The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

A second type of protein methylation reaction in bacterial chemotaxis

CheZ is the product of one of six genes required for sensory processing in Escherichia coli and Salmonella typhimurium chemotaxis. This 24-kDa cytoplasmic protein is modified by a posttranslational methylation reaction. The modified residue has been identified by analysis of radioactively labeled protein from two-dimensional electrophoretograms and Edman degradation of CheZ protein isolated by immunoaffinity chromatography using anti-CheZ monoclonal antibodies. The methylated group is an N-monomethylmethionine residue at the amino terminus of CheZ. L16, a ribosomal protein that is required for peptidyltransferase activity during protein synthesis, is also methylated at its amino-terminal methionine (Chen, R., Brosius, J., and Wittmann-Liebold, B. (1977) J. Mol. Biol. 111, 173-181). Homologous sequences at the amino termini of L16 and CheZ raise the possibility that a single S-adenosylmethionine-dependent methyltransferase modifies both proteins.     

Interaction of the 4 S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene

The induction of cytochrome P-450c, the isozyme most closely associated with aryl hydrocarbon hydroxylase activity in the rat, is mediated through a cytosolic polycyclic aromatic hydrocarbon (PAH)-binding protein(s). We have reported on the purification and characterization of a 4 S protein that interacts in a specific and saturable manner with [3H]benzo[a]pyrene and other PAHs. (W. H. Houser et al. (1985) Biochemistry 24, 7839-7845). We have also reported on the specific and saturable interaction of the 4 S protein with a plasmid containing 1.9 kbp of cloned rat P-450c sequence including exon 1, the 5' half of intron 1, and approximately 882 bp upstream information. Our investigations now show that incubation of this protein with a portion of the rat P-450c gene, followed by digestion with either lambda exonuclease or exonuclease III, tentatively identified two protected regions at -225 and -455 bp 5' from exon 1. To further study the significance of these protected regions, a 3.4-kbp fragment containing cytochrome P-450c promoter and 5'-upstream sequences (-882 to +2545) was fused to the chloramphenicol acetyl transferase (CAT) reporter gene and transfected into either rat epithelial RL-PR-C cells or rat hepatoma H-4-II-E cells. Both cell lines expressed CAT activity in response to induction by 3-methylcholanthrene (3MC), indicating that important regulatory regions responsive to 3MC are present in these constructs. However, neither cell line expressed CAT activity in response to 3MC when transfected with plasmids containing deletions (-95 to -724 or -240 to -720) in the regions shown to be protected by our footprinting studies. These results corroborate previous studies which indicated that the 4 S PAH-binding protein interacts in a specific manner with regions of the rat cytochrome P-450c gene. We conclude that the 4 S protein may play an important role in the regulation of expression of cytochrome P-450c in the rat.     

Extraction of monocrotaline from Crotalaria spectabilis using supercritical carbon dioxide and carbon dioxide-ethanol mixtures

Monocrotaline, a pyrrolizidine alkaloid of chemotherapeutic interest, was successfully extracted from the crushed seeds of Crotalaria spectabilis using supercritical carbon dioxide and ethanol mixtures. Overall solubilities of the plant material in the supercritical fluid phase were as high as 1.1 mass percent, and monocrotaline solubilities were as high as 0.07 mass percent. The solubility of monocrotaline in the presence of other plant material was smaller by 50 to 98% compared with the solubility of pure monocrotaline in the supercritical fluid. Also, it was found that the extraction of the complex plant material was time-dependent after approximately one percent of the original mass of the material had been extracted.     

Translational modification of an 18 kilodalton polypeptide by spermidine in rice cell suspension cultures

When rice (Oryza sativa) cell suspension cultures are grown in the presence of [terminal methylenes-³H]spermidine, label is incorporated in a single polypeptide with a molecular mass of 18 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Preincubation of cell cultures with polyamine biosynthesis inhibitors difluoromethylarginine and difluoromethylornithine, resulted in increased incorporation of the label into the 18 kilodalton polypeptide. In cells in which protein synthesis was arrested by cycloheximide, no label was detected in the 18 kilodalton polypeptide, suggesting a requirement for de novo protein synthesis.     

Identification and localization of pre-s-encoded polypeptides from woodchuck and ground squirrel hepatitis viruses.

A segment from the pre-s region of the woodchuck hepatitis virus (WHV) was inserted into an open reading frame vector allowing for the expression in Escherichia coli of viral determinants as part of a fusion protein. The bacterially synthesized fusion molecule contained eight amino acids from beta-galactosidase (beta-gal) at the N terminus, followed by 89 pre-s-encoded amino acids and 219 amino acids of chloramphenicol acetyltransferase (CAT) at the C terminus (beta-gal:pre-s:CAT). This tribrid protein was used to generate antiserum which had a significant titer to the viral portion of the fusion polypeptide. Anti-beta-gal:pre-s:CAT was used in Western blot analysis to identify viral proteins containing pre-s-encoded determinants. Antiserum to the tribrid molecule recognized four WHV polypeptides with molecular masses of 33, 36, 45, and 47 kilodaltons, each of which was also recognized by a monoclonal antibody to WHV surface antigen. Using the same anti-tribrid serum, we also identified analogous polypeptides from ground squirrel hepatitis virus. The antiserum was also used to immunoprecipitate virus particles containing endogenous DNA polymerase activity, indicating that pre-s determinants are found on the surface of mature virions. Based on previous computer studies and the location of pre-s-encoded molecules on the surface of virus particles, a role in hepadnavirus host cell entry is suggested for these polypeptides.     

Cytoplasmic suppression of malignancy

Summary Using both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished. This work was supported in part by United States Public Health Service grant CA12056, and grant CA09100 from the National Cancer Institute, Bethesda, MD. This work is partial fulfillment for the degree of Doctor of Philosophy for B.A.I.     

Anther and tissue culture-induced grain chalkiness and associated variants in rice

Rice, Oryza sativa, plants regenerated from anther culture with and without in vitro selection pressure were evaluated for chalky seed. Progeny evaluated included 21 spontaneously doubled haploids selfed 4 times, progeny from plants regenerated from S-aminoethylcysteine resistant callus selfed 4 times and backcrosses of both types to the parental type. All lines with in vitro histories had higher seed chalkiness than the controls both in the intensity of chalkiness and in the number of seeds expressing the character. The full range of intensity and amount of chalkiness was expressed in the progeny. The average intensity of anther/tissue culture-derived progeny was 4–5, based on a scale of 1 (translucent) to 10 (fully opaque), and the average amount of chalkiness within plants sampled was 50–75 percent. The chalky characteristic is transmitted from parent to offspring into a range of identifiable F₂ segregants. Disclaimer statement Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.     

Interference by Trypsin in the Interaction of Staphylococcal Enterotoxin B and Cell Cultures of Human Embryonic Intestine

The inhibitory effect of trypsin on the cytotoxicity of staphylococcal enterotoxin B acting with human embryonic intestine cell cultures was examined. Trypsin treatment of the cells rendered them resistant to enterotoxin for a period of 48 hr. The resistance increased proportionally with increased time of exposure of the cells to trypsin. Neither ethylenediaminetetraacetic acid nor scraping, which were used as alternate means of cell suspension, caused any resistance to the toxin. The effect is enzymatic and appears to be similar to the inhibitory action of trypsin and chymotrypsin on the attachment of polioviruses and coxsackieviruses to HeLa cells.     

Partial Purification and Characterization of the Vacuolar H+-ATPase of Mammalian Synaptic Vesicles

Several major proteins of synaptic vesicles from rat or cow brain sediment as a large complex on sucrose density gradients when solubilized in nonionic detergents. A vacuolar H(+)-ATPase identified by sensitivity to bafilomycin A1 appears to be associated with this oligomeric protein complex. Two subunits of this complex, synaptic vesicle proteins S and U, correspond to the 57-kDa (B) and 39-kDa accessory (Ac39) subunits, respectively, of bovine chromaffin granule vacuolar H(+)-ATPase as shown by Western immunoblot analysis. The five subunits of the oligomeric complex constitute approximately 20% of the total protein of rat brain synaptic vesicles. Taken together, these results strongly suggest that the abundant, multisubunit complex partially purified from brain synaptic vesicles by density gradient centrifugation is a vacuolar H(+)-ATPase. Bafilomycin A1 completely blocks proton pumping in rat brain synaptic vesicles as measured by [14C]methylamine uptake and also blocks catecholamine accumulation measured by [3H]dopamine uptake. Moreover, ATPase activity, [14C]methylamine uptake, and [3H]dopamine uptake are inhibited by bafilomycin A1 at similar I50 values of approximately 1.7 nmol/mg of protein. These findings indicate that the vacuolar H(+)-ATPase is essential for proton pumping as well as catecholamine uptake by mammalian synaptic vesicles.