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At least 811 records · Page 45Linked to original sources
PubMed2002

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta).

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.

Alleles↗
PubMed1990

Somatic cell hybrid and long-range physical mapping of 11p13 microdissected genomic clones.

Microdissection and microcloning of banded human metaphase chromosomes have been used to construct a genomic library of 20,000 clones that is highly enriched for chromosome 11p13 DNA sequences. Clones from this library have been mapped on a panel of human-rodent somatic cell hybrids that divides the region from distal p12 to proximal p14 into seven physical intervals, A total of 1500 clones has been isolated, 250 clones have been characterized, and 58 clones have been mapped. Six of the clones were used to complete a long-range physical map of 7.5 megabases through the region. Two of the clones are localized to the Wilms tumor (WT) region, three are localized to the aniridia (AN2) region, and two are localized to the region between WT and AN2. The library represents DNA sequences spanning a distance of approximately 13 x 10(6) base pairs, with an average density of one clone per 37,000 base pairs.

Animals↗
PubMed2000

Genomic organization of the human metabotropic glutamate receptor subtype 3.

In this study, the genomic organization of the human metabotropic glutamate receptor subtype 3 (mGluR3) gene has been determined. We have identified two transcription initiation sites and the polyadenylation signal by using 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE, respectively. The exon/intron organization of the human mGluR3 gene revealed the presence of 6 exons separated by 5 introns. The size of introns varied from 10.4 to 120 kbp that contained consensus sequences for repetitive elements such as Alu and long interspersed elements. A putative promoter region flanking the 5' sequence of exon 1 was identified by computer-aided analysis. The putative promoter region was characterized by the presence of a CAAT and GC box, and the absence of a TATA box or CpG islands. Several putative binding sites for transcription factors were also identified. In addition, we have isolated, from a mouse genomic library, part of the mouse mGluR3 gene and found it to correspond to exon 2 in the human mGluR3 gene. The mouse mGluR3 gene was then mapped by fluorescent in situ hybridization analysis to chromosome 5qA2.

Animals↗
PubMed1997

Physical mapping of the murine casein locus reveals the gene order as alpha-beta-gamma-epsilon-kappa.

The murine casein locus has been characterized by long-range restriction mapping and the analysis of large fragment genomic clones. Cloned sequences from five mouse casein genes (alpha, beta, gamma, epsilon, kappa) were used to screen a murine (strain 129) genomic library in a bacterial artificial chromosome vector (BAC). Of the nine clones isolated, two contained three casein genes alpha, beta, gamma and gamma, epsilon, kappa, respectively. The following combinations were found in other clones: alpha + beta, beta + gamma; and gamma + epsilon. Thus, the gene order in the locus can be deduced to be alpha-beta-gamma-epsilon-kappa. This order was confirmed by restriction analysis of the clones. A contig map of the clones and flanking sequences has been established by characterizing seven BAC clones, which together span approximately 470 kb. Long-range restriction analysis of genomic DNA indicates that the murine casein locus is confined to a 250-kb partial Xho I fragment. The alpha and beta casein genes were shown to be arranged in a tail-to-tail orientation.

Animals↗
PubMed1995

Microdissection and microcloning of chromosomal alterations in human breast cancer.

The recognition of recurring sites of chromosome changes in malignancies has greatly facilitated the identification of genes implicated in the pathogenesis of human cancers. Based especially upon recent studies [1-4], it appears increasingly likely that a subset of recurring chromosome alterations will be recognized in human breast cancer. Currently recognized chromosome changes characterizing breast carcinoma include the recognition of cytologic features of gene amplification (e.g. double minutes [dmins] and homogeneously staining regions [HSRs]) [5-8]. As these and other chromosome regions are implicated in recurring abnormalities in breast cancer, it will become increasingly important to have band- or region-specific genomic libraries and probes in order to facilitate high resolution physical mapping and ultimately to clone breast cancer related genes [9]. Toward this end an important recent development in physical mapping has been the establishment of chromosome microdissection as a rapid and reproducible approach to rapidly isolate and characterize chromosome region-specific DNA, greatly facilitating the initial steps in positional cloning of disease-related genes [10-13]. In this brief report, we will highlight the application of chromosome microdissection to the generation of region-specific probes for both fluorescent in situ hybridization (FISH) and the generation of genomic microclone libraries. Additionally, efforts using this methodology to generate a microclone library encompassing the early onset breast/ovarian cancer (BRCA1) gene will be presented.

Breast Neoplasms↗
PubMed1990

Isolation of a gene coding for a major heat shock protein HSP71 in rat.

A rat gene closely related to a human heat-inducible/cell cycle-dependent hsx70 gene was cloned from a rat genomic library. Northern blot analysis showed that it encodes a 2.5 kb transcript, one of the two heat-inducible mRNAs (2.5 and 2.7 kb) detected in rat tissues. Comparison of the known expression pattern of rat major heat shock proteins and mRNAs suggests that the isolated rat gene most probably codes for HSP71 protein.

Animals↗
PubMed1998

Complex probes for high-throughput parallel genetic mapping of genomic mouse BAC clones.

We describe a novel approach for the identification and mapping of polymorphic markers. Amplicons are generated by ligation of double-stranded adaptor molecules to genomic DNA cleaved with a restriction enzyme. Using primers that extend beyond the restriction site, reduced-complexity subsets of fragments are generated by PCR. Differences in the composition of complex probes generated from DNA of different strains are revealed through hybridization against high-density filter grids of large-insert genomic clones. Genetic mapping of genomic clones is achieved by hybridizing complex probes derived from backcross animals against the polymorphic clones. The mouse was chosen as a model system to test the feasibility of this technique because of the general availability of backcross resources and genomic libraries. Nevertheless, we would expect the method to be of particular use to generate markers for species that have not yet been extensively studied, because a substantial number of easy-to-use markers can be recruited in a relatively short period of time.

Animals↗
PubMed1991

Genomic organization of Drosophila choline acetyltransferase.

Choline acetyltransferase (ChAT; acetyl-CoA:choline-O-acetyltransferase; EC 2.3.1.6) is the enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is thus the genetic determinant of neurons with a cholinergic phenotype. We have screened a Drosophila genomic library using a cRNA probe, transcribed from Drosophila ChAT cDNA, and isolated three independent clones representing all the exons of this gene. The gene spans more than 26 kb of DNA and is organized into eight exons containing 594, 80, 192, 759, 408, 147, 201, and 1,612 nucleotides. All inserts that hybridized with a cRNA probe have been subcloned and the sequence of intron/exon boundaries determined. The only part of the ChAT gene not represented in our clones is a part of the first intron. A minimum size for this uncloned DNA has been deduced from Southern analysis of Drosophila genomic DNA. We also have probed the transcripts of the ChAT gene by northern analysis of total Drosophila RNA using two different exon-specific antisense RNA probes. An exon I probe detected two bands of RNA whereas an exon VIII probe hybridized with only the smaller band, previously identified as ChAT mRNA. These results indicate a complex transcription pattern for the ChAT locus in Drosophila.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of the first heat shock protein 70 from a reef coral.

The branching coral Stylophora pistillata, one of the most abundant hermatypic corals along the coasts of the Red Sea, has been used for many years as a model species for coral biological studies. Here we characterize the first coral heat shock protein 70 gene (SP-HSP70), cloned from S. pistillata, to be used as a tool for studying coral stress response. The cloning was carried out by a combination of PCR methods using heterologous, degenerate HSP70-based primers, followed by plaque-lift screening of a genomic library. The sequenced clone (5212 bp), contains a complete 1953 bp, intronless open reading frame, and 5' and 3' flanking regions of 1,935 and 1,324 bp, respectively. TATA, CAAT, and ATF boxes as well as 11 putative heat shock elements were identified in the SP-HSP70 5' flanking region. A polyadenylation site was identified in the 3' flanking region. SP-HSP70 protein sequence resembles the cytosolic/nuclear HSP70 cluster. RT-PCR studies confirmed SP-HSP70 mRNA expression in corals grown within their normal physiological conditions. Furthermore, SP-HSP70 has been shown to belong to the coral genome and not to its symbiotic algae one, as revealed by SP-HSP70 PCR amplification, using purified algal and coral DNA templates.

Amino Acid Sequence↗
PubMed1999

The isolation and mapping of 19 tetranucleotide microsatellite markers in the chicken.

Microsatellites consisting of tetranucleotide repeats are more easily, and consequently efficiently, scored than loci consisting of dinucleotides. However, they are much less frequent in the genome. A hybridisation selection protocol was therefore employed to generate a chicken genomic library enriched for inserts containing the tetranucleotide repeat motif (TTTC)n. Forty-five new microsatellite sequences were isolated that mainly consisted of perfect repeats of the tetranucleotide (TTTC) motif. Nineteen markers were mapped in one or both of the East Lansing and Compton international chicken reference populations.

Animals↗
PubMed2000

Cloning of acyl-acyl carrier protein (ACP) thioesterase gene from Brassica juncea.

Acyl-acyl carrier protein (ACP) thioesterase is the chain-length-determining enzyme in de novo biosynthesis of plant fatty acids. For cloning the gene encoding acyl-ACP thioesterase from Brassica juncea, genomic DNA was used as a template to amplify a 0.7 kb thioesterase fragment in a PCR with the primers designed from the known sequences available in the GenBank. This 0.7 kb fragment was used as a probe to study the expression of the gene in developing seeds and also to screen a genomic library of B. juncea constructed in lambdaEMBL-3 to get the full length of the gene. A 4.0 kb BamHI fragment containing the full gene was finally cloned in a plasmid vector from a recombinant phage clone lambda5.12 after a series of screening, sub-cloning and Southern hybridization.

Brassica↗
PubMed1990

Structure of genomic DNA for rat platelet phospholipase A2.

A genomic DNA for rat platelet phospholipase A2 was isolated by screening a rat genomic library with oligonucleotide probes based on its published amino acid sequence. The rat platelet phospholipase A2 gene had a total length of about 2.5 kb and contained five exons and four introns. The intron-exon structure of the rate gene was similar to that of human non-pancreatic phospholipase A2.

Amino Acid Sequence↗
PubMed1997

A tandem array of 5S ribosomal RNA genes in Pythium irregulare.

The 5S ribosomal RNA genes of the oomycete Pythium irregulare exist in tandem arrays unlinked to the rDNA repeat unit. A clone with a 9.2-kb insert containing an array of 5S genes was identified in a lambda genomic library and was characterized by restriction mapping and partial sequencing. The array consisted of 9 apparently identical 5S genes and their spacers in tandem, followed by a diverged 5S-like sequence that is likely to be a pseudogene. This gene arrangement, although almost universal in plants and animals, is rare in fungi and protists.

Base Sequence↗
PubMed1999

The L-gulono-gamma-lactone oxidase gene (GULO) which is a candidate for vitamin C deficiency in pigs maps to chromosome 14.

Vitamin C deficient pigs, when fed a diet lacking L-ascorbic acid (AscA), manifest deformity of the legs, multiple fractures, osteoporosis, growth retardation and haemorrhagic tendencies. This trait was shown by others to be controlled by a single autosomal recessive allele designated as od (osteogenic disorder). The inability of AscA biosynthesis in primates and guinea pigs that exhibit similar symptoms, when they are not supplemented with AscA in the food, was traced to the lack of L-gulono-gamma-lactone oxidase, which catalyzes the terminal step in the biosynthesis of AscA. The non-functional GULOP was mapped to human chromosome 8p21 that corresponds to an evolutionarily conserved segment on either porcine chromosome 4 (SSC4) or 14 (SSC14). We investigated linkage between OD and SSC4- and 14-specific microsatellite loci in order to map the OD locus. Twenty-seven informative meioses in families from one sire and three dams revealed linkage of od with microsatellites SW857 and S0089, located in the subcentromeric region of SSC14. We isolated part of the GULO gene of the pig by screening a porcine genomic library using a pig GULO cDNA as a probe, and mapped it to SSC14q14 by fluorescence in situ hybridization (FISH). Thus, the porcine GULO gene is both a good physiological and positional candidate gene for vitamin C deficiency in pigs.

Animals↗
PubMed2003

Microsatellite identification and characterization in peanut ( A. hypogaea L.).

A major constraint to the application of biotechnology to the improvement of the allotetraploid peanut, or groundnut ( Arachis hypogaea L.), has been the paucity of polymorphism among germplasm lines using biochemical (seed proteins, isozymes) and DNA markers (RFLPs and RAPDs). Six sequence-tagged microsatellite (STMS) markers were previously available that revealed polymorphism in cultivated peanut. Here, we identify and characterize 110 STMS markers that reveal genetic variation in a diverse array of 24 peanut landraces. The simple-sequence repeats (SSRs) were identified with a probe of two 27648-clone genomic libraries: one constructed using PstI and the other using Sau3AI/ BamHI. The most frequent, repeat motifs identified were ATT and GA, which represented 29% and 28%, respectively, of all SSRs identified. These were followed by AT, CTT, and GT. Of the amplifiable primers, 81% of ATT and 70.8% of GA repeats were polymorphic in the cultivated peanut test array. The repeat motif AT showed the maximum number of alleles per locus (5.7). Motifs ATT, GT, and GA had a mean number of alleles per locus of 4.8, 3.8, and 3.6, respectively. The high mean number of alleles per polymorphic locus, combined with their relative frequency in the genome and amenability to probing, make ATT and GA the most useful and appropriate motifs to target to generate further SSR markers for peanut.

Agriculture↗
PubMed1996

The Phytophthora sojae genome contains tandem repeat sequences which vary from strain to strain.

The oömycete Phytophthora sojae causes root and stem rot of soybean. In P. sojae, 37 cultivarspecific physiological races have been reported with different reactions against 13 single dominant resistance genes in soybean. Recent genetic studies have demonstrated that the P. sojae-soybean interaction follows Flor's gene-for-gene model. Genomic subtraction was carried out to isolate race-specific DNA sequences possibly including avirulence genes. DNA from a race 1 isolate was subtraction-enriched with the DNAs from race 19 and race 22 isolates. The enriched DNAs were used to differentially screen a P. sojae genomic library. Characterization of the clones obtained identified repetitive sequences with variable copy numbers among isolates. Five of these repetitive sequences were tandemly repeated and were localized on single chromosomes. The sixth corresponded to the ribosomal RNA genes. These observations suggest that gene amplification could contribute to the generation of genetic diversity in P. sojae. Enriched sequences corresponding to avirulence genes were not detected.

Base Sequence↗
PubMed1996

Cloning of the Rhodobacter sphaeroides hisL gene: unifunctionality of the encoded protein and lack of linkage to other his genes.

The Rhodobacter sphaeroides 2.4.1 hisI gene, which encodes a phosphoribosyl-AMP-cyclohydrolase that catalyses the third step in the histidine biosynthetic pathway, has been isolated from a genomic library of this phototrophic bacterium by complementation of an Escherichia coli hisI mutant. Analysis of the nucleotide sequence of the R. sphaeroides hisI gene reveals that it encodes a deduced product of 119 aa with a predicted molecular mass of 13.4 kDa. In contrast to the situation in E. coli, the R. sphaeroides hisI gene encodes a unifunctional protein and it is not linked to the hisE gene. The absence of a single histidine operon like that of E. coli was confirmed by PFGE experiments and complementation analysis of a R. sphaeroides hisI mutant that was constructed by marker exchange. The location of hisI in the R. sphaeroides genome has been determined to be at map co-ordinate 2275 +/- 20 of chromosome l.

Amino Acid Sequence↗