Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of humanmouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p ⁺ (7qter>7p22::Xq21>Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-q21.Preliminary report of this research was presented at Ninth International Workshop on Human Gene Mapping, Abstract supplement p. 5 (1987).     

Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.

Messenger RNA of rat ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, was enriched by immunoprecipitation of rat liver free polysomes, and recombinant plasmids were prepared from the enriched mRNA by a vector-primer method. The cDNA clones for ornithine carbamoyltransferase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pOTC-1, contained a 1.6-kilobase insert and hybridized to a mRNA of approximately equal to 1.8 kilobases in rat liver. The cDNA clone was subjected to nucleotide sequence analysis. The deduced amino acid sequence indicates that the ornithine carbamoyltransferase precursor consists of the mature enzyme of 322 amino acid residues and an NH2-terminal peptide extension (presequence) of 32 amino acid residues. The presequence contains 8 basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The amino acid sequence of the rat ornithine carbamoyltransferase was compared with the recently reported sequence of the human enzyme [Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W. & Rosenberg, L. E. (1984) Science 224, 1068-1074]. The sequences of the mature enzyme portion are 93% identical, whereas those of the presequences are 69% identical. There are two highly conserved segments in the presequences of the rat and human enzymes. One of the two conserved segments is significantly similar to a segment of the presequence of yeast mitochondrial elongation factor EF-Tu. These results suggest that the homologous segments are important for the proteins that are synthesized in the cytosol to be transported into the mitochondrial matrix.     

Depression of liver-specific gene expression in regenerating rat liver: a putative cause for liver dysfunction after hepatectomy.

We carried out studies on the expression of liver-specific genes during regeneration of the liver and searched for changes in the expression of oncogenes and housekeeping genes. Albumin and ornithine transcarbamylase genes were the liver-specific genes examined by Northern blot analysis, using total RNAs isolated from residual livers of Sprague-Dawley rats subjected to a 68% partial hepatectomy. The mRNA levels of both genes began to decrease 8 hr after hepatectomy, both reaching the lowest levels at 24 hr, and then recovered to some extent at 48 hr. In contrast, these levels in the housekeeping and growth-related genes were augmented during this period. This would suggest that there is a selective expression of growth-related and housekeeping genes, in preference to liver-specific genes during liver regeneration. The expression of these genes in the regenerating liver was simulated in primary cultured hepatocytes during the dedifferentiation processes. It would appear that the first step in regeneration of the residual liver is dedifferentiation, in which the depression of liver-specific genes may be linked to liver dysfunction following hepatectomy.     

Disruption of the vascular prosthesis caused by aortic calcification after replacement of the thoracoabdominal aortic aneurysm

A 52-year-old man underwent a replacement of the thoracoabdominal aorta. The aorta was severely calcified, and was replaced by a 24-mm woven Dacron (Vascutek, Renfrewshire, Scotland) graft wrapped with the calcificated aneurysmal wall. His postoperative course was uneventful; however, he collapsed on the 18th postoperative day. He underwent an emergent thoracotomy and the wrapped aneurysmal wall was taken down. The prosthesis graft had a 1-mm disruption in the middle portion, which did not relate to the anastomoses. Experimental study ex vivo showed that disruption of the prosthesis could have occurred after a 3-week pulsatile force caused by a seashell simulating aortic calcification.     

Control of pyrimidine biosynthesis in the Ascaris ovary: regulatory properties of glutamine-dependent carbamoyl-phosphate synthetase and copurification of the enzyme with aspartate carbamoyltransferase and dihydroorotase

Glutamine-dependent carbamoyl-phosphate synthetase, the first enzyme of the de novo biosynthetic pathway for pyrimidine nucleotides, was purified about twenty-fold from 105 000 x g supernatant of the Ascaris ovary homogenate. The enzyme activity was feedback-inhibited by UDP and UTP while it was stimulated by 5-phosphoribosyl 1-pyrophosphate. Most of the catalytic and regulatory properties of the Ascaris synthetase were similar to those of the mammalian synthetase. A significant difference is that the Ascaris enzyme was more strongly inhibited by UDP than by UTP whereas the mammalian enzyme is more sensitive to UTP than to UDP. The Ascaris enzyme was also inhibited by other various nucleoside diphosphates, such as dUDP, dADP and CDP, generally more strongly than by the corresponding nucleoside triphosphates. Aspartate carbamoyltransferase and dihydroorotase, the second and third enzymes of the pathway, were also demonstrated in the supernatant fraction. These two enzymes were copurified with the synthetase and the relative activities of the three enzymes remained nearly constant (1:850-890:50-60) throughout the purification. In a sucrose gradient centrifugation, the enzymes cosedimented as a single peak with a sedimentation coefficient (s20,w) of about 32 S under the condition used. These results strongly suggest that the enzymes exist as a multienzyme complex similar to those found in higher animals. The activity of the carbamoyltransferase was insensitive to nucleotides and related compounds. These results indicate that the synthetase plays a key role in the control of pyrimidine biosynthesis in the Ascaris ovary.     

Carbamyl phosphate synthetase I deficiency with no detectable mRNA activity

In the autopsied liver of a neonate with carbamyl phosphate synthetase (CPS)-I deficiency, the activity of CPS-I was about 9% of the normal neonatal control. The enzyme protein of CPS-I was hardly detectable by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) and an immunoblotting method using anti-rat liver CPS-I. The level of translatable mRNA for CPS-I was markedly decreased in a cell-free protein synthesis system consisting of rabbit reticulocyte lysate and total RNA extracted from the autopsied liver of the patient. These observations indicate that the enzyme deficiency in this case is probably mainly due to a diminished level of translatable mRNA, which would lead to a decrease in the synthesis of the CPS-I precursor.Abbreviations CPS carbamyl phosphate synthetase - SDS/PAGE sodium dodecyl sulphate/polyacrylamide gel electrophoresis - OTC ornithine transcarbamylase - NAGS N-acetyglutamate synthetase     

Detection of Paracoccidioides brasiliensis gp43 Gene in Sputa by Loop‐Mediated Isothermal Amplification Method

The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop‐mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMPmethod had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune‐suppressed patients. J. Clin. Lab. Anal. 23:139–143 2009. © 2009 Wiley‐Liss, Inc.     

Ornithine carbamoyltransferase deficiency: coexistence of active and inactive forms of enzyme

Kinetic and molecular properties of liver ornithine carbamoyltransferase from a female patient with the enzyme deficiency were studied. The enzyme activity in the patient was 9% of that in controls. Kinetic properties of the enzyme from the patient appeared to be identical to those of the control enzyme; apparent K_m values for L-ornithine and carbamoyl-phosphate at pH 7.2 were 1.3 mmol/l and 7.9 μmol/1, respectively. Patient and control livers contained similar amounts of protein cross-reactive with antibody to the bovine enzyme. No difference in the subunit size was observed between the patient enzyme and the control enzyme. When extracts of control liver were analyzed by isoelectric focusing, a major peak of immunoreactive protein with a pI value of 7.3 and a minor peak with a pI value of 6.8 were observed, and both forms were enzymatically active. On the other hand, extracts of the patient's liver gave a major peak of immunoreactive protein with a pI value of 7.0 and a minor one with a pI of 6.8; the minor form was enzymatically active while the major one showed little or no activity. These results indicate that the patient's liver contained an inactive form of ornithine carbamoyltransferase in addition to an active form of the enzyme, and may reflect the X-linkage of the enzyme at the molecular level.