Characterization of null and hypomorphic alleles of the Drosophilal(2)dtl/cdt2gene

Abstract

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that "denticleless" is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.

Identification of Male Gametogenesis Expressed Genes from the Scallop Nodipecten subnodosus by Suppressive Subtraction Hybridization and Pyrosequencing

“…Another meiotic checkpoint gene, denticleless protein homolog ( denticleless E3 ubiquitin protein ligase homolog ), or cdt2 in yeast, is known to control re-replication during the S-phase by promoting degradation of a protein required for pre-replication complex formation: CDT1 (chromatin licensing and DNA replication factor1). But it is also known as an essential component of the G2/M checkpoint in vertebrates [87], in agreement with this gene being overexpressed in the developing gonads of Drosophila [88].…”

Behavior of replication origins in Eukaryota – spatio-temporal dynamics of licensing and firing

“…49 Similar down regulation also occurs in Drosophila for its Cdt1 homolog -DUP gene. 50 CDKs in general prevent re-replication inhibiting pre-RC components. 39,40,42,51 An active Cdc6 protein is present in cells only during early G1 phase and is rapidly degraded from late G1 to S phase.…”

Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication

“…BrdU labeling of embryos was performed as previously described (Sloan et al, 2012). For co-detection of S phase and Dap, embryos were fixed for 15 min in 3.7% formaldehyde after antibody staining prior to BrdU detection.…”