Positional enhancer-blocking activity of the chicken β-globin insulator in transiently transfected cells

It is thought that insulators demarcate transcriptionally and structurally independent chromatin domains. Insulators are detected by their ability to block enhancer–promoter interactions in a directional manner, and protect a transgene from position effects. Most studies are performed in stably transformed cells or organisms. Here we analyze the enhancer-blocking activity of the chicken β-globin insulator in transient transfection experiments in both erythroid and nonerythroid cell lines. We show that four tandem copies of a 90-bp fragment of this insulator were able to block an enhancer in these experiments. In circular plasmids, placement on either side of the enhancer reduced activity, but when the plasmid was linearized, the enhancer-blocking activity was observed only when the insulator was placed between the promoter and the enhancer. These observations are consistent with the position-dependent enhancer-blocking activity of the insulator observed in stable transformation experiments.

Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila.

Boundary or insulator elements set up independent territories of gene activity by establishing higher order domains of chromatin structure. The gypsy retrotransposon of Drosophila contains an insulator element that represses enhancer-promoter interactions and is responsible for the mutant phenotypes caused by insertion of this element. The gypsy insulator inhibits the interaction of promoter-distal enhancers with the transcription complex without affecting the functionality of promoter-proximal enhancers; in addition, these sequences can buffer a transgene from chromosomal position effects. Two proteins have been identified that bind gypsy insulator sequences and are responsible for their effects on transcription. The suppressor of Hairy-wing [su(Hw)] protein affects enhancer function both upstream and downstream of its binding site by causing a silencing effect similar to that of heterochromatin. The modifier of mdg4 [mod(mdg4)] protein interacts with su(Hw) to transform this bi-directional repression into the polar effect characteristic of insulators. These effects seem to be modulated by changes in chromatin structure.

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.