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At least 127 records · Page 7Linked to original sources
PubMed2006

A library of gene expression signatures to illuminate normal and pathological lymphoid biology.

Genomics has provided a lever to pry open lymphoid cells and examine their regulatory biology. The large body of available gene expression data has also allowed us to define the of coordinately expressed genes, termed gene expression signatures, which characterize the states of cellular physiology that reflect cellular differentiation, activation of signaling pathways, and the action of transcription factors. Gene expression signatures that reflect the action of individual transcription factors can be defined by perturbing transcription factor function using RNA interference (RNAi), small-molecule inhibition, and dominant-negative approaches. We have used this methodology to define gene expression signatures of various transcription factors controlling B-cell differentiation and activation, including BCL-6, B lymphocyte-induced maturation protein-1 (Blimp-1), X-box binding protein-1 (XBP1), nuclear factor-kappaB (NF-kappaB), and c-myc. We have also curated a wide variety of gene expression signatures from the literature and assembled these into a signature database. Statistical methods can define whether any signature in this database is differentially expressed in independent biological samples, an approach we have used to gain mechanistic insights into the origin and clinical behavior of B-cell lymphomas. We also discuss the use of genomic-scale RNAi libraries to identify genes and pathways that may serve as therapeutic targets in B-cell malignancies.

Gene Expression↗
PubMed2004

Organization of genes required for gellan polysaccharide biosynthesis in Sphingomonas elodea ATCC 31461.

Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [-->4)-alpha- l-rhamnose-(1-->3)-beta- d-glucose-(1-->4)-beta- d-glucuronic acid-(1-->4)-beta- d-glucose-(1-->]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP- l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes ( gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.

Biotechnology↗
PubMed2006

An integrated approach to functional genomics: construction of a novel reporter gene fusion library for Sinorhizobium meliloti.

As a means of investigating gene function, we developed a robust transcription fusion reporter vector to measure gene expression in bacteria. The vector, pTH1522, was used to construct a random insert library for the Sinorhizobium meliloti genome. pTH1522 replicates in Escherichia coli and can be transferred to, but cannot replicate in, S. meliloti. Homologous recombination of the DNA fragments cloned in pTH1522 into the S. meliloti genome generates transcriptional fusions to either the reporter genes gfp(+) and lacZ or gusA and rfp, depending on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298 clones were identified by DNA sequence analysis, and the plasmid clones were recombined into S. meliloti. Reporter enzyme activities following growth of these recombinants in complex medium (LBmc) and in minimal medium with glucose or succinate as the sole carbon source allowed the identification of genes highly expressed under one or more growth condition and those expressed at very low to background levels. In addition to generating reporter gene fusions, the vector allows Flp recombinase-directed deletion formation and gene disruption, depending on the nature of the cloned fragment. We report the identification of genes essential for growth on complex medium as deduced from an inability to recover recombinants from pTH1522 clones that carried fragments internal to gene or operon transcripts. A database containing all the gene expression activities together with a web interface showing the precise locations of reporter fusion junctions has been constructed (www.sinorhizobium.org).

Bacterial Proteins↗
PubMed2002

High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library.

BACKGROUND: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant. RESULTS: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics. CONCLUSIONS: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

Bryopsida↗
PubMed1994

Screening of a mouse/human Y-chromosomal cosmid library for gene candidates and markers by using (short) oligonucleotide probes.

A comprehensive approach is described for the identification of sequences of interest from a human Y chromosomal cosmid library via (short) consensus oligonucleotide probes. It involves the ordering of cosmid clones grown in microtiter plates onto small filter membranes by a robot workstation. A high number of the clones are characterized by their repetitive sequence content, either by ubiquitously interspersed simple tandem blocks or by Y-specific elements. The Y chromosomal repeat (DYZ2) appears underrepresented in the library. In contrast many novel microsatellite marker systems can now be developed for the Y chromosome on the basis of the simple repeat blocks described here. Though novel genes were not yet delineated so far, a number of candidate sequences with high coding potential and other interesting characteristics are described.

Animals↗
PubMed1997

Cell display library for gene cloning of variable regions of human antibodies to hepatitis B surface antigen.

A novel cell display system was developed for cloning the variable region (V) genes of antigen-specific human antibodies. The system is based on an antibody library displayed on the surface of COS cells, using a plasmid vector designed to direct expression of membrane-bound antibodies. COS cells expressing antigen-specific antibodies were separated using a flow cytometer for their binding to a fluorescent dye-labeled antigen. To test the performance of this system. We cloned V genes of 4 antibodies directed against hepatitis B surface antigen (HBsAg) from a library prepared from peripheral blood lymphocytes of a vaccinated donor. These membrane-bound anti-HBsAg antibodies were easily converted to soluble forms, all of which showed a size similar to human serum IgG in SDS-PAGE and the same specific binding to HBsAg as membrane-bound forms in ELISA. All VH and VK gene segments of the 4 clones isolated in this study belonged to VHIII and VKI subgroups, respectively. These findings demonstrate the potential and selection capabilities of our cell display system for cloning the V genes of antigen-specific human antibodies.

Amino Acid Sequence↗
PubMed2000

Full-length single-gene cDNA libraries: applications in splice variant analysis.

Alternative splicing of pre-mRNA may generate many distinct proteins from a single gene: regulation of alternative exon selection constitutes control of molecular structure downstream of transcription. Identifying natural splice variants among hundreds or thousands of theoretical alternatives, and examining the regulation of exon selection at multiple sites, may require screening many full-length cDNAs. We describe methods for preparing full-length cDNA libraries comprising the splice variants from single genes. The methods employ robust long distance reverse transcription, gene-specific second strand synthesis, long PCR, and cloning: with these methods cDNAs coding full-length open reading frames were prepared for 21 ion channels (1.2-15 kb). Exon combinations in isolated clones are determined by multiplex PCR. Approximately 85% of the clones contain full-length inserts. Screening can detect even rare variants (0.1%) in linear proportion to their abundance in initial mRNA pools. Tissue-specific expression patterns are reproducible. We describe methods for quantifying and minimizing artifactual exon recombination by template switching. These methods can be used to generate thousands of full-length clones of even large transcripts (>8 kb) for the systematic identification of splice variants and the analysis of regulation of alternative exon selection.

Alternative Splicing↗
PubMed1992

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

An enzyme with alpha-galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of alpha-galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a lambda EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa alpha-galactosidase is absent from a strain with a disruption of the aglA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa alpha-galactosidase A represents a minor extracellular alpha-galactosidase activity in A. niger.

Amino Acid Sequence↗
PubMed1995

Clustering of the trp genes in Burkholderia (formerly Pseudomonas) cepacia.

Chromosomal location of trp genes of a strain Burkholderia cepacia (formerly Pseudomonas cepacia) has been determined by transduction using a generalized transducing phage CP75 and by molecular analysis for a cosmid plasmid clone with trp genes isolated from a genomic gene library of the strain. The trp genes were classified into three linkage groups and they all were closely linked on a short chromosomal region probably in the order (trpA, trpB, trpF)-(trpC, trpD)-trpE.

Bacteria↗
PubMed2005

Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis.

Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.

Cartilage, Articular↗
PubMed1997

Epitope mapping by phage display: random versus gene-fragment libraries.

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.

Amino Acid Sequence↗
PubMed1999

Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues.

A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.

Animals↗
PubMed2003

VH3 gene usage in neutralizing human antibodies specific for the Entamoeba histolytica Gal/GalNAc lectin heavy subunit.

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the V kappa 1 family, in which the closest V kappa germ line gene was 02/012 or L5, with the J kappa 1, J kappa 2, J kappa 4, or J kappa 5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

Acetylgalactosamine↗
PubMed1992

Molecular characterization and expression of the cell-associated glucosyltransferase gene from Streptococcus mutans.

A gene encoding cell-associated glucosyltransferase (CA-GTase) was cloned from Streptococcus mutans MT8148 into Escherichia coli DH5 alpha by using a low-copy-number plasmid, pMW119. After screening of a gene library with the oligonucleotide probe designed on the basis of a partial amino acid sequence of CA-GTase, a recombinant plasmid, pSK6, that had a 5.6 kb insert carrying the CA-GTase gene was selected. The gene product (recombinant CA-GTase) of pSK6 was expressed by using a lac promoter in pMW119. Western blotting revealed that rCA-GTase reacted with antibody to CA-GTase. rCA-GTase was found to synthesize water-insoluble glucans. Southern blotting indicated that the MT8148 chromosome contained another gene which was homologous to pSK6. A plasmid harboring this gene (pSK16) was also isolated from the gene library, the gene product of pSK16 exhibited GTase activity but ten times lower than that of pSK6.

Base Sequence↗
PubMed1982

Isolation of rat major histocompatibility genes expressed in pancreatic beta-cells.

The probable role of cell surface antigens in the development of juvenile diabetes necessitates a detailed study of the proteins expressed on the surface of pancreatic beta-cells. A gene library composed of genes expressed in beta-cells was constructed to provide a source of low abundance genes from those cells. Initial studies with this library have led to the isolation of three genes which share nucleotide and amino acid sequence homology with mouse and human class I major histocompatibility antigens.

Animals↗
PubMed1989

Cloning of a new bidirectionally selectable marker for Aspergillus strains.

Mutants that lack adenosine triphosphate sulfurylase (ATPsase; EC 2.7.7.4) are unable to use sulfate as sole source of sulfur and are also resistant to selenate. These mutants, denoted sC-, are readily obtained from any strain of Aspergillus niger or Aspergillus nidulans by the strong selection for selenate resistance. We have cloned the gene encoding ATPsase from A. nidulans by complementation of an sC mutant strain of A. nidulans with a gene library and show that plasmids containing this gene transform both A. niger and A. nidulans sC- strains, restoring their ability to grow on sulfate as sole sulfur source. The fact that strong selection for either sC+ or sC- can be applied provides a simple way of delivering genetically engineered constructs to any strain of A. niger including strains of industrial importance. In addition, this system is useful for gene replacements and other genomic DNA manipulations in Aspergillus species.

Aspergillus nidulans↗