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At least 19 recordsLinked to original sources
PubMedDate not supplied

[Construction of libraries of structural genes (cDNA) and "jumping" gene libraries in lambda vectors].

Main approaches and methods used for constructing "jamping" and cDNA libraries, including novel ones, that omit the employment of methylases and linkers, are presented. The advantages and drawbacks of the well-known and new lambda vectors, suitable for the purposes mentioned, are discussed. Special attention is paid to diphasmids lambda ZAP, lambda SK12, lambda SK15, that combine features of lambda and M13 phages and of plasmids. The convenience and difficulties of "jumping" libraries for physical mapping of chromosomes are briefly considered.

Bacteriophage lambda↗
PubMed1980

Isolation of two closely related vitellogenin genes, including their flanking regions, from a Xenopus laevis gene library.

A gene library of Xenopus laevis was constructed from embryonic DNA partially digested with restriction endonucleases Hae III and Alu I and joined to the phage lambda Charon 4 cloning vector with EcoRI linkers. Nucleotide sequences from three of the four related vitellogenin genes have been isolated. Two of the genes (called A1 and A2) were isolated in their entirety together with long stretches of flanking sequences. These two closely related vitellogenin genes have lengths of about 21 and 16 kilobases, but both produce a vitellogenin mRNA of 6.3 kilobases.

Animals↗
PubMed2004

Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries.

To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage Mu minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL and EMBL ), were used for rapid open reading frame (ORF) identification and prediction of protein localisation in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library to be tagged. It is suited for identifying genes encoding extracytosolic proteins in gene libraries of a wide range of prokaryotic organisms.

Amino Acid Sequence↗
PubMed1987

A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae.

We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae. The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites. Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique. This vector has advantages over similar yeast-E. coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.

Cloning, Molecular↗
PubMed1980

Analysis of mouse embryonic gene library for the frequency of single and multiple copy genes.

A gene library was constructed from embryonic mouse DNA by ligating DNA fragments generated by partial Eco RI digestion with Charon 4A vector and in vitro packaging. A special consideration was given to randomization of target DNA. The general applicability of a gene library prepared in this manner was assessed through cloning a variety of genes of known reiteration frequency in the mouse genome. The survey included a single copy gene--C region of the immunoglobulin heavy chain, and genes that appear in more than one copy--V region of the immunoglobulin light chain genes and the endogenous retrovirus related genes. In all cases tested the frequency of clone isolation was in good agreement with the expected incidence based on the number of genome equivalents screened and the reiteration frequency of that particular gene. Moreover, we found no preference with regard to the clonability of genes contained in fragments of a wide-size range.

Animals↗
PubMed2005

An episomal expression vector for screening mutant gene libraries in Pichia pastoris.

Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.

Bleomycin↗
PubMed1992

Cell selection strategies for making antibodies from variable gene libraries: trapping the memory pool.

The B cells of immunized animals can be used as a source of variable region (V) gene libraries. Such libraries offer a way of making antibodies directly in bacteria: rearranged V genes are amplified using the polymerase chain reaction, cloned and expressed as soluble fragments in bacteria, and then screened for antigen binding. Here we have used a model system to investigate antigen-selected B cells as a source of V gene libraries. Mice were immunized with (4-hydroxyl-3-nitrophenyl)acetyl (NP)-chicken gammaglobulin, and the splenocytes harvested seven days after primary immunization. We prepared a heavy chain variable (VH) gene library from the DNA of cells selected on antigen-coated magnetic beads, and two other libraries from the DNA or mRNA of unselected cells. The VH gene libraries were combined with the V lambda 1 gene (as this light chain dominates the primary response to NP), expressed as Fv fragments in Escherichia coli and screened for binding to (4-hydroxy-3-iodo-5-nitrophenyl)acetyl-bovine serum albumin. The frequency of antigen-binding clones was much greater (greater than 50 fold) in the library from the DNA of antigen-selected cells (17/282) or from the mRNA of unselected cells (29/282) compared to the DNA from unselected cells (0/940). Sequencing of the antigen-binding clones revealed that they almost invariably used the V-186.2 heavy chain, as expected from analysis of primary response hybridomas. The D segments from the mRNA library were entirely DFL16.1 (29/29), as in primary response hybridomas, whereas those from the DNA of selected cells were more diverse, using in addition to DFL16.1, other D segments (5/17) as in later response hybridomas. This suggests that the DNA library from selected cells is derived at least in part from cells destined for the memory compartment. Given the long life of memory cells, they may prove a useful source of antibody libraries in the absence of recent immunization.

Amino Acid Sequence↗
PubMed1982

Constructions of gene libraries of Rana nigromaculata and Mus musculus with cosmid pHC79.

Gene libraries of Rana nigromaculata and Mus musculus (C57J6) have been constructed by the cosmid system developed by Collins using BamHl and Pstl as cloning restriction enzymes. A simplified formula, which is a modification of Clarke and Carbon derived from approximation, was used for calculating the number of colonies or recombinants necessary for the construction of a gene library. Pro+ and leu+ complementary to the recipient cells (HB101) with the genetic constitution of pro- and leu- have been selected from the BamH1 gene libraries of both Rana nigromaculata and the mouse in the present work.

Animals↗
PubMedDate not supplied

Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification.

A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.

Bacterial Proteins↗
PubMed2002

Analysis of shuffled gene libraries.

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.

Base Sequence↗
PubMed2003

Removing the redundancy from randomised gene libraries.

Amino acid substitution plays a vital role in both the molecular engineering of proteins and analysis of structure-activity relationships. High-throughput substitution is achieved by codon randomisation, which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NN(G)(CorT) (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly; for example, methionine is encoded just once within NNN, whilst arginine is encoded six times. Finally, the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Here, we present a novel solution to randomisation where genetic redundancy is eliminated; the number of different genes equals the number of encoded proteins, regardless of codon number. There is no inherent amino acid bias and any required subset of amino acids may be encoded in one experiment. This generic approach should be widely applicable in studies involving randomisation of proteins.

Amino Acid Substitution↗
PubMed1985

[Construction of a gene library using partial filling of DNA sticky ends].

To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites.

Animals↗
PubMed1996

[Construction of gene library of attenuated liver hepatitis A vaccine (H2 strain)].

OBJECTIVE: To determine the complete gene library of attenuated live hepatitis A vaccine (H2 strain) from cloned cDNA by application of reverse transcription (RT) and polymerase chain reaction (PCR) technique. METHODS: Attenuated HAV H2 strain that was selected and studied in China was proven attenuated for human beings and proved to be effective in preventing hepatitis A. RESULTS: Ten overlapping cDNA clones were obtained in addition to spanned the entire genome. The restriction enzyme mapping was also determined. CONCLUSION: The gene library will benefit for the sequence of HAV cDNA and study on attenuation.

DNA, Viral↗
PubMed1993

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library.

Autonomously-replicating, marker-less "helper" plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.

Aspergillus nidulans↗
PubMed1986

An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends.

For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.

Cloning, Molecular↗
PubMed1987

Construction of a gene library from Azospirillum brasilense and characterization of a recombinant containing the nif structural genes.

1. We have constructed a gene library, from Azospirillum brasilense using the vector EMBL4. 2. A recombinant containing the nif structural genes from A. brasilense was isolated and characterized. This recombinant contains a DNA insert of about 15 kilobases (kb) which gives rise to five fragments after cleavage with EcoRI. Only one of the DNA fragments (6.5 kb) hybridized to the nifHDK genes of Klebsiella pneumoniae. 3. The organization of the nif genes in this DNA fragment was determined using different DNA segments containing the nifH, nifK or nifD genes of K. pneumoniae as probes.

Chromosome Mapping↗
PubMed1990

[Construction and analysis of the tularemia pathogen gene library in Escherichia coli].

The library of tularemia causative agent genes cloned on the pHC79 plasmid and the partial clonotek of these agents genes in Escherichia coli cells have been constructed. The immunochemical analysis has revealed seven clones of Escherichia coli harbouring the recombinant plasmids and expressing francisella antigens. The cloned sequences of francisella DNA as well as the recombinant plasmids containing them and coding for francisella antigens are capable of specific hybridization with the DNA from Francisella tularensis strains and Francisella novicida strain U112. The cloned DNA sequences have the properties of the genetic radiospecific molecular DNA probe.

Blotting, Southern↗
PubMed2006

Diversity of the Clostridium coccoides group in human fecal microbiota as determined by 16S rRNA gene library.

Fecal microbiota were analyzed in seven healthy individuals by 16S rRNA gene libraries (universal library) using universal primers, and the Clostridium coccoides group libraries using the universal primer 27F and the C. coccoides group-specific primer Erec482. The universal libraries were used in our previous studies. The 972 clones obtained from two different primer set libraries belonged to the C. coccoides group and were classified into 139 operational taxonomic units (OTU) (at least 98% sequence similarity). Of these, 41 OTU were detected commonly from universal libraries and C. coccoides group libraries. One hundred and ten OTU were detected from the C. coccoides group libraries. Fifteen new OTU were isolated from the C. coccoides group libraries in human gut. Most of the OTU did not correspond to known species, thus representing as-yet-uncultured bacteria. We also detected OTU that related to the butyrate-producing bacteria. The C. coccoides group consisted of an average of 35 OTU, although there were differences in the number and the type of species in each individual. When fecal microbiota were analyzed using universal libraries, the OTU belonging to the C. coccoides group detected in elderly individuals were fewer than those detected in adult individuals. When C. coccoides group libraries were used, the numbers of OTU in elderly and adult individuals were not different. Interindividual differences in the composition of OTU belonging to the C. coccoides group were also observed in fecal microbiota.

Adult↗