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At least 667 records · Page 37Linked to original sources
PubMed1997

Rapid isolation of genomic clones for individual members of human multigene families: identification and localisation of UBE2L4, a novel member of a ubiquitin conjugating enzyme dispersed gene family.

We describe a rapid, PCR-based, screening procedure for the isolation of human genomic clones in lambda bacteriophage, containing sequences coding for individual homologous members of a multigene family. The approach is based upon the identification, by dilution, of sub-pools of the genomic library that contain members of the gene family, prior to phage isolation. The presence of specific genes is established by PCR of aliquots of individually amplified library pools, using consensus primers and subsequent sequencing. We have used the approach to isolate a fourth member of the UBE2L gene family, UBE2L4, and located it on chromosome 19q13.1-->q13.2. This PCR-based approach to library screening has wider applicability in that it could be used to isolate alternate-spliced products from cDNA libraries.

Bacteriophage lambda↗
PubMed1992

Rat copper/zinc superoxide dismutase gene: isolation, characterization, and species comparison.

A 13 kb rat Cu/ZnSOD genomic clone has been purified from a rat liver genomic library and completely characterized by restriction mapping, detailed sequencing and Southern blot analysis. This gene spans approximately 6 kb and contains five exons and four introns. Comparison of rat, mouse, and human Cu/ZnSOD genes reveals a high conservation in genomic organization and exon-intron junctions, including an unusual 5'GC donor sequence at the first intron. The gene contains a TATA box as well as an inverted CCAAT box, a feature common to both the mouse and human genes. Furthermore, several repeats were identified in the 5' promoter region of this gene, and these regulatory elements are also strikingly conserved in these three species.

Amino Acid Sequence↗
PubMed2005

PLAUF is a novel P. lividus sea urchin RNA-binding protein.

Preliminary results have shown that various proteins bind long 3'UTR of the transcript for Paracentrotus lividus sea urchin H3.3 histone variant and are probably implicated in mRNA instability. In order to identify these RNA-binding proteins, we screened a lambda-ZAPII cDNA expression library prepared from poly(A) mRNA extracted from sea urchin embryos at blastula stage. We isolated a cDNA that codes for a novel RNA-binding protein homologous to rat and human AUF1 family proteins and we refer to it as PLAUF. Proteins present in the whole lysate of the phages expressing PLAUF bound specifically in vitro the 3'UTR of the H3.3 histone transcript. Northern blot analysis revealed three PLAUF transcripts that are already present in unfertilized eggs; during development their amount increased starting from 4-blastomere embryos and reached the plateau at blastula stage. While the transcription start point was unique, longer 3'UTRs were revealed by 3'RACE approach and further cDNA library screening. Moreover RT-PCR showed the presence of at least one alternative spliced mRNA that codes for a protein with different COOH terminus. The structure of the PLAUF gene was determined by screening a P. lividus sea urchin genomic library with the PLAUF cDNA as probe. Analysis of the positive clones showed that the PLAUF gene is split in 10 exons and 9 introns spanning a distance of about 10 kb. Moreover we demonstrated that the exon 9 was alternative spliced during mRNA processing.

3' Untranslated Regions↗
PubMed1994

Occurrence and inheritance of microsatellites in Pinus radiata.

Microsatellites are an important class of DNA marker because of their abundance and length hypervariability. As part of a project mapping the Pinus radiata genome, we have characterized some of the microsatellites in this species. Southern blots were screened with oligonucleotide probes [(CA)10, (GA)10, (GAA)9, (CAA)8, (CAC)5, (GACA)4] to assess their abundance. CA and GA were the most abundant microsatellites, while GAA was least abundant. A genomic library in lambda ZAP, covering 9 x 10(4) kb, was screened with a combined poly(CA) + poly(GA) probe and yielded 120 positives, approximately one CA or GA microsatellite every 750 kb of the P. radiata genome. It was found that 25% of the positives were embedded within highly repetitive DNA. Four of the five subclones sequenced contained compound microsatellites, with TA predominating as the additional repeat. Segregation analysis of PCR products for two microsatellites, PR4.6 and PR9.3, in 96 progeny of a controlled outcross verified simple Mendelian inheritance. Both loci are highly polymorphic with Polymorphism Information Content values of 0.63 and 0.70 for PR4.6 and PR9.3, respectively. These results indicate that microsatellites are abundant in a conifer genome and can be valuable markers for pine mapping, fingerprinting, and population genetic studies.

Base Sequence↗
PubMed1998

Two-hybrid systematic screening of the yeast proteome.

The yeast two-hybrid system is a genetic method that detects protein-protein interactions. One application is the detection by library screening of new interactors of a protein of known function. In the August issue of Nature Genetics, Fromont-Racine et al. showed for the first time that the construction of the protein interaction map of a complex pathway, such as that of the mRNA splicing machinery, is now possible, because of the combination of recent technical improvements elaborated in several laboratories. With a yeast cell mating procedure that increases screen efficiency, they used their complex yeast genomic library of 5 x 10(6) clones to test 700 x 10(6) interactions against 15 proteins. They identified and classified 170 potential interactors, including approximately 70 proteins of previously unknown function. More than 25% of the interactors are probably biologically relevant. The achievements of Fromont-Racine et al. have opened the way to the systematic analysis of the protein interaction networks of the 6,000 open reading frames-yeast proteome. This task requires, however, automation of the library screens and creation of a two-hybrid library database.

Fungal Proteins↗
PubMedDate not supplied

[Mutual location of the rRNA operon and tuf gene in the Mycoplasma gallisepticum strain S6 genome].

The genomic library of Mycoplasma gallisepticum was constructed and two clones, selected by hybridization on E. coli 16S rRNA, were analyzed. The restriction map of the clones indicate that both clones belong to the same region of the M. gallisepticum genome. The results of Southern hybridization with either E. coli 16S rRNA, or E. coli 23S rRNA, or oligonucleotide synthesized as a part of M. gallisepticum 5S rRNA, led to the conclusion that the unspliced rRNA operon was cloned. The order of genes in the operon is common for eubacteria: 16S-23S-5S. The tuf gene of M. gallisepticum was mapped inside the cloned region 5 kb upstream of 16S gene by hydridization on E. coli tufA gene and oligonucleotide synthesized on the basis of M. gallisepticum tuf gene sequence. The direction of transcription of the gene and the expected direction of transcription of the rRNA operon coincide.

Bacteriophages↗
PubMedDate not supplied

A 1.3 kb satellite DNA from Bubalus bubalis not conserved evolutionarily is transcribed.

A 1.3 kb satellite DNA from a size defined genomic library of mammal Bubalus bubalis was cloned and sequenced. The clone pSB1 is AT rich with 447 A (33.6%), 262 C (19.7%), 240 G (19.0%) and 383 T (28.8%). There were about 1400 copies of contig in the bubaline genome but it did not uncover allele length variation when used as probe in conjunction with a number of restriction enzymes. The contig pSB1 is not conserved evolutionarily and cross hybridizes only with the Bovideae family. A set of primers from 5' (nt 422 to 441) and 3' (nt 962 to 947) deduced from the clone used for PCR amplification with four members of the Bovideae family gave the expected 530 bp band of equal intensity indicating a similar number of copies in all the four species namely Bos indicus, Capra hircus, Ovis aries and Bubalus bubalis. Expression studies with pSB1 following slot-blot hybridization with total RNA isolated from ovary, testes, kidney, lung and spleen revealed varying signal intensities in all the tissues with a most prominent signal in spleen but a faint one in ovary. Further sequence analysis revealed the presence of several eukaryotic transcriptional elements such as NF-E1, Poly-A signal, lariat consensus sequences, and CTF/NF1 binding sites. Blast search showed 90% sequence similarity with the reverse transcriptase gene of Bos taurus and sequences from nt 283 to 636 within the contig showed highly conserved reverse transcriptase like signatures along with N-glycosylation and protein kinase C phosphorylation sites. From the data we conclude that the pSB1 representing satellite DNA is associated with transcribing sequences. The prospect of identifying functional genes linked with the satellite fraction in higher vertebrates is discussed.

Animals↗
PubMed1995

Construction of a porcine YAC library and mapping of the cardiac muscle ryanodine receptor gene to chromosome 14q22-q23.

Large-scale physical mapping of the porcine genome has been limited because up to now no suitable genomic libraries for this purpose have been available. Therefore, we have constructed a yeast artificial chromosome (YAC) library from porcine lymphocytes. The library was cloned in the amplifiable vector pCGS966. A total of 10080 YAC clones was obtained and has been ordered into 105 96-well microtiter plates. An average insert size of 300 kb was calculated from the analysis of 78 randomly selected clones, giving a one-fold coverage of the porcine genome. To analyze the complexity, we have screened the library for five different genes and isolated four different clones containing parts of three of these genes. One YAC clone harboring parts of the porcine cardiac muscle ryanodine receptor (RYR2) gene allowed us to assign this locus to Chromosome (Chr) 14q22-q23. The data were confirmed by PCR analysis of a rodent-porcine hybrid cell panel.

Animals↗
PubMed2004

A microsatellite map of white clover.

The white clover ( Trifolium repens) nuclear genome (n = 2x = 16) is an important yet under-characterised genetic environment. We have developed simple sequence repeat (SSR) genetic markers for the white clover genome by mining an expressed sequence tag (EST) database and by isolation from enriched genomic libraries. A total of 2,086 EST-derived SSRs (EST-SSRs) were identified among 26,480 database accessions. Evaluation of 792 EST-SSR primer pairs resulted in 566 usable EST-SSRs. Of these, 335 polymorphic EST-SSRs, used in concert with 30 genomic SSRs, detected 493 loci in the white clover genome using 92 F1 progeny from a pair cross between two highly heterozygous genotypes--364/7 and 6525/5. Map length, as estimated using the joinmap algorithm, was 1,144 cM and spanned all 16 homologues. The R (red leaf) locus was mapped to linkage group B1 and is tightly linked to the microsatellite locus prs318c. The eight homoeologous pairs of linkage groups within the white clover genome were identified using 96 homoeologous loci. Segregation distortion was detected in four areas (groups A1, D1, D2 and H2). Marker locus density varied among and within linkage groups. This is the first time EST-SSRs have been used to build a whole-genome functional map and to describe subgenome organisation in an allopolyploid species, and T. repens is the only Trifolieae species to date to be mapped exclusively with SSRs. This gene-based microsatellite map will enable the resolution of quantitative traits into Mendelian characters, the characterisation of syntenic relationships with other genomes and acceleration of white clover improvement programmes.

Chromosome Mapping↗
PubMed1996

Comparison of the canine and human acid beta-galactosidase gene.

Several canine cDNA libraries were screened with human beta-galactosidase cDNA as probe. Seven positive clones were isolated and sequenced yielding a partial (2060 bp) canine beta-galactosidase cDNA with 86% identity to the human beta-galactosidase cDNA. Preliminary analysis of a canine genomic library indicated conservation of exon number and size. Analysis by Northern blotting disclosed a single mRNA of 2.4 kb in fibroblasts and liver from normal dogs and dogs affected with GM1 gangliosidosis. Although incomplete, these results indicate canine GM1 gangliosidosis is a suitable animal model of the human disease and should further efforts to devise a gene therapy strategy for its treatment.

Amino Acid Sequence↗
PubMed1992

The Atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA.

Screening for the gene encoding salmon gonadotropin releasing hormone (sGnRH) in an Atlantic salmon (Salmo salar) genomic library resulted in isolation of a positive clone designated lambda sGnRH-1. An anchor polymerase chain reaction (PCR) technique was used to amplify GnRH cDNA derived from salmon hypothalamic mRNA. The cDNA sequence was aligned to the 7607 base pair genomic sequence which was shown to encode the entire prepro-GnRH gene. The cDNA proved that the cloned gene is expressed in the hypothalamus of mature salmon. The coding domain of sGnRH differs from the mammalian GnRH by six nucleotide changes which allow the two amino acid differences between the two GnRH variants. Salmon GnRH associated peptide (GAP) differs extensively in sequence and size from the mammalian counterpart. Compared to the GnRH cDNA of a cichlid species the similarity is 69.3% in the protein coding sequence.

Amino Acid Sequence↗
PubMed1999

Isolation, characterization, molecular cloning and amplification of a species-specific M. leprae antigen.

A polyclonal serum sample from a lepromatous leprosy (LL) patient, which presented a specific recognition pattern for leprosin, was used to screen a Mycobacterium leprae genomic library constructed with DNA isolated from human lepromas. One clone, designated ML4-1, which expressed a specific antigenic determinant of M. leprae as part of a beta-galactosidase fusion protein, was isolated. The 1.932 bp M. leprae-derived genomic fragment was sequenced, and it had an incomplete open-reading frame shown to code for a 644 amino-acid polypeptide (72.3 kDa). Some partial nucleotide homology to the M. tuberculosis MTCY9C4 cosmid and the M. leprae B1913 cosmid were found. Southern blot assays using the 584 bp Eco RI-Bam HI fragment excised from the ML4-1 clone revealed that this sequence is present only in the M. leprae genome and not in the 24 different mycobacterial DNA tested. Two oligonucleotides based on the genomic sequence were also synthesized and used as amplifiers for a polymerase chain reaction (PCR) test, giving a positive signal exclusively in M. leprae DNA. Furthermore, 32 sequential synthetic peptides, 20 amino-acids long, spanning the entire protein corresponding to the hypothetical ML4-1 clone sequence, were synthesized and evaluated by ELISA. A peptide included in the 221-240 region was significantly recognized by either lepromatous leprosy or healthy tuberculosis contact patient sera. Thus, PCR amplification of this fragment, along with the recognition of its protein sequence by leprosy patient sera, could be a useful tool for a potential diagnostic method in the detection of M. leprae infection in the future.

Amino Acid Sequence↗
PubMed2002

Identification of simple sequence repeats (SSRs) in olive ( Olea europaea L.).

A small insert genomic library of Olea europaea L., highly enriched in (GA/CT) n repeats, was obtained using the procedure of Kandpal et al. (1994). The sequencing of 103 clones randomly extracted from this library allowed the identification of 56 unique genomic inserts containing simple sequence repeat regions made by at least three single repeats. A sample of 20 primer pairs out of the 42 available were tested for functionality using the six olive varieties whose DNA served for library construction. All primer pairs succeeded in amplifying at least one product from the six DNA samples, and ten pairs detecting more than one allele were used for the genetic characterisation of a panel of 20 olive accessions belonging to 16 distinct varieties. A total of 57 alleles were detected among the 20 genotypes at the ten polymorphic SSR loci. The remaining primer pair allowed the amplification of a single SSR allele for all accessions plus a longer fragment for some genotypes. Considering the simple sequence repeat polymorphism, 5.7 alleles were scored on average for each of the ten SSR loci. A genetic dissimilarity matrix, based on the proportion of shared alleles among all the pair-wise combinations of genotypes, was constructed and used to disentangle the genetic relationships among varieties by means of the UPGMA clustering algorithm. Graphical representation of the results showed the presence of two distinct clusters of varieties. The first cluster grouped the varieties cultivated on the Ionian Sea coasts. The second cluster showed two subdivisions: the first sub-cluster agglomerated the varieties from some inland areas of Calabria; the second grouped the remaining varieties from Basilicata and Apulia cultivated in nearby areas. Results of cluster analysis showed a significant relationship between the multilocus genetic similarities and the geographic origin of the cultivars.

Journal Article↗
PubMed2000

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product.

S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t0.5 approximately 10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K0.5 approximately 100 microM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine.

Adenosylmethionine Decarboxylase↗
PubMed1990

Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA 'fingerprinting' analysis.

A minisatellite probe, MZ 1.3, detecting hypervariable fragment patterns was isolated from a human genomic library. A repetitive sequence of 27 bp length was identified which is contained in the probe approx. 40 times. The MZ 1.3 repeat shows variable homology of 53-73% to the repetitive sequence of the protein III gene of the bacteriophage M13 genome. Polymorphic restriction fragment patterns were found with MZ 1.3 using the enzymes Hinf I, BstN I, Hae III, Mbo I, PstI/Pvu II, and Rsa I. An average of 18 polymorphic fragments was observed using Hinf I as enzyme. The band sharing frequency after Hinf I digestion among unrelated individuals was determined to be 23.8 +/- 7.2%. An example for the application of MZ 1.3 to paternity testing in an incest case is given. The probe can be used with radioactive or non-radioactive detection systems. An approach is presented to compare polymorphic fragment patterns from individuals obtained by independent gel runs on the basis of relative band positions (RBP) and calculated in a computerized analysis.

Base Sequence↗
PubMed2002

First insight into the genome of an uncultivated crenarchaeote from soil.

Molecular phylogenetic surveys based on the characterization of 16S rRNA genes have revealed that soil is an environment particularly rich in microbial diversity. A clade of crenarchaeota (archaea) has frequently been detected among many other novel lineages of uncultivated bacteria. In this study we have initiated a genomic approach for the characterization of uncultivated microorganisms from soil. We have developed a procedure based on a two-phase electrophoresis technique that allows the fast and reliable purification of concentrated and clonable, high molecular weight DNA. From this DNA we have constructed complex large-insert genomic libraries. Using archaea-specific 16S rRNA probes we have isolated a 34 kbp fragment from a 900 Mbp fosmid library of soil DNA. The clone contained a complete 16S/23S rRNA operon and 17 genes encoding putative proteins. Phylogenetic analyses of the rRNA genes and of several protein encoding genes (e.g. DNA polymerase, FixAB, glycosyl transferase) confirmed the specific affiliation of the genomic fragment with the non-thermophilic clade of the crenarchaeota. Content and structure of the genomic fragment indicated that the archaea from soil differ significantly from their previously studied uncultivated marine relatives. The protein encoding genes gave the first insights into the physiological potential of these organisms and can serve as a basis for future genomic and functional genomic studies.

Crenarchaeota↗
PubMed2003

Advances in encoding of colloids for combinatorial libraries: applications in genomics, proteomics and drug discovery.

The creation of enormous libraries of chemicals and their subsequent screening for bioactivity has been accelerated through recent developments in encoding solid supports. The ability to accurately identify the structure of a biomolecule that has exhibited activity is invaluable and is closer to realisation in the advent of smart nanoscience. In this review the evolution of encoding solid supports as platforms for combinatorial synthesis is traced. Current approaches to encoding solid supports are reviewed and their potential for use as supports for the high-throughput screening of split and mix libraries explored. Finally, a brief consideration of the status of the application of encoded libraries is provided including creative chemical and colloidal encoding.

Biopolymers↗
PubMed1993

Comparative mapping of the Pseudomonas aeruginosa PAO genome with rare-cutter linking clones or two-dimensional pulsed-field gel electrophoresis protocols.

The Spe1 map of the Pseudomonas aeruginosa PAO (DSM 1707) chromosome was constructed by utilizing two-dimensional pulsed-field gel electrophoresis (PFGE) and rare-cutter linking clones. After end-labeling and fluorescence staining of macrorestriction fragments had been combined, the two-dimensional PFGE analyses of partial-total digests and reciprocal double digests were sufficient for the placement of all fragments on the genomic map. Spe1 linking fragments were isolated from BamH1, Pst1, and EcoR1 genomic libraries of P. aeruginosa PAO. After separation of the heterogeneously sized populations of Spe1-linearized and uncut circular plasmid DNAs by field inversion polyacrylamide gel electrophoresis, the gel-eluted linear DNAs were recircularized and subcloned. The 46 analyzed Spe1 linking clones recognized 16 of the 38 fragment links on the Spe1 genome map of P. aeruginosa PAO. The alignment with linking clones was consistent with that obtained from two-dimensional PFGE mapping protocols.

DNA, Bacterial↗