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 : 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.     

Cytogenetic Analysis of the Chromosomal Region Immediately Adjacent to the Rosy Locus in DROSOPHILA MELANOGASTER

This report describes the genetic analysis of a region of the third chromosome of Drosophila melanogaster extending from 87D2–4 to 87E12–F1, an interval of 23 or 24 polytene chromosome bands. This region includes the rosy (ry, 3–52.0) locus, carrying the structural information for xanthine dehydrogenase (XDH). We have, in recent years, focused attention on the genetic regulation of the rosy locus and, therefore, wished to ascertain in detail the immediate genetic environment of this locus. Specifically, we question if rosy is a solitary genetic unit or part of a larger complex genetic unit encompassing adjacent genes. Our data also provide opportunity to examine further the relationship between euchromatic gene distribution and polytene chromosome structure.——The results of our genetic dissection of the rosy microregion substantiate the conclusion drawn earlier (Schalet, Kernaghan and Chovnick 1964) that the rosy locus is the only gene in this region concerned with XDH activity and that all adjacent genetic units are functionally, as well as spatially, distinct from the rosy gene. Within the rosy micro-region, we observed a close correspondence between the number of complementation groups (21) and the number of polytene chromosome bands (23 or 24). Consideration of this latter observation in conjunction with those of similar studies of other chhromosomal regions supports the hypothesis that each polytene chromosome band corresponds to a single genetic unit.     

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.     

Heterochromatic Position Effect at the Rosy Locus of DROSOPHILA MELANOGASTER: Cytological, Genetic and Biochemical Characterization

This report describes cytological, genetic and biochemical studies designed to characterize two γ-radiation induced, apparent "underproducer" variants of the rosy locus (ry:3-52.0), ry^ps1149 and ry^ps11136. The following observations provide a compelling basis for their diagnosis as heterochromatic position effect variants. (1) They are associated with rearrangements that place heterochromatin adjacent to the rosy region of chromosome 3 (87D). (2) The effect of these mutations on rosy locus expression is subject to modification by abnormal Y chromosome content. (3) The rearrangement alters only the expression of the rosy allele on the same chromosome (cis-acting). (4) The Y chromosome modification is only on the position-affected allele's expression. (5) The recessive lethality associated with the rearrangements relate to specific rosy region vital loci, and for ry^{ps 11136}, the lethality is not Y chromosome modified. (6) The peptide product of the position-affected allele is qualitatively normal by several criteria. (7) Heterozygous deletion of 87E2-F2 is a suppressor of the rosy position effect. (8) The rosy position effect on XDH production may be assayed in whole larvae and larval fat body tissue as well as in adults.     

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.     

The effect of chromosomal position on the expression of the Drosophila xanthine dehydrogenase gene

Thirty-six isogenic D. melanogaster strains that differed only in the chromosomal location of a 7.2 or an 8.1 kb DNA segment containing the (autosomal) rosy gene were constructed by P-element-mediated gene transfer. Since the flies were homozygous for a rosy- allele, rosy gene function in these indicated the influence of flanking sequences on gene expression. The tissue distribution of XDH activity in all the strains was normal. Each line exhibited a characteristic level of adult XDH-specific activity. The majority of these values were close to wild-type levels; however, the total variation in specific activity among the lines was nearly fivefold. Thus position effects influence expression of the rosy gene quantitatively but do not detectably alter tissue specificity. X-linked rosy insertions were expressed on average 1.6 times more activity in males than in females. Hence the gene acquires at least partial dosage compensation upon insertion into the X chromosome.     

Post-Translational Modification as a Potential Explanation of High Levels of Enzyme Polymorphism: Xanthine Dehydrogenase and Aldehyde Oxidase in DROSOPHILA MELANOGASTER

Xanthine dehydrogenase (XDH) and aldehyde oxidase (AO) in Drosophila melanogaster require for their activity the action of another unlinked locus, maroon-like (mal). While the XDH and AO loci are on chromosome 3, mal maps to the X chromosome. Although functional mal gene product is required for XDH and AO activity, it is possible to examine the effects of mutant mal alleles in those cases when pairs of mutants complement to produce a partial restoration of activity. To test whether mal mediates a post-translational modification of the XDH and AO proteins, we constructed several mal heteroallelic complementing stocks of Drosophila in which the third chromosomes were co-isogenic. Since all lines were co-isogenic for the XDH and AO structural genes, any variation in these enzymes seen when comparing these stocks must have been produced by post-translational modification by mal. We examined the XDH and AO proteins in these stocks by gel-sieving electrophoresis, a procedure that permits independent characterization of a protein's charge and shape, and is capable of discriminating many variants not detected in routine electrophoresis. In every mal heteroallelic combination, there is a significant alteration in protein shape, when compared to wild type. The magnitude of differences in shape of XDH and AO is correlated both with differences in their enzyme activities and with differences in their thermal stabilities. As the body of this variation appears heritable, any functional differences resulting from these variants are of real genetic and evolutionary interest. A similar post-translational modification of XDH and AO by yet another locus, lxd, was subsequently documented in an analogous manner. The pattern of electrophoretic differences produced by mal and lxd modification is similar to that reported for electrophoretic "alleles" of XDH in natural populations. The implication is that heritable variation in electrophoretic mobility at these two enzyme loci, and potentially at other loci, is not necessarily allelic to the structural gene loci.     

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese

Background

To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.

Methods and Principal Findings

We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10⁻¹⁰). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.

Conclusions and Significance

In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.     

Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata

Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.     

Influence of Maternal Phenotype on Metabolic Differentiation of Agouti Locus Mutants in the Mouse

The results of extensive breeding experiments indicate that the phenotypic differentiation of embryos carrying the viable yellow, A ^vy, or mottled, a^m, mutations is influenced to a major extent by the agouti locus genotype and the strain genome of the dam. The A^vy/a and a^m/a genotypes are each expressed in a spectrum of coat color phenotypes. These can be grouped into two classes, mottled and pseudoagouti.—In a reciprocal cross of C57BL/6JNIcrWf and AM/Wf-a^m/a^m mice, 29.5% of the offspring of C57BL/6 dams were of the pseudoagouti phenotype, whereas no pseudoagouti offspring were produced by AM strain dams.—Mottled yellow A^vy/a mice become obese and tumor formation is enhanced in these mice in comparison with the lean pseudoagouti A^vy/a siblings.—In two different reciprocal crosses using four different inbred strains, the proportion of pseudoagouti A^vy/a offspring differed according to the strain of the dam. Regardless of strain, mottled yellow A ^vy/a dams produced significantly fewer pseudoagouti A ^vy/a offspring than did black a/a dams.—The data suggest that metabolic differentiation of A^vy/a zygotes into phenotypic classes with different susceptibilities to obesity and tumor formation is influenced to a considerable degree by the metabolic characteristics of the oviductal and uterine environment of the dam.     

CD1 gene polymorphisms and phenotypic variability in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is characterized by marked phenotypic variation ranging from adrenomyeloneuropathy (AMN) to childhood cerebral ALD (CCALD). X-ALD is caused by mutations in the ABCD1 gene, but no genotype-phenotype correlation has been established so far and modifier gene variants are suspected to modulate phenotypes. Specific classes of lipids, enriched in very long-chain fatty acids that accumulate in plasma and tissues from X-ALD patients are suspected to be involved in the neuroinflammatory process of CCALD. CD1 proteins are lipid- antigen presenting molecules encoded by five CD1 genes in human (CD1A-E). Association studies with 23 tag SNPs covering the CD1 locus was performed in 52 patients with AMN and 87 patients with CCALD. The minor allele of rs973742 located 4-kb downstream from CD1D was significantly more frequent in AMN patients (χ² = 7.6; P = 0.006). However, this association was no longer significant after Bonferroni correction for multiple testing. The other polymorphisms of the CD1 locus did not reveal significant association. Further analysis of other CD1D polymorphisms did not detect stronger association with X-ALD phenotypes. Although the association with rs973742 warrants further investigations, these results indicate that the genetic variants of CD1 genes do not contribute markedly to the phenotypic variance of X-ALD.     

A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol

Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ∼25% of the heritability of the phenotypes. To date, no unbiased screen for gene–environment interactions for circulating lipids has been reported. We screened for variants that modify the relationship between known epidemiological risk factors and circulating lipid levels in a meta-analysis of genome-wide association (GWA) data from 18 population-based cohorts with European ancestry (maximum N = 32,225). We collected 8 further cohorts (N = 17,102) for replication, and rs6448771 on 4p15 demonstrated genome-wide significant interaction with waist-to-hip-ratio (WHR) on total cholesterol (TC) with a combined P-value of 4.79×10⁻⁹. There were two potential candidate genes in the region, PCDH7 and CCKAR, with differential expression levels for rs6448771 genotypes in adipose tissue. The effect of WHR on TC was strongest for individuals carrying two copies of G allele, for whom a one standard deviation (sd) difference in WHR corresponds to 0.19 sd difference in TC concentration, while for A allele homozygous the difference was 0.12 sd. Our findings may open up possibilities for targeted intervention strategies for people characterized by specific genomic profiles. However, more refined measures of both body-fat distribution and metabolic measures are needed to understand how their joint dynamics are modified by the newly found locus.     

A common complement C3 variant is associated with protection against wet age-related macular degeneration in a Japanese population

Background

Genetic variants in the complement component 3 gene (C3) have been shown to be associated with age-related macular degeneration (AMD) in Caucasian populations of European descent. In particular, a nonsynonymous coding variant, rs2230199 (R102G), is presumed to be the most likely causal variant in the C3 locus based on strong statistical evidence for disease association and mechanistic functional evidence. However, the risk allele is absent or rare (<1%) in Japanese and Chinese populations, and the association of R102G with AMD has not been reported in Asian populations. Genetic heterogeneity of disease-associated variants among different ethnicities is common in complex diseases. Here, we sought to examine whether other common variants in C3 are associated with wet AMD, a common advanced-stage manifestation of AMD, in a Japanese population.

Methodology/Principal Findings

We genotyped 13 tag single nucleotide polymorphisms (SNPs) that capture the majority of common variations in the C3 locus and tested for associations between these SNPs and wet AMD in a Japanese population comprising 420 case subjects and 197 controls. A noncoding variant in C3 (rs2241394) exhibited statistically significant evidence of association (allelic P = 8.32×10⁻⁴; odds ratio = 0.48 [95% CI = 0.31–0.74] for the rs2241394 C allele). Multilocus logistic regression analysis confirmed that the effect of rs2241394 was independent of the previously described loci at ARMS2 and CFH, and that the model including variants in ARMS2 and CFH plus C3 rs2241394 provided a better fit than the model without rs2241394. We found no evidence of epistasis between variants in C3 and CFH, despite the fact that they are involved in the same biological pathway.

Conclusions

Our study provides evidence that C3 is a common AMD-associated locus that transcends racial boundaries and provides an impetus for more detailed genetic characterization of the C3 locus in Asian populations.     

An analysis of xanthine dehydrogenase negative mutants of the rosy locus in Drosophila melanogaster.

Eighteen alleles of the rosy locus in Drosophila melanogaster were characterized to identify putative nonsense mutants. Seven alleles exhibited no evidence of intragenic complementation, no evidence of immunological complementation, no evidence of immunological cross-reactivity to antibodies elicited by wild type xanthine dehydrogenase (XDH), and of course were completely deficient in measurable XDH activity. It is possible that one or more of these highly negative ry alleles are nonsense mutants. The remaining eleven ry alleles code for XDH molecules that retain some antigenic similarities to the wild type enzyme as assessed by immunoelectrophoresis and six of these eleven were capable of intragenic complementation.     

Molybdenum cofactor deficiency causes translucent integument,male-biased lethality,and flaccid paralysis in the silkworm Bombyx mori

Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants.