Elsevier

Gene

Volume 250, Issues 1–2, 30 May 2000, Pages 77-84
Gene

Drad21, a Drosophila rad21 homologue expressed in S-phase cells

https://doi.org/10.1016/S0378-1119(00)00166-9Get rights and content

Abstract

Cohesin is an evolutionarily conserved multiprotein complex required to establish and maintain sister chromatid cohesion. Here, we report the cloning and initial characterization of the Drosophila homologue of the fission yeast rad21 cohesin subunit, called Drad21. The Drad21 coding region was localized to centromeric heterochromatin and encodes a 715 amino acid (aa) protein with 42% aa identity to vertebrate Rad21p-homologues, 25% with Scc1p/Mcd1p (S. cerevisiae) and 28% with Rad21p (S. pombe). Sequences with similarity to the sites of proteolytic cleavage identified in Scc1p/Mcd1p are not evident in DRAD21. Northern blot and mRNA in-situ studies show that Drad21 is developmentally regulated, with high levels of expression in early embryogenesis, in S-phase cells of proliferating imaginal tissues, and in the early endocycling cells of the embryonic gut.

Introduction

Establishment, maintenance and coordinated loss of sister-chromatid cohesion are essential processes that ensure faithful chromosome partitioning in all eukaryotes. Recent studies have identified an evolutionarily conserved protein complex, called cohesin, which is instrumental in regulating sister chromatid cohesion in both meiosis and mitosis (for reviews, see Hirano, 1999, Nasmyth, 1999, Orr-Weaver, 1999). Cohesin is required in S phase to link newly replicated sister chromatids together and to maintain sister cohesion up until the metaphase–anaphase transition (Uhlmann and Nasmyth, 1998). An essential component and key regulator of cohesin complexes in both higher and lower eukaryotes is a phosphoprotein of presently unknown biochemical function related by homology to Schizosaccharomyces pombe Rad21p and Rec8p.

Originally identified by complementation of a radiation sensitive mutant, S. pombe rad21 encodes a 630 aa cell-cycle-regulated protein expressed in late G1/S phase (Birkenbihl and Subramani, 1992, Birkenbihl and Subramani, 1995). Subsequent genetic studies in Saccharomyces cerevisiae showed Scc1p/Mcd1p (a homologue of Rad21p) to be a key regulator of sister chromatid cohesion in yeast (Guacci et al., 1997, Michaelis et al., 1997). In S. cerevisiae, anaphase is initiated by site-specific cleavage of Scc1p/Mcd1p, which is coincident with cohesin dissociation from chromosomes and sister chromatid separation (Uhlmann et al., 1999). The S. pombe rec8 gene is a meiosis-specific paralogue of rad21 first identified in a screen for mutants with reduced recombination (Ponticelli and Smith, 1989). In meiosis, Scc1p/Mcd1p-containing cohesins dissociate from chromosomes at anaphase I, whereas centromeric Rec8p-containing cohesins persist until the onset of anaphase II (Klein et al., 1999, Watanabe and Nurse, 1999). Thus, meiotic cohesin dynamics can account for the sequential loss of chromatid arm and centromeric cohesion in the two meiotic divisions.

Rad21p/Rec8p homologues have been identified from a number of higher eukaryotic species (McKay et al., 1996, Parisi et al., 1999), and studies in Xenopus and mice indicate that cohesins are regulated differently in metazoa (Darwiche et al., 1999, Losada et al., 1998). Vertebrate cohesins remain at relatively constant levels throughout the cell cycle and display an unexpected cell-cycle-dependent localization pattern. They associate with chromosomes from telophase until prometaphase, then dissocciate and redistribute into the whole cell volume during metaphase. The proteolytic cleavage sites, identified in Mcd1p/Scc1p and conserved among Rad21p- and Rec8p-homologues in lower eukaryotes (Uhlmann et al., 1999), are not present in the vertebrate proteins. It is presently unclear how cohesion is maintained in vertebrates immediately prior to the metaphase–anaphase transition as vertebrate cohesins appear not to associate with condensed chromosomes in metaphase. To further investigate the regulation and cell-cycle dynamics of metazoan cohesin complexes in a genetically tractable organism, we have cloned and commenced characterization of the Drosophila homologue of the rad21 cohesin subunit, which we call Drad21.

Section snippets

Molecular techniques

All molecular techniques were performed using standard procedures (Sambrook et al., 1989) unless indicated otherwise. Genomic clones were isolated from an EMBL3 SP6/T7 Drosophila genomic library (Clontech). Genomic DNA was prepared as described by Bender et al. (1983). Total RNA was blotted to charged nylon membrane (GeneScreen Plus, NEN) and hybridized in accordance with the manufacturer's recommendations with full-length Drad21 cDNA labelled with 32P using random primers. Southern blot

Identification of Drad21

The Drad21 gene was initially identified by homology searches of the Berkeley Drosophila Genome Project (BDGP) EST database (http://www.fruitfly.org) using the BLAST algorithm. The full-length Drad21 cDNA was obtained by sequencing two EST clones (LD02527 and LD09474), which, apart from differing in length by 3 bp, were otherwise identical (Fig. 1). Subsequently, two additional full-length EST sequences have been deposited in GenBank, which are >99.5% identical to our cDNA data.

Conceptual

Discussion

rad21- and rec8-homologues have been identified in a number of eukaryotes ranging from yeasts to mammals. We have identified a Drosophila gene, Drad21, belonging to the Rad21p/Rec8p family of cohesin subunits. DRAD21 is more similar to vertebrate Rad21p-related proteins than those from lower eukaryotes and, based on sequence analysis and expression, belongs to the rad21 (mitosis-specific) subfamily rather than the rec8 (meiosis-specific) subfamily. Although we cannot rule out the possiblity

Acknowledgements

We thank Bert Collard for assistance with genomic clones, Marie Phillips for assistance with chromosome in-situ hybridization and Margarete Heck, Claudio Sunkel and Terry Orr-Weaver for open discussions and sharing of results prior to publication. This work was supported in part by a grant from the Australian Research Council to W.D.W.

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    Present address: Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.

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    Present address: Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Victoria 3052, Australia.

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