An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org      

Actin Disruption by Latrunculin B Causes Turgor-Related Changes in Tip Growth of Saprolegnia ferax Hyphae

Hyphae of Saprolegnia ferax growing under normal or low-turgor conditions were exposed to 0.1-10 &mgr;g/ml latrunculin B, an actin inhibitor. In the first 10 s of addition, hyphae with normal turgor levels accelerated while those with low turgor decelerated, consistent with the suggestion that actin restrains or protrudes tips under these respective turgor conditions. Both sets of hyphae then decelerated and eventually ceased extension within 60 s. These changes were reflected in rhodamine-phalloidin staining patterns, which showed that actin caps were disrupted progressively under both conditions in a time-dependent manner. After 60 s, normal-turgored hyphae started to swell rapidly while low-turgored hyphae showed little or no swelling. Swelling was characteristically subapical, which is best explained by tip growth models which incorporate actin-mediated exocytosis.     

Quantitative analysis of the relationship between nucleotide sequence and functional activity.

Matrices can be used to evaluate sequences for functional activity. Multiple regression can solve for the matrix that gives the best fit between sequence evaluations and quantitative activities. This analysis shows that the best model for context effects on suppression by su2 involves primarily the two nucleotides 3' to the amber codon, and that their contributions are independent and additive. Context effects on 2AP mutagenesis also involve the two nucleotides 3' to the 2AP insertion, but their effects are not independent. In a construct for producing beta-galactosidase, the effects on translational yields of the tri-nucleotide 5' to the initiation codon are dependent on the entire triplet. Models based on these quantitative results are presented for each of the examples.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.      

Polarized Growth of Fungal Hyphae Is Defined by an Alkaline pH Gradient

Robson, G. D., Prebble, E., Rickers, A., Hosking, S., Denning, D. W., Trinci, A. P. J., and Robertson, W. 1996. Polarized growth of fungal hyphae is defined by an alkaline pH gradient. Fungal Genetics and Biology 20, 289-298. Polarized cell growth is exhibited by a diverse range of eukaryotic and prokaryotic cells. The events which are responsible for this growth are poorly understood. However, the existence of ion gradients may play an important role in establishing and driving cell polarity. Using a pH-sensitive, ratiometric fluorescent dye to monitor intracellular pH in growing fungal hyphae, we report a gradient at the extending hyphal tip that is up to 1.4 pH units more alkaline than more distal regions. Both the magnitude and the length of the pH gradient were strongly correlated with the rate of hyphal extension and eradication of the gradient-arrested growth. These results suggest that alkaline pH gradients may be integral to hyphal extension in fungi.     

Characterization of translational initiation sites in E. coli.

We characterize the Shine and Dalgarno sequence of 124 known gene beginnings. This information is used to make "rules" which help distinguish gene beginning from other sites in a library of over 78,000 bases of mRNA. Gene beginnings are found to have information besides the initiation codon and Shine and Dalgarno sequence which can be used to make better "rules".     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Chromatin structure and evolution in the human genome

Background  

Evolutionary rates are not constant across the human genome but genes in close proximity have been shown to experience similar levels of divergence and selection. The higher-order organisation of chromosomes has often been invoked to explain such phenomena but previously there has been insufficient data on chromosome structure to investigate this rigorously. Using the results of a recent genome-wide analysis of open and closed human chromatin structures we have investigated the global association between divergence, selection and chromatin structure for the first time.