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PubMed1991

Direct selection: a method for the isolation of cDNAs encoded by large genomic regions.

We have developed a strategy for the rapid enrichment and identification of cDNAs encoded by large genomic regions. The basis of this "direct selection" scheme is the hybridization of an entire library of cDNAs to an immobilized genomic clone. Nonspecific hybrids are eliminated and selected cDNAs are eluted. These molecules are then amplified and are either cloned or subjected to further selection/amplification cycles. This scheme was tested using a 550-kilobase yeast artificial chromosome clone that contains the EPO gene. Using this clone and a fetal kidney cDNA library, we have achieved a 1000-fold enrichment of EPO cDNAs in one cycle of enrichment. More significantly, we have further investigated one of the "anonymous" cDNAs that was selectively enriched. We confirmed that this cDNA was encoded by the yeast artificial chromosome. Its frequency in the starting library was 1 in 1 x 10(5) cDNAs and after selection comprised 2% of the selected library. DNA sequence analysis of this cDNA and of the yeast artificial chromosome clone revealed that this gene encodes the beta 2 subunit of the human guanine nucleotide-binding regulatory proteins. Restriction mapping and hybridization data position this gene (GNB2) to within 30-70 kilobases of the EPO gene. The selective isolation and mapping of GNB2 confirms the feasibility of this direct selection strategy and suggests that it will be useful for the rapid isolation of cDNAs, including disease-related genes, across extensive portions of the human genome.

Base Sequence↗

PubMed2006

Microbial source tracking by DNA sequence analysis of the Escherichia coli malate dehydrogenase gene.

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.

Animals↗

PubMed1995

Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen.

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins.

Acrosome↗

PubMed1992

An ocular melanoma-associated antigen. Molecular characterization.

Monoclonal antibody (MAb) 8-1H defines a highly conserved ocular melanoma-associated antigen. Based on a potential binding site of MAb8-1, we identified, molecularly cloned, and sequenced the gene, encoding this melanoma-associated antigen from a uveal melanoma copy DNA library. Nucleotide sequence analysis of the melanoma-associated antigen revealed characteristics of a membrane-bound protein of 238 amino acids (approximately 25 kd) with four transmembrane domains. The sequence of the ocular melanoma-associated antigen is identical to the sequence of a recently reported cutaneous melanoma-associated antigen, substantiating our previous studies regarding common antigens between ocular and cutaneous melanoma. In addition, the melanoma-associated antigen shares sequence homology with a number of transmembrane proteins that appear to be involved in growth regulation. The implication of the biological function of this ocular melanoma-associated antigen, its relevance to the malignant phenotype, and clinical applications of the molecular characterization of this melanoma-associated antigen are described.

Amino Acid Sequence↗

PubMed1991

Dictyostelium discoideum gene family contains a long internal amino acid repeat.

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.

Amino Acid Sequence↗

PubMed1997

Human monocyte chemotactic protein-2: cDNA cloning and regulated expression of mRNA in mesenchymal cells.

Stimulated MG-63 osteosarcoma cells have been used as a source to purify and identify the monocyte chemokines MCP-1, MCP-2 and MCP-3. In comparison with MCP-1, the production yields of MCP-2 and MCP-3 in these cells are rather low and variable. Although the protein sequence of human MCP-2 is identified, its DNA sequence remains elusive. A degenerate primer set was used to isolate an MCP-2 gene fragment from the chemokine YAC contig on human chromosome 17. Based on the gene sequence of MCP-2, a unique primer set was synthesized and used to screen cDNA libraries for the presence of MCP-2 transcripts by PCR. The complete MCP-2 cDNA was cloned from a human bone marrow cDNA library and sequenced. The cDNA-derived protein sequence was identical to that of purified natural MCP-2, except for Gln46 which replaced Lys46. There seem thus to exist two MCP-2 allelic variants because at position 46 the codons of two residues (Lys46 and Gln46) were detected in individual genomes. As shown by Northern hybridization, the MCP-2 steady-state mRNA levels in normal diploid fibroblasts were increased by IL-1 beta, IFN-gamma and the double-stranded RNA poly rI:rC. RT-PCR analysis showed induction of MCP-2 mRNA in MG-63 cells by IFN-gamma and IL-1 beta. The regulated production of MCP-2 by tumor cells and normal mesenchymal cells is indicative of a role in neoplasia and inflammatory host responses.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of a gene encoding porcine interferon-beta.

A gene encoding the porcine interferon-beta (poIFN-beta) was cloned from a genomic library. The sequence of a potential intronless coding region as well as 1,265 bp of the 5'- and 277 bp of the 3'-flanking regions is presented. The gene is predicted to encode a mature protein of 165 amino acids and a signal peptide of 21 amino acids. This probable poIFN-beta shows high homology (63%) with human (hu) IFN-beta at the amino acid level, but less with porcine (po) IFN-alpha 1 (32%). It contains three cysteines and three potential N-glycosylation sites. A region of the 5' flank (-116 to -159) of the gene is homologous to the IFN gene regulatory element (IRE) of the huIFN-beta gene which mediates virus inducibility. Southern blot analysis indicates that the poIFN-beta gene is present as a single copy in the porcine genome. Its expression in porcine peripheral blood leukocytes stimulated in vitro by pseudorabies virus (PRV) was demonstrated at the RNA level both by Northern blot analysis and by in situ hybridization. The latter approach in addition detected only about one IFN-beta mRNA-containing cell per 2,000 PRV-stimulated porcine leukocytes, a frequency in the same range as that for leukocytes containing IFN-alpha mRNA.

Amino Acid Sequence↗

PubMed2000

BCAR1, a human homologue of the adapter protein p130Cas, and antiestrogen resistance in breast cancer cells.

BACKGROUND: Treatment of breast cancer with the antiestrogen tamoxifen is effective in approximately one half of the patients with estrogen receptor-positive disease, but tumors recur frequently because of the development of metastases that are resistant to tamoxifen. We have previously shown that mutagenesis of human estrogen-dependent ZR-75-1 breast cancer cells by insertion of a defective retrovirus genome caused the cells to become antiestrogen resistant. In this study, we isolated and characterized the crucial gene at the breast cancer antiestrogen resistance 1 (BCAR1) locus. METHODS/RESULTS: Transfer of the BCAR1 locus from retrovirus-mutated, antiestrogen-resistant cells to estrogen-dependent ZR-75-1 cells by cell fusion conferred an antiestrogen-resistant phenotype on the recipient cells. The complete coding sequence of BCAR1 was isolated by use of exon-trapping and complementary DNA (cDNA) library screening. Sequence analysis of human BCAR1 cDNA predicted a protein of 870 amino acids that was strongly homologous to rat p130Cas-adapter protein. Genomic analysis revealed that BCAR1 consists of seven exons and is located at chromosome 16q23.1. BCAR1 transcripts were detected in multiple human tissues and were similar in size to transcripts produced by retrovirus-mutated ZR-75-1 cells. Transfection of BCAR1 cDNA into ZR-75-1 cells again resulted in sustained cell proliferation in the presence of antiestrogens, confirming that BCAR1 was the responsible gene in the locus. CONCLUSIONS: Overexpression of the BCAR1 gene confers antiestrogen resistance on human ZR-75-1 breast cancer cells. Overexpression of BCAR1 in retrovirus-mutated cells appears to result from activation of the gene's promoter. The isolation and characterization of this gene open new avenues to elucidating mechanisms by which the growth of human breast cancer becomes independent of estrogen.

Amino Acid Sequence↗

PubMed2002

Dasheng and RIRE2. A nonautonomous long terminal repeat element and its putative autonomous partner in the rice genome.

Dasheng is one of the highest copy number long terminal repeat elements and one of the most recent elements to amplify in the rice (Oryza sativa) genome. However, the absence of any significant coding capacity for retroviral proteins, including gag and pol, suggests that Dasheng is a nonautonomous element. Here, we have exploited the availability of 360 Mb of rice genomic sequence to identify a candidate autonomous element. RIRE2 is a previously described gypsy-like long terminal repeat retrotransposon with significant sequence similarity to Dasheng in the regions where putative cis factors for retrotransposition are thought to be located. Dasheng and RIRE2 elements have similar chromosomal distribution patterns and similar target site sequences, suggesting that they use the same transposition machinery. In addition, the presence of several RIRE2-Dasheng element chimeras in the genome is consistent with the copackaging of element mRNAs in the same virus-like particle. Finally, both families have recently amplified members, suggesting that they could have been co-expressed, a necessary prerequisite for RIRE2 to serve as the source of transposition machinery for Dasheng. Consistent with this hypothesis, transcripts from both elements were found in the same expressed sequence tag library.

Base Sequence↗

PubMed2003

Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose.

Strains of Thermus thermophilus accumulate primarily trehalose and smaller amounts of mannosylglycerate in response to salt stress in yeast extract-containing media (O. C. Nunes, C. M. Manaia, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 61:2351-2357, 1995). A 2.4-kbp DNA fragment from T. thermophilus strain RQ-1 carrying otsA (encoding trehalose-phosphate synthase [TPS]), otsB (encoding trehalose-phosphate phosphatase [TPP]), and a short sequence of the 5' end of treS (trehalose synthase [TreS]) was cloned from a gene library. The sequences of the three genes (including treS) were amplified by PCR and sequenced, revealing that the genes were structurally linked. To understand the role of trehalose during salt stress in T. thermophilus RQ-1, we constructed a mutant, designated RQ-1M6, in which TPS (otsA) and TPP (otsB) genes were disrupted by gene replacement. Mutant RQ-1M6 accumulated trehalose and mannosylglycerate in a medium containing yeast extract and NaCl. However, growth in a defined medium (without yeast extract, known to contain trehalose) containing NaCl led to the accumulation of mannosylglycerate but not trehalose. The deletion of otsA and otsB reduced the ability to grow in defined salt-containing medium, with the maximum salinity being 5% NaCl for RQ-1 and 3% NaCl for RQ-1M6. The lower salt tolerance observed in the mutant was relieved by the addition of trehalose to the growth media. In contrast to trehalose, the addition of glycine betaine, mannosylglycerate, maltose, and glucose to the growth medium did not allow the mutant to grow at higher salinities. The results presented here provide crucial evidence for the importance of the TPS/TPP pathway for the synthesis and accumulation of trehalose and the decisive contribution of this disaccharide to osmotic adaptation in T. thermophilus RQ-1.

Adaptation, Physiological↗

PubMed1999

Isolation of fibroblast growth factor receptor binding sequences using evolved phage display libraries.

A fusion protein construct consisting of the short form of human fibroblast growth factor (FGFR) fused to the heavy chain of mouse IgG1 was used to screen four phage display libraries displaying 8, 13, 38 and 43 amino acids at the amino terminus of the bacteriophage M13 gene III minor coat protein. Phage with specific FGFR binding activity were isolated from the 13, 38 and 43 mer libraries. One of the highest affinity phage clones from the 13mer library was chosen to be further evolved by oligonucleotide saturation mutagenesis. We have isolated evolved sequences that have approximately 8 times the relative binding affinity of the parent sequence. The phage clones have a minimum consensus sequence of CR/SXLLXGAPFXXXXC, where X represents positions tolerant of several amino acids. A synthetic peptide based on this sequence specifically inhibits FGF from binding to its receptor in an in vitro ELISA.

Amino Acid Sequence↗

PubMed2003

[Molecular cloning of mTSARG3 gene related to apoptosis in mouse spermatogenic cells].

Beginning from a mouse EST (GenBank Accession No: BE644537) which was significantly changed in cryptorchidism and represented a novel gene, GeneScan program was performed and a predicted mouse novel gene full-length cDNA sequence containing the BE644537 sequence was attained. Gene-specific primers were designed for PCR in mouse testis cDNA library. The sequencing result of the PCR product showed that we obtain a new gene mTSARG3 (GenBank Accession No: AF419292) whose full cDNA length is 1328 bp containing 8 exons and 7 introns. The predicted open reading frame encodes a protein of 316 amino acid residues. The protein encoded by the new gene is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. mTSARG3 protein displays a 46% identity in a 336-amino-acid overlap with DJB4-MOUSE protein. The result of RT-PCR analysis and Northern blot showed that mTSARG3 is specifically expressed in mouse testis. Southern blot showed that there were no loss and rearrangement in cryptorchid. Based upon all these observations, it is considered that the function of the new gene is related to inhibit mouse testis spermatogenesis apoptosis.

Amino Acid Sequence↗

PubMed1996

Molecular cloning and tissue-specific expression of mouse kidney 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

A 1932 bp cDNA clone encoding a novel isozyme of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFK-2/ FBPase-2) was isolated from a mouse kidney cDNA library. The sequence encodes 519 amino acids and, based on homology to rat heart genomic sequence, appears to be the product of alternative splicing from PFK-2/FBPase-2 gene B with an extended version of exon 15. Northern blot analysis indicated that this clone corresponds to an 8 kb mRNA expressed in multiple tissues, with the strongest signal in kidney, and detects several additional transcripts which may be alternatively spliced from gene B.

Alternative Splicing↗

PubMed2005

Targeted oligonucleotide-mediated microsatellite identification (TOMMI) from large-insert library clones.

BACKGROUND: In the last few years, microsatellites have become the most popular molecular marker system and have intensively been applied in genome mapping, biodiversity and phylogeny studies of livestock. Compared to single nucleotide polymorphism (SNP) as another popular marker system, microsatellites reveal obvious advantages. They are multi-allelic, possibly more polymorphic and cheaper to genotype. Calculations showed that a multi-allelic marker system always has more power to detect Linkage Disequilibrium (LD) than does a di-allelic marker system. Traditional isolation methods using partial genomic libraries are time-consuming and cost-intensive. In order to directly generate microsatellites from large-insert libraries a sequencing approach with repeat-containing oligonucleotides is introduced. RESULTS: Seventeen porcine microsatellite markers were isolated from eleven PAC clones by targeted oligonucleotide-mediated microsatellite identification (TOMMI), an improved efficient and rapid flanking sequence-based approach for the isolation of STS-markers. With the application of TOMMI, an average of 1.55 (CA/GT) microsatellites per PAC clone was identified. The number of alleles, allele size distribution, polymorphism information content (PIC), average heterozygosity (HT), and effective allele number (NE) for the STS-markers were calculated using a sampling of 336 unrelated animals representing fifteen pig breeds (nine European and six Chinese breeds). Sixteen of the microsatellite markers proved to be polymorphic (2 to 22 alleles) in this heterogeneous sampling. Most of the publicly available (porcine) microsatellite amplicons range from approximately 80 bp to 200 bp. Here, we attempted to utilize as much sequence information as possible to develop STS-markers with larger amplicons. Indeed, fourteen of the seventeen STS-marker amplicons have minimal allele sizes of at least 200 bp. Thus, most of the generated STS-markers can easily be integrated into multilocus assays covering a broader separation spectrum. Linkage mapping results of the markers indicate their potential immediate use in QTL studies to further dissect trait associated chromosomal regions. CONCLUSION: The sequencing strategy described in this study provides a targeted, inexpensive and fast method to develop microsatellites from large-insert libraries. It is well suited to generate polymorphic markers for selected chromosomal regions, contigs of overlapping clones and yields sufficient high quality sequence data to develop amplicons greater than 250 bases.

Alleles↗

PubMed1989

Isolation of single-copy human genes from a library of yeast artificial chromosome clones.

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.

Chromosomes, Fungal↗

PubMed1992

Association of hY4 pseudogenes with Alu repeats and abundance of hY RNA-like sequences in the human genome.

Three loci having homology with the small human cytoplasmic RNA, hY4, were isolated from human genomic DNA libraries and sequenced. Each sequence contains dispersed mismatches as compared with hY4 RNA, is followed by an A-rich or A + T-rich sequence, and is bordered by direct repeats. Each of these loci, therefore, appears to constitute a small RNA class-III pseudogene. Surprisingly, two of the three loci are associated with Alu repeats. In the hY4.B7 locus, the hY4 sequence has integrated into the tail of an Alu element and in the hY4.F2 locus, an Alu sequence has inserted into the hY4 tail, confirming that A-rich tracts are preferential targets for retroposition. In addition, Southern blots with probes for each of the four hY RNAs indicate that hY RNA-like sequences are abundant in the human genome.

Base Sequence↗

PubMed1994

The Drosophila melanogaster ribosomal S6 kinase II-encoding sequence.

A cDNA encoding the Drosophila melanogaster p90 ribosomal S6 kinase II (RSK) was isolated from an eye-antennal imaginal disc library and sequenced. The conceptually translated protein is 60-63% identical to vertebrate RSK homologs and contains a perfectly conserved mitogen-activated protein kinase phosphorylation site. The gene was mapped to the base of the X chromosome in division 20 by in situ hybridization to polytene chromosomes.

Amino Acid Sequence↗

PubMed1995

Cloning, sequence, and expression of mouse protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin. A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria. We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase. The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library. The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp. This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870. The protein as expressed in E. coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen. Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

Amino Acid Sequence↗