Efficient recovery of centric heterochromatin P-element insertions in Drosophila melanogaster

Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.     

The Rosy Locus in Drosophila Melanogaster: Xanthine Dehydrogenase and Eye Pigments

The rosy gene in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. Xanthine dehydrogenase is not synthesized in the eye, but rather is transported there. The present report describes the ultrastructural localization of XDH in the Drosophila eye. Three lines of evidence are presented demonstrating that XDH is sequestered within specific vacuoles, the type II pigment granules. Histochemical and antibody staining of frozen sections, as well as thin layer chromatography studies of several adult genotypes serve to examine some of the factors and genic interactions that may be involved in transport of XDH, and in eye pigment formation. While a specific function for XDH in the synthesis of the red, pteridine eye pigments remains unknown, these studies present evidence that: (1) the incorporation of XDH into the pigment granules requires specific interaction between a normal XDH molecule and one or more transport proteins; (2) the structural integrity of the pigment granule itself is dependent upon the presence of a normal balance of eye pigments, a notion advanced earlier.     

Germ line and somatic instability of a white mutation in Drosophila mauritiana due to a transposable genetic element

A spontaneous white mutation recovered in Drosophila mauritiana is unstable and reverts to normal eye color at a frequency greater than 4 per 1,000 X-chromosomes. Germ line reversion occurs at a high rate in D. mauritiana males and in interspecific hybrid females, while the rate is depressed in D. mauritiana females. These events are not restricted to the germ line, as cases of variegated patterns of eye pigmentation, indicating somatic reversion, are recovered at a frequency comparable to that of the male germ line reversion rate. Germ line reversion events are genetically stable, while the somatic variegation patterns are not heritable. The patterns of eye pigment variegation produced suggests that reversion events are occurring throughout development. Whole genome DNA digests blotted and probed with the cloned D. melanogaster white gene indicate that this unstable white mutation in D. mauritiana is associated with an insertion of DNA that is lost upon reversion to wild type, indicating that this DNA insert is in fact a transposable element.     

Mosaic: a position-effect variegation eye-color mutant in the mosquito Anopheles gambiae

The Mosaic (Mos) mutation, isolated in the F1 of 60Co-irradiated mosquitoes, confers variegated eye color to third and fourth instar larvae, pupae, and adults of the mosquito Anopheles gambiae. Mos is recessive in wild pink eye (p+) individuals, but is dominant and confers areas of wild-type pigment in mutant pink eye backgrounds. Mos is located 14.4 cM from pink eye on the X chromosome and is associated with a duplication of division 2B euchromatin that has been inserted into division 6 heterochromatin. Various combinations of Mos, pink eye alleles, and the autosomal mutation red eye were produced. In all cases, the darker pigmented regions of the eye in Mos individuals show the phenotypic interactions expected if the phenotype of those regions is due to expression of a p+ allele. Expression of Mos is suppressed by rearing larvae at 32 degrees C relative to 22 degrees C. All of these characteristics are consistent with Mos being a duplicated wild copy of the pink eye gene undergoing position-effect variegation.     

Germ line and somatic instability of a white mutation in Drosophila mauritiana due to a transposable genetic element

A spontaneous white mutation recovered in Drosophila mauritiana is unstable and reverts to normal eye color at a frequency greater than 4 per 1,000 ×-chromosomes. Germ line reversion occurs at a high rate in D. mauritiana males and in interspecific hybrid females, while the rate is depressed in D. mauritiana females. These events are not restricted to the germ line, as cases of variegated patterns of eye pigmentation, indicating somatic reversion, are recovered at a frequency comparable to that of the male germ line reversion rate. Germ line reversion events are genetically stable, while the somatic variegation patterns are not heritable. The patterns of eye pigment variegation produced suggests that reversion events are occurring throughout development. Whole genome DNA digests blotted and probed with the cloned D. melanogaster white gene indicate that this unstable white mutation in D. mauritiana is associated with an insertion of DNA that is lost upon reversion to wild type, indicating that this DNA insert is in fact a transposable element.     

Esrom, an ortholog of PAM (protein associated with c-myc), regulates pteridine synthesis in the zebrafish

Zebrafish esrom mutants have an unusual combination of phenotypes: in addition to a defect in the projection of retinal axons, they have reduced yellow pigmentation. Here, we investigate the pigment phenotype and, from this, provide evidence for an unexpected defect in retinal neurons. Esrom is not required for the differentiation of neural crest precursors into pigment cells, nor is it essential for cell migration, pigment granule biogenesis, or translocation. Instead, loss of yellow color is caused by a deficiency of sepiapterin, a yellow pteridine. The level of several other pteridines is also affected in mutants. Importantly, the cofactor tetrahydrobiopterin (BH4) is drastically reduced in esrom mutants. Mutant retinal neurons also appear deficient in this pteridine. BH4-synthesizing enzymes are active in mutants, indicating a defect in the regulation rather than production of enzymes. Esrom has recently been identified as an ortholog of PAM (protein associated with c-myc), a very large protein involved in synaptogenesis in Drosophila and C. elegans. These data thus introduce a new regulator of pteridine synthesis in a vertebrate and establish a function for the Esrom protein family outside synaptogenesis. They also raise the possibility that neuronal defects are due in part to an abnormality in pteridine synthesis.     

Evolution of gene sequence in response to chromosomal location

Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution.     

Butyrate suppression of position-effect variegation in Drosophila melanogaster

Summary The strain of Drosophila melanogaster carrying the inversion In(1)w ^m4, which juxtaposes the normal w ⁺ gene to the centromeric heterochromatin, variegates for pigmentation in the eye. This strain was treated with various concentrations of n-butyrate and n-proprionate during the embryonic and larval stages. Concentrations as low as 70mM markedly suppress the variegated eye phenotype. This suggests that non-acetylated histones play a major role in the phenomenon of position-effect variegation.This research was supported by Natural Sciences and Engineering Research Council Canada team grant A-1764 to T.A.G. and D.T. Suzuki, and Natural, Applied & Health Sciences grant 9704 to T.A.G.     

The maternal and zygotic control of development by cinnamon, a new mutant in Drosophila melanogaster

A genetic analysis of the X-linked mutant cinnamon (cin) in Drosophila melanogaster demonstrates that this locus controls a process that is essential for zygote survival. Survival of a zygote is shown to be ensured by either the maternal expression of the cin⁺ allele or by the presence of a cin⁺ allele in the zygotic genome. The cin locus is also shown to affect pteridine pigment metabolism. The presence of a cin⁺ allele in either the mother or zygote is requisite for normal zygotic eye pigmentation. It is shown that cin flies lack xanthine dehydrogenase, an enzyme involved in pteridine metabolism, and accumulate the enzyme's pteridine and purine substrates. The relationship between the effects of cin on viability and xanthine dehydrogenase activity is discussed.     

The structure of heterochromatic DNA is altered in polyploid cells of Drosophila melanogaster.

DNA sequences within heterochromatin are often selectively underrepresented during development of polyploid chromosomes, and DNA molecules of altered structure are predicted to form as a consequence of the underrepresentation process. We have identified heterochromatic DNAs of altered structure within sequences that are underrepresented in polyploid cells of Drosophila melanogaster. Specifically, restriction fragments that extend into centric heterochromatin of the minichromosome Dp(1;f)1187 are shortened in polyploid cells of both the ovary and salivary gland but not in the predominantly diploid cells of the embryo or larval imaginal discs and brains. Shortened DNA molecules were also identified within heterochromatic sequences of chromosome III. These results suggest that the structure of heterochromatic DNA is altered as a general consequence of polyploid chromosome formation and that the shortened molecules identified form as a consequence of heterochromatic underrepresentation. Finally, alteration of heterochromatic DNA structure on Dp(1;f)1187 was not correlated with changes in the variegated expression of the yellow gene located on the minichromosome.     

The pugilistDominant Mutation of Drosophila melanogaster: A Simple-Sequence Repeat Disorder Reveals Localized Transport in the Eye

The pugilist-Dominant mutation results from fusion of a portion of the gene encoding the tri-functional Methylene Tetrahydrofolate Dehydrogenase (E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3) to approximately one kb of a heterochromatic satellite repeat. Expression of this fusion gene results in an unusual ring pattern of pigmentation around the eye. We carried out experiments to determine the mechanism for this pattern. By using FLP-mediated DNA mobilization to place different pug^D transgenes at pre-selected sites we found that variation in repeat length makes a strong contribution to variability of the pug phenotype. This variation is manifest primarily as differences in the thickness of the pigmented ring. We show that similar phenotypic variation can also be achieved by changing gene copy number. We found that the pug^D pattern is not controlled by wingless, which is normally expressed in a similar ring pattern. Finally, we found that physical injury to a pug^D eye can lead to pigment deposition in parts of the eye that would not have been pigmented in the absence of injury. Our results are consistent with a model in which a metabolite vital for pigment formation is imported from the periphery of the eye, and pug^D limits the extent of its transport towards the center of the eye, thus revealing the existence of a hitherto unknown mechanism of localized transport in the eye.     

Islands of Complex DNA Are Widespread in Drosophila Centric Heterochromatin

Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three ``islands' of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed.     

An investigation of heterochromatin domains on the fourth chromosome of Drosophila melanogaster

The banded portion of Drosophila melanogaster chromosome 4 exhibits euchromatic and heterochromatic characteristics. Reminiscent of heterochromatin, it contains a high percentage of repetitive elements, does not undergo recombination, and exhibits high levels of HP1 and histone-3 lysine-9 dimethylation. However, in the distal 1.2 Mb, the gene density is typical of euchromatin, and this region is polytene in salivary gland nuclei. Using P-element reporters carrying a copy of hsp70-white, alternative chromatin packaging domains can be distinguished by the eye color phenotype. Mapping studies identified the repetitive element 1360 as a candidate for heterochromatin targeting in the fourth chromosome Hcf region. We report here two new screens using this reporter to look for additional heterochromatin target sites. We confirm that reporter elements within 10 kb of 1360 are usually packaged as heterochromatin; however, heterochromatin packaging occurs in the sv region in the absence of 1360. Analyses of the sequences adjacent to P-element reporters show no simple association between specific repeated elements and transgene expression phenotype on a whole chromosome level. The data require that heterochromatin formation as a whole depends on a more complex pattern of sequence organization rather than the presence of a single sequence element.     

Somatic Reversion of Chromosomal Position Effects in Drosophila Melanogaster

A transgene was inserted at several different chromosomal sites in Drosophila melanogaster, where its expression was subject to genomic position effects. Quantitative position effects and variegated and constant patterned position effects were observed. We investigated the status of the affected gene in the somatic cells where it normally functions. The FLP site-specific recombinase was used to remove the gene from the chromosome and its expression was then evaluated. We show that the FLP recombinase functions in cells that have finished their developmental program of mitoses. When FLP acts on directly repeated copies of its target site (FRT), the DNA flanked by those FRTs is excised from the chromosome as a closed circle. The extrachromosomal circle is maintained in nondividing cells, and a gene located on such a circle can be expressed. We then demonstrate that a gene subject to either variegated or constant position effect can be relieved of that effect by excision of the gene from the chromosome in cells where it would otherwise be inactive. We also observed a strong inhibition of FLP-mediated recombination for target sites located near centric heterochromatin.     

The transposon A(R)4-24P[white, rosy] in Drosophila melanogaster is subject to position-effect variegation at a non-centromeric insertion site

The white gene within the transposon A(R)4-24P[white,rosy] inserted at cytological location 24D1-2 in the euchromatic portion of the Drosophila melanogaster genome exhibits a mosaic pattern of expression which is modified by temperature and Y-chromosome number, as in cases of classical position-effect variegation (PEV). The eye colour of the flies in this variegated stock remains mosaic in the presence of the PEV modifier Su(var)3-6, slightly less so with Su(var)3-9 and Su(var)2-5, and full suppression of variegation occurs in the presence of Su(var)3-7. We have induced further transposition of A(R)4-24 and isolated two mosaic stocks with this transgene at new cytological locations. In these stocks, the A(R)4-24 transposon was flanked by the same genomic DNA fragments as in the original location. Spontaneous loss of these fragments leads to reversion of the variegated eye colour to wild-type. We suggest that the flanking DNA fragments from 24D1-2 are capable of inducing position-effect variegation without any association with centromeric heterochromatin. In situ hybridisation and Southern analysis demonstrate that the 5' flanking genomic fragment contains repeated sequences which are abundantly present in heterochromatin.     

Biological and biochemical characterization of a red-eye mutant in Nilaparvata lugens （Hemiptera： Delphacidae）

A red-eye colony was established in our laboratory in brown planthopper （BPH）, Nilaparvata lugens （Stal）, a major rice pest in Asia. Except for the red-eye phenotype, no other differences were observed between the wild-type （brown eye） and the mutant-type （red eye） in external characters. Genetic analysis revealed that the red-eye phenotype was controlled by a single autosomal recessive allele. Biological studies found that egg produc- tion and egg viability in the red-eye mutant colony were not significantly different from those in the wild-type BPH. Biochemical analysis and electronic microscopy examination revealed that the red-eye mutants contained decreased levels of both xanthommatin （brown） and pteridine （red） and reduced number of pigment granules. Thus, the changes of amount and ratio of the two pigments is the biochemical basis of this red-eye mutation. Our results indicate that the red-eye mutant gene （red） might be involved in one common gene locus shared by the two pigments in pigment transportation, pigment granule formation or some other processes.     

Xanthine Dehydrogenase Is Transported to the Drosophila Eye

The rosy (ry) locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase. Mutants that have no enzyme activity are characterized by a brownish eye color phenotype reflecting a deficiency in the red eye pigment. This report demonstrates that enzyme which is synthesized in some tissue other than the eye is transported and sequestered at the eye. Previous studies find that no leader sequence is associated with this molecule but a peroxisomal targeting sequence has been noted, and the enzyme has been localized to peroxisomes. This represents a rare example of an enzyme involved in intermediary metabolism being transported from one tissue to another and may also be the first example of a peroxisomal protein being secreted from a cell.