Biomedical subjects

T Tamm

Publications and source records attributed to T Tamm.

7 recordsLinked to original sources

PubMed2000

RNA-binding activities of cocksfoot mottle sobemovirus proteins.

Cocksfoot mottle virus (CfMV) has a positive-sense ssRNA genome containing four open reading frames (ORFs). ORF1 encoded protein (P1) is the putative movement protein; the product of ORF2a (P2a) contains VPg and the motifs characteristic of serine proteases. P2b, encoded by ORF2b, is the putative RNA-dependent RNA polymerase. P3, the coat protein, is encoded by ORF3. CfMV P1, P2a, P2b, and P3, containing a six histidine tag at the amino terminus, were expressed in Escherichia coli, purified and their RNA-binding activities were analysed. The northwestern blot assay showed that His-tagged P1, P2a, P2b, and P3 were able to interact with ssRNA transcripts in a sequence-nonspecific manner. The filter-binding assay confirmed the ssRNA-binding capacity of recombinant P1, P2a, and P3. The RNA-binding activities of His-tagged P3 and native coat protein were similar. P1 and P2a binding to ssRNA decreased markedly by increasing NaCl concentrations. In contrast, P3 had the RNA-binding optimum at 100-200 mM NaCl. We discuss the possible amino acid motifs involved in the RNA-binding of CfMV proteins.

Binding, Competitive↗

PubMed2000

Regulation of -1 ribosomal frameshifting directed by cocksfoot mottle sobemovirus genome.

The polyprotein of Cocksfoot mottle virus (CfMV) is encoded by two overlapping open reading frames (ORFs). The putative replicase of CfMV is produced as a part of the polyprotein from ORF2b by the -1 ribosomal frameshifting mechanism. The signals leading to -1 ribosomal frameshifting directed by CfMV RNA are the slippery heptamer UUUAAAC and a stem-loop structure starting seven nucleotides downstream from the heptamer. We studied the effect of different parts of the CfMV genome on the -1 ribosomal frameshifting efficiency using a wheat germ extract transcription/translation system. A point mutation in the slippery heptamer and a mutation deleting the stem-loop structure prevented frameshifting. Seventy nucleotides of CfMV sequence, including the slippery sequence and the stem-loop structure, was found to act as a minimal region for frameshifting. Interestingly, a termination codon introduced into the -1-frame 27 nucleotides downstream of the stem-loop structure increased frameshift efficiency threefold, while a similarly located termination codon in the 0-frame had no effect. Even fourfold to fivefold efficiencies were observed when the polyprotein encoding ORFs were fused together, which led simultaneously to the formation of a termination codon downstream of the frameshift signal. Possible reasons underlying these observations are discussed.

Base Sequence↗

PubMed2000

Sobemoviruses.

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Genome, Viral↗

PubMed1999

Identification of genes encoding for the cocksfoot mottle virus proteins.

Cocksfoot mottle sobemovirus (CfMV) has a monopartite single-stranded positive-sense RNA genome. In wheat germ extract, in vitro translation of CfMV virion RNA resulted in the production of four major proteins of 100, 71, 34 and 12 kDa. In this paper we show the individual genes from which these polypeptides are synthesised. Polyclonal antisera against the proteins encoded by all open reading frames (ORFs) of CfMV were produced. Antibodies were used to immunoprecipitate the in vitro translation products of CfMV genomic RNA as well as the coupled in vitro transcription/translation products of individual viral genes. We demonstrate that the 12, 71, and 100 kDa CfMV proteins are synthesised from the bicistronic genomic RNA of the virus. The CfMV 12 kDa protein is produced from ORF1, the 71 kDa protein from ORF2a (and does not result from the proteolytic cleavage of the polyprotein) and the 100 kDa protein is a polyprotein encoded by ORFs 2a and 2b by a -1 ribosomal frameshift. ORF2b alone does not direct any in vitro protein synthesis. CfMV 34 kDa protein is a coat protein synthesised from the virion-packed subgenomic RNA. The translational strategies of different sobemoviruses are also discussed in this paper.

Animals↗

PubMed1995

The putative replicase of the cocksfoot mottle sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift.

The polyprotein of cocksfoot mottle sobemovirus (CfMV) is encoded by two overlapping open reading frames (ORF). The ORF 2a codes for the putative VPg and serine protease and the ORF 2b codes for the putative replicase. The consensus signals for a -1 ribosomal frameshifting event are found at the very beginning of the overlapping region of these ORFs. The shifty heptanucleotide in CfMV is UUUAAAC, and the secondary structure after the shifty sequence is predicted to be a stem-loop. In vitro translation of the CfMV RNA in wheat germ extract produced proteins of several sizes, including one of 100 kDa. According to the nucleotide sequence data, no single ORF is capable of directing the synthesis of a 100-kDa protein. A chimeric beta-glucuronidase-CfMV cDNA containing the entire ORF 2a and 2b overlap region including frameshift signals was constructed. A trans-frame protein of 108 kDa was produced from this construct with an efficiency of 26-29% by in vitro translation in wheat germ extract. CfMV is the first sobemovirus in which the putative replicase is reported to be produced as a part of a polyprotein by a -1 frameshift event. The replicases of the sobemoviruses are related to the luteovirus subgroup II replicases, which are known to be produced by -1 ribosomal frameshift. The reported amino acid sequences of the putative replicases of sobemo- and subgroup II luteoviruses were compared to that of the putative replicase of CfMV. This comparison revealed more extensive homology between these groups than previously reported.

Amino Acid Sequence↗

PubMed1995

Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses.

The genome of cocksfoot mottle virus (CfMV) is a positive-sense ssRNA molecule of 4082 nucleotides as revealed by sequencing the entire genome. The 5'-untranslated region of the genome is 69 nucleotides and the 3'-untranslated region is 225 nucleotides in length. The coding region contains four open reading frames (ORFs). The organization of CfMV ORFs differs significantly from that of the previously sequenced sobemoviruses southern bean mosaic virus and rice yellow mottle virus. ORF1 encodes a protein having a calculated molecular mass of 12.3 kDa. The function of this protein is unknown. The next ORF codes for the putative VPg and serine protease. The ORF2a product consists of 568 amino acids, with a calculated molecular mass of 60.9 kDa. The replicase of CfMV is translated as part of a polyprotein by--1 ribosomal frameshifting in ORF2a. The calculated molecular mass of the transframe protein is 103.4 kDa. ORF3 encodes the 27.6 kDa coat protein. This has been verified by amino acid sequencing of the CfMV coat protein N terminus. Northern blots of total RNA from CfMV-infected barley leaves reveal the 4.1 kb genomic RNA band and one virus-specific band of 1.2 kb, which may represent a subgenomic RNA for coat protein synthesis.

Amino Acid Sequence↗

PubMed1991

About the mutagenicity of chlorine-substituted furanones and halopropenals. A QSAR study using molecular orbital indices.

Electron affinities, frontier molecular orbital energies and electron densities at individual carbon atoms were calculated for 11 chlorofuranones including the strong mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and for 5 halopropenals by semi-empirical AM1 and ab initio STO-3G methods. Significant correlations were found between Ames TA100 mutagenicity and the following AM1 electronic parameters of chlorofuranones: electron affinity (r = 0.9556). LUMO energy (r = 0.9332) and frontier electron density of LUMO at the alpha-carbon (r = 0.8882). In halopropenals only LUMO electron density at the beta-carbon correlates well with mutagenicity. The observed correlations suggest a reaction mechanism in which chlorofuranones and halopropenals act as electron acceptors in the interaction with DNA.

Aldehydes↗