White Locus Research Articles

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

We used the UAS/GAL4 two component system to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi we demonstrate that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi. Our results demonstrate that inducible RNAi is a powerful tool to study the role of essential genes throughout development.

Gene Constitution of Egg White Proteins of Native Chicken in Asian Countries

Genetic variations of seven egg white protein loci in 1,112 samples from eight Asian countries (Yunnan province of China, Mongolia, Nepal, Vietnam, Laos, Thailand, Myanmar, Indonesia) and 360 samples from two improved breeds (Isa Brown, Boris Brown) were investigated by using starch gel and polyacrylamide gel electrophoresis. Five egg white protein loci (Oν, G 3 , G 2 , G 1 and Tf E W ) were found to be polymorphic in Asian native chicken populations. The proportion of polymorphic loci (P p o l y ) and average heterozygosity (H) of Asian native populations varied from 0.143 to 0.714 and 0.014 to 0.225, respectively, and were higher than those of improved breeds. The subdivision index (G S T ) value among 18 native chicken populations in Asia is lower (0.0827) than among improved chicken populations (0.1693). This value means that the degree of subdivision among Asian native populations is lower than among improved breeds and gene constitutions among populations in Asia are similar.

Genomic organization and characterization of the white locus of the Mediterranean fruitfly, Ceratitis capitata.

An approximately 14-kb region of genomic DNA encoding the wild-type white eye (w+) color gene from the medfly, Ceratitis capitata has been cloned and characterized at the molecular level. Comparison of the intron-exon organization of this locus among several dipteran insects reveals distinct organizational patterns that are consistent with the phylogenetic relationships of these flies and the dendrogram of the predicted primary amino acid sequence of the white loci. An examination of w+ expression during medfly development has been carried out, displaying overall similarity to corresponding studies for white gene homologues in Drosophila melanogaster and other insects. Interestingly, we have detected two phenotypically neutral allelic forms of the locus that have arisen as the result of an apparently novel insertion or deletion event located in the large first intron of the medfly white locus. Cloning and sequencing of two mutant white alleles, w1 and w2, from the we,wp and M245 strains, respectively, indicate that the mutant conditions in these strains are the result of independent events--a frameshift mutation in exon 6 for w1 and a deletion including a large part of exon 2 in the case of w2.

Localizing genes in Drosophila melanogaster polytene chromosomes by fluorescence in situ hybridization.

This paper describes a method for the identification of single copy genes in Drosophila melanogaster polytene chromosomes, using fluorescence in situ hybridization (FISH). We demonstrate the detection of white (w), a gene previously mapped to 1-1.5 region of the linkage map, and to 3C2 region of the cytogenetic map of X chromosome. Squash preparations of polytene chromosomes from salivary glands dissected out from third instar larvae of Drosophila melanogaster were denatured and subjected to hybridization with a digoxigenin labeled probe, corresponding to mini-white gene. The preparations were then washed and incubated with anti-digoxigenin-fluorescein antibodies. After removal of the nonspecifically bound antibodies, the polytene chromosomes were counterstained with propidium iodide. Fluorescence microscopy revealed white locus in the X chromosome in a subterminal location, in agreement with the above mentioned maps. The protocol is efficient and adaptable for simultaneously multiple signal detection.

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster.

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.

Sequence analysis of the boundaries of the tandem duplication from the white-ivory mutant of Drosophila melanogaster and two chemically induced revertants.

We have previously shown that revertants obtained from the white-ivory mutants of Drosophila melanogaster, both spontaneous and induced, have lost a DNA fragment of 2.9 kb that is duplicated in tandem in the white-ivory mutation. To prove the accuracy of the deletion in revertants obtained after treatment with alkylating agents, we have sequenced DNA fragments previously amplified by PCR. These fragments correspond to the ends of the remaining 2.9 kb copy of these revertants and the internal region of the junction of both copies, which constitutes the duplication in the white-ivory mutant. These sequences are compared with those from white-ivory mutants. Our results show slight differences from the published sequence of the white-ivory mutation and with the wild-type sequence of the white locus. The sequences of the two revertants analysed show that excision of the duplicated fragment is very precise. We hypothesize the mechanism of excision in terms of intrachromosomal recombination induced by double-strand break repair after treatment with alkylating agents.

Impact of multiple insertions of two retroelements, ZAM and Idefix at an euchromatic locus.

Transposable elements represent a large fraction of eukaryotic genomes and they are thought to play an important role in chromatin structure. ZAM and Idefix are two LTR-retrotransposons from Drosophila melanogaster very similar in structure to vertebrate retroviruses. In all the strains where their distribution has been studied, ZAM appears to be present exclusively in the intercalary heterochromatin while Idefix copies are mainly found in the centromeric heterochromatin with very few copies in euchromatin. Their distribution varies in a specific strain called RevI in which the mobilization of ZAM and Idefix is highly induced. In this strain, 15 copies of ZAM and 30 copies of Idefix are found on the chromosomal arms in addition to their usual distribution. Amongst the loci where new copies are detected, a hotspot for their insertion has been detected at the white locus where up to four elements occurred within a 3-kb fragment at the 5' end of this gene. This property of ZAM and Idefix to accumulate at a defined site provides an interesting paradigm to bring insight into the effect exerted by multiple insertions of transposable elements at an euchromatic locus.

The Belt mutation in pigs is an allele at the Dominant white (I/KIT) locus.

A white belt is a common coat color phenotype in pigs and is determined by a dominant allele (Be). Here we present the result of a genome scan performed using a Hampshire (Belt)/Pietrain (non-Belt) backcross segregating for the white belt trait. We demonstrate that Belt maps to the centromeric region of pig Chromosome (Chr) 8 harboring the Dominant white (I/KIT) locus. Complete cosegregation between Belt and a single nucleotide polymorphism in the KIT gene was observed. Another potential candidate gene, the endothelin receptor type A gene (EDNRA), was excluded as it was assigned to a different region (SSC8q21) by FISH analysis. We argue that Belt is a regulatory KIT mutation on the basis of comparative data on mouse KIT mutants and our previous sequence analysis of the KIT coding sequence from a Hampshire pig. Quantitative PCR analysis revealed that Belt is not associated with a KIT duplication, as is the case for the Patch and Dominant white alleles. Thus, Belt is a fourth allele at the Dominant white locus, and we suggest that it is denoted I(Be).

Overcoming false negatives due to the genomic context in polymerase chain reaction amplification

In some instances defined genomic regions are so poorly amplified that they seem to be unamplifiable. A protocol was developed which allows good PCR amplifications by the use of restriction digestion combined with the elution of a pool of restriction fragments of defined size range from agarose gel after electrophoresis. We describe the application of the method in the PCR amplification of a region of the white locus of Drosophila melanogaster that otherwise may be considered as a negative result.

RBE-LET relationships of high-LET radiations in Drosophila mutations.

The relative biological effectiveness (RBE) of 252Cf neutrons and synchrotron-generated high-energy charged particles for mutation induction was evaluated as a function of linear energy transfer (LET), using the loss of heterozygosity for wing-hair mutations and the reversion of the mutant white-ivory eye-color in Drosophila melanogaster. Loss of heterozygosity for wing-hair mutations results predominantly from mitotic crossing over induced in wing anlage cells of larvae, while the reverse mutation of eye-color is due to an intragenic structural change (2.96 kb-DNA excision) in the white locus on the X-chromosome. The measurements were performed in a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-color clone can be detected simultaneously in the same individual. Larvae were irradiated at the age of 3 days post oviposition with 252Cf neutrons, carbon beam or neon beam. For the neutron irradiation, the RBE values for wing-hair mutations were larger than that for eye-color mutation by about 7 fold. The RBE of carbon ions for producing the wing-hair mutations increased with increase in LET. The estimated RBE values were found to be in the range 2 to 6.5 for the wing-hair. For neon beam irradiation, the RBE values for wing-hair mutations peak near 150 keV/micron and decrease with further increase in LET. On the other hand, the RBE values for the induction of the eye-color mutation are nearly unity in 252Cf neutrons and both ions throughout the LET range irradiated. We discuss the relationships between the initial DNA damage and LET in considering the mechanism of somatic mutation induction.

Oncoviral DNAs induce transposition of endogenous mobile elements in the genome of Drosophila melanogaster

Previously, we have shown that particles of Rous sarcoma virus or cloned fragments of RSV cDNA as well as DNA of oncogenic simian adenovirus Sa7, injected into the polar plasm of early Drosophila melanogaster embryos, were able to induce, with high frequency, unstable visible mutations in different groups of genetic loci. The genetic instability of the recovered mutations, i.e., their ability to revert to normal state or to generate new mutant alleles at the affected locus, was manifest in mutant lines through several generations. The molecular analysis undertaken in this study of the yellow-scute loci region which is highly sensitive to the microinjected Sa7 DNA, and of the white locus, that frequently mutates under the influence of RSV cDNA, clearly shows that the induced mutations and reversions are accompanied by insertion/excision of endogenous mobile elements. This conclusion is confirmed by in situ hybridization experiments which demonstrate that the adenovirus DNA is able to change, though with different efficiency, the chromosomal localization of certain Drosophila retrotransposons. These results partially elucidate the molecular mechanism of the genetic instability in D. melanogaster induced by microinjection of oncoviruses into early embryos, implying that it results from mobilization of endogenous transposons which play the role of insertional elements directly causing unstable mutations.

Mutation at the anophthalmic white locus in Syrian hamsters: haploinsufficiency in the Mitf gene mimics human Waardenburg syndrome type 2.

Mutations in MITF (microphthalmia transcription factor) cause Waardenburg syndrome type 2 (WS2A) in humans, an autosomal dominant disorder consisting of deafness and hypopigmentation. Phenotypes vary significantly within WS2 pedigrees, and there is generally no correlation between the predicted biochemical properties of mutant MITF proteins and disease severity. We have identified a nonsense mutation in the Mitf gene of the anophthalmic white Wh) Syrian hamster that destabilizes its mRNA and prevents the encoded basic helix-loop-helix leucine zipper (bHLHzip) protein from dimerizing or binding DNA target sites. Although the resulting polypeptide does not act as a dominant-negative species in vitro , the Wh mutation is inherited as a semi-dominant trait. It thus more closely resembles WS2 than comparable Mitf alleles in laboratory mice and rats, which are expressed as purely recessive traits.

Induced somatic and germinal reversion of the white-spotted-1 insertional mutant phenotype in Drosophila melanogaster.

The white-spotted-1 (Wsp1) mutant of Drosophila melanogaster is characterized by the presence of an 8.7 kb retrotransposon (B104) inserted in the regulatory region of the white locus. The frequency of reversion in both somatic tissue and the germline after exposure to three different alkylating agents has been analysed. To determine if germinal revertants were induced by precise excision of the insertional element we analysed several phenotypic revertants using PCR and Southern blot techniques. The results indicate that, under our experimental conditions, the mutagens used did not induce excision of B104 in the white gene. In addition, the revertant phenotypes obtained were due to the existence of second site modifiers acting on expression of white. Such modifiers map near the white locus and, at least in one case, may correspond to suppressor-of-white-spotted.

Head spot and dilute mutations in the Norway rat.

A mutant allele of a new white spotting locus in the Norway rat is described, which is designated as head spot (hs). The allele is inherited as a recessive to normal. The expression is regularly manifested and has the form of a white sport of variable size in the middle of the forehead just above the eyes. A probable reoccurrence of blue dilution coat color is also reported. Tests for genetic linkage for hs indicate that it is independent of the agouti, dilute, and hooded loci.

Invertebrate retroviruses: ZAM a new candidate in D.melanogaster

ZAM, a new retroelement of Drosophila melanogaster, was identified as a mutational insertion at the white locus. It displays all the structural features of a vertebrate retrovirus. Its three open reading frames encode predicted products resembling the products of the gag, pol and env genes of retroviruses. Its transcription gives rise to an 8.6 kb full-length RNA and a 1.7 kb spliced message for the env gene. The latter encodes an envelope protein that is typical of elements having an extracellular phase of the life cycle. The identification of a ZAM envelope retrogene provides evidence that ZAM is mobilized through a reverse trancriptional process in the germ line of flies. We report that ZAM is distributed differently among D.melanogaster strains. Two stocks out of >15 tested display a ZAM high copy number, with numerous copies distributed on chromosomal arms. This high copy number is associated with a high transcriptional rate of ZAM. The existence of these two categories of strains offers a new genetic system in which the properties of a potential invertebrate retrovirus can be tested.

Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster.

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophila melanogaster. Double-strand breaks were made by excision of P-w(hd), a P-element insertion in the white gene. A nested set of repair templates was generated that contained the 8 kilobase (kb) yellow gene embedded within varying amounts of white gene sequence. Repair with unlimited homology was also analyzed. Files were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yellow gene was abolished when all of the 3' homology was removed. Increases in template homology up to 51 base pairs (bp) did not significantly promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequency when homology extended to the tips of the chromosome. These results demonstrate that the homology requirements for targeting a large heterologous insertion are quite different than those for a point mutation. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several aberrant conversion tracts, which arose from templates that contained reduced homology, also were examined and characterized.

A new inherited ocular anomaly in pigmented white Leghorn chickens.

Inherited ocular anomalies in chickens include several types of microphthalmia, retinal dysplasia, retinal degeneration, cataract, buphthalmos and pop-eye, or keratoconus in White Leghorns.2–5,7,8,12,15–18 In this report, a new ocular anomaly that appeared in pigmented White Leghorns homozygous for a mutation at the dominant white (I) locus is described with emphasis on clinical, gross, and histologic findings to aid in the diagnosis of the ocular lesions. An incompletely dominant mutation, called Smoky Joe (SJ), allows the production of feather pigment, which the I allele inhibits. The SJ mutation originally appeared in ADOL Line 0, a noninbred White Leghorn line maintained at the USDA Avian Disease and Oncology Laboratory (ADOL) in East Lansing, Michigan. Females homozygous for SJ have dark grey feathers with barring and homozygous males have much lighter colored plumage. This dimorphism in color is the result of the dilution that occurs with two copies of sexlinked barring in the males. The plumage of heterozygous birds (I, SJ) is intermediate in color compared to either parental type. Phthisis bulbi was noticed in adult females of the second generation of the homozygous SJ population. None of the parents of these females had apparent ocular lesions. Approximately 30% of the 85 surviving females had phthisis bulbi, while none of the 100 surviving males were affected. There was no clinical evidence of bacterial or viral infection, and the chickens were housed in pens isolated from chickens with experimental viral infections. Because the ocular anomaly appeared to be recessive and sex-linked, 4 test matings were designed to further study the inheritance. In matings 1 and 2, sighted SJ males, which were suspected of being carriers of the syndrome, were mated to either affected or sighted SJ females. These same male birds were then mated to either sighted females of Line 0 (II) (mating 3) or sighted females of ADOL Line 15I5 (II) and Line 0 (II) (mating 4). All chicks had ophthalmic examinations at hatch and 2, 4, and 8 weeks of age. In mating 1,197 chicks were examined. Of the females, 62% were affected, and 10% of the males were affected.

Bulked segregant analysis using microsatellites: mapping of the dominant white locus in the chicken

In order to perform a linkage study, the genotypes of a large number of individuals from a segregating population need to be determined. In case the phenotype to be mapped is influenced by a single locus or a major gene, sampling of the DNA from individual animals with the same phenotype into a single pool (bulked segregant) can reduce the number of typings. In this study we used bulked segregant analysis in order to map the Dominant White locus in the chicken. In a pilot experiment, we showed that allele frequencies can be accurately estimated from pooled samples using fluorescently labeled microsatellite markers. A segregating population for the Dominant White locus was obtained by performing a cross between a white male chicken (Genotype li for Dominant White) and a black female chicken (ii). The resulting progeny of 21 white and 18 black chickens were divided in two pools. Genotypes for both the parents and the pools were determined using 168 fluorescently labeled microsatellite markers, of which 68 were informative. The relative allele frequencies between the pools were estimated for these 68 informative markers. One marker (MCW188) was found to segregate with the Dominant White locus. Subsequent typing of all individuals from this cross and an additional 148 animals from five different families showed only two recombinants between the marker and the Dominant White locus, resulting in a LODlinkage score (log10 of odds) of 36. Using the pooled DNA approach, the Dominant White locus was successfully mapped on linkage group 22 of the East Lansing reference family at a distance of 2 cM from MCW188.

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation.

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus.

Effect of genetic background on the contribution of New Zealand black loci to autoimmune lupus nephritis.

Autoimmune diseases such as systemic lupus erythematosus are complex genetic traits with contributions from major histocompatibility complex (MHC) genes and multiple unknown non-MHC genes. Studies of animal models of lupus have provided important insight into the immunopathogenesis of disease, and genetic analyses of these models overcome certain obstacles encountered when studying human patients. Genome-wide scans of different genetic crosses have been used to map several disease-linked loci in New Zealand hybrid mice. Although some consensus exists among studies mapping the New Zealand Black (NZB) and New Zealand White (NZW) loci that contribute to lupus-like disease, considerable variability is also apparent. A variable in these studies is the genetic background of the non-autoimmune strain, which could influence genetic contributions from the affected strain. A direct examination of this question was undertaken in the present study by mapping NZB nephritis-linked loci in backcrosses involving different non-autoimmune backgrounds. In a backcross with MHC-congenic C57BL/6J mice, H2z appeared to be the strongest genetic determinant of severe lupus nephritis, whereas in a backcross with congenic BALB/cJ mice, H2z showed no influence on disease expression. NZB loci on chromosomes 1, 4, 11, and 14 appeared to segregate with disease in the BALB/cJ cross, but only the influence of the chromosome 1 locus spanned both crosses and showed linkage with disease when all mice were considered. Thus, the results indicate that contributions from disease-susceptibility loci, including MHC, may vary markedly depending on the non-autoimmune strain used in a backcross analysis. These studies provide insight into variables that affect genetic heterogeneity and add an important dimension of complexity for linkage analyses of human autoimmune disease.