis. Transient transfections of an ER beta expression construct together with an ERE-based reporter construct in CHO cells clearly demonstrated transactivation of ER beta by 17 beta-estradiol. In addition, the ER alpha antagonist ICI-164384 is a potent antagonist for ER beta as well. Interestingly, the level of transactivation by 17 beta-estradiol is higher for ER alpha than for ER beta, which may reflect suboptimal conditions for ER beta at the level of the ligand, responsive element or cellular context.

Amino Acid Sequence↗
PubMed1994

The crystal structure and biological function of leukemia inhibitory factor: implications for receptor binding.

The structure of murine leukemia inhibitory factor (LIF) has been determined by X-ray crystallography at 2.0 A resolution. The main chain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony-stimulating factor and growth hormone (GH). Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved surface character. Analysis of the biological function and receptor specificity of a series of human-mouse LIF chimeras implicate two regions of receptor interaction that are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the GH ligand-receptor complex requires additional, novel features to account for these data.

Amino Acid Sequence↗
PubMed1993

Abundance of heteronuclear and messenger RNA for internal Ca pump in stomach smooth muscle and myocardium.

The sarcoplasmic reticulum Ca pump gene SERCA2 is alternatively spliced to express mRNA encoding the protein SERCA2a in heart and SERCA2b in stomach smooth muscle. The expression of SERCA2 protein in heart is 70 +/- 10 fold that in stomach smooth muscle. To understand the mechanism underlying this tissue difference in the expression level, a comparison is made of their mRNA and heteronuclear RNA (hn-RNA) contents. A 72 bp intron present in the gene encoding SERCA2a/b was cloned and sequenced. By reverse transcription of total RNA followed by PCR using primers based on the sequence of this intron and the cDNA sequences flanking it, a value of 0.37 +/- 0.03 was obtained for the ratio heart hn-RNA/mRNA:stomach hn-RNA/mRNA. Similarly, a ratio of 3.4 +/- 0.5 was obtained for heart:stomach mRNA/28S values. This value was slightly lower but statistically similar to a value of 5.7 +/- 1.8 obtained for the heart:stomach mRNA/poly A+ RNA obtained by Northern blot analysis using a conserved region SERCA2 cDNA probe. Based on mRNA/28S ratio of 3.4 +/- 0.5 and hn/mRNA ratio of 0.37 +/- 0.03, the ratio of heart:stomach for hn-RNA/28S was 1.2 +/- 0.2. Thus, heart which expresses SERCA2a contains 70 +/- 10 times more protein, 3.4 +/- 0.5 times more mRNA and only 1.2 +/- 0.2 times more hn-RNA for this message than the stomach smooth muscle which expresses SERCA2b.

Animals↗
PubMed1995

Molecular cloning of equine interleukin-1 alpha and -beta cDNAs.

Equine interleukin-1 alpha (IL-1 alpha) and IL-1 beta were molecularly cloned to establish a basis for research on inflammatory and immune responses in the horse. Equine peripheral blood mononuclear cells (PBMC) were stimulated with lipopolysaccharide (LPS), and cDNA clones of equine IL-1 alpha and IL-1 beta covering the whole coding sequences were isolated from them. These equine IL-1 alpha and IL-1 beta clones contained open reading frames encoding 271 and 269 amino acids, respectively. The deduced amino acid sequence of equine IL-1 alpha showed 71.6% and 60.2% similarity with that of human and murine IL-1 alpha, respectively. Similarly, the amino acid sequence of equine IL-1 beta showed 66.7% and 61.8% similarity with that of human and murine IL-1 beta, respectively. In both equine IL-1 alpha and IL-1 beta, amino acids at miristoylation sites were well conserved. Dot blot analysis indicated that the expression of IL-1 beta was predominant to that of IL-1 alpha in equine PBMC stimulated with LPS or phorbol myristate acetate.

Amino Acid Sequence↗
PubMed1995

Rapid genotyping of hepatitis C virus isolates by dideoxy fingerprinting.

A number of distinct hepatitis C virus (HCV) types and subtypes have been identified by DNA sequencing of multiple genome regions. It has been postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship between HCV genotype and alpha-interferon response in patients with chronic hepatitis C. A simplified method of genotyping in comparison to direct DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid-phenol extraction and peripheral blood lymphocytes using RNAzol B (Cinna-Biotecx). The RNA was reverse transcribed and a 287 bp segment of the 5' non-coding region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly sequenced by cycle sequencing. Dideoxy termination analysis was carried out by cyclic extension of a 33P-labelled primer by Tth polymerase with termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction products were analysed by electrophoresis on denaturing 7 M urea/6% acrylamide gels followed by autoradiography. Computer aided sequence analysis indicated that conserved 5' NCR sequence variation alone was sufficient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerprinting improved greatly the efficiency of genotyping with an approximate four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a & 1b); a ddC reaction confirmed 1a and 1b typing.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1986

Differentiation of Schistosoma haematobium from related species using cloned ribosomal RNA gene probes.

The ribosomal RNA (rRNA) gene units of Schistosoma mansoni (lateral spined eggs) and six species of schistosomes with terminal spined eggs (S. haematobium, S. curassoni, S. bovis, S. intercalatum, S. margrebowiei and S. mattheei) have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit of the species studied is demonstrated and takes three forms: First, variation in DNA sequence leads to both reduced homology in the spacer regions between species and loss or gain of restriction sites. Second, variation in the length of the transcribed spacer is demonstrated and DNA insertions of 0.2 kilobases (kb) and 0.1 kb are observed in S. mattheei and S. margrebowiei, respectively. Third, the length of the non-transcribed spacer region varies between species. S. haematobium has a 0.5 kb deletion in this region, while that of S. margrebowiei contains varying numbers of a 0.4 kb DNA insert. These interspecific variations have been shown to be conserved within species. Analysis of the rRNA genes by DNA hybridisation techniques therefore serves as a means of species identification, whereby it is possible to differentiate S. haematobium from other schistosome species with terminal spined eggs. Similarly, S. margrebowiei and S. mattheei may be clearly distinguished, although no major variation has been detected between S. curassoni, S. bovis and S. intercalatum. All these species differ from S. mansoni by the absence of certain restriction sites in the non-transcribed spacer.

Animals↗
PubMed1989

Molecular cloning, genomic structure and localization in a blood stage antigen of Plasmodium falciparum characterized by a serine stretch.

Two short DNA segments were isolated by screening of a lambda gt11 library from Plasmodium falciparum schizont cDNA with an antiserum against the 140 kDa protein, which confers protective immunity to monkeys. The segments were used to identify a genomic fragment which carries the entire coding sequence for a protein of 113 kDa characterized by a stretch of serine residues (SERP I). We present the complete nucleotide and deduced amino acid sequence as well as the structure of the SERP I gene. The gene consists of four exons interrupted by three short introns located at the amino-terminal half. Exon 1 and the first part of exon 2 code for hydrophobic amino acids of a putative signal sequence. Exon 2 contains two repetitive segments, the first encoding six glycine rich octapeptides and a second region coding for 37 consecutive serine residues. Southern blot analysis demonstrated the conservation of the SERP I gene in four different parasite strains. SERP I could be localized in the parasitophorous vacuole and in the surrounding membranes. We discuss the relationship of this protein to the recently described P126 polypeptide and the possible role of this antigen as a vaccine candidate.

Amino Acid Sequence↗