DNA sequences flanking an E. coli insertion element IS2 in a cloned yeast TRP5 gene

The insertion of an Escherichia coli IS2 element upstream from a cloned yeast TRP5 gene results in an increased level of active tryptophan synthase in trpAB E. coli host cells. This insertion occurs about 60 bp upstream from the first AUG of the TRP5 gene and is associated with a duplication of the sequence TTACA at the target site. The nucleotide sequence corresponding to the first 173 amino acids of the yeast TRP5 gene has also been determined. The N-terminal region of the yeast tryptophan synthase includes areas of strong homology with the alpha-subunit of the corresponding E. coli enzyme. Sequences from the 5' untranslated region upstream from the TRP5 gene are compared to homologous areas of other yeast genes.     

Recombination of a eukaryotic DNA in bacteria

Single, 824 bp repeating units of Xenopus laevis oocyte-type 5S DNA were inserted into the recombination vectors, λrva and λrvb. When the inserts had the same orientation with respect to the λ chromosomes, Spi^-imm434 recombinants were recovered by selection on a P2, λ double lysogenic host. Because of the structure of the vectors, the crossover point in each recombinant must lie completely within the 5S DNA insert. The physical characteristics of these recombinants were determined by examination of restriction enzyme digests. By use of RecA mutant hosts and the Red^- vector, λrvc, recombination frequencies were measured separately for the bacterial and phage systems.Some of the recombination events resulted in 5S DNA inserts of altered length due to unequal crossovers within repeated sequences in the 5S DNA spacer. The occurrence of just such events in frog 5S DNA had been predicted, based on the structure of 5S DNA and evolutionary considerations.     

Enhancement of UDP-galactose:mucin galactosyltransferase activity by spermine

A microsomal preparation prepared from the mucosal lining of canine trachea catalyzed the transfer of galactose from its uridine diphosphate derivative to sialidase-treated ovine submaxillary mucin. Maximal incorporation occurred at 30 mm mn²⁺. When the concentration of mn²⁺ in the reaction mixture was reduced to 2.5 mm, approximately two-thirds of the enzymatic activity was lost, but full activity could be restored by the addition of 1 mm spermine. Under these conditions spermine did not affect the K_m for UDP-galactose, but lowered the K_m for sialidase-treated ovine submaxillary mucin and Mn²⁺ by a factor of 10. The effect of spermine was abolished with increasing concentrations of Mn²⁺, and in the absence of the metal, enzymatic activity was lost and could not be restored by the addition of spermine. Spermidine also stimulated activity at low levels of Mn²⁺, but to a lesser degree than spermine. A slight stimulatory effect was consistently derived from putrescine as well, while cadaverine, putreanine, and monoamines were ineffectual. Spermine had a similar effect on the enzymatic transfer of GalNAc to a protein core acceptor but had little or no effect on the enzymatic transfer of sialic acid to sialidase-treated ovine submaxillary mucin, galactose to N-acetylglucosamine, or fucose to sialidase-galactosidase-treated fetuin. Similar results were obtained with enzyme preparations prepared from canine submaxillary glands. Other polycationic compounds such as protamine, histone, and polylysine also stimulated enzymatic activity at suboptimal concentrations of mn²⁺.     

Recovery of DNA fragments inserted by the "tailing" method: regeneration of PstI restriction sites

A general method has been developed for the recovery of any DNA fragment inserted into a cloning vehicle containing a single endonuclease PstI site. Endonuclease PstI sites are regenerated by the addition of one or more deoxyguanosine residues to the 3' termini of the PstI-cleaved vehicle by terminal deoxynucleotidyl transferase. Chain elongation by terminal deoxynucleotidyl transferase is then continued with dITP, dATP or dGTP. A plasmid vehicle, pAO1, containing a single PstI site has been constructed. Insertional (foreign) DNA fragments that were "tailed" with dCTP have been annealed to PstI-cleaved pAO1 that was "tailed" with dGTP. When the annealed fragments were used to transform competent Escherichia coli cells, the single-stranded DNA gaps in the recombinant plasmids were repaired. Plasmids recovered from transformed bacteria could be cleaved by PstI into the insertional DNA with dG:dC tracts and linear pAO1 molecules.     

NP-Sticky: A Web Server for Optimizing DNA Ligation with Non-Palindromic Sticky Ends

High-efficiency DNA ligation is vital for many molecular biology experiments, and it is best achieved using reactants with non-palindromic sticky ends to maximize specificity. However, optimizing such multi-parametric ligation reactions often involves extensive trial and error. We have developed a freely available Web-based ligation calculator, NP-Sticky (http://sarkarlab.umn.edu/npsticky/), that predicts product distribution for given reactant concentrations, thus enabling straightforward computational optimization of these reactions. Built-in schemes include two-piece and three-piece linear ligation, as well as insert-vector circular ligation. The only parameters needed for the underlying thermodynamic model are the free energies of ligation for each sticky end, which can be estimated by the calculator from the overhang sequences or provided by the user from direct experimental measurement. Free energies of sticky-end mismatches are also calculated for determining the extent of byproduct formation. This ligation calculator allows rapid identification of the optimal conditions for maximizing incorporation, efficiency, and/or accuracy, based on specific needs.     

Identification of a new strain of adenovirus type 2 by restriction endonuclease analysis

Restriction endonuclease analysis has been used to identify a new strain Ad2a of human adenovirus type 2 (Ad2). The sites for six restriction endonucleases have been mapped on the Ad2a genome and compared with those for the Ad2 prototype.     

Comparison of left-end DNA sequences of bacteriophages Mu and D108

The nucleotide sequences of the left ends of bacteriophage Mu DNA and that of its close relative D108 have been determined. The first 100 bp of phages Mu and D108 are substantially the same except for an octanucleotide change from bp 53 to 61 and other small interspersed base-pair changes from bp 61 to 200. The first five host nucleotides preceding the host-phage junction are generally, but not always, G + C-rich and these five nucleotides display no obvious consensus sequence. Both phages Mu and D108 share striking similarity in their end DNA sequences to the end sequences of the newly described Escherichia coli movable genetic element IS30.     

Restriction maps of plasmids pUB110 and pBD9

Restriction-enzyme cleavage site maps for 12 and 14 enzymes have been constructed for the Bacillus subtilis plasmids pUB110 and pBD9, respectively.     

In vitro and in vivo manipulations of bacteriophage Mu DNA: cloning of Mu ends and construction of mini-Mu's carrying selectable markers

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.     

A novel deletion found during cloning of a synthetic palindromic DNA

A 212-bp palindromic DNA comprising two copies of the left end of bacteriophage Mu was assembled from chemically synthesized oligonucleotides and inserted into plasmid pUC9. When cloned and propagated in Escherichia coli, the palindrome was found to be unstable and was generally lost. However, in a few cases, a precise, asymmetric deletion of one half of the insert was observed. This pattern of deletion suggests that the symmetry axis region of the palindrome was involved as recognition site in the deletion process.     

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.     

The ori sequences of the mitochondrial genome of a wild-type yeast strain: number,location, orientation and structure

We have investigated the number, the location, the orientation and the structure of the seven ori sequences present in the mitochondrial genome of a wild-type strain, A, of Saccharomyces cerevisiae. These homologous sequences are formed by three G + C-rich clusters. A, B and C, and by four A + T-rich stretches. Two of the latter, p and s, are located between clusters A and B; one, l between clusters B and C; and one r, either immediately follows cluster C (in ori 3–7), or is separated from it by an additional A + T-rich stretch, r', (in ori 1 and ori 2). The most remarkable differences among ori sequences concern the presence of two additional G + C-rich clusters,β and γ, which are inserted in sequence l of ori 4 and 6 and in the middle of sequence r of ori 4,6 and 7, respectively. Neglecting clusters /gb and γ and stretch r', the length of on sequences is 280 ± 1 bp, and that of the l stretch 200 ± 1 bp. Hairpin structures can be formed by the whole A-B region, by clusters β and γ, and (in ori 2–6) by a short AT sequence, lp, immediately preceding cluster /gb. An overall tertiary folding of ori sequences can be obtained. Some structural features of ori sequences are shared by the origins of replication of the heavy strands of the mitochondrial genomes of mammalian cells.     

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases BglII, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.