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Biomedical subjects

S D Senanayake

Publications and source records attributed to S D Senanayake.

9 recordsLinked to original sources
PubMed2006

Photocatalysis and the origin of life: synthesis of nucleoside bases from formamide on TiO2(001) single surfaces.

We report the conversion of a large fraction of formamide (NH(2)CHO) to high-molecular-weight compounds attributed to nucleoside bases on the surface of a TiO(2) (001) single crystal in ultra-high vacuum conditions. If true, we present previously unreported evidence for making biologically relevant molecules from a C1 compound on any single crystal surface in high vacuum and in dry conditions. An UV light of 3.2 eV was necessary to make the reaction. This UV light excites the semiconductor surface but not directly the adsorbed formamide molecules or the reaction products. There thus is no need to use high energy in the form of photons or electrical discharge to make the carbon-carbon and carbon-nitrogen bonds necessary for life. Consequently, the reaction products may accumulate with time and may not be subject to decomposition by the excitation source. The formation of these molecules, by surface reaction of formamide, is proof that some minerals in the form of oxide semiconductors are active materials for making high-molecular-weight organic molecules that may have acted as precursors for biological compounds required for life in the universe.

Carbon↗
PubMed2005

Coupling of carbon monoxide molecules over oxygen-defected UO2(111) single crystal and thin film surfaces.

While coupling reactions of carbon-containing compounds are numerous in organometallic chemistry, they are very rare on well-defined solid surfaces. In this work we show that the reductive coupling of two molecules of carbon monoxide to C2 compounds (acetylene and ethylene) could be achieved on oxygen-defected UO2(111) single crystal and thin film surfaces. This result allows in situ electron spectroscopic investigation of a typical organometallic reaction such as carbon coupling and extends it to heterogeneous catalysis and solids. By using high-resolution photoelectron spectroscopy (HRXPS) it was possible to track the changes in surface states of the U and O atoms as well as identify the intermediate of the reaction. Upon CO adsorption U cations in low oxidation states are oxidized to U4+ ions; this was accompanied by an increase of the O-to-U surface ratios. The HRXPS C 1s lines show the presence of adsorbed species assigned to diolate species (-OCH=CHO-) that are most likely the reaction intermediate in the coupling of two CO molecules to acetylene and ethylene.

Journal Article↗
PubMed2001

Downstream sequences influence the choice between a naturally occurring noncanonical and closely positioned upstream canonical heptameric fusion motif during bovine coronavirus subgenomic mRNA synthesis.

Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5' and 3' coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (-17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5' end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.

Animals↗
PubMed1999

Translation from the 5' untranslated region (UTR) of mRNA 1 is repressed, but that from the 5' UTR of mRNA 7 is stimulated in coronavirus-infected cells.

Viral gene products are generally required in widely differing amounts for successful virus growth and assembly. For coronaviruses, regulation of transcription is a major contributor to these differences, but regulation of translation may also be important. Here, we examine the possibility that the 5' untranslated regions (UTRs), unique for each of the nine species of mRNA in the bovine coronavirus and ranging in length from 70 nucleotides (nt) to 210 nt (inclusive of the common 5'-terminal 65-nt leader), can differentially affect the rate of protein accumulation. When the natural 77-nt 5' UTR on synthetic transcripts of mRNA 7 (mRNA for N and I proteins) was replaced with the 210-nt 5' UTR from mRNA 1 (genomic RNA, mRNA for viral polymerase), approximately twofold-less N, or (N) CAT fusion reporter protein, was made in vitro. Twofold less was also made in vivo in uninfected cells when a T7 RNA polymerase-driven transient-transfection system was used. In coronavirus-infected cells, this difference surprisingly became 12-fold as the result of both a stimulated translation from the 77-nt 5' UTR and a repression of translation from the 210-nt 5' UTR. These results reveal that a differential 5' UTR-directed regulation of translation can occur in coronavirus-infected cells and lead us to postulate that the direction and degree of regulation is carried out by viral or virally induced cellular factors acting in trans on cis-acting elements within the 5' UTR.

5' Untranslated Regions↗
PubMed1997

Bovine coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA.

The mRNA encoding the 49-kDa nucleocapsid protein (N) of the bovine coronavirus is bicistronic. A 23-kDa protein, termed the I protein for the 'internal' open reading frame (ORF), is also synthetized but in the +1 reading frame beginning 61 nt downstream of the N start codon. Sequences flanking the N and I start codons suggest that the I ORF might be accessed by scanning ribosomes passing over the N start codon. Here we test this idea and demonstrate with translation studies both in vitro and in vivo that the I protein is synthesized according to the leaky scanning model for initiation of translation on the subgenomic N mRNA molecule.

Animals↗
PubMed1995

Precise large deletions by the PCR-based overlap extension method.

The authors describe an efficient method for generating large deletions (> 200 nts) of precise length using the PCR-based method of gene splicing by overlap extension (1). This method is technically simple and less time consuming than conventional loop-out mutagenesis techniques requiring preparation of a single-stranded DNA template.

Base Sequence↗
PubMed1993

A translation-attenuating intraleader open reading frame is selected on coronavirus mRNAs during persistent infection.

Short open reading frames within the 5' leader of some eukaryotic mRNAs are known to regulate the rate of translation initiation on the downstream open reading frame. By employing the polymerase chain reaction, we learned that the 5'-terminal 5 nt on the common leader sequence of bovine coronavirus subgenomic mRNAs were heterogeneous and hypervariable throughout early infection in cell culture and that as a persistent infection became established, termini giving rise to a common 33-nt intraleader open reading frame were selected. Since the common leader is derived from the genomic 5' end during transcription, a common focus of origin for the heterogeneity is expected. The intraleader open reading frame was shown by in vitro translation studies to attenuate translation of downstream open reading frames in a cloned bovine coronavirus mRNA molecule. Selection of an intraleader open reading frame resulting in a general attenuation of mRNA translation and a consequent attenuation of virus replication may, therefore, be a mechanism by which coronaviruses and possibly other RNA viruses with a similar transcriptional strategy maintain a persistent infection.

Amino Acid Sequence↗
PubMed1992

The nucleocapsid protein gene of bovine coronavirus is bicistronic.

For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another.

Animals↗