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PubMed2001

Genotype-phenotype linkage for directed evolution and screening of combinatorial protein libraries.

The technologies for screening peptide and protein libraries for studies in the fields of directed protein evolution and functional genomics have advanced with astonishing speed. For screening of functional proteins, three technologies are required: (i) the construction of a gene library (genotype), (ii) the establishment of a linkage between each protein (phenotype) and its encoding gene (genotype), and (iii) the selection of desired proteins (phenotype) from the library. This review highlights the genotype-phenotype linkage technologies, which can be classified into three types; that is, cell-type linkage, virus-type linkage, and array-type linkage methods. These methods are summarized, and their advantages and disadvantages are discussed.

Animals↗
PubMed1986

Regulation of nitrogen metabolism in Azotobacter vinelandii: isolation of ntr and glnA genes and construction of ntr mutants.

The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric bacteria. The presence of a similar system in the non-enteric bacterium Azotobacter vinelandii is reported here. Genes analogous to ntrA and ntrC were isolated from an A. vinelandii gene library by complementation of Escherichia coli mutants. The gene encoding glutamine synthetase, glnA, was also isolated and found to be adjacent to ntrC but distant from ntrA, as it is in enteric organisms. The cloned Azotobacter genes also complemented Klebsiella pneumoniae mutants and hybridized to K. pneumoniae ntrA, ntrC and glnA gene probes. The role of ntrA and ntrC in A. vinelandii was established by using Tn5 insertions in the cloned genes to construct mutants by marker exchange. These mutants show that both ntrA and ntrC are required for the utilization of nitrate as a nitrogen source. However, ntrC is not required for nitrogen fixation by A. vinelandii, in contrast with K. pneumoniae where both ntrA and ntrC are essential.

Azotobacter↗
PubMed1988

Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans.

The isolation and characterization of the highly expressed glyceraldehyde-3-phosphate dehydrogenase (GPD)-coding gene (gpdA) of Aspergillus nidulans is described. The gene was isolated from an A. nidulans lambda gene library with a Saccharomyces cerevisiae GPD-coding gene as a probe. Unlike many other eukaryotes, A. nidulans contains only one GPD-coding gene. At the amino acid level, homology with other GPD enzymes is extensive. The A. nidulans gene contains seven introns, one of which is positioned in the 5'-untranslated part of the gene. The major transcription start point is found at 172 bp upstream from the start codon. Polyadenylation occurs at several sites about 200 bp downstream from the stop codon. Comparison of 5' and 3' flanking sequences with flanking sequences of other highly expressed (glycolytic) genes shows several regions of similar sequence.

Amino Acid Sequence↗
PubMed2001

Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin.

Identification of expanding roles for matrix metalloproteinases (MMPs) in complex regulatory processes of tissue remodelling has stimulated the search for genes encoding proteinases with unique functions, regulation and expression patterns. By using a novel cloning strategy, we identified three previously unknown human MMPs, i.e. MMP-21, MMP-26 and MMP-28, in comprehensive gene libraries. The present study is focused on the gene and the protein of a novel MMP, MMP-26. Our findings show that MMP-26 is specifically expressed in cancer cells of epithelial origin, including carcinomas of lung, prostate and breast. Several unique structural and regulatory features, including an unusual 'cysteine-switch' motif, discriminate broad-spectrum MMP-26 from most other MMPs. MMP-26 efficiently cleaves fibrinogen and extracellular matrix proteins, including fibronectin, vitronectin and denatured collagen. Protein sequence, minimal modular domain structure, exon-intron mapping and computer modelling demonstrate similarity between MMP-26 and MMP-7 (matrilysin). However, substrate specificity and transcriptional regulation, as well as the functional role of MMP-26 and MMP-7 in cancer, are likely to be distinct. Despite these differences, matrilysin-2 may be a suitable trivial name for MMP-26. Our observations suggest an important specific function for MMP-26 in tumour progression and angiogenesis, and confirm and extend the recent findings of other authors [Park, Ni, Gerkema, Liu, Belozerov and Sang (2000) J. Biol. Chem. 275, 20540--20544; Uría and López-Otín (2000) Cancer Res. 60, 4745--4751; de Coignac, Elson, Delneste, Magistrelli, Jeannin, Aubry, Berthier, Schmitt, Bonnefoy and Gauchat (2000) Eur. J. Biochem. 267, 3323--3329].

Amino Acid Sequence↗
PubMed2004

Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation.

We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.

Animals↗
PubMed2004

A group II intron-type open reading frame from the thermophile Bacillus (Geobacillus) stearothermophilus encodes a heat-stable reverse transcriptase.

The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75 degrees C.

Amino Acid Sequence↗
PubMed2001

Analysis of a complete library of putative drug transporter genes in Escherichia coli.

The complete sequencing of bacterial genomes has revealed a large number of drug transporter genes. In Escherichia coli, there are 37 open reading frames (ORFs) assumed to be drug transporter genes on the basis of sequence similarities, although the transport capabilities of most of them have not been established yet. We cloned all 37 putative drug transporter genes in E. coli and investigated their drug resistance phenotypes using an E. coli drug-sensitive mutant as a host. E. coli cells transformed with a plasmid carrying one of 20 ORFs, i.e., fsr, mdfA, yceE, yceL, bcr, emrKY, emrAB, emrD, yidY, yjiO, ydhE, acrAB, cusA (formerly ybdE), yegMNO, acrD, acrEF, yhiUV, emrE, ydgFE, and ybjYZ, exhibited increased resistance to some of the 26 representative antimicrobial agents and chemical compounds tested in this study. Of these 20 ORFs, cusA, yegMNO, ydgFE, yceE, yceL, yidY, and ybjYZ are novel drug resistance genes. The fsr, bcr, yjiO, ydhE, acrD, and yhiUV genes gave broader resistance spectra than previously reported.

ATP-Binding Cassette Transporters↗
PubMed1981

Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.

Animals↗
PubMed1999

Representative cDNA libraries and their utility in gene expression profiling.

An increasing interest in gene expression profiles in human diseases has led to the use of microdissected tumors and biopsies in gene discovery approaches. Since many of these clinical samples yield extremely small amounts of RNA, reproducible methods are needed to amplify this RNA while maintaining the original message profile. Using the SMART cDNA Synthesis Method, we show that high-, medium- and low-abundance transcripts can be amplified in a representative fashion and that the resulting cDNA can also be used as a complex probe to confirm gene expression differences identified by other techniques.

DNA, Complementary↗
PubMed1992

Expression of a partial synthetic human TNF cDNA in E. coli.

A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exon codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fermentator was used to produce rhTNF. About 20 g (wet weight) of bacterial pellet per liter medium and 10(6)-10(7) units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer. rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.

Base Sequence↗
PubMed1992

Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis.

Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG. The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting). One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M. tuberculosis gene library (R. A. Young, B. R. Bloom, C. M. Grosskinsky, J. Ivanji, D. Thomas, and R. W. Davis, Proc. Natl. Acad. Sci. USA 82:2583-2587, 1985). Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced. The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli. The entire M. tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones. A phage isolated from this screening was able to express the native protein in E. coli when introduced as a lysogen. A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast.

Amino Acid Sequence↗
PubMed2000

A gene-enriched BAC library for cloning large allele-specific fragments from maize: isolation of a 240-kb contig of the bronze region.

A generic bacterial artificial chromosome (BAC) library from a complex plant genome like maize may not be suitable for some types of genomic analysis, for example, for establishing correlations between the genetic and the physical organization of a given chromosome region. Previously, we carried out extensive genetic analysis of the bronze (Bz) region in Zea mays using a W22 inbred line carrying the Bz-McC allele; however, BAC libraries of that line are neither available nor under construction. Here, we report the isolation of large, adjacent BAC clones of this region from a partial BAC library of W22. We developed a BAC vector suitable for cloning NotI fragments and used it to clone size-fractionated genomic DNA that had been cut to completion with the methylation-sensitive, rare-cutting enzyme NotI. This strategy resulted in a very significant enrichment of large genic DNA. From a library of about 20,000 BACs, containing just two-thirds of a maize genome, we isolated 16 BAC clones of the 110-kb distal Bz fragment and 10 BAC clones of the 130-kb proximal Bz fragment. This recovery means that our strategy resulted in a 15- to 24-fold enrichment of specific sequences. The order of the BAC clones in the 240-kb contig, predetermined from an internal NotI site in the Bz-McC allele was confirmed by hybridization with sequences from sites previously mapped proximal and distal to Bz and by sequencing. To show the general utility of our approach and the value of our partial BAC library, we also isolated BAC clones of other sequences, such as tub4 and the complex R-r allele, contained in the same size fraction of DNA. This is the first report of the use of a BAC vector to clone allele-specific large DNA fragments from a plant with a large genome, circumventing the need to construct a complete BAC library.

Alleles↗
PubMed1990

Molecular cloning and characterization of retrovirus-like intracisternal type A particle genes (IAP) present in the Chinese hamster genome.

Endogenous retrovirus-like sequences that are homologous to the multigenic murine and Syrian hamster intracisternal type A particle (IAP) genes can be detected in very few copies in the Chinese hamster (CH) genome. They were cloned from a CHO gene library and two recombinants, harboring the major IAP-like DNA genes characterized by Southern blot hybridization after DNA digestion with several restriction enzymes. The IAP DNA inserts of the two clones analyzed were 4.70 and 8.04 kb respectively, allowing us to construct a physical map of our Chinese hamster clone that represents an almost complete IAP element.

Animals↗
PubMed2001

A set of 840 mouse oocyte genes with well-matched human homologues.

GenBank contains 14 477 expressed sequence tags (EST) derived from mouse oocyte cDNA libraries: 3499 of these are from two unfertilized oocyte libraries and 10 978 are from two fertilized oocyte libraries. Gene expression profiles were obtained for these libraries by matching library EST to UniGene clusters. The 14 477 EST identified 4226 UNIGENES: These were screened using HomoloGene to identify 1386 homologous UniGene clusters in two other species with one of the matches being human. Within these human matches, 840 encoded named proteins, 223 encoded hypothetical proteins, and 323 encoded clustered EST. The set of named genes provides the first step in establishing a database of genes expressed in mouse oocytes and, by extension, human oocytes.

Animals↗
PubMed1992

Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis.

A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42 degrees C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli secY24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.

Adenosine Triphosphatases↗