Variables Influencing Differences in Sequence Conservation in the Fission Yeast Schizosaccharomyces pombe

Which variables determine the constraints on gene sequence evolution is one of the most central questions in molecular evolution. In the fission yeast Schizosaccharomyces pombe, an important model organism, the variables influencing the rate of sequence evolution have yet to be determined. Previous studies in other single celled organisms have generally found gene expression levels to be most significant, with numerous other variables such as gene length and functional importance identified as having a smaller impact. Using publicly available data, we used partial least squares regression, principal components regression, and partial correlations to determine the variables most strongly associated with sequence evolution constraints. We identify centrality in the protein-protein interactions network, amino acid composition, and cellular location as the most important determinants of sequence conservation. However, each factor only explains a small amount of variance, and there are numerous variables having a significant or heterogeneous influence. Our models explain more than half of the variance in dN, raising the possibility that future refined models could quantify the role of stochastics in evolutionary rate variation

Molecular genetic analysis of the maize terminal ear1 gene and in silico analysis of related genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand

Mutants of the maize terminal ear1 (te1) gene have shortened internodes, abnormal phyllotaxy, leaf pattern defects and partial feminisation of tassels. The te1 gene encodes an RNA recognition motif (RRM) protein, and is expressed in the vegetative shoot apex in semicircular rings that laterally oppose the positions of leaf primordia (Veit 1998). This project aimed to further characterise the molecular biology and function of the te1 gene. Molecular genetic studies aimed to further characterise the genes structure and expression. Genomic clones were sequenced revealing the intron exon structure. 5' RACE was used to predict a 5' transcription start site. Competitive RT-PCR showed that te1 transcripts were highest in vegetative shoot meristems and embryos, lower in ears, roots and tassels, and undetectable in leaves. Two te1 mutant alleles were cloned and the junctions sequenced, a further five alleles were characterised incompletely. The TE1 peptide belongs to a subclass of RRM proteins which includes the Schizosaccharomyces pombe protein MEI2. More than 30 putative plant Mei2-like genes were identified in Genbank, no examples have been found in metazoans. Seven Mei2-like genes were predicted from the completed Arabidopsis genome. Exon structure and amino acid sequence supported three groupings of Mei2-like genes. Structural predictions of Mei2-like proteins indicate that the third RRM contained some novel structural features not present in canonical RRM proteins. Attempts to study the function of the TE1 protein in vitro were limited by the inability of both E. coli and Pichia pastoris expression systems to express the full length protein, probably due to codon bias. Antibodies produced to a C-terminal portion of the protein did not specifically detect the TE1 protein in plant extracts without incurring non-specific activity. The te1 cDNA was ectopically expressed in Arabidopsis from a copper-inducible promoter both with and without the SV40 nuclear localisation signal (NLS). Although both te1 and NLS:te1 transgenes were detected in transformants no phenotypes consistently correlated with transgene expression

Direct isolation of poly(A)(+) RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture

LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)(+) RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)(+) RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T–A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)(+) RNA in a low salt concentration range of 50–100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH(2)-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles