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At least 811 records · Page 45Linked to original sources
PubMed2004

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

BACKGROUND: The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. RESULTS: Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. CONCLUSIONS: Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online.

Ambystoma↗
PubMed2000

Human proton/oligopeptide transporter (POT) genes: identification of putative human genes using bioinformatics.

The proton-dependent oligopeptide transporters (POT) gene family currently consists of approximately 70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat histidine/peptide transporter (rPHT1). Because the Candida elegans genome contains five putative POT genes, we searched the available protein and nucleic acid databases for additional mammalian/human POT genes, using iterative BLAST runs and the human expressed sequence tags (EST) database. The apparent human orthologue of rPHT1 (expression largely confined to rat brain and retina) was represented by numerous ESTs originating from many tissues. Assembly of these ESTs resulted in a contiguous sequence covering approximately 95% of the suspected coding region. The contig sequences and analyses revealed the presence of several possible splice variants of hPHT1. A second closely related human EST-contig displayed high identity to a recently cloned mouse cDNA encoding cyclic adenosine monophosphate (cAMP)-inducible 1 protein (gi:4580995). This contig served to identify a PAC clone containing deduced exons and introns of the likely human orthologue (termed hPHT2). Northern analyses with EST clones indicated that hPHT1 is primarily expressed in skeletal muscle and spleen, whereas hPHT2 is found in spleen, placenta, lung, leukocytes, and heart. These results suggest considerable complexity of the human POT gene family, with relevance to the absorption and distribution of cephalosporins and other peptoid drugs.

Blotting, Northern↗
PubMed2005

Chromosome loss followed by duplication is the major mechanism of spontaneous mating-type locus homozygosis in Candida albicans.

Candida albicans, which is diploid, possesses a single mating-type (MTL) locus on chromosome 5, which is normally heterozygous (a/alpha). To mate, C. albicans must undergo MTL homozygosis to a/a or alpha/alpha. Three possible mechanisms may be used in this process, mitotic recombination, gene conversion, or loss of one chromosome 5 homolog, followed by duplication of the retained homolog. To distinguish among these mechanisms, 16 spontaneous a/a and alpha/alpha derivatives were cloned from four natural a/alpha strains, P37037, P37039, P75063, and P34048, grown on nutrient agar. Eighteen polymorphic (heterozygous) markers were identified on chromosome 5, 6 to the left and 12 to the right of the MTL locus. These markers were then analyzed in MTL-homozygous derivatives of the four natural a/alpha strains to distinguish among the three mechanisms of homozygosis. An analysis of polymorphisms on chromosomes 1, 2, and R excluded meiosis as a mechanism of MTL homozygosis. The results demonstrate that while mitotic recombination was the mechanism for homozygosis in one offspring, loss of one chromosome 5 homolog followed by duplication of the retained homolog was the mechanism in the remaining 15 offspring, indicating that the latter mechanism is the most common in the spontaneous generation of MTL homozygotes in natural strains of C. albicans in culture.

Base Sequence↗
PubMed2003

PCR-assisted contig extension: stepwise strategy for bacterial genome closure.

Finishing is rate limiting for genome projects, and improvements in the efficiency of complete genome-sequence compilation will require improved protocols for gap closure. Here we report a novel approach for extending shotgun contigs and closing gaps that we termed PCR-assisted contig extension (PACE). PACE depends on the capture of rare mismatched interactions that occur between arbitrary primers and template DNA of unknown sequence, even under highly stringent conditions, by means of elevated PCR-cycle repetition and the use of specific anchoring primers corresponding to adjacent regions of known sequence. Using PACE, we have generated extensions with an average of 1 kb from all contigs generated from the shotgun sequencing of a 5-Mb genome, which closed the majority of gaps with a single round of experimentation. This included the generation of multiple extensions for contigs that terminated in one of the eight copies of the rRNA operon. We calculate that the switch from shotgun sequencing to PACE should occur between 5- and 8-fold genome coverage for maximum benefit and minimum overall cost. PACE is a robust and straightforward strategy that should simplify the finishing phase of bacterial genome projects.

Chromobacterium↗
PubMed1999

Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis.

In this report, we present a simple nonradioactive labeling procedure for DNA fragments of high specific labeling density that can be used for a variety of applications. The protocol is based on the universal mono-functional platinum reagent for chemical digoxigenin (DIG) labeling of nucleic acids. The labeling protocol was optimized for large DNA templates as complete bacterial artificial chromosomes (BAC). Variations of incubation time and temperature improved the labeling density such that about 30% of the nucleotides were DIG-modified within 30 min. Furthermore, the refined procedure generates in a single-tube reaction and without prior digestion-labeled DNA fragments of 0.5-4.0 kb from a 130-kb template. Hybridization experiments were performed on Southern and northern blots and allowed the detection of single copy genes in 2.5 micrograms genomic DNA from Arabidopsis thaliana, which has a haploid genome size of 0.13 pg (ca. 120 Mb) and medium expressed transcripts from 0.8 microgram poly(A)+ RNA, respectively. The extremely high specific labeling density, the stability and the universal application of the probe generated with the platinum reagent makes this method a useful alternative to classical radioactive nuclei acids labeling techniques.

Alleles↗
PubMed2000

Complete genomic sequence of 195 Kb of human DNA containing the gene GABRG2.

GABA (gamma-aminobutyric acid), as the main inhibitory neurotransmitter in the brain, plays an essential role for the overall balance between neuronal excitation and inhibition by acting on GABAA receptors, which are ligand-gated chloride channels. Impaired GABAergic function contributes to certain forms of epilepsy, schizophrenia, Alzheimer's Disease, and other neurological disorders. In order to identify possible genetic features and to further study biological regulation of GABAA receptor genes whose promoter elements and sequence anomalies may contribute to epileptic disorders, as an initial step, we shot-gun sequenced a BAC clone, dj082c10 (195,909-bp in size), encompassing human gamma(2) subunit of GABAA receptor (GABRG2). It is, we believe, the first genomic sequence of the GABA receptor gamma subunit family. Four contigs were assembled from 2950 reads prior to gap in an average redundancy of eight folds over the entire region. The precision of the consensus sequence was predicted to be 99.999% after closing gaps and finishing weak regions. The nine exons of GABRG2 spans an 85-kb region that had 81 SINEs comprising 22.32%, and nine L1 elements comprising 3.40%, respectively. However, the density of L1 in the regions flanking GABRG2 gene (29.45% by 45 elements) is significantly higher than that within the gene. The length of GABRG2 introns varies in the range of 1.5 kb to 38.1 kb.

Amino Acid Sequence↗
PubMed2000

High-level rearrangement and transcription of yeast artificial chromosome-based mouse Ig kappa transgenes containing distal regions of the contig.

The mouse Ig kappa L chain gene locus has been extensively studied, but to date high-level expression of germline transgenes has not been achieved. Reasoning that each end of the locus may contain regulatory elements because these regions are not deleted upon V kappa-J kappa joining, we used yeast artificial chromosome-based techniques to fuse distal regions of the contig to create transgene miniloci. The largest minilocus (290 kb) possessed all members of the upstream V kappa 2 gene family including their entire 5' and 3' flanking sequences, along with one member of a downstream V kappa 21 gene family. In addition, again using yeast artificial chromosome-based technology, we created Ig kappa miniloci that contained differing lengths of sequences 5' of the most distal V kappa 2 gene family member. In transgenic mice, Ig kappa miniloci exhibited position-independent and copy number-dependent germline transcription. Ig kappa miniloci were rearranged in tissue and developmental stage-specific manners. The levels of rearrangement and transcription of the distal and proximal V kappa gene families were similar to their endogenous counterparts and appeared to be responsive to allelic exclusion, but were differentially sensitive to numerous position effects. The minilocus that contained the longest 5' region exhibited significantly greater recombination of the upstream V kappa 2 genes but not the downstream V kappa 21 gene, providing evidence for a local recombination stimulating element. These results provide evidence that our miniloci contain nearly all regulatory elements required for bona fide Ig kappa gene expression, making them useful substrates for functional analyses of cis-acting sequences in the future.

Alleles↗
PubMed2002

The IgH locus of the channel catfish, Ictalurus punctatus, contains multiple constant region gene sequences: different genes encode heavy chains of membrane and secreted IgD.

The delta-chain of catfish IgD was initially characterized as a unique chimeric molecule containing a rearranged VDJ spliced to C micro 1, seven C domain-encoding exons (delta1-delta7), and a transmembrane tail. The presence of cDNA forms showing splicing of delta7 to an exon encoding a secretory tail was interpreted to indicate that membrane (deltam) and secreted (deltas) forms were likely expressed from a single gene by alternative RNA processing. Subsequent cloning and sequence analyses have unexpectedly revealed the presence of three delta C region genes, each linked to a micro gene or pseudogene. The first (IGHD1) is located 1.6 kb 3' of the functional C micro (IGHM1). The second (IGHD3) is positioned immediately downstream of a pseudo C micro (IGHM3P), approximately 725 kb 5' of IGHM1. These two delta genes are highly similar in sequence and each contains a tandem duplication of delta2-delta3-delta4. However, IGHD1 has a terminal exon encoding the transmembrane region, whereas IGHD3 has a single terminal exon encoding a secreted tail. The occurrence of IGHD3 immediately downstream of a micro pseudogene indicates that the putative deltas product may not be expressed as a chimeric micro delta molecule. Western blots and protein sequencing data indicate that an IGHD3-encoded protein is expressed in catfish serum. Thus, catfish deltam transcripts appear to originate from IGHD1, whereas deltas transcripts originate from IGHD3 rather than, as previously inferred, from a single expressed delta gene. The third delta (IGHD2) is associated with a pseudo C micro (IGHM2P); its presence is inferred by Southern blot analyses.

Amino Acid Sequence↗
PubMed1999

Analysis of a 26-kb region linked to the Mhc in zebrafish: genomic organization of the proteasome component beta/transporter associated with antigen processing-2 gene cluster and identification of five new proteasome beta subunit genes.

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for beta subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-gamma. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became associated with it.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Deletions of chromosome 5q13.3 and 17p loci cooperate in myeloid neoplasms.

Nonrandom interstitial deletions and monosomy of chromosomes 5, 7, and 17 in refractory myelodysplasia (MDS) and acute myelogenous leukemia (AML) suggest a multistep pathway that culminates in aggressive clinical course. Because cytogenetic studies frequently identify chromosome 5 and 17 deletions within a single clone, we searched for allele loss for 5q loci and TP53 gene mutations in the same leukemic samples. Cosegregating deletions of chromosomes 5 and 17 were found to specifically include the 5q13.3 interval between the loci D5S672 and D5S620/D5S626, a locus hypothesized to harbor a tumor suppressor gene(1) and the TP53 gene on 17p. A rare patient with secondary refractory MDS and an unbalanced translocation [der(5;17)], which resulted in deletions of the 5q13.3-qter and 17p loci, provided clues on the sequence of genetic alterations. Serial molecular analysis of this patient revealed a dysplastic clone with der(5;17), which gave rise to a leukemic clone on acquiring an inactivating mutation of TP53. Our findings are consistent with functional cooperation between a putative tumor suppressor gene at 5q13.3 that contributes toward the progression of early stages of MDS, and the TP53 gene when mutated, causes transformation to AML. (Blood. 2000;95:2138-2143)

Alleles↗
PubMed2003

Identification and characterization of human SNAIL3 (SNAI3) gene in silico.

Human SNAIL1 (SNAI1) protein encoded by SNAI1/SNA gene represses transcription of E-cadherin/CDH1 gene. Human SNAIL2 (SNAI2) protein encoded by SNAI2/SLUG gene induces the first phase of epithelial-mesenchymal transition (EMT), including desmosome dissociation, cell spreading, and initiation of cell separation. Here, we have identified human SNAIL3 (SNAI3) gene using bioinformatics. Human SNAI3 gene, consisting of at least three exons, spans around the nucleotide position 320214-328221 of human reference genomic contig NT_010404.8 in the reverse orientation. SNAI3 gene, was located between KIAA0233 gene and CBFA2T3 gene in human chromosome 16q24.3, a region affected in breast cancer, gastric cancer, hepatocellular carcinoma, ovarian cancer, and therapy-related myeloid leukemia with t(16;21)(q24;q22) translocation. Human SNAI3 gene was found to encode 292-amino-acid polypeptide with the N-terminal SNAG domain and five zinc finger domains. N-terminal SNAG domain was identified in zinc finger proteins SNAI1, SNAI2, SNAI3, SCRATCH (SCRT1), GFI1, and GFI1B. ATP/GTP binding site was identified in SCRT1, GFI1 and GFI1B, but not in SNAI1, SNAI2 and SNAI3. Phylogenetic analysis of human zinc finger proteins with SNAG domain revealed that SNAI1, SNAI2 and SNAI3 were more closely related. These results clearly indicate that SNAI1, SNAI2 and SNAI3 constitute a subfamily among SNAG zinc-finger proteins. Human SNAI3 mRNA was expressed in skin melanotic melanoma, lung epidermoid carcinoma, and germ cell tumor. Because SNAG zinc-finger proteins are transcriptional repressors implicated in carcinogenesis and embryogenesis, SNAI3 gene might be a potent target of pharmacogenomics in the field of oncology and regenerative medicine.

Amino Acid Sequence↗