Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

Extension of the Limits of the Xdh Structural Element in DROSOPHILA MELANOGASTER

Experiments expanding the array of mutants affecting the xanthine dehydrogenase (XDH) structural element in Drosophila melanogaster are described. These include rosy eye color mutants which exhibit interallelic complementation, and mutants with normal eye color but lowered levels of XDH. Evidence is presented which argues that these are structural alterations in the enzyme. Recombination experiments were performed using these mutants as well as some electrophoretic variants. The two ends of the rosy locus are marked with mutant sites which are clearly structural in nature; the XDH structural element and the rosy null mutant map are completely concordant. A possible procedure to recover control element mutants is described.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER: Further Evidence in Support of a CIS-Acting Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

The present report summarizes our recent progress in the genetic dissection of an elementary genetic unit in a higher organism, the rosy locus (ry:3--52.0) in Drosophila melanogaster. Pursuing the hypothesis that the rosy locus includes a noncoding control region, as well as a structural element coding for the xanthine dehydrogenase (XDH) peptide, experiments are described that characterize and map a rosy locus variant associated with much lower than normal levels of XDH activity. Experiments are described that fail to relate this phenotype to alteration in the structure of the XDH peptide, but clearly associate this character with variation in number of molecules of XDH per fly. Large-scale fine-structure recombination experiments locate the genetic basis for this variation in the number of molecules of XDH per fly to a site immediately to the left of the XDH structural element within a region previously designated as the XDH control element. Moreover, experiments clearly separate this "underproducer" variant site from a previously described "overproducer" site within the control region. Examination of enzyme activity in electrophoretic gels of appropriate heterozygous genotypes demonstrates the cis-acting nature of this variation in the number of molecules of XDH. A revision of the map of the rosy locus, structural and control elements is presented in the light of the additional mapping data now available.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER : Evidence for a Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

From a collection of electrophoretic variants of XDH obtained from laboratory strains and natural populations, a stock was isolated that was associated with much greater than normal levels of XDH activity. Preliminary recombination experiments demonstrated that this character maps to the rosy locus. While a series of observations failed to relate this phenotype to alteration in the structure of the XDH polypeptide, kinetic and immunological experiments did succeed in associating this character with variation in number of molecules of XDH/fly. Large scale fine structure recombination experiments locate the genetic basis for this variation in number of molecules of XDH/fly to a site very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Observations are described which eliminate the possibility that we are dealing with a tandem duplication of the XDH structural element. Turning to a regulatory role for this genetic element located adjacent to the XDH structural information, a simple experiment is described which demonstrates that it functions as a "cis-acting" regulator of the XDH structural element.     

On the Identification of the ROSY Locus DNA in DROSOPHILA MELANOGASTER: Intragenic Recombination Mapping of Mutations Associated with Insertions and Deletions

DNA extracts of several rosy-mutation-bearing strains were associated with large insertions and deletions in a defined region of the molecular map believed to include the rosy locus DNA. Large-scale, intragenic mapping experiments were carried out that localized these mutations within the boundaries of the previously defined rosy locus structural element. Molecular characterization of the wild-type recombinants provides conclusive evidence that the rosy locus DNA is localized to the DNA segment marked by these lesions. One of the mutations, ry2101, arose from a P-M hybrid dysgenesis experiment and is associated with a copia insertion. Experiments are described which suggest that copia mobilizes in response to P-M hybrid dysgenesis. Relevance of the data to recombination in higher organisms is considered.     

The Effect of DNA Sequence Polymorphisms on Intragenic Recombination in the Rosy Locus of Drosophila Melanogaster

The effect of simple DNA sequence polymorphisms on intragenic recombination in the rosy locus of Drosophila melanogaster was assayed. Two crosses were performed involving nearly identical molecular distances between selective ry null mutations (3778 nucleotides and 3972 nucleotides). In one heterozygote (ry606/ry531), in addition to the nucleotide substitution ry- mutations, there were 11 simple nucleotide polymorphisms between the selective markers as well as additional flanking simple nucleotide polymorphisms within the rosy locus. In the other heterozygote (ry606/ry609), there were no additional polymorphisms because the two rosy nucleotide substitution mutations were induced on the same rosy isoallele (ry+6). From ry606/ry531 heterozygous females, 27 intragenic crossovers and five marker conversions were seen among 4.53 x 10(5) progeny. From ry606/ry609 heterozygous females, 23 intragenic crossovers and eight marker conversions were seen among 4.18 x 10(5) progeny. The intragenic crossover frequencies per kilobase of DNA were very similar, 1.6 x 10(-5) for ry606/ry531 and 1.4 x 10(-5) for ry606/ry609. Thus, simple DNA sequence polymorphisms neither inhibit nor promote intragenic recombination in D. melanogaster.     

Induction of alcohol dehydrogenase null mutants in the mediterranean fruit fly Ceratitis capitata

Alcohol dehydrogenase null mutants have been induced with X rays in Ceratitis capitata, for use in a genetic sexing system. A combination of selective medium and electrophoretic screening was used to identify these mutants. The results indicate that they are probably large deletions since no homozygotes were found for any of the induced mutants.     

Isozyme variability in species of the genus Drosophila. I. A multiple allelic isozyme system in Drosophila busckii: Inheritance and general considerations

Starch gel electrophoretic analysis of a triallelic leucine aminopeptidase polymorphism in a laboratory population of Drosophila busckii is described. The three alleles involved are expressed without dominance. A series of single-pair matings revealed an excess of heterozygous types in most segregating families, suggesting selection against the homozygous genotypes. A few cases of heterogeneity among progeny extracted from a single family were the result of matings that produced Mendelian ratios. These few cases had no clear genetic explanation, but there is a suggestion of two isoalleles for the electrophoretically intermediate enzyme.The research reported here was begun at the University of Hawaii and completed at the University of Texas and was supported (in part) by Public Health Service Research Grant No. GM 11609 to W. S. Stone and M. R. Wheeler and by Training Grant No. 2 T1-GM-337-06 and GM 00337-07 to R. P. Wagner et al., from the National Institutes of Health.     

Dysgenesis-Induced Instability of Rosy Locus Transformation in DROSOPHILA MELANOGASTER: Analysis of Excision Events and the Selective Recovery of Control Element Deletions

Utilizing the method of P-M hybrid dysgenesis-mediated gene transfer to insert rosy locus DNA into various chromosomal locations, we recovered a transformed strain that carries an ry+ transposon inserted in or near the scalloped locus in polytene section 13F on the X chromosome. The resultant product, when stabilized, behaves as a homozygous and hemizygous viable and fertile extreme scalloped allele associated with wild-type expression of the rosy locus. We have labeled this allele, sdry+. This allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scalloped phenotypes. Representative samples of all phenotypic classes have been characterized by Southern blot analyses of restricted DNA. The most common events are excisions of DNA wholly internal to the transposon and representing sections of rosy DNA. In addition to loss of rosy locus function, such excisions affect the scalloped locus expression.--A second dysgenesis experiment was carried out involving an ry+ transposon inserted in polytene section 16D on the X chromosome. A minimal estimate of the relative frequency of imprecise excisions, determined in this experiment is 75%.--A successful pilot experiment is described that utilizes dysgenic perturbation of the sdry+ allele to select for small deletions of the 5' noncoding region of the rosy locus.     

Biochemical and molecular analysis of spontaneous and induced mutations at the mouse Mod-1 locus

We have analyzed five Mod-1 (malic enzyme) mutants at the molecular and biochemical level. Four of these mutants, three electrophoretic variants and one null mutant, were induced by ethylnitrosourea (ENU). Another null mutant was the result of a spontaneous mutation. All of these mutations were heritable in a Mendelian fashion and viable in the homozygous condition. Restriction endonuclease and Southern blot analysis revealed that the spontaneous null mutant possessed an altered restriction fragment banding pattern. All of the ENU-induced mutants possessed normal restriction fragment banding patterns. All 5 mutants produced normal levels of Mod-1-specific mRNA. Only the spontaneous null mutant produced mRNA with altered size, which was consistent with the altered DNA-banding pattern. MOD-1 enzyme activity levels were normal in the three ENU-induced mutants with altered electrophoretic mobility. Enzyme activity was significantly lower than normal in tissues from animals homozygous for the null alleles, however, using Western blot analysis, low but significant levels of MOD-1 protein in Mod-1 null homozygotes were detected.     

Isolation of induced mutations in E. coli affecting the electrophoretic mobility of enzymes

A screening method was developed for detection of bacterial mutants having active enzymes with altered electrophoretic mobility. The method is based on the use of a mixture of several clones, and examination of an extract of the mixture electrophoretically. A variant enzyme will thus be detectable by its position apart from the mixture of wild-type enzymes.Following exposure to a mutagenic agent, five mutants of E. coli K12 were detected and isolated. Two of these have variant MDH (malate dehydrogenates), the others have variant forms of 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and esterase.Preliminary mapping of the MDH locus has been performed.     

Genetic Recombination in Micromonospora

Biochemical mutants were obtained from Micromonospora chalcea, M. purpurea, and M. echinospora by using ultraviolet radiation or nitrosoguanidine. Crosses carried out between complementary nutritional mutants of the same species showed positive genetic interaction. Data are reported which indicate that the interaction between the crossed strains is due to genetic recombination. No evidence for interspecific genetic recombination was found.     

Absence of Detectable Mitochondrial Recombination in Paramecium

An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

Intramolecular homologous recombination in cells infected with temperature-sensitive mutants of vaccinia virus.

I have used a plasmid containing two copies of the Saccharmyces cerevisiae his3 gene to study intramolecular homologous recombination in vaccina virus-infected cells. Recombination of the plasmid was monitored by restriction enzyme digestion and Southern blot hybridization in cells infected with representatives from each of 32 complementation groups of temperature-sensitive mutants ts42 and ts17 did not replicate nor detectably recombine the input plasmid. All except one of the mutants that synthesized normal amounts of viral DNA and protein replicated and recombined the plasmid in a manner indistinguishable from wild-type virus. The remaining mutant, ts13, only poorly replicated and recombined the input plasmid. Thus, the processes of replication and recombination could not be separated by using this battery of mutants. Viral mutants defective in late protein synthesis were unable to resolve the vaccinia virus concatemer junction in plasmids but carried out intramolecular homologous recombination with plasmids as efficiently as did wild-type virus at the conditionally lethal temperature. This result distinguishes homologous recombination, which requires early gene products, from resolution of concatemer junctions, which requires additional late gene products.     

Marker Effects in the Genetic Transduction of Tryptophan Mutants of E. COLI

Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.-Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.-Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).     

Xanthine dehydrogenase from Drosophila melanogaster: purification and properties of the wild-type enzyme and of a variant lacking iron-sulfur centers.

Xanthine dehydrogenase has been purified to homogeneity by conventional procedures from the wild-type strain of the fruit fly Drosophila melanogaster, as well as from a rosy mutant strain (E89----K, ry5231) known to carry a point mutation in the iron-sulfur domain of the enzyme. The wild-type enzyme had all the specific properties that are peculiar to the molybdenum-containing hydroxylases. It had normal contents of molybdenum, the pterin molybdenum cofactor, FAD, and iron-sulfur centers. EPR studies showed its molybdenum center to be quite indistinguishable from that of milk xanthine oxidase. As isolated, only about 10% of the enzyme was present in the functional form, with most or all of the remainder as the inactive desulfo form. It is suggested that this may be present in vivo. Extensive proteolysis accompanied by the development of oxidase activity took place during isolation, but dehydrogenase activity was retained. EPR properties of the reduced iron-sulfur centers, Fe-SI and Fe-SII, in the enzyme are very similar to those of the corresponding centers in milk xanthine oxidase. The E89----K mutant enzyme variant was in all respects closely similar to the wild-type enzyme, with the exception that it lacked both of the iron-sulfur centers. This was established both by its having the absorption spectrum of a simple flavoprotein and by the complete absence of EPR signals characteristic of iron-sulfur centers in the reduced enzyme. Despite the lack of iron-sulfur centers, the mutant enzyme had xanthine:NAD+ oxidoreductase activity indistinguishable from that of the wild-type enzyme. Stopped-flow measurements indicated that, as for the wild-type enzyme, reduction of the mutant enzyme was rate-limiting in turnover. Thus, the iron-sulfur centers appear irrelevant to the normal turnover of the wild-type enzyme with these substrates. However, activity to certain oxidizing substrates, particularly phenazine methosulfate, is abolished in the mutant enzyme variant. This is one of the first examples of deletion by genetic means of iron-sulfur centers from an iron-sulfur protein. The relevance of our findings both to the roles of iron-sulfur centers in other systems and to the nature of the oxidizing substrate for the Drosophila enzyme in vivo are briefly discussed.