A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila.

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.

Molecular cloning and transcript mapping of the dihydroorotate dehydrogenase dhod locus of Drosophila melanogaster.

The dhod locus encodes dihydroorotate dehydrogenase, the fourth enzymatic step of de novo pyrimidine biosynthesis. This locus was cloned previously by a chromosome walk in cytogenetic region 85A. The location of dhod within 85A DNA has been determined by mapping two rearrangement mutations to a small DNA region. A nearly full-length cDNA clone of the dhod transcript was isolated and partially sequenced, to confirm its identity. The cDNA clone was also used to map the transcribed DNA. A 1.5 kb dhod RNA is described which is most abundant in embryos and displays minor length heterogeneity in pupae and adults. The developmental expression of this transcript is discussed relative to the expression of dihydroorotate dehydrogenase activity and other genes of the pyrimidine biosynthetic pathway.

High efficiency expression of transfected genes in a Drosophila melanogaster haploid (1182) cell line.

Drosophila tissue culture cells have been important in the study of homologous promoters and more recently in the study of mammalian transcriptional factors such as CTF and SP1 which bind and stimulate transcription from transfected genes. In this paper we show that a Drosophila melanogaster haploid cell line (1182-4), not previously used for transfection studies, is capable of taking up and expressing DNA without the use of a facilitating agent such as calcium phosphate. Furthermore expression from a variety of Drosophila promoters such as copia, heatshock and rudimentary as well as a mammalian promoter RSV-LTR, show between 20 and over 100 times more activity in 1182-4 cells than in D.hydei DH33 or D.melanogaster S3, or D1 cell lines. This cell line should prove to be particularly useful for the analysis of weak promoters and heterologous transcription factors.

Genetic and molecular mapping of chromosome region 85A in Drosophila melanogaster.

Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups.

Organization of transcription units around the Drosophila melanogaster rudimentary locus and temporal pattern of expression.

The molecular organization of 90 kb of DNA derived from a region of the X chromosome that encompasses the rudimentary locus of D. melanogaster is presented. This segment spans the cytogenetic region 14F2-3 to 15A1-2, and there are, in addition to the rudimentary gene several transcription units present, whose functions are still unknown. We have determined the pattern of expression of all these genes at several stages of development, and found that they all show a different temporal modulation of their activity. The accumulation of the r product correlates well with the enzymatic activity determined for the protein product of the gene, being highest in very early embryos and adult females.

The rudimentary locus of Drosophila melanogaster.

In the studies reported here, we have examined the molecular organization of the rudimentary gene of Drosophila melanogaster. rudimentary encodes a 220,000 Mr polypeptide catalyzing the first three steps in pyrimidine biosynthesis: carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase. We have determined the direction of transcription of the gene relative to the genetic map of the locus. The 5' end maps to the distal end of the locus (relative to the centromere) and transcription proceeds through the domains encoding dihydroorotase, carbamyl phosphate synthetase and finally aspartate transcarbamylase. The rudimentary transcription unit spans a DNA segment of 13.2 X 10(3) base-pairs and encodes a mature messenger RNA of 7.3 X 10(3) base-pairs. Three intervening sequences have been identified, one of which is over 4 X 10(3) base-pairs in length. Finally, we have compared the DNA sequence organization of the Drosophila rudimentary gene with the corresponding loci of yeast and hamster.

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

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.

Analysis of the phenotypes exhibited by rudimentary-like mutants of Drosophila melanogaster.

Flies mutant for one or both of the last two enzymes of de novo pyrimidine biosynthesis express a number of phenotypes that are also expressed by mutants of the first four pathway enzymes (r and Dhod-null mutants). However, r-1 flies also express two phenotypes, mottled eyes and poor viability, that are not usually expressed by r and Dhod-null flies. Chemical determinations show that orotic acid, a substrate for the fifth pathway enzyme, accumulates in r-1 individuals but not in r and wild-type individuals. Moreover, flies simultaneously mutant for r and r-1 do not express the mottled-eye phenotype, showing that r is epistatic to r-1 for this r-1-specific phenotype. When genotypically wild-type flies are cultured on a medium containing 6-azauracil, the base of a potent inhibitor of the last enzyme of de novo pyrimidine biosynthesis, phenocopies are obtained that include the mottled-eye as well as the wing phenotypes of r-1 flies. These results support hypotheses that the phenotypes common to r, Dhod-null, and r-1 flies are consequences of uridylic acid deficiency, whereas the r-1-specific phenotypes result from orotic acid accumulation in flies lacking either or both of the last two enzymes of de novo pyrimidine biosynthesis.

A small genetic region that controls dihydroorotate dehydrogenase in Drosophila melanogaster.

A locus is described that controls levels of mitochondrial dihydroorotate dehydrogenase (EC 1.3.3.1) in Drosophila melanogaster. The effects of alleles of the locus, Dhod, are manifest in preparations from whole organisms as well as in partially purified mitochondrial preparations; however, other mitochondrial functions do not appear to be appreciably affected by Dhod genotypes. The locus maps near p in the proximal portion of the right arm of chromosome 3. Flies trisomic for a chromosome segment including that region display elevated enzyme levels, implying that an enzyme structural gene is in that vicinity. Furthermore, Dhod alleles are semidominant in heterozygotes, suggesting that the dosage-sensitive element detected in the trisomics is actually the Dhod locus. These findings are discussed relative to the role of dihydroorotate dehydrogenase in the de novo pyrimidine biosynthetic pathway and relative to other pathway mutants that have been described in Drosophila.

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

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 (OPRTase) 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 consist of two mutants that each specify severely reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and ODCase 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 ODCase in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.

Identification of a small genetic region that encodes orotate phosphoribosylatransferase and orotidylate decarboxylase in Droxophila melanogaster.

A variety of tests indicate that a small genetic region in chromosome 3R encodes at least a part of the bienzyme complex containing the last two enzymes of the pyrimidine biosynthetic pathway in Drosophila melanogaster, Complex U. Electrophoretic variants of the last pathway enzyme map to this region and both Complex U activities are proportionally reduced in flies heterozygous for specific deficiencies of the region. Complex U mutants have been isolated that lack both activities. Characteristics of these mutants are discussed relative to other pyrimidine pathway mutants known in Drosophila melanogaster and in mammals.

Organization of the Rudimentary Wing Locus in DROSOPHILA MELANOGASTER. I. the Isolation and Partial Characterization of Mutants Induced with Ethyl Methanesulfonate, Icr-170 and X Rays.

New rudimentary (r) mutants have been isolated following mutagenesis with ethyl methanesulfonate (r(LE)), ICR-170 (r(LI)) and X rays (r(LX)). From wing phenotype measurements on homoallelic females, it has been shown that the r(LE) mutant series includes several leaky alleles, as well as alleles that produce moderate and strong r phenotypes. All of the tested r(LI) alleles yielded strong r phenotypes in homoallelic females, whereas the r(LX) series was found to include both moderate and strong alleles. Based on allele complementation for the wing phenotype, it was found that all three mutant series include both complementing and noncomplementing alleles, but the relative frequencies of these two types of alleles differ considerably among the three series. Complementing alleles comprise most of the r(LE) mutant series (19 of 25) and almost one-half of the r(LX) series (five of 12), while only one of 16 r(LI) mutants is a complementing allele. Data from enzyme assays of mutants mostly support the direct correlation of genetic complementation units with the activities of the first three enzymes in the de novo pyrimidine biosynthetic pathway. All of these findings are discussed in light of evidence that these three enzymes are contained within a trienzyme complex in animals. We conclude that the available genetic evidence supports the contention that the trienzyme complex is encoded by a single mRNA.