BIOLOGICAL OVERVIEW

The cramped (crm) gene of Drosophila can be classified as a Polycomb-group (Pc-G) gene. crm mutants exhibit typical Pc-G mutant phenotypes, reminiscent of ectopic homeotic gene expression, with additional sex comb teeth found on the mesothoracic and metathoracic legs, and proximodistal transformations of the tarsal segments. Mutants show a typical altered morphology of the antennae: swollen antennae, short aristae with fewer branches than in wild type (Yamamoto, 1997 and references).

Staining of Crm protein indicates a cell-cycle-dependent localization: for example, other Polycomb-group proteins, such as Polycomb and Posterior sex-combs remain associated with condensed chromatin during mitosis. In contrast, Crm disappears from the cell in prophase, after the nuclear envelope breakdown occurs. This cell cycle dependent localization for Crm staining is particularly evident during the first 13 zygotic division cycles, a time when cells divide synchronously. Crm immunostaining reappears at telophase, concomitant with formation of two daughter nuclei. By the time of gastrulation, mitosis is no longer synchronous, with groups of cells (the so-called mitotic domains) entering mitosis at different times. This results in precise and reproducible patterned mitosis replacing the prior global mitotic waves. Cell cycle-dependent nuclear localization of Crm protein can be seen in single embryos at this stage, with cells in interphase staining strongly while cells in metaphase show no Crm immunostaining. It is concluded that Crm protein localizes to the nucleus mainly during S-phase in early embryogeneisis and is not associated with chromatin during mitosis (Yamamoto, 1997).

Several lines of evidence tie Cramped to Proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and repair. PCNA, together with Replication factor C (RFC), function as processivity factors for DNA polymerases delta and epsilon. DNA polymerase delta is the functional DNA polymerase on the leading strand of the eucaryotic DNA replication fork. RFC carries out multiple activities, including the ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template allowing processive DNA chain elongation (Uhlmann, 1997 and references). The S-phase-specific nuclear localization of Crm is reminiscent of PCNA. During the first 13 nuclear division cycles, PCNA is present in all interphase nuclei and absent from metaphase chromosomes. Double immunostaining of Crm and PCNA reveals that the timing of the appearance and disappearance of the nuclear signal is identical for both proteins during the preblastoderm cycles; by gastrulation, both proteins show overlapping patterns of expression. These two proteins are clearly observed in polytene tissues, and their staining pattens overlap (Yamamoto, 1997).

cramped genetically interacts with mus209, the gene that codes for PCNA. In trans-heterozygotes, a strong enhancement of both posterior-to-anterior and distal-to proximal crm transformation phenotypes takes place. crm mutation also suppresses position effect variagation (PEV), the variation of expression of a gene depending on its chromosomal position. This suppression of PEV, along with the phenotypic effects of crm mutation, allies Crm with the Polycomb-group (Yamamoto, 1997).

The evidence supports a possible interaction between Pc-G mediated silencing (through CRM) and DNA replication (crm interaction with mus209) in Drosophila. Evidence for the involvement of DNA replication in silencing comes from studies of silencing of the mating-type loci in the budding yeast Saccharomyces cerevisiae. The silencer elements flanking the silent HMR and HML loci have the ability to allow autonomous replication of plasmids. Proteins of the Origin recognition complex 2 (ORC) are required for both silencing and DNA replication. Whether Crm might be involved in some aspects of DNA replication is still unknown. Nevertheless, preliminary data suggest that the Crm protein partially co-localizes with sites of DNA replication, visualized by BrdU staining in early embryos. Since heterochromatin is known to be late replicating, and Crm and PCNA are implicated in chromatin dynamics by virtue of the ability of crm and mus209 mutations to suppress PEV, it can be argued that interactions of Crm and PCNA regulate the timing of replication of repressed genes (Yamamoto, 1997).

GENE STRUCTURE

Bases in 5' UTR - 76

Bases in 3' UTR - 1622

PROTEIN STRUCTURE

Amino Acids - 693

Structural Domains

Crm is a novel protein. PEST sequences, thought to be involved in protein degradation, are present in the N-terminal region and the central region. There are three putative nuclear localization signals and several consensus sites for phosphorylation by MAP kinase, cyclin dependent kinases and casein kinase II. The C-terminal region is rich in serine and threonine residues. Crm contains a region with high density of alanine residues, with a stretch of 36 residues containing 14 alanines. Alanine-rich regions are thought to be involved in transcriptional repression (Yamamoto, 1997).

date revised: 20 October 97

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