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Posttransplantation diabetes mellitus (PTDM) is a major complication in renal transplant recipients. Some studies have demonstrated that tumor necrosis factor alpha (TNF-α) expression and its genetic polymorphism are associated with diabetes mellitus. We investigated this association in Asian renal transplant recipients. Polymerase chain reaction-restriction-fragment length polymorphism was used to measure TNF-α G-238A and G-308A gene polymorphisms among 241 nonposttransplantation diabetic subjects and 73 PTDM patients. PTDM patients showed higher values of body weight and body mass index (BMI) than the non-PTDM group. However, no significant association was observed between TNF-α G-238A and TNF-α G-308A polymorphisms with PTDM incidence, gender, age at transplantation, follow-up duration, BMI, or type of immunosuppression., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Brome mosaic virus (BMV) genomic minus-strand RNA synthesis requires an RNA motif named stem-loop C (SLC). An NMR-derived solution structure of SLC was reported by Kim et al. (Nature Struc Biol, 2000, 7:415-423) to contain three replicase-recognition elements, the most important of which is a stable stem with a terminal trinucleotide loop, 5'AUA3'. The 5'-most adenine of the triloop is rigidly fixed to the stem helix by interactions that require the 3'-most adenine, which is called a clamped adenine motif. However, a change of the 3' adenine to guanine (5'AUG3') unexpectedly directed RNA synthesis at 130% of wild type (Kim et al., Nature Struc Biol, 2000, 7:415-423). To understand how RNA with the AUG mutation maintains interaction with the BMV replicase, we used NMR and other biophysical techniques to elucidate the solution conformation of a 13-nt RNA containing the AUG triloop, called S-AUG. We found that S-AUG has a drastically different loop conformation in comparison to the wild type, as evidenced by an unusual C x G loop-closing base pair. Despite the conformational change, S-AUG maintains a solution-exposed adenine similar to the clamped adenine motif found in the wild type. Biochemical studies of the 5'AUG3' loop with various substitutions in the context of the whole SLC construct confirm that the clamped adenine motif exists in S-AUG remains a primary structural feature required for RNA synthesis by the BMV replicase.

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.

The 3' end of brome mosaic virus RNA contains a tRNA-like sequence that directs its RNA synthesis. A stem loop structure in this sequence, stem loop C (SLC), was investigated using NMR, and correlated with its ability to direct RNA synthesis by its replicase. SLC consists of two discrete domains, a flexible stem with an internal loop and a rigid stem containing a 5'-AUA-3' triloop. Efficient RNA synthesis requires the sequence on only one side of the flexible stem and a specific compact conformation of the triloop. A high resolution structure of the triloop places the 5' adenine out in solution, and the 3' adenine within the triloop, held tightly through stacking and unusual hydrogen bonds. This high resolution structure of an RNA promoter from a (+)-strand RNA virus provides new insights into how the RNA-dependent RNA polymerase binds to the RNA to initiate synthesis.

Viral RNA replication provides a useful system to study the structure and function of RNAs and the mechanism of RNA synthesis from RNA templates. Previously we demonstrated that a 27 nt RNA from brome mosaic virus (BMV) can direct correct initiation of genomic plus-strand RNA synthesis by the BMV replicase. In this study, using biochemical, nuclear magnetic resonance, and thermodynamic analyses, we determined that the secondary structure of this 27 nt RNA can be significantly altered and retain the ability to direct RNA synthesis. In contrast, we find that position-specific changes in the RNA sequence will affect replicase recognition, modulate the polymerization process, and contribute to the differential accumulation of viral RNAs. These functional results are in agreement with the phylogenetic analysis of BMV and related viral sequences and suggest that a similar mechanism of RNA synthesis takes place for members of the alphavirus superfamily., (Copyright 1999 Academic Press.)

The approximately 150 nt tRNA-like structure present at the 3' end of each of the brome mosaic virus (BMV) genomic RNAs is sufficient to direct minus-strand RNA synthesis. RNAs containing mutations in the tRNA-like structure that decrease minus-strand synthesis were tested for their ability to interact with RdRp (RNA-dependent RNA polymerase) using a template competition assay. Mutations that are predicted to disrupt the pseudoknot and stem B1 do not affect the ability of the tRNA-like structure to interact with RdRp. Similarly, the +1 and +2 nucleotides are not required for stable template-RdRp interaction. Mutations in the bulge and hairpin loops of stem C decreased the ability of the tRNA-like structure to interact with RdRp. Furthermore, in the absence of the rest of the BMV tRNA, stem C is able to interact with RdRp. The addition of an accessible initiation sequence containing ACCA3' to stem C created an RNA capable of directing RNA synthesis. Synthesis from this minimal minus-strand template is dependent on sequences in the hairpin and bulged loops., (Copyright 1998 Academic Press.)

RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (-17, -14, -13, and -11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238-11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides -17 and -11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides -11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

Each of the brome mosaic virus (BMV) genomic RNAs contains a conserved tRNA-like structure that is sufficient to direct minus-strand RNA synthesis in vitro. The tRNA-like promoters, tB1 and tB3, direct approximately equal amounts of synthesis in vitro. However, 5' sequences were found to affect the amount of minus-strand synthesis, suggesting that sequences beyond the tRNA-like structure are important in moderating minus-strand synthesis. Consistent with this, sequences upstream the tRNA-like structure are able to partially suppress mutations at or near the initiation site. This activity is observed in the 5' sequences of both BMV and CMV (cucumber mosaic virus) templates. However, a chimeric RNA containing the CMV tRNA-like promoter fused to the 5' sequences of BMV was not able to suppress mutations at the initiation site, suggesting that homologous 5' and 3' sequences are required to affect initiation. The ability to suppress mutations at the initiation site was correlated with a slight increase in the ability of the BMV RNA-dependent RNA polymerase to interact with the RNA., (Copyright 1998 Academic Press.)

Both the brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) RNA-dependent RNA polymerases (RdRps) were found to recognize the BMV core subgenomic promoter in the same manner, requiring specific functional groups at positions -17, -14, -13, and -11 relative to the subgenomic initiation site (+1). For CCMV subgenomic RNA synthesis, both RdRps required the same nucleotides and four additional nucleotides at positions -20, -16, -15, and -10. The -20 nucleotide is partially responsible for the differential mode of recognition of the two promoters. These data provide evidence that the RNA can induce RdRps to alter the mode of promoter recognition.

RNAs 33 nucleotides in length can direct accurate initiation of subgenomic RNA synthesis by the brome mosaic virus RNA-dependent RNA polymerase (RdRp), provided that the native sequences are maintained at five positions: -17, -14, -13, -11, and the +1 initiation site. The functional groups in the bases of these essential nucleotides required to interact with RdRp were examined by using chemically synthesized RNAs containing base analogs at each of the five positions. Analysis using a template competition assay revealed that the mode of recognition for the initiation nucleotide (+1) is distinct from that of the other essential nucleotides in the promoter. Competition experiments also determined that three template nucleotides are sufficient for stable interaction with RdRp. These results identify base moieties in the brome mosaic virus subgenomic promoter required for efficient RNA synthesis and support the hypothesis that the recognition of a RNA promoter by a viral RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions -17, -14, -13, and -11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position -17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animal-infecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.

Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.