In vitro insertion of the lambda attachment site into the plasmid RP4.

Summary The region of the phage lambda chromosome containing the attachment site (P · P) and the genes int and xis, excised by the action of endonuclease R.EcoRI, has been inserted into the unique site for that enzyme on the promiscuous conjugative plasmid, RP4, generating the recombinant plasmid RP4att. Transformants containing the hybrid plasmid were recognised by their ability to allow efficient lysogenization by phage b2 (Weil and Signer, 1968; Echols et al., 1968) containing the mutant attachment site · P. The construction and properties of the hybrid plasmid RP4att are described.     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

Molecular analysis of plants regenerated from embryogenic cultures of hybrid sugarcane cultivars (Saccharum spp.)

Summary The genomic stability of tissue culture regenerants of sugarcane (Saccharum spp. hybrids, cvs CP721210, CP68-1067 and B43-62) was analyzed by DNA restriction fragment length polymorphism (RFLP). Plants regenerated from calli, cell suspensions, cryopreserved cell suspensions and protoplasts were used. Total DNA isolated from 19 different sources was digested with EcoRI, HindIII, BamHI, BamHI, EcoRI and PstI and probed with six known maize mitochondrial genes (coxI, coxII, atpA, atp6, atp9 and rrn18-rrn5), three random maize mitochondrial cosmid clones, two random maize chloroplast cosmid clones and a wheat Nor locus clone. Hybridization patterns indicated that the variation observed was minor and appeared only in the secondcycle regenerants. No differences were observed among the three cultivars and the regenerants from calli, suspension culture, cryopreserved suspension culture and protoplasts. Mitochondrial DNA (mtDNA) isolated from CP72-1210 plants and its embryogenic cell suspensions, and bulk samples from all CP72-1210 regenerants pooled together were digested with EcoRI, HindIII, PstI, BamHI and SalI and probed with three recombinationally active wheat mtDNA clones, K, K3 and X2. No variation in the mtDNA restriction patterns was observed between the CP72-1210 plants and its regenerants. However, restriction pattern variation was observed only from EcoRI digestion, and hybridization patterns of K3, K and X2 revealed minor variations in the mtDNA of cell suspensions when compared with the DNA of the CP72-1210 plant. Except for a qualitative variation detected by the X2 probe and minor stoichiometric variations detected by the K3 probe, sugarcane DNAs were found to be stable after plant regeneration.Florida Agriculture Experiment Station Journal Series No. R-02703     

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.     

Optimization of an electroporation procedure for Kluyveromyces lactis transformation

Summary An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into Kluyveromyces lactis yeasts. The plasmid pCR1, derived from a native Kluyveromyces plasmid, was used to transform K. lactis. This plasmid produces a wheat -amylase and contains both the biosynthetic marker URAA and G418 resistance genes. Transformation was optimal at 4500 V/cm, 25 F, and with 0.2 g plasmid DNA. Transformation efficiencies in the range 10⁴–10⁵ transformants/10⁷ cells/g DNA were obtained.     

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.     

Cloning of an EcoRI fragment carrying E. coli tufA gene.

Summary EcoRI fragments of the transducing phage fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Ap^r) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA·DNA hybridization of RNA transcribed from phage rif ^d18 carrying tufB with DNA from pTUAl.     

UV-induced allevation of lambda restriction in Escherichia coli K-12: kinetics of induction and specificity of this SOS function

Summary In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage is observed when the cells are recA ⁺ lexA ⁺. We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis. Maximum expression was reached within 60 to 90 min after irradiation. Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed. This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol. The decay kinetics were similar in uvr ⁺ and uvrA mutants. RA appeared to be specific for EcoK insofar as no allevation of restriction by EcoRI, EcoRII and EcoP1 occurred. During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells. Since, in fully expressed cells, up to 75% of the infecting DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of infections may undergo RA.     

Isolation and characterization of a genomic sequence of maize coding for a zein gene

Summary An EcoRI restriction endonuclease fragment of maize DNA coding for the 19,000 dalton zein protein was cloned in phage gt WES. The zein gene was identified by the electron microscopic analysis of RNA-DNA hybrids (R-loops) and DNA-DNA hybrids (D-loops). The R-loops were formed with poly(rA)-containing RNA isolated from 18 days post-pollination maize endosperm and showed no intervening non-hybridizing sequences (introns) within their 800 base length. A cDNA clone specific for the 19,000 dalton zein protein formed D-loops in the same position and orientation as the R-loops. The cloned fragment measured 4.4 kilobases (kb), the same size as an EcoRI fragment of maize DNA revealed by Southern analysis.     

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage

Summary The position of the metJBLF gene cluster in the transducing phage dmet102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the BCDEF (nin5) EcoRI fragment of gtl (BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.     

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome

To date, vertebrate DNA has been found methylated at the 5 position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5-GGCC-3 sequences in the teleost Sparus aurata EcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E21 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E21 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E21 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.     

Molecular cloning of fragments of bacteriophage T4 DNA

Summary Non-glucosylated T4 DNA was digested with R. EcoRI and the resulting fragments covalently joined to vectors. The genetic content of each -T4 hybrid was determined by marker-rescue tests. The isolation of many recombinants containing partialdigestion products of T4 DNA provided the overlapping sequences necessary to order fragments within the T4 genome. The present analyses include parts of the early region between genes 42 and 46, and much of the late region between genes 50 and 29. T4 cytosine-DNA digested to completion by R.EcoRI was used to identify the fragments of DNA within the -T4 recombinants. The T4 cytosine-DNA was also sensitive to R.HindIII and R.Xho but not to R.BamH1.     

Construction and some properties of packageable plasmid F

Summary A derivative of plasmid F which is packageable in phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus Plasmid that carries the -lactamase gene, and a portion of guaA (B) transducing phage DNA carrying cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7×10⁷ daltons, about 84% size of phage genome, and can be packaged in coat when helper phage replicates in the plasmid-carrier cell. The package plasmid and the helper phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F^- cell and establishes a plasmid state with normal efficiency. In F⁺ or Hfr cells, the resident F factor hinders this process.     

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli

Summary A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage host-vector system was shown to direct the synthesis of a thermostable -amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000.The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused -lactamase--amylase protein with amylase activity.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Stablizing crude and ultrafiltrated α-amylase and α-glucosidase from Aspergillus oryzae by trehalose

Thermal resistance of freeze-dried -amylase and -glucosidase in trehalose matrices (1 to 20 % w/v) stored at 90 °C and relative humidities (RH) between 0 and 44 % was studied. At RH values up to 33 %, 10 % (w/v) trehalose was necessary to retain at least 50 % of -amylase activity. For -glucosidase, 10 % (w/v) trehalose was effective only at 0 % RH. Ultrafiltration of the crude enzymatic fermentation extracts enhanced enzyme stability per se. However, ultrafiltration in combination with 1 % (w/v) trehalose retained 74 % of -glucosidase and 95 % of -amylase activities. © Rapid Science Ltd. 1998