Functional analysis of the cis-acting elements responsible for the induction of the Cyp6a8 and Cyp6g1 genes of Drosophila melanogaster by DDT, phenobarbital and caffeine.

Many Drosophila cytochrome P450 or Cyp genes are induced by caffeine and phenobarbital (PB). To understand the induction mechanism, we created Drosophila S2 cell lines stably transformed with different luciferase reporter plasmids carrying upstream DNAs of Cyp6a8 allele of the resistant 91-R strain, and the 1.1-kb upstream DNAs of Cyp6g1 of the 91-R and the susceptible 91-C strains. Following 24 h treatment with dichlorodiphenyltrichloroethane (DDT), caffeine or PB, luciferase activity of all cell lines was determined. Results showed that the 0.1-kb DNA of Cyp6a8 and the upstream DNAs of Cyp6g1 from both strains are not induced by these chemicals in S2 cells. However, the 0.2-, 0.5- and 0.8-kb DNAs of Cyp6a8 showed 13-24-, 4-5- and 2.2-2.7-fold induction with caffeine, PB and DDT, respectively. These DNAs also showed a 2-3-fold synergistic effect of caffeine and PB but not of caffeine and DDT. The results suggest that the cis-regulatory elements for all three chemicals are located within the -11/-199 DNA of Cyp6a8. Furthermore, caffeine and PB inductions appear to be mediated via different cis-elements, whereas caffeine and DDT induction may involve common regulatory elements. These stably transformed cell lines should help understand the mechanism of resistance-associated Cyp gene overexpression in Drosophila.

Association of a protease with polytene chromosomes of Drosophila melanogaster.

Incubation of Drosophila salivary glands with radioactive diisopropyl fluorophosphate results in the uniform labeling of polytene chromosomes. Extensive labeling is seen only when chromosome squashes are prepared by a formaldehyde fixation procedure and not by standard acetic acid techniques. The labeling is inhibited in the presence of tosylphenylalanine chloromethyl ketone and phenylmethane sulfonylfluoride but not by tosyllysine chloromethyl ketone, suggesting that a chymotrypsin-like serine protease is associated with the chromosomes. Protease inhibitors show no apparent effect on heat-shock specific puffing.

Gene dosage compensation and the evolution of sex chromosomes.

Dosage compensation is a mechanism by means of which the activity of X-linked or Z-linked genes is made equal in the two sexes of organisms with an XX compared to XY or ZZ compared to ZW basis of sex determination. In mammals, compensation is achieved by the inactivation of one X chromosome in somatic cells of females. In Drosophila, compensation does not involve inactivation. The two X chromosomes in females as well as the single X in males are regulated, and individual genes are thought to respond independently to the regulatory mechanism. It is proposed that in both groups of organisms the evolution of heteromorphic sex chromosomes was gradual and occurred as the direct result of the evolution of dosage compensation rather than the reverse.

Genetic rescue of a lethal "null" activity allele of 6-phosphogluconate dehydrogenase in Drosophila melanogaster.

While a null activity mutant allele of the structural gene for 6-phosphogluconate dehydrogenase in Drosophila melanogaster is lethal, a similar mutation for glucose-6-phosphate dehydrogenase is not. Double mutant combinations lacking both enzyme activities, obtained either by recombination or by mutagen treatment of a chromosome bearing the lethal allele, result in a restoration of viability. The indispensability of the pentose phosphate shunt in Drosophila appears to depend upon the specific position of the block within the pathway.

Dosage compensation in flies and worms: the ups and downs of X-chromosome regulation.

Dosage compensation ensures that individuals with a single X chromosome have the same amount of most X-linked gene products as those with two. In Drosophila, this equalization is achieved by a two-fold enhancement of the level of transcription of the X in males (XY) relative to each X chromosome in females (XX). In Caenorhabditis, equalization of X-linked gene products between hermaphrodites (XX) and males (XO) is achieved by decreasing the activity of genes in the former. These two different solutions to the common problem of unequal dosage of X-linked genes in different sexes provide invaluable paradigms for the study of gene regulation at the level of chromatin remodeling.

Recycling to remodel: evolution of dosage-compensation complexes.

In diploid species where sex determination involves heteromorphic sex chromosomes, a mechanism has evolved to compensate for gene-dosage differences in sex-linked genes between the sexes. This regulatory mechanism, which is based on chromatin remodeling, is the function of complexes that include components themselves involved in other cellular functions or with homologs that are involved in such functions. Directing these complexes to the correct chromosome in the appropriate sex relies on pioneer or novel components as well as on the presence of sequence-dependent target sites.

Dosage compensation: roX marks the spot.

The roX genes of Drosophila produce a transcript that is spliced and polyadenylated but not translated. Recent work has shown that these genes provide an element that the dosage compensation complex of Drosophila uses to initiate its association with the X chromosome.

The drosophila MSL complex acetylates histone H4 at lysine 16, a chromatin modification linked to dosage compensation.

In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.

A new human member of the MYST family of histone acetyl transferases with high sequence similarity to Drosophila MOF.

We have identified a novel human gene product, hMOF, which exhibits significant similarity to the Drosophila dosage compensation regulator, MOF. A recombinant C-terminal portion of hMOF has histone acetyltransferase activity directed toward histones H3, H2A and H4, a specificity characteristic of other MYST family histone acetyltransferases. Based on hMOF's chromodomain, we discuss possible interactions with other proteins.

A novel DEAD-box RNA helicase exhibits high sequence conservation from yeast to humans.

We have identified a novel Drosophila protein, DBP80, that exhibits significant similarity to mouse mDEAD5, yeast TIF1/2, and mammalian eIF-4A. DBP80 is a member of a subclass of DEAD-box proteins that contains a distinct domain, PX(I/R)ILLKR(E/D)EETLEGIKQ(F/Y)(F/Y), in addition to the seven canonical helicase domains.

Analysis of the dosage compensation of a specific transcript in Drosophila melanogaster.

The amount of steady-state level RNA complementary to the X-linked salivary gland secretion polypeptide gene Sgs-4 was measured in male and female third-instar larvae carrying one or two doses of a wild-type allele of the gene. RNA levels were found to be compensated in normal one-dose males and two-dose females and to be dosage-dependent within each sex. The presence of mutant alleles of male-less (mle) was found to reduce the level of Sgs-4 transcripts in males. These results support the contentions that dosage compensation is mediated by regulating the level of X-linked gene transcripts and that a product of the mle+ gene is involved in this process.

Lethal effects of low and "null" activity alleles of 6-phosphogluconate dehydrogenase in Drosophila melanogaster.

EMS-induced "null" and low activity alleles for 6-phosphogluconate dehydrogenase were characterized with respect to enzymatic activity, relative viability, fertility, and the effective lethal phase. It was determined that flies hemizygous and homozygous for the low activity allele, Pgd minus, possessed a depressed development rate, diminished viability, and loss of female female fertility. Heterozygotes for Pgd minus and a deficiency for Pgd+ were lethal. The "null" activity allele demonstrated a lethal phenotype in both the hemizygous and homozygous condition. The effective lethal phase for the "null" allele occurs during late embryonic development (20-22 hr).

Regulation of gene function: a comparison of X-linked enzyme activity levels in normal and intersexual triploids of Drosophila melanogaster.

We have measured gene function in normal and male-like intersexual triploids of Drosophila melanogaster by assaying crude extracts of whole flies or thoraces for levels of an X-linked (6-phosphogluconate dehydrogenase) and an autosomal (NADP-dependent isocitrate dehydrogenase) enzyme activity. Our observations lead us to conclude that each dose of the X-linked gene is more active in the cells of these intersexes than it is in normal triploid or diploid female cells. These results indicate that a level of activity intermediate between the normal male and female levels is possible for X-linked genes.

Dosage compensation of genes on the left and right arms of the X chromosome of Drosophila pseudoobscura and Drosophila willistoni.

We have investigated the occurrence of dosage compensation in D. willistoni and D. pseudoobscura, two species whose X chromosome is metacentric with one arm homologous to the X and the other homologous to the left arm of chromosome 3 of D. melanogaster. Crude extracts were assayed for isocitrate dehydrogenase (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?), and alpha-glycerophosphate dehydrogenase (chromosome 2) in D. willistoni, and for esterase-5 (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?) and amylase (chromosome 3) in D. pseudoobscura. Our results indicate that a mechanism for dosage compensation is operative in both arms of the X chromosome of these two species.

Male-specific lethal mutations of Drosophila melanogaster.

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mlets, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0----The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.

Mutational Events in the Triplo- and Haplo-Lethal Region (83de) of the DROSOPHILA MELANOGASTER Genome.

The Drosophila melanogaster genome contains a single region (at 83DE on the polytene chromosome map) for which both heterozygous deficiency and heterozygous duplication are inviable. Seven EMS-induced mutations have been recovered that are viable in combination with a duplication of this region. Two classes of mutations are reported: (1) Mutations that allow survival of flies with either a duplication or a normal third chromosome. These mutations retain Ki, a closely linked marker on the mutagenized chromosome. They fail to complement, and one has been mapped to the vicinity of 83DE. (2) Mutations that allow survival only in heterozygous combination with a duplication and have lost the Ki marker. These mutations represent new deletions of the dose-sensitive information. The possible structural organization of the 83DE region is discussed in light of these two classes of mutations.

Transposition of the Responder element (Rsp) of the Segregation distorter system (SD) to the X chromosome in Drosophila melanogaster.

In order to test whether the meiotic drive system Segregation distorter (SD) can operate on the X chromosome to exclude it from functional sperm, we have transposed the Responder locus (Rsp) to this element. This was accomplished by inducing detachments of a compound-X chromosome in females carrying a Y chromosome bearing a Rsps allele. Six Responder-sensitive-bearing X chromosomes, with kappa values ranging from 0.90 to 1.00, were established as permanent lines. Two of these have been characterized more extensively with respect to various parameters affecting meiotic drive. SD males with a Responder-sensitive X chromosome produce almost exclusively male embryos, while those with a Rsp-Y chromosome produce almost exclusively female embryos. This provides a genetic system of great potential utility for the study of early sex-specific differentiation events as it allows the collection of large numbers of embryos of a given sex.

Lack of dosage compensation for an autosomal gene relocated to the X chromosome in Drosophila melanogaster.

Aldehyde oxidase activity has been measured in flies with the structural gene for this enzyme translocated to the X chromosome. These measurements are presented as experimental evidence that, in Drosophila melanogaster, an autosomal gene relocated to the X chromosome is not dosage compensated.

Regulation of enzyme activities in Drosophila: genetic variation affecting induction of glucose 6-phosphate and 6-phosphogluconate dehydrogenase in larvae.

The genetic basis of modulation by dietary sucrose of the enzyme activities glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of cross-reacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 mM sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well.

The male-specific lethal-one (msl-1) gene of Drosophila melanogaster encodes a novel protein that associates with the X chromosome in males.

Male-specific lethal-one (msl-1) is one of four genes that are required for dosage compensation in Drosophila males. To determine the molecular basis of msl-1 regulation of dosage compensation, we have cloned the gene and characterized its products. The predicted msl-1 protein (MSL-1) has no significant similarity to proteins in the current data bases but contains an acidic N terminus characteristic of proteins involved in transcription and chromatin modeling. We present evidence that the msl-1 protein is associated with hundreds of sites along the length of the X chromosome in male, but not in female, nuclei. Our findings support the hypothesis that msl-1 plays a direct role in increasing the level of X-linked gene transcription in male nuclei.