Otu and Rif1 double mutant enables analysis of satellite DNA in polytene chromosomes of ovarian germ cells in Drosophila melanogaster

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Abstract

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation were similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

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About the authors

T. D. Kolesnikova

Institute of Molecular and Cellular Biology of the Siberian Branch of the RAS

Author for correspondence.
Email: doliolida@gmail.com
Russian Federation, Novosibirsk

A. R. Nokhova

Novosibirsk State University

Email: doliolida@gmail.com
Russian Federation, Novosibirsk

A. S. Shatskikh

National Research Centre “Kurchatov Institute”

Email: doliolida@gmail.com
Russian Federation, Moscow

M. S. Klenov

Institute of Molecular Genetics RAS

Email: doliolida@gmail.com
Russian Federation, Moscow

I. F. Zhimulev

Institute of Molecular and Cellular Biology of the Siberian Branch of the RAS

Email: doliolida@gmail.com

Academician

Russian Federation, Novosibirsk

References

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Supplementary files

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2. Fig. 1. The Rif11 mutation leads to additional polytenization of the pericentromeric regions in the ACC. Shown is the pericentromeric region of chromosome 2 in the ACC of otu11 mutants (A-B) and otu11 mutants; Rif11 (D, E). In the Rif1+ background, the polytenized arms of chromosome 2 can be connected by thin strands of heterochromatin (A). In other cases, the left (B) and right (C) arms of chromosome 2 lie separately from each other. White arrows point to the ends of polytenized chromosome regions. Against the background of the Rif11 mutation, the zone flanked by white arrows becomes polytenized, and chromosome 2 looks like a single whole and is represented by blocks of compact chromatin, intensely stained with aceto-orcein. Stained with acetoorcein, phase contrast. Scale: 10 µm.

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3. Fig. 2. The Rif11 mutation leads to polytenization of the Prodsat satellite on chromosome 2 of the PPC. Shown are phase-contrast images stained with acetoorcein (top row), the result of in situ hybridization of the probe to the Prodsat satellite (middle row), and the signal superimposed on the phase-contrast image (bottom row). Scale: 10 µm.

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4. Fig. 3. DAPI-stained pericentromeric regions of chromosome 3 in polytene chromosomes SG (A) and ACC (B) against the background of the Rif11 mutation. The area bounded by regions 80A and 81F corresponds to the mitotic heterochromatin of chromosome 3. Arrows and lines indicate regions that have similar morphology in the two types of cells, and also indicate the expected DNA composition for some regions according to [4]. The white triangle indicates a DAPI-positive block corresponding to block h48 of the mitotic heterochromatin map. The position of the Prodsat satellite flanking the centromere of chromosome 3 is indicated by a bracket. The approximate position of the centromere is indicated by a dotted line crossing the chromosome in accordance with [4] for polytene chromosomes of the SG and by the similarity of the morphology of the corresponding regions in the ACC. V.G. United pericentromeric regions of polytene chromosomes X and 4 in SG (B) and ACC (D). Staining – DAPI, scale – 10 µm.

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5. Fig. 4. FISH results with probes to satellites 1.688 and Prodsat in the polytene chromosomes of the PPC (A, B) and SG (B) otu11 mutants; Rif11. A. One of the two blocks of DAPI-positive material at the base of chromosome 4 hybridizes to the probe to satellite 1.688, consistent with the conclusion drawn from morphological analysis. Identification of the Prodsat satellite flanking the centromere of chromosome 3 in the SG (B) and ACC (C). The position of the signal is fully consistent with the prediction made based on the morphology analysis. Staining – chromosomes with DAPI. Signal positions are indicated by arrows. Scale: 10 µm.

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