Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins.

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57 Kip2 , as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57Kip2 during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57 Kip2, and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57Kip2 have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57Kip2 is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).

Body shape determination represents a critical aspect of morphogenesis. In the course of investigating body shape regulation in Drosophila, we have identified a dominant mutation, TweedleD 1 (TwdlD 1 ), that alters overall dimensions at the larval and pupal stages. Characterization of the affected locus led to the discovery of a gene family that has 27 members in Drosophila and is found only among insects. Analysis of gene expression at the RNA and protein levels revealed gene-specific temporal and spatial patterns in ectodermally derived tissues. In addition, light microscopic studies of fluorescently tagged proteins demonstrated that Tweedle proteins are incorporated into larval cuticular structures. This demonstration that a mutation in a Drosophila cuticular protein gene alters overall morphology confirms a role for the fly exoskeleton in determining body shape. Furthermore, parallels between these findings and studies of cuticle collagen genes in Caenorhabditis elegans suggest that the exoskeleton influences body shape in diverse organisms.arthropod ͉ morphogenesis ͉ tandem duplication ͉ Tubby

Genomic imprinting, the unequal expression of gene alleles on the basis of parent of origin, is a major exception to mendelian laws of inheritance. By maintaining one allele of a gene in a silent state, imprinted genes discard the advantages of diploidy, and for this reason the rationale for the evolution of imprinting has been debated. One explanation is the parent-offspring conflict model, which proposes that imprinting arose in polyandrous mammals as the result of a parental conflict over the allocation of maternal resources to embryos. This theory predicts that there should be no selection for imprinting in a monogamous species. Crosses between the monogamous rodent species Peromyscus polionotus and the polyandrous Peromyscus maniculatus yield progeny with parent-of-origin growth defects that could be explained if imprinting was absent in the monogamous species. We find, however, that imprinting is maintained in P. polionotus, but there is widespread disruption of imprinting in the hybrids. We suggest that the signals governing genomic imprinting are rapidly evolving and that disruptions in the process may contribute to mammalian speciation.