The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

Crystallization and preliminary analysis of the deoxyoligonucleotide d(CGTAGATCTACG)

Two crystal forms of the self-complementary DNA 12-mer d(CGTAGATCTACG) were grown by the vapour diffusion technique. Form I is in space group C2 with a = 64.8 A, b = 35.4 A, c = 24.4 A and beta = 92.2 (1 A = 0.1 nm). The crystals are grown as monoclinic blocks or hexagonal plates. There are two strands (one duplex) in the asymmetric unit. Form II crystallizes as monoclinic blocks, space group P21 with a = 64.5 A, b = 35.1 A, c = 25.2 A and beta = 91.8 degrees. This form contains four strands (2 duplexes) in the asymmetric unit. Both forms are suitable for high resolution X-ray analysis. The diffraction patterns suggest that the DNA is in a B-type conformation and that the packing in the two forms is very similar.     

Peroxisomal oxidation of thiazolidine carboxylates in firefly fat body, frog retina, and rat liver and kidney.

D-amino acid oxidase is a widely distributed peroxisomal enzyme whose principal natural substrates are still unknown. Thiazolidine carboxylates, their derivatives and relatives, and the intermediates in their metabolism are among the more plausible substrate candidates. Using a cytochemical procedure, we have explored the distribution of peroxide-generating enzymatic activity against two thiazolidine carboxylates. We find that these compounds are effective substrates for peroxisomal oxidation in a variety of tissues that contain peroxisomal D-amino acid oxidase. Reaction was seen in the "classical" peroxisomes of rat liver and kidney, the peroxisomes of the fat body of firefly and of Drosophila and the peroxisomes of frog retina. Interestingly, both with the thiazolidine compounds and with more traditional D-amino acid oxidase substrates, the fireflies' photocyte granules, which are peroxisomes, lack activity.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

Refined crystal structure of an octanucleotide duplex with G . T mismatched base-pairs

Single crystal X-ray diffraction techniques have been used to determine the structure of the DNA octamer d(G-G-G-G-C-T-C-C) at a resolution of 2.25 A. The asymmetric unit consists of two strands coiled about each other to produce an A-type DNA helix. The double helix contains six G . C Watson-Crick base-pairs and two G . T mismatched base-pairs. The mismatches adopt a "wobble" type structure in which both bases retain their major tautomer forms. The double helix is able to accommodate this G . T pairing with little distortion of the overall helical conformation. Crystals of this octamer melt at a substantially lower temperature than do those of a related octamer also containing two G . T base-pairs. We attribute this destabilization to disruption of the hydration network around the mismatch site combined with changes in intermolecular packing. Full details are given of conformational parameters, base stacking, intermolecular contacts and hydration involving 52 solvent molecules.     

The crystal structure of d(G-G-G-G-C-C-C-C). A model for poly(dG).poly(dC)

The structure of the DNA oligomer d(G-G-G-G-C-C-C-C) has been determined at a resolution of 2.5 A by single-crystal X-ray methods. There are two strands in the asymmetric unit, and these coil about each other to form a right-handed double-helix of the A-type with Watson-Crick hydrogen bonds between base-pairs. The helix has a shallow minor groove and a deep, water-filled major groove; almost all exposed functional groups on the DNA are hydrated, and 106 ordered solvent molecules have been found. The two d(G-G-G-G).d(C-C-C-C) segments in the octamer exhibit similar and uniform structures, but there is a slight discontinuity at the GpC step between them. A recurring feature of the structure is the overlap of adjacent guanine bases in each GpG step, with the five-membered ring of one guanine stacking on the six-membered ring of its neighbour. There is little or no overlap between adjacent cytosine rings. Conformational parameters for these GpG steps are compared with those from other single-crystal X-ray analyses. In general, GpG steps exhibit high slide, low roll and variable twist. Models for poly(dG).poly(dC) were generated by applying a simple rotation and translation to each of the unmodified d(G-G-G-G).d(C-C-C-C) units. Detailed features of these models are shown to be compatible with various assays of poly(dG).poly(dC) in solution, and are useful in understanding the polymorphic behaviour of this sequence under a variety of experimental conditions.     

Structural characterisation of the bromouracil.guanine base pair mismatch in a Z-DNA fragment.

The deoxyoligonucleotide d(BrU-G-C-G-C-G) was crystallised at pH 8.2 and its structure analysed by X-ray diffraction. The unit cell, of dimensions a = 17.94, b = 30.85, c = 49.94A contains four DNA duplexes in space group P2(1)2(1)2(1). The duplexes are in the Z conformation, with four Watson-Crick G.C base pairs and two BrU.G base pairs. The structure was refined to an R factor of 0.16 at a resolution of 2.2A with 64 solvent molecules located. The BrU.G base pair mismatch is of the wobble type, with both bases in the major tautomer form and hydrogen bonds linking 0-2 of BrU with N-1 of G and N3 of BrU with 0-6 of G. There is no indication of the presence of ionised base pairs, in spite of the high pH of crystallisation. The results are discussed in terms of the mutagenic properties of 5- bromouracil.     

Structural features and hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a double helix containing two G.A mispairs

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5A resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-1 and O-6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

Ordered water structure in an A-DNA octamer at 1.7 A resolution

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.     

Genetic analysis of morphological variation in Brassica oleracea using molecular markers

A cross between the open-pollinated Brassica oleracea cabbage cultivar Wisconsin Golden Acre and the hybrid broccoli cultivar Packman was used with molecular markers to investigate the genetic control of morphological variation. Twenty-two traits derived from leaf, stem, and flowering measurements were analyzed in 90 F₂ individuals that were also classified for genotype by restriction fragment length polymorphism (RFLP) markers. Seventy-two RFLP loci, which covered the mapped genome at an average of 10 map-unit intervals on all nine linkage groups, were tested individually for associations to phenotypic measurements by single factor ANOVA, and markers with significant associations (P<0.05) were used to develop multilocus models. These data were utilized to describe the location, parental contribution of alleles, magnitude of effect, and the gene action of trait loci. Single marker loci that were significantly associated (P<0.05) with trait measurements accounted for 6.7–42.7% of the phenotypic variation. Multilocus models described as much as 60.1% of the phenotypic variation for a given trait. In some cases, different related traits had common marker-locus associations with similar gene action and genotypic class ranking. The numbers, action, and linkages, of genes controlling traits estimated with marker loci in this population corresponded to estimates based on classical genetic methods from other studies using similar, or similarly-wide, crosses. There was no evidence that genome duplication accounted for a significant portion of multiple genes controlling trait loci over the entire genome, but possible duplications of trait loci were identified for two regions with linked, duplicated marker loci.