An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression inChlamydomonas reinhardtii

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular algaChlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show thatChlamydomonasVIG1, an ortholog of theDrosophila melanogasterVasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. AVIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.

Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle

Cardiac muscle expresses sarcoplasmic/endoplasmic-reticulum Ca2+ pump isoform SERCA2a; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100–200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (mean±S.E.M.) were (29±4)-fold higher in the heart. In both tissues the SERCA2 mRNA was associated with polyribosomes. In a sucrose-density-gradient sedimentation velocity experiment on polyribosomes, there was no difference in the sedimentation pattern of SERCA2 mRNA between the two tissues, suggesting that the translation efficiency of SERCA2 RNA in the two tissues is quite similar. Thus the main difference in the control of SERCA2 expression in the two tissues is post-transcriptional and pretranslational.

Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein

ABSTRACT Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.

Isolation of plasma membrane fractions from the intestinal epithelial model T84

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.

Solubilization, characterization, and detergent interactions of lymphocyte 5′-nucleotidase

5′-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5′-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5′-nucleotidase showed "break points" for all detergents in the temperature range 30–37 °C. SDS-PAGE of pure 5′-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200–630 kDa, suggesting that lymphocyte 5′-nucleotidase may be present in high molecular mass aggregates in its native state.Key words: 5′-nucleotidase, plasma membrane, detergents, solubilization, stability, activation energy.

Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.

Biochemical characterization of an aryl acetic ester hydrolase isolated from human monocytes.

A carboxylic-ester hydrolase was isolated from the leukocytes of a patient with myelomonocytic leukemia. Its relative molecular mass as estimated by sucrose density-gradient sedimentation is about 70 000. The purified enzyme is specific for acetyl esters of aromatic alcohols. It is inhibited by fluoride, but insensitive to eserine or p-chloromercuriphenylsulfonate. Hydrolysis of 1-naphthyl acetate was optimal above pH 6.0; of o-nitrophenyl acetate, above 8.0. The common catalytic site for the two types of substrates on the enzyme was confirmed by competitive inhibition data.