Structural evolution of the germ line-limited chromosomes in Acricotopus

Abstract

The elimination of chromatin or whole chromosomes from the future somatic nuclei during germ line-soma differentiation in early embryogenesis is a genetic phenomenon found in a wide variety of animal species. Less is known about the origin, structure, and function of the germ line-limited chromosomes. In the chironomid Acricotopus lucidus fluorescence in situ hybridization (FISH) with labeled soma DNA to "Keimbahn" chromosomes (Ks) and soma chromosomes (Ss) of spermatogonial mitoses revealed that each of the nine different K types possesses large S-homologous sections, mostly in the distal parts of both chromosome arms. Painting probes of the three Ss and of each of their chromosome arms were generated by microdissection of polytene salivary gland chromosomes and subsequent amplification by the degenerate oligonucleotide-primed polymerase chain reaction. Multicolor FISH demonstrated that each of the Ks, with the exception of one K type, was painted by only one of the three S probes. Furthermore, in seven Ks, one chromosome arm was painted by the long-arm probe and the other by the short-arm probe of the S concerned. The hybridization pattern strongly suggests that each of these K types is derived from a specific S. One function of the S-homologous K sections is thought to be determination of the regular occurrence of crossover events, with the resulting chiasmata in these sections ensuring correct segregation of the K homologs during meiosis. Reverse chromosome painting on polytene S sets with a probe generated from metaphase Ks corroborates the above results and produces conclusive evidence for the hypothesis that during evolution the Ks have developed from the Ss by endopolyploidization and rearrangements followed by the accumulation of germ line-specific repetitive DNA sequences in the centromeric regions.

Isolation of a new germ line-specific repetitive DNA family in <i>Acricotopus</i> by microdissection of polytenized germ line-limited chromosome sections from a permanent larval salivary gland preparation

“…In A. lucidus, a probe of a specific K type was made by microdissection of ten K4s of freshly prepared spermatogonial metaphases for reverse painting onto polytene salivary gland Ss to identify the S-homologous sections of K4 (Staiber and Schiffkowski, 2000). Microdissection of defined small sections of metaphase Ks results in only very little amounts of DNA.…”

“…1B were collected for probe generation. Chromosome microdissection and treatment of isolated chromatin with proteinase K was performed as described in Staiber and Schiffkowski (2000). Spermatocyte preparations were made via the dry ice method from prepupal testes previously treated with 0.5 % sodium citrate for 20 min and fixed in ethanol:acetic acid (3:1, v/v) for 2 h.…”

“…In the Orthocladiinae, a subfamily of the Chironomidae, the germ line-limited chromosomes (Ks; K being derived from "Keimbahn") and the soma chromosomes (Ss) pass through a complex chromosome cycle with interesting genetic features such as elimination mitoses and monopolar movements of chromosome complements (Bauer and Beermann, 1952;Bauer, 1970). In the last few years, chromosome painting analyses with S-specific probes, and the isolation and localization of tandem repetitive DNA families clearly demonstrated that in the orthocladiid Acricotopus lucidus the Ks have developed from the Ss by duplication, rearrangments and accumulation of germ line-specific repetitive sequences (Staiber et al, 1997;Staiber and Schiffkowski, 2000;Staiber and Wahl, 2002). First indications that the Ks are composed of S-homologous sections and heterochromatic segments resulted from X-ray induced K-S-rearrangments analysed in the polytene larval salivary gland chromosomes (Staiber and Thudium, 1986;Staiber, 1991).…”

FISH analysis and cytogenetic characterization of male meiotic prophase I in Acricotopus lucidus (Diptera, Chironomidae)

“…The Ks are composed of large eu chroma tic S-homologous sections, which are painted by the SI-SIII probes, and of hetero chromatic segments containing germ line-specific repetitive DNA sequences (Staiber and Schiffkowski, 2000). The dissected testis cells were treated with a hypotonic sodium citrate solution prior to their fixation.…”

“…They were then washed in 2X SSC, pH 7.0, at room temperature for 5 min and twice in 2X SSC at 37°C for 5 min each. The biotinylated SI probe was detected with Cy5-conjugated strept avidin (Dako), the digoxigenated SII probe with rhodamine-conjugated anti-digoxigenin Fab fragments (Roche) and the fluorescein-labeled SIII probe by layers of a rabbit anti-fluorescein antibody (Dako) and fluorescein conjugated goat anti-rabbit antibody (Vector Laboratories) as described in Staiber and Schiffkowski (2000). Chromosomes were counter stained with 0.2 µg/mL 4',6-diamidino-2-phenylindole (DAPI) in phosphate-buffered saline, pH 7.4, for 5 min and mounted in Vectashield antifade solution (Vector Laboratories).…”