Hans-Joachim Breter

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. II. Advantages of a variable-wavelength HPLC spectrophotometric detector for the determination of 6-thiopurines.

The advantages of a variable-wavelength spectrophotometric detector for use in high-performance liquid chromatography are demonstrated with the detection of 6-mercaptopurine metabolites in cell extracts. 6-Methylthiopurines, unsubstituted 6-thiopurines and 6-thioguanines are most sensitively detected at 291, 322, and 342 NM, respectively. Compared with detection at 254 nm, the sensitivity at these wavelengths is about one to two orders of magnitude greater. Furthermore, in the 291-355-nm range, common purines, which are normally contained in cell extracts as free bases, nucleosides and nucleotides exhibit only minute absorbances and so do not interfere in quantitative determinations of 6-thiopurine compounds.

Single-step separation of major and rare ribonucleosides and deoxyribonucleosides by high-performance liquid cation-exchange chromatography for the determination of the purity of nucleic acid preparations

A method is described for the separation of 13 major and minor ribo- and deoxyribonucleosides in a single chromatographic run using high-performance liquid chromatography on strongly acidic cation-exchange columns. The method proved useful for the routine determination of small amounts of ribonucleic acid impurities in deoxyribonucleic acid preparations and vice versa. About 3% or even less of nucleic acid contamination in a given sample can be easily detected and quantitatively determined under the conditions used.

Differential polyadenylation pattern of ovalbumin precursor RNAs during development

The expression of the ovalbumin gene encoding for the major hen oviduct protein slows down with age. Analysis of Northern blots of electrophoretically separated total and poly(A)+ RNA from oviducts of hens of different age with an ovalbumin-specific probe (nick-translated 9.5 kb ovalbumin gene DNA cloned into pBR322) revealed that the largest high molecular weight ovalbumin RNA precursor (7.9 kb band, representing the putative primary transcript of the ovalbumin gene) was most intense if total RNA from non-egg-laying old hen oviduct was checked as compared to that from egg-laying mature animals. On the other side, the 7.9 kb RNA precursor band was readily detected in the poly(A)+ RNA from mature hen oviduct where-as it was invisible in the old hen oviduct poly(A)+ RNA fraction. The lack of detection of the 7.9 kb RNA species within the poly(A)+ RNA fraction and its increased concentation within the total oviduct RNA, both from old animals, suggest that age-dependent impairment of ovalbumin mRNA processing may be caused by altered polyadenylation of distinct RNA precursors.

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. I. A separation method for purine and 6-thiopurine bases and nucleosides using high-pressure liquid cation-exchange chromatography.

Abstract In the course of determining 6-mercaptopurine (6MP) metabolites formed in 6MP-treated cultured cells or in animal tissues, on the base and (deoxy-)-ribonucleoside levels a method has been established for the separation and quantitative determination of picomole amounts of 15 purine and 6-thiopurine compounds and of 2 oxidized 6-thiopurines using high-pressure liquid cation-exchange chromatography. A 0.18 × 100-cm column filled with strongly acidic Beckman M71 exchange resin is eluted with 0.4 m ammonium formate, pH 4.6, at a flow velocity of 5.2 cm·min −1 and at 50°C column-oven temperature. After 75 min, the flow velocity is doubled. A separation is finished within 140 min. The compounds are eluted in the series: 6-thiouric acid + 8-hydroxy-6MP (within the void fraction), uric acid, xanthosine, inosine, xanthine, 6MP ribonucleoside, hypoxanthine, 6MP deoxyribonucleoside, 6-thioguanosine, 6MP, guanine, 6-methylmercaptopurine ribonucleoside, 6-thioguanine, 6-methylthioguanine, and 6-(1-methyl-4-nitro-imidazole-)MP. Using a 0.18 × 40-cm column at a flow velocity of 8.7 cm·min −1 , either the 6-thiopurine bases or the 6-thiopurine ribonucleosides are separated from each other within 30 and 80 min, respectively. About 30 pmol of 6MP and 15 pmol of 6-thioguanosine have quantitatively been detected with a variablewavelength uv detector. The most sensitive determination is achieved at 254 nm for common purines, at 291 nm for 6-methylthiopurines, at 322 nm for 6-thiopurines, and at 342 nm for 6-thioguanines.

A highly sensitive method for the estimation of pyrimidine dimers in DNA by high-pressure liquid cation-exchange chromatography.

Abstract We have developed a rapid, highly sensitive method for the separation of thymine-dimers from thymine on a cation-exchange resin, which is capable of detecting one dimer out of 5 × 103 to 104 thymine molecules (depending on the specific activity of the used material) by the means of high-pressure liquid chromatography. The assay has been very useful in DNA-hybridization studies using ( TT ) -containing DNA-strands and there is evidence that the method will be valuable for photoreactivation studies of uv-damaged DNA and for the enzymes involved in dimer-excision studies.

Evidence for age-dependent impairment of ovalbumin heterogeneous nuclear RNA (HnRNA) processing in hen oviduct

The expression of the ovalbumin gene in hen oviduct decreases during ageing. Northern transfer of electrophoretically fractionated total RNA from oviducts of mature egg-laying and old non-egg-laying hens, and hybridization to nick-translated plasmid pOV230 (ovalbumin complementary DNA cloned into pMB9) revealed with age increasing concentrations of distinct high-molecular-weight ovalbumin pre-mRNA species as compared with that of functional 18S ovalbumin mRNA. The accumulation of ovalbumin sequence-containing processing intermediates in old hen oviducts indicates lowered rates of RNA splicing and/or altered steady state concentrations of splicing intermediates during post-transcriptional maturation of heterogeneous nuclear RNA in old animals.

The quantitative determination of metabolites of 6-mercaptopurine in biological materials III. The determination of 14C-labeled 6-thiopurines in L5178Y cell extracts using high-pressure liquid cation-exchange chromatography

A method is presented for the separation of 6-thiopurine bases and ribonucleosides, of sulphate anions and of common purine bases and oxidized purines by means of high-pressure liquid cation-exchange chromatography using a 0.18 X 100 cm column, filled with Beckman M71 resin, and eluted with 0.4M ammonium formate, pH 4.6, at a linear flow velocity of 5.2 cm/min at 50 degrees C. The method has been applied to the separation and quantitative determination of 14C-labeled 6-mercaptopurine metabolites in HClO4 extracts of L5178Y murine lymphoma cells. Distribution patterns of 14C radioactivity within the cells after a 24 h incubation period with (8-14C)-labeled 6-mercaptopurine have been established. The indentification of 6-mercaptopurine metabolites, such as 6-thioxanthosine ribonucleotide, 6-thioinosinic acid, 6-thioguanylic acid, 6-methylthioinosinic acid, and 6-thiouric acid, after the digestion of the extracts with alkaline phosphatase has been confirmed using the behaviour of each compound in enzymatic peak-shifting analyses with purine nucleoside phosphorylase and the corresponding elution volumes of 6-thiopurine bases and ribonucleosides as proofs. According to the specific radioactivity of the (8-14C)-labeled 6-mercaptopurine batch, the amounts of the various 6-mercaptopurine metabolites in about 6% of the total HClO4 extract of 1.6 . 10(8) labeled cells have quantitatively been determined as 1--130 pmol. The intracellular concentration of 6-thiopurines was determined at 1.4 . 10(-5)mol/1.

The mapping of chromosomes in Saccharomyces cerevisiae. I. A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome.

A series of vectors for cosmid cloning in yeast has been derived from cosmid pHC79. Vectors pMT4 through pMT6 contain two tandemly arranged cohesive end sites (cos) from the genome of bacteriophage lambda. Their design allows the rapid and simple preparation of cosmid arms by linearizing a vector at the unique PvuII-restriction site located between the two cos-sequences and then cutting the linearized molecule at one of its unique cloning sites for BamHI, ClaI, PvuI, SalI or ScaI. Cosmids generated with arms from the most advanced vector, pMT6, carry the origin of replication (ori) and the ApR gene from pBR322 and the TRP1/ARS1 and URA1 genes from Saccharomyces cerevisiae. A yeast genomic DNA library was established by packaging in vitro, into bacteriophage lambda preheads, of partially restricted yeast DNA fragments ligated to cosmid arms of vector pMT6. About 80% of the clones thus obtained comprise inserts of contiguous genomic DNA over 30 kb in length. Unique DNA probes for the yeast genes CDC10, CDC39, HIS4, LEU2, and PGK1 have successfully been applied when testing for completeness of this library by isolating a series of overlapping cosmid clones that carry the respective genes. The library will thus be useful for the selection of cosmid clones which carry CDC genes from yeast by complementing first, with the vectorial yeast gene URA1, the pyrimidine auxotrophy of most cdc-strains and then, with the respective CDC wild-type genes, of the temperature-sensitive mutant alleles. Most CDC clones thus obtained will provide unique DNA probes which serve as randomly distributed start sequences within the yeast genome for overlap hybridization screening in chromosome mapping studies.

The Determination of the DNA Base Composition in 19 Species of Adriatic Sponges with High-Pressure Liquid Cation-Exchange Chromatography

Abstract The (adenine + thymine)/(guanine + cytosine) base ratios of 19 species of adriatic sponges have been determined by high-pressure liquid cation-exchange chromatography. The base ratios vary from 1.49 (Mycale massa) to 0.63 (Hippospongia communis) according to an (A+T) content of 59.7 and 38.6 mol%, respectively. The DNAs of sponges of the order Keratosa showed marked differences in their (A +T) contents (39.5 to 58.8 mol%) whereas those of Tetractinellida and Halichondrina were nearly identical (39.3 to 40.8 and 49.5 to 49.8 mol%, respectively). The 5-methylcytosine (5MC) content was determined in 8 sponge DNAs by a semiquantitative method. The values differed from 0.8 to 2.2 mol% of 5MC.

Alteration of DNA reassociation kinetics due to base mismatch caused by thymine dimerisation

Abstract Base mismatch was introduced into Escherichia coli DNA by photodimerisation between contiguous thymine molecules. With increasing dimer content both the melting temperature and the reassociation velocity of the DNA decrease. The reassociation rate is reduced by a factor of approx. 2 when the melting temperature is lowered by 5°C. Since the dimers are distributed statistically over the DNA molecules, in a DNA sample, molecules with different dimer content and hence with different reassociation rates will occur. Consequently, molecules with a dimer content below the average will reassociate preferentially early during the course of reassociation while molecules with high dimer content will reassociate later. During the progress of the reaction the average dimer content will, therefore, increase in the single-stranded fraction. Base mismatch does not only reduce the rate of reassociation but also causes a deviation from second-order kinetics.