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Results for “positional cloning”

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At least 343 records · Page 19Linked to original sources

PubMed2005

[Proliferation inhibition of human lung adenocarcinoma cell line A549 transfected by RASSF1A gene].

OBJECTIVE: To determine the effects of exogenous RASSF1A gene on the proliferation and expression of P65 and subunit of NF-kappaB, in lung adenocarcinoma cell line A549. METHODS: pcDNA3.0-RASSF1A and pcDNA3. 0 were introduced into A549 cell line by lipofectin transfection, and the A549 cells stably expressing RASSF1A gene were established by G418 selection. The expression of RASSF1A was detected by Western blotting. The cytobiologic characterizations of the positive clone were analyzed by methythiazoletertraolium (MTT) assay and cytometry. The expressing of P65 was analyzed by RT-PCR and Western blotting. RESULTS: A549 cells stably expressing RASSF1A protein were established by lipofection mediated transfection and selected for further study. Compared with the nontransfected and vector transfected cells, the positive clone cells grew more slowly. Flow cytometric data showed that more positive clone cells went into phase G0/G1 and fewer cells went into phase S. The expression of P65 in nuclear protein in positive clone cells was lower than that of the control group while there was no obvious difference between the expression of p65 mRNA and P65 protein in total protein among the 3 groups. CONCLUSION: RASSF1A gene might suppress the proliferation of A549 cells through blocking the activity of P65 protein.

Adenocarcinoma↗

PubMed2002

Cloning of full length cDNA sequence of the mouse ameloblastin.

OBJECTIVE: Screening for special genes of matrix proteins of dentin and enamel of mouse dental germ. METHODS: A cDNA library of dental germ of mouse was screened by differential display. The interesting clones were sequenced. RESULTS: Six positive clones were isolated from the cDNA library. The sequence of one of the six positive clones was homologous with the ameloblastin sequence of rat. There are 497 homologous base pairs between the 526 base pairs sequenced by pTriplEX 3' primer of this clone and the 32-580 sequence of the rat ameloblastin gene; and there are 533 homologous base pairs between the 567 base pairs sequenced by pTriplEX 5' primer of this clone and the 1285-1854 sequence of the rat ameloblastin gene. CONCLUSIONS: The full length cDNA sequence of the mouse ameloblastin was cloned.

Amino Acid Sequence↗

PubMed2004

[Screening hepatitis B virus X-interactive proteins by yeast two-hybrid system].

BACKGROUND & OBJECTIVE: Hepatitis B virus-encoded X protein is a promiscuous transactivator and contributes to the development of hepatocellular carcinoma. Protein-protein interaction seems to be crucial for HBx transactivation. The aim of this study was to screen and identify the proteins which interact with hepatitis B virus (HBV) X protein by yeast two-hybrid system. METHODS: HBV X gene was amplified by polymerase chain reaction (PCR). HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system 3 and verified by sequencing. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was confirmed by Western blot analysis. Yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were cultured in selective SC/-trp-leu-his-ade medium. The second screening was performed with beta-gal activity detection. The false positive clones were eliminated by segregation analysis and mating experiment. The real positive clones were amplified, sequenced, and analyzed with bioinformatics. RESULTS: Bait plasmid pAS2-1-X was successfully constructed. The result of Western blot analysis confirmed that pAS2-1-X correctly expressed X-BD fusion protein in the transformed yeast AH109. Ninety-seven clones grew in the selective SC/-trp-leu-his-ade medium; however, only one clone past through the beta-gal activity detection, segregation analysis, and mating experiment. The inserted cDNA fragment of positive clone showed high homology with Fis gene. CONCLUSION: Fis protein is a novel protein which can interact with X protein in vivo by yeast two-hybrid system.

Gene Library↗

PubMed1996

Hypervariable region sequence in cryoglobulin-associated hepatitis C virus in sera of patients with chronic hepatitis C: relationship to antibody response against hypervariable region genome.

Essential mixed cryoglobulinemia is frequently associated with hepatitis C virus (HCV) infection, with the formation of HCV antigen/antibody complexes. The hypervariable region (HVR) of the HCV E2/NS1 region is thought to include epitopes for neutralizing antibodies, but it remains uncertain whether cryoglobulins (CGs) contain such antibody-bound HCV. Thus, we studied HVR clones isolated from cryoprecipitate and supernatant in the sera of four chronic hepatitis C patients with cryoglobulinemia, and expressed as fusion proteins with glutathione S-transferase (GST). Patients' sera were tested for antibody binding to the proteins. The rate of anti-HVR antibody-positive clones was significantly higher in cryoprecipitate (89% +/- 13%, P < .05) than in supernatant (41% +/- 25%). Both HCV RNA and anti-HVR antibody were more concentrated in cryoprecipitates compared with those of serum and supernatant in two patients tested. Anti-HVR antibody-positive clones in cryoprecipitate showed common amino acid (aa) sequences in each of the four patients. Similarly, all the antibody-positive clones in supernatant showed the same aa sequences for three of the four patients. When aa sequences were compared with those of reported isolates with genotype 1b, the mean percentage of aa difference was greater in the clones from supernatant and in anti-HVR antibody-negative clones than in the clones from cryoprecipitate and in the antibody-positive clones, respectively. These findings indicate that serum CG contains anti-HVR antibody-bound HCV in patients with chronic hepatitis C. Anti-HVR antibody-free individual clones, which were more frequently noted in supernatant, showed closely related sequences, but which were of a heterogeneous quasispecies nature.

Adult↗

PubMed2000

[Preliminary report on immunoscreening of Schistosoma japonicum adult worm cDNA library by sera from infected Microtus fortis].

Microtus fortis (Mf) is resistant to Schistosoma japonicum (Sj) infection. To explore if Mf responses to special Sj antigens after infection, Sj adult worm cDNA library was screened with sera from infected Mf. Twelve positive clones were obtained after rescreening. After automatically excised with the helper phage, positive clones were further identified by PCR. The size of Sj cDNA fragments in positive clones ranged from 300 bp to 1.8 kb. Six of these fragments were 300 bp, one was 1 kb, and five were 1.8 kb in size. The results indicate that the infected Mf serum could recognize Sj specific antigens. It is valuable to further study the immune protection of these cloned molecules.

Animals↗

PubMed1995

High-resolution genetic mapping of complex traits.

Positional cloning requires high-resolution genetic mapping. To plan a positional cloning project, one needs to know how many informative meioses will be required to narrow the search for a disease gene to an acceptably small region. For a simple Mendelian trait studied with linkage analysis, the answer is straightforward. In this paper, we address the situation of a complex trait studied with affected-relative-pair methods. We derive mathematical formulas for the size of an appropriate confidence region, as a function of the relative risk attributable to the gene. Using these results, we provide graphs showing the number of relative pairs required to narrow the gene hunt to an interval of a given size. For example, we show that localizing a gene to 1 cM requires a median of 200 sib pairs for a locus causing a fivefold increased risk to an offspring and 700 sib pairs for a locus causing a twofold increased risk. We discuss the implications of these results for the positional cloning of genes underlying complex traits.

Alleles↗

PubMed2006

[Screening of human anti-gamma-seminoprotein light chain guided with the murine Fd fragment].

AIM: To screen human anti-gamma-sm (gamma-seminoprotein) light chain (Lc) with guided selection of murine Fd fragment. METHODS: The human Lc repertorie genes were amplified by RT-PCR from PBMC in patients with prostate cancer, and cloned into the phagemid vector pComb3X with murine Fd gene against gamma-seminoprotein to construct the human-mouse hybrid Fab antibody library. The size of the library, antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified gamma-sm was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected by ELISA with pIII-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST. RESULTS: A 1.2x10(7) CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds' panning with gamma-sm, 5 positive clones were selected by ELISA and 2 clones with higher affinity were selected. Sequence analysis suggested these two positive clones contained the same light gene with high V(L) homology to human germline gene IGKV4-1*01. CONCLUSION: Human anti-gamma-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.

Animals↗

PubMed2001

Genomewide scans of complex human diseases: true linkage is hard to find.

Many "complex" human diseases, which involve multiple genetic and environmental determinants, have increased in incidence during the past 2 decades. During the same time period, considerable effort and expense have been expended in whole-genome screens aimed at detection of genetic loci contributing to the susceptibility to complex human diseases. However, the success of positional cloning attempts based on whole-genome screens has been limited, and many of the fundamental questions relating to the genetic epidemiology of complex human disease remain unanswered. Both to review the success of the positional cloning paradigm as applied to complex human disease and to investigate the characteristics of the whole-genome scans undertaken to date, we created a database of 101 studies of complex human disease, which were found by a systematic Medline search (current as of December 2000). We compared these studies, concerning 31 different human complex diseases, with regard to design, methods, and results. The "significance" categorizations proposed by Lander and Kruglyak were used as criteria for the "success" of a study. Most (66.3% [n=67]) of the studies did not show "significant" linkage when the criteria of Lander and Kruglyak (1995) were used, and the results of studies of the same disease were often inconsistent. Our analyses suggest that no single study design consistently produces more-significant results. Multivariate analysis suggests that the only factors independently associated with increased study success are (a) an increase in the number of individuals studied and (b) study of a sample drawn from only one ethnic group. Positional cloning based on whole-genome screens in complex human disease has proved more difficult than originally had been envisioned; detection of linkage and positional cloning of specific disease-susceptibility loci remains elusive.

Asthma↗

PubMed2005

[Study on the internal control on polymerase chain reaction in Yersinia pestis detection].

OBJECTIVE: For the detection of Yersinia pestis by polymerase chain reaction (PCR), internal control (IC) is required in order to prevent false negative results that might be caused by PCR inhibitors. METHODS: F1 antigen was amplified by PCR with primer F1 and the PCR product of primer F1 were cloned with TOPO TA cloning Kit. The plasmid of positive clone was then digested with HpaI. The digested plasmid and the PCR products of 16S rRNA were ligated with T(4) DNA ligase before the ligated products were transformed. Isolate plasmid DNA on positive clone and its concentration were measured. Plasmid DNA on different concentration by PCR amplification with primer F1 was analyzed and the standard concentration of IC was determined. RESULTS: Constructing an IC by inserting a 16S rRNA amplicon to the original target DNA between the two primer F1 sites, the size was longer than the target DNA. The standard concentration of IC was determined. CONCLUSION: An optimal IC concentration to increase the reliability of the PCR assays might be used to prevent false negative results and appeared to be useful for detection of Yersinia pestis.

Bacterial Proteins↗

PubMedDate not supplied

Effect of interferon-alpha in patients with chronic myelogenous leukemia in the accelerated phase: cytogenetic and molecular studies.

Human interferon-alpha (IFN-alpha) has been shown to be effective in the treatment of Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in the benign stable phase. The present study indicates that IFN-alpha may have a suppressive effect on Ph1-positive clones not only in the early stable phase but also in the accelerated phase with additional chromosomal abnormalities in some patients. In this study, in addition to 5 benign-phase patients, 3 patients with CML in the accelerated phase who had additional chromosomal abnormalities were treated with IFN-alpha. The presence of the Ph1-positive clone was estimated by chromosomal analysis and by Southern analysis at the DNA level using a 3' breakpoint cluster region (bcr) probe. Hematological remission and the suppression of proliferation of Ph1-positive clone to various extents were achieved by IFN-alpha treatment in 2 benign-phase patients and 3 patients with additional chromosomal abnormalities. Interestingly, in one of the latter three patients, Ph1-positive clones with or without additional chromosomal abnormalities were completely suppressed judging from chromosomal analysis and from the disappearance of bcr gene rearrangements.

Adult↗

PubMed2000

[Cloning of a new catechol 1,2-dioxygenase gene (tfd C) from Plesiomonas and its expression in the E. coli].

A new catechol-1,2-dioxygenase gene (tfd C) was cloned from the Plesiomonas using the PCR method. Primers were designed according to the reported sequence of Catechol-1,2-dioxygenase (C120) gene from Alcaligenes eutroplus. The amplified fragment contained a 765 bp open reading frame (ORF), encoding a protein of 255 amino acids. The new tfd C gene shared a high homology with the one cloned from Alcaligenes eutroplus, showing only one base difference at 693 site (C-->A) and consequently one amino acid difference at 228 site (P-->T). The ORF was cloned to the plasmid pBluescriptII KS, which was transferred to E. coli JM109 and a positive clone, pBt2G, was then selected. A significant activity of C120 was detected in the positive clone. When the ORF was cloned to the plasmid pET-30a, which was transferred to E. coli BL21(DE3) plysS, the expected 33 kD protein was detected from a positive clone, pET30A, by SDS-PAGE. The C120 is a key enzyme in degrading aromatic pollutants in the environment. In order to use plants to degrade aromatic pollutants, the gene will be introduced into the turfgrass. To express the gene properly in plants, its translation initiation codon was modified from GTG to ATG. A similar activity of C120 was obtained following the modification.

Amino Acid Sequence↗

PubMed2000

Construction of a binary bacterial artificial chromosome library of Petunia inflata and the isolation of large genomic fragments linked to the self-incompatibility (S-) locus.

The Solanaceae family of flowering plants possesses a type of self-incompatibility mechanism that enables the pistil to reject self pollen but accept non-self pollen for fertilization. The pistil function in this system has been shown to be controlled by a polymorphic gene at the S-locus, termed the S-RNase gene. The pollen function is believed to be controlled by another as yet unidentified polymorphic gene at the S-locus, termed the pollen S-gene. As a first step in using a functional genomic approach to identify the pollen S-gene, a genomic BAC (bacterial artificial chromosome) library of the S2S2 genotype of Petunia inflata, a self-incompatible solanaceous species, was constructed using a Ti-plasmid based BAC vector, BIBAC2. The average insert size was 136.4 kb and the entire library represented a 7.5-fold genome coverage. Screening of the library using cDNAs for the S2-RNase gene and 13 pollen-expressed genes that are linked to the S-locus yielded 51 positive clones, with at least one positive clone for each gene. Collectively, at least 2 Mb of the chromosomal region was spanned by these clones. Together, three clones that contained the S2-RNase gene spanned approximately 263 kb. How this BAC library and the clones identified could be used to identify the pollen S-gene and to study other aspects of self-incompatibility is discussed.

Chromosomes, Artificial, Bacterial↗

PubMed1995

Persistence of immunoglobulin heavy chain/c-myc recombination-positive lymphocyte clones in the blood of human immunodeficiency virus-infected homosexual men.

We studied blood lymphocytes of human immunodeficiency virus (HIV)-seropositive and -negative homosexual men for the presence of T(8;14) translocations that recombine c-myc and immunoglobulin heavy-chain (IgH) mu/IgH alpha switch regions. Clones with T(8;14) translocations were detected in 10.5% (12/114) of the HIV-positive and in 2.0% of the 99 uninfected patients. The majority of recombinations were found at a single time point only. Four patients, however, harbored multiple (up to four) and persistent (up to 9 years) translocation-positive cell clones. No correlation between the presence of these aberrant lymphocytes and a later lymphoma could be established. The exon 1/intron 1 region of the recombined c-myc was investigated for the presence of point mutations and these were found in the nonpersistent clones. Additional alterations detected in these clones included duplications and a deletion in the c-myc gene. The pattern of base substitution indicates that they were introduced after the translocation event.

Base Sequence↗

PubMed1990

Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction.

We have developed an approach for screening ordered arrays of yeast artificial-chromosome (YAC) clones containing human DNA that is based on the polymerase chain reaction (PCR). This approach is designed to determine the locations of positive clones within a YAC library that is stored as individual clones in 96-well microtiter plates. The high sensitivity and specificity of the PCR allow the detection of target sequences in DNA prepared from pools of 1920 or more YAC clones. The PCR-based screening protocol is performed in two successive stages, which effectively limit the location of a positive clone to four microtiter plates (384 clones). Final localization of each positive clone is accomplished by conventional DNA.DNA hybridization using a single filter containing the YAC clones from the appropriate four microtiter plates. This PCR-based screening strategy has proven highly efficient, allowing the identification and isolation of numerous YAC clones containing specific human genes. The prospects of developing a strategy for screening YAC libraries based completely on PCR assays are discussed, as are the potential applications of this approach to the systematic analysis of the human genome.

Base Sequence↗

PubMed1984

Change in functional phenotype of cloned human alloreactive cytolytic T cells.

Alloreactive T cell clones primed in vivo were tested for the expression of T cell differentiation antigens CD2, CD3, CD4, and CD8. Each of 29 different clones were found to express CD2 and CD3, but were variable in their expression of CD4 (7 positive clones) and CD8 (15 positive clones). Six clones were positive for both CD4 and CD8. One of the 29 clones expressed neither CD4 or CD8. Over a period of 12-18 weeks of culture, these clones began to lose their alloreactivity but acquired NK-like activity. By changing the concentration of TCGF, the "allo" and "NK-like" lytic activities could be modulated. After 18 weeks of culture, these clones lost their alloreactive specificity, but not their NK activity. The expression of surface markers was unchanged. CD2 and CD3 molecules were determined to play a role in both the alloreactive and NK activity of these clones.

Adult↗

PubMed1991

Immunocytochemical localization of xanthine oxidase in rat myocardium.

Monoclonal antibodies (MAb's) to xanthine oxidase (XO) (from bovine milk) were produced by hybridoma technique. Culture supernatants were initially screened using enzyme-linked immunoabsorbant assay (ELISA). Forty four positive clones were subcloned and further characterized by ELISA and immunoblot analysis. Out of fifteen clones, which were positive in immunoblot analysis, one clone N2-26 was also positive in immunocytochemical studies. Indirect immunoperoxidase and enzyme histochemistry staining showed that XO activity is present in endothelial cells of capillaries, small blood vessels and also in interstitial cells. Electron microscopy revealed that diaminobenzidine reaction product was distributed in the cytoplasm of interstitial cells and endothelial cells of capillaries and small blood vessels. This is the first report of the presence of XO in interstitial cells and endothelial cells of small blood vessels. Allopurinol, which inhibits the xanthine oxidase activity, did not have any effect on the immunocytochemical staining. Our results in normal rat heart suggest that XO activity is confined to interstitial cells, endothelial cells of capillaries and small blood vessels.

Animals↗

PubMed2001

[Cloning and characterization of three novel genes encoding transmembrane proteins of Schistosoma japonicum].

OBJECTIVE: To clone and analyze novel antigen molecules of Schistosoma japonicum (Sj), and to provide effective vaccine candidate antigens against schistosomiasis japonica. METHODS: Sj adult cDNA library was screened using sera of mice infected with Trichinella spiralis (Ts) and the inserts of positive clones were specifically amplified by PCR. The positive clones were sequenced and the sequence data were analyzed using Nucleotide BLAST software of NCBI and Expert Protein Analysis System of Swiss Institute of Bioinformatics. RESULTS: Nine positive clones were obtained after three rounds of immunoscreening. The size of these inserts ranged from 0.6 kb to 2.1 kb. Among five novel genes, Sj-Ts1, Sj-Ts3 and Sj-Ts5 (GenBank accession number: AY005816, AF299080 and AY024352, respectively) encode trans-membrane proteins with 83, 83 and 233 amino acids, respectively. Sj-Ts1 protein predicted contains one possible trans-membrance helix, one N-myristoylation site, two phosphorylation sites for protein kinase C and one for tyrosine kinase, Sj-Ts3 protein contains two possible transmembrance helices and one casein kinase II phosphorylation site, whereas Sj-Ts5 protein has five possible transmembrance helices, one N-glycosylation site, one N-myristoylation site, two phosphorylation sites for cAMP- and cGMP-dependent protein kinase and four for protein kinase C and one for casein kinase II. CONCLUSION: Three novel genes encoding three transmembrane proteins might be developed as new vaccine candidates against Sj infection.

Amino Acid Sequence↗

PubMed2005

[Construction and preliminary identification of subtracted cDNA library of leukemia cell line K562].

OBJECTIVE: Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes. METHODS: cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A I-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced. RESULTS: The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes. CONCLUSION: Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.

Cloning, Molecular↗