A convenient approach to the synthesis of medium size oligodeoxyribonucleotides by improved new phosphite method.

Improvement of the new phosphite method for the synthesis of oligodeoxyribonucleotides using the deoxyribonucleoside 3'-bis(1,1,1,3,3,3- hexafluoro-2-propyl) phosphite unit has been carried out via the hydrolysis and capping steps, without any side reaction products. The new phosphite unit and capping agent, bis(1,1,1,3,3,3-hexafluoro-2-propyl)-2-propyl phosphite, is readily activated by N-methylimdazole under very mild condition on a solid support. This operation involves a one pot reaction, which is an advantage over both the phosphite and H- phosphonate approaches. The mechanism of internucleotidic bond formation of the new phosphite method is also discussed.     

Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.     

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.     

Polyol pathway in tissues of spontaneously diabetic Chinese hamsters (Cricetulus griseus) and the effect of an aldose reductase inhibitor, ONO-2235

1. Sorbitol and fructose levels were significantly elevated in the lens, the sciatic nerve, the retina and the kidney of diabetic Chinese hamsters and inositol level was significantly decreased in the lens and sciatic nerve of diabetics. 2. The activity of an aldose reductase in the kidney was not different between normal and diabetic Chinese hamsters. 3. An aldose reductase inhibitor (ONO-2235) had no effect in sorbitol, fructose and inositol contents of all these tissues from diabetic Chinese hamsters. 4. These results suggest that diabetic Chinese hamsters produce polyol accumulation in tissues but that there is a clear species-specific difference to inhibition of aldose reductase.     

Interstrain differences in red cell enzyme activities in mice and rats.

1. Interstrain differences in red blood cell enzyme activities were studied in mice (BALB/c, C57BL/6, C3H/He, DBA/2 and ddY) and rats (Donryu, F344/N, SD, Wistar and Wistar/ST), and were also compared with hamster, guinea-pig and rabbit. 2. The enzyme activities measured were: glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD), NADPH-diaphorase (ND), hexokinase (Hx), glutamate oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE). 3. There were marked variations in the activities of some red cell enzymes (e.g. GST, Hx, ND), while others (e.g. G-6-PD, 6-PGD) were much less variable both within different strains and species.     

A YAC contig across the fragile X site defines the region of fragility.

The fragile X syndrome is a common cause of mental retardation and is associated with a fragile site at Xq27.3 (FRAXA). Recently, evidence has been presented for the role of methylation and genomic imprinting in the expression of the disease. We have identified a site of methylation in patients by long range restriction mapping of the region. In this paper we present a YAC contig of this area, localise the CpG sequences which are methylated, and show by in situ hybridisation that the site of fragility lies within this region.