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 1,315 records · Page 73Linked to original sources
PubMed1984

Molecular cloning and analysis of a gene coding for the Bowman-Birk protease inhibitor in soybean.

We have constructed cDNA clones from size-selected mRNA of developing soybean seeds to identify genes encoding the Bowman-Birk protease inhibitor. Recombinant clones containing sequences coding for the Bowman-Birk inhibitor were identified by mRNA hybrid selected translation and immunoprecipitation. The nucleotide sequence of the insert of plasmid pB38 corresponds to the mRNA for the Bowman-Birk inhibitor, although it is missing a portion of the coding sequence at the 5' end. Northern hybridization analysis of total RNA isolated from developing soybean seeds showed that the mRNA for the inhibitor is approximately 450 nucleotides long and that it accumulates early in embryogeny. Southern hybridization analysis of restriction enzyme-digested soybean nuclear DNA indicated that the gene encoding the Bowman-Birk inhibitor is not highly reiterated in the genome. We have isolated a gene encoding the Bowman-Birk inhibitor from a soybean genomic library constructed in Charon 4A. DNA sequence analysis of the genomic clone reveals that it is similar, although not identical, to the cDNA clone and that it contains no intervening sequences.

Amino Acid Sequence↗
PubMed1984

Molecular cloning of cDNA for Avena phytochrome.

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)(+) RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)(+) RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)(+) RNA hybridized with (32)P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.

Journal Article↗
PubMed1991

Hybridization selections, using immobilized driver DNA, to enrich for clones unique to a library.

We have developed a technique which allowed us to isolate sex-specific repeats of chickens (Gallus g. domesticus) from a genomic library originally containing one sex-specific repeat clone per 300 clones. Using this plus/minus selection method, we were able to enrich a sub-library in which the sex-specific repetitive clones made up over half of the population (170-fold increase in representation). This enrichment technique used hybridization kinetics to collect clones which are bound (plus selection) or not bound (minus selection) to their respective driver DNAs immobilized on nitrocellulose paper. Plus selections enriched for repetitive sequences and removed from the library most unique sequences as well as vectors containing no inserted sequences. These plus-selected sub-libraries were enriched for repetitive clones, yet still contained sequences representing as little as 10(-4) of the genome. Minus selection removed repetitive sequences shared between the driver and the library. When a plus-selected sub-library was minus selected, the doubly selected sub-library was enriched in repetitive sequences present in the library but not the minus driver.

Animals↗
PubMed1991

Abundance and DNA sequence of two-base repeat regions in tropical tree genomes.

Tandem DNA repeats of two-base pairs are potentially important tools for population genetic studies because of their abundance and length variation. As part of our research into the ecology of tropical forest plants, we began a study of dinucleotide repeat regions in several genera of tropical trees. Genomic libraries in bacteriophage lambda were screened with the oligonucleotide probes poly(GT) and poly(AG). Both types of repeat regions were abundant in the genomes of all six plant species examined. Using the size of inserts in the phage libraries and number of phage screened, we estimated that there were 5 x 10(3) to 3 x 10(5) poly(AC) sites per genome, with slightly more AG than AC sites. When libraries were made from smaller fragments of genomic DNA, abundance estimates were higher, suggesting that two-base repeat sites were clustered in the genome. Poly(AC) sites were 16-22 bp in length, and four of the five sequenced were adjacent to either poly(AG)or poly(AT) sites. Other repeat region s appeared in DNA flanking the AC sites. This further demonstrated that two-base repeats and other repetitive DNA were clustered in the genome. Two-base repeats are abundant in plant genomes and could provide a large number of polymorphic markers for studies of plant population genetics.

Bacteriophage lambda↗
PubMed2002

Cloning and sequence analysis of Mucor circinelloides glyceraldehyde-3-phosphate dehydrogenase gene.

A genomic library of Mucor circinelloides ATCC 1216b has been constructed in Lambda Fix II vector. The library has an average insert site of 10 kb and covers the genome 12 times. The M. circinelloides gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The promoter region, containing a consensus TATA and CAAT box and a 298 nucleotid long termination region were also determined.

Amino Acid Sequence↗
PubMed1999

Molecular cloning of the guinea pig GRO gene and its rapid expression in the tissues of lipopolysaccharide-injected guinea pigs.

BACKGROUND: CXC chemokines, IL-8 and GRO, play a role in the recruitment of neutrophils in the human. The functional orthologues in the rat and mouse are CINC/KC and MIP-2. The lack of IL-8 made these animals less useful to study the role of IL-8 and GRO. METHODS: Guinea pig (gp) cDNA libraries were screened for GRO and IL-1beta. A gp genomic library was screened with a gpGRO cDNA probe. Expression of gpIL-8, gpGRO, gpTNFalpha, and gpIL-1beta was investigated by Northern analysis and/or by in situ hybridization. RESULTS: Two gpGRO cDNAs, a 3.0-kb gpGRO genomic DNA, and a gpIL-1beta cDNA were cloned. gpGRO and gpIL-8 mRNA were detected in different tissues including lungs 1 h after intraperitoneal injection of lipopolysaccharide (LPS) into guinea pigs. gpGRO, gpIL-8, gpTNFalpha, and gpIL-1beta expression peaked at 3 h in the lungs. Both gpGRO and gpIL-8 mRNA were detected in the cells in alveolar spaces and bronchial epithelial cells. However, gpGRO mRNA, but not gpIL-8, was also expressed in endothelial cells and vascular smooth muscle cells. CONCLUSIONS: gpGRO and gpIL-8 mRNA rapidly accumulated in the lungs of guinea pigs after LPS injection. Expression of gpIL-8 and gpGRO mRNA appeared to be independent from TNFalpha- or IL-1beta-stimulation in this model. A high level expression of gpGRO in vascular cells suggest an important role of GRO in the sequestration of neutrophils and multi-organ injuries induced by LPS. The guinea pig will provide an excellent model to study the roles of IL-8 and GRO, important inflammatory mediators in the human.

Amino Acid Sequence↗
PubMed1997

Sequence and chromosomal localization of the mouse brevican gene.

Brevican is a brain-specific proteoglycan belonging to the aggrecan family. Phage clones containing the complete mouse brevican open reading frame of 2649 bp and the complete 3'-untranslated region of 341 bp were isolated from a mouse brain cDNA library, and cosmid clones containing the mouse brevican gene were isolated from a genomic library using a PCR-generated DNA fragment as probe. The obtained genomic sequence of 13,700 nucleotides revealed that the murine gene has a size of approximately 13 kb and contains the sequence of the mRNA for the secreted brevican isoform on 14 exons. The exon-intron structure reflected the structural organization of the multidomain protein brevican. No consensus TATA sequence was found upstream of the first exon, and RNase protection experiments revealed multiple transcriptional start sites for the brevican gene. The first part of the sequence of intron 8 corresponded to an alternative brevican cDNA, coding for a GPI-linked isoform. Single strand conformation polymorphism analysis mapped the brevican gene (Bcan) to chromosome 3 between the microsatellite markers D3Mit22 and D3Mit11.

Amino Acid Sequence↗
PubMed1988

Genes and pseudogenes for calmodulin in the spontaneously hypertensive rat.

The structural organization of calmodulin genes in the spontaneously hypertensive rat (SHR) was extensively studied to search for alterations in calmodulin. We constructed genomic libraries of SHR and cloned all calmodulin-related genes in the genome. We also cloned and sequenced calmodulin complementary (c)DNA from a rat brain (Sprague-Dawley) cDNA library. We cloned three distinct calmodulin genes, naming them CaM I, II and III. Three distinct cDNA clones corresponding to these genes (pRCM1, pRCM3 and pRCM4) were also cloned. These SHR calmodulin genes all encoded normal calmodulin, and no alteration was found. Four processed pseudogenes, lambda SC9 for CaM I gene and lambda SC8, lambda SC19 and lambda SC27 genes for CaM II genes were also cloned and analysed.

Animals↗
PubMedDate not supplied

[Isolation of nucleotide sequences, possessing enhancer functions from the Saccharomyces cerevisiae genetic library].

We propose a method for isolation of enhancer-like sequences from a yeast genomic library. The method was used to identify DNA inserts capable of increasing bacterial bla gene expression when located downstream from the transcription initiation site. Plasmids carrying different fragments of one such insert were used to localize the enhancer-like function on a 1.2 kb fragment.

Bacteriophage lambda↗
PubMed1995

The mouse angiogenin gene family: structures of an angiogenin-related protein gene and two pseudogenes.

Angiogenin, a homologue of pancreatic ribonuclease, is a potent inducer of blood vessel formation. As an initial step toward investigating the in vivo functional role of this protein via gene disruption, we undertook the isolation of the angiogenin gene (Ang) from the 129 strain mouse, which will be used for generating targeting constructs. Unexpectedly, screening of a genomic library with an Ang gene probe obtained previously from the BALB/c strain yielded two new genes closely similar to Ang rather than Ang itself. One of these encodes a protein with 78% sequence identity to angiogenin and is designated "Angrp" for "angiogenin-related protein." The ribonucleolytic active site of angiogenin, which is critical for angiogenic activity, is completely conserved in Angrp, whereas a second essential site, thought to bind cellular receptors, is considerably different. Thus, the Angrp product may have a function distinct from that of angiogenin. The second gene obtained by library screening is a pseudogene, designated "Ang-ps1," that contains a frameshift mutation in the early part of the coding region. Although the Ang gene was not isolated from this library, it was possible to amplify this gene from 129 mouse genomic DNA by the polymerase chain reaction (PCR). Sequence analysis showed that the 129 strain Ang gene is identical to the BALB/c gene throughout the coding region. PCR cloning also yielded a second Ang-like pseudogene, designated "Ang-ps2." Southern blotting of genomic DNA confirmed the presence of Ang, Angrp, and at least one of the pseudogenes in an individual mouse and suggested that the mouse Ang gene family may contain more than the four members identified here.

Amino Acid Sequence↗
PubMed1994

Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation.

Yeast artificial chromosomes (YACs) enable the cloning and analysis of large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli. However, the construction of genome libraries in YAC vectors is beset by a number of technical problems, not least of which is the creation of cloned fragments which are not true representatives of the donor genome. These artefactual clones arise mainly due to intra-fragment rearrangements or inter-fragment chimaera formation, both phenomena resulting from the activity of the host yeast's mitotic recombination system. We demonstrate that this system is significantly stimulated by the spheroplasting step of the standard YAC transformation system. In contrast, the transformation of intact yeast cells by either the lithium method or a new lithium-free protocol is much less recombinagenic. It is not possible to introduce high molecular weight YACs into yeast using the lithium protocol, but we find that such molecules may be introduced into pde2-mutants using the lithium-free approach. Since intact cells are transformed by this method, automation of post-transformation steps in the construction of YAC libraries is facilitated. Moreover, the frequency of cotransformation (and, therefore, chimera formation) is significantly reduced. However, these advantages do incur a penalty. Yields of YAC transformants by this simplified intact cell approach are reduced some 25- to 30-fold compared to those obtained by the spheroplast transformation route. Nevertheless, the considerable advantages of the new system recommend it for a number of applications.

Chromosomes, Artificial, Yeast↗
PubMed2001

Identification of a new human CYP2A gene fragment with close linkage to CYP2GP1.

Human genomic libraries were screened to identify CYP2G-related cytochrome P450 genes. A genomic fragment comprising exons 7 through 9 of CYP2GP1 and exons 6 through 9 of a previously unidentified CYP2A gene, designated CYP2A7P2, was isolated from an EMBL3 library; the two genes were arranged in outward opposite directions with about 8 kbp of intervening sequence. The same structure was also detected in a bacteriophage P1 clone, which contained a full-length CYP2GP1 gene, exons 6 through 9 of CYP2A7P2, and the CYP2B7 gene. However, additional CYP2A-related exons as well as other CYP2A genes, CYP2A7P1, CYP2B6, CYP2F1, and CYP2GP2 were not detected. These results indicate that CYP2A7P2 is located near CYP2B7 in the middle of the CYP2A-2B-2F gene cluster on chromosome 19. Furthermore, an analysis of CYP2A sequence alignment suggests that CYP2A7P2 may be derived from the same ancestral gene that gave rise to CYP2A7P1, which was corrupted by a large insertion at intron 5.

Adult↗
PubMed1994

Molecular cloning and structural analysis of the human gene encoding cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) appears to play a role in the vertebrate visual process as a substrate-routing protein, influencing the enzymatic partitioning of 11-cis-retinol at a key branch point in the visual cycle. Genomic clones spanning 29 kilobases and encompassing the human CRALBP gene have been isolated by screening two human genomic libraries and from polymerase chain reaction amplification of human leukocyte DNA. The sequence of 13,647 contiguous nucleotides has been determined, including 3130 and 516 bases from the 5'- and 3'-flanking regions, respectively. The human CRALBP gene exists as a single copy in the genome based on Southern analyses and localization to a single site on human chromosome 15 (Sparkes, R. S., Heinzmann, C., Goldflam, S., Kojis, T., Saari, J. C., Mohandes, T., Klisak, I., Bateman, J. B., and Crabb, J. W. (1992) Genomics 12, 58-62). The gene is composed of eight exons and seven introns with average lengths of 198 base pairs and 1.2 kilobases, respectively, and which exhibit conventional vertebrate splicing. Alu repetitive sequences exist in introns 4 and 5 as well as in the 5'- and 3'-flanking regions of the gene. RNase protection and primer extension analyses indicate that the human CRALBP gene transcription start site is 922 bases upstream of the initiation codon. The first exon is entirely untranslated and both exon 2 and exon 8 contain untranslated regions. The proximal 5'-flanking region lacks GC boxes and consensus TATA and CCAAT boxes at the usual positions. The 3'-untranslated region of CRALBP exon 8 is essentially identical to a partial cDNA clone reportedly isolated from a human hippocampus cDNA library, suggesting that the protein may be expressed in a wider spectrum of tissues than previously recognized. The human CRALBP genomic clones and structure provide valuable tools for studying the physiological role of the protein in vision and visual disorders.

Animals↗
PubMed1991

DNA probes to identify members of the Anopheles farauti complex.

DNA probes have been constructed to distinguish between the members of the Anopheles farauti complex of mosquitoes known as species numbers 1, 2 and 3. Partial genomic libraries of the three known species were exposed to labelled total genomic DNA from each species. Colonies showing differential hybridization were selected for further testing. These probes were found which allow identification of the three known species: probe pAf1 (160 bp fragment) hybridizes to DNA from An. farauti nos. 1 and 2; probe pAf2 (95 bp fragment) hybridizes to DNA from An. farauti no. 2 only; and probe pAf3 (1.3 kb fragment) hybridizes strongly to DNA from An. farauti no. 3, less to no. 1 and faintly to no. 2. Increasing the stringency of hybridization reduced the cross-hybridization of probes pAf1 and pAf3. Only radioactively labelled probes were tested. Males and females and individuals from diverse habitats and localities showed the same species/probe hybridization characteristics. This technique allows faster identification of the sibling species than previous methods, and has the added advantage that it allows air-dried and alcohol stored specimens to be identified.

Animals↗
PubMed1992

Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA.

Five monoclonal antibodies raised against rat PCNA cross-reacted with a similar protein in the fission yeast Schizosaccharomyces pombe. One of these was used to screen an S.pombe cDNA expression library. An incomplete cDNA was isolated and used to screen a genomic library, identifying a single gene, designated pcn1+ (proliferating cell nuclear antigen). The gene encodes a protein of 260 amino acids, with a deduced sequence 52% identical to human and rat PCNAs, which are 98.5% identical to each other. The budding yeast PCNA homologue POL30 is only 35% identical to the human and rat proteins. Pcn1 has a region near the C-terminus of particularly high homology to higher eukaryotic PCNA proteins. pcn1+ is essential for viability and delta pcn1 cells undergo aberrant DNA replication before cell cycle arrest. Overproduction of the protein leads to cell cycle delay in G2. Disruption of pcn1+ is complemented by the human PCNA gene, demonstrating that these genes are functional homologues.

Amino Acid Sequence↗
PubMed1998

Analysis of distribution in the human, pig, and rat genomes points toward a general subtelomeric origin of minisatellite structures.

We have developed approaches for the cloning of minisatellites from total genomic libraries and applied these approaches to the human, rat, and pig genomes. The chromosomal distribution of minisatellites in the three genomes is strikingly different, with clustering at chromosome ends in human, a seemingly almost even distribution in rat, and an intermediate situation in pig. A closer analysis, however, reveals that interstitial sites in pig and rat often correspond to terminal cytogenetic bands in human. This observation suggests that minisatellites are created toward chromosome ends and their internalization represents secondary events resulting from rearrangements involving chromosome ends.

Animals↗
PubMed1989

Molecular cloning, nucleotide sequence, and abscisic acid induction of a suberization-associated highly anionic peroxidase.

A highly anionic peroxidase induced in suberizing cells was suggested to be the key enzyme involved in polymerization of phenolic monomers to generate the aromatic matrix of suberin. The enzyme encoded by a potato cDNA was found to be highly homologous to the anionic peroxidase induced in suberizing tomato fruit. A tomato genomic library was screened using the potato anionic peroxidase cDNA and one genomic clone was isolated that contained two tandemly oriented anionic peroxidase genes. These genes were sequenced and were 96% and 87% identical to the mRNA for potato anionic peroxidase. Both genes consist of three exons with the relative positions of their two introns being conserved between the two genes. Primer extension analysis showed that only one of the genes is expressed in the periderm of 3 day wound-healed tomato fruits. Southern blot analyses suggested that there are two copies each of the two highly homologous genes per haploid genome in both potato and tomato. Abscisic acid (ABA) induced the accumulation of the anionic peroxidase transcripts in potato and tomato callus tissues. Northern blots showed that peroxidase mRNA was detectable at 2 days and was maximal at 8 days after transfer of potato callus to solid agar media containing 10(-4) M ABA. The transcripts induced by ABA in both potato and tomato callus were identical in size to those induced in wound-healing potato tuber and tomato fruit. The anionic peroxidase peptide was detected in extracts of potato callus grown on the ABA-containing media by western blot analysis. The results support the suggestion that stimulation of suberization by ABA involves the induction of the highly anionic peroxidase.

Abscisic Acid↗
PubMed2002

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3.

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM +/- 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.

Animals↗