Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda

The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages.     

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.     

New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries

A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.     

Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.     

Plasmid and bacteriophage vectors for excision of intact inserts

Plasmid (pPolyIII) and bacteriophage lambda (EMBL301) vectors are described in which sites for the rare-cutting enzymes SfiI and NotI (8-bp, recognition sequences) flank the polylinker cloning region. Intact DNA inserts for introduction into cultured cells or into the early embryo are readily excised from the vectors. General-purpose miniplasmid cloning vectors pPolyI and pPolyII are also described, and the utility of the bacteriophage lambda vector is demonstrated in the construction of a bovine genomic library.     

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 and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase

The Lactococcus lactis subsp. cremoris SK11 plasmid-located prtP gene, encoding a cell-envelope-located proteinase (PrtP) that degrades alpha s1-, beta- and kappa-casein, was identified in a lambda EMBL3 gene library in Escherichia coli using immunological methods. The complete prtP gene could not be cloned in E. coli and L. lactis on high-copy-number plasmid vectors. However, using a low-copy-number vector, the complete prtP gene could be cloned in strains MG1363 and SK1128, proteinase-deficient derivatives of L. lactis subsp. lactis 712 and L. lactis subsp. cremoris SK11, respectively. The proteinase deficiency of these hosts was complemented to wild-type (wt) levels by the cloned SK11 prtP gene. The caseinolytic specificity of the proteinase specified by the cloned prtP gene was identical to that encoded by the wt proteinase plasmid, pSK111. The expression of recombinant plasmids containing 3' and 5' deletions of prtP was analyzed with specific attention directed towards the location of the gene products. In this way the expression signals of prtP were localized and overproduction was obtained in L. lactis subsp. lactis. Furthermore, a region at the C terminus of PrtP was identified which is involved in cell-envelope attachment in lactococci. A deletion derivative of prtP was constructed which specifies a C-terminally truncated proteinase that is well expressed and fully secreted into the medium, and still shows the same capacity to degrade alpha s1-, beta- and kappa-casein.     

Cloning and sequencing of the genes of 2-hydoxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the lambda vectors gt11 and EMBL 3 were screened with antisera raised against 2-hydroxyglutaryl-CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a lambda gt11 clone with an insert of 2050 bp and one was a lambda EMBL-3 clone with an insert of approximately 11,000 bp. Escherichia coli cells infected with the lambda gt11 clone expressed the alpha subunit of the dehydratase (Mr, 53,870), whereas with the lambda EMBL-3 clone, the alpha and beta subunits (Mr, 41,857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (alpha) and hgdB (beta) were obtained. The genes have the following order: A-B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the alpha and beta subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in alpha, including a CNC and two CC clusters, and nine in beta) but no similarities to other known protein sequences were found.     

Vectors--derivatives of phage lambda for construction and analysis of genome libraries

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.     

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.     

Cloning of the saliva-interacting protein gene from Streptococcus mutans.

Genomic libraries from Streptococcus mutans OMZ175 were constructed in bacteriophage vectors. DNA fragments 1 to 2 kilobases in length were cloned in expression vector lambda gt11. S. mutans DNA fragments 15 to 20 kilobases in length were inserted in the BamHI site of phage EMBL3. Rabbit antiserum raised against an S. mutans saliva-interacting protein with a molecular weight of 74,000, designated 74K SR, was used to screen the lambda gt11 library. A recombinant phage carrying an S. mutans DNA sequence of 1.45 kilobases, lambda SmAD2, was detected and isolated. This fragment, named SmAD2, was used to construct the recombinant expression plasmid pSAD2-4 which encoded for the expression of a 60,000-molecular-weight protein controlled by the beta-galactosidase promoter from plasmid pUC8. The SmAD2 fragment and polyclonal anti-74K SR antibodies were used to screen the EMBL3 library. A total coincidence between the screening with antibodies and the DNA probe was observed, and two phages, lambda SmAD9 and lambda SmAD10, were isolated. They contained a common S. mutans DNA sequence of about 11.8 kilobases and coded for a protein with a molecular weight of about 195,000, which comigrated with a protein of an S. mutans cell wall extract. The expressed protein was purified, and a very strong relationship with the S. mutans 74K SR protein was found by competitive enzyme-linked immunosorbent assay. Thus, cloning of the 74K SR gene allowed us to demonstrate that the saliva receptor appears to be a part of an S. mutans precursor molecule with a molecular mass of 195,000 daltons.     

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

Phasmid lambda pMYF131, a hybrid of phage lambda 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 lambda pMYF131 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 lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 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 lambda pMYF131. 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.     

Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA.     

Cloning of clusters of erythromycin biosynthesis genes of Streptomyces erythraeus (Saccharopolyspora erythraea)

The erythromycin resistance gene (ermE) and part of erythromycin biosynthesis genes located in the same cluster with the ermE gene were cloned from S. erythraeus 3 subjected to improvement with respect to erythromycin production. For isolating the erythromycin biosynthesis genes, the plasmid vector pUC18 and the phage vector lambda EMBL3 were used. The ermE gene DNA was used as a labeled probe for analysis of the recombinant plasmids and phages. The recombinant phages lambda ermE1 and ermE4 containing fragments of the chromosomal DNA collinear to the genome DNA of S. erythraeus 3 were analyzed. The size of the cloned fragment of the chromosomal DNA of S. erythraeus 3 was about 20 kb. Subcloning with the vector pUS18 resulted in isolation of plasmids pSU235-pSU244 containing BamHI fragments of chromosomal DNA from S. erythraeus 3. The restriction map of the chromosomal region of S. erythraeus 3 containing the ermE gene was constructed. The cloned genes of erythromycin biosynthesis are useful in the study of their structure and functions, construction of integrative vectors, improvement of cultures producing macrolide antibiotics and isolation of genes responsible for biosynthesis of other polyketide antibiotics.     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.     

p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning.

We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.     

Rapid isolation and characterization of hybridization selected recombinants from lambda genomic libraries

This paper describes an efficient protocol for the screening of lambda genomic libraries, plaque and DNA purification, and probe characterization by a combination of new and recently described techniques. The protocol has allowed large numbers of human subchromosome-specific probes to be rapidly generated from an EMBL3 library of human-mouse somatic cell hybrid DNA. The protocol affords considerable savings in time and effort over previous procedures.     

Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes.

Genomic libraries have been constructed from bovine C. parvum DNA in the lambda ZAP and lambda DASH vectors. Based on an estimated genome size of 2 x 10(4) kilobases (kb), each recombinant library contains greater than 10 genomic equivalents. The average recombinant size for the lambda ZAP library is 2.1 kb and for the lambda DASH library is 14 kb. We have identified genes to major antigens recognized by hyperimmune bovine antiserum. These recombinants are currently being purified and characterized. Limited DNA sequence analysis of random C. parvum clones confirms suggestions that the genome is quite AT-rich. The DNA sequence of random lambda ZAP fusion proteins has identified a potential ATPase, a structural protein and a DNA-binding protein.