The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens

Ehrlichiae are responsible for important tick-transmitted diseases, including anaplasmosis, the most prevalent tick-borne infection of livestock worldwide, and the emerging human diseases monocytic and granulocytic ehrlichiosis. Antigenic variation of major surface proteins is a key feature of these pathogens that allows persistence in the mammalian host, a requisite for subsequent tick transmission. In Anaplasma marginale pseudogenes for two antigenically variable gene families, msp2 and msp3, appear in concert. These pseudogenes can be recombined into the functional expression site to generate new antigenic variants. Coordinated control of the recombination of these genes would allow these two gene families to act synergistically to evade the host immune response.

Functional neuroanatomical double dissociation of mnemonic and executive control processes contributing to working memory performance

We used event-related functional MRI to investigate the neural bases of two categories of mental processes believed to contribute to performance of an alphabetization working memory task: memory storage and memory manipulation. Our delayed-response tasks required memory for the identity and position-in-the-display of items in two- or five-letter memory sets (to identify load-sensitive regions) or memory for the identity and relative position-in-the-alphabet of items in five-letter memory sets (to identify manipulation-sensitive regions). Results revealed voxels in the left perisylvian cortex of five of five subjects showing load sensitivity (as contrasted with alphabetization-sensitive voxels in this region in only one subject) and voxels of dorsolateral prefrontal cortex in all subjects showing alphabetization sensitivity (as contrasted with load-sensitive voxels in this region in two subjects). This double dissociation was reliable at the group level. These data are consistent with the hypothesis that the nonmnemonic executive control processes that can contribute to working memory function are primarily prefrontal cortex-mediated whereas mnemonic processes necessary for working memory storage are primarily posteriorly mediated. More broadly, they support the view that working memory is a faculty that arises from the coordinated interaction of computationally and neuroanatomically dissociable processes.

Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G1 cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB−/−, p107−/−, p130−/−, pRB−/−/p107−/−, pRB−/−/p130−/−, and p107−/−/p130−/−). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G1 and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G1 and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.