Ann Jacob Stocker

The association between inversion In(3R)Payne and clinally varying traits in Drosophila melanogaster.

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.

Banding studies on the polytene chromosomes of Rhynchosciara hollaenderi

C-, Q-, H-, and Ag-banding have been carried out on the polytene chromosomes of Rhynchosciara hollaenderi. The results of these techniques are presented and are correlated with the molecular data which exists for Rhynchosciara polytene chromosomes. By systematic organization of both banding and molecular data, we have attempted to give as complete a picture as possible of the characteristics of the differentially banding regions. This presents an organized method of approaching mechanisms of banding in terms of structural and functional aspects of the intact chromosome. Polytene chromosomes are particularly suited for this type of analysis and with them, both developmental and evolutionary changes can be conveniently utilized for additional insights into the functions of banding regions.

Correspondence of silver banding with rRNA hybridization sites in polytene chromosomes of Rhynchosciara hollaenderi

Abstract The ammonical silver banding technique has been used to stain the polytene chromosomes of Rhynchosciara hollaenderi . The initial regions to stain black with this technique are regions which hybridize with rRNA. The pattern of silver staining in the NOR of the X-chromosome corresponds closely with the regional clumping of grains observed after in situ hybridization with rRNA. This region of the X-chromosome is much more active than any other rRNA hybridizing region in the formation of nucleoli and is also Ag-banded more frequently. This implies that regions most active in rRNA production may preferentially Ag-band. The use of this technique to study the production of nucleoli and micronucleoli in sciarid polytene chromosomes is discussed as are the potential contributions of these chromosomes to arriving at a better understanding of the mechanisms of silver banding.

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi.

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both RNA and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. — Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puffs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the Drosophila-based model may not be completely applicable to Rhynchosciara.

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson–Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

DNA bending in the replication zone of the C3 DNA puff amplicon of Rhynchosciara americana (Diptera: Sciaridae).

Intrinsic bent DNA sites were identified in the 4289xa0bp segment encompassing the replication zone which directs DNA amplification and transcription of the C3-22 gene of Rhynchosciara americana. Restriction fragments showed reduced electrophoretic mobility in polyacrylamide gels. The 2D modeling of the 3D DNA path and the ENDS ratio values obtained from the dinucleotide wedge model of Trifonov revealed the presence of four major bent sites, positioned at nucleotides −6753, −5433, −5133 and −4757. Sequence analysis showed that these bends are composed of 2–6xa0bp dA·dT tracts in phase with the DNA helical repeat. The circular permutation analysis permitted the verification that the fragments containing the bending sites promote curvature in other sequence contexts. Computer analyses of the 4289xa0bp sequence revealed low helical stability (ΔG values), negative roll angles indicating a narrow minor groove and a putative matrix attachment region. The data presented in this paper add to information about the structural features involved in this amplified segment.

The chromosomes of Rhynchosciara baschanti (Diptera: Sciaridae): molecular cytogenetic comparisons with taxa in the americana-like group.

Polytene chromosome analysis is presented for Rhynchosciara baschanti, a species belonging to the americana-like group of Rhynchosciara. R. baschanti chromosomes show morphological differences in centromeric and telomeric regions compared to two other members within the group, R. americana and R. hollaenderi. In addition, fixed band and autosomal inversion differences were noted. Physical mapping data showed synteny among the taxa under study for DNA puffs and single-copy or histone gene probes, whereas rDNA and poly-(r)A probes showed different diagnostic patterns. The activity of developmentally active genes and the pattern of thymidine incorporation into DNA puff sites of R. baschanti are consistent with those found in the two previously studied species, except for lower levels of expression at some of these sites. These results suggest that differential duplication of specific DNA sequences, in particular repetitive and homopolymeric DNA, has played a role in the chromosomal evolution of these Rhynchosciara species. Inversions and band dimorphisms have also occurred, but the processes leading to their maintenance and fixation appear to have been slow, since these three species are in general chromosomally monomorphic.

The localization of ribosomal DNA in Sciaridae (Diptera: Nematocera) reassessed

The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites.

C-banding and chromosome evolution in some related species of Australian chironominae

C-banding of the chromosomes from members of a group of related Australian Chironomus species showed that most of these species have little C-banded heterochromatin. C ~ regions were usually observed to correlate with single thin to thick bands at centromeres, telomeres, nucleoli, and a few interstitial regions. Despite the generally low amount of C ~ heterochromatin, each species showed some differences in amount and pattern. Such differences could be significant, even among species within the same evolutionary line. In some species, certain of the above regions did not C-band, the most obvious being two centromeric bands of Ch. duplex. In Ch. australis three C-bands, including that at the centromere, were linked to the X chromosome. The greatest amount of C ~ heterochromatin was observed in one of the two forms of Ch. nepeanensis which had thick C + blocks at all centromeres except chromosome 1, where a thick-block/thin-band heterozygosity was present. The low amount of C ÷ heterochromatin in cells of high degrees of polyteny such as those of salivary glands and Malphigian tubules, explains why no C-bands could be observed in interphase, mitotic or meiotic cells of most species. The breakpoints of inversions which have previously been mapped in these species do not coincide with the position of C-bands. Therefore, among the species of this group, changes in C ~ heterochromatin appear to have occurred through addition or deletion of small amounts of this material at various chromosomal regions. The possibility that duplication could

Nucleolar relationships in some Australian chironomus species.

Members of a group of Australian Chironomus species in the pseudothummi complex show wide variation in number and location of nucleolar organizing regions (NORs). The structure of these regions has been examined by phase contrast microscopy and silver banding of salivary gland polytene chromosomes. Presence of nucleoli was also checked on other types of chromosomes in some species. The contribution of the silver banding technique to nucleolar studies in these chironomid chromosomes is discussed. Nucleoli often seem to emerge from groups of (up to 9) bands. Further studies are necessary to confirm the presence of rRNA cistrons in all of these bands. Banding differences, in particular absence of bands from homologous regions of some species which have smaller nucleoli or lack particular nucleoli, have been found. In the case of Ch. tepperi, however, little banding difference is apparent in the 16B region between the N(IV)+ and N(IV)− chromosomes, although in situ hybridization (Eigenbrod 1978) shows a deletion of rRNA cistrons in the N(IV)− stock. Differences in heterochromatin amount have also been observed at different NORs. A scheme for the evolution of nucleolar-producing regions in this Chironomus group in terms of these and other known chromosomal changes is presented and discussed.