Partial characterization of 5-fluoropyrimidine-resistant mutants of Neurospora crassa

Summary The primary lesion in a number of 5-fluoropyrimidine resistant mutants of Neurospora crassa has been identified. ud-1 mutants, previously designated fdu-2, are deficient in nucleoside uptake and show extensive intragenic complementation. uc-4 mutants lack uracil phosphoribosyl transferase with no complementation between 23 alleles. udk mutants lack uridine kinase activity. fdu-2 mutants affect the repression of the first two de novo pyrimidine biosynthetic enzymes, have no detectable uridine kinase activity and show decreased uridine uptake. Accordingly, fdu-2 may be involved in the regulation of pyrimidine uptake, salvage and de novo synthesis.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Genetic mosaics of the rudimentary locus of Drosophila melanogaster: a genetical investigation into the physiology of pyrimidine synthesis.

Genetic mosaics have been utilized to investigate each of the three pleiotropic effects which result from mutation of the rudimentary locus. The wing abnormality results from a requirement for endogenous pyrimidine synthesis within the wing imaginal disk itself. Mosaic analysis indicates the defect in the wing can be rescued by pyrimidine synthesis any place within the imaginal disk and that no more than 10% of the cells of the disk need be genetically capable of producing pyrimidines. This requirement for endogenous synthesis is probably the result of a reduction or complete elimination of pyrimidine uptake at a particular stage during development. Ovary transplantations indicate that the sterility of homozygous rudimentary females results from a requirement for pyrimidine synthesis within the ovary itself, and mosaic analysis of the auxotrophic phenotype indicates that survival of larvae on pyrimidine-free medium depends on pyrimidine synthesis within a variety of different tissues.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998     

Apparent Genetic Complexity Generated by Developmental Thresholds: The Apterous Locus in DROSOPHILA MELANOGASTER

Mutations at the apterous (ap) locus in Drosophila melanogaster give rise to three distinct phenotypes: aberrant wings, female sterility and precocious adult death. The wing phenotype includes five types of abnormality: blistering, deficiencies, duplications, high-order repetitions and transformation of structures. The mildest phenotype is seen with homozygous apblt animals which have either normal or slightly blistered wings. Most alleles produce, in the homozygote, a deficient wing in which part or all of the wing margin and wing blade is missing, but wing hinge and notum regions are normal. Animals hemizygous for each of 20 ap alleles, as well as apID/apXa heterozygotes, show duplication of parts of the notum associated with complete wing deficiency. Animals heterozygous for apc and the other tested ap alleles show repetitions of parts of the anterior wing margin, an engrailed-like transformation of posterior wing margin into anterior margin or both. Both apblt and apc show similar phenotypes in homozygotes and hemizygotes, yet both produce a less extreme phenotype than that of the other hemizygotes, suggesting that neither mutation causes loss of the entire ap+ function. The 15 alleles that cause precocious death and female sterility occur in six complementation groups based on complementation for these phenotypes. This supports the previous conclusion that the effects of apterous mutations on the wing do not correlate with their effects on viability and fertility. We propose an explanation for the effects of apterous mutations on the wing in which quantitative reductions in the activity of gene product give rise to qualitatively different phenotypes because of different threshold requirements of the ap+ function for critical events in wing disc development.     

Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster

Summary Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPR-Tase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consists of two mutants that each specify severly reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and OD-Case reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and OD-Case in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

A further characterization of the cinnamon gene in Drosophila melanogaster

Summary Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2⁺:2^- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.The mutations map on the leaft arm of chromosome I and form a single complementation group with five other pyruvate kinase mutations in the pyk1 gene that was earlier suggested to be a regulatory locus controlling the synthesis of this enzyme. A comparative study of these mutants has been made with the structural mutants described here.     

Identification of nonsense mutations in Neurospora: Application to the complex arg-6 locus

Summary The arg-6 locus of Neurospora crassa encodes two enzymes of arginine synthesis, acetylglutamate kinase and acetylglutamyl phosphate reductase. Mutants lacking one or the other enzyme fall into two different complementation groups; a large non-complementing group lacks both enzymes. We wished to survey over 50 alleles for suppressibility by a nonsense suppressor. We compared two methods of assessing suppressibility. One, based on trans-action of a nonsense suppressor, was simple, but not efficient in detecting all suppressible alleles. The other, based on crosses involving a marker linked to the locus surveyed was very efficient in methodology and is suited to all cases, such as the arg-6 locus, in which allelic crosses are sterile. The data indicate that the arg-6 locus encodes a bifunctional protein.     

The Dhod locus of Drosophila: mutations and interrelationships with other loci controlling de novo pyrimidine biosynthesis

Summary Mutations at the Dhod locus have been isolated following ethylmethanesulfonate mutagenesis. These mutants express those phenotypes common to other mutations of the de novo pyrimidine pathway: specific wing and leg defects and female sterility. Dihydroorotate dehydrogenase activity is severely reduced in all Dhod mutants, whereas levels of the other pathway enzymes are largely unaffected. The twelve Dhod mutations described here comprise a single complementation group. All of these mutations are nonlethal and the collection includes apparent amorphic as well as hypomorphic alleles. These results are discussed relative to the properties of the complex loci that encode the other steps of de novo pyrimidine biosynthesis.Abbreviations DHOase dihydroorotase (EC 3.5.2.3.) - DHOdehase dihydroorotate dehydrogenase (EC 1.3.3.1.) - EMS ethylmethanesulfonate - ODCase orotidylate decarboxylase (EC 4.1.1.23) - OPRTase orotate phosphoribosyltransferase (EC 2.4.2.10) - UMP uridine 5-monophosphate     

A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase. Received: 25 April 1998 / Accepted: 27 May 1998     

Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.     

Temperature-sensitive yellow mutants of Chlamydomonas reinhardtii

Summary We have isolated and genetically characterized 10 mutants of Chlamydomonas reinhardtii carrying single, mendelian, temperature-sensitive yellow mutations. The mutants have a yellow phenotype at the restrictive temperature (33°C), but have a wildtype phenotype at the permissive temperature (25°C). Based on complementation and recombination tests, the ten mutations include alleles of two previously described yellow loci (y-1 and y-6) and three new yellow loci (y-8, y-9, and y-10). At the restrictive temperature, y-8, y-9, and y-10 are physiologically similar to other yellow mutants. They accumulate small amounts of protochlorophyllide when grown under dim light, but synthesize normal amounts of chlorophyll when grown in the light. Linkage tests indicate that the three new mutations are not linked to each other. y-8 is linked to y-7 on linkage group III, and y-10 is linked to y-5 and y-6 on linkage group I. y-9 is located on linkage group II. We conclude that the control of light-independent protochlorophyllide reduction is complex, involving several genetic loci which are scattered in the genome and which code for gene products able to complement in trans. Temperature-sensitive alleles at several of the yellow loci suggest that the gene products made by these loci are proteins.     

Genetic studies of mutations at two loci of Drosophila melanogaster which cause a wide variety of homeotic transformations

Summary The ash-1 locus is in the proximal region of the left arm of the third chromosome of Drosophila melanogaster and the ash-2 locus is in the distal region of the right arm of the third chromosome. Mutations at either locus can cause homeotic transformations of the antenna to leg, proboscis to leg and/or antenna, dorsal prothorax to wing, first and third leg to second leg, haltere to wing, and genitalia to leg and/or antenna. Mutations at the ash-1 locus cause, in addition, transformations of the posterior wing and second leg to anterior wing and second leg, respectively. A similar spectrum of transformations is caused by mutations at yet another third chromosome locus, trithorax. One extraordinary aspect of mutations at all three of these loci is that they cause such a wide variety of transformations. For mutations at both of the loci that we have studied the expression of the homeotic phenotype is both disc-autonomous (as shown by injecting mutant discs into metamorphosing larvae) and cell autonomous (as shown by somatic recombination analysis). The original mutations which identified these two loci, although lethal, manifest variable expressivity and incomplete penetrance of the homeotic phenotype suggesting that they are hypomorphic. The phenotype of double mutants which were synthesized by combining different pairs of those original mutations manifest for two of the four pairs a greater degree of expressivity and slightly more penetrance of the homeotic transformations. This mutual enhancement suggests that the products of both loci interact in the same process. A third double mutant expresses a discless phenotype.Additional alleles have been recovered at both the ash-1 and the ash-2 loci. Some of these alleles as homozygotes or transheterozygotes express the wide range of transformations revealed first by double mutants. One of the alleles at the ash-1 locus when homozygous and several transheterozygous pairs can cause either the homeotic transformation of discs or the absence of those discs. The fact that these two defects, absence of specific discs and homeotic transformations of those same discs can be caused by mutations within a single gene suggests that the activity of the product of this gene is essential for normal imaginal disc cell proliferation. Loss of that activity leads to the absence of discs, whereas, reduction of that activity leads to homeotic transformations.     

Analysis of pyrimidine catabolism in Drosophila melanogaster using epistatic interactions with mutations of pyrimidine biosynthesis and beta-alanine metabolism

The biochemical pathway for pyrimidine catabolism links the pathways for pyrimidine biosynthesis and salvage with beta-alanine metabolism, providing an array of epistatic interactions with which to analyze mutations of these pathways. Loss-of-function mutations have been identified and characterized for each of the enzymes for pyrimidine catabolism: dihydropyrimidine dehydrogenase (DPD), su(r) mutants; dihydropyrimidinase (DHP), CRMP mutants; beta-alanine synthase (betaAS), pyd3 mutants. For all three genes, mutants are viable and fertile and manifest no obvious phenotypes, aside from a variety of epistatic interactions. Mutations of all three genes disrupt suppression by the rudimentary gain-of-function mutation (r(Su(b))) of the dark cuticle phenotype of black mutants in which beta-alanine pools are diminished; these results confirm that pyrimidines are the major source of beta-alanine in cuticle pigmentation. The truncated wing phenotype of rudimentary mutants is suppressed completely by su(r) mutations and partially by CRMP mutations; however, no suppression is exhibited by pyd3 mutations. Similarly, su(r) mutants are hypersensitive to dietary 5-fluorouracil, CRMP mutants are less sensitive, and pyd3 mutants exhibit wild-type sensitivity. These results are discussed in the context of similar consequences of 5-fluoropyrimidine toxicity and pyrimidine catabolism mutations in humans.     

Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus

Summary The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) were analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long period (31–39 h), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. This is a unique phenotype that had not previously been observed for mutants at the per locus. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms. The clock phenotypes of 3B1-2 chromosome aberrations, the three per mutant alleles, and per ⁺ duplications suggest that mutations at the per locus shorten, lengthen, or eliminate periodicity by respectively increasing, decreasing, or eliminating per activity.     

GENETIC BLOCKS IN THE THIAMINE SYNTHESIS OF THE ANGIOSPERM ARABIDOPSIS,,

Five thiamine-requiring mutants were obtained at two loci. Two are blocked in the synthesis of the pyrimidine part of the vitamin, the other three have lost the ability to make the thiazole moiety. None of the tested substances suggested as possible or likely precursors of the pyrimidine or the thiazole components of thiamine displayed any activity in the mutants. These conditional lethals responded to remarkably small supplements of thiamine. The pyrimidine-requiring mutants utilized to some extent the anti-vitamin neopyrithiamine. The thiazole-less mutants grew on basal media supplemented only with the analog, oxythiamine. Thiamine deficiency, irrespective of the position of the genetic block in the synthesis, results in a characteristic anomaly of pigmentation. The position of the py locus in the second linkage group has been determined. Allelic complementation has not been detected. The frequency of mutations affecting thiamine synthesis appears about the same in Arabidopsis as in fungi. The general frequency of reparable genetic lesions is, however, one to two orders of magnitude lower in Arabidopsis than that in fungi or bacteria.     

The dominant mutation Suppressor of black indicates that de novo pyrimidine biosynthesis is involved in the Drosophila tan pigmentation pathway

A deficiency in the production of -alanine causes the black (b) phenotype of Drosophila melanogaster. This phenotype is normalized by a semi-dominant mutant gene Su(b) shown previously to be located adjacent to or within the rudimentary (r) locus. The r gene codes for three enzyme activities involved in de novo pyrimidine biosynthesis. Pyrimidines are known to give rise to -alanine. However, until recently it has been unclear whether de novo pyrimidine biosynthesis is directly coupled to -alanine synthesis during the tanning process. In this report we show that flies carrying Su(b) can exhibit an additional phenotype, resistance to toxic pyrimidine analogs (5-fluorouracil, 6-azathymine and 6-azauracil). Our interpretation of this observation is that the pyrimidine pool is elevated in the mutant flies. However, enzyme assays indicate that r enzyme activities are not increased in Su(b) flies. Genetic mapping of the Su(b) gene now places the mutation within the r gene, possibly in the carbamyl phosphate synthetase (CPSase) domain. The kinetics of CPSase activity in crude extracts has been studied in the presence of uridine triphosphate (UTP). While CPSase from wild-type flies was strongly inhibited by the end-product, UTP, CPSase from Su(b) was inhibited to a lesser extent. We propose that diminished end-product inhibition of de novo pyrimidine biosynthesis in Su(b) flies increases available pyrimidine and consequently the -alanine pool. Normalization of the black phenotype results.     

Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis

Summary Heme-deficient mutants of Saccharomyces cerevisiae have been isolated from two isogenic strains with the use of an enrichment method based on photodynamic properties of Zn-protoporphyrin. They defined seven non-overlapping complementation groups. A mutant representative of each group was further analysed. Genetic analysis showed that each mutant carried a single nuclear recessive mutation. Biochemical studies showed that the observed accumulation and/or excretion of the different heme synthesis precursors by the mutant cells correlated well with the enzymatic deficiencies measured in acellular extracts. Six of the seven mutants were blocked in a different enzyme activity: 5-aminolevulinate synthase, porphobilinogen synthase, uroporphyrinogen I synthase, uroporphyrinogen decarboxylase, coproporphyrinogen III oxidase and ferrochelatase. The other mutant had the same phenotype as the mutant deficient in ferrochelatase activity. However, it possessed a normal ferrochelatase activity when measured in vitro, so this mutant was assumed to be deficient in protoporphyrinogen oxidase activity or in the transport and/or reduction of iron.The absence of PBG synthesis led to a total lack of uroporphyrinogen I synthase activity. The absence of heme, the end product, led to an important increase of coproporphyrinogen III oxidase activity, while the activity of 5-aminolevulinate synthase, the first enzyme of the pathway, was not changed. These results are discussed in terms of possible modes of regulation of heme synthesis pathway in yeast.     

Complementation pattern of lexB and recA mutations in Escherichia coli K12; mapping of tif-1, lexB and recA mutations

Summary Three lexB mutations, whose phenotypes have been previously characterized, are studied here in relation to a few recA mutations as to their complementation pattern and relative location.The restoration of resistance to UV-light and to X-rays in the hetero-allelic diploid bacteria was used as a test for dominance and complementation. The wild type allele was always dominant over the mutant allele. Only partial complementation was found between lexB and two recA alleles. There was no complementation between the recA alleles. All the data taken together strongly suggest that the complementations found are intragenic: lexB and recA mutations are in one gene.Mapping of lexB, recA and tif-1 mutations in relation to srl-1 and cysC by phage P1 transduction shows that lexB and the tif-1 mutations form a cluster proximal to srl-1 whereas recA mutations are located at the other extremity of the gene. Variability with temperature of cotransduction frequencies as well as their extended range of values prevent a meaningful calculation of the length of the recA gene.Our hypothesis is that the recA protein has two functional regions called A and B respectively defined at the genetical level by recA and lexB mutations and that it is, in vivo, an oligomeric protein forming a complex with the lexA protein. This complex is postulated to be multifunctional: recombination and control of exonuclease V are effected by the A region while the B region and lexA protein effect induced DNA repair and lysogenic induction.     

A Genetic and Biochemical Analysis of the Temperature Sensitive, Normal-Winged Alleles of the Rudimentary Locus of DROSOPHILA MELANOGASTER

The genetic and biochemical characteristics of a particular class of mutants at the rudimentary locus are described. The mutants are pyrimidine auxotrophs, like classical rudimentary alleles, but they are unique in that they do not alter the size or shape of the wing (Falk and Nash 1974b). Aspartate transcarbamylase and dihydroorotase activities have been measured in seven different normal-winged mutants, and the results indicate that these strains are enzymologically "leaky" mutants. Previous studies have shown that three genetic functions (corresponding to the first three enzymes of pyrimidine synthesis) are associated with the rudimentary locus. Four of the seven mutants appear to affect all three of these functions. Each of the four is temperature sensitive, and a biochemical analysis of the temperature sensitivity of one of these mutants, (r)pyr1-3, suggests that a process affecting the synthesis or assembly of these enzymes is altered at high temperatures.