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V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 and RAG2 proteins was previously shown to demand only a single signal sequence. Here, we establish conditions where 12- and 23-spacer signal sequences are both necessary for cleavage. Coupled cutting at both sites requires only the RAG1 and RAG2 proteins, but depends on the metal...
S. cerevisiae inherits its vacuole by projecting vacuole-derived membrane vesicles and tubules into the bud, where they fuse to establish the daughter vacuole. This homotypic fusion event can be assayed in vitro. It requires Sec17p and Sec18p, the homologs of the mammalian α-SNAP and NSF, which cooperate in multiple steps of membrane trafficking. We now report that Sec17p, Sec18p, and ATP are only...
Telomeres, the nucleoprotein complexes at the ends of eukaryotic chromosomes, are essential for chromosome stability. In the yeast S. cerevisiae, telomeric DNA is bound in a sequence-specific manner by RAP1, a multifunctional protein also involved in transcriptional regulation. Here we report the crystal structure of the DNA-binding domain of RAP1 in complex with a telomeric DNA site at 2.25 Å resolution...
The cell cycle inhibitor p16 INK4a is inactivated in many human tumors and in families with hereditary melanoma and pancreatic cancer. Tumor-associated alterations in the INK4a locus may also affect the overlapping gene encoding p19 ARF and the adjacent gene encoding p15 INK4b , both negative regulators of cell proliferation...
To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic...
In Drosophila imaginal wing discs, the achaete-scute (ac-sc) proneural genes and rhomboid (veinlet) are expressed in highly resolved patterns that prefigure the positions of sensory organs and wing veins, respectively. It is thought that these patterns are generated by a combination of factors (a prepattern) regulating these genes. We provide evidence for the existence of this prepattern by identifying...
Using a yeast one-hybrid screen with a DNA-bound Ets-1 protein, we have identified MafB, an AP-1 like protein, as a direct interaction partner. MafB is specifically expressed in myelomonocytic cells and binds to the DNA-binding domain of Ets-1 via its basic region or leucine-zipper domain. Furthermore, it represses Ets-1 transactivation of synthetic promoters containing Ets binding sites and inhibits...
A yeast gene homologous to bacterial RNase III (RNT1) encodes a double-strand-specific endoribonuclease essential for ribosome synthesis. Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent A0 site in the 5' ETS and cleavage in the 3' ETS. Recombinant RNT1 protein accurately cleaves a synthetic 5' ETS RNA at the A0 site in vitro, in the...
CDC16 and CDC27 were identified as genes in S. cerevisiae necessary to limit DNA replication to once per cell cycle. A screen for mutants that overreplicated their DNA uncovered new conditional alleles that cause accumulation of up to 8C DNA. DNA overreplication involves all chromosomes and does not require passage through mitosis or another START. It occurs within a single cell cycle and can cause...
E. coli preprotein translocase comprises SecA and a SecY/E/G complex. SecA delivers the preprotein to the putative protein-conducting channel formed by SecY/E by undergoing ATP-driven cycles of membrane insertion and deinsertion. SecG renders the translocase highly efficient. An antibody raised against the C-terminal region of SecG inhibits preprotein translocation into everted membrane vesicles despite...
Expression of a constitutively active R-ras converted two cell lines that grow in suspension into highly adherent cells. There was little change in cell surface expression of integrins, but attachment to surfaces coated with the integrin ligands was greatly enhanced. Cells transfected with activated R-ras bound integrin ligands from solution with higher affinities and assembled severalfold more fibronectin...
Operation of the secretory pathway in eukaryotic cells requires the selective docking and fusion of transport vesicles with the appropriate target organelle. This is mediated in part by integral membrane proteins termed v-SNAREs (on vesicles) and t-SNAREs (on the target membranes). We describe a novel yeast t-SNARE that resides on the endoplasmic reticulum and mediates retrograde traffic from the...
VP39 is a bifunctional vaccinia virus protein that acts as both an mRNA cap–specific RNA 2′-O-methyltransferase and a poly(A) polymerase processivity factor. Here, we report the 1.85 Å crystal structure of a VP39 variant complexed with its AdoMet cofactor. VP39 comprises a single core domain with structural similarity to the catalytic domains of other methyltransferases. Surface features and mutagenesis...
The KH module is a sequence motif found in a number of proteins that are known to be in close association with RNA. Experimental evidence suggests a direct involvement of KH in RNA binding. The human FMR1 protein, which has two KH domains, is associated with fragile X syndrome, the most common inherited cause of mental retardation. Here we present the three-dimensional solution structure of the KH...
The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90)...
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