Biomedical subjects

P Jahnke

Publications and source records attributed to P Jahnke.

6 recordsLinked to original sources

PubMed1979

Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens.

The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274--4284 of bacteriophage phi X174 viral DNA , and pCCCAGCCTAAA, which corresponds to the same sequence with a C leads to A change at the ninth nucleotide, were synthesized enzymatically. The second of these oligonucleotides was used as a primer for E. coli DNA polymerase I, from which the 5'-exonculease has been removed by proteolysis (Klenow enzyme), on wild-type phi X174 viral DNA template. After ligation, this yielded closed circular heteroduplex DNA with a G, A mismatch at nucleotide 5276. Transfection of E. coli spheroplasts with the heteroduplex DNA produced phage mutated at this nucleotide (G leads to T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am16, was reverted (T leads to G) by the wild type oligonucleotide with an efficiency of 19%. The nucleotide changes were established by sequence determination of the mutated viral DNA using the enzymatic terminator method. The production of specific transversion mutations, together with a previous demonstration of specific transition mutations (1), established that short enzymatically synthesized oligodeoxyribonucleotides can be used to induce any class of single nucleotide replacement with high efficiency and thus provide a powerful tool for specific genetic manipulations in circular genomes like that of phi X174.

Bacteriophage phi X 174↗

PubMed1978

Mutagenesis at a specific position in a DNA sequence.

Predefined changes in a known DNA sequence were introduced by a general method. Oligodeoxyribonucleotides complementary to positions 582 to 593 of the viral DNA strand of the bacteriophage phiX174 am3 mutant (pGTATCCTACAAA), and to the wild type sequence in this region (pGTATCCTACAAA), were synthesized and used as specific mutagens. Each of these oligonucleotides was incorporated into a complete circular complementary strand when used as primer on a genetically heterologous viral strand template, by the combined action of subtilisin-treated Escherichia coli DNA polymerase I and T4 DNA ligase. Incomplete duplexes were removed or were inactivated by nuclease S1 and the products were used to transfect spheroplasts of E. coli. Both oligonucleotides induced specific mutations at high efficiency when used with heterologous template (15% mutants among progeny phage). The am phages isolated by this procedure are phenotypically gene E mutants, and contain A at position 587 of the viral strand. They thus appear identical with am3 and provide evidence that the change G leads to A at position 587 is sufficient to produce a defective E function. Since the template for the induction of am mutants carried another genetic marker (sB1), the strains carrying the induced mutations have the new genotype am3 sB1. It should be possible to introduce the am3 mutation into any known mutant strain of phi174 using this same oligonucleotide. Both possible transition mutations were induced in these experiments. In principle, the method could also induce transversions, insertions, and deletions. The method should be applicable to other circular DNAs of similar size, for example recombinant DNA plasmids.

Base Sequence↗

PubMed1978

Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence.

Procedures for the controlled addition of one or more deoxyribonucleotide residues to the 3' end of an oligodeoxyribonucleotide primer are described. Polynucleotide phosphorylase (EC 2.7.7.8), purified from Escherichia coli B, catalyzes the reaction using a deoxyribonucleoside 5'-diphosphate as substrate, with Mn2+ as cofactor. Reaction occurs rapidly in aqueous solution, and no protecting groups are required, simplifying recovery and purification of the products. The concentrations of sodium chloride and manganous chloride in the incubation mixture are critical to obtaining good yield of the required product. Primers of chain length from 3 to 12 have been extended by up to 9 deoxyribonucleotide residues to obtain oligodeoxyribonucleotides of chain length up to 13. Yields of single addition products varied from 8 to 59%. Factors which influence these yields are discussed. The effects of added polyamines and some organic solvents on the reaction are described. Spermidine or dimethylsulfoxide in the incubation medium tend to favor the addition of several residues of deoxyribonucleotide to the primer.

Base Sequence↗

PubMed1977

Enzymatic synthesis of oligonucleotides of defined sequence: synthesis of a segment of yeast iso-1-cytochrome c gene.

The deoxyribooligonucleotide, d(pT-T-A-G-C-A-G-A-A-C-C-G-G), constituting a segment of yeast iso-1-cytochrome c gene, has been synthesized by a combination of chemical and primarily enzymatic methods. The starting primer, d(pT-T-A-G1, was chemically synthesized by the phosphodiester method and was extended stepwise, by reactions catalyzed by polynucleotide phosphorylase.

Cytochrome c Group↗

PubMed1975

The synthesis of the internucleotide (phosphodiester) bond by a base-catalysed reaction.

Potassium tert-butoxide in hexamethyl phosphoramide and dimethyl formamide provides an excellent catalyst for the reaction of a 3'-hydroxyl or a 5'-hydroxyl group of a nucleoside with an appropriate nucleoside phosphorofluoridate to yield the dinucleoside phosphate. This paper describes the experiments leading to the development of this reaction together with the synthesis of thymidylyl-(5' leads to 3')-thymidine (dT-dT).

Methods↗

PubMed1975

Application of base-catalysed reaction to the synthesis of dinucleotides containing the four common deoxyribonucleosides and of oligodeoxythymidylates.

The phosphorylation of nucleosides by a nucleoside phosphorofluoridate in the presence of potassium tert-butoxide is a very effective reaction for internucleotide bond synthesis in the case of pyrimidine deoxynucleosides. However, for the purine deoxynucleosides, yields are reduced due to competing reactions. The method was applied to the stepwise synthesis of oligothymidylates. The yield of the trinucleotide was good whereas that of the tetranucleotide was reduced due to the insolubility of the intermediate trinucleotide in the presence of potassium tert-butoxide.

Fluorides↗