Interactions of Polyhomeotic with Polycomb Group Genes of Drosophila Melanogaster

The Polycomb (Pc) group genes of Drosophila are negative regulators of homeotic genes, but individual loci have pleiotropic phenotypes. It has been suggested that Pc group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic (ph) immunoprecipitates in a multimeric complex that includes Pc. Here, we show that duplications of ph suppress homeotic transformations of Pc and Pcl, supporting a mass-action model for Pc group function. We crossed ph alleles to all members of the Polycomb group, and to E(Pc) and Su(z)2 to look for synergistic effects. We observed extragenic noncomplementation between ph(503) and Pc, Psc(1) and Su(z)2(1) in females, and between ph(409) and Sce(1), Scm(D1) and E(z)(1) mutations in males, suggesting that these gene products might interact directly with ph. Males hemizygous for a temperature-sensitive allele, ph(2), are lethal when heterozygous with mutants in Asx, Pc, Pcl, Psc, Sce and Scm, and with E(Pc) and Su(z)2. Mutations in trithorax group genes were not able to suppress the lethality of ph(2)/Y; Psc(1)/+ males. ph(2) was not lethal with extra sex combs, E(z), super sex combs (sxc) or l(4)102EFc heterozygotes, but did cause earlier lethality in embryos homozygous for E(z), sxc and l(4)102EFc. However, ph(503) did not enhance homeotic phenotypes of esc heterozygotes derived from homozygous esc(-) mothers. We examined the embryonic phenotypes of ph(2) embryos that were lethal when heterozygous or homozygous for other mutations. Based on this phenotypic analysis, we suggest that ph may perform different functions in conjunction with differing subsets of Pc group genes.     

The Autosomal Flp-Dfs Technique for Generating Germline Mosaics in Drosophila Melanogaster

The production of female germline chimeras is invaluable for analyzing the tissue specificity of recessive female sterile mutations as well as detecting the maternal effect of recessive zygotic lethal mutations. Previously, we developed the ``FLP-DFS' technique to efficiently generate germline clones. This technique uses the X-linked germline-dependent dominant female sterile mutation ovo(D1) as a selection for the detection of germline recombination events, and the FLP-FRT recombination system to promote site-specific chromosomal exchange. This method allows the efficient production of germline mosaics only on the X chromosome. In this paper we have built chromosomes that allow the use of this technique to the autosomes. We describe the various steps involved in the development of this technique as well as the properties of the chromosomes utilized.     

A Role of Polycomb Group Genes in the Regulation of Gap Gene Expression in Drosophila

Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.     

The Transposable Element Mariner Mediates Germline Transformation in Drosophila Melanogaster

A vector for germline transformation in Drosophila melanogaster was constructed using the transposable element mariner. The vector, denoted pMlwB, contains a mariner element disrupted by an insertion containing the wild-type white gene from D. melanogaster, the β-galactosidase gene from Escherichia coli and sequences that enable plasmid replication and selection in E. coli. The white gene is controlled by the promoter of the D. melanogaster gene for heat-shock protein 70, and the β-galactosidase gene is flanked upstream by the promoter of the transposable element P as well as that of mariner. The MlwB element was introduced into the germline of D. melanogaster by co-injection into embryos with an active mariner element, Mos1, which codes for a functional transposase and serves as a helper. Two independent germline insertions were isolated and characterized. The results show that the MlwB element inserted into the genome in a mariner-dependent manner with the termini of the inverted repeats inserted at a TA dinucleotide. Both insertions exhibit an unexpected degree of germline and somatic stability, even in the presence of an active mariner element in the genetic background. These results demonstrate that the mariner transposable element, which is small (1286 bp) and relatively homogeneous in size among different copies, is nevertheless capable of promoting the insertion of the large (13.2 kb) MlwB element. Because of the widespread phylogenetic distribution of mariner among insects, these results suggest that mariner might provide a wide hostrange transformation vector for insects.     

The Accumulation of P-Element-Induced Recombinants in the Germline of Male Drosophila Melanogaster

P-element-induced recombination in Drosophila melanogaster occurs premeiotically. Recombinants are therefore expected to accumulate in the stem cells of the germline of P-element-carrying males. We show that both the recombination frequency and the incidence of ``clustering' increase with the age of males carrying various P-element derivatives. The combination of end-deleted elements can lead to average recombination frequencies >50% with individual instances of 100% recombination. These elements also lowered the fertility of the carriers. We investigated these features by constructing an analytical and a computer simulation model of the course of events in the germline, incorporating the recently proposed hybrid element insertion (HEI) model of P-element activity. The model is able to predict extreme recombination levels, segregation ratio biases and lowered fertility through cell death in a single analysis.     

Introduction of the Transposable Element Mariner into the Germline of Drosophila Melanogaster

A chimeric white gene (wpch) and other constructs containing the transposable element mariner from Drosophila mauritiana were introduced into the germline of Drosophila melanogaster using transformation mediated by the P element. In the absence of other mariner elements, the wpch allele is genetically stable in both germ cells and somatic cells, indicating that the peach element (i.e., the particular copy of mariner inserted in the wpch allele) is inactive. However, in the presence of the active element Mos1, the wpch allele reverts, owing to excision of the peach element, yielding eye-color mosaics and a high rate of germline reversion. In strains containing Mos1 virtually every fly is an eye-color mosaic, and the rate of wpch germline reversion ranges from 10 to 25%, depending on temperature. The overall rates of mariner excision and transposition are approximately sixfold greater than the rates in comparable strains of Drosophila simulans. The activity of the Mos1 element is markedly affected by position effects at the site of Mos1 insertion. In low level mosiac lines, dosage effects of Mos1 are apparent in the heavier level of eye-color mosaicism in Mos1 homozygotes than in heterozygotes. However, saturation occurs in high level mosaic lines, and then dosage effects are not observed. A pBluescribe M13+ plasmid containing Mos1 was injected into the pole plasm of D. melanogaster embryos, and the Mos1 element spontaneously integrated into the germline at high efficiency. These transformed strains of D. melanogaster presently contain numerous copies of mariner and may be useful in transposon tagging and other applications.     

Essential Genes in Autosomal Heterochromatin of Drosophila Melanogaster

We are taking two approaches to understanding the structure, function and regulation of essential genes within Drosophilaheterochromatin. In the first, we have undertaken a genetic and molecular characterization of essential genes within proximal 3L heterochromatin. The expression of such ‘resident’ genes within a heterochromatic environment is paradoxical and poorly understood, given that the same environment can inactivate euchromatic sequences (position effect variegation, or PEV). A second approach involves the study of the local chromosomal environment of heterochromatic (het) genes, as assayed both biochemically, and via the effects of genetic modifiers of PEV, the latter being putative components important for het gene expression. Our results to date suggest that the three most proximal genes in 3L heterochromatin have key roles in development, and indicate strong effects of combinations of genetic modifiers of PEV on het gene expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.     

Altered Expression of Polycomb Group Genes in Glioblastoma Multiforme

The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.     

黑腹果蝇种组五种核型的报道

通过传统的敲片、Giemsa染色的方法, 本文首次对果蝇属黑腹果蝇种组的5种果蝇 （D. constricta、D. ohnishii、D. ogumai、D. pseudobaimaii、D. tani）染色体的数目和形态进行了分析报道。分析发现：这5个种具有相同的染色体数目（2n=8）和不同的形态。D. pseudobaimaii和D. tani 为2V,1R,1D型;D. constricta染色为2V,1R,1D型且其点状染色体难以辨认;D. ohnishii和D. ogumai 具有相同形态为2V,2R。另外,还发现核型与亲缘关系之间有一定的对应性。     

Duplicated Proteasome Subunit Genes in Drosophila Melanogaster Encoding Testes-Specific Isoforms

Using the previously cloned proteasome α-type subunit gene Pros28.1, we screened a Drosophila melanogaster genomic library using reduced stringency conditions to identify closely related genes. Two new genes, Pros28.1A (map position 92F) and Pros28.1B (map position 60D7), showing high sequence similarity to Pros28.1, were identified and characterized. Pros28.1A encodes a protein with 74% amino acid identity to PROS28.1, while the Pros28.1B gene product is 58% identical. The Pros28.1B gene has two introns, located in exactly analogous positions as the two introns in Pros28.1, while the Pros28.1A gene lacks introns. Northern blot analysis reveals that the two new genes are expressed only in males, during the pupal and adult stages. Tissue-specific patterns of expression were examined using transgenic flies carrying lacz-fusion reporter genes. This analysis revealed that both genes are expressed in germiline cells during spermatogenesis, although their expression patterns differed. Pros28.1A expression is first detected at the primary spermatocyte stage and persists into the spermatid elongation phase of spermiogenesis, while Pros28.1B expression is prominent only during spermatid elongation. These genes represent the most striking example of cell-type-specific proteasome gene expression reported to date in any system and support the notion that there is structural and functional heterogeneity among proteasomes in metazoans.