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PubMed2001

Molecular cloning and characterization of human WNT5B on chromosome 12p13.3 region.

WNT signaling pathway plays a key role in carcinogenesis and embryogenesis. We have cloned and characterized the human WNT5B. Overlapping WNT5B cDNAs, containing 1080-bp ORF, were isolated. WNT5B encoded a 359-amino-acid polypeptide with the N-terminal signal peptide, four N-linked glycosylation sites, and consensus amino-acid residues conserved among the WNT family. WNT5B showed 80.5% total-amino-acid identity with WNT5A. Comparison between nucleotide sequence of WNT5B cDNA and human genome draft sequences revealed that the WNT5B gene, consisting of 4 exons, was located on human chromosome 12p13.3 region. Northern blot analyses with W5B2 probe detected the 2.8- and 2.4-kb WNT5B mRNAs. WNT5B was moderately expressed in adult prostate and fetal brain, and weakly expressed in fetal lung, kidney, adult liver, ovary, and small intestine. Among human cancer cell lines, WNT5B was expressed in gastric cancer cell lines MKN7, MKN45, KATO-III, and a teratocarcinoma cell line NT2. WNT5B might be implicated in human carcinogenesis through activation of the WNT-beta-catenin-TCF signaling pathway, just like Wnt5a.

Amino Acid Sequence↗
PubMed2001

Molecular cloning and characterization of human WNT7B.

WNT signaling molecules are implicated in carcinogenesis and embryogenesis. Only partial coding sequence of human WNT7B is reported so far, and human genome draft sequence corresponding to the WNT7B gene in human chromosome 22q13 region is not available at present. Here, we have cloned human WNT7B cDNAs, spanning the complete coding sequence, by using rapid amplification of cDNA ends (RACE) and cDNA-PCR. WNT7B encoded a 349-amino-acid polypeptide with three N-linked glycosylation sites and consensus amino-acid residues conserved among members of the WNT family. WNT7B showed 77.1% total-amino-acid identity with WNT7A. The 4.0-kb WNT7B was moderately expressed in fetal brain, weakly expressed in fetal lung and kidney, and faintly expressed in adult brain, lung and prostate. Expression levels of WNT7B mRNA in a lung cancer cell line A549, esophageal cancer cell lines TE2, TE3, TE4, TE5, TE6, TE7, TE10, TE12, a gastric cancer cell line TMK1, and pancreatic cancer cell lines BxPC-3, AsPC-1 and Hs766T were significantly higher than that in fetal kidney. In addition, WNT7B was up-regulated in 5 out of 10 cases of primary gastric cancer. These results strongly suggest that WNT7B might play important roles in various types of human cancer.

Amino Acid Sequence↗
PubMed2001

MRP8, a new member of ABC transporter superfamily, identified by EST database mining and gene prediction program, is highly expressed in breast cancer.

BACKGROUND: With the completion of the human draft genome sequence, efforts are now devoted to identifying new genes. We have developed a computer-based strategy that utilizes the EST database to identify new genes that could be targets for the immunotherapy of cancer or could be involved in the multistep process of cancer. MATERIALS AND METHODS: Utilizing our computer-based screening strategy, we identified a cluster of expressed sequence tags (ESTs) that are highly expressed in breast cancer. Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses demonstrated the tissue specificity of the computer-generated cluster and comparison with the human genome sequence assisted in isolating a full-length cDNA clone. RESULTS: We identified a new gene that is highly expressed in breast cancer. This gene is expressed at moderate levels in normal breast and testis and at very low levels in liver, brain, and placenta. The gene has two major transcripts of 4.5 kb and 4.1 kb. The 4.5-kb transcript is very abundant in breast cancer, and has an open reading frame of 1382 amino acids. The predicted protein sequence of the 4.5-kb transcript reveals that it has high homology with MRP5, a member of multidrug resistant-associated protein family (MRP). There are seven reported members in the MRP family; we designate this gene as MRP8 (ABCC11). The 4.5-kb MRP8 transcript consists of 31 exons and is located in a genomic region of over 80.4 kb on chromosome 16q12.1. The smaller 4.1-kb transcript of MRP8 is found in testis and may initiate within intron 6 of the gene. CONCLUSION: The selective expression of MRP8 (ABCC11), a new member of ATP-binding cassette transporter superfamily could be a molecular target for the treatment of breast cancer.

ATP-Binding Cassette Transporters↗
PubMed2002

Molecular cloning and characterization of mouse Wnt14b, clustered with mouse Wnt3 in mouse chromosome 11.

WNTs are a family of secreted-type glycoproteins implicated in embryogenesis and carcinogenesis. We have previously cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, and WNT14B/WNT15. WNT14B gene is clustered with WNT3 gene in human chromosome 17q21, and mRNA expression of WNT14B is significantly up-regulated by retinoic acid during the early phase of neuronal differentiation in human NT2 cells. Here, we identified mouse Wnt14b gene fragments in mouse genome draft sequence AL596108.5 by using bioinformatics, and isolated mouse Wnt14b cDNAs by using cDNA-PCR. Mouse Wnt14b was found to encode a 359-amino-acid WNT family protein with the N-terminal signal peptide, an N-linked glycosylation site, and 24 conserved cysteine residues. Mouse Wnt14b showed 92.5% total-amino-acid identity with human WNT14B, and 64.2% total-amino-acid identity with human WNT14. Mouse Wnt14b gene, consisting of 4 exons, was clustered with mouse Wnt3 gene in mouse chromosome 11. Mouse Wnt14b mRNA was relatively highly expressed in 17-day embryo, and also expressed in adult brain, kidney, liver, 7-day embryo, and 11-day embryo. This is the first report on molecular cloning and characterization of mouse Wnt14b.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and characterization of human SOX17.

SOX proteins are a family of transcription factors with high-mobility-group DNA-binding domain (HMG box) homologous to SRY, which are implicated in embryogenesis. Xenopus Sox17 alpha, Sox17 beta, and Sox3 are reported to negatively modulate the WNT - beta-catenin - TCF signaling pathway. Here, human SOX17 gene fragments were identified in human genome draft sequences by using bioinformatics, and SOX17 cDNAs were isolated by using cDNA-PCR. Human SOX17 was found to encode a 414-amino-acid protein with a HMG box, which was homologous to SOX18 and SOX7. SOX17 gene, consisting of 2 exons, was located in human chromosome 8q12-q13 region. SOX17 mRNAs of 2.5- and 2.2-kb in size were detected in adult heart, lung, spleen, testis, ovary, placenta, fetal lung, and kidney. In normal gastrointestinal tract, SOX17 mRNA was preferentially expressed in esophagus, stomach and small intestine than in colon and rectum. SOX17 mRNA was almost undetectable in human cancer cell lines HL-60, HeLa S3, K-562, MOLT-4, Raji, SW480, A549, G-361, and also in 66 cases of human primary tumors derived from various tissues, except one case of primary cervical cancer. This is the first report on molecular cloning and characterization of human SOX17.

Amino Acid Sequence↗
PubMed2002

Up-regulation of WNT8B mRNA in human gastric cancer.

WNT - beta-catenin - TCF signaling pathway is activated by Xenopus wnt-8 (Xwnt-8) during Xenopus early development, and dysregulated activation of beta-catenin - TCF signaling pathway in mammalian cells leads to carcinogenesis. We have previously cloned and characterized human WNT8A, one of human orthologues of Xwnt-8. Here, we cloned and characterized human WNT8B by using bioinformatics, cDNA-PCR, and RACE. WNT8B gene of about 23-kb in size consisted of six exons, and encoded a 351-amino-acid polypeptide with the N-terminal signal peptide and two N-linked glycosylation sites. C-terminal region of WNT8B, WNT8A, WNT2, and WNT2B were longer than that of other human WNTs. Thirty-five nucleotide changes between WNT8B isolated by us and WNT8B isolated by another group resulted in Gly230Ala and Arg284Leu amino-acid substitutions. Gly230 and Arg284 of WNT8B were conserved in WNT8A. Gly230-Arg284 WNT8B allele was also identified in human genome draft sequences AL133352.10, AL359759.18, and human EST BF732616. These results indicate that the Gly230-Arg284 WNT8B cDNA isolated in this study is derived from the more common WNT8B allele. WNT8B mRNAs of 4.4- and 3.5-kb in size were weakly detected in a colorectal cancer cell line SW480, but were undetectable in any normal human tissues by using Northern blot analyses. WNT8B was significantly up-regulated in gastric cancer cell lines KATO-III (signet-ring cell carcinoma) and MKN45 (poorly differentiated adenocarcinoma), and also in 5 out of 10 cases of primary gastric cancer. WNT8B might play key roles in gastric cancer through activation of the beta-catenin - TCF signaling pathway.

Alleles↗
PubMed2002

Molecular cloning and expression of proto-oncogene FRAT1 in human cancer.

FRAT1 and FRAT2 genes, clustered in human chromosome 10q24, are human homologues to mouse proto-oncogene Frat1, which promotes carcinogenesis through activation of the WNT - beta-catenin - TCF signaling pathway. FRAT1 and FRAT2 mRNAs are up-regulated together in a gastric cancer cell line TMK1, and also in 2 out of 10 cases of primary gastric cancer. Here, we isolated FRAT1 cDNA (AB074890), which showed two amino-acid substitutions (Gln57X and His58Asp) compared with human FRAT1 cDNA previously reported by another group (U58975). The Gln57-His58 FRAT1 allele isolated in this study was also identified in human genome draft sequences. FRAT1 mRNA was almost ubiquitously expressed in human pancreatic cancer cell lines. Expression level of FRAT1 mRNA was relatively higher in esophageal cancer cell lines TE2, TE3, TE4, a cervical cancer cell line SKG-IIIa, and breast cancer cell lines MCF-7 and T-47D. Expression level of FRAT1 mRNA was not significantly changed after all-trans retinoic-acid treatment in NT2 cells with the potential of neuronal differentiation. Expression of FRAT1 mRNA in MCF-7 cells derived from breast cancer was down-regulated by beta-estradiol. This is the first report on isolation of FRAT1 cDNA derived from the more common FRAT1 allele, and also on regulation of FRAT1 mRNA in human cancer cells.

Adaptor Proteins, Signal Transducing↗
PubMed2002

[Screening of novel genes differentially expressed in human renal cell carcinoma by suppression subtractive hybridization].

BACKGROUND AND OBJECTIVE: Identifying the differentially expressed genes in renal cell carcinoma (RCC) contributes to the elucidation of its genetic basis. However, the above knowledge has not yet been fully understood. The aims of this experiment were to screen novel genes differentially expressed in RCC tissues by suppression subtractive hybridization (SSH) and clone RCC-specific related genes. METHODS: To construct SSH library of RCC by using the mRNA from RCC tissues and matched normal kidney tissues as tester and driver, respectively. Partial positive clones in the library were selected randomly and sequenced, then analyze the sequences with the BLAST software. To confirm the location of the fragments of interest in human chromosome through comparing their sequences with the human genome draft. mRNA levels of the novel genes in RCC and matched normal kidney tissues were determined by Northern blot and semi-quantitative RT-PCR analysis. RESULTS: The SSH library contained 414 positive clones. Random analysis of 280 clones with enzyme restriction showed that 265 clones contained cDNA fragments distributed mainly between 300-900bp. Among 80 arbitrary clones with were derived from above 265 clones and sequenced, No. 28, 158, 170, and 249 clones are previously unknown genes and located in human chromosome 21q22, 4p15.3, 9q34, and 22q11.2 by electronic mapping, respectively. The consequence of semi-quantitative RT-PCR demonstrated that mRNA levels of the two novel genes were overexpressed in RCC compared to matched normal tissues by more than 2-6 folds. Northern blot analysis confirmed the above results. CONCLUSIONS: SSH is a reliable strategy for screening novel genes differentially expressed in RCC. The novel gene fragments can be used to clone their full length and further to study their functions.

Blotting, Northern↗
PubMed2003

Identification and characterization of human PRICKLE1 and PRICKLE2 genes as well as mouse Prickle1 and Prickle2 genes homologous to Drosophila tissue polarity gene prickle.

Drosophila prickle is implicated in tissue polarity or planar polarity. Here, human PRICKLE1 gene corresponding to FLJ31937 cDNA and human PRICKLE2 gene corresponding to DKFZp686D143 cDNA were identified to be homologous to Drosophila prickle gene by using bioinformatics. PRICKLE1 gene was mapped to human chromosome 12p11-q12, and PRICKLE2 gene was mapped to human chromosome 3p14. Mouse Prickle1 and Prickle2 genes were next identified in mouse genome draft sequences NW_000106.1 and NW_000262.1, respectively. Human PRICKLE1, PRICKLE2, Xenopus Prickle, and Drosophila prickle were homologous in the PET domain, three LIM domains, and the C-terminal Prickle homologous (PKH) domain. LMO6 and TESTIN, containing the PET domain and three LIM domains, were found to lack the PKH domain. Therefore, PRICKLE1 and PRICKLE2 rather than LMO6 and TESTIN were found to be human homologs of Drosophila prickle. PRICKLE1 and PRICKLE2 mRNAs were expressed together in brain, eye and testis. PRICKLE1 mRNA was expressed in fetal heart and hematological malignancies, while PRICKLE2 mRNA in fetal brain, adult cartilage, pancreatic islet, gastric cancer with signet-ring cell features, and uterus tumors. Because tissue polarity genes frizzled, dishevelled, flamingo, and Vang are evolutionary and functionary conserved from Drosophila to human, PRICKLE1 and PRICKLE2 might be implicated in the localization of Frizzled and Dishevelled proteins, just like Drosophila prickle. This is the first report on identification and characterization of human PRICKLE1, PRICKLE2, mouse Prickle1, and Prickle2 genes.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Identification and characterization of human DAPPER1 and DAPPER2 genes in silico.

WNT signals play key roles in carcinogenesis and embryogenesis through the specification of cell fate and polarity. Dishevelled (DVL) proteins are WNT signaling molecules implicated in beta-catenin pathway and PCP pathway. Xenopus Dapper and Frodo are Dvl-binding proteins, showing 89.8% total-amino-acid identity. Here, we identified and characterized human homologs of Xenopus Dapper and Frodo using bioinformatics. Human DAPPER1 gene was located within human genome draft sequence NT_025892.9 (nucleotide position 39378960-39387891 in the forward orientation), and human DAPPER2 gene within NT_007302.10 (nucleotide position 660279-672480 in the reverse orientation). DAPPER1 (799-amino-acids) and DAPPER2 (774-amino-acids) showed 28.8% total-amino-acid identity. Seven DAPPER homologous (DAPH) domains, including DAPH2 (leucine zipper), DAPH3 (serine rich) and DAPH7 (PDZ binding), were conserved between DAPPER1 and DAPPER2. Phylogenetic analysis of vertebrate Dapper proteins revealed that Xenopus Dapper and Frodo are orthologs of human DAPPER1. DAPPER1 mRNA was expressed in amnion, fetal brain, eye, heart, adult brain medulla, gastric cancer (signet ring cell features), RER+ colon tumor, acute lymphoblastic leukemia, germ cell tumor, chondrosarcoma, and parathyroid tumor. DAPPER2 mRNA was expressed in placenta, genitourinary tract tumor, and endometrial adenocarcinoma. DAPPER1 and DAPPER2 genes were mapped to human chromosome 14q22.3 and 6q27, respectively. Human chromosome 14q22.3 is deleted in astrocytoma, while human chromosome 6q27 is deleted in breast, ovarian, and gastric cancer. Based on evolutionary and functional conservation of WNT signaling molecules as well as human chromosomal localization, DAPPER1 and DAPPER2 genes are predicted to be potent cancer-associated genes.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Identification and characterization of human TIPARP gene within the CCNL amplicon at human chromosome 3q25.31.

Array CGH combined with mRNA microarray analyses was successfully applied for genome-wide screening of proto-oncogenes as well as tumor suppressor genes in 2002. It has been reported that an uncharacterized gene, corresponding to a 5'-truncated partial cDNA DKFZp434J214, is amplified and up-regulated together with CCNL in head-and-neck squamous cell carcinoma (HNSCC). Here, we identified that the novel gene, corresponding to the 5'-truncated DKFZp434J214 cDNA, is the human ortholog of mouse Tiparp gene, by using bioinformatics. Complete coding sequence of human TIPARP mRNA was determined in silico by assembling nucleotide sequences of BC034397 and DKFZp434J214 cDNAs. Human TIPARP gene, consisting of six exons, was located between SSR3 and CCNL genes at human chromosome 3q25.31. Fugu tiparp gene was located around nucleotide position 14280-23355 of fugu genome draft sequence CAAB01003597.1. Human TIPARP showed 91.8% and 52.7% total amino-acid identity with mouse Tiparp and fugu tiparp, respectively. TPH domain (codon 242-296 of human TIPARP), WWE domain (codon 329-378) and PARP-like domain (codon 465-648) were evolutionarily conserved among TIPARP proteins. CCCH-type zinc finger was located within the N-terminal part of the TPH domain. Human TIPARP showed 27.5% and 26.0% total-amino-acid identity with human FLJ22693 and ZAP, respectively. TIPARP, FLJ22693 and ZAP, sharing the common structure with the TPH, WWE and PARP-like domains, were found to constitute the TIPARP family. This is the first report on human TIPARP and fugu tiparp genes as well as on the TIPARP family.

Amino Acid Sequence↗
PubMed2002

The hepatic stellate cell in the post-genomic era.

The draft human genome sequence was published on February 15, 2001, which will provide a huge amount of information on human genetics, human disease, and human cell biology. Now, medical scientists and cell biologists are turning their attention to illustrating gene expression pattern using gene microarray and to identifying the functions and the expression patterns of proteins encoded by the genes. Hepatic stellate cell is one of the sinusoid-constituent cells that play multiple roles in the liver pathophysiology. Transformation of stellate cells from the vitamin A-storing phenotype to the "myofibroblastic" one closely correlates to hepatic fibrosis during chronic liver trauma. Analyses of the molecular mechanisms of stellate cell activation have made a great progress, in particular, in the field of intracellular signal transduction of transforming growth factor-beta and platelet-derived growth factor, integrin signaling related to cell-adhesion, and cell motility-associated Rho and focal-adhesion kinase. Accumulation of the information on the stellate cell activation would shed light on the establishment of a novel therapeutic strategy against fibrosis of human liver disease.

Animals↗
PubMed2001

Assembly of the working draft of the human genome with GigAssembler.

The data for the public working draft of the human genome contains roughly 400,000 initial sequence contigs in approximately 30,000 large insert clones. Many of these initial sequence contigs overlap. A program, GigAssembler, was built to merge them and to order and orient the resulting larger sequence contigs based on mRNA, paired plasmid ends, EST, BAC end pairs, and other information. This program produced the first publicly available assembly of the human genome, a working draft containing roughly 2.7 billion base pairs and covering an estimated 88% of the genome that has been used for several recent studies of the genome. Here we describe the algorithm used by GigAssembler.

Algorithms↗
PubMed2026

Characterization of a draft chromosome-scale genome assembly for the mutton snapper, Lutjanus analis.

BACKGROUND: The mutton snapper (Lutjanus analis) is a reef fish commonly found in tropical waters of the Western Atlantic Ocean. Genomic studies of this species are needed to support conservation efforts and breeding programs. OBJECTIVE: Here, we report the development of a chromosome-scale reference assembly for the mutton snapper and conduct an initial comparative genomic analysis with other lutjanids. METHODS: The genome of one mutton snapper specimen was sequenced using PAC-Bio HiFi long reads and Illumina short reads. Contigs and scaffolds were assembled in the Flye pipeline and anchored using Hi-C proximity guided assembly. Gene prediction and functional annotations were obtained in AUGUSTUS and eggNOG-mapper, respectively. The mutton snapper genome was compared to those of other lutjanids to infer gene family evolution and chromosome synteny conservation. RESULTS: Assembly and polishing yielded 946 contigs and 926 scaffolds (N50 of 3.16 Mb, complete BUSCO score 98.1%) that were anchored using Hi-C scaffolding in 24 draft chromosomes. The anchored assembly featured a N50 of 42.47 Mb and contained 97.6% of the unanchored assembly length. The 24 mutton snapper chromosomes showed a one-to-one syntenic relationship with their counterparts in medaka, and other Lutjanids. AUGUSTUS predicted 29,023 genes, 24,335 of which (83.85%) could be functionally annotated. Gene family evolution analysis revealed 1,014 significantly expanded or contracted hierarchical ortholog groups in mutton snapper. Expansions and contractions were linked to several biological functions including growth, oocyte maturation, and response to exogenous stressors. CONCLUSION: The draft genome will be a valuable tool for forthcoming applied genomic studies of mutton snapper.

Animals↗
PubMed2001

Cancer and genomics.

Identification of the genes that cause oncogenesis is a central aim of cancer research. We searched the proteins predicted from the draft human genome sequence for paralogues of known tumour suppressor genes, but no novel genes were identified. We then assessed whether it was possible to search directly for oncogenic sequence changes in cancer cells by comparing cancer genome sequences against the draft genome. Apparently chimaeric transcripts (from oncogenic fusion genes generated by chromosomal translocations, the ends of which mapped to different genomic locations) were detected to the same degree in both normal and neoplastic tissues, indicating a significant level of false positives. Our experiment underscores the limited amount and variable quality of DNA sequence from cancer cells that is currently available.

Base Sequence↗
PubMed2026

Annotated genome assemblies of two temperate North American dung beetles, Canthon chalcites and Phanaeus vindex.

Dung beetles serve as cultivators of their natural habitats, improving soil health and functions in both natural and anthropogenic environments. Despite their ecological importance, whole genome sequences for Scarabaeinae are limited. Here, we present the draft annotated genome assemblies for 2 temperate species of North American dung beetles collected from eastern Tennessee: Canthon chalcites and Phanaeus vindex. Both genome assemblies were generated from PacBio long reads and have high completeness, with BUSCO scores of 98.1% and 98.6% for C. chalcites and P. vindex, respectively. For C. chalcites, the BRAKER3 pipeline predicted 12,799 genes, and the gene set was 93.7% complete. For P. vindex, the BRAKER3 predicted 12,252 genes, and the gene set was 94.9% complete. From the annotated gene sets, orthologous protein sequence analyses among C. chalcites, P. vindex, the dung beetle species Onthophagus taurus, and the more evolutionarily distant beetle Tribolium castaneum indicated that there are 260 unique protein clusters for C. chalcites and 210 unique protein clusters for P. vindex. These 2 draft genomes provide valuable data for comparative genomics, evolution, and phylogenic studies for dung beetle species.

Animals↗