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.

We have performed single-molecule studies of GFP-LacI repressor proteins bound to bacteriophage DNA containing a 256 tandem lac operator insertion confined in nanochannels. An integrated photon molecular counting method was developed to determine the number of proteins bound to DNA. By using this method, we determined the saturated mean occupancy of the 256 tandem lac operators to be 13, which constitutes only 2.5% of the available sites. This low occupancy level suggests that the repressors influence each other even when they are widely separated, at distances on the order of 200 nm, or several DNA persistence lengths.binding coefficient ͉ multiple sites ͉ transcription factors O ne of the great challenges in protein-DNA interaction studies is to obtain information from single protein-DNA molecules, rather than ensembles of millions of molecules. To understand protein-DNA interactions at the single-molecule level, single protein molecules must be imaged with high resolution to resolve two key fundamental issues: the number of bound proteins and their locations, which requires that the DNA be extended in a linear manner. We have developed an integrated photon molecular counting (IPMC) method to determine the number of proteins bound to DNA and a platform incorporating nanofluidic channels and total internal reflection fluorescence (TIRF) microscopy to determine the location of the lactose repressor protein (LacI) bound to DNA carrying multiple tandem lac operator (lacO) insertions.Since its use by Jacob and Monod (1) in Escherichia coli as the fundamental model for gene regulation, the LacI repressor has been one of the most intensively studied proteins in molecular biology (2-4). LacI normally exists as a tetramer consisting of two tightly coupled dimers with a dimer-dimer dissociation constant K d of Ϸ10 Ϫ13 M (5). Because, in principle, tetramers can bind to two remote sites and, thus, form protein-DNA complexes, in this experiment we worked with LacI monomers to avoid aggregation problems. LacI monomers bind strongly to lacO sequences (K d Ϸ 10 Ϫ10 M; ref. 6), and the expected off rate k off is on the order of hours (6). This slow rate allows us ample time for observation of the repressor-DNA complexes. We used a DNA construct containing 256 contiguous copies of the lacO target sequence (7,8) (Fig. 1a). Each binding unit consists of a palindromic 21-bp lacO binding site, followed by a 15-bp nonspecific sequence. Because of this 15-bp spacer, we might expect that at the 60 nM monomer protein concentration used in our experiments, all 256 sites would be occupied by two GFP-LacI monomers (Fig. 1b), and we might expect no steric interaction between the contiguously bound GFPLacI molecules. As described below, this is not the case (Fig. 1c).The key idea of the IPMC method is that a fluorescent molecule will emit a mean number of photons N 0 before it irreversibly bleaches. If the emitted number of photons N t from an illuminated region is integrated until the region is bleached, the total number of molecules tha...

One of the great advantages of Drosophila melanogaster as model organism is the availability of balancer chromosomes. These chromosomes suppress recombination with their homologues, allowing the maintenance of lethal and sterile mutants as balanced heterozygotes. All balancers carry dominant markers that are visible in adult flies, but only a subset have markers that unambiguously distinguish homozygous mutant larvae from their heterozygous siblings. The latter balancers include those that express high levels of the GFP protein under the indirect control, via the UAS/GAL4 system, of either the Kruppel (Kr), 1 or hsp70, 2 promoter. In addition, there are direct-drive balancers that express GFP under the control of the actin A5c promoter 3 or YFP under the control of Deformed (Dfd HZ2.7rev) or glass (GMR) enhancer elements. 4 Each of the GFP-or YFP-expressing balancers has specific advantages, but all share a common drawback: These balancers require the use of a dissecting microscope equipped with an UV light source, which for reliable fluorescence detection is preferably used in the dark.The TM6B balancer 5 carries the Tb 1 dominant mutation, which results in squat larvae and pupae. We have been using this balancer for many years to unambiguously distinguish homozygous mitotic mutants dying at late larval stages from their heterozygous siblings (reviewed in ref. 6). This balancer proved particularly useful when mutant larvae are rare and one has to examine several vials (or bottles) to find third instar larvae suitable for dissection and cytological analysis. To generate new tools for easy detection of larvae homozygous for lethal mutations on the X or the second chromosome, we decided to generate males to w/w females (TMS is a Δ2-3-bearing third chromosome balancer expressing the P-element transposase described in ref. 10); from approximately 1,000 progeny we recovered two FM7 chromosomes that co-segregated with Tb (we propose to name these balancers FM7-TbA and FM7-TbB). To generate a second chromosome balancer with a P{Tb 1 } insertion we used a CyO balancer bearing the additional markers S and bw 1 (designated as CyO-Sbw in FlyBase). We crossed w/w; P{Tb 1 }/CyOSbw; TMS/+ females to w; CyO/Sco males and recovered three CyO-Sbw-P{Tb 1 } chromosomes from approximately 1,000 progeny (we propose to name these balancers CyO-TbA, CyO-TbB and CyO-TbC). To assess the utility of these FM7a and CyO Tb-bearing balancers, we compared their Tb phenotype with the Tb 1 mutant phenotype associated with TM6B. 5 To quantify the squat phenotype elicited by the balancers we measured the axial ratio 11 of larvae and pupae (AR, length/width) heterozygous for each balancer. It has been previously shown that the AR does not depend on larval and pupal size and provides a reliable measure of the Tb phenotype.9 As shown in figure 1, the ARs observed in FM7-TbA, FM7-TbB, CyO-TbA, CyO-TbB, CyOTbC and TM6B heterozygotes are fully comparable and significantly different from those of wild-type or non-Tb-bearing larvae and pupae. We thus co...

Genes subject to genomic imprinting exist in large chromosomal domains, probably reflecting coordinate regulation of the genes within a cluster. Such regulation has been demonstrated for the H19,Igf2, and Ins2 genes that share a bifunctional imprinting control region. We have identified the Dlk1 gene as a new imprinted gene that is paternally expressed. Furthermore, we show that Dlk1 is tightly linked to the maternally expressedGtl2 gene. Dlk1 and Gtl2 are coexpressed and respond in a reciprocal manner to loss of DNA methylation. These genes are likely to represent a new example of coordinated imprinting of linked genes.

The mouse Fused locus encodes a protein that has been implicated in the regulation of embryonic axis formation. The protein, which has been named Axin to distinguish it from the product of the unrelated Drosophila melanogaster gene fused, contains regions of similarity to the RGS (regulators of G-protein signaling) family of proteins as well as to dishevelled, a protein that acts downstream of Wingless in D. melanogaster. Loss-of-function mutations at Fused lead to lethality between days 8 and 10 of gestation. Three dominant mutations result in a kinked tail in heterozygotes. Two of the dominant mutations, Fused and Knobbly, result from insertions of intracisternal A particle retrotransposons into the gene. The insertion in Fused, within the sixth intron, creates a gene that produces wild-type transcripts as well as mutant transcripts that initiate at both the authentic promoter and the 3′-most long terminal repeat of the insertion. Knobbly, an insertion of the retrotransposon into exon 7, precludes the production of wild-type protein. Thus the Fused homozygote is viable whereas Knobbly is a recessive embryonic lethal. In both mutants the dominant kink-tailed phenotype is likely to result from the synthesis of similar amino-terminal fragments of Axin protein that would contain the RGS domain, but lack the dishevelled domain.