Identification of Mutations in Three Genes That Interact with Zeste in the Control of White Gene Expression in Drosophila Melanogaster

Three previously described genes, enhancer of yellow, 1, 2 and 3, are shown to cooperate with the zeste gene in the control of white gene expression. The mutations e(y)1(u1), e(y)3(u1), and to a lesser extent e(y)2(u1), enhance the effect of the zeste null allele z(v77h). Different combinations of e(y)1(u1), e(y)2(u1) and e(y)3(u1) mutations with several other z alleles also enhance the white mutant phenotype, but only to levels characteristic of white alleles containing a deletion of the upstream eye enhancer. Loss of zeste protein binding sites from the white locus does not eliminate the effect of e(y)1(u1) and e(y)3(u1) mutations, suggesting that the products of these genes interact with some other nucleotide sequences. Combinations of either e(y)1(u1) or e(y)2(u1) mutations with e(y)3(u1) are lethal. The products of these three genes may represent, together with zeste, a group of proteins involved in the organization of long-distance interactions between DNA sequences.     

Genetic instability in Drosophila melanogaster

Summary An unstable long tandem duplication which includes the white locus twice, marked with w ^sp in the left and w ^17G in the right locus, when kept in males has been found to produce red-eyed sons which have lost the long duplication and with it the w ^sp and w ^17G mutants. Such exceptions were produced also when w ^17G had been exchanged for w ^a.Stocks originating from these exceptions are unstable, producing: 1) zeste males, also unstable, 2) w ^- deletions, stable, 3) transpositions of the white locus to sites in other chromosomes.The instability is interpreted as the effect of an IS element, within or adjacent to the white locus, which is supposed to retain a duplication of the proximal zeste interacting part of this locus. According to the orientation of the IS element the duplicated part can be active or inactive, giving a zeste or red eye phenotype.The frequency of exceptional offspring after X-ray treatment of the red and zeste unstable stocks have been compared to stable stocks with corresponding genotypes.     

Mutagenic effects of some water-soluble metal compounds in a somatic eye-color test system in Drosophila melanogaster

Nickel, cadmium, lead, arsenic, manganese and chromium salts as well as MeHgOH were screened for mutagenicity, using a sensitive somatic eye-color test system in Drosophila melanogaster. The test is based on the insertion of a mobile element which causes instability in the white locus that is somatically enhanced by mutagens. This white locus expression is combined with a mutation, zeste, in another gene, to produce a light yellow eye color. Larval feeding with mutagens causes somatic mutations in the eye imaginal disc cells that develop into easily detectable red spots in the yellow eyes of adult males. Survival tests showed large differences in the toxicity of different metals, but only hexavalent chromium increased the frequency of somatic mutations above the control level. When combined treatments were carried out with MMS and various metals, sodium arsenite caused a reduction of the MMS-induced mutation frequency while methylmercury increased the frequency of somatic spots.     

A Fragment of Engrailed Regulatory DNA Can Mediate Transvection of the White Gene in Drosophila

We have found a fragment of engrailed regulatory DNA that has an unusual effect on expression of a linked marker gene, white, in the P element transposon CaSpeR. Normally, flies homozygous for a given CaSpeR insertion have darker eyes than heterozygotes. However, when a particular engrailed DNA fragment is included in that transposon, homozygotes often have lighter eyes than heterozygotes. Thus, engrailed DNA appears to cause white expression to be repressed in homozygotes. The suppression of white is dependent on the proximity of the two transposons in the genome-either in cis (i.e., on the same chromosome) or in trans (i.e., on homologous chromosomes). Thus, the engrailed fragment is mediating a phenomenon similar to that mediated by the zeste gene at the white locus. However, the interactions we observe do not require, nor are influenced by, mutations of zeste. We suggest that the engrailed DNA contains one or more binding sites for a protein that facilitates interactions between transposons. The normal function of these sites may be to mediate interactions between distant cis-regulatory regions of engrailed, a large locus that extends over 70 kilobases.     

The product of the Drosophila zeste gene binds to specific DNA sequences in white and Ubx.

Three different segments of the zeste coding sequence were inserted in an expression vector and antibodies were raised against the resulting zeste-beta galactosidase hybrid proteins. The antibodies were used to analyse the zeste protein produced in bacteria from a different expression vector containing the entire zeste coding region. The major products made in bacteria as well as the products of in vitro translation of zeste RNA migrate anomalously upon SDS--acrylamide gel electrophoresis. Specific DNA fragments from the white and Ubx gene co-immunoprecipitate with zeste protein. At least two independent zeste binding sites are found in a 250-bp interval of the white regulatory region that contains also the sites of wsp mutations, which are known to be deficient in zeste interaction.     

Stepwise assembly of hyperaggregated forms of Drosophila zeste mutant protein suppresses white gene expression in vivo.

The zeste gene is involved in two chromosome pairing-dependent phenomena: transvection and the suppression of white gene expression. Both require the ability of zeste protein to multimerize, dependent on three interlaced hydrophobic heptad repeats in the C-terminal domain. The first step is dimerization through a leucine zipper. Two other heptad repeats are then required to form higher multimers. The zeta(1) mutation, which causes the pairing-dependent suppression of white, creates a new hydrophobic nucleus that allows the formation of a new and larger aggregate. The zeta(op6) mutation, which suppresses even unpaired copies of white, makes even larger aggregates. The phenotypic suppression of white by a series of mutants is strictly correlated with hyperaggregation and the larger the hyperaggregates, the weaker the requirement for the pairing of white. Hyperaggregation of the Z1 protein in vitro is suppressed by co-translation with the C-terminal peptide of wild-type protein, lacking the DNA-binding domain. This C-Z+ peptide also complements the zeta(1) allele in vivo and restores normal color, demonstrating that zeste product also exists in a multimeric complex in the cell. Complementation in vivo is strictly correlated with the prevention of hyperaggregation of the zeste mutant products in vitro, supporting the interpretation that excessive association of zeta(1) and zeta(op6) proteins is responsible for their repression of white gene expression.     

In vivo functional analysis of in vitro protein binding sites in the immunoglobulin heavy chain enhancer.

We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.     

Self-association of the Drosophila zeste protein is responsible for transvection effects.

The zeste gene product is required for transvection effects that imply the ability of regulatory elements on one chromosome to affect the expression of the homologous gene in a somatically paired chromosome. The z1 mutation causes a pairing dependent inhibition of the expression of the white gene. Both of these phenomena can be explained by the tendency of zeste protein, expressed in bacteria or in flies, to self-associate, forming complexes of several hundred monomers. These large aggregates bind to DNA and are found in nuclear matrix preparations, probably because they co-sediment with the matrix. The principal determinants of this self-association are located in the C-terminal half of the protein but some limited aggregation is obtained also with the N-terminal half, which contains the DNA binding domain. The z1 and zop2 mutant proteins aggregate to the same degree as the wild type but the z11G3 product, a pseudorevertant of z1, has a reduced tendency to aggregate. This mutation, which in vivo is antagonistic to z1 and does not support transvection effects, can be made to revert its phenotype when the mutant protein is over-produced under the control of the heat shock promoter. These results indicate that both the zeste-white interaction and transvection effects require the formation of high order aggregates. When the z1 protein is over-produced in vivo, it reduces the expression of an unpaired copy of white, indicating that the normal requirement for chromosome pairing is simply a device to increase the size of the aggregate bound to the white regulatory region.     

A Genetic Analysis of the Suppressor 2 of Zeste Complex of Drosophila Melanogaster

The zeste(1) (z(1)) mutation of Drosophila melanogaster produces a mutant yellow eye color instead of the wild-type red. Genetic and molecular data suggest that z(1) achieves this change by altering expression of the wild-type white gene in a manner that exhibits transvection effects. There exist suppressor and enhancer mutations that modify the z(1) eye color, and this paper summarizes our studies of those belonging to the Suppressor 2 of zeste complex [Su(z)2-C]. The Su(z)2-C consists of at least three subregions called Psc (Posterior sex combs), Su(z)2 and Su(z)2D (Distal). The products of these subregions are proposed to act at the level of chromatin. Complementation analyses predict that the products are functionally similar and interacting. The alleles of Psc define two overlapping phenotypic classes, the hopeful and hapless. The distinctions between these two classes and the intragenic complementation seen among some of the Psc alleles are consistent with a multidomain structure for the product of Psc. Psc is a member of the homeotic Polycomb group of genes. A general discussion of the Polycomb and trithorax group of genes, position-effect variegation, transvection, chromosome pairing and chromatin structure is presented.