Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae)

Abstract

In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line.

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

“…The first germ line-specific highly repeated DNA family isolated in A. lucidus was named AlKe family (from A. lucidus and "Keimbahn", Staiber et al, 1997). Two subfamilies have been differentiated, subfamily A, repeat units 183-184 bp, and subfamily B, repeat units 175-178 bp, being nonhomologous in a section of about 75 bp.…”

“…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).…”

“…family exhibits partial homologies to earlier isolated germ linespecific highly repeated DNA sequence families of Acricotopus (Staiber et al, 1997). Therefore, the collection of permanent preparations of X-ray induced polytene K-S-rearrangements can be used for obtaining DNA from small defined K sections, e.g.…”

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

“…In A. lucidus, no telomere-specific DNA sequences are available to label and to visualize the chromosome ends by FISH. K centromere-specific AlKeRe1 DNA probes (from A. lucidus "Keimbahn" Repetitive element; Staiber et al, 1997) were therefore used to check whether specific arrangements of the centromeric regions of the Ks occur in pachytene nuclei.…”

“…A digoxigenized probe of the germ line-specific tandem repetitive AlKeRe1 family (Staiber et al, 1997) localized in the centromeric heterochromatin of the Ks was used together with fluorescein-conjugated anti-digoxigenin Fab fragments (Roche) to label the centromeric regions of the Ks in the meiosis preparations. Chromosomes were stained by mounting the preparations in Vectashield fluorescence antifade solution (Vector Laboratories) containing 2.5 μg/mL propidium iodide.…”