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.     

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.     

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.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.