We describe a new human isoform, GFAP⑀, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP⑀ mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP⑀ has a new C-terminal protein sequence. This provides GFAP⑀ with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP⑀ is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP⑀ should be taken into consideration when studying neurodegenerative diseases.
Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.
The peptide-chain elongation rate of Saccharomyces cerevisiae at two different growth rates was estimated by the kinetics of radioactive labelling of nascent and finished polypeptides as described by Gausing, 1972, and Young and Bremer, 1976. The elongation rates of a diploid strain cultured in yeast nitrogen base supplemented with glucose or acetate were 9.3 amino acids/s and 5.5 amino acids/s at 30 degrees C, respectively. These data together with published values on the "ribosomal efficency" as a function of growth rate (Waldron and Lacroute, (1975) enable us to estimate the rate of synthesis of ribosomal proteins as a function of the rate of total protein synthesis, alpha r, and the fraction of ribosomes that one active in protein synthesis. We conclude that in S. cerevisiae alpha r is largely independent of the growth rate while the fraction of active ribosomes decreases with decreasing growth rate.
and 1Gesellschaft fir Biotechnologische Forschung (GBF), D-3300 Braunschweig, FRG Communicated by U.K.LaemmliPreviously, we have demonstrated that in Tetrahymena DNA topoisomerase I has a strong preference in situ for a hexadecameric sequence motif AZACTTAGAZAAATTA present in the non-transcribed spacers of r-chromatin. Here we characterize more extensively the interaction of purified topoisomerase I with specific hexadecameric sequences in cloned DNA. Treatment of topoisomerase I-DNA complexes with strong protein denaturants results in single strand breaks and covalent linkage of DNA to the 3' end of the broken strand. By mapping the position of the resulting nicks, we have analysed the sequence-specific interaction of topoisomerase I with the DNA. The experiments demonstrate that: (i) the enzyme cleaves specifically between the sixth and seventh bases in the hexadecameric sequence; (ii) a single base substitution in the recognition sequence may reduce the cleavage extent by 95%; (iii) the sequence specific cleavage is stimulated 8-fold by divalent cations; (iv) 30% of the DNA molecules are cleaved at the hexadecameric sequence while no other cleavages can be detected in the 1.6-kb fragment investigated;(v) the sequence specific cleavage is increased 2-to 3-fold in the presence of the antitumor drug camptothecin; (vi) at high concentrations of topoisomerase I, the cleavage pattern is altered by camptothecin; (vii) the equilibrium dissociation constant for interaction of topoisomerase I and the hexadecameric sequence can be estimated as -10-10 M.
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