REC8 is a key component of the meiotic cohesin complex. During meiosis, cohesin is required for the establishment and maintenance of sister-chromatid cohesion, for the formation of the synaptonemal complex, and for recombination between homologous chromosomes. We show that REC8 has an essential role in mammalian meiosis, in that Rec8 null mice of both sexes have germ cell failure and are sterile. In the absence of REC8, early chromosome pairing events appear normal, but synapsis occurs in a novel fashion: between sister chromatids. This implies that a major role for REC8 in mammalian meiosis is to limit synapsis to between homologous chromosomes. In all other eukaryotic species studied to date, REC8 phenotypes have been restricted to meiosis. Unexpectedly, Rec8 null mice are born in sub-Mendelian frequencies and fail to thrive. These findings illuminate hitherto unknown REC8 functions in chromosome dynamics during mammalian meiosis and possibly in somatic development.

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were indentified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/− animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/− animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues.

The evolutionarily conserved cohesin complex is required for the establishment and maintenance of sister chromatid cohesion, in turn essential for proper chromosome segregation. RAD21/SCC1 is a regulatory subunit of the mitotic cohesin complex, as it links together all other subunits of the complex. The destruction of RAD21/SCC1 along chromosomal arms and later at centromeres results in the dissociation of the cohesin complex, facilitating chromosome segregation. Here, we report for the first time that mammalian RAD21/SCC1 associates with the axial/lateral elements of the synaptonemal complex along chromosome arms and on centromeres of mouse spermatocytes. Importantly, RAD21/ SCC1 is lost from chromosome arms in late prophase I but persists on centromeres. The loss of centromeric RAD21/SCC1 coincides with the separation of sister chromatids at anaphase II. These findings support a role for mammalian RAD21/SCC1 in maintaining sister chromatid cohesion in meiosis.

The structural maintenance of chromosome (SMC) proteins are required for a number of essential nuclear processes, including those of chromosome condensation, chromatid cohesion, and DNA repair. Eukaryotic SMC proteins form heterodimers capable of binding DNA and possess a DNA-stimulated ATPase activity. They have a characteristic structure of terminal globular domains with two internal arms that are predicted to form a coiled-coil structure interspaced with a globular "hinge" domain. We report here that the predicted coiled-coil arms are disrupted at conserved sites in SMC proteins. These disruptions, which vary in length and sequence identity, abolish the otherwise symmetrical secondary structure of antiparallel SMC heterodimers and provide the first evidence for a possible functional orientation of eukaryotic SMC complexes. The retention of these breaks between evolutionarily distant, yet related, SMC members indicates that they may have a fundamental role in SMC heterodimer function.

Conventional antibody surface display requires fusion protein export through at least one cellular membrane, constraining the yield and occasioning difficulties in achieving scaled production. To circumvent this limitation, we developed a novel cytoplasmic display platform, Retained Display (ReD), and used it to screen for human scFv frameworks that are highly soluble and stable in the bacterial cytoplasm. ReD, based on the retention of high-molecular weight complexes within detergent-permeabilized Escherichia coli, enabled presentation of exogenous targets to antibodies that were expressed and folded in the cytoplasm. All human λ and κ light chain family genes were expressed as IGHV3-23 fusions. Members of the λ subfamilies 1, 3 and 6 were soluble cytoplasmic partners of IGHV3-23. Contrary to previous in vivo screens for soluble reduced scFvs, the pairings identified by ReD were identical to the human germline sequences for the framework, CDR1 and CDR2 regions. Using the most soluble scFv scaffold identified, we demonstrated tolerance to CDR3 diversification and isolated a binding scFv to an exogenous protein target. This screening system has the potential to rapidly produce antibodies to target threats such as emerging infectious diseases and bioterror agents.