Analysis of the Expression of Repetitive DNA Elements in Osteosarcoma.

Osteosarcoma (OS) is a rare malignant bone tumor. It affects mostly young persons and has poor outcome with the present treatment. No improvement was observed since the introduction of chemotherapy. The better understanding of osteosarcoma development could indicate better management strategy. Repetitive DNA elements were found to play a role in cancer mechanism especially in epithelial tumors but not yet analyzed in osteosarcoma. We conducted the study to analyse the expression profile of repetitive elements (RE) in osteosarcoma. Methods: Fresh bone paired (tumor and normal bone) samples were obtained from excised parts of tumors of 18 patients with osteosarcoma. We performed sequencing of RNA extracted from 36 samples (18 tumor tissues and 18 normal bone for controls), mapped raw reads to the human genome and identified the REs. EdgeR package was used to analyse the difference in expression of REs between osteosarcoma and normal bone. Results: 82 REs were found differentially expressed (FDR < 0.05) between osteosarcoma and normal bone. Out of all significantly changed REs, 35 were upregulated and 47 were downregulated. HERVs (THE1C-int, LTR5, MER57F and MER87B) and satellite elements (HSATII, ALR-alpha) were the most significantly differential expressed elements between osteosarcoma and normal tissues. These results suggest significant impact of REs in the osteosarcoma. The role of REs should be further studied to understand the mechanism they have in the genesis of osteosarcoma.

Biosynthesis of tetrahydrobiopterin: a sensitive assay of 6-pyruvoyltetrahydropterin synthase using [2'-3H]dihydroneopterin 3'-triphosphate as substrate.

Pyruvoyltetrahydropterin synthase catalyzes the release of tritiated water from [2'-3H]dihydroneopterin 3'-triphosphate. The tritiated water formed during the enzymatic reaction is separated from substrate by adsorption of the latter to activated charcoal. The sensitivity and specificity of the assay allows the determination of the enzyme in crude cell extract.

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase.

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.

Biosynthesis of riboflavin: mechanism of formation of the ribitylamino linkage.

Feeding experiments with Ashbya gossypii followed by NMR analysis of the resulting riboflavin showed incorporation of deuterium from D-[2-2H]ribose at C-2' and from D-[1-2H]ribose in the pro-R position at C-1' of the ribityl side chain. The results rule out an Amadori rearrangement mechanism for the reduction of the ribosylamino to the ribitylamino linkage and point to formation of a Schiff base that is reduced stereospecifically opposite to the face from which the oxygen has departed. As prerequisite for the analysis, the 1H NMR signals for the pro-R and pro-S hydrogens at C-1' of 6,7-dimethyl-8-ribityllumazine and riboflavin and its tetraacetate were assigned with the aid of synthetic stereospecifically deuteriated samples.

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.

Biosynthesis of riboflavin. Incorporation of 13C-labeled precursors into the xylene ring.

Growing cultures of Ashbya gossypii were supplemented with various 13C-labeled precursors including [1-13C]acetate, [2-13C]acetate, [1-13C]ribose, [1-13C]glucose, [6-13C]glucose, and [2-13C]glycerol. Riboflavin was isolated from the culture medium, chemically converted to riboflavin tetraacetate, and analyzed by 13C NMR spectroscopy. The xylene ring of riboflavin is formed by dismutation of 6,7-dimethyl-8-ribityllumazine, thus the 8 carbon atoms of the riboflavin xylene ring are composed of 4 biochemically different carbon atoms which are duplicated in the dismutation. The formation of the lumazine from a pyrimidine precursor requires the addition of these 4 carbon atoms which constitute C-6 alpha, C-6, C-7, and C-7 alpha of the lumazine. Results from the present work indicate that these 4 carbon atoms do not arise from acetate, diacetyl, acetoin, or a tetrose, nor from loss of 1 or 2 carbon atoms from either end of a pentose or hexose. Additionally, the data do not support the recent contention that these 4 atoms arise via a dismutation of the pyrimidine precursor of the lumazine. The findings show that there exists a close correspondence between the lumazine carbons 6 alpha, 6, and 7 with C-1, C-2, and C-3, respectively, of a pentose, while C-7 alpha corresponds to C-5 rather than C-4 of a pentose. This in conjunction with results reported earlier indicates an intramolecular rearrangement involving carbons 3, 4, and 5 of a pentose or its biochemical equivalent.