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PubMed1998

The mouse Y chromosome: enrichment, sizing, and cloning by bivariate flow cytometry.

In this report, we demonstrate the utility of interleukin-2 (IL-2) stimulation of spleen cell cultures and bivariate flow cytometry in the analysis and purification of the C57BL/6J mouse Y Chromosome. We determined that the DNA content of the C57BL/6J Y Chromosome is approximately 94.7 Mb, making it similar in size to human Chromosome 16 and significantly larger than previous estimates. In addition, we describe the bulk isolation of mouse Y Chromosomes and demonstrate enrichment of the isolated material using a fluorescence in situ hybridization strategy. We detail the construction of two small insert Y Chromosome-specific libraries, ideal for sampling Y Chromosome sequences. From these libraries 1566 clones were analyzed. We provide a detailed characterization of 103 clones, generating nearly 50 kb of sequence. For 30 of these clones, we identify regions of homology to known Y chromosomal sequences, confirming the enrichment of the sorted DNA. From the remaining characterized clones, we describe the development of 15 male-specific PCR assays and 19 male-female PCR assays potentially originating from the pseudoautosomal region or other areas of X-Y or autosome-Y homology.

Animals↗

PubMed2000

Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene.

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5' flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon-intron boundaries display the canonical GT/AG sequences. Using promoter-luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.

ATP-Binding Cassette Transporters↗

PubMed1978

The isolation of structural genes from libraries of eucaryotic DNA.

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of beta-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.

Base Sequence↗

PubMed1990

Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2000

The prostate expression database (PEDB): status and enhancements in 2000.

The Prostate Expression Database (PEDB) is an online resource designed to access and analyze gene expression information derived from the human prostate. PEDB archives >55 000 expressed sequence tags (ESTs) from 43 cDNA libraries in a curated relational database that provides detailed library information including tissue source, library construction methods, sequence diversity and sequence abundance. The differential expression of each EST species can be viewed across all libraries using a Virtual Expression Analysis Tool (VEAT), a graphical user interface written in Java for intra- and inter-library species comparisons. Recent enhancements to PEDB include: (i) the functional categorization of annotated EST assemblies using a classification scheme developed at The Institute for Genome Research; (ii) catalogs of expressed genes in specific prostate tissue sources designated as transcriptomes; and (iii) the addition of prostate proteome information derived from two-dimensional electrophoreses and mass spectrometry of prostate cancer cell lines. PEDB may be accessed via the WWW at http://www.mbt.washington.edu/PEDB/

Databases, Factual↗

PubMed2003

Identification of a second member of the ponticulin gene family and its differential expression pattern.

We have identified a homologue (ponB) of the ponticulin gene (ponA), an F-actin binding protein, in the expressed sequence tag library generated to mRNA isolated from fusion-competent cells of Dictyostelium discoideum. PonB is predicted to have many of the same characteristics as ponticulin. Both proteins are predicted to possess a cleaved signal peptide, a glycosyl anchor, an amphipathic beta-strand structure and six conserved cysteines. Because of the sequence similarity and predicted conserved structures, this gene constitutes the second member of a ponticulin gene family. Unlike ponticulin, ponB is not expressed in axenically grown cells or during the asexual reproductive phase of D. discoideum. PonB is expressed by cells grown on bacterial lawns and by cells induced to be fusion-competent, i.e., gametes. The expression of ponB correlates with the appearance of a new F-actin binding activity in cell lysates of bacterially grown ponA(-) cells. By immunofluorescence microscopy, ponB appears to be localized to vesicles and to the plasma membrane of bacterially grown cells. Because ponticulin is the major high-affinity link between the plasma membrane and the cytoskeleton, the ponticulin gene family is likely to be part of the redundant system of proteins involved in connecting the cytoskeleton to the plasma membrane.

Amino Acid Sequence↗

PubMed1992

[The construction of genomic library of Alcaligenes faecalis and cloning of nifH gene sequence homologous to Klebsiella pneumoniae].

A genomic library of Alcaligenes faecalis A-15Hl which possesses rather high nitrogenase activity has been constructed. The total DNA of A. faecalis A-15 Hl was partially digested with Sau3AI. 13-20 kb of fragments recovered from agarose gel were cloned in bacteriophage EM-BL4 vector. A total number of 1.2 x 10(6) of recombinants was obtained. It is much beyond the desired capacity of a library. By using nifH gene of K. pneumoniae from plasmid pGB1 as probe, we have successfully screened the clone containing its homologous sequence. The recombinant bacteriophage DNA was digested with EcoRI. A 3.5 kb of hybridizing band appeared after southern blotting and then was cloned in pUC19 DNA. The result of southern blotting indicated that the recombinant plasmid clone contained nifH gene sequence of A. faecalis. This clone was named as pAFH.

Alcaligenes↗

PubMed2005

Potato expressed sequence tag generation and analysis using standard and unique cDNA libraries.

To help develop an understanding of the genes that govern the developmental characteristics of the potato (Solanum tuberosum), as well as the genes associated with responses to specified pathogens and storage conditions, The Canadian Potato Genome Project (CPGP) carried out 5' end sequencing of regular, normalized and full-length cDNA libraries of the Shepody potato cultivar, generating over 66,600 expressed sequence tags (ESTs). Libraries sequenced represented tuber developmental stages, pathogen-challenged tubers, as well as leaf, floral developmental stages, suspension cultured cells and roots. All libraries analysed to date have contributed unique sequences, with the normalized libraries high on the list. In addition, a low molecular weight library has enhanced the 3' ends of our sequence assemblies. Using the combined assembly dataset, unique tuber developmental, cold storage and pathogen-challenged sequences have been identified. A comparison of the ESTs specific to the pathogen-challenged tuber and foliar libraries revealed minimal overlap between these libraries. Mixed assemblies using over 189,000 potato EST sequences from CPGP and The Institute for Genomics Research (TIGR) has revealed common sequences, as well as CPGP- and TIGR-unique sequences.

DNA, Complementary↗

PubMed2001

Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.

Animals↗

PubMed1996

Regional assignment and tissue expression of twenty-three expressed sequence tags (ESTs) from human chromosome 5.

Regional localization and expression patterns are reported for 19 expressed sequence tags (ESTs) from human chromosome 5, two of which were derived from the same transcript. Two of the ESTs correspond to genes not previously characterized in humans: a stress-activated protein kinase and nicotinamide nucleotide transhydrogenase. Expression was determined by three methods: Northern blots, PCR from tissue-specific cDNA libraries, and sequence sampling from EST sequencing projects. Six of the ESTs show no expression, and EST01986 appears to be expressed predominantly in the brain by all methods tested.

Chromosome Mapping↗

PubMed2003

cDNAs generated from individual epidermal cells reveal that differential gene expression predicting subsequent resistance or susceptibility to rust fungal infection occurs prior to the fungus entering the cell lumen.

As the cowpea rust fungus penetrates the wall of a cowpea epidermal cell, resistant and susceptible plants exhibit different ultrastructural and cytochemical changes within the epidermal protoplast. To examine plant gene expression at this stage of infection, cytoplasm was extracted from individual inoculated or uninoculated epidermal cells before the fungal penetration peg reached the cell lumen. Initial differential colony hybridization screening of an expressed sequence tag library constructed from globally amplified cDNAs generated from the inoculated resistant cells resulted in 80 clones (out of 835) with a differential hybridization pattern. Further slot-blot screening and screening of the amplified cDNAs generated from inoculated or uninoculated, resistant or susceptible cells revealed 28 separate genes, mostly with no matching sequences in the databases, that were up-regulated in response to the growth of the fungus through the wall of resistant or susceptible cells. Five genes, including those coding for beta- and alpha-tubulin, were found to be down-regulated specifically in inoculated, susceptible cells, and five were specifically up-regulated in inoculated, resistant cells, including a PR-10 homolog and a phenylalanine ammonia-lyase gene. Probing the amplified cDNAs from each cell type for the expression of cell death-related genes revealed that an LLS1 homolog (vuLLS1), cloned from cowpea, was up-regulated by infection in both resistant and susceptible cells and that a homolog of HSR203J was differentially up-regulated in resistant cells. These data show that changes in gene expression predicting the subsequent expression of susceptibility or hypersensitive resistance to fungal infection occur prior to the fungus entering the cell lumen.

Amino Acid Sequence↗

PubMed2001

Libraries of hybrid proteins from distantly related sequences.

We introduce a method for sequence homology-independent protein recombination (SHIPREC) that can create libraries of single-crossover hybrids of unrelated or distantly related proteins. The method maintains the proper sequence alignment between the parents and introduces crossovers mainly at structurally related sites distributed over the aligned sequences. We used SHIPREC to create a library of interspecies hybrids of a membrane-associated human cytochrome P450 (1A2) and the heme domain of a soluble bacterial P450 (BM3). By fusing the hybrid gene library to the gene for chloramphenicol acetyl transferase (CAT), we were able to select for soluble and properly folded protein variants. Screening for 1A2 activity (deethylation of 7-ethoxyresorufin) identified two functional P450 hybrids that were more soluble in the bacterial cytoplasm than the wild-type 1A2 enzyme.

Amino Acid Sequence↗

PubMed1992

Genomic DNA sequence and organization of a TL-like gene in the grc-G/C region of the rat.

Genes in the grc-G/C region, which is linked to the rat major histocompatibility complex, influence the control of growth, development, and susceptibility to chemical carcinogens. As an initial approach to analyzing the structure and organization of these genes, a class I hybridizing fragment designated RT(5.8) was isolated from an R21 genomic DNA library and sequenced from overlapping restriction enzyme fragments. The RT(5.8) clone has 5788 base pairs and contains the eight exons characteristic of a class I gene. There are CAAT and TATA boxes upstream of the signal peptide, and the recognition sequence that precedes the site of polyadenylation is located downstream from the third cytoplasmic domain. Comparison of the RT(5.8) gene with representative class I genes from the rat and other species shows that the nucleotide sequences of RT(5.8) have a high level of similarity to those of TL region genes of several strains of mice. The peptide sequence deduced from the RT(5.8) clone is distinct from all previously published class I gene sequences, and at many positions there are amino acid residues that are unique to the RT(5.8) sequence. Probes have been isolated from the third exon and from the 5' and 3' flanking regions of the RT(5.8) clone, and Southern blot analysis with genomic DNA of various rat strains shows that these probes are specific for the RT(5.8) fragment. Northern blot analysis shows that the gene is transcribed in the thymus but not in the liver or spleen. The RT(5.8) sequence is more similar to some mouse TL genes (especially in the alpha 2 and cytoplasmic domains and in the 5' and 3' untranslated regions) than it is to other rat class I genes. Hence, TL-like genes are not restricted to the mouse.

Amino Acid Sequence↗

PubMed2004

Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov.

The sequences of five conserved genes, in addition to the 16S rRNA gene, were investigated in 30 members of the Geobacteraceae fam. nov. All members of the Geobacteraceae examined contained nifD, suggesting that they are capable of nitrogen fixation, which may explain their ability to compete effectively in nitrogen-poor subsurface environments undergoing remediation for petroleum or metal contamination. The phylogenies predicted from rpoB, gyrB, fusA, recA and nifD were generally in agreement with the phylogeny predicted from 16S rRNA gene sequences. Furthermore, phylogenetic analysis of concatemers constructed from all five protein-coding genes corresponded closely with the 16S rRNA gene-based phylogeny. This study demonstrated that the Geobacteraceae is a phylogenetically coherent family within the delta-subclass of the Proteobacteria that is composed of three distinct phylogenetic clusters: Geobacter, Desulfuromonas and Desulfuromusa. The sequence data provided here will make it possible to discriminate better between physiologically distinct members of the Geobacteraceae, such as Pelobacter propionicus and Geobacter species, in geobacteraceae-dominated microbial communities and greatly expands the potential to identify geobacteraceae sequences in libraries of environmental genomic DNA.

Bacterial Proteins↗

PubMed1998

Cloning of two Bombyx homologues of the Drosophila rosy gene and their relationship to larval translucent skin colour mutants.

To clone the Bombyx xanthine dehydrogenase (XDH) gene as a dominant marker for silkworm transgenesis, we performed nested reverse transcriptase-polymerase chain reaction (RT-PCR) using embryonic mRNA and primers designed from the conserved region of Drosophila and rat XDH genes. Sequencing of amplified 180 bp fragments showed that two different sequences were present in the fragments. Since both possessed striking similarity to XDH genes of other organisms, we considered these to be portions of silkworm XDH genes and designated them BmXDH1 and BmXDH2. Subsequently we cloned separately the entire region of the two cDNAs by PCR using phage DNA of an embryonic cDNA library and sequenced them. The two cDNAs were around 4 kb in size and possessed complete open reading frames. The deduced amino acid sequences of the two BmXDHs were very similar to each other and to those of other organisms. The expression pattern of wild-type larvae basically followed the tissue specificity of the enzyme and no significant difference was observed between the two XDH genes. The expression of both genes was detected in the XDH-deficient mutants, oq and og, but non-synonymous substitutions were specifically detected in the BmXDH1 of the oq mutant. In addition, a length polymorphism of the second intron of the BmXDH1 co-segregated with the oq translucent phenotype, suggesting that deficiency in BmXDH1 is the cause of the oq translucent phenotype.

Amino Acid Sequence↗

PubMed2004

Regulation of fumonisin biosynthesis in Fusarium verticillioides by a zinc binuclear cluster-type gene, ZFR1.

Fusarium verticillioides, a pathogen of maize, produces a class of mycotoxins called fumonisins in infected kernels. In this study, a candidate regulatory gene, ZFR1, was identified in an expressed sequence tag library enriched for transcripts expressed by F. verticillioides during fumonisin B(1) (FB(1)) biosynthesis. ZFR1 deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain. ZFR1 encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression of ZFR1 in colonized germ and degermed kernel tissues correlated with FB(1) levels. Overexpression of ZFR1 in zfr1 mutants restored FB(1) production to wild-type levels; however, FB(1) was not restored in an fcc1 (Fusarium C-type cyclin) mutant by overexpression of ZFR1. The results of this study indicate that ZFR1 is a positive regulator of FB(1) biosynthesis in F. verticillioides and suggest that FCC1 is required for ZFR1 function.

DNA-Binding Proteins↗

PubMed1994

Cone arrestin identified by targeting expression of a functional family.

High acuity, color vision in humans is initiated in cones by a receptor/G-protein-linked phototransduction cascade. G-protein-linked receptors are rapidly deactivated by receptor phosphorylation and the binding of a member of the "arrestin" family of proteins. Divergence in amino acid sequence at the carboxyl terminus of S-antigen (rod photoreceptor arrestin) and beta-arrestin 1 and beta-arrestin 2 (beta-adrenergic receptors) suggests that receptor specificity may be coded within this region. An anchor primer strategy was utilized to screen for carboxyl-terminal variability with a rat pineal library, identifying three known arrestins plus three unknowns (C-arrestin, D-arrestin, and E-arrestin). cDNA was prepared by reverse transcription of mRNA from 12 rat tissues, and members of the arrestin family were amplified by polymerase chain reaction using the anchor primer and customized 3'-primers for the individual arrestins. The amplified arrestins were then digested by selected restriction endonucleases, producing a pattern of characteristic cleavage products for each arrestin isoform. The procedural combination of epitope domain anchor and tissue screening demonstrated that C-arrestin is enriched in the retina. C-arrestin was isolated from a lambda MAX1 human retinal cDNA library and sequenced, revealing significant identity to known arrestins and divergence within the 3'-region. The mRNA for C-arrestin was visualized by in situ hybridization, localizing in the retina with cone photoreceptors and in the pineal to a subpopulation of pinealocytes. A gene for human C-arrestin was mapped to the X chromosome, making C-arrestin a candidate for several inherited X-linked retinopathies. The localization of C-arrestin to cone photoreceptors suggests that it, like others in the arrestin family, may bind to phosphorylated receptors and participate in deactivation of the phototransduction cascade.

Amino Acid Sequence↗

PubMed2005

Amsacta moorei Entomopoxvirus inhibitor of apoptosis suppresses cell death by binding Grim and Hid.

Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.

Animals↗