Importance of ornithine transcarbamylase (OTC) deficiency in small intestine for urinary orotic acid excretion: analysis of OTC-deficient spf-ash mice with OTC transgene.

We report the effect of the ornithine transcarbamylase (OTC) transgene composed of 1.3 kb of the 5' flanking region of the rat OTC gene fused to rat OTC cDNA on urinary orotic acid excretion in OTC-deficient spf-ash (sparse-fur with abnormal skin and hair) mice during overnight-starvation and nitrogen loading. During starvation, spf-ash mice with about 6% and 2% of control levels of OTC activity in the liver and small intestine excreted a large amount of orotic acid in the urine. Transgenic spf-ash mice with about 10% and 30% of the control OTC activities in the liver and small intestine did not excrete more than the normal level of orotic acid. Accidental parasitization of transgenic spf-ash mice with ticks (Myocoptes musculinus) resulted in decrease of the OTC activities in the liver and small intestine to the levels in spf-ash mice, and increased excretion of orotic acid. During extermination of the ticks, the mice showed varied levels of OTC activity and orotic acid excretion. On nitrogen loading, transgenic spf-ash mice as well as spf-ash mice excreted larger amounts of orotic acid, while control mice showed no increase in its excretion. The levels of urinary orotic acid were inversely correlated to the logarithms of the OTC activities in the liver and small intestine, the correlation being significantly higher with intestinal OTC than with hepatic OTC activity. These results suggest that the level of OTC activity in the small intestine is important for production of orotic acid.

Purification and characterization of an acidophilic xylanase from Aureobasidium pullulans var. melanigenum and sequence analysis of the encoding gene.

An extracellular endo-1,4-beta-xylanase was purified from the culture supernatant of Aureobasidium pullulans var. melanigenum (ATCC 20524) grown on oat-spelt xylan. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 24 kDa and had an isoelectric point of 6.7. Xylanase activity was optimal at pH 2.0 and 50 degrees C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. Southern blot analysis indicated that the xylanase gene (xynI) was present as a single copy in the genome. An open reading frame, consisting of 663 bp, encoded a presumed prepropeptide of 34 amino acids and a mature protein of 187 amino acid. The DNA region encoding the prepeptide was interrupted by a 59-bp intron. A single transcription start point was observed at position -46 (A) from the start codon. The 5'-noncoding region had a putative TATA box at -91 (TATATAA) and two possible CCAAT boxes at -247 (CAAT) and -283 (CCAAT). A cloned xynI cDNA was expressed in Saccharomyces cerevisiae. The deduced amino acid sequence showed 94% identity with that of a previously reported equivalent gene (xynA) encoding a xylanase with an optimal pH of 4.8 from a color variant strain, NRRL Y-2311-1, of A. pullulans. A neighbor-joining tree showed that the Aureobasidium enzymes are closely related to several other family-11 xylanases from black aspergilli and Penicillium purpurogenum.