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Results for “positional cloning”

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At least 289 records · Page 16Linked to original sources

PubMed2003

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells.

OBJECTIVE: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). METHODS: Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. RESULTS: These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. CONCLUSIONS: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.

Cells, Cultured↗

PubMed2002

Identification of genes induced by BRCA1 in breast cancer cells.

Inherited mutations of the BRCA1 gene predispose to breast, ovarian, and other cancers. The role of the BRCA1 gene in the maintenance of chromosomal integrity is linked to a number of biological properties of its protein product, including transcriptional regulation. In the present study, we have used suppression subtractive hybridisation (SSH) to identify genes induced by BRCA1 by comparing control MCF7 breast carcinoma cells (driver) with MCF7 cells ectopically expressing BRCA1 (tester) and generated a forward subtracted cDNA library. We screened 500 putative positive clones from this library. Two hundred and ten of these clones were positive by differential screening with forward and reverse subtracted probes and the 65 cDNA clones which showed more than fivefold increase were selected for sequencing analysis. We clustered 46 different genes that share high homology with sequences in the GenBank/EMBL databases. Among these, 30 were genes whose function had been previously identified while the remaining 16 clones were genes with unknown functions. Of particular interest, BRCA1 gene induces the expression of genes encoding DNA repair proteins RAD21 and MSH2, ERBB2/HER2 interacting protein ERBIN, meningioma-associated protein MAC30, and a candidate ovarian tumour-suppressor OVCA1. Northern and Western blot analyses confirmed that the expression of these five genes are up-regulated following BRCA1 overexpression in MCF7 and UBR60-bcl2 cells. This is the first study reporting a set of BRCA1-induced genes in breast carcinoma cells by the SSH technique. We suggest that some known genes identified in this study may provide new insights into the tumour-suppressor function of BRCA1.

BRCA1 Protein↗

PubMed2003

[Screening of mimotope of Salmonella typhimurium lipopolysaccharide from phage-displayed peptide].

OBJECTIVE: To identify and characterize the mimotope of lipopolysaccharide (LPS) from cyclic 7-mer phage peptide library. METHODS: Cyclic 7-mer phage-displayed peptide library was screened using monoclonal antibody 2F4 (mAb 2F4) against Salmonella typhimurium LPS as the target, and the selected clones were tested by sandwich enzyme-linked immunosorbent assay (ELISA) and specific antigen inhibition ELISA. RESULTS: After 3 round of screening, 34 of the 38 selected clones were identified as positive for binding to mAb 2F4. Salmonella typhimurium LPS was capable of inhibiting the binding between the cones and mAb 2F4, with the 50% inhibitory concentration of all the positive clones within the range of 0.125-0.250 microg/ml. Sequence analysis was performed for 10 of the positive clones, whose amino acid sequences were subsequently deduced, and 7 of them had conservative amino acid of P-X-WAS-X-W with the mean hydrophobic amino acid content of 71.4% in all the sequences. CONCLUSION: The phage-displayed peptide is capable of simulating the epitope of Salmonella typhimurium LPS.

Antibodies, Monoclonal↗

PubMed2001

Comparative analysis of a BAC contig of the porcine RN region and the human transcript map: implications for the cloning of trait loci.

The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.

Animals↗

PubMed2004

[A study on the mutation of P gene region including YMDD motif in hepatitis B virus].

OBJECTIVE: To investigate the primary structure and heterogenenity of P gene region including YMDD motif in hepatitis B virus. METHODS: From serum samples collected from 4 patients who had never been treated with anti-viral drugs, DNA fragments of 1057bp long of P gene were amplified and cloned into pUC19. Twenty positive clones were chosen randomly from each sample. The YMDD motif mutation was detected by mismatched PCR-RFLP. Finally last ten positive clones of two samples were sequenced. RESULTS: Nucleotide mutation rates among clones of Sample 1 and 2 were 0.3% - 1.1%, 0.4% - 1.7%, respectively. Among 80 clones, the variations from YMDD to YMGD were revealed in two clones. CONCLUSION: There are HBV quasispecies in the P gene region including YMDD motif of hepatitis B virus and a novel mutation of YMDD motif in the sera of patients without being therapied by anti-viral drugs.

Adult↗

PubMed1993

Sequence of ovine Ig gamma 2 constant region heavy chain cDNA and molecular modelling of ruminant IgG isotypes.

Ovine mesenteric lymph node mRNA was used for PCR amplification of DNA coding for immunoglobulin gamma 1 and gamma 2 heavy chain constant regions. Primers complementary to regions of CH1 conserved between ruminants were used for upstream priming, with downstream priming on the poly-A segment. PCR products of the appropriate length were cloned and gamma positive clones selected with a CH1 conserved-region probe. Of these, gamma 1 clones were positively selected and gamma 2 clones negatively selected with a gamma 1 hinge-specific probe. Ovine gamma 2 cDNA has 93% identity of nucleotides with ovine gamma 1. Both ovine gamma 1 and gamma 2 CH1 domains encoded two consecutive cysteine residues (Cys-127, -128, Kabat numbering), an arrangement which is deduced to form a pair of disulphide bridges, one to the L chain and one as an intra-chain bridge to the uppermost Cys of the hinge, as in rabbit and goat IgG. The majority of the differences between the isotypes occur in the hinge region and an evolutionary pattern for ruminant IgG hinges can now be identified. IgG1 isotypes are typical, with hinges containing the C-terminal Cys-Pro motif, but deletion and replacement of nucleotides (in the ancestral gene) of ruminant gamma 2 has shortened the IgG2 hinge, removing the Cys-Pro motif and the consensus high affinity Fc gamma RI receptor motif at the start of CH2. An N-terminal glycosylation site and the peptide motif for complement C1q binding are present in CH2 of both isotypes. The hinge regions of gamma 1 and gamma 2 and predicted structures for ovine IgG1 and IgG2 have been modelled. Close apposition of Fab and Fc in IgG2 produces steric hindrance at the normally accessible Fab/hinge/Fc interface; the structural differences between the ruminant isotypes form a basis for understanding some of the differences in their effector properties.

Amino Acid Sequence↗

PubMed2007

[Human esophageal carcinoma antigens screened by serologic analysis of recombinant cDNA expression libraries (SEREX)].

BACKGROUND & OBJECTIVE: In malignant transformation, mutant gene products and dysregulated proteins can become tumor antigens and activate immunoreactions. Therefore, auto-antibodies exist in sera of cancer patients. Serologic analysis of recombinant cDNA expression libraries (SEREX) using autologous and allogenic patient sera provides a powerful approach to identify tumor antigens. This study was to identify esophageal cancer antigens with SEREX for serologic diagnosis, gene therapy, and immune therapy. METHODS: Expression library of cDNA from esophageal squamous cell carcinoma was constructed. SEREX screened out 21 positive clones from the 1.6x10(6) clones in the established library. The 21 positive clones were subcloned to monoclonality and submitted to in vivo excision of pBluescript phagemids. The nucleotide sequences of cDNA inserts were analyzed with DNASIS and BLAST software on EMBL and GenBank. According to the bioinformatics analyses, serologic immunoreactions of 4 colons in 10 samples of esophageal cancer serum and 10 samples of normal control serum were further detected by SADA. RESULTS: Of the 21 positive clones, 4 had no homology to any known genes, 17 were known fragments which were defined as antigens of esophageal cancer for the first time. The serologic immunoreaction rates of 4 selected antigens, including Ribosomal protein S4, and so on, were 40%, 60%, 70%, and 30%, respectively, in cancer sera, and 0%, 10%, 20%, and 20%, respectively, in normal sera. CONCLUSIONS: Antigens, such as Ribosomal protein S4, are frequently involved in serologic immunoreactions of esophageal cancer. The 21 antigens identified by the present study can be used as potential targets for gene therapy and serologic biomarkers of esophageal cancer.

Antibodies, Neoplasm↗

PubMed2004

Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer.

A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

Bacillus subtilis↗

PubMed2003

[Screening the hepatitis B virus PreS1 associated protein by the yeast two-hybrid system].

OBJECTIVE: To screen the hepatitis B virus PreS1 associated protein from normal human liver cDNA library by the yeast two-hybrid system and explore the role of PreS1 protein in the infection of hepatitis B virus (HBV). METHODS: PCR was preformed to amplify the PreS1 gene containing EcoRI and PstI from HBV positive serum, and the production was inserted into plasmid pAS2-1 after digesting with the former two restricted endonuclease, then the bait vector pAS2-1-PreS1 was verified by auto-sequencing assay. The PreS1-BD fusion protein expressed in the yeast cells was confirmed by western blot, after pAS2-1-PreS1 was transfected into the yeast cell AH109. Yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade2 medium, and the second screening was performed with LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive, then the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed Site NCBI to seek homologous sequence. RESULTS: Bait vector pAS2-1-PreS1 included the anticipated fragment of PreS1 gene. Western blot showed that pAS2-1-PreS1 could correctly express PreS1-BD fusion protein in the yeast cells. After yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library, 97 colonies grew in the selective SC/-trp-leu-his-ade2 medium, only one clone was positive and showed high homology with Homo sapiens nascent-polypeptide-associated complex alpha polypeptide. CONCLUSION: Bait vector pAS2-1-PreS1 is successfully constructed, and nascent-polypeptide-associated complex alpha polypeptide protein expressed in hepatocyte can interact with PreS1 by the yeast two-hybrid system.

Base Sequence↗

PubMed2007

Genetic analysis of the innate immune responses in wild-derived inbred strains of mice.

The vertebrate immune system has evolved to recognize nucleic acids of bacterial and viral origin. Microbial DNA, as well as synthetic oligonucleotides based on these motifs, activates innate immune pathways mediated by the family of Toll-like receptors (TLR) initiating a cascade of signals in immune cells necessary for responses to pathogens. However, not all of the proteins that participate in TLR-mediated responses have been identified. In studies described herein, we observed significant variation in innate immune responses among selected wild-derived strains of mice. Specifically, we show that mice of MOLF/Ei, Czech/Ei, and MSM/Ms strains are hypo-responsive to polyinosinic-polycytidylic acid (poly(I:C)) because of a mutation in Tlr3. In addition, we discovered a hypo-response to cytosine guanine dinucleotide in MOLF/Ei mice and established that it is not linked to Tlr9, but to another locus. Further inquiry revealed that this hypo-response is transmitted as a monogenic dominant trait that can be mapped and cloned through positional cloning methods. These results suggest the existence of a novel molecule that can alter pro-inflammatory signals or activate additional signal transduction pathways. In addition, they support the wild-derived mouse strain as a forward genetic tool for the identification of novel immunological phenotypes.

Amino Acid Sequence↗

PubMed1995

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences.

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers.

Animals↗

PubMed2002

Development of a high-throughput yeast two-hybrid screening system to study protein-protein interactions in plants.

We have developed a high-throughput yeast two-hybrid screening system (HTP-YTH) that incorporates yeast gap-repair cloning, multiple positive ( ADE2, HIS3, lacZ) and negative ( URA3-based) selection schemes to reduce the incidence of negative and false positive clones, and automation of laboratory procedures to increase throughput. This HTP-YTH system has been applied to the study of protein-protein interactions that are involved in rice defense signal transduction pathways. More than 100 genes involved in plant defense responses were selected from DuPont's rice expressed sequence tag (EST) databases as baits for HTP-YTH screening. Results from YTH screening of eight of these rice genes are presented in this paper. Not only have we identified known protein-protein interactions, but we have also discovered novel interactions, which may ultimately reveal the regulatory network of host defense signal transduction pathways. We have demonstrated that our HTP-YTH method can be used to map protein-protein interaction networks and signal transduction pathways in any system. In combination with other approaches, such efficient YTH screens can help us systemically to study the functions of known and unknown genes in the genomics era.

Base Sequence↗

PubMed1992

Isolation of a cDNA encoding a glutathione S-transferase (GST) class-pi from the bovine ocular ciliary epithelium.

We have used a polyclonal antiserum to bovine ciliary epithelium, a secretory tissue involved in the formation of aqueous humor, to immunoscreen a directional lambda gt11 Sfi-Not cDNA expression library prepared from bovine ciliary epithelium poly(A)+ RNA. After immunoscreening 6 x 10(5) independent clones, 41 cDNA clones positive for ciliary epithelium were isolated and characterized. About one-third of the positive cDNA clones were found to be identical and to encode a glutathione S-transferase (GST) class-pi. The largest bovine GST cDNA clone isolated, pCN11, contains an open reading frame of 630 bases, encoding a protein of 210 amino acids with a calculated molecular weight of 23,335 Da. The corresponding amino acid sequence showed an overall identity of 85.6% with the human, and 85.2% with the rat and mouse GST class-pi. Northern analysis of bovine ocular tissues revealed that the GST class-pi gene encodes a 0.8-kilobase mRNA which is expressed most abundantly in cornea, ciliary epithelium and retina, and in lower levels in iris and lens. Cell lines derived from non-pigmented or pigmented bovine ciliary epithelium also showed high levels of GST-pi mRNA expression. These results provide additional evidence for differential gene expression of GST class-pi mRNA in various areas of the bovine eye.

Animals↗

PubMed1991

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.

Amino Acid Sequence↗

PubMed1999

Utilization of mouse models in the discovery of human disease genes.

The mouse is a powerful model organism for studying human disease. By the use of various breeding strategies in combination with positional cloning and positional candidates, many genes underlying human disease have been and will continue to be discovered through the use of mouse genetics. The author illustrates how the mouse can be used to discover human disease genes underlying simple mendelian and complex polygenic diseases and genes involved in the modification of disease.

Journal Article↗

PubMed1997

From genetic research into clinical practice.

The present genome era is characterized by speedy progress and prompt transfer of results into clinical practice. This creates the need for rapid disclosure of results and renewal of laboratory's protocols. Molecular cytogenetics has provided and increased ability to identify chromosomes, correlate chromosome structure with gene location, find out cryptic aberrations, and detect specific DNA sequences. These advances have allowed the confident discovery of a number of contiguous gene syndromes. The positional cloning and positional candidate strategies have greatly expedited the search process of disease genes, and become relevant methods for genes' discovery. Understanding the molecular basis of diseases has shown an unpredicted wide genetic heterogeneity, which has splitted single disorders into many clinically similar conditions, and added complexity to the nosology of human diseases. The opposite process, allelism, where clinical diversity results from allelic mutations, has lumped together many distinct disorders, by showing that different clinical entities are not necessarily due to mutations in different genes. Dynamic mutations have provided the molecular understanding of interindividual and intrafamilial variability including anticipation, in a number of diseases. The discovery of distinct correlations between the molecular pattern and disease severity is providing a unique opportunity for using molecular results to assess the clinical outcome. Diagnostic, presymptomatic and predictive molecular testing are becoming widely used and provide enormous opportunities for improving the lot of our patients.

Genetic Diseases, Inborn↗

PubMed2001

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Animals↗

PubMed1999

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas.

3T3 Cells↗