Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae

Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase — aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase⁺ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.     

Correlation between restriction map, genetic map and catalytic functions in the gene complex URA2

Summary We replaced the URA2 gene by six different deleted alleles constructed in vitro by Bg/II digestion in order to correlate the genetic map with the restriction map and to define the regions coding for the different functions of the carbamylphosphate synthetase — aspartate transcarbamylase complex (CPSase-ATCase). We also enlarged the collection of ura2 point mutations by using a positive selection method based on resistance to the toxic accumulation of ureidosuccinic acid (USA). Of the new independent mutations nine mapped in the intermediary zone, a previously defined mutationless region localized between regions coding for CPSase and ATCase. This shows that the former definition resulted from analysis of a limited number of mutants (40). The study of an allele deleted in the intermediary zone shows that this sequence codes for a protein region necessary for the feedback inhibition of the CPSase-ATcase enzyme complex. The CPSase^- ATCase^- phenotype of 26 mutants resistant to USA accumulation shows the importance of the in vivo channelling of carbamylphosphate in the CPSase-ATCase complex for USA and subsequent pyrimidine biosynthesis. Finally, our results confirm that the CPSase and ATCase activities are separate functions.     

Fine structure of the URA2 locus in Saccharomyces cerevisiae. II. Meiotic and mitotic mapping studies.

Summary The URA2 locus codes for a multifunctional enzyme complex carrying aspartate transcarbamylase (ATCase) and carbamyl phosphate synthetase (CPSase) activities. Three different types of ura2 mutants were tested in meiotic and mitotic recombination experiments: ura2A mutants devoid of ATCase activity, ura2C mutants devoid of CPSase activity and ura2B mutants devoid of both activities. All the ura2C mutations were found to be clustered at one end of the URA2 locus, called zone A, while the ura2C mutations were localized in a region at the other end, called zone C. All but two ura2B mutations (most of them suppressible) were distributed throughout zone C; the two ura2B exceptions which are small deletions, mapped in zone A. On the meiotic as well as on the mitotic map an intermediary or dead-space zone is located between zones A and C. No mutation has yet been found to map in this zone. The relative lengths of the three zones A, intermediary and C are 1:2–3:3–4, respectively.These data are consistent with the hypothesis that the URA2 locus consisting of at least two cistrons: C (CPSase) and A (ATCase), is transcribed into a single polycistronic message in the direction C to A. However, alternative hypotheses in reference to Peterson and MacLaughlin's observations (1973) are discussed.This paper is dedicated to the memory of our friend and colleague Huguette de Robichon-Szulmajster     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

Characterization of recA genes and recA mutants of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae

Summary DNA fragments carrying the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar viciae were isolated by complementing a UV-sensitive recA ^– Escherichia coli strain. Sequence analysis revealed that the coding region of the R. meliloti recA gene consists of 1044 by coding for 348 amino acids whereas the coding region of the R. leguminosarum bv. viciae recA gene has 1053 bp specifying 351 amino acids. The R. meliloti and R. leguminosarum bv. viciae recA genes show 84.8% homology at the DNA sequence level and of 90.1% at the amino acid sequence level. recA ^– mutant strains of both Rhizobium species were constructed by inserting a gentamicin resistance cassette into the respective recA gene. The resulting recA mutants exhibited an increased sensitivity to UV irradiation, were impaired in their ability to perform homologous recombination and showed a slightly reduced growth rate when compared with the respective wild-type strains. The Rhizobium recA strains did not have altered symbiotic nitrogen fixation capacity. Therefore, they represent ideal candidates for release experiments with impaired strains.The accession numbers: X59956 R. LEGUMINOSARUM REC A ALAS-DNA; X59957 R. MELITOTI REC A ALAS-DNA     

Drosophila GABAergic systems II. Mutational analysis of chromosomal segment 64AB, a region containing the glutamic acid decarboxylase gene

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter -aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

Molecular organization of master mind, a neurogenic gene of Drosophila melanogaster

Summary The gene master mind (mam) is located in bands 50C23-D1 of the second chromosome of Drosophila melanogaster. mam is one of the neurogenic genes, whose function is necessary for a normal segregation of neural and epidermal lineages during embryonic development. Loss of function of any of the neurogenic genes results in a mis-routeing into neurogenesis of cells that normally would have given rise to epidermis. We describe here the molecular cloning of 198 kb of genomic DNA containing the mam gene. Ten different mam mutations (point mutants and chromosomal aberrations) have been mapped within 45 kb of the genomic walk. One of the mutations, an insertion of a P-element, was originally recovered from a dysgenic cross. Four different wild-type revertants of this mutation were characterized at the molecular level and, although modifications of the insertions were found, in no case was the transposon completely excised. An unusually high number of the repetitive opa sequence, and of an additional previously unknown element, which we have called N repeat, are scattered throughout the 45 kb where the mam mutations map. The functional significance of these repeats is unknown.     

Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene

Summary Insertion mutations in yeast caused by the transposable element Ty have been identified at several genetic loci. In all cases so far, the site of Ty insertion has been in the 5 non-coding region of the affected gene. Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene. This Ty insertion within a gene has a similar structure to those in non-coding regions.     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase

Summary The URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5-triphosphate to cytidine 5-triphosphate, the last step of the pyrimidine biosynthetic pathway. We have cloned and sequenced the URA 7 gene. The coding region is 1710 by long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases. Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast. This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

A translocation activating the cryptic nitrogen regulation gene areB inactivates a previously unidentified gene involved in glycerol utilisation in Aspergillus nidulans

Summary The chromosome VIII translocation breakpoint of the areB-404 translocation, selected for its ability to activate the cryptic nitrogen metabolism regulatory gene areB, and the mutation glcD-100 both lead to loss of mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase in Aspergillus nidulans. These two lesions therefore define glcD, a second gene (in addition to glcB) where mutation can result in loss of this enzyme. The glcD gene has been localised to a centromere-proximal region of the right arm of chromosome VIII. Although all six known areB-activating mutations involve chromosomal rearrangements and presumably therefore gene fusions, areB-404 is the first such rearrangement where the gene involved in an areB fusion has been identified.     

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.     

Isolation of stable aroA mutants of Salmonella typhi Ty2: properties and preliminary characterisation in mice

Summary Derivatives of the Salmonella typhi strain Ty2 carrying stable mutations in the aroA gene were isolated. The mutations were generated by transducing an aroA::Tn10 marker into Ty2 and selecting for derivatives which were tetracyline sensitive and dependent on aromatic compounds for growth. Isolates that did not revert to aromatic compound independence at a detectable frequency were obtained. An S. typhimurium derived aroA specific DNA probe was used to demonstrate the presence of DNA rearrangements in the aroA region of the chromosome of some of the S. typhi aroA mutants. Most of these isolates still expressed Vi antigen. Aromatic compound dependent mutants of S. typhi were less virulent in mice than S. typhi Ty2 following intraperitoneal challenge with bacteria suspended in mucin. Mice immunised with one of these mutants, named WBL85-1, were protected against a potentially lethal challenge of S. typhi Ty2.     

Study of the ref(2)P locus of Drosophila melanogaster

The ref(2)P gene of Drosophila melanogaster is implicated in sigma rhabdovirus multiplication. Two common alleles of ref(2)P are known, ref(2)P ⁰ which permits sigma virus multiplication and ref(2)P ^pwhich is restrictive for most sigma virus strains. This gene maps to the cytogenetic region 37E3-F3. Using Df(2L)E55 (=Df(2L)37D2-El;37F5-38A1), we have screened for lethal, semi-lethal and visible mutations following diepoxybutane (DEB) or ethyl methanesulfonate (EMS) mutagenesis. Our data confirm than DEB is mor efficient than EMS at inducing deletions. The mutations obtained in this region define 14 complementation groups. One of them, l(2)37Dh, appears to be a general enhancer of Minute and Minute-like mutations. None of the mutations were allelic to the ref(2)P locus. Loss-of-function alleles of ref(2)P (called null) were selected following DEB mutagenesis. Homozygous or hemizygous ref(2)P ^nullflies are male sterile. These flies, like homozygous or hemizygous ref(2)P ⁰flies, are fully permissive for sigma virus replication. We suggest that the ref(2)P products interact with viral products, but that this interaction is not necessary for an efficient viral cycle.