Both the 68-base 5' leader (f0) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within 0 is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to 0 than to the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between Q and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation. colinear with the genomic mRNA at the 3' terminus; consequently, all TMV mRNAs contain the same 205-base 3' UTR. The TMV 3' UTR is the functional equivalent to a poly(A) tail in that it promotes efficient translation and increases mRNA stability of chimeric mRNA constructs (12). The 3' UTR is composed of two domains. Located at the 3' terminus, a 105-base tRNA-like domain mimics the three-dimensional structure of true tRNAs to such an extent that many tRNA-specific enzymes will also recognize and modify it (reviewed in references 20 and 21). Immediately upstream of the tRNA-like domain is a 72-base domain composed solely of three RNA pseudoknots (40). This upstream pseudoknot domain (UPD) is responsible for the regulation of translation associated with the TMV 3' UTR (12). The fact that the tRNA-like domain, which itself contains two RNA pseudoknots, does not increase translational efficiency (12) demonstrates that the mere presence of RNA pseudoknots within the 3' UTR is not the basis for enhanced translation.To precisely determine the sequences within the UPD that are essential for the regulation of translation, mutations were made throughout the region and their effects on translation of chimeric mRNA constructs were measured in vivo. The 5'-proximal pseudoknot was not required for UPD function. The secondary and tertiary structure and primary sequence at several positions within the 3'-proximal pseudoknot were critical for regulatory function. The higher-order structure of the middle pseudoknot of the UPD may be needed as well. We also demonstrate that protein-binding activity from both wheat germ and carrot extracts specifically recognizes the UPD. The same protein activity also recognized ...

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The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP sc , a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP sc was detected in the blood of 55% of scrapie agent-infected animals (n ‫؍‬ 80) and 71% of animals with bovine spongiform encephalopathy (n ‫؍‬ 7). PrP sc was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP sc is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.