Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Genomic Library”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources
PubMed2001

Human glycosaminoglycan glucuronyltransferase I gene and a related processed pseudogene: genomic structure, chromosomal mapping and characterization.

Here we describe the characterization of the human glycosaminoglycan glucuronyltransferase I gene (GlcAT-I) and a related pseudogene. The GlcAT-I gene was localized to human chromosome 11q12-q13 by in situ hybridization of metaphase chromosomes. GlcAT-I spanned 7 kb of human genomic DNA and was divided into five exons. Northern blot analysis showed that GlcAT-I exhibited ubiquitous but markedly different expressions in the human tissues examined. The GlcAT-I promoter was approx. 3-fold more active in a melanoma cell line than in a hepatoma cell line, providing evidence for the differential regulation of the gene's expression. Stepwise 5' deletions of the promoter identified a strong enhancer element between -303 and -153 bp that included binding motifs for Ets, CREB (cAMP-response-element-binding protein) and STAT (signal transducers and activators of transcription). Screening of a human genomic library identified one additional distinct genomic clone containing an approx. 1.4 kb sequence region that shared an overall 95.3% nucleotide identity with exons 1-5 of GlcAT-I. However, a lack of intron sequences, as well as the presence of several nucleotide mutations, insertions and deletions that disrupted the potential GlcAT-I reading frame, suggested that the clone contained a processed pseudogene. The pseudogene was localized to chromosome 3. The human genome therefore contains two related GlcAT-I genes that are located on separate chromosomes.

Base Sequence↗
PubMed1991

The Q300 gene: a novel transcription unit induced in simian virus 40-infected and -transformed mouse cells.

The Q300 element is a single-copy 233-bp genomic mouse DNA fragment carrying a high-affinity binding site for the simian virus 40 (SV40) large T antigen. This element was used to screen an EMBL3 mouse genomic library. We could identify a genomic clone containing an approximately 500-bp transcribed region flanking the Q300 element. The transcribed region, termed the Q300 transcription unit or Q300 gene, is overexpressed in acutely SV40-infected or SV40-transformed mouse and rat cells. The Q300 gene includes an open reading frame which has the coding potential for a small polypeptide with an extremely hydrophobic N terminus and a hydrophilic C terminus. The deduced polypeptide has some similarity with the papillomavirus E5 oncoprotein.

Amino Acid Sequence↗
PubMed1991

There appear to be conserved constraints on the distribution of nucleotide sequences in cellular genomes.

The data from a genomic library can be sorted into the frequencies of every possible tetranucleotide in the sequence. This tabulation, a short sequence distribution, contains the frequency of occurrence of the 256 tetranucleotides and thus seems to serve as a vehicle for averaging sequence information. Two such distributions can be readily compared by correlation. Reported here are correlations (Spearman rs) of the distributions from all of the genomic libraries in GenBank 44.0 with sizes equal to or larger than that of Salmonella typhimurium, except for the data for mouse and humans. All of the organisms examined showed highly significant correlations between the two DNA strands (not the complementarity expected from base pairing). Of 155 comparisons between libraries, 132 showed significant correlations at the 99% confidence level. Application of the correlation coefficients as a similarity matrix clustered most organisms in a phenogram in a pattern consistent with other hypotheses. This suggests a highly conserved pattern underlying all other genetic information in cellular DNA and affecting both DNA strands, perhaps caused by interaction with conserved factors necessary for DNA packaging.

Animals↗
PubMed2004

Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility.

Gene dosage has frequently been exploited to select for genetic interactions between a particular mutant and clones from a random genomic library at high copy. We report here the first use of multicopy suppression as a forward genetic method to determine cellular targets and potential resistance mechanisms for novel antibacterial compounds identified through high-throughput screening. A screen of 8640 small molecules for growth inhibition of a hyperpermeable strain of Escherichia coli led to the identification of 49 leads for suppressor selection from clones harboring an E. coli genomic library. The majority of suppressors were found to encode the multidrug efflux pump AcrB, indicating that those compounds were substrates for efflux. Two leads, which produced clones containing the gene folA, encoding dihydrofolate reductase (DHFR), proved to target DHFR in vivo and were competitive inhibitors in vitro.

Anti-Bacterial Agents↗
PubMed2002

Chemical regulated production of cDNAs from genomic DNA fragments in plants.

We have developed a new chemical inducible genetic system that allows for the isolation of any cDNA molecule from in vitro generated genomic transgenes in transgenic plants. This system, termed regulated in vivo cDNA generation (RIDE), permits both targeted isolation of individual full-length cDNA molecules and random isolation of any partial or full-length cDNA from in planta genomic libraries. The RIDE system makes use of the 17-beta estradiol-inducible promoter system linked to intron donor and acceptor sites in a new binary vector configuration. In transgenic Arabidopsis and tobacco plants, we show that the RIDE system can isolate low-abundance full-length cDNAs previously unattainable by conventional means at high efficiencies (75-85%). The ability to randomly isolate individual exons and exons spliced together from genomic libraries in planta suggest that this system can be used for the isolation of any cDNA molecules. The RIDE system thus appears to be an efficient and versatile system for the generation of potentially any cDNA molecule. Moreover, the ORF structural data generated will be of value in both verifying and correcting computational ORF predications in the databases available to the scientific community.

Arabidopsis↗
PubMed1997

Identification of putative transmembrane receptor sequences homologous to the calcium-sensing G-protein-coupled receptor.

The sensing of extracellular calcium is a general paradigm for regulating diverse cellular functions in many tissues. A calcium-sensing receptor (Casr) belonging to the metabotropic glutamate family of G-protein-coupled receptors (GPCR) that transduces the effects of extracellular calcium in the parathyroid gland as well as other tissues has been identified. The diversity of GPCR families and the recent finding of calcium sensing in cells lacking the known Casr suggest the existence of additional receptors related to Casr. By polymerase chain reaction (PCR) amplification and screening of genomic libraries, we have identified multiple Casr-related sequences (Casr-rs) in the mouse. Using primers designed to regions of the first and third intracellular loops of Casr, we initially PCR amplified a 497-bp Casr-related sequence (Casr-rs1) with high homology to Casr. The deduced protein sequence of Casr-rs1 is 63% similar and 40% identical to Casr over the available transmembrane region. We screened a mouse genomic library with a Casr-rs1 probe and identified two additional Casr-related sequences (Casr-rs2 and Casr-rs3). In the predicted transmembrane domain, Casr-rs2 and Casr-rs3 are 95% identical to Casr-rs1. We mapped Casr-rs1 to mouse Chromosome (Chr) 7 by interspecific backcross analysis, whereas the known Casr localizes to mouse Chr 16. By fluorescence in situ hybridization, Casr-rs2 also localized to mouse Chr 7 and Casr-rs3 mapped to mouse Chr 4. We were able to distinquish Casr-rs1 from Casr-rs2 by PCR using specific primers, suggesting that they are distinct genes clustered on Chr 7. By RT-PCR, we identified additional Casr-rs transcripts in mouse kidney, brain, testis, embryo, and MC3T3-E1 osteoblasts, but not in lung or liver. The homologous sequence in mouse kidney, embryo, and MC3T3-E1 osteoblasts, designated Casr-rs4, has a deduced amino acid sequence that is 100% similar and 97% identical to that of Casr-rs1. The sequence amplified from mouse brain, Casr-rs5, has a deduced protein sequence that is 96% similar and 92% identical to that of Casr-rs1. Our findings establish the existence of a novel multimembered family of Casr-related sequences in the mouse which may encode receptors that transduce responses to diverse extracellular cations.

Amino Acid Sequence↗
PubMed1993

A PCR-based method for high stringency screening of DNA libraries.

A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described. A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria. Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled. The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers. A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe. This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol. A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR. Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques. PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert. This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal. Similar methodology has also been used to clone a cDNA gene contained within a plasmid library.

Animals↗
PubMed2006

Development and characterization of a normalized canine retinal cDNA library for genomic and expression studies.

PURPOSE: Identification of causative mutations for retinal blinding disorders is often limited by restricted understanding of gene expression and underlying molecular mechanisms that trigger degenerative processes. This study was conducted to develop a catalog of canine retina-expressed genes that would provide a unique tool to investigate normal and altered function in the adult retina. Because of the conserved syntenies between the dog and human, this approach would identify new potential disease candidate genes for both species. METHODS: A canine normalized retinal cDNA library was produced and analyzed by using a modified PhredPhrap algorithm. Computerized annotation provided gene homology and chromosomal location for individual clones and contigs in a Web-accessible database. RESULTS: From 6316 cDNA clones, 3980 retinal expressed sequence tags (ESTs) were derived. Homology to the canine genome draft sequence was found for more than 99% of all ESTs, but only for 32% when compared with annotated canine cDNAs. Functional analysis suggests an enrichment of this library for genes involved with eye function and development, chaperone, or ribosomal functions when compared with mouse and human National Center for Biotechnology Information (NCBI) RefSeq entries. CONCLUSIONS: A combination of annotation approaches with ongoing mapping and expression studies provide functional data covering at least 27% to 30% of the currently proposed canine catalog of genes expressed in the retina. This is an essential first step toward establishing an integrated network for gene identification and expression patterns suitable for functional genetics, comparative genomics and evolutionary analysis of genes and gene families with respect to the developmental and degenerative processes of the retina.

Animals↗
PubMed1999

Cross-reactive epitope mimics in a fragmented-genome phage display library derived from the rickettsia, Cowdria ruminantium.

BACKGROUND: Epitopes can be mapped by comparing immunoaffinity-selected peptides from fragmented-gene display libraries with the target gene. With larger libraries derived from unsequenced genomes, this is not possible. Spurious epitope mimics may be created by expressing DNA in a variety of meaningless reading frames and orientations. OBJECTIVES: To determine empirically whether panning a large fragmented-genome phage display library with antibodies to MAP1, the major antigenic protein of the rickettsial parasite Cowdria ruminantium, would result in the selection of irrelevant, cross-reactive mimotopes. STUDY DESIGN: A gene III phage library displaying peptides derived from C. ruminantium was constructed using cloned DNA from a bacteriophage lambda genomic library. After in vivo excision, plasmids were cleaved with PvuII followed by PCR. Genes with a PvuII site, including MAP1 were therefore not amplified. DNA was sonicated, partially digested with DNase and cloned into the display vector fUSE2. Affinity-purified MAP1 antibodies were used for panning. Peptides expressed by panned phages were tested for recognition in Western blot and ELISA. Oligonucleotides representing antigenic sequences were used to locate their encoding DNA sequences in the original lambda library. The phage display library was also panned with two monoclonal antibodies (Mabs) against bluetongue virus (BTV). RESULTS: Five different peptide sequences were selected from the MAP1-deficient phage display library. None was identical to MAP1, but four peptides had regions that were similar, both to each other, and to the parasite protein. They produced strong signals in ELISA and Western blot. None could be located to any C. ruminantium open reading frame. Two BTV Mabs recognised a sequence similar to their authentic epitope. CONCLUSION: Large genome-targeted phage display libraries may be sufficiently diverse to allow the selection of peptides that mimic actual antigenic determinants. This diversity may be exploited in the search for useful epitopes.

Amino Acid Sequence↗
PubMed2025

Expansion of the functional genomics GRACE library reveals genes relevant for temperature-dependent fitness in Candida albicans.

A small percentage of species in the fungal kingdom can cause devastating infections in humans, with Candida albicans reigning as a leading cause of systemic disease. One of the key virulence phenotypes for pathogenic fungi is the ability to survive at host body temperature; however, a comprehensive understanding of the mechanisms that orchestrate thermal adaptation in fungi remains incomplete. In this study, we expand the largest functional genomics resource in C. albicans, reaching 71.3% coverage of the entire genome, and perform screens under six different temperatures to identify genes important for temperature-dependent fitness. We describe the function of genes involved in translation (GAR1), splicing (C1_11680C or YSF3), and cell cycle progression (C6_00110C or RHT1) in enabling fungal survival at both low and high temperatures. Through experimental evolution, we also show that C. albicans can rapidly overcome deleterious mutations and adapt to extreme temperature environments. Overall, our study highlights the transformative potential of genome-wide functional genomics to uncover critical vulnerabilities in pathogenic fungi.

Genomics↗
PubMed1985

Identification of a human clustered G + C-rich DNA family of repeats (Sau3A family).

Sau3A digestion of human G + C-rich DNA molecules yields discrete bands of approximately 70 and 140 base-pairs, under-represented in A + T-rich DNA molecules and in total DNA. We have cloned the 70 base-pair band in a plasmid vector and isolated a representative recombinant clone that identifies a new human family of repeats, the Sau3A family. The new family has been characterized for a number of parameters: genomic organization; reiteration frequency; sequence analysis; and distribution in a human genomic library. The Sau3A sequence (68 base-pairs in length, 53% G + C) is present in approximately 4 X 10(4) copies/haploid genome; the family is characterized by a cluster organization and is confined to a limited fraction (0.5%) of phages of a human genomic library. Southern blot hybridizations of the cloned sequence to restriction digests of total human DNA and of isolated genomic clones does not show the involvement of Sau3A blocks in long-range periodicities for any of the enzymes tested. The data suggest either a high sequence variability in the family or a complex organization of Sau3A sequence domains.

Cloning, Molecular↗
PubMedDate not supplied

[Immunization with Leishmania amazonensis subgenomic libraries protects BALB/c mice against the challenge].

A genomic library of Leishmania amazonensis in expression vector of eukaryote cells (pEF1HisA, pEF1HisB, pEF1HisC) was prepared. Also two subgenomic libraries having each 500 clones approximately were created and BALB/c mice were immunized with 50 mg/0,1 mL of DNA from each. Two immunizations were administered intramuscularly at 15-day interval. Groups of control mice were immunized with DNA from empty plasmid pEF1His, with soluble parasite antigen (100 mg/0,1 mL) and saline solution. The size of lesions was measured for 12 weeks and at the end of the experiment, the parasite load at lesion sites was determined by plaque microtitration method. In mice immunized with subgenomic library DNAI and with soluble antigens,the size of lesions was controlled, which reached an statistical difference (p< 0,05) in relation to the rest of groups whose lesions increased. The parasite load found in lesion sites confirmed the previous results; the number of promastigots was significantly lower in those mice already protected. It was concluded that in subgenomic library DNA1 there should be genes or gene fragments whose in vivo expression induces protective immune response against the challenge in the murine model used.

Animals↗
PubMed1989

Phasmids as effective and simple tools for construction and analysis of gene libraries.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

Bacteriophage lambda↗
PubMed1990

Structural analysis of multiple bovine P-450(11 beta) genes and their promoter activities.

A bovine genomic library was constructed using a cosmid vector, pHC79, and bovine DNA partially digested by EcoRI. Bovine P-450(11 beta) cDNA, pcP-450(11 beta)-2 [Morohashi et al. (1987) J. Biochem. 102,559-568], was used as a probe for screening the genomic library. Ten clones carrying P-450(11 beta) genomic DNA were isolated from 8 x 10(4) colonies and classified into five groups (CB11 beta-1, CB11 beta-3, CB11 beta-7, CB11 beta-20, and CB11 beta-21) according to differences in the restriction endonuclease sites. Nucleotide sequences of amino acid coding regions of the five clones were determined by the dideoxy sequencing method using synthetic nucleotides corresponding to various parts of the cDNA as primers. The nucleotide sequences revealed that three clones, CB11 beta-1, CB11 beta-3, and CB11 beta-21, were pseudogenes. Amino acid sequences coded by the other two clones, CB11 beta-7 and CB11 beta-20, were identical with that coded by a previously described cDNA, pcP-450(11 beta)-3 [Kirita et al. (1988) J. Biochem. 104, 683-686]. The promoter regions of the five clones were introduced in front of chloramphenicol acetyltransferase (CAT) gene of pSV00CAT and used to examine P-450(11 beta) gene regulation in cultured cells. The five recombinant plasmids showed cAMP-responsive CAT activities in Y-1 cells, a cell strain derived from adrenal tumor. The induction rates of the recombinant plasmids carrying the promoters of normal genes, CB11 beta-7 and -20, were larger than those of pseudogenes, CB11 beta-1, -3, and -21. CAT activities expressed by the promoter regions of the normal genes in the presence or absence of cAMP in Y-1 cells were almost equal to that by the promoter region of human P-450(SCC) gene. Though the promoter of the P-450(SCC) gene also showed cAMP-responsive CAT activity in I-10 cells, a cell strain derived from Leyding cell tumor, P-450(11 beta) gene promoter did not express the activity in I-10 cells.

Amino Acid Sequence↗
PubMed1991

Construction and characterization of a NotI linking library of human chromosome 21.

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.

Cell Line↗
PubMed1985

Plasmodium falciparum: expression of the adenine phosphoribosyltransferase gene in mouse L cells.

Genomic libraries of Plasmodium falciparum were constructed in the pBR322 plasmid. Using the DNA-mediated gene transfer technique, the genomic libraries were introduced into tissue-cultured mouse cells lacking the enzyme adenine phosphoribosyltransferase. Following selection for the adenine phosphoribosyltransferse phenotype, several colonies were isolated. All clones were shown to possess adenine phosphoribosyltransferase activity and pBR322 sequences. In addition, the Km value of adenine phosphoribosyltransferase (for adenine) from a transformant was found to be identical to that from P. falciparum. These results indicate that the adenine phosphoribosyltransferase gene of P. falciparum was successfully cloned and expressed in a mammalian system.

Adenine Phosphoribosyltransferase↗
PubMedDate not supplied

Isolation and characterization of beta- and gamma-crystallin genes from rat genomic cosmid libraries.

Two libraries, together containing about 10(6) colonies, have been constructed by cloning different size fractions of a partial Sau3A digest of rat genomic DNA in the cosmid vector pTM. Upon screening with two cDNA clones, one containing alpha A2-crystallin and one containing beta B1-crystallin sequences, 14 cosmid clones were isolated which were beta B1-crystallin-specific; none was found which contained alpha A2-crystallin sequences. The inserts of the beta B1 clones, which range from 35 to 45 kb in length, contain overlapping DNA segments covering more than 60 kb of rat genomic DNA. The composite BamHI restriction map of this region shows a single beta B1-crystallin gene, which is interrupted by several intronic sequences. Five recombinants hybridizing with two different rat lens gamma-crystallin cDNA clones were also isolated from these libraries. Four of these contain 31- to 41-kb inserts, whereas the fifth recombinant contains a 12.2-kb insert. Hybridization analysis with 5' and 3'-specific cDNA fragments indicates that altogether these inserts contain six gamma-crystallin genes, three of which are located on one insert of only 31 kb.

Animals↗