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Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum: genomic characterization.

The G + C contents of 25 strains of Dipodascus capitatus, Dipodascus spicifer and Geotrichum clavatum were found to be heterogeneous on basis of derivative graphs of the melting profiles. Strains showing similar derivative graphs of the melting curve exhibited high levels of DNA homology (80-100%); strains showing dissimilar derivative graphs exhibited low levels of DNA homology (5 to 45%). Being considered separate taxa on basis of these parameters, D. capitatus, D. spicifer and G. clavatum could be identified by a combination of the key characteristics growth on xylose, cellobiose, salicin and arbutin.

Base Composition↗

Biochemical and genomic characterization of muscovy duck parvovirus.

A duck parvovirus (DPV) isolated from muscovy ducks during the epizootic in France in 1989 was purified from inoculated allanto-amniotic fluids by CsCl density gradient centrifugation and characterized. Full and empty non-enveloped icosahedral viral particles were observed banding at densities of 1.39 to 1.42 and 1.38 respectively, with a diameter of 22 to 23 nm. Viral proteins were analyzed by SDS-PAGE and the estimated molecular weights of the 3 major proteins were 91, 78 and 58 kDa. The nucleic acid was shown to be a single-stranded DNA of about 5,300 bases with terminal palindromic hairpins. These results confirm the previous classification of the virus in the family Parvoviridae established by Jestin et al. [14] on morphological and serological bases. The DPV DNA was reannealed indicating that complementary DNA strands were encapsidated. A partial restriction endonuclease map was also established. This work constitutes the first biochemical and genomic description of a muscovy duck parvovirus.

Animals↗

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. Yidianhong [corrected].

Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yidianhong [corrected] with yellowing at the leaf edge in China. Purified viral particles of 28-30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5' untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3' UTR, with an identical terminal motif (5'-AAAAUAAAACC-3'). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.

3' Untranslated Regions↗

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes.

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.

Base Sequence↗

Comparative immunological and genomic characterization of fowlpox virus isolates.

Chickens infected with fowlpox virus (FPV) IVRI vaccine strain and two field isolates collected from clinical cases of disease (Bareilly isolate and Panchmahal isolate) produced humoral antibody response after 2nd week post-infection, with a noticeable variation in degree of immune response. Serum antibody titre peaked at 4th week post-infection with a titre of 25,600, 25,600 and 51,200 being detected in ELISA and neutralization index of 2.75, 2.43 and 3.12 in serum neutralization test (SNT) with IVRI vaccine strain, Panchmahal isolate and Bareilly isolate, respectively. Cellular immune response was detected as early as 1st week post-infection by leukocyte migration inhibition test (LMIT). Per cent migration inhibition too peaked at 4th week with a value of 40.30 +/- 3.45, 36.93 +/- 4.11 and 45.45 +/- 3.66 being detected with the three viruses respectively. The Hind III and Hae III restriction fragment profile of viral DNA showed almost similar pattern both in vaccine strain and two field isolates. Hind III digestion produced 47 well resolved fragments of sizes between 24.30 and 1.20 kb and the total genomic size was estimated to be between 305.81 and 306.06 kb. Hae III digestion revealed 34 well resolved fragments of sizes between 27.55 and 1.32 kb. The three viruses could not be differentiated on the basis of their genomic restriction pattern. However immunogenic and antigenic differences were noticed by ELISA and SNT tests.

Animals↗

Genomic characterization of type 2 polioviruses isolated from vaccine-associated cases in Brazil.

Twenty strains of P2/Sabin-related polioviruses isolated in Brazil were analyzed; ten from persistent paralytic poliomyelitis cases, three from suspected polio cases with transient paralysis, and seven from healthy contacts. The serotypes of the viral isolates were identified by the neutralization test with hyperimmune equine sera. The relationship of the isolates to the P2/Sabin strain was demonstrated by molecular hybridization and polymerase chain reaction (PCR). Partial sequencing demonstrated mutations at nucleotide 481 in the 5' noncoding region and at amino acid 143 of the capsid protein VP1 in most of these isolates from vaccine-associated cases in Brazil. These data support previous studies on the importance of mutations at these attenuated determinants in the establishment of the disease. However, the existence of isolates without mutations at these positions suggests that they are not essential. The results also strengthen the possibility of the participation of a mutation at nucleotide 398 in the establishment of the disease, and suggest that a mutation at nucleotide 491 or 500 may also be involved in this process. The isolates from healthy contacts presented the same mutations as the isolates from vaccine-associated cases with which they were in contact. This strengthens the observation that, although mutations in the genome of the P2/Sabin strain are important for the establishment of the disease, host factors are also involved.

Base Sequence↗

Genomic characterization of human DSPG3.

DSPG3, the human homolog to chick PG-Lb, is a mejrkp6of the small leucine-rich repeat proteoglycan (SLRP) family, including decorin, biglycan, fibromodulin, and lumican. In contrast to the tissue distribution of the other SLRPs, DSPG3 is predominantly expressed in cartilage. In this study, we have determined that the human DSPG3 gene is composed of seven exons: Exon 2 of DSPG3 includes the start codon, exons 4-7 code for the leucine-rich repeats, exons 3 and 7 contain the potential glycosaminoglycan attachment sites, and exon 7 contains the potential N-glycosylation sites and the stop codon. We have identified two polymorphic variations, an insertion/deletion composed of 19 nucleotides in intron 1 and a tetranucleotide (TATT)n repeat in intron 5. Analysis of 1.6 kb of upstream promoter sequence of DSPG3 reveals three TATA boxes, one of which is 20 nucleotides before the transcription start site. The transcription start site precedes the translation start site by 98 nucleotides. There are 14 potential binding sites for SOX9, a transcription factor present in cartilage, in the promoter, and in the first intron of DSPG3. We have examined the evolution of the SLRP gene family and found that gene products clustered together in the evolutionary tree are encoded by genes with similarities in genomic structure. Hence, it appears that the majority of the introns in the SLRP genes were inserted after the differentiation of the SLRP genes from an ancestral gene that was most likely composed of 2-3 exons.

Amino Acid Sequence↗

Human ARX gene: genomic characterization and expression.

Arx is a homeobox-containing gene with a high degree of sequence similarity between mouse and zebrafish. Arx is expressed in the forebrain and floor plate of the developing central nervous systems of these vertebrates and in the presumptive cortex of fetal mice. Our goal was to identify genes in Xp22.1-p21.3 involved in human neuronal development. Our in silico search for candidate genes noted that annotation of a human Xp22 PAC (RPCI1-258N20) sequence (GenBank Accession No. AC002504) identified putative exons consistent with an Arx homologue in Xp22. Northern blot analysis showed that a 3.3kb human ARX transcript was expressed at high levels in fetal brain. A 5.9kb transcript was expressed in adult heart, skeletal muscle, and liver with very faint expression in other adult tissues, including brain. In situ hybridization of ARX in human fetal brain sections at various developmental stages showed the highest expression in neuronal precursors in the germinal matrix of the ganglionic eminence and in the ventricular zone of the telencephalon. Expression was also observed in the hippocampus, cingulate, subventricular zone, cortical plate, caudate nucleus, and putamen. The expression pattern suggests that ARX is involved in the differentiation and maintenance of specific neuronal cell types in the human central nervous system. We also mapped the murine Arx gene to the mouse genome using a mouse/hamster radiation hybrid panel and showed that Arx and ARX are orthologues. Therefore, investigations in model vertebrates may provide insight into the role of ARX in development. The recent identification of ARX mutations in patients with various forms of mental retardation make such studies in model organisms even more compelling.

Amino Acid Sequence↗

Molecular cloning of the Mason-Pfizer monkey virus genome: biological characterization of genome length clones and molecular comparisons to other retroviruses.

The molecular cloning of the DNA provirus of Mason-Pfizer monkey virus (M-PMV) is described. Fourteen independent clones of integrated M-PMV proviruses were isolated from a human embryo kidney cell line that had been previously derived from a single cell clone infected with M-PMV. Characterization of these clones for size of insert, restriction pattern of flanking DNA, and presence of repetitive DNA in the flanking sequences revealed that 10 of the isolates were identical while the four remaining clones were unique. Three independent clones of unintegrated M-PMV proviruses containing a single copy of the long terminal repeat (LTR) were cloned from acutely infected human embryo kidney cells, Transfection assays revealed that 13 of 14 integrated proviruses and 2 of 3 unintegrated proviruses were capable of producing infectious virus. One of the integrated provirus clones (clone 6A) produced consistently higher titers of virus than all of the other clones in all assays used and in two different cell lines, indicating that it contained a mutation that enhances virus replication. The virus recovered after transfection was shown to be capable of inducing cell fusion in nontransformed cell lines, confirming that this property is associated with M-PMV. One of the clones was hybridized under conditions of varying stringency, to molecular clones of type B, C, and D retroviruses. These studies revealed M-PMV to be most closely related to squirrel monkey retrovirus (D-type virus) and more distantly related to mouse mammary tumor virus (B-type virus). Hybridization was also detected with clones from the pol gene region of a family of human endogenous sequences. No homology was detected with Rous sarcoma virus or most mammalian C-type viruses tested. The exceptions were baboon endogenous virus and RD114 in which previously identified homology in the env gene was confirmed. These results suggest that the type D and type B viruses can be linked together in a group of viruses of similar ancestral origin analogous to that recently proposed for the human T-cell leukemia viruses and bovine leukemia virus.

Animals↗

Serological and genomic characterization of porcine rotaviruses in Thailand: detection of a G10 porcine rotavirus.

A total of 557 fecal specimens collected from piglets with diarrhea in Thailand were examined for rotavirus RNA by polyacrylamide gel electrophoresis. Twenty-three, one, and two samples were positive for group A, group B, and group C rotaviruses, respectively. Two samples exhibited two segments found in picobirnavirus RNA. RNA electropherotyping of 23 group A rotaviruses showed that they were classified into five patterns. By serotyping by enzyme-linked immunosorbent assay and PCR, viruses in 3 and 14 specimens were found to be serotype G3 and serotype G10, respectively. For one specimen, containing a serotype G10 virus (strain P343), virus was isolated in MA-104 cells, and the nucleotide sequences of the VP7 and VP4 genes were determined. Comparative sequence analysis and cross-neutralization tests showed that strain P343 has B223-like G10 and UK-like P7 serotype (or VP4 genotype 5) specificities. Rotaviruses having such antigenic specificities have not been detected in piglets. Thus, the interspecies transmission of rotaviruses between cows and pigs was suggested.

Amino Acid Sequence↗

Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis.

The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells. It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER). Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants. In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity). Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A. thaliana. Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development. Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress.

Amino Acid Sequence↗

Genomic characterization and regulation of CYP3a13: role of xenobiotics and nuclear receptors.

We report that CYP3a13 gene, located on mouse chromosome 5, spans 27.5 Kb and contains 13 exons. The transcription start site is 35 bp upstream of the coding region and results in a 109 bp 5' untranslated region. CYP3a13 promoter shows putative binding sites for retinoid X receptor, pregnane X receptor, and estrogen receptor. CYP3a13 shows a broad tissue distribution with predominant expression in liver. Although CYP3a13 shares 92% nucleotide identity with the female-specific rat CYP3A9, its expression does not exhibit sexual dimorphism. Ligand activation of peroxisomal proliferator-activated receptor-gamma and retinoid X receptor inhibit expression of CYP3a13 at the transcription level in a tissue-specific manner. Another novel finding is hepatic induction of CYP3a13 by dexamethasone occurring only in pregnane X receptor null mice. We also report that pregnane X receptor is essential to maintain robust in vivo basal levels of CYP3a13 in contrast to CYP3a11. CYP3a13 protein expressed in vitro can metabolize clinically active drugs ethylmorphine and erythromycin, as well as benzphetamine. We conclude that CYP3a13 is regulated differentially by various nuclear receptors. In humans this may lead to altered drug metabolism, as many of the newly synthesized ligands/drugs targeted toward these nuclear receptors could influence CYP3A gene expression.

Animals↗

Genomic characterization of oxacillin-resistant Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from Brazilian medical centres.

Studies on the genetic diversity of oxacillin-resistant coagulase-negative staphylococcal (CNS) isolates are important for the control and prevention of infections. The present study evaluated the clonal diversity of oxacillin-resistant Staphylococcus epidermidis (ORSE) and Staphylococcus haemolyticus (ORSH) strains, isolated from patients in nine Brazilian medical centres by using pulsed-field gel electrophoresis (PFGE) after digestion of bacterial DNA using SmaI. PFGE analysis of ORSE (N=44) and ORSH (N=25) strains showed the presence of 29 restriction profiles clustered in 16 PFGE types, and 21 distinct profiles in 15 PFGE types, respectively, indicating a large genetic diversity among isolates of both of these species. Among the ORSE isolates, 23 (52%) strains belonged to two predominant PFGE types (named A and B), which were observed in most of the hospitals assessed, indicating the spread of these PFGE types in hospitals located in Rio de Janeiro. The spread of PFGE types of ORSH was also detected in some of the hospitals investigated. The results show that PFGE is a suitable tool for epidemiological studies of oxacillin-resistant CNS, and can be used as a basis for infection control procedures for these multiresistant organisms.

Bacterial Proteins↗

Genomic characterization of type 1 Sabin-related polioviruses isolated in Brazil.

Eight strains of P1/Sabin-derived polioviruses isolated in Brazil from paralysis cases were analyzed. The serotypes of the viral isolates were identified by neutralization test with hyperimmune equine sera. The relationship of the isolates to the P1/Sabin strain was demonstrated by molecular hybridization and PCR. The isolates were partially sequenced with the objective of finding mutations at nucleotides (nt) 480 and 525 of the 5'-noncoding region (5' NCR) and at nt 6203 of the 3Dpol coding region (3Dpol), which are important for reversion towards neurovirulence. Four isolates from paralysis cases classified as Guillain Barré Syndrome (GBS; three with sequels) were analyzed; one presented G-->A (480) and C-->U (6203) mutations, one G-->A (480) mutation, one G-->A (480) and U-->C (525) mutations, and one did not mutate at the analyzed positions. Two isolates from transient facial paralysis cases were analyzed; one presented U-->C (525) mutation and the other G-->A (480) mutation. One isolate from a transient paralysis case classified as a neuroviral disease and one isolate from a paralysis case with sequels were analyzed and none mutated at the analyzed positions. Although the isolates may not be the causative agent of the disease, a temporal association between the isolation of the P1/Sabin-derived isolates and the disease was observed. The possibility that GBS and the facial paralysis were caused by these isolates could not be excluded.

Base Sequence↗

Functional genomic characterization of delipidation elicited by trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) in a polygenic obese line of mice.

Gene expression was measured during t10c12-CLA-induced body fat reduction in a polygenic obese line of mice. Adult mice (n = 185) were allotted to a 2 x 2 factorial experiment consisting of either nonobese (ICR-control) or obese (M16-selected) mice fed a 7% fat, purified diet containing either 1% linoleic acid (LA) or 1% t10c12-CLA. Body weight (BW) by day 14 was 12% lower in CLA- compared with LA-fed mice (P < 0.0001). By day 14, t10c12-CLA reduced weights of epididymal, mesenteric, and brown adipose tissues, as a percentage of BW, in both lines by 30, 27, and 58%, respectively, and increased liver weight/BW by 34% (P < 0.0001). Total RNA was isolated and pooled (4 pools per tissue per day) from epididymal adipose (days 5 and 14) of the obese mice to analyze gene expression profiles using Agilent mouse oligo microarray slides representing > 20,000 genes. Numbers of genes differentially expressed by greater than or equal to twofold in epididymal adipose (days 5 and 14) were 29 and 125, respectively. It was concluded that, in adipose tissue, CLA increased expression of uncoupling proteins (1 and 2), carnitine palmitoyltransferase system, tumor necrosis factor-alpha (P < 0.05), and caspase-3 but decreased expression of peroxisome proliferator-activated receptor-gamma, glucose transporter-4, perilipin, caveolin-1, adiponectin, resistin, and Bcl-2 (P < 0.01). In conclusion, this experiment has revealed candidate genes that will be useful in elucidating mechanisms of adipose delipidation.

Animals↗

Serological and genomic characterization of human rotaviruses detected in China.

A total of 1,385 stool specimens were collected from children with diarrhea at two hospitals in Wuhan, Hubei Province, China, in 1994 and 1995, and screened for rotavirus by polyacrylamide gel electrophoresis of viral RNA. Group A rotavirus was detected with high frequency; 56.5% (87/154) and 40.8% (502/1,231) of the specimens collected in 1994 and 1995, respectively, were positive for rotavirus. Assignment of G serotype and P type (VP4 genotype) of group A rotavirus by ELISA with monoclonal antibodies and/or PCR, respectively, showed that strains of G2-P[4] and G1-P[8] specificity were predominant in 1994 and in 1995, respectively. In contrast, a single strain was found to have a P[9] type specificity, and no G4 strain was detected. Unusual combinations of RNA pattern-subgroup-G serotype-P type, such as long pattern-subgroup I-G1-P[8], short pattern-subgroup II-G3-P[4] and short pattern-subgroup I-G1-P[4], were detected in four specimens. Nucleotide sequences of the VP8* and/or NSP5 genes from two Chinese P[8] strains 470 and 582 and one Chinese P[9] strain 512 as well as five Japanese P[9] strains (K8, AU1, M318, 0264, and 0265) were determined and compared with the published sequences of the corresponding gene. In the phylogenetic tree of VP8* sequences of P[9] strains, which formed two clusters each having strain K8 or AU-1 as the representative strain, the Chinese P[9] strain was found in the cluster represented by AU-1, although it was most distantly related to other strains. While NSP5 sequences of human strains with P[9] specificity were related to simian and bovine strains, that of Chinese P[8] strains was most closely related to those of porcine strains. A single group C rotavirus (No. 208) was detected. Nucleotide sequences of its VP4, VP6, VP7, and NSP4 genes were very similar to those of group C human rotaviruses detected worldwide.

Amino Acid Sequence↗

Antigenic and S-1 genomic characterization of the Delaware variant serotype of infectious bronchitis virus.

A previously unrecognized infectious bronchitis virus (IBV) serotype, referred to hereafter as the Delaware variant (DE var), was isolated from commercial broiler chickens during a severe, widespread respiratory disease epornitic in the Delmarva peninsula region of the United States in January-March 1992. The DE var serotype was found to be antigenically unrelated by virus-neutralization (VN) test to nine reference IBV serotypes from North America. Additional VN tests indicated that the DE var isolates (DE/072/92, DE/121/ 92, DE/152/92, and DE/174/92) from broilers were fully or partially neutralized by monospecific antisera prepared against themselves and against two IBV field isolates (DE/492/90 and DE/903/90) recovered from a Delmarva commercial layer flock experiencing egg production losses in 1990. Antigenic relatedness values determined by VN indicated layer isolate DE/492/90 was more closely related to the broiler DE var isolates than was layer isolate DE/903/90. Cross-challenge tests performed in specific-pathogen-free chickens also demonstrated the antigenic similarity of the broiler (DE/072/92 and DE/174/92) and the layer isolates (DE/492/90 and DE/903/90), with heterologous strain protection values ranging from 55% to 100%. Protection values of DE var isolates vs. Massachusetts 41 and Arkansas DPI were considerably lower (0-60%). The S-1 gene of the US/DE/072/92 isolate of the DE var serotype was amplified by reverse transcription polymerase chain reaction, cloned, and sequenced. The DE var S-1 gene sequence was compared with the S-1 gene sequences of IBV serotypes from North America, Europe, and Australia. A dendrogram based on this analysis supported the conclusion that the DE var serotype is highly novel among IBV. A high degree of similarity (> 88%) was observed between the S-1 genes of the DE var broiler isolates (DE/072/92 and DE/174/92) and layer isolates (DE/492/90 and DE/903/90). These data, taken with the VN and cross-challenge results, establish a genetic as well as an antigenic link between the isolates from layers and broilers and indicate the DE var serotype was responsible for both infectious bronchitis outbreaks.

Animals↗

Human KLRF1, a novel member of the killer cell lectin-like receptor gene family: molecular characterization, genomic structure, physical mapping to the NK gene complex and expression analysis.

The human NK gene complex localized on chromosome 12p12.3 - p13.2 codes for several lectin-like receptor genes expressed by NK cells as well as by other hematopoietic cells. In this study, by using the expressed sequence tag database we identified a novel receptor gene, designated as killer cell lectin-like receptor, subfamily F, member 1 (KLRF1), encoding a putative type II transmembrane glycoprotein. The KLRF1 gene has been localized on the high-resolution physical map of chromosome 12p. The genomic structure of the KLRF1 gene and the existence of one spliced variant are also described. KLRF1 was expressed at the mRNA level in peripheral blood leukocytes, activated NK cells, monocytes and NK and myeloid cell lines. The presence of two immunoreceptor tyrosine-based inhibitory-like motifs within the cytoplasmic tail of KLRF1 suggests an inhibitory role in NK cell and monocyte activity.

Amino Acid Sequence↗