Construction Of Gene Libraries Research Articles

Evaluation of different lymphoid tissue sources for the construction of human immunoglobulin gene libraries

Background: Phage display technology allows the isolation of novel human monoclonal antibodies. The technology relies on the construction of a recombinant antibody library and its display on phage particles. The quality of an antibody library is affected by several factors including the size, diversity and source of immunoglobulin genes.Objective: The aim of the project was to determine the best tissue source for the construction of antibody libraries.Study design: Three tissue sources were used in this study: peripheral blood mononuclear cells from a healthy donor, Epstein-Barr virus (EBV) transformed peripheral blood mononuclear cells and lymph node tissue from individuals with breast cancer. The quality of each tissue source was assessed using two criteria: (1) the number of mature and activated B cells in each source; (2) the amount of immunoglobulin heavy and light chain genes amplifiable by polymerase chain reaction (PCR). Results: EBV-transformed peripheral blood mononuclear cells and lymph node tissue were shown to contain more B cells than peripheral blood mononuclear cells. A relatively larger amount of immunoglobulin gene products could be amplified from EBV-transformed peripheral blood mononuclear cells and the lymph node. However, immunoglobulin containing γ1 chains could not be amplified from EBV-transformed mononuclear cells, and the resultant pattern of gene amplification suggests a possible selection bias. Conclusion: This study indicates that among the three tissue sources examined, lymph node tissue is the most suitable source for the construction of antibody libraries.

Instability of pneumococcus library in pHC79 and pACYC184

S. pneumoniae (pneumococcus) gene cloning and library construction inE. coli multicopy plasmid has been hampered, in part, by instability problems. In this study, stability of pneumococcus gene library in cosmid vector and pACYC184 was examined. Pneumococcus library in the cosmid vector pHC79 was extremely unstable that most of the recombinant clones were degenerated rapidly. Only 2 out of 849 clones were stable and had appropriate insert size. Pneumococcus library in pACYC184 was also so unstable that the pneumococcal insert and/or part of the vector were deleted. However, the instability problems seemed to be resolved when transcription terminator plasmid was employed for pneumococcus library construction.

An “instant gene bank” method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA

We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis. This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple. The method relies on recombination between a fungal replicating vector pHELP1 and linear G. graminis genomic DNA during co-transformation. We were able to complement two out of seven A. nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli. Complementation of the A. nidulans argB mutation resulted from integration of 8-10 kb segments of G. graminis DNA into pHELP1. The complementation of the A. nidulans pyrG mutation resulted from a complex rearrangement. Complementing DNA was shown to originate from G. graminis, and was capable of retransforming the original mutants to give the expected phenotype.

The sequence of Saccharomyces cerevisiae cloning vector pCS19 allowing direct selection for DNA inserts

The complete nucleotide sequence of plasmid pCS19 has been determined. This centromeric Saccharomyces cerevisiae cloning vector was designed for the construction of gene libraries, since it allows direct selection for DNA inserts.

A rapid and simple method for DNA preparation fromCandida utilis

A method for the extraction of genomic DNA from the industrial yeast Candida utilis is described. The method is rapid, simple and produces DNA that is sufficiently pure for restriction analysis and should be suitable for Southern blotting and the construction of gene libraries.

Characterization of somatic cell hybrids by bivariate flow karyotyping and fluorescencein situ hybridization

We report on the use of flow karyotyping and fluorescence in situ hybridization (FISH) to characterize the human chromosomes in somatic cell hybrids. The identity, DNA content, and relative frequency of human chromosomes are derived from flow karyotypes, i.e., measurements of Hoechst and chromomycin fluorescence intensities of chromosomes by dual beam flow cytometry. Chromosome integrity is assessed by comparing the peak position of a human chromosome in the flow karyotypes of a hybrid cell line and its human donor. When human donor cells are unavailable, the peak position of a human chromosome in a hybrid line is compared to the range of peak positions among normal individuals. The relative frequency of human chromosomes in subclones or hybrids grown in culture is monitored using the volumes of peaks in flow karyotypes. FISH with biotinylated human genomic DNA or chromosome-specific repeat sequence as probe is used in conjunction with flow karyotyping to confirm the number of human chromosomes in hybrids. Some small rearrangements are detected by flow karyotyping and not by FISH. On the other hand, translocations between human and rodent chromosomes are detected by FISH and not always by flow karyotyping. Flow karyotyping and FISH were used to characterize over 100 hybrid lines donated by other laboratories. A hybrid set useful for the construction of chromosome-enriched gene libraries is presented. In this set, each of the 24 human chromosome types is present and intact, as judged by these techniques, in a line containing little or no other human material.

A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, λpGY97

It is shown here that the phosphate groups at the cos ends of phage λ DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native λ DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector λpGY97 have been used to construct a representative gene bank of alfalfa in a Mcr − (5-methylcytosine resstriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA − E. coli , to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5–19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by λ terminase just before in vitro packaging.

Construction of gene libraries for each human chromosome

We describe the construction of two complete sets of small insert, complete digest DNA libraries for each of the 24 human chromosomal types by the National Laboratory Gene Library Project. Flow sorting was used to purify the chromosomes which provided the DNA for cloning. One set of libraries was cloned into the HindIII site of the lambda vector Charon 21A, and the other set was cloned into the EcoRI site of the same vector. Characterization information from both in-house experiments and user feedback is presented. These chromosome-specific libraries are available to the general scientific community from a repository at the American Type Culture Collection, Rockville, MD. The second phase of the project, the construction of large insert, partial digest libraries in both lambda and cosmid vectors, is underway.

Phasmids as effective and simple tools for construction and analysis of gene libraries

Phasmid ApMYFlSl, a hybrid of phage λ vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The λpMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector λpMYF131, compared to phage vector λL47.1AB, and attained 3 × 10 6 clones per μg DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. λpMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for λpMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts ( Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50–150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.

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 μ origin of replication, and a region from pUC18 that includes the amp r 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.

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 λEMBL3 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 (Pollk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and Pollk. 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 λ particles per μg of the genomic DNA (as compared to approx. 10 7 per μg for the wild-type λ DNA). This procedure is very rapid and requires only μg 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.

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.

Construction of human gene libraries from small amounts of peripheral blood: analysis of beta-like globin genes.

We describe a rapid procedure for constructing cloned human genomic libraries from small amounts of peripheral blood. High molecular weight DNA is isolated from 5-20 ml peripheral blood, partially cleaved with Eco R1, and 8-22 kb fragments are cloned using bacteriophage Charon 4A and suitable E. coli host. Using the approach we have isolated and characterized several non-alpha globin clones from a Kurdish Jew with homozygous beta thalassemia. The ability to isolate suitable amounts of high molecular weight DNA from peripheral blood provides a relatively simple means of constructing human gene libraries representing a variety of hemoglobin disorders.

Isolation of a Xenopus laevis α-globin gene

We report here the construction of a Xenopus laevis gene library and the isolation and characterization by nucleotide sequence analysis of a clone containing an α-globin gene.