John C. Lucchesi

The link between dosage compensation and sex differentiation in Drosophila melanogaster

The rate of 3H-uridine incorporation into X-chromosome and autosomal RNA was measured as an indicator of relative transcription activity in larvae carrying various Sxl mutant alleles. Hyperactivity of X chromosomes was found in heteroallelic Sxlf#1/Sxlfhv#1 and homozygous Sxlf#2 female larvae. Sxlfhv#1 homozygotes, Sxlf#1/Sxl+ heterozygotes, heteroallelic Sxlf#2/Sxlf#2 as well as homozygous Sxlf#ba female larvae exhibited normal X chromosome transcription. Except for Sxlf#ba, there is a correlation between the viability of the mutants and the degree to which X-chromosome activity is elevated. Male larvae carrying the dominant male-specific lethal mutation SxlM#1 displayed X chromosomes only half as wide as those of control larvae. However, it could not be determined whether this property is the result of a lower transcription rate or of underreplication of the mutated X chromosomes. The results demonstrate that the Sxl gene plays an important role in controlling X-chromosome activity. The relationship among the various genes known to act in sex differentiation and dosage compensation is discussed.

α-Glycerophosphate dehydrogenase in Drosophila melanogaster: Kinetic differences and developmental differentiation of the larval and adult isozymes☆

Abstract The isozymes of α-glycerophosphate dehydrogenase (α-GPDH) differ markedly with respect to their kinetic and stability parameters. Adult-limited GPDH-1 is stable at 50°C but decays at 57°C, while GPDH-3 is labile at 50°C under similar experimental conditions. By extrapolation of the thermal denaturation curves of crude adult extracts, we estimate GPDH-1 to constitute 76 per cent of the adult α-GPDH activity. Substrate kinetic studies revealed that, at pH 9·5, GPDH-3 exhibits an affinity for α-glycerophosphate which is twofold higher than that of GPDH-1, while Kms for NAD+ are indistinguishable. The apparent Km of GPDH-3 for dihydroxyacetone phosphate is consistently lower than that of GPDH-1 at pH 7·5, whereas at pH 6·7 the latter isozymes apparent Km approximates that of GPDH-3 at pH 7·5. Indistinguishable molecular weights of 66,000 were estimated by gel filtration for both GPDH-1 and 3. Gene dosage studies indicate that all three α-GPDH isozymes are simultaneously affected by dosage of the Gdh+ locus. These observations support a homomultimeric model of α-GPDH and the isozymes just discussed arise through epigenetic modification of the product of a single structural gene locus.

Regulation of gene function: A comparison of enzyme activity levels in relation to gene dosage in diploids and triploids of Drosophila melanogaster

A study of gene activity in diploid and triploid Drosophila melanogaster females has been performed. Levels of enzyme activity of X-linked glucose 6-phosphate and 6-phosphogluconate dehydrogenases and autosome-linked α-glycerophosphate and NADP-dependent isocitrate dehydrogenases were measured and correlated with DNA content, in crude extracts of whole flies or thoraces. The results show that the contribution of each dose of a given gene to the level of enzyme activity is equal in diploid and triploid cells. These findings are discussed in the context of the phenomenon of dosage compensation.

Regulation of enzyme activities in Drosophila: I. The detection of regulatory loci by gene dosage responses

In order to detect regulatory genetic sites in the autosomes of Drosophila melanogaster , the levels of X-linked glucose-6-phosphate dehydro-genase and autosomally linked α-glycerophosphate and isocitrate dehydrogenases have been monitored in extracts of flies aneuploid for regions of chromosomes II and III. In addition to expected structural gene dosage responses of α-GPDH and IDH, flies hyperploid for several autosome regions were found to display altered levels of one or more of the enzymes studied. While IDH activity was increased in flies hyperploid for segments of both chromosomes II and III, α-GPDH activity was decreased in specific hyperploids for chromosome II regions only. The latter group of segmental aneuploids were normal with respect to levels of chromosome II-linked alcohol dehydrogenase. To test if the observed responses were due to dosage changes of discrete genes lying within the larger effective segments, flies aneuploid for subdivisions of the chromosome segments 21A-25CD, 35A–40, and 70CD–71B were assayed. For two of these large segments so analysed, the apparent effects were attributable to specific small subdivisions, suggesting the presence of discrete regulatory sites within the latter. For the 35A–40 region the α-GPDH effect observed for subdivisions was not sufficient to account for the large α-GPDH decrease seen in flies hyperploid for the large, inclusive region. These observations are discussed with respect to the possible bases of effect of regulatory elements on enzyme activity.

Analysis of a dispersed repetitive DNA sequence in isogenic lines of Drosophila

The location of sequences homologous to a cloned D. melanogaster DNA segment, Dm 25, has been examined in polytene chromosomes by hybridization in situ. Dm 25 localizes to multiple sites and shows variation in patterns between different strains and among individuals within wild-type laboratory strains. Analysis of numerous geographically distinct isogenic lines suggests that Dm 25 patterns are determined by germ-line factors and are not the product of strictly somatic events. In general there is wide variation in Dm 25 patterns among different lines, but a significant number of sites are common to two or more distinct lines. Hybridization to restriction digests of genomic DNA suggests that Dm 25 is a moderately repetitive, conserved sequence whose copies are dispersed throughout the genome. Analysis of species other than melanogaster indicates a significant divergence in structure of sequences homologous to Dm 25 as well as a drastic reduction in amount of homology to the melanogaster sequence.

X-chromosome transcription in Drosophila.

Duplications involving the X chromosome of Drosophila were used to produce individuals with 1.25, 1.50, 1.62 and 1.85 X chromosomes. Rates of transcription in polytene chromosomes were measured autoradiographically after pulse-labeling salivary glands with 3H-uridine. We conclude that: (1) the sum of all transcription occurring on the X elements is constant (relative to autosomal transcription) regardless of how much X-chromosome material is present; (2) this constancy is apparently achieved through a uniform reduction of the rate of synthesis over all X-chromosomal segments as the size of the duplication increases. -/3 Thus, the transcription of a given segment of the X is dependent not just on the number of copies of that segment but also on the number of copies of other regions of the chromosome.

X-Linked gene activity in metamales (XY; 3A) of Drosophila

In order to determine the level of X-linked gene activity in metamales (XY; 3A) of Drosophila melanogaster, RNA synthesis was monitored as a function of 3H-uridine incorporation in nuclei of isolated larval salivary glands by standard autoradiographic means. These data show that the rate of RNA synthesis by the single X chromosome in metamales is approximately 90% of the rate achieved by the three X chromosomes of triploid females (3X; 3A). As an independent measure of chromosome activity, two X-linked enzymes and two autosomal enzymes were monitored in crude extracts by spectrophotometric assay procedures. The results reveal that X-linked enzyme activity levels in metamales constitute 80% of the levels exhibited by control triploid females or intersexes (XXY; 3A). We conclude that metamales exhibit substantial compensation beyond that which occurs in regular, diploid males. This finding establishes a new level of transcriptional activity possible for the X chromosome of Drosophila melanogaster.

Dosage compensation of X-linked heat-shock puffs in Drophila pseudoobscura

Four major puffs are inducible by heat shock in the larval salivary gland chromosomes of D. pseudoobscura. Two of these puffs are present at 23 and 39–40 on the right arm of the X chromosome and two are present at 53 and 58 on chromosome 2. By means of in situ hybridization, residual homologies were demonstrated between the puffs at 23 in D. pseudoobscura and at 63C in D. melanogaster, and between the two chromosome 2 puffs of D. pseudoobscura and 87A and 87C of D. melanogaster. RNA synthesis was monitored as a function of 3H-uridine incorporation in the major heat-induced puffs of D. pseudoobscura and was found to be equivalent in males and females indicating dosage compensation of the two X-linked loci. The evolution of the regulatory controls of these genes is discussed.

Origin of ?-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster and their functional relationship in the ?-glycerophosphate cycle

The basis for the differentiation of l-glycerol-3-phosphate dehydrogenase (α-GPDH) into larval and adult isozymes in Drosophila melanogaster was investigated by the correlation of a lack of appearance of each isozyme during development within Drosophila bearing α-GPDH “null” alleles and by the study of a putative conversion factor. Conversion studies indicate the presence of a heat-labile RNase-resistant conversion factor present in crude larval extracts with the ability to convert GPDH-1 to GPDH-2 and GPDH-3 but not vice versa. In addition, “null” mutations at the Gpdh locus obliterate all isozymatic species of α-GPDH in all developmental stages. These observations suggest that all α-GPDH isozymes are the product of a single structural gene and that the multiple forms of this enzyme arise during successive developmental stages through an epigenetic modification of the primary Gpdh+ polypeptide. Finally, observations are reported which bear on the functional divergence of the α-glycerophosphate cycle in the adult and larval stage of development.

Intersexuality resulting from the interaction of sex-specific lethal mutations in Drosophila melanogaster.

Abstract Male specific lethals mle, msl-1 , and msl-2 interact with female specific mutations at the Sxl locus to bring about intersexuality in double mutant 2X;2A individuals. The frequency and degree to which females are sexually transformed vary among different combinations of alleles. All sexually dimorphic structures as well as the external and internal reproductive organs were found to be affected in some individuals. In general there do not appear to be substantial viability interactions between these mutants; however, under certain marginally permissive genetic and environmental conditions, lethality in double-mutant individuals did seem to be significantly less than expected. The sex-specific genes dealt with in this study were chosen because of their participation in the establishment of the level of X-chromosome function corresponding to the X/A ratio of the karyotype. The unexpected interactions of these genes leading to intersexuality are interpreted as supporting the hypothesis of a close relationship between the genetic control of sex determination and dosage compensation.