Neurospora pyrimidines

NCF, Neurospora crassa, forward mutation to pyrimidine - NT NG Reissig (1963) Q... 

Early clues to the nature of the pyrimidine pathway were provided by the observations that orotic acid (6-carboxyuracil, fig. 23.12) can satisfy the growth requirement of mutants of the fungus Neurospora that are unable to make pyrimidines, and that isotopically labeled orotate is an immediate precursor of pyrimidines in Neurospora and a number of bacteria. 

R2. Radford, A., Information from I.C.R-170-induced mutations on the structure of the pyrimidine-3 locus in neurospora. Muiat. Res. 8, 537-544 (1969). 

W5. Williams, L. G., and Davis, R. H., Pyrimidine-specific carbamyl phosphate synthetase in neurospora crassa. J. Bacteriol. 103, 335-341 (1970). 

Orotic acid, Oro uracil 4-carboxylic acid, M, 156.1, m.p. 344 347°C (d.). Oro is an intermediate in Pyrimidine biosynthesis (see), and it is secreted in large quantities by mutants of Neurospora crassa that show a growth requirement for uridine, cytidine or uracil. 

Orotidine, Onb orotic acid-3-P-D-tibofiiranoade, a P-glycosidic Nucleoside (see) of o-ribose and the pyrimidine, orotic acid. M, 288.21, m.p. > 400°C Cyclo-hexylamine salt m.pi 184°C, [ajc -1-14.3 (c = 1, water). O. does not appear to be a normal intermediate of pyrimidine metabolism, but it is prodnced by mutants of Neurospora crassa. 

PURINE AND PYRIMIDINE BIOSYNTHESIS IN NEUROSPORA CRASSA AND HUMAN SKIN PIBROBIASTS.ALTERATION BY RIBOSIDES AND RIBOTIDES OP ALIOPURINOL AND OXIPURINOL... 

We studied the effects of allopurinol, allopurinol-1-ribonucleoside, allopurinol-1-ribonucleotide and oxipurinol, oxipurinol-7-rlbonucleoside, oxipurinol-7-ribonucleotide on purine and pyrimidine biosynthesis in cultures of Neurospora crassa (wild strain 74 a) and human fibroblasts. 

I. Influence of allopurinol- or oxipurinolribo-nucleosides and -ribonucleotides on purine and pyrimidine biosynthesis in Neurospora crassa. 

Pig. 1 demonstrates the effects of all these tested substances on pyrimidine biosynthesis in cultures frcm Neurospora crassa. Allopurinol and oxipurinol have marked inhibitory effects. Allopurinol-1-ribonuclecside inhibits pyrimidine biosynthesis in the order of 55 to 6o percent in concentrations from 10 5 to 10 during our incubation period of 45 minutes. Allopurinol-1-ribonucleotide shows influences comparable to those of allopurinol. The inhibitory effect of oxipirinol-7-ribonucleoside is in straight parallelity to the effect of oxipurinol, whereas oxipurinol-7-ribonucleotide begins with its marked effect (8o - 90 percent inhibition) at concentrations between 10 to 10 M. 

At the same time that the early studies mentioned above were carried out, it was shown by Loring and Pierce that certain Neurospora mutants utilized uracil and cytosine very poorly in comparison to uridine (uracil riboside) and cytidine (cytosine riboside). These observations led to the suggestion that free pyrimidines were not normal intermediates in nucleic acid biosynthesis. Similar conclusions have been reached by Fries with pyrimidine-deficient strains of the mold Ophiostoma muUiannulatum. ... 

Oxalacetate and Related Compounds. Mitchell and Houlahan have suggested several plausible intermediates that may be involved in pyrimidine biosynthesis in Neurospora. Oxalacetic acid, aminofumaric acid, and aminofumaric acid diamide (Fig. 10) stimulated the growth of mutants with partial blocks in pyrimidine synthesis. Lagerkvist et al. compared the incorporation of N -labeled aspartic acid and (8,7-carbon-labeled aspartate into the pyrimidines of regenerating rat liver. Since nitrogen-labeled aspartate was less effective than carbon-labeled aspartate as a pyrimidine precursor, the authors concluded that aspartic acid was... 

In 1905, orotic acid was discovered and isolated from cow s milk. This acid has also been found in appreciable quantities in the culture media of certain Neurospora mutants by Mitchell and co-workers they believed that it arose from a side reaction during nucleic acid biosynthesis and was not a normal intermediate in pyrimidine formation by Neuro-sporad ° Wright and associates have studied the distribution of orotic acid and have found large amounts in natural materials, e.g., 2670 y per gram of yeast extract. 

The riboside of orotic acid, orotidine, was isolated subsequently from the culture medium of a uridine-requiring Neurospora mutant (350). Orotidine was readily split to orotic acid because of unusually great acid lability this offered a plausible explanation for previous failures to isolate a conjugated form of orotic acid from those natural sources that 3oelded the free acid (338, 342, 343). With the current knowledge of the individual steps in pyrimidine biosynthesis (Section VI, D, 2), it is likely that orotidine was derived from orotidylic acid (orotidine 5 -phosphate), and that a genetic block in the mutant organism prevented decarboxylation of oro-... 

Uracil was degraded similarly via dihydrouracil and 8-ureidopropionic acid to 9-alanine (Fig. 26). These findings have also been demonstrated under a variety of experimental conditions, using isotopes in vivo and in vitro (415-419) and in studies with isolated enzyme systems (430, 431)-In support of the importance of reduced pyrimidines, dihydrouracil has been foimd in beef spleen (433). A similar reductive pathway for uracil catabolism was found in bacteria (43S 43B) and in Neurospora crasaa (436). 

Further elaboration of the route of thiamine syntheas is due principfdly to Harris who used five thiamine-requiring mutants of Neurospora obtained by Beadle and Tatum in 1941 (6) and Tatum and Bell in 1946 6). Harris has concluded on the basis of the growth requirements of these strains that in Newrospora, at least, there are two alternative pathways of thiamine bios3mtheds (7). The favored pathway consists of condensation of the pyrimidine moiety with a precursor of the tbiazole moiety to form an intermediate which is then converted to thiamine (Fig. 1). In an alternative pathway the tbiazole moiety itself combines with the pyrimidine to form thiamine directly. 

Purine and Pyrimidine Biosynthesis in Neurospora Crassa and Human Skin Fibroblasts. Alteration by Ribosides and Ribotides of... 

There is genetic evidence for the presence of two forms of CPS in Neurospora, one apparently involved in ai nine synthesis and the other providing carbamyl phosphate for pyrimidine synthesis (Davis, 1972). Carbamoyl-phosphate synthetase has been partially purified from peas (O Neal and Naylor, 1969,1976 Kolloffel and Verkerk, 1982) and O N and Naylor (1969, 1976) found no evidence for isozymes of CPS in this series. Carbamoyl-phosphate synthetase purified 45-fold from pea seedlings exhibited values for glutamine and ATP... 

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