Paul Unrau

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Retired genetics/molecular biology with interests in recombination models and radiation damage and reapir. Also interests in genetic factors in endogenous radiation sensitivity, and endogamy in repair sensitivity and segregation in interbreeding demes.

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Non-cloning amplification of specific DNA fragments from whole genomic DNA digests using DNA ‘indexers’

Gene, 1994

A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class... more A highly systematic, non-cloning method of distinguishing and isolating every fragment in a class-IIS or interrupted palindrome restriction digest has been developed in our laboratory. These enzymes produce informative, non-identical cohesive ends which can be selectively modified by ligation to individual synthetic oligodeoxyribonucleotides with the corresponding complementary ends. In this way, polymerase chain reaction and sequencing primer sites and labels can be introduced specifically into a single fragment in a total genomic digest. Known and unknown fragments from genomes of the complexity of Escherichia coli can be isolated directly in sequencable form without the necessity of synthesizing unique primers. Human DNA has also been assessed in this way. Problems intrinsic to cloning (selective fragment loss, mutation and sequence rearrangement) are avoided. Systematic characterization of DNA fragments by their cohesive ends and length provides tremendous power and flexibility for analysis of any DNA molecule without specific clones, probes or libraries. We report proof of principle of this remarkable system and indicate potential applications in DNA sequence tagged site and restriction mapping, sequencing, restriction-fragment-length polymorphism analysis and DNA diagnostics.

The excision of pyrimidine dimers from DNA of ultraviolet irradiated yeast

Molecular and General Genetics MGG, 1971

It is shown that pyrimidine dimers formed by ultraviolet light in the DNA of haploid Saccharomyce... more

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Chapter 24 Induction and Isolation of Cold-Sensitive Lines of Chinese Hamster Cells

Methods in Cell Biology, 1976

Publisher Summary It is possible, using modifications of standard procedures for the selection of... more Publisher Summary It is possible, using modifications of standard procedures for the selection of heat-sensitive variants, to obtain cold-sensitive mammalian cell lines. The frequency of ribosomal subunit assembly defective mutants among these lines is apparently not as high as in prokaryotes. These cold-sensitive mammalian lines should however, provide an additional source of cells with conditional defects in essential cellular functions. Cold-sensitive mutants of prokaryotes have been a valuable source of cells with defects in essential cellular processes. Some of these cold-sensitive mutations are in genes for which no heat-sensitive counterparts have been discovered. Cold-sensitive strains of bacteria include an especially high frequency of ribosomal subunit assembly defective mutants. This phenomenon is probably explained by the observation that ribosome assembly is a highly temperature-dependent process. Subunit assembly defective mutants also appear among cold-sensitive strains of fungi, but with a much lower frequency than in bacteria. The mutagenesis and selection temperatures were reversed, and the times for each treatment were altered to compensate for the difference in the growth rates of wild-type cells at high and low temperatures.

The formation of pyrimidine dimers in the DNA of fungi and bacteria

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1973

The dose-related yields of the three kinds of pyrimidine dimer induced in DNA by ultraviolet ligh... more The dose-related yields of the three kinds of pyrimidine dimer induced in DNA by ultraviolet light of 254 nm have been measured in three species of bacteria (Escherichia coli, Micrococcus luteus and Bacillus subtilis) and two species of fungi (Saccharomyces cerevisiae and Ustilago maydis). The yield in each organism is related to its base composition. When allowance is made for differing nearest-./N /'~ ./N neighbour frequencies, it is found that CC, CT and TT dimers form in the approximate ratios of 1 : 1:2 at doses of ultraviolet light below 2000 ergs/mm 2. Dimers are formed in native DNA at twice the rate they are formed in cells.

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The REC1 Gene of Ustilago maydis, Which Encodes a 3′ → 5′ Exonuclease, Couples DNA Repair and Completion of DNA Synthesis to a Mitotic Checkpoint

Genetics, 1996

Mutation in the REC1 gene of Ustilago maydis results in extreme sensitivity to killing by ultravi... more Mutation in the REC1 gene of Ustilago maydis results in extreme sensitivity to killing by ultraviolet light. The lethality of the rec1-1 mutant was found to be partially suppressed if irradiated cells were held artificially in G2-phase by addition of a microtubule inhibitor. This mutant was also found to be sensitive to killing when DNA synthesis was inhibited by external means through addition of hydroxyurea or by genetic control in a temperature-sensitive mutant strain defective in DNA synthesis. Flow cytometric analysis of exponentially growing cultures indicated that wild-type cells accumulated in G2 after UV irradiation, while rec1-1 cells appeared to exit from G2 and accumulate in G1/S. Analysis of mRNA levels in synchronized cells indicated that the REC1 gene is periodically expressed with the cell cycle and reaches maximal levels at G1/S. The results are interpreted to mean that a G2-M checkpoint is disabled in the rec1-1 mutant. It is proposed that the REC1 gene product fun...

Isolation of cold-sensitive Chinese hamster cells

Molecular and General Genetics, 1975

Six cold-sensitive variants have been isolated from Chinese hamster ovary cells by the BUdR-visib... more Six cold-sensitive variants have been isolated from Chinese hamster ovary cells by the BUdR-visible light selection technique. The properties of one of these lines have been studied in detail. This line stops dividing immediately after a shift from 39° C to 33° C though its doubling time at 39° C is only slightly longer than that of wild-type cells. The rates of DNA and protein synthesis are severely reduced at 33° C, but the rate of RNA synthesis is not significantly different from wild-type cells. This line may be defective in protein synthesis, but the results of sedimentation analysis indicate that it probably has normal ribosomal subunit assembly.

Isolation of cold-sensitive Chinese hamster cells

Molecular and General Genetics, 1975

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

Molecular and General Genetics, 1977

The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutan... more The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungusUstilago maydis after incubation at the restrictive temperature (32° C) for eight hours. Mutantsts-220,ts-207,ts-432 andts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutantsts-20,tsd 1-1,ts-84 andpol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutantpol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutantts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32°C.tsd 1-1 andts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis with correlates to increasing UV sensitivity of these strains on incubation at 32° C. Apol 1-1ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

format_quoteTemperature-sensitive mutants show reduced nuclear and mitochondrial DNA synthesis, highlighting mitochondrial enrichment despite DNA replication blocks.format_quote

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Recombination during blocked chromosome replication in temperature-sensitive strains of Ustilago maydis

Genetics Research, 1972

A search for temperature-sensitive mutants of Ustilago maydis blocked in DNA synthesis

Genetics Research, 1970

Methods for the assay of ultraviolet light-induced pyrimidine dimers in Saccharomyces cerevisiae

Biochimica Et Biophysica Acta (bba) - Nucleic Acids and Protein Synthesis, 1972

Pyrimidine dimers induced in DNA by ultraviolet light were detected and assayed in yeast labelled... more Pyrimidine dimers induced in DNA by ultraviolet light were detected and assayed in yeast labelled with [14Cluracil. The method required an efficient extraction system, prior to the hydrolysis of acid-precipitable DNA, which involved both the complete removal of labelled RNA and a pronase digestion. Ultraviolet light photoproducts of three different kinds were separated from one another and from all native pyrimidines in suitable chromatographic systems. They were shown to be dimers of UU, UT and TT by comparing the RF values with artificial preparations of dimers and by the recovery of monomers in the appropriate quantities from reirradiated eluates of the photoproduct regions of the chromatograms. The methods described are likely to be suitable for the analysis of repair processes in yeast and other organisms which do not incorporate exogenous thymine as a precursor specific to DNA.

format_quoteNo yield difference in pyrimidine dimers across extraction methods, demonstrating the robustness of the extraction procedure used in the study.format_quote

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Paul Unrau

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