Light regulation of the abundance of mRNA encoding a nucleolin-like protein localized in the nucleoli of pea nuclei.

A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced. The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%). It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain. By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA. In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found. Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size and the cDNA. After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h. Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control. This indicates that phytochrome may regulate the expression of this gene.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.     

Host factor for coliphage Qbeta RNA replication is present in Pseudomonas putida.

Summary Host Factor (HF)¹, is a 12000 molecular weight polypeptide that is found in uninfected Escherichia coli and is required as a hexamer along with Q replicase for in vitro replication of Q phage RNA. It has recently been found to be associated with ribosomes and to bind tightly to poly(A).We report here the identification and purification of HF from Pseudomonas putida. HF can be detected in crude extracts by both functional activity in the Q RNA replication assay and by immunodiffusion with antibody made against E. coli HF. HF from E. coli and P. putida chromatograph similarly on DEAE-cellulose and phosphocellulose. They have similar but not identical molecular weights as judged by SDS-polyacrylamide gel electrophoresis. Like E. coli HF, P. putida HF was found to be associated with ribosomes and to bind tightly to poly (A). Furthermore, the pure protein from P. putida has full functional activity in the in vitro Q RNA replication assay.The findings that HF has been conserved during evolution, is associated with ribosomes, and binds poly(A), suggest that HF may be an important translational element in uninfected cells and that its role involves an interaction with RNA.Research supported by National Institutes of Health Grant GM 21024.     

pH-dependent fusion of vesicular stomatitis virus with Vero cells. Measurement by dequenching of octadecyl rhodamine fluorescence

We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.     

Amino acid sequence of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of the amino-terminal, 235-residue segment of rabbit skeletal muscle myosin light chain kinase has been determined. Together with the carboxyl-terminal segment previously described [Takio, K., Blumenthal, D. K., Edelman, A. M., Walsh, K. A., Krebs, E. G., & Titani, K. (1985) Biochemistry 24, 6028], the present work completes the 603-residue sequence of this protein. The amino-terminal segment that has been analyzed herein corresponds to a domain reported to be of highly asymmetrical shape and as yet unknown function. Secondary structure calculations failed to provide any evidence of alpha-helix or beta-structures, but polyproline II like helical structure is possible. Sequence analysis indicates the presence of approximately equal quantities of two isoforms differing in a single amino acid replacement. Unexpected difficulties were encountered in the present sequence analysis due to the presence of acid-labile Asp-Pro bonds and to five separable variants of a blocked 21-residue amino-terminal peptide, arising from rearrangement at an Asn-Gly bond.     

Coordinate production by a T cell hybridoma of gamma interferon and three other lymphokine activities: multiple activities of a single lymphokine?

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.