Essential genes in proximal 3L heterochromatin of Drosophila melanogaster.

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.

Identifying a single-copy DNA sequence associated with the expression of a heterochromatic gene, the light locus of Drosophila melanogaster.

Although little is known about the molecular organization of most genes within heterochromatin, the unusual properties of these chromosomal regions suggest that genes therein may be organized and expressed very differently from those in euchromatin. We report here the cloning, by P transposon tagging, of sequences associated with the expression of the light locus, an essential gene found in the heterochromatin of chromosome 2 of Drosophila melanogaster. We conclude that this DNA is either a segment of the light locus, or a closely linked, heterochromatic sequence affecting its expression. While other functional DNA sequences previously described in heterochromatin have been repetitive, light gene function may be associated, at least in part, with single-copy DNA. This conclusion is based upon analysis of DNA from mutations and reversions induced by P transposable elements. The cloned region is unusual in that this single-copy DNA is embedded within middle-repetitive sequences. The in situ hybridization experiments also show that, unlike most other sequences in heterochromatin, this light-associated DNA evidently replicates in polytene chromosomes, but its diffuse hybridization signal may suggest an unusual chromosomal organization.

Genetic analysis of the heterochromatin of chromosome 3 in Drosophila melanogaster. I. Products of compound-autosome detachment.

The heterochromatin of the third chromosome is the largest uncharacterized region of the Drosophila melanogaster genome, and the last major block of D. melanogaster heterochromatin to be thoroughly analyzed. In the present study, this region was genetically dissected by generating and analyzing a series of attached, detached and reattached third chromosomes. Separate detachment experiments were conducted for all 12 possible combinations of four newly synthesized sister-strand compound-3L and three newly synthesized sister-strand compound-3R chromosomes. A total of 443 recessive lethal detachment products carrying putative heterochromatic deficiencies were tested for complementation in a several-stage complementation analysis. The results revealed the presence of seven separable vital regions in the heterochromatin of chromosome 3. Attempts to reattach deficiency-carrying detachment products established that six of these vital regions are on the left arm, but only one is on the right arm. An analysis of the types and frequencies of detachment-product deficiencies generated in each detachment experiment permitted the genetic characterization of the progenitor compounds. It was also possible to determine the proximal-distal orientation of the genes on each arm, and to identify possible breakpoints for each lethal detachment product produced. The results of this study suggest that vital genes in the heterochromatin of the third chromosome are not randomly distributed between, nor within, the heterochromatic blocks of the left and right arms.

Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis.

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.

The influence of whole-arm trisomy on gene expression in Drosophila.

The biochemical consequences of extensive aneuploidy in Drosophila have been examined by measuring the levels of specific proteins in larvae trisomic for entire chromosome arms. By far the most common effect is a reduction in gene product levels (per gene template) by one-third from the diploid quantity, consistent with the model that concentration-dependent repressors of these loci reside on the duplicated chromosome arms. Most loci appear sensitive to such repression in one or more of the trisomies examined, suggesting that such regulatory loci might be quite common. Repression of gene-product levels in trisomies may significantly contribute to their inviability. Few loci are activated in trisomies implying that most factors necessary for gene expression are in excess. While autosomal trisomies can repress the expression of both X-linked and autosomal loci, X-chromosomal trisomies have little effect on most autosomal genes. A family of genes coding for larval serum proteins do not respond similarly in trisomies, suggesting that regulation operates on a process which is not common to their coordinate regulation. Finally, Adh genes transposed to new chromosomal positions maintain their ability to be repressed in 3L trisomies suggesting that this response to regulation involves a closely linked cis-acting regulatory element.

Further Characterization of Genetic Elements Associated with the Segregation Distorter Phenomenon in DROSOPHILA MELANOGASTER.

Segregation Distorter, SD, associated with the second chromosome of Drosophila melanogaster, is known to cause sperm bearing the non-SD homologue to dysfunction in heterozygous males. In earlier studies, using different, independently derived, SD chromosomes, three major loci were identified as contributing to the distortion of segregation ratios in males. In this study the genetic components of the SD-5 chromosome have been the subjects of further investigation, and our findings offer the following information. Crossover analysis confirms the mapping of the Sd locus to a position distal to but closely linked with the genetic marker pr. Spontaneous and radiation-induced recombinational analyses and deficiency studies provide firm support to the notion that the Rsp (Responder) locus lies within the proximal heterochromatin of chromosome 2, between the genetic markers lt and rl and most likely in the heterochromatin of the right arm. The major focus of this study, however, has been on providing a better definition of the genetic properties of the Enhancer of SD [E(SD)]. Our findings place this locus within the region of the two most proximal essential genes in the heterochromatin of the left arm of chromosome 2. Moreover, our analysis reveals a probable association of the E(SD) locus with a meiotic drive independent of that caused by Sd.

Autosomal dosage compensation Drosophila melanogaster strains trisomic for the left arm of chromosome 2.

Drosophila melanogaster individuals trisomic for an entire chromosome arm can survive to late stages of pupal development. We have examined the levels of five enzymes whose structural genes are located on the left arm of chromosome 2 both in trisomy 2L and in diploid strains. In trisomies, three distally mapping loci showed compensated levels of expression close to that observed in the diploid strains. Analysis of electrophoretic variants of a compensated locus revealed that all three alleles are active in trisomies. The two proximally located loci displayed dose-dependent levels of expression. Therefore, at the level of the individual gene, autosomal compensation appears to be an all-or-none phenomenon. Furthermore, the compensatory response may be regionally distributed along the chromosome arm. The presence of both autosomal and sex-linked dosage compensation prompts us to speculate that there phenomenon are similar homeostatic mechanisms that modulate gene expression both in euploid and aneuploid genomes.

Meiotic behavior of compound autosomes in females of Drosophila melanogaster: interchromosomal effects and the source of spontaneous nonsegregation.

In females of Drosophila melanogaster, compound autosomes enter the repulsion phase of meiosis uncommitted to a particular segregation pattern because their centromeres are not restricted to a bivalent pairing complex as a consequence of crossing over. Their distribution at anaphase, therefore, is determined by some meiotic property other than exchange pairing, a property that for many years has been associated with the concept of nonhomologous pairing. In the absence of heterologous rearrangements or a free Y chromosome, C(3L) and C(3R) are usually recovered in separate gametes, that is as products of meiotic segregation. Nevertheless, there is a regular, albeit infrequent, recovery of reciprocal meiotic products (the nonsegregational products) that are disomic and nullosomic for compound thirds. The frequency of these exceptions, which is normally between 0.5 and 5.0%, differs for the various strains examined, but remains constant for any given strain. Since previous studies have not uncovered a cause for this base level of nonsegregation, it has been referred to as the spontaneous frequency. In this study, crosses between males and females whose X chromosomes, as well as compound autosomes, are differentially marked reveal a highly significant positive correlation between the frequency of compound-autosome nonsegregation and the frequency of X-chromosome nondisjunction. However, an inverse correlation is found when the frequency of nondisjunction is related to the frequency of crossing over in the proximal region of the X-chromosome. These findings have been examined with reference to the distributive pairing and the chromocentral models and interpreted as demonstrating (1) that nonsegregational meiotic events arise primarily as a result of nonhomologous interactions, (2) that forces responsible for the segregation of nonhomologous chromosomes are properties of the chromocentral region, and (3) that these forces come into expression after the exchange processes are complete.

Chromosome replacement in mixed populations of compound-2L; free-2R and standard strains of Drosophila melanogaster : An example of unstable genetic isolation.

Crosses between compound-2L; free-2R (free-arm) and standard strains of Drosophila melanogaster produce two classes of inviable aneuploid hybrids in equal proportions: monosomic 2L and trisomic 2L. The lethal period for monosomics occurs during embryogenesis while the trisomics survive to late pupae. Since the hybrids are inviable, standard and free-arm strains within a mixed population remain genetically isolated. Genetic isolation in the absence of mating isolation offers an extreme example of unstable equilibrium. Relative fitness data indicate that an unstable equilibrium will be established between free-arm and standard strains at a ratio of 2.5∶1. Indeed, in three cage experiments established at initial ratios of 3∶1, free arms to standards, laboratory (Oregon R) or native (Okanagan S) standard strains were completely replaced in approximately 100 days by free-arm lines derived either from laboratory or from native genetic background. In contrast, one cage established at an initial ratio of 4∶1 failed to show replacement and for 92 days remained at approximately the initial ratio. Subsequent genetic analysis of flies removed from this cage identified the presence of an anomalous strain through which genetic information was transferred reciprocally between the free-arm and standard lines. The second chromosomes carried by this strain consisted of a free-2R and a standard second on the right arm of which was attached a duplication for all of 2L. While the origin of the 2L·2R+2L chromosome was uncertain, genetic and cytological examinations revealed that it represented the reciprocal crossover product expected from an exchange that generated a F(2R). Additional crosses disclosed that the transmission frequency of the asymmetrical pair of second chromosomes, as well as their right-arm crossover products, was disproportionately in favor of the short arm. Since unequal transmission was invariably greater from female parents, this phenomenon was viewed as further evidence in support of the drag hypothesis.

Exploring the Potential of Compound;free-Arm Combinations of Chromosome 2 in DROSOPHILA MELANOGASTER for Insect Control and the Survival to Pupae of Whole-Arm Trisomies.

Genetic tests of second-chromosome compound;free-arm combinations ("free arms") in Drosophila melanogaster indicate that the egg hatch is approximately 50% that of standard lines and adult recovery is approximately 40%. Free-arm strains are genetically isolated from both compound-chromosome lines and standards.A large proportion of the hybrid progeny arising from crosses between free arms and standards or free arms and compounds, survive to the pupal stage. Cytological examinations reveal that these hybrids are trisomic for one arm of chromosome 2. Such hybrid progeny may place an added constraint upon the competition between free-arm and standard strains by competing for food, but not contributing to the adult population. The fitness data, the genetic isolation characteristic and the possible impact of hybrid progeny all suggest that free arms may prove to be a valuable genetic tool for insect population control. Preliminary cage-competition experiments to test this prediction have demonstrated that free arms are able to displace standards at ratios as low as 3:1, which is close to the theoretical equilibrium predicted by the fitness data (2.5:1).

Genetic analysis of the proximal region of chromosome 2 of Drosophila melanogaster. I. Detachment products of compound autosomes.

To examine the genetic composition of proximal heterochromatin in chromosome 2, the detachment of compound second autosomes, for generating proximal deficiencies, appeared a promising method. Compound seconds were detached by gamma radiation. A fraction of the detachment products were recessive lethals owing to proximal deficiencies. Analysis by inter se complementation, pseudo-dominance tests with proximal mutations and allelism tests with known deficiencies provided evidence for at least two loci between the centromere and the light locus in 2L and one locus in 2R between the rolled locus and the centromere. The data further demonstrate that rolled, and probably light, are located within the proximal heterochromatin. Thus, functional genetic loci are found in heterochromatin, albeit at low density.

Compound autosomes in Drosophila melanogaster: The meiotic behavior of compound thirds.

Studies of the meiotic distribution of compound-3 chromosomes in males and females of Drosophila melanogaster provided the following results. (1) From females homozygous for the standard arrangement of all chromosomes other than C(3L) and C(3R), less than 5% of the gametes recovered were nullosomic or disomic for compound-3 chromosomes. The frequency of nonsegregation differed between strains, but within a given strain it remained relatively constant. (2) According to egg-hatch frequencies, C(3L) and C(3R) segregate independently during spermatogenesis. (3) In females, structurally heterozygous second chromosomes occasion a marked increase in the recovery of nonsegregational progeny; in males, rearranged seconds have no apparent influence on the distribution of compound thirds. (4) The highest frequencies of nonsegregational progeny were recovered from C(3L);C(3R) females carrying compound-X (plus free Y) chromosomes. (5) In comparing the recovery of nonsegregating compound thirds to the recovery of rearranged heterologs, a definite nonrandom distribution was realized in several crosses. These results are examined in reference to the concepts of distributive pairing (Grell 1962). Moreover, considering the structural nature of compound autosomes, we propose that nonhomologous (distributive) pairing is a property of the centromeric region and suggest that rearrangements involving breaks in this region possibly alter the effectiveness of distributive pairing forces.

Population control of caged native fruitflies in the field by compound autosomes and temperature-sensitive mutants.

A genetic technique for insect population control has been tested in cages under field conditions at two different locations in British Columbia. The method entails the population replacement of standard insects by those bearing compound autosomes using the principle of negative heterosis, thus permitting control or elimination through conditional mutations. Both native- and laboratory-derived compound strains of the fruitfly Drosophila melanogaster were tested in population cages against standards in the laboratory and at the two field sites. Those compound-bearing insects originating from the wild were the most successful, both in the laboratory and the field, in displacing standards from the cages down to a minimum initial ratio of 5 compounds to 1 standard. The importance is stressed of collecting strains from the wild, and performing the necessary genetic manipulations as rapidly as possible, prior to releasing the rearrangement in the field for control purposes.

Genetic control of insect population. I. Cage studies of chromosome replacement by compound autosomes in Drosophila melanogaster.

A genetic method for insect control was evaluated using the test organism, Drosophila melanogaster. The technique involved the displacement under a system of continuous reproduction, of standard strains by those carrying compound autosomes. The eradication of the replacements could subsequently be achieved through the use of temperature-sensitive lethal mutations.-While certain compound autosome strains failed to displace standards in population cages, even at the initial release ratio of 25:1, others were highly successful. Indeed, for some strains when the ratio of compounds to standards was as low as 9:1, the population rapidly went to fixation in favor of the compound line.-Hatchability was found to be an insufficient index of fitness to estimate the initial ratios of compounds to standards that would guarantee fixation of the former. Differences in other fitness components, such as development time, were detected that could seriously modify displacement, especially with continuous overlapping generations. The importance of examining the fitness of various compound lines and selecting the most competitive in cages, prior to field tests, cannot be overemphasized.