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.

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.

During germ line-soma differentiation in early syncytial embryonic development of the chironomid Acricotopus lucidus, a complement of supernumerary chromosomes, the so-called germ line limited chromosomes (Ks), is excluded from the future somatic nuclei in the course of elimination mitoses. The Ks lag behind in the equatorial plane, while the somatic chromosomes (Ss) segregate equally. After elimination mitoses, the Ks are only present in the pole cells, the primary germ cells. In the divisions before their elimination, the Ks frequently showed delayed separation of sister chromatids with high-frequency formation of anaphasic bridges and lagging in pole movement as detected in 4',6-diamidino-2-phenylindole (DAPI)-stained squash preparations of early embryos. To determine if all of the Ks are eliminated in one step during a single mitosis, a fluorescence in situ hybridization (FISH) analysis of early embryonic divisions was performed using probes of germ line specific repetitive DNA sequences, which specifically label the Ks in their centromeric regions. In most cases, all of the Ks are lost in one mitosis; however, occasionally one or several of the Ks can escape their elimination by segregating and moving poleward together with the Ss. The escaping Ks will then be eliminated in one of the following mitoses. This clearly indicates that the specific conditions to eliminate Ks are not restricted to only one division. Possible mechanisms of elimination of Ks are discussed.

Additional chromosomes present only in the germ line are a specific feature of the Orthocladiinae, a subfamily of the Chironomidae. During the complex chromosome cycle in the orthocladiid Acricotopus lucidus, about half of the germ-line-limited chromosomes (Ks) are eliminated in the first division of the primary germ cells. Following normal gonial mitoses, the reduction in the number of Ks is compensated for, in the last mitosis prior to meiosis, by a monopolar movement of the unseparated Ks, while the somatic chromosomes (Ss) segregate equally. This differential mitosis produces daughter cells with different chromosome constitutions and diverse developmental fates. A preferential segregation of mitochondria occurs to one pole associated with an asymmetric formation of the mitotic spindle. This has been detected in living gonial cells in both sexes by using MitoTracker probes and fluorochrome-labelled paclitaxel (taxol). In males, the resulting unequal partitioning of mitochondria to the daughter cells is equalised by the transport of mitochondria through a permanent cytoplasmic bridge from the aberrant spermatocyte to the primary spermatocyte. This asymmetry in the distribution and in the segregation of cytoplasmic components in differential gonial mitosis in Acricotopus may be involved in the process of cell-fate determination.

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