Rudimentary Locus Research Articles

Interallelic complementation for pyrimidine requirement in rudimentary mutants of Drosophila melanogaster.

Complementation at the rudimentary locus in Drosophila melanogaster is reported for the nutritional requirement observed earlier in the rudimentary mutants. It is demonstrated that this complementation parallels that for wing phenotype, and that at the same time it is a more potent and quantitative test for complementation at this locus. The possible molecular basis for the observed complementation is discussed in the light of current knowledge of the genetic regulation of pyrimidine biosynthesis in other organisms.

The biochemical genetics of rudimentary mutants of Drosophila melanogaster

Aspartate carbamoyltransferase (ACT) levels were determined in larvae from two completely complementing and one non-complementing rudimentary strain, as well as in wild-type larvae. The levels were low in the non-complementing and in one of the complementing strains, whereas the other complementing strain had wild-type levels. When viewed together with the previous findings of a nutritional requirement for pyrimidines in the non-complementing strain, that can be satisfied by carbamoyl aspartate, these results indicate that the rudimentary locus determines the first two enzyme activities in the pyrimidine biosynthetic pathway; carbamoyl phosphate synthetase and aspartate carbamoyltransferase. Addition of uracil to the culture medium caused no decrease in the level of ACT in wild type or ACT-positive rudimentary larvae indicating that end product repression of this enzyme does not take place in Drosophila.

The phylogenetic relationships of flies in the superfamily Empidoidea (Insecta: Diptera)

We conducted a molecular phylogenetic study of the Empidoidea, a diverse group of 10,000 species of true flies, with two major goals: to reconstruct a taxonomically complete and robustly supported phylogeny for the group and to use this information to assess several competing classifications for the clade. We amassed 3900+ nucleotides of coding data from the carbamoylphosphate synthase domain of the rudimentary locus (CAD) and 1200+ nucleotides from the large nuclear ribosomal subunit (28S) from 72 and 71 species, respectively, representing several orthorrhaphan and cyclorrhaphan families and all previously recognized empidoidean subfamilies. Independent and combined phylogenetic analyses of these data were conducted using parsimony, maximum likelihood, and Bayesian criteria. The combined matrix included 61 taxa for which both CAD and 28S sequences were obtained. Analyses of CAD first and second codon positions alone and when concatenated with 28S sequences yielded trees with similar and largely stable topologies. Analyses of 28S data alone supported many clades although resolution is limited by low sequence divergence. The following major empidoid clades were recovered with convincing support in a majority of analyses: Atelestidae, Empidoidea exclusive of Atelestidae, Hybotidae sensu lato, Dolichopodidae + Microphorinae (including Parathallassius), and Empididae sensu lato (including Brachystomatinae, Ceratomerinae, Clinocerinae, Empidinae, Hemerodromiinae, Oreogetoninae, and Trichopezinae). The branching arrangement among these four major clades was Atelestidae, Hybotidae, Dolichopodidae/Microphorinae, Empididae. Previously recognized subclades recovered with robust support included Hybotinae, Brachystomatinae, Tachydromiinae, Clinocerinae (in part), Hemerodromiinae, Empidinae, and Empidiini.

Phylogeny and Bayesian divergence time estimations of small-headed flies (Diptera: Acroceridae) using multiple molecular markers

The first formal analysis of phylogenetic relationships among small-headed flies (Acroceridae) is presented based on DNA sequence data from two ribosomal (16S and 28S) and two protein-encoding genes: carbomoylphosphate synthase (CPS) domain of CAD (i.e., rudimentary locus) and cytochrome oxidase I (COI). DNA sequences from 40 species in 22 genera of Acroceridae (representing all three subfamilies) were compared with outgroup exemplars from Nemestrinidae, Stratiomyidae, Tabanidae, and Xylophagidae. Parsimony and Bayesian simultaneous analyses of the full data set recover a well-resolved and strongly supported hypothesis of phylogenetic relationships for major lineages within the family. Molecular evidence supports the monophyly of traditionally recognised subfamilies Philopotinae and Panopinae, but Acrocerinae are polyphyletic. Panopinae, sometimes considered “primitive” based on morphology and host-use, are always placed in a more derived position in the current study. Furthermore, these data support emerging morphological evidence that the type genus Acrocera Meigen, and its sister genus Sphaerops, are atypical acrocerids, comprising a sister lineage to all other Acroceridae. Based on the phylogeny generated in the simultaneous analysis, historical divergence times were estimated using Bayesian methodology constrained with fossil data. These estimates indicate Acroceridae likely evolved during the late Triassic but did not diversify greatly until the Cretaceous.

Molecular phylogeny of the green lacewings (Neuroptera: Chrysopidae)

Abstract The first quantitative analysis of phylogenetic relationships of green lacewings (Chrysopidae) is presented based on DNA sequence data. A single nuclear and two mitochondrial genes are used in the analysis: carbomoylphosphate synthase (CPS) domain of carbamoyl‐phosphate synthetase‐aspartate transcarbamoylase‐dihydroorotase (CAD) (i.e. rudimentary locus), large subunit ribosomal gene (16S) and cytochrome oxidase I (COI). This study represents the first use of the CAD gene to investigate phylogenetic relationships of the lacewings. DNA sequences for 33 chrysopid species from 18 genera, representing all subfamilies and tribes, were compared with outgroups sampled from families Hemerobiidae, Osmylidae and Polystoechotidae. Parsimony analyses of the combined data set recovered all of the previously established subfamilial and tribal groups as monophyletic clades (although relatively weakly supported) except Apochrysinae sensu lato. The enigmatic Nothancyla verreauxi Navás has historically been difficult to place in a subfamily group based on morphological characteristics; molecular data presented herein do not adequately resolve this problem.

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.

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 beta-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 beta-alanine. However, until recently it has been unclear whether de novo pyrimidine biosynthesis is directly coupled to beta-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 beta-alanine pool. Normalization of the black phenotype results.

Two non-gypsy rudimentary mutations and their suppression by mutations of suppressor of Hairy-wing in Drosophila.

Two spontaneous mutations of rudimentary, the gene encoding the first steps of de novo pyrimidine biosynthesis in Drosophila, are suppressed by mutant alleles of the suppressor of Hairy-wing locus. This interaction differs from typical su(Hw) suppression in that neither rudimentary allele is associated with an insertion of the gypsy retrotransposon. One allele, rsP1, appears to be a point mutation. Adult rsP1 homozygous females accumulate substantially less 7.3 kb rudimentary transcript than do wild-type females. The other allele, rsP2, is an insertion of an mdg3 retrotransposon in the sixth exon of rudimentary and in the opposite transcriptional orientation. This insertion divides the rudimentary locus into two separate, yet functional, transcription units by truncating transcription from the rudimentary promoter and promoting transcription of downstream rudimentary sequences. Phenotypic suppression of both rsP1 and rsP2 by mutant alleles of the suppressor of Hairy-wing locus correlates with enhanced levels of the rsP1 and rsP2 transcripts.

Developmental expression pattern of a 800 kb DNA continuum cloned from the Drosophila X chromosome 14B–15B region

The expression throughout development of a long DNA segment from the Drosophila genome was investigated. A near continuum of 800 kb DNA was cloned by extending a 90 kb previous chromosome walk, spanning the rudimentary locus (r), and by initiating 2 chromosome walks from an isolated DNA clone located next to the r locus. Hexanucleotide-primed single strand cDNAs were synthesized from poly(A) +RNAs prepared from staged embryos, larvae, pupae, males, females, heads from adults and thoraces plus abdomens from adults. The representativity of the cDNAs with regard to the whole length and the molar abundance of a selected gene mRNA was checked. These cDNAs were then used to probe Southern transfers of the cloned DNA after digestion with combinations of restriction enzymes. The limits of the expressed parts of the cloned DNA and their developmental profiles were then deduced. Data show that, in contrast to what was known on the basis of genetic analysis, this region of the Drosophila genome is densely transcribed. Numerous parts of the cloned DNA are found to be stage-, or sex-or territory-specifically expressed. Data are consistent with a view of the organization of this representative part of the Drosophila genome according to which the expressed regions tend to be clustered, whereas no tendency for transcription units displaying comparable specific spatial, temporal or sex expression patterns to be arranged in groups is noted.

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.

P-element-induced control mutations at the r gene of Drosophila melanogaster.

The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non-P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression.

P-element-induced control mutations at the r gene of Drosophila melanogaster.

The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non-P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression.

Amplification of the rudimentary gene in a PALA-resistant Drosophila cell line

In Drosophila melanogaster the rudimentary locus encodes for a multifunctional protein catalyzing the first three enzymatic activities of pyrimidine biosynthesis. Cell lines were selected which were resistant to PALA ( N-(phosphonoacetyl)-L-aspartate), a specific inhibitor of aspartate transcarbamylase, the second enzyme of this pathway. In a cell line where the enzyme production is increased 5 times, Southern blot analyses show that the rudimentary gene and surrounding regions are amplified about 5 times. In this case gene amplification could therefore account for the observed enzyme overproduction.

Isolation of the rudimentary locus of Drosophila melanogaster

In Drosophila melanogaster the rudimentary locus encodes the first three enzymes in de novo pyrimidine biosynthesis: CPSase, ATCase and DHOase. rudimentary is located towards the proximal end of the X chromosome at the cytogenetic locus 15A1. A variety of genetic and molecular studies have suggested that all three enzymatic activities are encoded in a single transcription unit whose mRNA is translated into a multifunctional polypeptide with a molecular weight of approximately 220,000 D. In this paper we describe the isolation of a 90 kb D. melanogaster DNA segment which contains the rudimentary gene. This was achieved by screening a D. melanogaster genomic phage library with a cDNA probe homologous to the corresponding genetic unit of hamster (pyr 1–3). One of the phage recombinants homologous to this hamster probe, BS313 was found to be derived from the 15A1 region of the X chromosome. Starting from BS313 we “walked” in both directions along the X chromosome to isolate the entire rudimentary locus plus flanking DNA segments. To positively identify the rudimentary gene we in situ hybridized the various phage recombinants obtained in this walk to an inversion chromosome that has one breakpoint at 15A1 which inactivates the gene. The phage immediately adjacent to BS313 was found to span the inversion breakpoint and we were able to determine the orientation of the phage walk on the basis of this in situ hybridization result. Finally we indentify a portion of the rudimentary mRNA coding region on the basis of homology first to the hamster cDNA probe and to total Drosophila RNA and second, to a 7.3 kb long poly A+ transcript which is found both in adult flies and in embryos.

Genetic and biochemical properties of revertants at the rudimentary locus in Drosophila melanogaster.

The rudimentary (r) locus of Drosophila melanogaster encodes the first three enzymes for the de novo synthesis of pyrimidines: carbamyl phosphate synthase (CPSase), aspartyl transcarbamylase (ATCase), and dihydroorotase (DHOase). Revertants of leaky r mutants deficient in CPSase have been recovered. Of those examined, most (28/30) appear to result from reversion of the original mutation or a second site mutation in the mutant gene: They map to the r locus, have levels of CPSase and Km's for glutamine similar to wild type, and have comparatively little change in ATCase and DHOase activity. Two of the revertants appear to involve regulatory mutations that lead to an increase in defective CPSase molecules: They map to the r locus, have Km's for glutamine like that of the CPSase in the progenitor stock, and lead to relatively small increases in CPSase activity that are roughly paralleled by increases in ATCase and DHOase. The recovery of these putative regulatory mutations increasing the activity of the three enzymes supports the conclusion that the three activities are part of a trifunctional polypeptide or that their genes are transcribed together as parts of a multicistronic transcript.

Characterization of an autosomal rudimentary-shaped wing mutation in Drosophila melanogaster that affects pyrimidine synthesis.

A new autosomal mutation, rudimental (ral), which causes rudimentary-shaped wings in Drosophila melanogaster, has been isolated following ethyl methanesulfonate (EMS) mutagenesis. The wing phenotype of rudimental is identical to that of the X-linked rudimentary (r) mutation, which affects the first three enzymes in the pyrimidine biosynthetic pathway. The autosomal mutant maps very close to ebony (3-70.7) at 70.42 on the right arm of chromosome 3. Analysis of the enzyme activities of orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) indicates that the rala26a allele has less than wild-type activity for both enzymes. This result is discussed in light of the fact that the OPRTase and ODCase activities are part of an enzyme complex, as are the carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which are encoded by the complex rudimentary locus. We suggest that rudimental is also a complex lucus.

A genetic and dietary study of the physiology of pyrimidine synthesis in Drosophila melanogaster

A combination of genetic and dietary manipulations have been utilized to investigate the physiology of pyrimidine metabolism throughout the Drosophila life cycle. We present evidence that the dietary sources of pyrimidines ingested at the larval stage are sufficient for all subsequent stages of the life cycle, except the process of oögenesis in adult females where a maternal supply of endogenously synthesized pyrimidines is normally required. Deprivation of dietary and de novo synthesis does not affect adult longevity; indicating that with the exception of oögenesis, all normal functions are carried out by recycling pyrimidines produced or ingested at the larval stage. In an enzymatic analysis, we have determined that Drosophila does not adjust for pyrimidine dietary deficiencies by significantly altering the level of synthesis of two tested enzymes encoded by the rudimentary locus, even though the pyrimidine deficiency is a rate limiting step in the completion of development.

The enzymes for de novo pyrimidine biosynthesis in Drosophila melanogaster: Their localization, properties and expression during development

1. 1. The first three and last two pyrimidine pathway enzymes are soluble, cytoplasmic activities in Drosophila melanogaster larvae and evidence is presented showing that the last two enzymes comprise a bifunctional enzyme complex. 2. 2. The fourth pathway enzyme, dihydroorotate dehydrogenase, is a particulate activity that co-sediments with mitochondrial cytochrome oxidase. 3. 3. During development, the soluble enzymes exhibit maximal values in embryos and young larvae and show minimal values in later larval stages, pupae and adult males: adult females show high activities due to oocytes and retained embryos. 4. 4. Mutations of the rudimentary locus destroy only the initial three pathway enzymes.

Genetical and cytological location of the structural parts coding for the first three steps of pyrimidine biosynthesis in Drosophila melanogaster.

The rudimentary locus (r; X--55.3) of Drosophila melanogaster is shown to contain the structural sequences for the enzymes CPSase, ATCase and DHOase. The enzyme concentration in adult flies is correlated with the number of r+ copies in the genome. The expression of the locus follows the rules of the gene dosage compensation hypothesis when extracts of newly emerged males and females are compared.

Developmental regulation of the first three enzymes of pyrimidine biosynthesis in Drosophila melanogaster

The developmental profiles of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, the first three enzymatic activities of the pyrimidine biosynthetic pathway, are presented. The three enzymes appear to share a common developmental regulation. In addition, it is shown that the three enzymes are isolated under two different states, either aggregated or dissociated, depending upon the particular developmental stage of the animal. Evidence is presented for the existence in larvae of a soluble factor, which in vitro dissociates the aggregate into its three components. Altogether, the results are in good accordance with the proposal that the three enzymes are encoded by the rudimentary locus [Jarry, B. P., and Falk, D. R. (1974). Mol. Gen. Genet. 135, 113–122].