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Results for “positional cloning”

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PubMed1999

[Molecular genetics of inherited chorioretinal dystrophy--strategy for identifying disease causing genes].

PURPOSE: To review the strategies for identifying the causative genes of inherited chorioretinal diseases. METHODS: Three techniques, namely functional cloning, positional cloning, and positional candidate gene approach, have been used for this purpose. Our strategy for identifying the causative genes of inherited chorioretinal diseases is to clone retina-enriched genes, learn their chromosomal map position, and identify their expression in the retina. RESULTS: In the past 10 years, one gene has been cloned by functional cloning, 9 genes by positional cloning, and 19 genes by the positional candidate gene approach. Our strategy has identified 3 novel genes expressed in the retina, but they have not been associated with chorioretinal diseases. CONCLUSION: In the next century when the Human Genome Project is finished and the human genome has been sequenced completely, the positional candidate gene approach will become the predominant method of disease gene discovery. The future success of this method is predicated on increasingly dense mapping of the responsible genes by linkage analysis of multiple affected families with inherited chorioretinal diseases.

Choroid Diseases↗

PubMed2003

Isolation and analysis of a novel gene over-expressed during liver regeneration.

AIM: To isolate and analyze a novel gene over-expressed during liver regeneration. METHODS: Total RNA of regenerating liver was extracted from liver tissue after 0-4-36-36-36 hr short interval successive partial hepatectomy (SISPH). Reverse transcription-polymerase chain reaction was used to synthesize double strand cDNA, after the tissue was digested by proteinase K and Sfi A/B. The double-strand cDNA was ligated to lambdaTriplEx2. lambdaphage packaging reaction was performed and E. coli XL1-Blue was infected for titering and amplifying. One expressed sequence tag was probed by Dig and phage in situ hybridization was carried out to isolate positive clones. Positive recombinant lambdaTriplEx2 was converted to the corresponding pTriplEx2, and bioinformatics was used to analyze full-length cDNA. RESULTS: We isolated a novel full-length cDNA during liver regeneration following SISPH. CONCLUSION: We have succeeded in cloning a novel gene, based on bioinformatics. We postulate that this gene may function in complicated network in liver regeneration. On the one hand, it may exert initiation of liver regeneration via regulating nitric oxide synthesis. On the other hand, it may protect damaged residue lobus following SISPH.

Amino Acid Sequence↗

PubMed1992

Molecular linkage of the human CTLA4 and CD28 Ig-superfamily genes in yeast artificial chromosomes.

CD28 and CTLA4 are structurally homologous single-V-domain molecules of the Ig superfamily, the genes of which comap on the same chromosomal bands in mouse and man. Using polymerase chain reactions, we isolated six yeast artificial chromosome (YAC) clones positive for CTLA4 and/or CD28 from a human-DNA-containing YAC library. Two double-positive clones, 365 and 550 kb long, respectively, were further studied. Detailed restriction enzyme maps showed that one of these YACs was nested in the other, that they both bore the same CD28- and CTLA4-hybridizing fragments, that similar fragments were seen in genomic DNA, and that the distance between the CD28 and CTLA4 genes was at most 150 kb and at least 25 kb. A CpG island was found between these genes. These results provide a high-resolution estimate of the physical distance between the CD28 and CTLA4 genes and constitute a basis for the isolation of neighboring structures.

Abatacept↗

PubMed2001

Identification of dopamine responsive mRNAs in glial cells by suppression subtractive hybridization.

Recent studies have established that glial cells are important targets of the neurotransmitter dopamine (DA), but the regulatory effects of DA on glial cells have not been extensively studied. In the present study, we have investigated the influence of DA on gene transcription in glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes cultured from rat cerebral tissues in standard media or in culture media treated with DA. PCR-select differential screening was used to further verify the differentially expressed cDNA clones, positive clones were sequenced, and the mRNAs were re-examined on Northern blots. Fourteen sequences were identified of which eleven are homologous to known genes, three are homologous to expressed sequence tags (ESTs). Three novel full-length cDNAs were isolated using the EST fragments as probes to screen a cDNA library constructed from human brain. Analysis of these sequences suggested that complex intracellular signaling pathways, involving crosstalk with growth factor pathways, steroid hormone pathways, and an interferon-regulated 2-5 A pathway, are responsive to DA in astrocytes. The responsive proteins downstream from the signaling pathways were found to fall into at least three groups, including a series of metabolic enzymes, stress proteins, transfer proteins, etc. In addition, several of them have established their relationships with specific neurodegenerative diseases, showing that there is overlap in the pathogenic mechanisms of different diseases. Our results have provided a foundation for better understanding of the molecular basis of glial cell functions in dopaminergic transmission and an approach to find possible medication for the related disorders.

Animals↗

PubMed1999

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

Amino Acid Sequence↗

PubMed1999

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Animals↗

PubMed2006

Differential gene expression in response to ventricular unloading in rat and human myocardium.

UNLABELLED: Left ventricular unloading by mechanical assist devices induces myocardial atrophy. We aimed to systematically identify differentially expressed genes in a model of physiological atrophy (unloading of healthy rat myocardium) and compare these changes to those in a unloaded, failing human heart. METHODS: Atrophy in rat hearts was induced by heterotopic transplantation of a donor heart into the abdomen of an isogenic recipient. After one week, donor and recipient RNA was isolated. Differential gene expression was assessed by subtractive hybridization. Two screens with radioactive probes were performed to verify differentially expressed clones. Positive clones were sequenced and cDNA of genes of known homology were used as probes for hybridization with RNA from separate atrophied rat hearts and human tissue from a normal, failing or failing and unloaded left ventricle. RESULTS: We picked 1880 clones from the subtractive hybridization procedure (940/940: forward/reverse runs assessing up- or down-regulation, respectively). The first screen verified 465/140 and the second screen verified 67/30 clones. 24/23 clones were sequenced and 14/10 homologies to known genes were found. In the atrophied heart, respiratory chain and metabolic genes were down-regulated (NADH-DH, cytochrome c oxidase, acetyl-CoA synthetase, myoglobin) and cellular recognition and stress genes were up-regulated (MHC1 and 2, HSP70). In the human heart, cytochrome c oxidase, acetyl-CoA synthetase, and myoglobin expression was increased in the failing heart and returned to normal with unloading. Unloading also resulted in up-regulation of HSP70. CONCLUSIONS: The genetic responses of failing human and healthy rat myocardium to mechanical unloading show similarities that appear to be independent of species differences and/or underlying disease. Thus, heterotopic heart transplantation is a relevant model for investigating the mechanisms of mechanical unloading.

Animals↗

PubMed1991

Transcripts from amplified sequences of an inherited homogeneously staining region in chromosome 1 of the house mouse (Mus musculus).

Several populations of the house mouse, Mus musculus, are polymorphic for the presence or absence of an inherited homogeneously staining region (HSR) in chromosome 1. The HSR consists of highly amplified DNA sequences, present in low copy numbers in the HSR- genome. A cloned HSR-derived genomic sequence detected transcripts of about 1.3 and 4.5 kb on blots of poly(A)+ RNA from liver of HSR+ mice but not from that of HSR- mice. A cDNA library was established from RNA of HSR+ mice and screened with the HSR-derived genomic clone. Positive clones were isolated and shown to be complementary to the 1.3-kb RNA species and to amplified DNA sequences in the HSR+ genome. The combined sequence of four overlapping cloned cDNAs is 959 nucleotides long and includes an open reading frame encoding a putative protein of 208 amino acids. The pertinent gene is unidentified. No homologous sequence is stored in the EMBL data base. A stretch of 109 nucleotides at the 3' end of the 1.3-kb RNA homology region in the same genomic fragment, as indicated by hybridization data and sequence motifs resembling promoter elements. Thus, our data suggest that at least two genes or gene families are encoded in the HSR.

Amino Acid Sequence↗

PubMed1995

[Strategies for the detection and the identification of tumor suppressor genes].

The methods for the detection and the identification of tumor suppressor genes are characterized and classified as follows: Firstly; by the comparison of expressed genes or proteins between tumor cells and normal cells. Secondly; by the gene transfer. Thirdly; by the identification of proteins which are involved in the biochemical process for cell growth suppression and DNA repair. Human genome contains dozens of tumor suppressor genes and tumor suppression-related genes which have not yet been cloned. Positional cloning is the most reliable method for the cloning of these genes. However, it cost huge amount of labor and time. New methods developed in recent years, such as differential mRNA display or two hybrids system, would facilitate the identification and the molecular cloning of novel tumor suppressor genes.

Cloning, Molecular↗

PubMed1996

Positive selection, cloning vectors for gram-positive bacteria based on a restriction endonuclease cassette.

Lactococcus lactis contains numerous restriction and modification (R/M) systems of different specificities. A novel IIS type R/M system encoded by the LlaI operon has previously been characterized from the L. lactis conjugative plasmid pTR2030. The LlaI operon is composed of six genes: First, a small regulatory gene llaIC precedes the methylase gene llaIM. The following three genes, llaI.1, llaI.2, llaI.3, are all essential for restriction endonuclease activity and are designed as the restriction cassette llaIR. The forth open reading frame of unknown function follows the llaIR gene cassette. We have successfully subcloned the three llaIR genes, llaI.1, llaI.2, and llaI.3, without llaIM, as a suicide cassette into the three shuttle vectors pTRKL2, pTRKH2, and pBV5030. A promoter (P6) from Lactobacillus acidophilus ATCC4356, which is functional in E. coli, lactococci, and lactobacilli (Djordjevic and Topisirovic, unpublished) was cloned upstream of the three gene cassette. Restriction activity was evaluated in Escherichia coli and several gram-positive bacteria. The llaIR restriction cassette was not functional in E. coli, but its presence was lethal to L. lactis, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus johnsonii, Lactobacillus acidophilus, Carnobacterium pisicola, Enterococcus faecalis, Bacillus subtilis, and Leuconostoc gelidum. Several novel, positive selection cloning vectors were developed that can exploit unique cloning sites within the llaIR cassette. Insertions in llaI.1 resulted in complete inactivation of restriction activity and provided unconditional selection for recombinant plasmids in surviving transformants. These positive selection cloning vectors are the first for gram-positive bacteria that are based on a restriction endonuclease cassette. Functional activity of the llaIR genes in various gram-positive bacteria would also enable use of these cloning vectors for positive selection of promoters, terminators, and regulatory sequences across these genera.

Cloning, Molecular↗

PubMed2005

Calpain 10 and type 2 diabetes: are we getting closer to an explanation?

PURPOSE OF REVIEW: A variation in the gene encoding the cysteine protease calpain 10 (CAPN10) was recently linked and associated with type 2 diabetes by positional cloning. This positional cloning was a follow-up investigation to the identification of a diabetes-linked region on human chromosome 2 identified by genome-wide scanning a few years earlier. In this paper we give a general background on the genetic studies performed on CAPN10 to date, and review the most recent studies on the functional role of calpain 10. RECENT FINDING: A haplotype or haplotype combination comprising three intronic single nucleotide polymorphisms (UCSNP-43, 19, and 63) were associated with a threefold increased risk of type 2 diabetes in the population in which linkage was first found. Another polymorphism, UCSNP-44, which is in linkage disequilibrium with a coding single-nucleotide polymorphism (Thr504Ala), has subsequently been associated with type 2 diabetes in extensive meta-analyses. Meanwhile, initial studies probing the possible role of calpain-10, completely unknown at the time, are now being pursued, both in isolated cells and humans. SUMMARY: The positional cloning of CAPN10 as a candidate gene for type 2 diabetes has been particularly fruitful. Not only has it identified an important and surprising piece of the puzzle underlying the development of diabetes, but it has also modelled and paved the way for investigations concerning complex genetic diseases other than type 2 diabetes.

Calpain↗

PubMed1990

[Screening and identification of apolipoprotein CIII cDNA clones from human liver cDNA library and preparation of apolipoprotein CIII cDNA probe from the clones].

Four of positive clones were obtained by three runs of screening the cDNA library of human liver with rabbit antihuman apolipoprotein (apo) CIII IgG. One of them was identified to be the positive clone of apoC III cDNA by restriction endonuclease map studies of the cDNA insert fragment and by Western blot analysis of the expression products. The apoCIII cDNA-gamma gt11 recombinant DNA prepared from the positive clone can be used in RNA-dot blot analysis as apoCIII cDNA probe. And the apoCIII cDNA clone can be also used to produced apoCIII peptide under the induction of isopropyl-beta-D-thiogalactoside. The apoCIII peptide, after purification, has a lot of use in further studies.

Apolipoprotein C-III↗

PubMed2002

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp.

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Animals↗

PubMed1992

Construction of a new Escherichia coli-Saccharomyces cerevisiae shuttle plasmid cloning vector allowing positive selection for cloned fragments.

A new E. coli-S. cerevisiae shuttle plasmid cloning vector (pPW263) with a positive type of selection, was constructed. The selection system, based on the regulatory region of lambda phage controlling the expression of tetracycline resistance, was derived from the cloning vector pUN121 (Nilsson et al. 1983). There are three cloning sites in the cI gene, EcoRI, HindIII and BglII, and, in addition, two unique sites in the neighborhood, BamHI and SalI. The size of the vector is 7.8 kb. The maintenance of the vector and the selection in yeast was ensured by the replication region of the 2 mu plasmid and by the URA3 marker gene, respectively.

Amino Acid Sequence↗

PubMed1984

Autoimmunity following viral infection: demonstration of monoclonal antibodies against normal tissue following infection of mice with reovirus and demonstration of shared antigenicity between virus and lymphocytes.

Splenic lymphocytes from adult C57BL/6 mice infected with purified reovirus type 1 or 3 particles were fused with NS1 myeloma cells. Approximately 300 clones were obtained from each fusion (type 1 or type 3) and the supernatants from these clones were screened by radioimmunoassay for their ability to bind virus, T lymphocytes, brain, liver, lung tissues and isolated oligodendrocytes and ependymal cells. Approximately 10% of clones (33 and 26 clones, respectively) were positive for each fusion. For reovirus type 1:21% of positive clones bound only virus, 64% bound one of the normal tissues but not virus, and 15% bound both virus and one or more of the normal tissues. For reovirus type 3: 19% of positive clones bound only virus, 73% bound normal tissue only, and 8% bound both virus and normal tissue. Only 3 positive clones were obtained from uninfected control animals. These experiments demonstrate that (a) during the course of an immune response to a virus, autoantibodies are generated which react with a large variety of normal tissues and that (b) there are shared antigenic structures between viral determinants and normal tissue that can be identified by monoclonal antibodies. Although these results suggest two mechanisms by which an autoimmune response may develop following viral infection, the biological significance of these autoreactive monoclonal antibodies remains to be elucidated.

Animals↗

PubMed1999

Establishment of a human fusion partner for making human T-T hybridomas which can grow in serum free medium.

A new human fusion partner, ICLU-T was established for making human T-T hybridomas. Some 6-thioguanine resistant (6-TGr) clones were separated from human T cell acute lymphocytic leukemia, PEER. The fastest growing clone of the 6-TGr cells was scaled up and was cultured in serum free medium. The clone that proliferated most quickly in the serum free medium was separated and named ICLU-T. When ICLU-T was fused with human peripheral blood lymphocytes by using only polyethylene glycol, the viability of fused cells was below 20% just after fusion and the fusion efficiency was 0.01-0.02 per 10(5) parent cells. Whereas, the combined use of 1% lecithin with polyethylene glycol increased the viability over 50% and the fusion efficiencies over 0.2 per 10(5) parent cells on the average. In a total of 34 clones of obtained hybridomas, there were 25 CD2 positive clones. CD4 positive or CD8 positive clones of the CD2 positive clones were 12 and 5 clones, respectively. Further, 4 clones of all hybridomas were CD19 positive, and one clone of these hybridomas was an Ig producer. These obtained hybridomas could proliferate in serum free medium.

Cell Division↗

PubMed1993

In vivo regulation of murine hair growth: insights from grafting defined cell populations onto nude mice.

The nude mouse graft model for testing the hair-forming ability of selected cell populations has considerable potential for providing insights into factors that are important for hair follicle development and proper hair formation. We have developed a minimal component system consisting of immature hair follicle buds from newborn pigmented C57BL/6 mice and adenovirus E1A-immortalized rat vibrissa dermal papilla cells. Hair follicle buds contribute to formation of hairless skin when grafted alone or with Swiss 3T3 cells, but produce densely haired skin when grafted with a fresh dermal cell preparation. The fresh dermal cell preparation represents the single cell fraction after hair follicles have been removed from a collagenase digest of newborn mouse dermis. It provides dermal papilla cells, fibroblasts, and possibly other important growth factor-producing cell types. Rat vibrissa dermal papilla cells supported dense hair growth at early passage in culture but progressively lost this potential during repeated passage in culture. Of 19 E1A-immortalized, clonally derived rat vibrissa dermal papilla cell lines, the four most positive clones supported hair growth to the extent of approximately 200 to 300 hairs per 1-2 cm2 graft area. The remaining clones were moderately positive (five clones), weakly positive (three clones), or negative (seven clones). Swiss 3T3 cells prevented contraction of the graft area but did not appear to affect the number of hairs in the graft site produced by dermal papilla cells plus hair follicle buds alone. The relatively low hair density (estimated 1-5% of normal) resulting from grafts of hair follicle buds with the most positive of the immortalized dermal papilla cell clones compared to fresh dermal cells suggests that optimal reconstitution of hair growth requires some function of dermal papilla cells partially lost during the immortalization process and possibly the contribution of other cell types present in the fresh dermal cell preparation, which is not supplied by the Swiss 3T3 cells. The current graft system, comprising hair follicle buds and immortalized dermal papilla cell clones, provides an assay for positive or negative influences on hair growth exerted by added selected cell types, growth factors, or other substances. Characterization of the phenotype of the dermal papilla cell lines, which differ in their ability to support hair growth when grafted with hair follicle buds, may provide insight into specific dermal papilla cell properties important for their function in this system.

Animals↗

PubMed1995

Molecular cloning of a group of mouse pancreatic islet beta-cell-related genes by random cDNA sequencing.

To understand the molecular basis of glucose concentration-responsive insulin synthesis and secretion from pancreatic islet beta cells, a group of pancreatic islet beta-cell-related cDNAs was cloned. A pair of cDNA libraries was constructed from a mouse pancreatic islet beta-cell line of MIN6, which was cultured in either high glucose or low glucose media. By applying a random cDNA sequencing approach, 503 and 395 independent species were obtained from a total of 1,011 and 762 clones in the high glucose and low glucose library, respectively. The unknown genes comprised the majority of about 70% independent clones in both libraries. In Northern blot analysis, 311 (69.4%) of 448 independent clones showed positive signals within 72 h of autoradiographic exposure. Surprisingly, 150 (48.2%) out of 311 positive clones showed positive signals to MIN6 cells, but not to NIH/3T3 fibroblasts. The expression level of three unknown clones were glucose-concentration dependent. Combination of a random cDNA sequencing approach and Northern blot analysis is useful to obtain a large number of novel genes and islet beta-cell-related genes.

Animals↗