Germline restricted DNA has evolved in diverse animal taxa, and is found in several vertebrate clades, nematodes, and flies. In these lineages, either portions of chromosomes or entire chromosomes are eliminated from somatic cells early in development, restricting portions of the genome to the germline. Little is known about why germline restricted DNA has evolved, especially in flies, in which three diverse families, Chironomidae, Cecidomyiidae, and Sciaridae exhibit germline restricted chromosomes (GRCs). We conducted a genomic analysis of germline restricted chromosomes in the fungus gnat Bradysia (Sciara) coprophila (Diptera: Sciaridae), which carries two large germline restricted L chromosomes. We sequenced and assembled the genome of B. coprophila, and used differences in sequence coverage and k-mer frequency between somatic and germ tissues to identify GRC sequence and compare it to the other chromosomes in the genome. We found that the GRCs in B. coprophila are large, gene-rich, and have many genes with paralogs on other chromosomes in the genome. We also found that the GRC genes are extraordinarily divergent from their paralogs, and have sequence similarity to another Dipteran family (Cecidomyiidae) in phylogenetic analyses, suggesting that these chromosomes have arisen in Sciaridae through introgression from a related lineage. These results suggest that the GRCs may have evolved through an ancient hybridization event, raising questions about how this may have occurred, how these chromosomes became restricted to the germline after introgression, and why they were retained over time.
Meiosis in male Sciara is unique with a single centrosome. A monopolar spindle forms in meiosis I, but a bipolar spindle forms in meiosis II. The imprinted paternal chromosomes are eliminated in meiosis I; there is non-disjunction of the X in meiosis II. Despite differences in spindle construction and chromosome behavior, both meiotic divisions are asymmetric, producing a cell and a small bud. Observations of live spermatocytes made with the LC-PolScope, differential interference contrast optics and fluorescence revealed maternal and paternal chromosome sets on the monopolar spindle in meiosis I and formation of an asymmetric monastral bipolar spindle in meiosis II where all chromosomes except the X congress to the metaphase plate. The X remains near the centrosome after meiosis I and stays with it as the spindle forms in meiosis II. Electron microscopy revealed amorphous material between the X and the centrosome. Immunofluorescence with an antibody against the checkpoint protein Mad2 stains the centromeres of the maternal X dyad in late meiosis I and in meiosis II where it fails to congress to the metaphase plate. Mad2 is also present throughout the paternal chromosomes destined for elimination in meiosis I, suggesting a possible role in chromosome imprinting. If Mad2 on the X dyad mediates a spindle checkpoint in meiosis II, it may delay metaphase to facilitate formation of the second half spindle through a non-centrosomal mechanism.
DNA replication in dividing eukaryotic cells imposes a requirement for the faithful recreation on the newly synthesized chromatids of the nucleoprotein architecture of parent chromosomes. Practically nothing is known about the structure of postreplicative immature chromatin (a very short-lived entity of <30 min.). We report here the unexpected discovery that during DNA amplification of locus II/9A in salivary gland polytene chromosomes of the fungus fly Sciara coprophila, DNA replication fork passage is uncoupled from postreplicative chromatin assembly; this enables visualization and analysis of chromatin fibers disassembled by DNA replication. We used electron microscopy to visualize a wealth of low nucleosome density immature chromatin fibers in preparations of Sciara chromatin from amplification-stage tissue. Remarkably, as gauged by high sensitivity to micrococcal nuclease and an unusually short length of DNA associated with each histone octamer, we found that locus II/9A which undergoes amplification and is replicated once every 4-6 hrs. (but not the bulk genome or a replicatively quiescent DNA stretch) was maintained in such an ummature fiber for ca. 24 hrs. Following amplification, locus II/9A assumed conventional chromatin organization, indicating that the epigenetic mark targeting nascent DNA to the chromatin assembly machinery is stable for several hours. We propose that this very unusual prolonged maintenance of a segment of the genome in immature chromatin facilitates access by the basal transcriptional machinery to the amplified DNA, and thus is an evolutionary adaptation to the demand for high transcription from genes that reside in the amplified loci.
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