Genomic Organization Analysis Research Articles

Topological Analysis of Enzymatic Actions on DNA Polyhedral Links

Current synthetic biology has witnessed a revolution that natural DNA molecule steps onto a broad scientific area by assembling a large variety of three-dimensional structures with the connectivity of polyhedra. Amathematical model of these biomolecules is crucial to clarify the biological self-assembly principle, and unravel a first-step understanding of biological regulation and controlling mechanisms. In this paper, mechanisms of two different enzymatic actions on DNA polyhedra are elucidated through theoretical models of polyhedral links: (1)topoisomerase that untangles DNA polyhedral links produces separated single-stranded DNA circles through the crossing change operation; (2)recombinase generates a class of polyhedral circular paths or polyhedral knots by applying the crossing smoothing operation. Furthermore, we also discuss the possibility of applying two theoretical operations in molecular design of DNA polyhedra. Thus, our research provides a new sight of how geometry and topology of DNA polyhedra can be manipulated and controlled by enzymes, as well as has implications for molecular design and structural analysis of structural genome organization.

Genomic organization of δ-guaiene synthase genes in Aquilaria crassna and its possible use for the identification of Aquilaria species

The resinous portions of Aquilaria plants, called agarwood, have been used as medicines and incenses. Agarwood contains a great variety of sesquiterpenes, and a study using cultured cells of Aquilaria crassna showed that the production of sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) was induced by treatment with methyl jasmonate, which led to the cloning of δ-guaiene synthases. In the present study, analyses of genomic organization and Southern blotting of δ-guaiene synthase in A. crassna were performed in order to examine the genomic background of δ-guaiene synthases in Aquilaria plants. Genomic cloning and sequencing revealed five types of sequence in putative δ-guaiene synthases sharing more than 96% identity in exon regions, and that these enzymes belonged to the class III TPS subfamily with seven exons and six introns. Furthermore, Southern blotting revealed that at least five copies of δ-guaiene synthase existed in A. crassna. The hybridization of digested DNA of A. crassna and A. sinensis with probes made with a δ-guaiene synthase cDNA fragment resulted in different banding patterns for these two species. It may be possible to identify Aquilaria species by restriction fragment length polymorphism analyses with δ-guaiene synthase cDNA probes.

Molecular Cloning and Phylogenetic Analysis of the Duck Enteritis Virus UL55 Gene

A 561-bp complete open reading frame of the duck enteritis virus(DEV) UL55 gene (GenBank accession No EU071034) was isolated in our laboratory and amplified by common PCR using a pair of specific primers .PCR product containing this ORF was cloned into the vector of PMD18-T. Four evolutionary analysis approaches were used to construct phylogenetic trees of DEV in relation to herpesviruses based on the nucleotide sequences. Bootstrap was used as statistic method to testify the reliablity of the constructed phylogenetic tree. Phylogenetic trees indicated that DEV and other herpesviruses generated from one ancestor and DEV were grouped into the subfamily Alphaherpesvirinae. In our result, DEV showed a close relationship with the genus Mardivirus, but formed a single branch. Partial genomic organization and phylogenetic analysis in the present study provides evidence that DEV was a member of the subfamily Alphaherpesvirinae and should be assigned as an individual genus or group.

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

BackgroundThe rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.ResultsThe cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat fut8 gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of fut8 in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 fut8 gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.ConclusionAltogether, these data contribute to a better knowledge of fut8 expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.

Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease-activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3-day-old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively.

Genomic organisation analysis of novel immunoglobulin-like transcripts in Atlantic salmon (Salmo salar) reveals a tightly clustered and multigene family

BackgroundSeveral novel immunoglobulin-like transcripts (NILTs) which have previously been identified in the salmonid species rainbow trout (Oncorhynchus mykiss) contain either one or two extracellular Ig domains of the V-type. NILTs also possess either an immunoreceptor tyrosine-based activating motif (ITAM) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region resulting in different signalling abilities. Here we report for the first time the genomic organisation and structure of the multigene family of NILTs in Atlantic salmon (Salmo salar) using a BAC sequencing approach.ResultsWe have identified six novel Atlantic salmon NILT genes (Ssa-NILT1-6), two pseudogenes (Ssa-NILTp1 and Ssa-NILTp2) and seven genes encoding putative transposable elements in one BAC covering more than 200 kbp. Ssa-NILT1, 2, 4, 5 and 6 contain one Ig domain, all having a CX3C motif, whereas Ssa-NILT3 contains two Ig domains, having a CX6C motif in Ig1 and a CX7C motif in Ig2. Atlantic salmon NILTs possess several ITIMs in the cytoplasmic region and the ITIM-bearing exons are in phase 0. A comparison of identity between the amino acid sequences of the CX3C Ig domains from NILTs varies from 77% to 96%. Ssa-NILT1, 2, 3 and 4 were all confirmed to be expressed either by their presence in EST databases (Ssa-NILT1) or RT-PCR (Ssa-NILT2, 3, and 4) using cDNA as template. A survey of the repertoire of putative NILT genes in a single individual revealed three novel genes (Ssa-NILT7-9) represented by the Ig domain, which together with Ig domains from Ssa-NILT1-6 could be divided into different groups based on specific motifs.ConclusionsThis report reveals a tightly clustered, multigene NILT family in Atlantic salmon. By screening a highly redundant Atlantic salmon BAC library we have identified and characterised the genomic organisation of six genes encoding NILT receptors. The genes show similar characteristics to NILTs previously identified in rainbow trout, having highly conserved cysteines in the Ig domain and several inhibitory signalling motifs in the cytoplasmic region. In a single individual three unique NILT Ig domain sequences were discovered at the genomic DNA level, which were divided into two different groups based on a four residue motif after the third cysteine. Our results from the BAC screening and analysis on the repertoire of NILT genes in a single individual indicates that many genes of this expanding Ig containing NILT family are still to be discovered in fish.

Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions.

Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy.

PmPPAE2, a new class of crustacean prophenoloxidase (proPO)-activating enzyme and its role in PO activation

The prophenoloxidase (proPO) activating system plays an important role in the defense against microbial invasion in invertebrates. In the present study, we report a second proPO-activating enzyme (designated PmPPAE2) from the hemocytes of the black tiger shrimp, Penaeus monodon. PmPPAE2 contained the structural features of the clip domain serine proteinase family and exhibited 51% amino acid sequence similarity to the insect Manduca sexta PAP-1. Amino acid sequence alignment with the available arthropod PPAE sequences demonstrated that PmPPAE2 is a new class of crustacean PPAE. Transcript expression analysis revealed that PmPPAE2 transcripts were mainly expressed in hemocytes. Double-stranded RNA-mediated suppression of PmPPAE2 transcript levels resulted in a significant decrease in the total hemolymph PO activity (41%) and also increased the shrimp's susceptibility to Vibrio harveyi infection. Genomic organization analysis revealed that PmPPAE1 and PmPPAE2 are encoded by different genomic loci. The PmPPAE1 gene consists of ten exons and nine introns, whilst PmPPAE2 comprises of eight exons interrupted by seven introns. Analysis of the larval developmental stage expression of the four key genes in the shrimp proPO system ( PmPPAE1, PmPPAE2, PmproPO1 and PmproPO2) revealed that PmPPAE1 and PmproPO2 transcripts were expressed in all larval stages (nauplius, protozoea, mysis and post-larvae), whilst PmPPAE2 and PmproPO1 transcripts were mainly presented in the late larval developmental stages (mysis and post-larvae). These results suggest that the PmPPAE2 functions as a shrimp PPAE and possibly mediates the activation of PmproPO1.

Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in Oreochromis mossambicus, O. urolepis hornorum and their hybrid

In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female x O. u. hornorum male. An identical karyotype ((2n = 44, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.

Human interphase chromosomes: a review of available molecular cytogenetic technologies.

Human karyotype is usually studied by classical cytogenetic (banding) techniques. To perform it, one has to obtain metaphase chromosomes of mitotic cells. This leads to the impossibility of analyzing all the cell types, to moderate cell scoring, and to the extrapolation of cytogenetic data retrieved from a couple of tens of mitotic cells to the whole organism, suggesting that all the remaining cells possess these genomes. However, this is far from being the case inasmuch as chromosome abnormalities can occur in any cell along ontogeny. Since somatic cells of eukaryotes are more likely to be in interphase, the solution of the problem concerning studying postmitotic cells and larger cell populations is interphase cytogenetics, which has become more or less applicable for specific biomedical tasks due to achievements in molecular cytogenetics (i.e. developments of fluorescence in situ hybridization -- FISH, and multicolor banding -- MCB). Numerous interphase molecular cytogenetic approaches are restricted to studying specific genomic loci (regions) being, however, useful for identification of chromosome abnormalities (aneuploidy, polyploidy, deletions, inversions, duplications, translocations). Moreover, these techniques are the unique possibility to establish biological role and patterns of nuclear genome organization at suprachromosomal level in a given cell. Here, it is to note that this issue is incompletely worked out due to technical limitations. Nonetheless, a number of state-of-the-art molecular cytogenetic techniques (i.e multicolor interphase FISH or interpahase chromosome-specific MCB) allow visualization of interphase chromosomes in their integrity at molecular resolutions. Thus, regardless numerous difficulties encountered during studying human interphase chromosomes, molecular cytogenetics does provide for high-resolution single-cell analysis of genome organization, structure and behavior at all stages of cell cycle.

HaloWeb: the haloarchaeal genomes database

BackgroundComplete genome sequencing together with post-genomic studies provide the opportunity for a comprehensive 'systems biology' understanding of model organisms. For maximum effectiveness, an integrated database containing genomic, transcriptomic, and proteomic data is necessary.DescriptionTo improve data access and facilitate functional genomic studies on haloarchaea in our laboratory, a dedicated database and website, named HaloWeb, was developed. It incorporates all finished and publicly released haloarchaeal genomes, including gene, protein and RNA sequences and annotation data, as well as other features such as insertion element sequences. The HaloWeb database was designed for easy data access and mining, and includes tools for tasks such as genome map generation, sequence extraction, and sequence editing. Popular resources at other sites, e.g., NCBI PubMed and BLAST, COG and KOG protein clusters, KEGG pathways, and GTOP structures were dynamically linked. The HaloWeb site is located at http://halo4.umbi.umd.edu, and at a mirror site, http://halo5.umbi.umd.edu, with all public genomic data and NCBI, KEGG, and GTOP links available for use by the academic community. The database is curated and updated on a regular basis.ConclusionsThe HaloWeb site includes all completely sequenced haloarchaeal genomes from public databases. It is currently being used as a tool for comparative genomics, including analysis of gene and genome structure, organization, and function. The database and website are up-to-date resources for researchers worldwide.

Identification and Characterization of Microsatellite Markers Useful for Genetic Analysis of Black Spruce (Picea mariana (Mill.) Populations

Summary Large - scale isolation of microsatellite and information in any conifer species is limited. Our knowledge of microsatellite in spruce (Picea spp.) is still sketchy. Genomic libraries of P. mariana were constructed and screened with (AC)15 probes. Over 200 positive clones were found for this dinucleotide and ten were analyzed in details. They were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on sequences flanking the microsatellites. All sequenced (AC)n clones had repeats of n > 23. Primer pairs were designed from seven sequences. These primer pairs along with 15 primer pairs from white spruce (Picea glauca) were tested on individual trees. Seven primer pairs from P. mariana and three from P. glauca (white spruce) amplified DNA from P. mariana and were used for genetic analysis of black spruce populations from uplands (drylands) and lowlands (wetlands). High levels of polymorphism and heterozygosity were observed in all the populations studied. Both highlands and lowlands showed similar levels of genetic variation. The selected microsatellites sequences are being used for genome organization analysis of black spruce.

Complete nucleotide sequence and genome organization of Calibrachoa mottle virus (CbMV)—A new species in the genus Carmovirus of the family Tombusviridae

Complete genomic sequence of the viral RNA of Calibrachoa mottle virus (CbMV) has been determined. The CbMV genome has a positive-sense single-stranded RNA of 3919 nucleotides in length and encodes five open reading frames (ORFs). ORF1 encodes a protein with predicted molecular weight of 28 kDa (p28). ORF2 extends through the amber stop codon of ORF1 to give a protein with a predicted molecular weight of 87 kDa (p87). The readthrough domain of p87 contains the GDD motif common to RNA-dependent RNA polymerases (RdRp). ORF3 and ORF4 encode two small overlapping polypeptides of 8 kDa (p8) and 9 kDa (p9), respectively. The 3′-proximal ORF5 encodes a capsid protein (CP) of 37 kDa (p37). The untranslated 5′- and 3′-terminal regions are composed of 34 and 234 non-coding nucleotides, respectively. Comparisons of amino acid sequences of the ORFs of CbMV with members of Tombusviridae show that CbMV is closely related to members of the genus Carmovirus. Phylogenetic analyses based on the amino acid sequences of RdRp and coat protein and nucleotide sequences of the whole genome reveal that CbMV forms a subgroup with several carmoviruses. Therefore, the genome organization, physico-chemical properties, sequence alignments and phylogenetic analysis support the classification of CbMV as a new species in the genus Carmovirus, family Tombusviridae.

Complete nucleotide sequence of double-stranded RNA viruses from Fusarium graminearum strain DK3

The complete genomes two different dsRNA mycoviruses, Fusarium graminearum virus 3 (FgV3) and Fusarium graminearum virus 4 (FgV4), was sequenced and analyzed. The viral genome of FgV3 is 9,098 base pairs (bp) long and contains two open reading frames (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) and a protein of unknown function. The FgV4 genome is composed of two dsRNA genome segments of 2,383 bp and 1,739 bp. FgV4 dsRNA-1 contains a single ORF, which has a conserved RdRp motif, while FgV4 dsRNA-2 contains two putative ORFs coding for products of unknown function. Both the genome organization and phylogenetic analysis indicated that FgV3 was closely related to members of the families Totiviriridae and Chrysoviridae, but it was placed outside of their main clusters, whereas FgV4 formed a distinct clade with the family Partitiviridae. This is the first report of the full-length nucleotide sequences of FgV3 and FgV4 infecting Fusarium graminearum.

Genomic organization and promoter analysis of the Trichomonas vaginalis core histone gene families

Core histone gene is a well-established model to study eukaryote gene transcription regulation mechanism. However, the protozoan core histone gene regulation mechanism remains largely unknown. In this study, we observed almost all protozoan Trichomonas vaginalis core histone genes (60/74) organize as gene pairs in a head-to-head manner, thus facilitating the divergent transcription of both partners. Additionally, the majority of both T. vaginalis core histone genes pairs (50/60) and solitary genes (10/14), contain three over-represented motifs with conserved positional architecture at their promoter regions. Notably of the three motifs, Motif I is highly similar to the Inr which mediates the transcription start site selection in T. vaginalis. Motif II and Motif III preferably locate at the promoter regions of the T. vaginalis genome. Those findings reveal that both genomic organization and cis-acting transcription elements facilitate these large number of T. vaginalis core histone genes under the control of the same transcription machine.

Genomic organization and expression analysis of Toll-like receptor 3 in grass carp ( Ctenopharyngodon idella)

Toll-like receptor 3 (TLR3) participates in the innate immune response by recognizing viral pathogens. To investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 ( CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp ( Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection ( P < 0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases.

A novel species-specific satellite DNA family in the invasive root-knot nematode Meloidogyne mayaguensis and its potential use for diagnostics

The root-knot nematode (RKN) Meloidogyne mayaguensis is considered as one of the most damaging RKN species because of its extremely wide host range. Recent surveys have shown the rapid spread of this parasite in agro-ecosytems, often making crop cultivation not viable in the heavily infested areas. Here, we report the identification, molecular cloning, genomic organisation and sequence analysis of a new satellite DNA (satDNA) family from M. mayaguensis (named pMmPet). It is comprised of two groups of A+T rich, tandemly repeated units of 174 and 180 bp, respectively. Using these sequences as targets, hybridisation and PCR experiments performed on a wide collection of 44 populations belonging to 15 RKN species showed that the pMmPet family could only be detected in the 16 M. mayaguensis populations tested. In addition, because of their repetitive nature, positive detection of pMmPet sequences was achieved in single individual nematodes. Therefore, the repeated sequence described here possesses features that make it an excellent candidate for use as a specific and extremely sensitive tool for the accurate detection and identification of this invasive pest on a routine basis. Clearly, monitoring the occurrence and spread of M. mayaguensis at the domestic and international levels are needed to avoid wholesale loss of agricultural resources in the infested regions.

Zwilling-A and -B, Two Related Myelin Proteins of Teleosts, Which Originate from a Single Bicistronic Transcript

Myelination, the ensheathment of axons by membranes of highly specialized glial cells, has been a crucial innovation during early vertebrate evolution. It enables high nerve signal conduction velocities, while maintaining nervous system size and energy requirements at moderate levels. Consequently, myelination has been conserved in all extant gnathostome vertebrates. In a genomewide mRNA expression screen, we identified several novel neural crest and myelin-specific transcripts in the zebrafish (Danio rerio). Here, we describe the characterization of two proteins, Zwilling-A and -B (ZwiA and ZwiB), which are exclusively expressed in myelinating glia of teleosts. They are structurally homologous and are translated from a common, bicistronic transcript. No similarities to sequences or domains of other proteins were detected. Analysis of phylogeny, genomic organization, and genomic syntenies suggests that the zwi gene has appeared soon after the teleost-specific genome duplication event and evolved under conservative selective pressure. We hypothesize that ZwiA and ZwiB serve important physiological functions in teleost myelin.

Biological and molecular characterization of a putative new sobemovirus infecting Imperata cylindrica and maize in Africa

A new virus was isolated from both the grass Imperata cylindrica and maize plants that had yellow mottle symptoms in Burkina Faso, West Africa. The virus has isometric particles ca. 32 nm in diameter. The experimental host range was restricted to Rottboellia exaltata. Virions were isolated from leaves of systemically infected maize plants. Koch's postulates were completed by mechanically inoculating uninfected Imperata or maize with either purified virus or sap from infected Imperata plants. Virion preparations were used to produce a specific polyclonal antiserum, and an enzyme-linked immunosorbent assay test was set up. The full genome of the virus was sequenced, and it comprised 4,547 nucleotides. Phylogenetic studies indicated that the virus is closely related to rice yellow mottle virus, a sobemovirus that infects monocotyledons in Africa, and is more distantly related to cocksfoot mottle virus, another sobemovirus that infects monocotyledons. Although the virus can infect R. exaltata experimentally, it differs from Rottboellia yellow mottle virus, a member of a tentative species of the genus Sobemovirus that also infects monocotyledons in Africa. Particle morphology, serological properties, genomic organization, and phylogenetic analysis are all consistent with assignment of the new virus to the genus Sobemovirus. The name Imperata yellow mottle virus is proposed.

Molecular cloning, genomic organization and expression analysis of the gene encoding bovine ( Bos taurus) B-cell activating factor belonging to the TNF family (BAFF)

A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine B-cell activating factor belonging to TNF family (bBAFF). The open reading frame (ORF) of this cDNA covers 843 bp, encoding 280 amino acids. The functional soluble part of bBAFF (bsBAFF) shows 96% and 91% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bBAFF genomic sequence consists of six exons and five introns, is approximately 30 kb in size, and maps to bovine chromosome 12q. Southern blotting analysis indicated that the bBAFF gene is a single copy gene. Real-time quantitative PCR (qPCR) analysis revealed that bBAFF is predominantly expressed in bovine lymphoid tissues PBLs and spleen. The predicted three dimensional (3D) structure of the bsBAFF monomer analyzed by “comparative protein modeling” revealed that it is very similar to its human counterpart. In western blotting analysis, His 6-tagged bsBAFF protein expressed in E. coli could be recognized not only by an anti-His 6.tag mAb but also by an anti-human sBAFF mAb, indicating immunological cross-reactivity occurs between bovine and human sBAFF protein.