A mutation defective in the xanthine alternative pathway of Aspergillus nidulans: its use to investigate the specificity of uaY mediated induction.

Summary In Aspergillus nidulans uric acid can be produced from xanthine via purine hydroxylase I (xanthine dehydrogenase) or via the xanthine alternative pathway (Darlington and Scazzocchio, Biochem. Biophys. Acta, 166, 569–571; 1968). A mutation defective in the xanthine alternative pathway of Aspergillus nidulans is described. By combining this mutation with hxB-20 which results in complete loss of purine hydroxylase I and II activities, but which conserves cross-reacting material, it is possible to block completely uric acid production and thus investigate which are the effective in vivo inducers of three enzymes under the control of the positive regulatory gene uaY: adenine deaminase, purine hydroxylase I (measured as cross-reacting material) and urate oxidase. It is concluded that uric acid is the only effective physiological inducer, while its 2 and 8 thio-analogues serve as gratuitous inducers.     

The genetic control of molybdoflavoproteins in Aspergillus nidulans. II. Use of NADH dehydrogenase activity associated with xanthine dehydrogenase to investigate substrate and product inductions

Summary An NADH dehydrogenase activity is induced together with xanthine dehydrogenase I in Aspergillus nidulans wild type strains. The two activities have the same mobility in polyacrylamide gels (Fig.1) and are immunologically indistinguishable (Fig.2). Several hxB mutants which lack xanthine dehydrogenase activity but conserve the associated NADH dehydrogenase activity were used to determine that uric acid, but not hypoxanthine, is an inducer of the enzyme (Figs. 3 and 4). This fact together with results reported previously (Scazzocchio and Darlington, 1968) indicate that the induction of xanthine dehydrogenase I and urate oxidase requires the product specified by the uaY gene, as well as the common effector, urie acid.Paper I of this series is Scazzocchio, Holl and Foguelman (1973).     

A rapid and simple radioassay for inosinic acid: pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa.

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10^?5m allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) into the reaction mixture, the [8-¹⁴C]hypoxanthine (Hx) is converted only to [8-¹⁴C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase.Using [8-¹⁴C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 mm ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8-¹⁴C]IMP or [8-¹⁴C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value.Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.     

Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans

Summary In this publication we report the identification of a protein likely to be coded by uaY, a regulatory gene in the ascomycete Aspergillus nidulans. uaY is a positive control gene necessary for the expression of at least eight unlinked structural genes involved in purine uptake and degradation (Scazzocchio and Gorton 1977). The physiological effector of the uaY system is uric acid, while some of its thioanalogs serve as gratuitous inducers. Effector binding proteins were detected by binding to 2-thiouric acid after phosphocellulose column chromatography, or as uric acid binding fractions after DNA-cellulose column chromatography. Two binding peaks are present in mycelial extracts purified by either method. These are missing in a putative small deletion of the uaY gene. A leaky mutation, uaY 109 described in detail elsewhere (Scazzocchio et al. 1980) shows only one peak. The wild type peaks are eluted at 55 mM NaCl and at 720 mM NaCl while the peak present in uaY109 is eluted at 120 mM NaCl. This implies that at least one peak represents a protein coded by the uaY gene. The major peak was analysed by equilibrium dialysis experiments. These establish a K_diss.2×10^-7 and a minimum number of binding sites of 3×10^-14 moles/mg of soluble protein in a crude extract derived from protoplast lysis. An extract from a strain carrying the uaY207 deletion, purified blind, lacks any binding activity in the equilibrium dialysis cell.     

The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Summary Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by dur0^hmutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The dur0^hmutations act only in cis and are a new case of cis effect strongly cancelled in /a diploid, similar to cargA ⁺0^hmutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity.The constitutivity produced by dur0^hmutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH ₄ ⁺ effect.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Thermostable Xanthine Oxidase Activity from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t_1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg²⁺, Ag⁺ and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.     

Methylammonium Resistance in Aspergillus nidulans

Mutants of Aspergillus nidulans resistant to methylammonium toxicity are simultaneously derepressed in the presence of ammonium for apparently all ammonium-repressible activities. Enzyme assays directly demonstrate derepression of nitrate, nitrite, and hydroxylamine reductases, xanthine dehydrogenase, urate oxidase, and allantoinase, whereas in vivo tests show that ammonium and methylammonium repression or inhibition (or both) is relieved in these mutants in pathways of nitrate assimilation, purine transport and degradation, and amino acid, amine, and amide catabolism. Ammonium and methylammonium uptake is apparently not defective in these mutants, for they grow normally on limiting levels of these ions as sole nitrogen source. There is no evidence that more than one gene can mutate to produce the methylammonium resistance (mea^R) phenotype. Such mutations are semidominant in both heterocaryons and diploids. The ability of mea^R mutations to effect derepression of activities specified by genes within another nucleus in a heterocaryon shows that the action of the mea product is not restricted to the nucleus. Three types of hypotheses might explain this generalized derepression. First, ammonium and methylammonium might not themselves be co-repressors but might require a metabolic conversion, blocked in these mutants, to become co-repressors. Secondly, the mea locus might specify an activity expressed in mea^R but not wild-type (mea^S) strains, which diminishes the concentration of ammonium and methylammonium participating in co-repression. Finally, ammonium repression might involve a macromolecular control element specified by the mea^R locus and common to many or all ammonium-repressible systems. The existence of “regulation reversal mutations” at the mea^R locus and the lack of uniformity and coordination with which different enzymatic activities respond to mutational derepression is most compatible with the last type of hypothesis.     

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd ⁺ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.     

UTX and UTY Demonstrate Histone Demethylase-Independent Function in Mouse Embryonic Development

UTX (KDM6A) and UTY are homologous X and Y chromosome members of the Histone H3 Lysine 27 (H3K27) demethylase gene family. UTX can demethylate H3K27; however, in vitro assays suggest that human UTY has lost enzymatic activity due to sequence divergence. We produced mouse mutations in both Utx and Uty. Homozygous Utx mutant female embryos are mid-gestational lethal with defects in neural tube, yolk sac, and cardiac development. We demonstrate that mouse UTY is devoid of in vivo demethylase activity, so hemizygous X^Utx− Y⁺ mutant male embryos should phenocopy homozygous X^Utx− X^Utx− females. However, X^Utx− Y⁺ mutant male embryos develop to term; although runted, approximately 25% survive postnatally reaching adulthood. Hemizygous X⁺ Y^Uty− mutant males are viable. In contrast, compound hemizygous X^Utx− Y^Uty− males phenocopy homozygous X^Utx− X^Utx− females. Therefore, despite divergence of UTX and UTY in catalyzing H3K27 demethylation, they maintain functional redundancy during embryonic development. Our data suggest that UTX and UTY are able to regulate gene activity through demethylase independent mechanisms. We conclude that UTX H3K27 demethylation is non-essential for embryonic viability.     

Effects of a purine inhibitor,allopurinol, on urate metabolism in the german cockroach,Blattella germanica L. (Dictyoptera: Blattellidae)

• 1.1. Adult male and female cockroaches (Blattella germanica) were maintained on a positive nitrogen balance diet (66% protein) containing various levels of allopurinol (0–3%) to determine the effects of allopurinol on urate synthesis and storage.
• 2.2. Each insect was injected with [¹⁴C]hypoxanthine and after 1 week was analyzed for whole-body hypoxanthine, xanthine and urate radiolabel.
• 3.3. There was a general trend of decreased whole-body radiolabel retention, radiolabeled body urates and total-body urate content in both sexes with increasing amounts of dietary allopurinol.
• 4.4. Virgin female adults were allowed to feed on diets containing 0, 25 and 66% protein plus 0.1% allopurinol and were injected with [¹⁴C]xanthine.
• 5.5. After 1 week radiolabel content in the whole-body xanthine and urate pools was determined.
• 6.6. Females on the 0% protein diets contained less radiolabel in the whole-body and body urates than those on either 25 or 66% protein diets.

     

Experimental Population Genetics of Meiotic Drive Systems I. Pseudo-Y Chromosomal Drive as a Means of Eliminating Cage Populations of DROSOPHILA MELANOGASTER

The experimental population genetics of Y-chromosome drive in Drosophila melanogaster is approximated by studying the behavior of T(Y;2),SD lines. These exhibit "pseudo-Y" drive through the effective coupling of the Y chromosome to the second chromosome meiotic drive locus, Segregation distorter (SD). T(Y;2),SD males consequently produce only male offspring. When such lines are allowed to compete against structurally normal SD⁺ flies in population cages, T(Y;2),SD males increase in frequency according to the dynamics of a simple haploid selection model until the cage population is eliminated as a result of a deficiency in the number of adult females. Cage population extinction generally occurs within about seven generations.—Several conclusions can be drawn from these competition cage studies:

(1) Fitness estimates for the T(Y;2),SD lines (relative to SD⁺ ) are generally in the range of 2–4, and these values are corroborated by independent estimates derived from studies of migration-selection equilibrium.

(2) Fitness estimates are unaffected by cage replication, sample time, or the starting frequency of T(Y;2),SD males, indicating that data from diverse cages can be legitimately pooled to give an overall fitness estimate.

(3) Partitioning of the T(Y;2),SD fitnesses into components of viability, fertility, and frequency of alternate segregation (Y + SD from X + SD⁺) suggests that most of the T(Y;2),SD advantage derives from the latter two components. Improvements in the system might involve increasing both the viability and the alternate segregation to increase the total fitness.

While pseudo-Y drive operates quite effectively against laboratory stocks, it is less successful in eliminating wild-type populations which are already segregating for suppressors of SD action. This observation suggests that further studies into the origin and rate of accumulation of suppressors of meiotic drive are needed before an overall assessment can be made of the potential of Y-chromosome drive as a tool for population control.

     

Triallelic complementation and hybrid enzyme formation in Chlamydomonas reinhardi

Summary In Chlamydomonas, the arg-7 cistron (linkage group I) is the structural gene for the multimeric (probably pentameric) enzyme argininosuccinate lyase. Most of the alleles of the cistron were previously shown to complement in some pair combinations, giving rise to phenotypically wild-type diploids.By crossing diploid (mt^-) and haploid (mt⁺) cells bearing different markers of auxotrophy, seven different presumptive triploid strains, phenotypically wild-type, were isolated. Each strain had 3 different arg-7 alleles or 2 mutant alleles associated with a wild one.The isolates were cytologically and biochemically analyzed: it could be concluded that they were triploid or ar least trisomic for the linkage group I.The specific activity and the thermosensitivity of the lyase were compared in the different triploids and in the diploids bearing two of the three corresponding arg-7 alleles. In most cases, the enzyme formed by triallelic complementation was more active and more heat resistant than the enzyme formed by diallelic complementation. These results can be interpreted by assuming that hybrid enzyme is formed by interaction between the products of the three different alleles. They provide a molecular basis for explaining the increased vigor often found in polyploids.     

Two independent mutational events in the loss of urate oxidase during hominoid evolution

Summary Urate oxidase was lost in hominoids during primate evolution. The mechanism and biological reason for this loss remain unknown. In an attempt to address these questions, we analyzed the sequence of urate oxidase genes from four species of hominoids: human (Homo sapiens), chimpanzee (Pan troglodytes), orangutan (Pongo pygmaeus), and gibbon (Hylobates). Two nonsense mutations at codon positions 33 and 187 and an aberrant splice site were found in the human gene. These three deleterious mutations were also identified in the chimpanzee. The nonsense mutation at codon 33 was observed in the orangutan urate oxidase gene. None of the three mutations was present in the gibbon; in contrast, a 13-bp deletion was identified that disrupted the gibbon urate oxidase reading frame. These results suggest that the loss of urate oxidase during the evolution of hominoids could be caused by two independent events after the divergence of the gibbon lineage; the nonsense mutation at codon position 33 resulted in the loss of urate oxidase activity in the human, chimpanzee, and orangutan, whereas the 13-bp deletion was responsible for the urate oxidase deficiency in the gibbon. Because the disruption of a functional gene by independent events in two different evolutionary lineages is unlikely to occur on a chance basis, our data favor the hypothesis that the loss of urate oxidase may have evolutionary advantages. Offprint requests to: C.T. Caskey     

Male-Sterilizing Interactions between Duplications and Deficiencies for Proximal X-Chromosome Material in DROSOPHILA MELANOGASTER

The genetic limits of sixty-four deficiencies in the vicinity of the euchromatic-heterochromatic junction of the X chromosome were mapped with respect to a number of proximal recessive lethal mutations. They were also tested for male fertility in combination with three Y chromosomes carrying different amounts of proximal X-chromosome-derived material (B^SYy⁺, y⁺Ymal¹²⁶ and y⁺Ymal⁺). All deficiencies that did not include the locus of bb and a few that did were male-fertile in all male-viable Df(1)/Dp(1;Y) combinations. Nineteen bb deficiencies fell into six different classes by virtue of their male-fertility phenotypes when combined with the duplicated Y chromosomes. The six categories of deficiencies are consistent with a formalism that invokes three factors or regions at the base of the X, one distal and two proximal to bb, which bind a substance critical for precocious inactivation of the X chromosome in the primary spermatocyte. Free duplications carrying these regions or factors compete for the substance in such a way that, in the presence of such duplications, proximally deficient X chromosomes are unable to command sufficient substance for proper control of X-chromosome gene activity preparatory to spermatogenesis. We conclude that there is no single factor at the base of the X that is required for the fertility of males whose genotype is otherwise normal.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Mapping of the polA Locus of ESCHERICHIA COLI K12: Genetic Fine Structure of the Cistron

The close linkage of the glnA gene with polA was exploited to construct a fine structure map of polA by means of generalized transduction with phage P1. Nine different polA^- alleles were mapped by recombinational crosses. The results indicate a gene order consistent with previous observations (Kelley and Grindley 1976a; Murray and Kelley 1979). Three mutations, polA5, polA6 and polA12 map within the "carboxy-terminal" or "large-fragment" portion of the gene in unambiguous order. Four alleles, known to affect the "aminoterminal" portion of the gene, polA107, polA214, polA480ex and polA4113, appear to be closely linked with certain ambiguities in their exact order. All four of these mutations are known to alter the 5''→3'' exonuclease activity of DNA polymerase I and three of them result in the conditional lethal polA^- phenotype. The polA1 nonsense mutation maps between these two groups in a position consistent with its known effect, production of an amber fragment that includes the 5''→3'' exonuclease. The final allele, resA1, is another nonsense mutation that maps at the extreme "amino-terminus" of the cistron.——A number of control experiments were conducted to determine the effects of polA^- mutations on the P1-mediated recombinational event. These experiments indicated that abortive transduction occurs quite frequently, but the formation of abortive transductants and segregation of unselected transduced markers among daughter progeny is like that observed by other investigators. There was no evidence that any individual polA^- allele behaved in an exceptional fashion during recombination.     

Specific induction of catabolism and its relation to repression of biosynthesis in arginine metabolism of Saccharomyces cerevisiae.

Experimental results are presented in support of the model previously proposed for specific induction of the synthesis of enzymes for arginine catabolism in Saccharomyces cerevisiae (Wiame, 1971a,b), and its connection with end-product repression of arginine biosynthetic enzymes. The data support the occurrence of negative regulation of metabolism in a eukaryote.Operator regions, one for arginase and another for ornithine transaminase, are identified. The operator mutations are fully constitutive. A mutation compatible with the occurrence of a catabolic represser, CARGR, leads to partial pleiotropic constitutivity.The connection between the induction process and the repression of biosynthetic enzymes is due to a common receptor of metabolic signals, an ambivalent repressor ARGR endowed with the property of a usual repressor for anabolic enzymes and playing the role of inducer at the level of CARGR; this cascade process simulates a positive control. argR^? mutations, by producing defective ARGR, “turn on” anabolic enzyme synthesis and “turn off” the synthesis of catabolic enzymes (Fig. 2). The dual role of ARGR is confirmed by the isolation of a mutation argRII^d which, in contrast to the defective properties caused by usual argR^? mutations, causes a dominant hyperactivity toward induction of a catabolic enzyme, but retains recessive hypoactivity toward repression of an anabolic enzyme. Such an ambivalent repressor is a function necessary for mutual, balanced exclusion between opposite metabolisms.Many operator constitutive mutations for arginase, cargA⁺O^?, change the level of enzyme to a similar value, thus defining a genetic function. One of these mutations, cargA⁺O^h, in addition to having unusual genetic behaviour, leads to production of twice as much arginase as cargA⁺O^?. This suggests the existence of another genetic region near the structural gene for this enzyme and an additional regulatory function to be analyzed in a separate paper (Dubois &; Wiame, 1978).