Site-Directed Mutagenesis of a Saccharomyces Cerevisiae Mitochondrial Translation Initiation Codon

We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

Model based dynamics analysis in live cell microtubule images

Background

The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data.

Results

In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior.

Conclusion

Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior.

     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome

A model of DNA sequence evolution applicable to coding regions is presented. This represents the first evolutionary model that accounts for dependencies among nucleotides within a codon. The model uses the codon, as opposed to the nucleotide, as the unit of evolution, and is parameterized in terms of synonymous and nonsynonymous nucleotide substitution rates. One of the model's advantages over those used in methods for estimating synonymous and nonsynonymous substitution rates is that it completely corrects for multiple hits at a codon, rather than taking a parsimony approach and considering only pathways of minimum change between homologous codons. Likelihood-ratio versions of the relative-rate test are constructed and applied to data from the complete chloroplast DNA sequences of Oryza sativa, Nicotiana tabacum, and Marchantia polymorpha. Results of these tests confirm previous findings that substitution rates in the chloroplast genome are subject to both lineage-specific and locus-specific effects. Additionally, the new tests suggest tha the rate heterogeneity is due primarily to differences in nonsynonymous substitution rates. Simulations help confirm previous suggestions that silent sites are saturated, leaving no evidence of heterogeneity in synonymous substitution rates.      

Origins of spatial working memory deficits in schizophrenia: an event-related FMRI and near-infrared spectroscopy study

Abnormal prefrontal functioning plays a central role in the working memory (WM) deficits of schizophrenic patients, but the nature of the relationship between WM and prefrontal activation remains undetermined. Using two functional neuroimaging methods, we investigated the neural correlates of remembering and forgetting in schizophrenic and healthy participants. We focused on the brain activation during WM maintenance phase with event-related functional magnetic resonance imaging (fMRI). We also examined oxygenated hemoglobin changes in relation to memory performance with the near-infrared spectroscopy (NIRS) using the same spatial WM task. Distinct types of correct and error trials were segregated for analysis. fMRI data indicated that prefrontal activation was increased during WM maintenance on correct trials in both schizophrenic and healthy subjects. However, a significant difference was observed in the functional asymmetry of frontal activation pattern. Healthy subjects showed increased activation in the right frontal, temporal and cingulate regions. Schizophrenic patients showed greater activation compared with control subjects in left frontal, temporal and parietal regions as well as in right frontal regions. We also observed increased 'false memory' errors in schizophrenic patients, associated with increased prefrontal activation and resembling the activation pattern observed on the correct trials. NIRS data replicated the fMRI results. Thus, increased frontal activity was correlated with the accuracy of WM in both healthy control and schizophrenic participants. The major difference between the two groups concerned functional asymmetry; healthy subjects recruited right frontal regions during spatial WM maintenance whereas schizophrenic subjects recruited a wider network in both hemispheres to achieve the same level of memory performance. Increased "false memory" errors and accompanying bilateral prefrontal activation in schizophrenia suggest that the etiology of memory errors must be considered when comparing group performances. Finally, the concordance of fMRI and NIRS data supports NIRS as an alternative functional neuroimaging method for psychiatric research.