Stage and organ specificity of the degree of variegation of the 6-phosphogluconate dehydrogenase gene in Drosophila melanogaster

Summary The effects of chromosomal rearrangements pn2, pn3, TE100 and TE101 on variegation of the gene Pgd, which controls the synthesis of 6-phosphogluconate dehydrogenase (PGD), were studied in Drosophila melanogaster. The electrophoretic patterns of PGD activity were first examined at different developmental stages. The degree of variegation of Pgd caused by pn2 and pn3 was higher in adult flies (the calculated percentage of cells with inactive Pgd was 70%–80%) as compared with larvae (about 50%). This difference can be explained by the tissue-specific mosaicism of Pgd expression; variegation was high in the neural ganglia, imaginal discs, and posterior gut but relatively low in the salivary glands, fat bodies and Malpighian tubes. In the case of TE100, neither tissue specificity, nor marked differences in the degree of variegation between larvae and adults were found. None of the rearrangements examined had an effect on the expression of Pgd in the ovary cells, but repression was seen in some cells of the male gonads. The data obtained suggest that the timing of clonal initiation is influenced by the rearrangements studied. The possible mechanisms preventing changes in the expression of the Pgd gene in the nurse cells caused by these rearrangements are discussed.     

Cloning and dosage compensation of the 6-phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X-linked gene Pgd+, which encodes the enzyme 6-phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd- phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.     

Demonstration of 6-phosphogluconolactonase activity in Drosophila melanogaster using a null allele of 6-phosphogluconate dehydrogenase

Using a double mutant strain, Pgdn Zwn, we have developed an assay for 6-phosphogluconolactonase activity and have demonstrated its occurrence in adult Drosophila melanogaster.     

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.     

Carnitine suppression of position-effect variegation in Drosophila melanogaster

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila melanogaster, we tested a further possible function of carnitine in the nucleus, using an assay for the suppression of position-effect variegation. We tested three physiological forms of carnitine (l-carnitine, l-propionylcarnitine, l-acetylcarnitine) for the ability to suppress two different chromosomal rearrangements, inducing variegation of the white ⁺ and brown ⁺ genes. The results show that the carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2)1 ⁰¹, a mutation that induces hyperacetylation of histones, whilst hyperacetylated histories accumulated in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level.     

Investigations on the organization of genetic loci in Drosophila melanogaster: lethal mutations affecting 6-phosphogluconate dehydrogenase and their suppression.

Summary The molecular nature of lethal and semilethal mutations in the Pgd locus of D. melanogaster coding for 6-phosphogluconate dehydrogenase (6PGD) was studied. All the 11 mutations affect the structural gene of the Pgd locus: 3 semilethal mutations resulted in altered 6PGD molecules with decreased catalytic activities; the rest 8 lethals were null alleles characterized by mutant polypeptides capable of reacting with antisera against highly purified 6PGD.Null or low activity alleles for glucose-6-phosphate dehydrogenase induced by ethyl methanesulfonate were shown to be suppressors for the lethal mutations in the Pgd locus.A monocistronic type of organization of the Pgd locus is suggested taking into account the biochemical mechanism of suppression of the Pgd-lethals and their location in the structural gene coding for 6PGD.     

Cytogenetic and molecular aspects of position-effect variegation in Drosophila melanogaster

Position-effect variegation in Drosophila melanogaster is accompanied by compaction of the corresponding chromosomal regions. The compaction can be continuous, so that bands and interbands located distal to the eu-heterochromatic junction fuse into one dense block, or discontinuous, when two or more zones of compaction are separated by morphologically and functionally normal regions. In this work it was found that in both continuous and discontinuous compaction the blocks of dense material contain the immunochemically detectable protein HP1, which has previously been characterized as specific for heterochromatin. The regions undergoing compaction do not contain HP1 when they have a normal banding pattern. Thus, it may be proposed that HP1 is one of the factors involved in compaction. If two different or two identical rearrangements are combined in the same nucleus, they variegate independently. The frequency of compaction of the two rearrangements in the same nucleus corresponds to the product of the frequencies of the compact state of the individual elements. The extent of compaction (i.e. the number of bands involved in heterochromatization) of each rearrangement does not depend on the compaction pattern of the other rearranged element.     

Characterization of mutations that enhance position-effect variegation in Drosophila melanogaster

Summary Several mutants that enhance the gene inactivation associated with position-effect variegation [E(var) mutants] have been characterized. These include three ethyl methanesulfonate (EMS)-induced lesions and a second chromosome duplication. Each of the EMS mutations maps to a discrete euchromatic site on the third chromosome. One is located within the chromosomal region occupied by a cluster of Su(var) mutations. All four E(var) mutants enhance the inactivation of several different variegators and therefore they appear to influence position-effect variegation generally. However, the enhancement caused by the single site E(var) mutations is less striking than that caused by the duplication or by loss of the Y chromosome. The interaction between the E(var) mutants and selected Su(var) mutations, as well as the effects of extra Y heterochromatin on E(var) expression, have also been investigated. Based on the results of these studies, various hypothetical functions of the E(var) ⁺ products are suggested.     

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.     

Importance in catalysis of the 6-phosphate-binding site of 6-phosphogluconate in sheep liver 6-phosphogluconate dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A chromosomal region subjected to position effect variegation was analysed for possible DNA under-replication. DNA clones from the vicinity of the euheterochromatin junction and from a distance of hundreds of kilobase pairs were used as probes. Formation of compact blocks of chromatin is regarded as a characteristic feature of position effect variegation. It was shown that in T (1;2) dor^var7 males undergoing position effect variegation clones representing the DNA nearest to the breakpoint in 2B7 hybridized normally in situ to the compact blocks, providing evidence against DNA underreplication. In females the same clones did not hybridize to the compact blocks. These variations in hybridization may be related to different degrees of compaction of chromosome regions in males and females. A correlation between the degree of underreplication and the level of cell polyteny was shown by Southern-blot hybridization of a DNA probe from the 2B region to DNA from an X/O strain carrying Dp (1;1)pn2b displaying position effect variegation and compaction in 94% of salivary gland cells. Almost complete underreplication of the DNA of this region was found in salivary gland cells (with a maximal degree of polyteny), intermediate underreplication was found in fat body cells (with an intermediate degree of polyteny), and replication was not disturbed in diploid cells of the larval cephalic complex.by W. Beermann     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A new genetic model system for studying position effect variegation in Drosophila melanogaster was found. It allows the analysis of genetic inactivation and changes in chromosome morphology in the same cells. In T(1;2)dor ^var7 strains the 2B5 early ecdysone puff, and the ecs locus which maps in this puff are translocated into the vicinity of centromeric heterochromatin. The ecs locus plays a key role in the system of ecdysone puffs: genetic damage to this locus results in loss of sensitivity of cells to the hormone and, as a consequence, ecdysone-induced puffs do not develop. In the T(1;2)dor ^var7 chromosome the ecs and at least five adjoining loci are inactivated in a variegated fashion. In the salivary gland cells of T(1;2)dor ^var7/ ecs^lt435 0 h prepupae which do not show the ecdysone puffs, the morphology of the 2B region was analysed. In all cases where the ecs locus was inactivated, a dense block of chromatin reminiscent of a solid band was found in the 2B region instead of the four bands 2B1–2, 3–4, 5 and 6. Sometimes compaction of the chromatin reached the 2A1–2 or even 1E1–4 bands. Formation of the compact block of chromatin coincided with late replication in this region. In situ hybridization of polytene chromosomes with a DNA clone from the ecs locus showed that when the dense chromatin block was present, no DNA was accessible for hybridization in 2B5. Hybridization of DNA of another clone located in the region of the translocation breakpoint (2B7–8) was found only in polytene chromosomes of larvae grown at 25° C, and never in those grown at 18° C, independently of the morphology of the 2B5 puff. The possibility that in the case of block formation both late replication and, as a consequence, underreplication of chromosome DNA take place, is discussed.Dedicated to Professor W. Beermann on the occasion of his 65th birthday