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R Y Walder

Publications and source records attributed to R Y Walder.

26 records · Page 2Linked to original sources
PubMed1983

Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII.

The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.

Cloning, Molecular↗
PubMed1981

Cloning and expression of the Pst I restriction-modification system in Escherichia coli.

Here we report the cloning and preliminary characterization of the Pst I restriction-modification system of Providencia stuartii 164. Transformants of Escherichia coli carrying the Pst I gene system inserted into the cloning vector pBR322 were selected on the basis of acquired resistance to bacteriophage lambda infection. Pst I endonuclease was detected in osmotic shock fluid from each of the resistant clones. Plasmid and chromosomal DNA from these clones could not be digested by Pst I, indicating that the gene for the corresponding modification enzyme had also been cloned and was being expressed. The smallest recombinant plasmid encoding both activities, pPst201, contains an insert of approximately 4000 base pairs. In vitro transcription studies indicate that this DNA fragment also contains the endogenous promoter(s) of the system. When pPst201 was introduced into a minicell-producing strain of E. coli, two new proteins, 32,000 and 35,000 daltons, were synthesized. We have assigned these to the Pst I modification (methylase) and restriction enzymes, respectively. The active form of the restriction enzyme is a dimer, as determined by gel filtration. Constructed transformants of P. stuartii 164 that carry the Pst I system inserted into pBR322 produce approximately 10 times more Pst I endonuclease activity than does the native strain.

Base Sequence↗
PubMed1980

Modification of hemoglobin with analogs of aspirin.

A variety of acyl esters of salicyclic acid and 3,5-dibromosalicylic acid have been prepared and examined for their ability to place the acyl group on hemoglobin. In general, short chain acyl groups (C2 and C3) are more reactive than longer chains (C4 to C10), but longer chains may be more effective with intact red cells because of their enhanced ability to permeate the erythrocyte membrane. The brominated salicyl esters also exhibit enhanced permeation of the membrane, as well as increased activity due to activation at the acyl site. Bis(salicyl) esters, nonbrominated and brominated, are more reactive than corresponding monoesters, and those from C4 dicarboxylic acids connect beta subunits by covalent bridges. These double-headed aspirins have the attractive features of being bound selectively by hemoglobin and of forming a covalent cross-link that may influence the conformation of the tetramer.

Aspirin↗
PubMed1979

Diaspirins that cross-link beta chains of hemoglobin: bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate.

Two double-headed aspirins, bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate, have been found to be potent acylating agents of intracellular hemoglobin (A or S) in vitro. Furthermore, each of these reagents cross-links beta chains of hemoglobin, probably at the beta cleft. The modified hemoglobins show increased oxygen affinities and reduced gelation or sickling tendencies.

Acylation↗
PubMed1979

Complementary carrier peptide synthesis: general strategy and implications for prebiotic origin of peptide synthesis.

A method for peptide synthesis is proposed based on a template-directed scheme that parallels that of the native ribosomal mechanism. In this procedure, peptide bond formation is facilitated by the juxtaposition of aminoacyl and peptidyl oligonucleotide carriers bound adjacent to one another on an oligonucleotide template. The general strategy of the synthesis and relevant model studies are described. The scheme provides an intrinsic mechanism by which oligonucleotides can direct the synthesis of polypeptides in the absence of protein or ribosomal machinery and, as such, suggests a model for the origin of prebiotic protein synthesis.

Biological Evolution↗