Location of the 18/28S ribosomal RNA genes in two Hawaiian Drosophila species by monoclonal immunological identification of RNA.DNA hybrids in situ.

Abstract

Using both heterologous rabbit antisera and mouse monoclonal antibody to RNA-DNA hybrids, we have mapped the in situ hybridization locus of the 18/28S ribosomal RNA fraction to a single large band on polytene autosome 3 in -Drosophila heteroneura and Drosophila silvestris. This portion of the chromosome is not physically connected with the nucleolus at the end of larval salivary gland development. In mature larvae, little or no hybridization with the material in the nucleolus can be detected. In younger larvae, hybridization of the ribosomal RNA probe to the nucleolus itself can be observed. The In the course of producing an antibody to RNA-DNA hybridduplexes, we have used in situ hybrids formed on polytene chromosomes as an antigenic test system (1). The reports that fused immunoglobulin-producing cells would subsequently produce both types ofparental immunoglobulins. have led to the development of monoclonal cell lines that produce large quantities of specific antibody in culture (2, 3). Recently, we developed a monoclonal cell line that produces antibody to RNA-DNA hybrids (4) as part of an experimental attempt to. identify in situ RNA-DNA hybrid-duplexes in human chromosomal preparations. As one assay for the presence of antibody, we used the salivary gland chromosomes of larvae of Drosophila silvestris and D. heteroneura, two members of the Hawaiian picturewinged species group, hybridized in situ with homologous ribosomal RNA. Antibodies reactive with RNA-DNA hybrids were obtained from immunized rabbits and from clonal.cultures of immunized mouse spleen cells fused with a mouse myeloma cell line.Using these immunological reagents in a secondary immunofluorescent assay, we have identified the in situ hybridization site for 18/28S rRNA. The genes are located in a chromosome region that is involved in an unusually large proportion of inversion breaks which have occurred in the polytene sequence during phylogeny of the Hawaiian picture-winged species. MATERIALS AND METHODSChromosomal Preparations. Salivary glands from larvae were excised in Grace's insect medium (GIBCO), fixed for 8-10 min in methanol/acetic acid, 3:1 (vol/vol), placed on acidwashed slides with one drop of 45% acetic acid, and squashed under siliconized coverslips. The preparations were held at -20°C for at least 30 min and then immersed in liquid nitrogen. After 1 min, the slides were withdrawn and the coverslips were removed by inserting a no. 10 surgical blade between the slide and the coverslip. The slides were immediately immersed in 95% ethanol. The slides remained in ethanol for at least 4 hr prior to the in situ hybridization. Preparations may be stored in ethanol for up to 2 weeks without deterioration of structure or hybridization results.RNA Probe. RNA was isolated from larvae quick-frozen in liquid nitrogen and stored at -70°C for up to 2 weeks. The isolation method of Kirby (5) was used with the following modifications: sodium triisopropylnaphthalenesulfonate was substituted for sodium p-toluenesulfonate; the phenol/creso...

Evolution of the chromosomal location of rDNA genes in two Drosophila species subgroups: ananassae and melanogaster

“…They are only available for 20 species, scattered across three of the 15 subgenera. The two members of the Idiomyia subgenus studied, D. heteroneura and D. sylvestris, exhibit only autosomal NORs (Stuart et al, 1981), whereas, in most of the species examined in the Sophophora and Drosophila subgenera, the NORs appear to be restricted to the sex chromosomes. In the subgenus Drosophila and in the repleta group, both D. repleta and D. hydei, as well as two of their closest relatives, possess one NOR on the X chromosome and two on the Y chromosome (Hennig et al, 1975(Hennig et al, , 1982.…”

Comparative Analysis of the Chromosomal Positions of rDNA Genes in Species of the <i>tripunctata</i> Radiation of <i>Drosophila</i>

“…However, there are few cases where rDNA is found on the autosomes, but not in the sex chromosomes [Parise-Maltempi and Avancini, 2001;Stuart et al, 1981;Roy et al, 2005]. In the Hawaiian Drosophilids, 2 species of the Idiomyia genus, I. sylvestris and I. heteroneura , show rDNA exclusively in the autosomes [Stuart et al, 1981].…”

“…However, there are few cases where rDNA is found on the autosomes, but not in the sex chromosomes [Parise-Maltempi and Avancini, 2001;Stuart et al, 1981;Roy et al, 2005]. In the Hawaiian Drosophilids, 2 species of the Idiomyia genus, I. sylvestris and I. heteroneura , show rDNA exclusively in the autosomes [Stuart et al, 1981]. Actually the taxonomic position of these species is still a controversial matter, and they are sometimes classified as Drosophila [Grimaldi, 1990;Da Lage et al, 2007;Magnacca and O'Grady, 2008].…”

Ribosomal intergenic spacer (IGS) length variation across the Drosophilinae (Diptera: Drosophilidae)

“…We only examined female flies to prevent any sex bias in our interpretation. In D. melanogaster , and a few other species, Nucleolar Organizer Regions (NORs) are found on the X and Y chromosomes but in other species the locations are not entirely clear [28,29]. Furthermore, previous studies have shown that the Y-linked IGS variants of D. melanogaster are not related to differences in development time [13].…”