Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

Identification and molecular analysis of rice somatic hybrids using foreign Transgenes

Summary Somatic fusion of rice [Oryza sativa L.] cell lines [hyg^r-philipino 28] and [geneticin^r(G418^r)-G171] was carried out using electrofusion methodology. Heterokaryons were identified in culture medium supplemented with both hygromycin B and G418. The hybrid character, of some of the putative somatic hybrid plants, was confirmed by Southern blot analysis which indicated the presence of the selectable marker genes [hpt, npt II] brought by the two parents of fusion.     

Identification and molecular analysis of superoxide dismutase isoforms in Helicobacter pylori

Three electromorphs of iron superoxide dismutase (FeSOD) were identified among 29 Helicobacter pylori isolates by native gel electrophoresis and activity staining. The electromorphs designated isoforms A, B, and C are characterized by slow, intermediate and fast electrophoretic migration, respectively, which was not observed under denaturing conditions. The isoforms were not associated with virulence determinants and with the outcome of disease. Sequence analysis of the sodB gene in strains producing different FeSOD isoforms and comparison of deduced protein sequences revealed that differences in the electric migration behavior are associated with exchange of charged amino acids, suggesting that faster migration is caused by a more negative total charge of the proteins. Electrophoretic migration of native FeSOD was not influenced by changes in the iron cofactor concentration, oxidative stress, and different media, indicating that FeSOD isoforms represent stable strain-specific markers.     

Identification and molecular analysis of the cuticle protein genes of Dacus oleae

A 11.9 Kb DNA segment of the Dacus oleae genome that contains three cuticle protein genes has been cloned and characterized. These three genes are clustered within 8.04 Kb of DNA; the restriction sites for eight enzymes and the organization of the cuticle genes in this 11.9 Kb cloned fragment were determined. Using this clone as a radioactive probe, it was shown that the three cuticle genes are expressed as poly(A) RNA in the epidermis of late third instar larvae but are not abundantly expressed in other tissues.     

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.     

Identification of potential drug target in malarial disease using molecular docking analysis

Malaria caused by genus Plasmodium, is a parasite which is the main health issue for humans and about half of the population were suffered. An every year, approximately 1.2–2.7 million people died due to malaria globally. Therefore to prevent the spreading of malaria from the glob novel active drugs with specific activities are necessary. The present study aimed to identify novel drug molecule together with the bioinformatic tools for the development of active malarial drugs. As the search for latest anti malarial compound was developed, this work determined six active blends from various drug databases which possess drug-like characteristics and presents a significant anti malarial actions in in-silico level. Compound ID 300238, 889, 76569, 87324, 45678, and Z185397112are a few of the ligands were got from the Toss lab, Maybridge, Cambridge, Life chem, Bitter, and Examine drug databases and docked against hexokinase 1 protein (PDB: 1CZA) with high throughput practical screening (HTVS) using Glide v6.6. Amid the 6 compounds, compound no: 300238 from Toss lab has the greatest docking score of −9.889 kcal/mol targeting 1CZA protein. The active sites of Hexokinase I of protein were determine by using superimposition of the destination and template structure showed similar structural folds and active sites which were decidedly conserved. The quality of hexokinase I protein was considered to be sterically stable where the protein was prepared by utilizing the software protein preparation execute in the Schrodinger suite. Prepared proteins were evaluated using SAVES and the studies of molecular dynamics of the hexokinase, and the GROMACS were performed for protein–ligand complex. The low HOMO-LUMO energy gaps of the compound verified the greater stability of the molecule. Here, the tested drug candidates have good absorption, distribution, metabolism, and excretion (ADME) properties which were established by using QikProp, version 3.4 of Schrodinger.     

Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae.

Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein.     

Identification of an olfactory receptor neuron subclass: cellular and molecular analysis during development

Development of olfactory receptor neuron populations was studied using the previously described monoclonal antibody (Mab) 2B8 which binds to cell surface glycoproteins of presumptive olfactory receptor neurons. In order to definitively demonstrate that the cells recognized were olfactory receptor neurons and to better characterize these cells during development, a well-established receptor cell marker, olfactory marker protein (OMP), was studied at the same time as the 2B8 antigens in double-label immunofluorescence analyses of olfactory structures in rats from Day 13 of gestation (E13) to the early postnatal period. Olfactory epithelium cryostat sections of E13 rats showed binding of the 2B8 Mab to bipolar cells in caudal regions of the nasal cavity. The 2B8 Mab also recognized a large number of cells in the vomeronasal organ (VNO) at this stage. No specific binding of anti-OMP was seen until E15. At this time approximately half of the 2B8 reactive cells also expressed OMP. By birth, greater than 90% of the 2B8 reactive cells expressed OMP. The percentage of total fluorescent labeled cells which are double labeled remained relatively constant at 23-33% as the total number of cells increased between E15 and 2 days postnatal. 2B8 immunoreactivity can be found in the olfactory nerve layer of the olfactory bulb and the presumptive accessory olfactory bulb at E15. In double-label experiments the 2B8 Mab did not bind to all anti-OMP-labeled glomeruli of postnatal to adult rats. In summary, the 2B8 Mab recognizes cells early during development and appears to recognize a subclass of olfactory receptor cells and their axon terminals. Developmental changes in the electrophoretic profile of the olfactory 2B8 antigens were also studied. In the olfactory epithelium a single band at Mr of 200,000 was seen at E19. After birth three bands at 220,000, 180,000 and 110,000 were observed but in adults only two bands of Mr 215,000 and 163,000 were detected. During olfactory bulb development the Mr of the two major 2B8 reactive bands did not change but remained the same as the two major bands seen in the adult olfactory epithelium. The olfactory bulb band at Mr of 215,000 showed a 3 to 4-fold increase and the band at 163,000 showed a 10-fold increase in specific activity from birth to adulthood.     

Identification and molecular analysis of transgenic potato chromosomes transferred to tomato through microprotoplast fusion

 Results are reported on the integration sites and copy number of alien marker genes neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA), introduced into diploid potato Solanum tuberosum through transformation by Agrobacterium tumefaciens. Also, the transgenic potato chromosomes 3 and 5 harbouring the nptII and uidA genes, which were transferred to tomato (wild species Lycopersicon peruvianum) by microprotoplast fusion, as revealed by genomic in situ hybridization (GISH), were identified by RFLP analysis using chromosome-specific markers. The data revealed three integration sites in the donor potato genome, each containing the uidA gene, and two also harbouring the nptII gene. Analysis of monosomic-addition hybrid plants obtained after microprotoplast fusion showed that each of these three integration sites is located on a different potato chromosome. The microprotoplast hybrid plants contained only the chromosomes that carried the selectable gene nptII. The data on sexual transmission of the donor potato chromosome carrying the uidA and nptII genes were obtained by analysing the first backcross progeny (BC₁) derived from crossing a monosomic-addition hybrid plant to tomato (L. peruvianum). The glucuronidase (GUS) assay and PCR analysis using primers for the uidA gene indicated the presence of the potato chromosome in GUS-positive and its absence in GUS-negative BC₁ plants. RFLP analysis confirmed sexual transmission of the potato chromosome carrying the nptII and uidA genes to the BC₁ plants. A few BC₁ plants contained the nptII and uidA genes in the absence of the potato additional chromosome, indicating that the marker genes were integrated into the tomato genome. The potential applications of the transfer of alien chromosomes and genes by microprotoplast fusion technique are discussed. Recieved: 1 September 1996 / Accepted: 20 September 1996     

Identification, molecular cloning, and phylogenetic analysis of a non-respiratory pseudo-hemocyanin of Homarus americanus

Copper-containing hemocyanins serve to transport oxygen in many arthropod species. Here I describe the identification and cDNA cloning of a structurally closely related non-respiratory pseudo-hemocyanin (PHc) of the American lobster, Homarus americanus. This protein has lost the ability to bind copper and, therefore, oxygen because a histidine residue in copper-binding site A is replaced by tyrosine. Like many arthropod hemocyanins, PHc forms a hexamer. It consists of two different subunit types of 660 and 661 amino acids, respectively, that share a 94.4% sequence identity. Whereas Homarus hemocyanin is produced in the hepatopancreas, PHc is synthesized by the ovaries and the heart tissue. Because different levels of PHc were observed in distinct individuals, I propose an association of the synthesis of this protein with the molting or reproduction cycle, similar to the hexamerins, insect storage proteins that are also related to the hemocyanins. However, phylogenetic analyses show that PHc derived independently from crustacean hemocyanins. Therefore, Homarus PHc is a member of a new class within the growing hemocyanin protein superfamily.     

Identification of Swedish mosquitoes based on molecular barcoding of the COI gene and SNP analysis

Mosquito‐borne infectious diseases are emerging in many regions of the world. Consequently, surveillance of mosquitoes and concomitant infectious agents is of great importance for prediction and prevention of mosquito‐borne infectious diseases. Currently, morphological identification of mosquitoes is the traditional procedure. However, sequencing of specified genes or standard genomic regions, DNA barcoding, has recently been suggested as a global standard for identification and classification of many different species. Our aim was to develop a genetic method to identify mosquitoes and to study their relationship. Mosquitoes were captured at collection sites in northern Sweden and identified morphologically before the cytochrome c oxidase subunit I (COI) gene sequences of 14 of the most common mosquito species were determined. The sequences obtained were then used for phylogenetic placement, for validation and benchmarking of phenetic classifications and finally to develop a hierarchical PCR‐based typing scheme based on single nucleotide polymorphism sites (SNPs) to enable rapid genetic identification, circumventing the need for morphological characterization. The results showed that exact phylogenetic relationships between mosquito taxa were preserved at shorter evolutionary distances, but at deeper levels, they could not be inferred with confidence using COI gene sequence data alone. Fourteen of the most common mosquito species in Sweden were identified by the SNP/PCR‐based typing scheme, demonstrating that genetic typing using SNPs of the COI gene is a useful method for identification of mosquitoes with potential for worldwide application.     

Identification and molecular analysis of a locus that regulates extracellular toxin production in Clostridium perfringens

The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb Hindlll fragment in the C. perfringens chromosome. A 4.3 kb Pstl fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb Hindlll fragment, or the 4.3 kb Pstl fragment resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The Pstl fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51 274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved In virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.     

Identification and molecular analysis of a ferritin subunit from red drum (Sciaenops ocellatus)

Ferritin is a conserved iron binding protein existing ubiquitously in prokaryotes and eukaryotes. In this study, the gene encoding a ferritin M subunit homologue (SoFer1) was cloned from red drum (Sciaenops ocellatus) and analyzed at expression and functional levels. The open reading frame of SoFer1 is 531 bp and preceded by a 5′-untranslated region that contains a putative Iron Regulatory Element (IRE) preserved in many ferritins. The deduced amino acid sequence of SoFer1 possesses both the ferroxidase center of mammalian H ferritin and the iron nucleation site of mammalian L ferritin. Expression of SoFer1 was tissue specific and responded positively to experimental challenges with Gram-positive and Gram-negative fish pathogens. Treatment of red drum liver cells with iron, copper, and oxidant significantly upregulated the expression of SoFer1 in time-dependent manners. To further examine the potential role of SoFer1 in antioxidation, red drum liver cells transfected transiently with SoFer1 were prepared. Compared to control cells, SoFer1 transfectants exhibited reduced production of reactive oxygen species following H₂O₂ challenge. Finally, to examine the iron binding potential of SoFer1, SoFer1 was expressed in and purified from Escherichia coli as a recombinant protein. Iron-chelating analysis showed that purified recombinant SoFer1 was capable of iron binding. Taken together, these results suggest that SoFer1 is likely to be a functional ferritin involved in iron sequestration, host immune defence against bacterial infection, and antioxidation.     

Identification of genetic factors for alachlor tolerance in maize by molecular markers analysis

Genetic factors controlling tolerance to the herbicide Alachlor in maize were localised by means of two different strategies. In the first approach, backcross (BC) plants, derived from pollen which had been subjected to selective pressure for resistance to the herbicide, were analysed for segregation distortion at 47 RFLP loci and compared to BC plants obtained from non-selected pollen. Preferential transmission of five chromosomal regions where putative QTLs (Quantitative Trait Loci) are localised was revealed in the BC plants from selected pollen. A second approach was based on a classical linkage analysis for segregation of the same set of RFLPs and factors controlling the trait, in a BC population of 210 individuals, by means of regression analysis. This study detected seven significant loci in four genomic regions. Overall, two loci revealed both segregation distortion and association with the expression of the trait, indicating linkage to genes expressed in both gametophytic and sporophytic phase. Three chromosomal regions appeared to carry factors involved in plant tolerance to Alachlor which are not expressed in pollen. Conversely, three loci were linked to factors selectable in pollen, but did not reveal significant association with tolerance in the plant in the segregating populations.