Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites.
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Biomedical subjects
Publications and source records attributed to B Gronenborn.
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A HindII restriction fragment comprising the Escherichia coli lac regulatory region and the genetic information for the alpha peptide of beta-galactosidase (beta-D-galactosidegalactohydrolase, EC. 3.2.1.23) has been inserted into 1 of the 10 Bsu I cleavage sites of M13 by blunt end ligation. A stable hybrid phage was isolated and identified by its ability to complement the lac alpha function. Further characterization of the hybrid phage includes retransformation studies, agarose gel electrophoresis, DNA-DNA hybridization, and heteroduplex mapping. The insertion point has been localized at 0.083 map unit on thewild-type circular map-i.e., within the intergenic region. The results prove that part of the intergenic region is nonessential and that the phage can be used as a cloning vehicle.
The gene coding for bacteriophage Lambda repressor (cI gene) has been fused to the lac operon of Escherichia coli. In some of the fusions Lambda repressor synthesis can be controlled by the lac operator and promoter. Upon induction of the lac operon the amount of Lambda repressor is increased by a factor of 7 over that found in a single lysogen. In combination with the polarity suppressor suA the induction factor rises to 20. Transducing phages of one fusion were constructed. After thermal induction of this phage the final level of Lambda repressor was enhanced by a factor of 150.
Lambda repressor was purified from an E. coli strain which produces 150 times more lambda repressor than a single lysogen. The sequence of the fifty N-terminal residues was determined by automated Edman degradation. It contains 43% of all arginine and lysine residues of the chain and constitutes according to the genetic data of Oppenheim et al. (1975) a substantial part of the operator-DNA-binding site of the repressor.
Selection methods for the isolation of i(-d) and i(s) mutants are described. Two hundred and forty-seven i(-d) and 98 i(s) mutations have been localized by deletion mapping. All i(-d) mutations map in the region of the i gene, which codes for the aminoterminal part of the lac repressor, whereas i(s) mutations map in the middle of the i gene and at the proximal end of the i(-d) cluster.
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We studied the host range of tomato yellow leaf curl virus (TYLCV) in some agronomically important tomato species. Transmission tests with the natural vector Bemisia tabaci from tomato to sweet pepper, eggplant, cucumber, melon, zucchini and spinach showed that these species did not develop symptoms and did not support viral replication. These species therefore do not constitutive a reservoir of the virus and can be cultivated as alternatives to tomato in the most affected areas. For host-range studies, we used a quick and sensitive dot-blot assay employing non-radioactive DNA probes. This technique, developed for detecting TYLCV in plant extracts, is easily used for diagnosis. The sensitivity of this non-radioactive test was comparable to that of radiolabelled probes.