In Drosophila melanogaster, inversion In(3R)Payne increases in frequency towards low latitudes and has been putatively associated with variation in size and thermal resistance, traits that also vary clinally. To assess the association between size and inversion, we obtained isofemale lines of inverted and standard karyotype of In(3R)Payne from the ends of the Australian D. melanogaster east coast cline. In the northern population, there was a significant association between In(3R)Payne and body size, with standard lines from this population being relatively larger than inverted lines. In contrast, the inversion had no influence on development time or cold resistance. We strengthened our findings further in a separate study with flies from populations from the middle of the cline as well as from the cline ends. These flies were scored for wing size and the presence of In(3R)Payne using a molecular marker. In females, the inversion accounted for around 30% of the size difference between cline ends, while in males the equivalent figure was 60%. Adaptive shifts in size but not in the other traits are therefore likely to have involved genes closely associated with In(3R)Payne. Because the size difference between karyotypes was similar in different populations, there was no evidence for coadaptation within populations.

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The polytene chromosomes of Rhynchosciara americana and R. hollaenderi, a pair of sibling species in the americana-like group of Rhynchosciara, were compared using a number of techniques, including in situ hybridization. With classical cytological techniques, the only differences observed were in the morphology of centromeric and telomeric heterochromatin, in the size of a DNA and RNA puff, and in the presence of an inversion polymorphism in R. hollaenderi. However, after in situ hybridization with rDNA and poly-r(A) probes, differences between the two species appeared at a number of sites. Differences in poly-r(A) sites were especially informative in establishing phylogenetic relationships between these two species and a third species currently being examined from this group. Chromosomal evolution between these species appears to have occurred mainly through differential amplification and transposition of repetitive sequence DNA, of which dA:dT tracts are an important component. The R. hollaenderi karyotype is tentatively considered more ancestral than that of R. americana because it has features present in the third Rhynchosciara species. Explanations for the monomorphisms observed in Rhynchosciara species and mechanisms of speciation in the group are considered within the context of the species' complex behavior.

Late in the fourth larval instar, several regions of the Rhynchosciara amercana salivary gland chromosomes undergo "DNA puffing. " We have constructed a library ofcloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands'during the period ofdevelopment when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puffsites C3 and C8and.encode 1.25-kilobase and 1.95-kldobase RNA molecules, respectively. We estimate -from the quantitation -of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.Gene amplification in somatic cells was first detected by morphological criteria in the larval salivary glands offlies ofthe family Sciaridae. Several regions of the Rhynchosciara americana polytene chromosomes were found to show a type ofpuffing in which, after puff regression, there was more DNA in the bands involved compared with neighboring bands as indicated by Feulgen staining (1). This was later confirmed. both by spectrophotometric measurements (2) and by autoradiographical studies on the incorporation of [3H]thymidine (3). Subsequently, similar observations-were made on the salivary chromosomes of larvae from the genus Sciara (4-6).The DNA puffs, which appear in late fourth instar in-Rhynchosciara salivary glands, are involved in the production of messenger RNAs (7,8). These encode several peptides of the communal cocoon, which are needed in large amounts over a short period of time (9, 10). Based on morphological and physiological criteria, the fourth instar of R. americana larvae has been divided into six periods (11). The first indication of DNA puff formation is the appearance of a fast-green staining band between two orcein (+) bands at the chromosomal sites ofthese puffs early in period 4 (ref. 12; unpublished data). Amplification and puffformation are subsequently maximal at different times for each site and are also dependent on the position of the cell within the gland (13, 14). The largest puffs are found in region 2 of the B chromosome and in regions 3 and 8 of the C chromosome. The B2 puffis formed preferentially in the first 50 cells of the gland in period 5, whereas C3 attains its largest size in the middle and distal section of the gland in period 6. The C8 puff is similar in all regions and is also maximal in period 6. As the larvae progress from period 3 to period 4; there is a dramatic change in the pattern of RNA and protein synthesis. This consists of an inhibition of rRNA synthesis (15) and the synthesis of new poly(A)+RNA species (7,8). This is accompanied by inhibition of the synthesis of certain peptides and the synthesis jtg of unfractionated RNA from the salivary glands of periods 3 and 5 larvae, was fractionated by electrophores...

We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.