Restriction mapping of genetic transfer factor pAP39

A model of calculation of molecular weights of fragments EcoRI, Hind III and PvuI is formulated. A restriction site map of factor pAP39 is constructed automatically. Sites to EcoRI and PvuI are distributed in the segment with molecular weight 9.1 MD.     

Range of the transmissivity of the genetic transfer factors pAP38, pAP39, pAP41 and pAP42

A study was made of the transmissivity range of the genetic transfer factors pAP38, pAP39, pAP41 and pAP42 identified in E. coli. It was demonstrated that these factors are not capable of transfer to the cells of P. putida, P. fluorescens, R. leguminosarum, A. lipoferum, A. tumefaciens. Factor pAP42 is mobilized to transfer to P. putida and R. leguminosarum with the aid of plasmid RP4. It is assumed that in the course of mobilization, the cointegrative structures are formed between plasmids pAP42 and RP4.     

Restriction map of the genetic transfer factor pAP42

Based on the calculated molecular weights of EcoR1, HindIII, and SalI fragments of the genetic transfer factor pAP42 the restriction map of this plasmid was designed. Sites recognizing restrictases are mostly located in the plasmid fragment with a molecular weight of 5.7 MD.     

Genetic transfer of pAP38, pAP39, and pAP41 plasmids into E. coli, Erwinia, and Hafnia strains

Genetic transfer factors pAP38, pAP39 and pAP41 can be transferred to E. coli, both typed and untypecd, as well as to Erwinia and Hafnia strains, with different frequency (10(-2) to 10(-8)). The transconjugates thus obtained possess donor activity and can transfer the factors they have received to recipient E. coli strains. After transfer these factors are stably maintained in a new host for at least 10 days. Under the action of ethidium bromide or elevated temperature the elimination of the transfer factors from host bacteria is observed. The studied transfer factors pAP38, pAP39 and pAP41 (F-like factors) are plasmids fi+.     

Atypical incompatibility of the F-like factors of pA22-4, pAP39 and pA41 genetic transfer with the F-group incompatibility plasmids

A study was made of compatibility of three F-like factors of the genetic transfer (pAP22-4, pAP39, pAP41) identified in the cells of serologically typed E. coli strains with F-group incompatibility reference plasmids. The factors of pAP22-4 and pAP41 transfer are partly incompatible with groups FII, FIII, FIV, and FI, FIV, respectively, while the factors of pAP39 transfer are completely incompatible both with groups FI and FIV plasmids.     

Cloning of F DNA fragments carrying the origin of transfer oriT and the fertility inhibition gene finP

The two SalI fragments derived from the F transfer region that are bounded at one end by the SalI cleavage site in traM were cloned into pBR322. From these, smaller (540 bases) SalI-BglII fragments were subcloned to give plasmids containing the origin of transfer oriT (pED806) and finP (pED812), respectively, but no entire tra genes. All four plasmids were characterized by genetic tests and by restriction endonuclease analysis. pED806 could not be used to search for an F oriT-related “relaxation complex” because of its unexpected instability in the presence of Flac, and extensive efforts to prepare such a complex using other oriT⁺ plasmids were unsuccessful. We therefore suggest that a cell-free F relaxation complex does not exist. Protein synthesis directed by pED812 in minicells allowed the finP product to be tentatively identified as a 4000 M_r, protein.     

Deletion mapping of 39 random isolated Y-chromosome DNA fragments

Summary Thirty-nine recombinants isolated from a Y chromosome-specific library were deletion mapped. Seven deletion intervals were defined by hybridization of probes to DNA of eight individuals with aberrant Y chromosomes. Extreme cytogenetic limits of the deletion intervals were determined by in situ hybridization of one probe per deletion interval. Five intervals, with a total of twenty-five probes, were allocated to the longarm euchromatic region. The probes described will be useful for characterization of aberrant Y chromosomes, in searching for expressed sequences on the Y chromosome, and for further study of the evolutionary relationship between the Y chromosome and other chromosomes.     

Cloning DNA restriction endonuclease fragments with protruding single-stranded ends

A new method of in vitro recombination was employed to construct plasmids containing lac promoter fragments 64 bp and 144 bp long. The 64 bp HpaII-HhaI fragment contains the binding site for the catabolite activator protein (CAP). The HpaII-HaeIII 144 bp fragment includes the binding sites for RNA polymerase, the lac repressor and CAP. The method utilizes the ability of T4 DNA polymerase to make flush-ended DNA either by filling in a recessed 3'-end or by exonucleolytic removal of a protruding 3'-end. The treated fragments were then blunt-end ligated to the filled-in EcoRI cloning sites of the plasmids pVH51 and pBR322 using T4 ligase. In this process, the EcoRI sites were regenerated on the fragment ends thus facilitating the subsequent isolation of the fragments from their cloning vectors.     

Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata

A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata.     

Cloning and characterization of restriction fragments of phage Mu DNA

Abstract The Eco RI-A and B, the Bam HI-C and the two Eco RI- Bam HI restriction fragments of transposing phage Mu DNA were inserted into vector plasmids pRSF2124 and pBR322. Quantitative marker rescue experiments for five genes located on the Eco RI-A fragment revealed complementation of phages carrying amber mutations in genes C , E , H , F and L . The in vitro coding capacity of the recombinant plasmids was assayed in a DNA-directed protein synthesizing system.     

Hydrodynamics-based transfer of PCR-amplified DNA fragments into rat liver

A high level of plasmid DNA expression in rat liver can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein, called the 'hydrodynamics-based procedure.' The preparation of PCR-amplified DNA fragments is easier than that of naked DNA. In this paper we evaluated the effects of expressing the erythropoietin (Epo) gene in the rat liver by injecting fCAGGS-Epo, an Epo-expressing PCR-amplified DNA fragment, via the tail vein. After injection of 5 pmol fCAGGS-Epo (10 microg) or pCAGGS-Epo (18.4 microg), plasmid DNA, the serum Epo levels peaked at week 1, then persisted for at least 12 weeks. Transgene-derived Epo secretion resulted in significant erythropoiesis. These results demonstrated that transfer of PCR-amplified DNA fragments into the rat liver via rapid tail vein injection can be achieved. This method may provide a useful means for studying the physiologic function of a putative gene.     

Directed transfer of large DNA fragments between Streptomyces species

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically amenable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene cluster from one Streptomyces species to another. The method takes advantage of tra gene-mediated conjugal transfer of chromosomal DNA between actinomycetes. As proof of principle, we demonstrate transfer of the entire approximately 22-kb actinorhodin gene cluster, and also the high-frequency cotransfer of two loci that are 150 to 200 kb apart, from Streptomyces coelicolor to an engineered derivative of Streptomyces lividans.     

Cloning of Herpesvirus saimiri DNA fragments representing the entire L-region of the genome

Purified particles of Herpesvirus saimiri, a potent tumor-eliciting virus of primates, contain genomic DNA molecules (145-170 kb) consisting of a unique L-DNA region (112 kb) which is flanked by variable stretches of repetitive sequences (H-DNA). Restriction fragments representing the entire L-DNA of H. saimiri strain No. 11 were cloned in plasmid and bacteriophage vectors. The internal fragments of L-DNA generated by the enzymes EcoRI and KpnI were inserted into plasmid pACYC184, cosmid pJC81, or bacteriophage lambda derivative Charon 4A. The terminal parts of L-DNA, including the junctions between repetitive DNA and unique sequences, were cloned between the cleavage sites for KpnI and SmaI in the plasmid vector pWD7, which was constructed for this purpose. Molecular cloning allowed us to confirm and modify, in part, the existing cleavage maps of H. saimiri DNA. It provides a basis for future studies on virus replication and oncogenic transformation.     

Cloning of restriction fragments of DNA from staphylococcal bacteriophage phi 11.

EcoRI fragments of Staphylococcus aureus bacteriophage phi 11 DNA were cloned in vector plasmid pSA2100 in S. aureus. The clones were analyzed in marker rescue experiments with suppressor- and temperature-sensitive mutants of phi 11 to correlate the genetic and physical map. Several mutants could be identified on the physical map, and a clone containing fragment EcoRI-B of phi 11 DNA expressed immunity to phage infection. In addition, it was found that recombinant plasmids containing phi 11 DNA sequences can be transferred by high-frequency transduction after phage phi 11 infection of host cells.     

Cloning high molecular weight DNA fragments by the bacteriophage P1 system.

The cloning of high molecular weight genomic DNA promises to provide the means of mapping chromosomes, isolating genes, and understanding long-range effects on gene expression. This review describes the background and some recent advances in cloning of high molecular weight DNA using the bacteriophage P1 system.     

Cloning of ten distinct DNA fragments of Clostridium thermocellum coding for cellulases

Abstract Ten distinct Eco RI fragments of Clostridum thermocellum DNA have been cloned in Escherichia coli and shown to express enzymatic activities related to cellulose degradation. Two of the cloned fragments appeared to carry the previously characterized celA and celB genes, which code for the endoglucanases (EG) A and B. Five other cloned fragments code for hitherto unidentified EGs, which can be detected by the Congo red test for hydrolysis of carboxymethylcellulose (CMC). In addition, three separate clones hydrolyzed methylumbelliferyl-β-cellobioside (MUC) but not CMC, hinting that they may express three different cellobiohydrolase genes.