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PubMed1992

Isolation and characterization of a gene encoding rat nucleoside diphosphate kinase.

Two overlapping genomic clones for a rat nucleoside diphosphate kinase (NDP kinase) have been isolated and characterized. Complete sequencing of the genomic segment including the whole coding region for the enzyme revealed that the gene consists of four exons spanning 5.5 kilobase pairs. Primer extension analyses and ribonuclease protection assays indicated that the transcription may start from multiple sites with the major initiation site at 3 base pairs upstream from the translation initiation site, Met-1. Neither CAAT-box nor TATA-box could be assigned for each transcription initiation site, whereas five putative Sp1-binding sites (GC-boxes) were present in the 5'-flanking region. These features of the NDP kinase gene represent those of housekeeping genes. In genomic Southern blotting using a full-length rat NDP kinase cDNA as a probe, many positively hybridized fragments were detected. In support of this, five possible processed pseudogenes were identified in different DNA segments although many other NDP kinase-related genomic fragments remained to be characterized. These results demonstrate that the NDP kinase gene may consist of a multiple gene family.

Amino Acid Sequence↗

PubMed2026

The Complete Mitochondrial Genome of a Newly Recorded Chinese Species of Diglyphus sabulosus (Hymenoptera: Eulophidae) and Insights into Its Phylogenetic Position.

Diglyphus Walker, 1844 is an economically important genus which many species acting as biocontrol agents against agromyzid leafminer pests, but there is a lack of mitogenomic data on the evolutionary relationships within this genus, hindering a comprehensive understanding of its evolutionary history. We used traditional morphological methods to identify species, and present the first complete mitochondrial genome sequence and characterization of features of Diglyphus sabulosus and further infer its phylogenetic position based on the amino acid sequences of 13 protein-coding genes (PCGs). The complete mitochondrial genome of D. sabulosus is 15,690 bp in length, including 13 PCGs, 22 transfer RNA genes, 2 ribosomal RNA genes and a control region. The AT content of the whole genome sequence was 81.0%, indicating a significant AT bias. All protein-coding genes have the typical ATN as the start codon and TAA as the stop codon. Phylogenetic analysis inferred from the amino acid sequences of 13 PCGs revealed that all species within the family Eulophidae constituted a monophyletic clade, supporting the monophyly of this family. D. sabulosus and D. poppoea form a well-supported sister group, representing the species with the closest phylogenetic relationship within the analyzed taxa. In this study, the mitogenome structure was analyzed and the taxonomic status of D. sabulosus was clarified, thus providing a theoretical basis for understanding the phylogenetic relationships of Diglyphus.

Animals↗

PubMed2005

Characterization of new members of the human type II keratin gene family and a general evaluation of the keratin gene domain on chromosome 12q13.13.

The recent completion of a reference sequence of the human genome now allows a complete characterization of the type II keratin gene domain on chromosome 12q13.13. This, domain, approximately 780 kb in size, is present on nine bacterial artificial chromosome clones sequenced by the Human Genome Sequencing Project. The type II keratin domain contains 27 keratin genes and eight pseudogenes. Twenty-three of these genes and four pseudogenes have been previously reported. This study describes, in addition to the genomic sequencing of the K2p gene and the bioinformatic identification of four keratin pseudogenes, the characterization of cDNA corresponding to three previously undescribed keratin genes K1b, K6l, and Kb20, as well as cDNA sequences for the previously described keratin genes hHb2, hHb4, and K3. Northern analysis of the new keratins K1b, K6l, K5b, and Kb20 using mRNA of major organs as well as of specific epithelial subtypes shows singular expression of these keratins in skin, hair follicles and, for K5b and Kb20, in tongue, respectively. In addition, the obvious discrepancies between the current reference sequence of the human genome and the previously described gene/cDNA sequences for K6c, K6d, K6e, K6f, K6h are investigated, leading to the conclusion that K6c, K6d as well as K6e, K6f are probably polymorphic variants of K6a and K6h, respectively. All 26 human type II keratins found on this domain as well as K18, dtype 1 Keratin, are identified at the genomic and transcriptional level. This appears to be the total complement of functional type II keratins in humans.

Chromosomes, Human, Pair 12↗

PubMed2000

Genomic characterization of the lysophosphatidic acid receptor gene, lp(A2)/Edg4, and identification of a frameshift mutation in a previously characterized cDNA.

To understand the regulation, evolution, and genetics of lp(A2)/Edg4, a second lysophosphatidic acid receptor gene, we characterized its complete cDNA sequence, genomic structure, and chromosomal location. The full-length mouse transcript sequence was determined using rapid amplification of cDNA ends. Southern blot and restriction fragment length polymorphism segregation analyses revealed that the mouse gene was present as a single copy and located at the middle of Chromosome 8 near the mutations for myodystrophy (myd) and "kidney-anemia-testes" (kat). This region is syntenic with human chromosome 19p12, where the human genomic clone containing the lp(A2) gene (EDG4) was mapped. Sequence analysis of genomic clones demonstrated that both mouse and human transcripts were encoded by three exons, with an intron separating the coding region for transmembrane domain VI. Reverse transcriptase-PCR demonstrated that the three exons were spliced in all mouse tissues shown to express the transcript. Finally, in a comparison of all human lp(A2) sequences present in the database, we identified several sequence variants in multiple tumors. One such variant (a G deletion) in the initially characterized Edg4 cDNA clone (derived from an ovarian tumor) results in a frameshift mutation near the 3' end of the coding region. In addition to increasing our understanding of the mechanisms underlying lysophosphatidic acid signaling and lysophospholipid receptor gene evolution, these results have important implications regarding the genomic targeting and oncogenic potential of lp(A2).

Animals↗

PubMed2001

Complete cytogenetic characterization of the human breast cancer cell line MA11 combining G-banding, comparative genomic hybridization, multicolor fluorescence in situ hybridization, RxFISH, and chromosome-specific painting.

The MA11 cell line was established from malignant cells isolated from the bone marrow of a breast cancer patient. It metastasizes selectively to the brain in athymic mice. Since the genomic rearrangements of only a few breast cancer cell lines have been characterized completely, we analyzed MA11 cytogenetically. Because the G-banding analysis revealed a very complex karyotype with several markers and chromosomes with additional material of unknown origin, we used multicolor fluorescence in situ hybridization (M-FISH), cross-species color banding (RxFISH), comparative genomic hybridization (CGH), and chromosome-specific probes to better characterize the chromosome abnormalities. The use of these FISH-based screening techniques allowed us to detect previously unsuspected chromosomal changes and determine the identity of chromosomal markers. Multicolor FISH was especially useful to identify the rearranged chromosomes, whereas RxFISH, G-banding, and CGH were instrumental in determining breakpoint positions, although some uncertainties were removed only after hybridization with chromosome-specific probes. The combined analysis revealed a near-triploid karyotype with no less than 20 chromosomes demonstrating structural rearrangements. The resulting imbalances included several of those known to be common in primary breast carcinomas (gain of 1q, 8q, and 20q and loss of 8p, 11q, and 13q), indicating that the MA11 cell line may serve as a good model to study breast carcinogenesis. The full cytogenetic characterization we present may guide future searches for the mechanism of organ-selective metastasis in this model system and, possibly, also in vivo.

Breast Neoplasms↗

PubMed2005

Isolation and characterization of a new Vesivirus from rabbits.

This report describes the isolation, cDNA cloning, complete genome nucleotide sequence, and partial characterization of a new cultivable calicivirus isolated from juvenile feeder European rabbits (Oryctolagus cuniculus) showing symptoms of diarrhea. Absence of neutralization by type-specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons of the open reading frame 1-encoded polyprotein with those of other caliciviruses demonstrate that this new calicivirus is a putative novel member of the Vesivirus genus which is closely related to the marine calicivirus subgroup. According to its putative classification, this new virus has been named rabbit vesivirus.

Animals↗

PubMed2006

Wheat EST resources for functional genomics of abiotic stress.

BACKGROUND: Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project. RESULTS: We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology. CONCLUSION: We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals.

Acclimatization↗

PubMed1998

Prediction of transcriptional regulatory sites in the complete genome sequence of Escherichia coli K-12.

MOTIVATION: As one of the best-characterized free-living organisms, Escherichia coli and its recently completed genomic sequence offer a special opportunity to exploit systematically the variety of regulatory data available in the literature in order to make a comprehensive set of regulatory predictions in the whole genome. RESULTS: The complete genome sequence of E.coli was analyzed for the binding of transcriptional regulators upstream of coding sequences. The biological information contained in RegulonDB (Huerta, A.M. et al., Nucleic Acids Res.,26,55-60, 1998) for 56 different transcriptional proteins was the support to implement a stringent strategy combining string search and weight matrices. We estimate that our search included representatives of 15-25% of the total number of regulatory binding proteins in E.coli. This search was performed on the set of 4288 putative regulatory regions, each 450 bp long. Within the regions with predicted sites, 89% are regulated by one protein and 81% involve only one site. These numbers are reasonably consistent with the distribution of experimental regulatory sites. Regulatory sites are found in 603 regions corresponding to 16% of operon regions and 10% of intra-operonic regions. Additional evidence gives stronger support to some of these predictions, including the position of the site, biological consistency with the function of the downstream gene, as well as genetic evidence for the regulatory interaction. The predictions described here were incorporated into the map presented in the paper describing the complete E.coli genome (Blattner,F.R. et al., Science, 277, 1453-1461, 1997). AVAILABILITY: The complete set of predictions in GenBank format is available at the url: http://www. cifn.unam.mx/Computational_Biology/E.coli-predictions CONTACT: ecoli-reg@cifn.unam.mx, collado@cifn.unam.mx

Bacterial Proteins↗

PubMed2003

Analysis of conserved non-rRNA genes of Tropheryma whipplei.

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.

Actinomycetales↗

PubMed2000

Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data.

Genomic diversity within and between populations is caused by single nucleotide mutations, changes in repetitive DNA systems, recombination mechanisms, and insertion and deletion events. The contribution of these sources to diversity, whether purely genetic or of phenotypic consequence, can only be investigated if we have the means to quantitate and characterize diversity in many samples. With the advent of complete sequence characterization of representative genomes of different species, the possibility of developing protocols to screen for genetic polymorphism across entire genomes is actively being pursued. The large numbers of measurements such approaches yield demand that we pay careful attention to the numerical analysis of data. In this paper we present a novel application of an Affymetrix GeneChip to perform genome-wide screens for deletion polymorphism. A high-density oligonucleotide array formatted for mRNA expression and targeted at a fully sequenced 4.4-million-base pair Mycobacterium tuberculosis standard strain genome was adapted to compare genomic DNA. Hybridization intensities to 111,000 probe pairs (perfect complement and mismatch complement) were measured for genomic DNA from a clinical strain and from a vaccine organism. Because individual probe-pair hybridization intensities exhibit limited sensitivity/specificity characteristics to detect deletions, data-analytical methodology to exploit measurements from multiple probes in tandem locations across the genome was developed. The TSTEP (Tandem Set Terminal Extreme Probability) algorithm designed specifically to analyze the tandem hybridization measurements data was applied and shown to discover genomic deletions with high sensitivity. The TSTEP algorithm provides a foundation for similar efforts to characterize deletions in many hybridization measures in similar-sized and larger genomes. Issues relating to the design of genome content screening experiments and the implications of these methods for studying population genomics and the evolution of genomes are discussed.

Algorithms↗

PubMedDate not supplied

Molecular structure of yoyo, a gypsy-like retrotransposon from the mediterranean fruit fly, Ceratitis capitata.

We have isolated and characterized a new LTR-retrotransposon in the genome of the Mediterranean fruit fly (Medfly), Ceratitis capitata. This retrotransposon, which we named yoyo, appears to be a member of the gypsy/Ty3 class of elements. The yoyo element was originally discovered on the Y chromosome of the Medfly. Although the Y chromosome copy appears to be truncated, at least two other apparently complete copies of yoyo from other genomic locations have been isolated and characterized. The complete element is approximately 7.7 kb in size. In addition to fairly typical GAG and POL coding regions, the yoyo element contains a potential ENV gene. The presence of an ENV gene is a key feature distinguishing potential retroviral-like elements, such as gypsy (and possibly yoyo), from many other invertebrate retrotransposons previously described. In addition to the structural features of yoyo, evidence is provided to show that yoyo is capable of movement in the genome, including RFLPs showing variability in genomic localization of copies of yoyo between strains, and differences among individuals in the presence of yoyo at a specific site in the genome.

Amino Acid Sequence↗

PubMed2005

Full genome analysis of hepatitis B virus genotype E strains from South-Western Africa and Madagascar reveals low genetic variability.

The eight genotypes of hepatitis B virus (HBV), A-H, have a characteristic geographical distribution. The high prevalence of genotype E within a wide expanse of Africa makes it important to characterize this genotype. Although the preS/S region of a large number of genotype E isolates has been sequenced, to date only eight complete genotype E genomes have been published. The aim of this study, therefore, was to sequence and characterize the complete genome of genotype E isolates obtained from geographically distinct regions in Africa, including Namibia, Angola, and Madagascar. The nine newly sequenced genotype E isolates were closely related with an intra-group nucleotide divergence of 1.2%, reflecting their close geographical origin. All genotype E strains have the same characteristics, an in-frame deletion of three nucleotides (one amino acid) in the 5' pre-S1, a signature pattern of amino acids in the pre-S1 region and the serological subtype ayw4. In addition, we have identified another unique attribute of genotype E strains, the introduction of another start codon Met(83) in the pre-S1 region that may result in the translation of an elongated middle hepatitis B surface protein (MHBs).

Adult↗

PubMed2026

Comparative mitogenomics of Ocnus glacialis reveals lineage-specific evolutionary rates and complex gene rearrangements in Dendrochirotida.

The order Dendrochirotida (Class Holothuroidea) is a species-rich echinoderm group, yet its internal evolutionary history remains poorly resolved due to limited mitogenomic resources. In this study, we characterized the first complete mitochondrial genome of Ocnus glacialis and conducted comparative analyses to elucidate its phylogenetic position and molecular evolutionary patterns. The circular mitogenome of O. glacialis is 16,776 bp in length, containing the canonical set of 37 genes. Among the analyzed dendrochirotids, O. glacialis exhibited the highest A + T content (70.88%) and a near-zero AT-skew, a compositional profile often linked to lineage-specific evolution in specialized environments. Selection pressure analyses, including branch-model tests, revealed that these compositional features are associated with relaxed purifying selection and an accelerated rate of sequence evolution. Branch-site analyses further identified specific codon sites in cytb, nad2, nad4l, nad5, and nad6 under positive or relaxed constraints. Structurally, O. glacialis displayed the most complex gene rearrangement pattern among the studied species, characterized by multiple tandem duplication-random loss (TDRL) events and extensive intergenic sequences. Furthermore, divergence time estimation suggests that these structural and compositional shifts occurred in tandem with the lineage's diversification. We propose that these mitogenomic signatures reflect a synergistic outcome of habitat transition toward Arctic cold-water and deep-sea environments, coupled with demographic factors such as reduced effective population sizes inherent to its benthic life history. By resolving taxonomic uncertainties, this study provides a robust temporal and molecular framework for understanding the evolutionary history and ecological diversification of the Ocnus lineage.

Animals↗

PubMed1994

Genome characterization and taxonomy of Plantago asiatica mosaic potexvirus.

The complete nucleotide sequence of Plantago asiatica mosaic virus (P1AMV) genomic RNA has been determined. The 6128 nucleotide sequence contains five open reading frames (ORFs) coding for proteins of M(r) 156K (ORF1), 25K (ORF2), 12K (ORF3), 13K (ORF4) and 22K (ORF5). The sequences of these P1AMV proteins exhibit strong homology to the proteins of the other potexviruses. Phylogenetic trees based on the multiple sequence alignments of three conserved domains in ORF1 product and capsid protein reveal a close relationship of P1AMV to papaya mosaic virus and clover yellow mosaic virus. The P1AMV genomic RNA and a major subgenomic RNA (sgRNA) of 0.9 kb have been detected in infected leaves by Northern blot hybridization. The latter sgRNA is the messenger for virus capsid protein and its 5' terminus has been located 23 nucleotides upstream of the initiator codon of the coat protein gene. The P1AMV virion RNA and RNA transcript resembling the 0.9 kb sgRNA have been translated in vitro giving rise to a single major 170K product and a major 22K product, respectively.

Amino Acid Sequence↗

PubMed2006

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera.

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.

Alternative Splicing↗

PubMed1999

Complete DNA sequence and characterization of a 330-kb VNTR-rich region on chromosome 6q27 that is commonly deleted in ovarian cancer.

We report the complete genomic DNA sequence and the characterization of a 330-kb region on chromosome 6q27 that is often deleted in ovarian cancers. Using computer programs to predict exonic sequences, we isolated four novel genes, HGC6.1-4, as well as the known AF-6 gene. None of the deduced products of the novel genes exhibited significant homology to previously known proteins. We also identified ten microsatellites and 12 different VNTR sequences within the target region. HGC6.3 contained a VNTR within a coding exon, each repeat consisting of 42 nucleotides; the predicted 14-amino-acid consensus unit is MTPTVFSSQHTAGG. At least nine different sizes of this VNTR locus were detected among 20 unrelated DNA samples from caucasians. The polymorphic markers and the transcript map documented here may contribute to identification of novel genes or allelic aberrations associated with the development of ovarian cancers.

Amino Acid Sequence↗

PubMed1994

Mouse homeobox gene Dbx: sequence, gene structure and expression pattern during mid-gestation.

Homeobox genes regulate multiple aspects of mouse development including the axial patterning in the central nervous system. Some of the more divergent members of this gene family are expressed in the anterior region of the central nervous system where the Hox genes are not expressed. We previously reported the isolation of a mouse homeobox gene, Dbx, from the forebrain. Here we describe its genomic structure, complete cDNA sequence, characterization of the basal promoter and the expression pattern at different stages of the developing embryo. During early and mid-gestation, Dbx expression is restricted to the telencephalon, diencephalon, dorsal mesencephalon and spinal cord. At later gestational stages, Dbx expression continues in the dorsal mesencephalon and diencephalon, in which expression is more restricted than at the earlier stages. Dbx transcripts were also detected in the primitive cerebellum. At all stages, the Dbx expressing regions contain a high proportion of proliferating cells, suggesting a role for Dbx in patterning the central nervous system during embryogenesis.

Amino Acid Sequence↗

PubMed1998

Genome characterization of a Korean isolate of cymbidium mosaic virus.

The complete nucleotide sequence of the genomic RNA of a Korean isolate of cymbidium mosaic virus (CymMV-K2) was determined. The genomic RNA is 6227 nucleotides in length, excluding the poly(A) tail. It contains a 5'-noncoding region (NCR) of 73 nucleotides, five open reading frames (ORFs 1 to 5) which encode proteins with M(r) 160 kDa RNA-dependent RNA polymerase (ORF1), 26 kDa movement protein 1 (ORF2), 13 kDa movement protein 2 (ORF3), 10 kDa movement protein 3 (ORF4), 24 kDa coat protein (OFR5), and a 3' NCR of 76 nucleotides. The 5'-end of the CymMV-K2 genome initiates with GGAAAA which contrasts to GAAAA at the 5'-ends of other potexviruses, including a Singapore isolate of CymMV (CymMV-S2). When compared with CymMV-S2, 171 base substitutions were observed in the CymMV-K2 genome. Substitutions in the overlapping ORFs (ORFs 2 to 4) occurred more frequently than those in 5' NCR, ORF1, and 3' NCR. In addition to substitutions, two single-base deletions, one in the intercistronic region between ORF1 and ORF2 and the other in the ORF2, were found on the CymMV-K2 genome. The deletion in the ORF2 induced a frameshift which altered the C-terminal domain of movement protein 1. ORF3 and ORF4 of the CymMV-K2 genome are partially different from those of another Singapore CymMV genome (CymMV-S1) which has four frameshifts due to nucleotide deletions within these ORFs. Interestingly, the frameshifts resulted in no change in the conserved sequences of the movement proteins but reconstructed their transmembrane domains.

Amino Acid Sequence↗