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PubMed2004

Genomic characterization of a slow/low maedi visna virus.

The complete genomic sequence of a sheep lentivirus isolate that presents a slow/low phenotype in vitro has been determined. The virus, designated P1OLV, was isolated from lung cells of a naturally infected sheep in Portugal. Three overlapping DNA fragments amplified by PCR, and encompassing the entire viral genome were cloned and sequenced. This isolate has an overall similarity of approximately 80% with the K1514 Maedi Visna virus (MVV) and approximately 70% with the caprine arthritis encephalitis virus (CAEV) Co strain. Phylogenetic analysis based on SU and RT nucleotide sequences grouped P1OLV with previously reported ovine MVV. To determine the virus replication rate, sheep choroid plexus (SCP) and lung cells, macrophages (MØ), and goat synovial membrane (GSM) cells were inoculated with either P1OLV or with the lytic North American strain WLC-1. Viral RNA in culture supernatants was measured by one-tube real time quantitative RT-PCR. Significant differences were observed between the replication rates of the two viruses, with WLC-1 growing rapidly and to high levels in all the cells tested, while P1OLV replicated more slowly and to lower levels inducing persistent infections in lung and SCP cells. The U3 region of the LTR of P1OLV lacks the sequence repeats that are present in the LTRs of WLC-1 and MVV prototype K1514 and that contain additional binding sites for the AML(vis) transcriptional factor. To evaluate the contribution of LTR in the virus replication rate in vitro, we measured the basal activity of the promoter from P1OLV and WLC-1 in a luciferase-driven gene expression assay and lower levels of expression were achieved for P1OLV. The genetic and biological properties of P1OLV will be useful for the study of virus transcriptional factors and genes that may be responsible for the slow/low phenotype.

Animals↗
PubMed2026

Characterization and comparative analysis of the complete chloroplast genomes of twelve Allium species from Kazakhstan.

The genus Allium L. represents one of the largest and taxonomically complex groups of monocots, with Central Asia recognized as a major center of its diversity. Despite the high species richness of Allium in Kazakhstan, genomic data for many native taxa remain limited. In this study, we sequenced, assembled, and analyzed the complete chloroplast genomes of 12 Allium species from Kazakhstan. All chloroplast genomes exhibited a conserved quadripartite structure, with genome sizes ranging from 152,029 to 153,521 bp and a uniform gene content of 137 genes, including 88 protein-coding genes, 38 tRNAs, 8 rRNAs, and 3 pseudogenes. Comparative analyses revealed high structural conservation, with most sequence divergence concentrated in intergenic regions. Several highly variable regions, including ycf1, matK, rpoC2, and ycf2, were identified as potential molecular markers. Phylogenetic analyses based on chloroplast genome sequences using Maximum Likelihood and Bayesian approaches recovered three major chloroplast genome-based lineages within Allium, largely consistent with previous phylogenomic studies. Divergence-time analyses suggested that major chloroplast lineage diversification events within the genus occurred during the early Eocene (ca. 47.97 Mya). Overall, this study expands the currently available chloroplast genomic resources for Allium from Kazakhstan, provides insights into chloroplast genome evolution and chloroplast genome-based relationships, and establishes a valuable foundation for future phylogenetic, taxonomic, and evolutionary studies of this diverse genus.

Genome, Chloroplast↗
PubMed1999

Complete cDNA cloning, genomic organization, chromosomal assignment, functional characterization of the promoter, and expression of the murine Bamacan gene.

Bamacan is a chondroitin sulfate proteoglycan that abounds in basement membranes. To gain insights into the bamacan gene regulation and transcriptional control, we examined the genomic organization and identified the promoter region of the mouse bamacan gene. Secondary structure analysis of the protein reveals a sequential organization of three globular regions interconnected by two alpha-helix coiled-coils. The N- and the C-terminal ends carry a P-loop and a DA box motif that can act cooperatively to bind ATP. These features as well as the high sequence homology with members of the SMC (structural maintenance of chromosome) protein family led us to conclude that bamacan is a member of this protein family. The gene comprises 31 exons and is driven by a promoter that is highly enriched in GC sequences and lacks TATA and CAAT boxes. The promoter is highly functional in transient cell transfection assays, and step-wise 5' deletions identify a strong enhancer element between -659 and -481 base pairs that includes Jun/Fos proto-oncogene-binding elements. Using backcrossing experiments we mapped the Bam gene to distal chromosome 19, a locus syntenic to human chromosome 10q25. Bamacan is differentially expressed in mouse tissues with the highest levels in testes and brain. Notably, bamacan mRNA levels are low in normal cells and markedly reduced during quiescence but are highly increased when cells resume growth upon serum stimulation. In contrast, in all transformed cells tested, bamacan is constitutively overexpressed, and its levels do not change with cell cycle progression. These results suggest that bamacan is involved in the control of cell growth and transformation.

Amino Acid Sequence↗
PubMed2004

Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain.

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

Alleles↗
PubMed2026

Characterization and comparative analysis of the complete mitochondrial genome sequence of Aucklandia lappa Decne.

BACKGROUND: Aucklandia lappa Decne, a precious medicinal herb in China, utilises the dried rhizome as its medicinal part, which riches in volatile oils, sesquiterpene compounds and other active constituents. Although the mitogenome of A. lappa has been assembled, related research remains in its preliminary stages, far from achieving comprehensive and in-depth understanding. RESULTS: In this paper, the complete mitogenome of A. lappa was assembled by employing a hybrid strategy that combined Illumina short-read and Nanopore long-read sequencing. The assembled mitogenome is 436,648 bp in size with a GC content of 45.06%. The mitogenome has only one chromosomal structure, It contains 54 genes, including 31 protein-coding genes(PCG), 19 tRNA genes, and 4 rRNA genes. A total of 32 high-frequency codons exhibiting significant AT-bias, 431 RNA editing sites, and 26 homologous fragments were transferred from the chloroplast to mitochondria genes (7,337 bp, 1.68%). Furthermore, we conducted a phylogenetic analysis involving in A. lappa and 27 other taxa to clarify its evolutionary and taxonomic status. These findings provide a foundation for further understanding the evolutionary relationships within Asteraceae plant. CONCLUSION: Through the assembly and comprehensive analysis of A. lappa mitogenome, This study has for the first time fully elucidated its mitogenomic structural characteristics.These results not only provide high-quality genetic resources for research on the Asteraceae mitogenome but also lay a solid foundation for in-depth exploration of the evolution and functional genomics of medicinal plants in the Asteraceae family.

Genome, Mitochondrial↗
PubMed2003

Full-length genome sequence of Mossman virus, a novel paramyxovirus isolated from rodents in Australia.

Mossman virus (MoV) was isolated on two occasions from wild rats trapped in Queensland, Australia, during the early 1970s. Together with Nariva virus and J-virus MoV belongs to a group of novel paramyxoviruses isolated from rodents during the last 40 years, none of which had been characterized at the molecular level until now. cDNA subtraction strategies used to isolate virus-specific cDNA derived from both MoV-infected cells and crude MoV pellets were pivotal steps in rapid characterization of the complete genome sequence. Analysis of the full-length genome and its encoded proteins confirmed that MoV is a novel member of the subfamily Paramyxovirinae which cannot be assigned to an existing genus. MoV appears to be more closely related to another unclassified paramyxovirus Tupaia paramyxovirus (TPMV), isolated from the tree shrew Tupaia belangeri. Together with Salem virus (SalV), a further unclassified paramyxovirus that was isolated from a horse, MoV and TPMV make up a new collection of paramyxoviruses situated evolutionally between the genus Morbillivirus and the newly established genus Henipavirus.

Amino Acid Sequence↗
PubMed2004

Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen.

Johne's disease, a chronic inflammatory disease caused by infection with Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), is one of the most prevalent and costly diseases of dairy cattle worldwide. This ruminant pathogen is closely related to the ubiquitous animal and human pathogen Mycobacterium avium subspecies avium (M. avium), confounding the development of specific diagnostic reagents. Exacerbating this problem further is that most existing microbiological, serological, and immunologic assays for the identification of infected animals are inadequate. This is primarily because of the slow-growing nature of the organism, genetic intractability and the previous lack of information on M. paratuberculosis subspecies-specific genes or proteins that may enable the development of specific and sensitive assays. New detection tools are critically needed to definitively answer questions surrounding M. paratuberculosis as a foodborne pathogen as well as aid in determining if it is a contributing factor in Crohn's disease. Thus, the recent characterization of the complete genome sequence of M. paratuberculosis in our laboratories has been a major step forward in meeting this need. We have performed studies that utilize genomic information for the identification of specific DNA sequences and protein antigens in M. paratuberculosis. Based on a preliminary in silico comparison of the M. paratuberculosis genome sequence with that of M. avium, we have now identified at least 35 novel coding sequences that are unique to M. paratuberculosis. These in silico data were then confirmed and expanded by PCR amplification analysis with DNA from several species and isolates of mycobacteria. Finally, these unique sequences have been incorporated into an antigen discovery project that may allow reliable detection of the bacterium in antigen-based diagnostic tests. Application of these new tools in addressing foodborne related issues of M. paratuberculosis is discussed.

Animals↗
PubMed2006

Characterization and multi-generational stability of the growth hormone transgene (EO-1alpha) responsible for enhanced growth rates in Atlantic Salmon.

Transgenic technologies provide a promising means by which desirable traits can be introduced into cultured fish species within a single generation thus accelerating the production of genetically superior broodstock for aquaculture. However, before such fish are allowed to be marketed as food they must receive government regulatory approval. Two pivotal regulatory requirements are: (1) complete characterization of the genomically integrated transgene and, (2) demonstration that the transgene remains stable over multiple generations. We have generated a stable line of growth hormone (GH) transgenic Atlantic salmon (Salmo salar) using an "all fish" gene construct (opAFP-GHc2) containing a growth hormone cDNA from chinook salmon whose expression is regulated by the 5' promoter and 3' termination regions derived from an ocean pout antifreeze protein (AFP) gene. In this study we show that a reorganized form of the opAFP-GHc2 construct (termed EO-1alpha) integrated as a single functional copy into a 35 bp repeat region of the genomic DNA. PCR based mapping revealed that the linear sequence of the EO-1alpha integrant was organized as follows: base pairs 1580-2193 of the ocean pout promoter region followed by the intact chinook salmon GH cDNA, the complete ocean pout antifreeze 3' region, and the first 1678 bp of the ocean pout antifreeze 5' region. Sequence analysis of the EO-1alpha integrant and genomic flanking regions in F2 and F4 generation salmon revealed that they were identical. In addition, apart from the disruption at the integration sites, the consensus sequences of the integrant in these two generations of salmon were identical to the sequence of the opAFP-GHc2 construct. These results indicate that the EO-1alpha transgene codes for the chinook salmon GH, and that the transgene and the integration site have remained stable over multiple generations.

Animals↗
PubMed2026

Assembly and comparative analysis of the mitochondrial genome of Pleione yunnanensis: genome structure and evolutionary insights.

BACKGROUND: Pleione yunnanensis a terrestrial or semi-epiphytic herbaceous plant belonging to the Orchidaceae family, is valued for both its medicinal uses and ornamental appeal. Although its chloroplast genomes have been sequenced, its complete mt genome had not previously been resolved, limiting genetic and evolutionary studies of the species. RESULTS: In this work, we assembled and characterized the first complete mt genome of P. yunnanensis, revealing a structurally complex, multibranched system composed of 14 circular-mapping molecules totaling 468,176 bp with a GC content of 44.32%. The genome encodes 44 annotated genes, including 28 protein-coding genes (PCGs), 15 tRNAs, and one rRNA. The multibranched architecture provides new evidence supporting the dynamic and recombinational nature of plant mt genomes. Repeat analysis uncovered 29 simple sequence repeats (SSRs), 19 tandem repeats, and 118 dispersed repeats, indicating a comparatively lower repeat abundance than that found in closely related orchids with similar mt genome sizes. Codon-usage profiling of PCGs showed a marked bias toward A/T-ending codons. Prediction of RNA editing sites identified 4,708 putative edits across mitochondrial PCGs. Most mitochondrial genes displayed Ka/Ks ratios close to 1.0, suggesting relaxed selective constraints or lineage-specific evolutionary patterns rather than strong positive selection. Moreover, we detected 69 chloroplast-derived homologous fragments, including 15 intact genes, suggesting ongoing plastid-mitochondrial DNA transfer. Phylogenetic reconstruction and collinearity comparisons demonstrated that P. yunnanensis clustered closely with Dendrobium species, including D. amplum and D. hancockii, within the Orchidaceae clade. CONCLUSIONS: This study provides the first complete mt genome of P. yunnanensis, providing a foundational genomic resource for the genus Pleione. The results not only improve our understanding of mt genome structure and evolution in Orchidaceae, but also offer valuable molecular evidence for phylogenetic inference, germplasm identification, and conservation of this endangered medicinal species.

Orchidaceae↗
PubMed2001

Annotation transfer for genomics: measuring functional divergence in multi-domain proteins.

Annotation transfer is a principal process in genome annotation. It involves "transferring" structural and functional annotation to uncharacterized open reading frames (ORFs) in a newly completed genome from experimentally characterized proteins similar in sequence. To prevent errors in genome annotation, it is important that this process be robust and statistically well-characterized, especially with regard to how it depends on the degree of sequence similarity. Previously, we and others have analyzed annotation transfer in single-domain proteins. Multi-domain proteins, which make up the bulk of the ORFs in eukaryotic genomes, present more complex issues in functional conservation. Here we present a large-scale survey of annotation transfer in these proteins, using scop superfamilies to define domain folds and a thesaurus based on SWISS-PROT keywords to define functional categories. Our survey reveals that multi-domain proteins have significantly less functional conservation than single-domain ones, except when they share the exact same combination of domain folds. In particular, we find that for multi-domain proteins, approximate function can be accurately transferred with only 35% certainty for pairs of proteins sharing one structural superfamily. In contrast, this value is 67% for pairs of single-domain proteins sharing the same structural superfamily. On the other hand, if two multi-domain proteins contain the same combination of two structural superfamilies the probability of their sharing the same function increases to 80% in the case of complete coverage along the full length of both proteins, this value increases further to > 90%. Moreover, we found that only 70 of the current total of 455 structural superfamilies are found in both single and multi-domain proteins and only 14 of these were associated with the same function in both categories of proteins. We also investigated the degree to which function could be transferred between pairs of multi-domain proteins with respect to the degree of sequence similarity between them, finding that functional divergence at a given amount of sequence similarity is always about two-fold greater for pairs of multi-domain proteins (sharing similarity over a single domain) in comparison to pairs of single-domain ones, though the overall shape of the relationship is quite similar. Further information is available at http://partslist.org/func or http://bioinfo.mbb.yale.edu/partslist/func.

Computational Biology↗
PubMed1995

Genomic organization and complete cDNA sequence of the human phosphoinositide-specific phospholipase C beta 3 gene (PLCB3).

We have characterized the complete cDNA sequence, genomic structure, and expression of the human phosphoinositide-specific phospholipase C beta 3 (PLC beta 3) gene (gene symbol PLCB3). PLC beta 3 plays an important role in initiating receptor-mediated signal transduction. Activation of PLC takes place in many cells as a response to stimulation by hormones, growth factors, neurotransmitters, and other ligands. The partial cDNA sequence of PLC beta 3, previously published, was extended with 876 bp in the 5' direction, giving a transcript of 4400 bp and a total open reading frame of 1234 amino acids. This was in accordance with expression analysis by Northern blotting that revealed a single 4.4-kb transcript in all tissues tested. Genomic data were obtained by sequencing plasmid subclones of a cosmid that contained the whole gene. The size of the complete transcription unit was estimated to be on the order of 15 kb. The gene contains 31 exons, with all splice donor and acceptor sites conforming to the GT/AG rule. No exon exceeds 571 bp in length, and the shortest exon spans only 36 bp. More than half of the introns are smaller than 200 bp, with the smallest being only 79 bp long. The transcription initiation site was determined to be within an 8-bp cluster 328-321 bp upstream of the translation initiation site. The 5'flanking region is highly GC rich, with multiple CpG doublets, and contains multiple binding sites for Sp1. Lacking typical transcriptional regulatory sequences such as TATA and CAAT boxes, the putative promoter region conforms to the group of housekeeping promoters.

Amino Acid Sequence↗
PubMed1991

Expression of the major inner capsid protein of the group B rotavirus ADRV: primary characterization of genome segment 5.

A complete cDNA copy of the fifth RNA segment of the human group B rotavirus, ADRV, has been cloned into plasmid AD512. Gene segment 5 contains 1269 bases and encodes one long open reading frame of 391 amino acids beginning at base 31 and terminating at base 1203. The gene 5 polypeptide, expressed in vitro in a rabbit reticulocyte lysate, comigrates with the 44-kDa major inner capsid protein present on EDTA treated ADRV virions. The gene 5 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum and by a group B-specific monoclonal antibody. In addition, this protein shares amino acid identity and similarity with the VP6 proteins from group C and group A rotavirus strains. The ADRV VP6 equivalent protein appears to be more closely related to the group C VP6 than the Group A VP6 polypeptide and a common ancestral rotavirus VP6 precursor protein is suggested. As a result, the fifth RNA segment of ADRV defines the major inner capsid protein, or VP6 equivalent, in the group B rotavirus. Expression of the ADRV VP6 equivalent protein is potentially useful for evaluating the prevalence of serum antibodies to group B rotavirus in human and animal populations as well as for generating antibodies for the direct detection of group B rotavirus antigen.

Amino Acid Sequence↗
PubMed2003

Relationships between fuselloviruses infecting the extremely thermophilic archaeon Sulfolobus: SSV1 and SSV2.

The fusellovirus SSV2 from an Icelandic Sulfolobus strain was isolated, characterized and its complete genomic sequence determined. SSV2 is very similar in morphology, replication, genome size and number of open reading frames (ORFs) to the type virus of the family, SSV1 from Japan, except in its high level of uninduced virus production. The nucleotide sequences are, however, only 55% identical to each other, much less than related bacteriophage, related animal viruses and the rudiviruses of Sulfolobus, SIRV1 and SIRV2. Nevertheless the genome architecture is very similar between the two viruses, indicating that despite this genomic dissimilarity the virus genomes are mostly homologous. Unlike SSV1, the sequence of SSV2 indicates integration into a glycyl tRNA gene and is completely missing a DNA packaging gene. There is a unique, perfectly tandemly directly repeated sequence of 62 nucleotides in SSV2 that has no similarity to known sequences or structures. By comparison to the SSV2 genome, an integrated partial fusellovirus genome was found in the Sulfolobus solfataricus P2 genome further confirming the dynamism of the Sulfolobus genome. Clustering of cysteine codon containing ORFs both in SSV1 and SSV2 indicates that these Fuselloviridae arose from a genome fusion event.

Fuselloviridae↗
PubMed2000

Genomic organization and regulation of the human interleukin-18 gene.

The human interleukin(IL)-18 is a key regulator of interferon(IFN)-gamma production and T-cell differentiation. Here we report the complete genomic structure and characterization of the 5'untranslated promoter region of the human IL-18 gene. The gene is composed of six exons and five introns, spanning approximately 19. 5kb. Promoter activity of the 5'-flanking region was investigated with a luciferase reporter gene assay. Transient transfection studies demonstrate a constitutive expression of the IL-18 gene in monocytic U937 and THP-1 cells. For this constitutive expression at least 92 base pairs of the promoter region are essential as shown by consecutive 5' promoter deletions in both cell types. DNA protein binding experiments revealed specific binding of activated signal transducer and activator of transcription factor-5 (STAT5) but not of STAT3 to three consensus sequences upstream in the promoter region. Cotransfection of STAT5 resulted in increased induction of the IL-18 promoter in the U937 and THP-1 cells.

Base Sequence↗
PubMed1996

Genomic structure and promoter activity of the mouse polysialic acid synthase gene (mST8Sia II). Brain-specific expression from a TATA-less GC-rich sequence.

The mouse ST8Sia II (mST8Sia II/STX) gene encodes a neural cell adhesion molecule-specific polysialic acid synthase whose expression is regulated during the developmental stages of mouse brain. To elucidate the molecular mechanism by which the expression is tissue-specifically and developmentally regulated, we isolated the complete genomic DNA and characterized the promoter of the gene for mST8Sia II. The gene encoding mST8Sia II was found to span about 80 kilobases and to be composed of six exons. Primer extension and S1 nuclease protection analyses revealed that the transcription started from 167 nucleotides upstream of the translational initiation site. Promoter analyses of the 5'-flanking region of the mST8Sia II gene using a luciferase gene reporter system revealed strong promoter activity in retinoic acid-induced differentiated P19 cells, which highly express the mST8Sia II gene. Deletion analyses demonstrated that the minimal promoter activity detected for the proximal region 325 base pairs upstream from the translational initiation codon (-158 to +167) could be modulated by various sequences within the 9. 5-kilobase 5'-flanking region. The minimal promoter was embedded in a GC-rich domain (74%, GC content), in which two Sp1 binding motifs as well as a long purine-rich region were found, but it lacked TATA and CAAT boxes. The positive regulatory region located between -159 and -659 contained two additional Sp1 binding motifs and a long pyrimidine-rich region. We also found that the minimal promoter region of the mST8Sia II gene was sufficient for expression of a reporter gene in mST8Sia II gene-expressing neural differentiated P19 cells but not in nonexpressing ones. Thus the TATA-less GC-rich minimal promoter region of mST8Sia II probably controls the cell type-specific expression of the mST8Sia II gene.

3T3 Cells↗
PubMed2006

[Study of animal viruses in yeast].

Yeast is often considered to be a model eukaryotic organism, in a manner analogous to E. coli as a model prokaryotic organism. Yeast has been extensively characterized and the genomes completely sequenced. Despite the small genome size, yeast displays most of features of higher eukaryotes. The facts that most of cellular machinery is conserved among different eukaryotes and that the powerful technologies of genetics and molecular biology are available have made yeast model eukaryotic cells in biological and biomedical sciences including virology. Cumulative data indicate that yeast can be a host for animal viruses. I briefly describe yeast gene expression and review viral replication in yeast. Great discovery include complete replication of animal viruses and production of virus-like particle vaccines in yeast. Current studies on yeast focus on identification of host factors and machinery used for viral replication. The studies are based on traditional yeast genetics and genome-wide identification using a complete set of yeast deletion strains.

Animals↗
PubMed2000

Biology of tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is a member of the ubiquitously expressed enzyme family of the acid phosphatases. Nearly 30 years ago, TRAP became known to hematologists as cytochemical marker enzyme of hairy cell leukemia. Physiologically, TRAP is primarily a cytochemical marker of macrophages, osteoclasts and dendritic cells. TRAP is localized intracellularly in the lysosomal compartment. Recent data suggest also secretion of TRAP by some cell types, in particular by osteoclasts. Human, mouse and rat TRAP are biochemically well characterized. While the complete genomic sequence of TRAP has been elucidated, only limited information on the genetic details of the gene and its regulation is available. It appears that the intracellular iron content is involved in the regulation of the enzyme. The physiological substrates for this enzyme have not been identified yet and consequently the functional role of TRAP remains completely unknown, though some hypotheses have been forwarded, e.g. involvement in bone resorption and iron homeostasis (transport, metabolism). Taken together, research on the biology of TRAP has been intensive and has led to considerable progress on a number of fronts, including the cloning of the gene. Further studies are, however, still required to determine the role of TRAP in vivo.

Acid Phosphatase↗