Secondary structure model for 23S ribosomal RNA.

A secondary structure model for 23S ribosomal RNA has been constructed on the basis of comparative sequence data, including the complete sequences from E. coli. Bacillus stearothermophilis, human and mouse mitochondria and several partial sequences. The model has been tested extensively with single strand-specific chemical and enzymatic probes. Long range base-paired interactions organize the molecule into six major structural domains containing over 100 individual helices in all. Regions containing the sites of interaction with several ribosomal proteins and 5S RNA have been located. Segments of the 23S RNA structure corresponding to eucaryotic 5.8S and 25 RNA have been identified, and base paired interactions in the model suggest how they are attached to 28S RNA. Functionally important regions, including possible sites of contact with 30S ribosomal subunits, the peptidyl transferase center and locations of intervening sequences in various organisms are discussed. Models for molecular 'switching' of RNA molecules based on coaxial stacking of helices are presented, including a scheme for tRNA-23S RNA interaction.     

A molecular characterization of the charismatic Faroe house mouse

Faroe house mice are a ‘classic’ system of rapid and dramatic morphological divergence highlighted by J. S. Huxley during the development of the Modern Synthesis. In the present study, we characterize these charismatic mice using modern molecular techniques, examining specimens from all Faroe islands occupied by mice. The aims were to classify the mice within the modern house mouse taxonomy (i.e. as either Mus musculus domesticus or Mus musculus musculus) using four molecular markers and a morphological feature, and to examine the genetic diversity and possible routes of colonization using mitochondrial (mt) control region DNA sequences and microsatellite data (15 loci). Mice on the most remote islands were characterized as M. m. domesticus and exhibited exceptionally low genetic diversity, whereas those on better connected islands were more genetically diverse and had both M. m. musculus and M. m. domesticus genetic elements, including one population which was morphologically M. m. musculus‐like. The mtDNA data indicate that the majority of the mice had their origins in south‐western Norway (or possibly southern Denmark/northern Germany), and probably arrived with the Vikings, earlier than suggested by Huxley. The M. m. musculus genetic component appears to derive from recent mouse immigration from Denmark. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 471–482.     

Identification of structural DNA variations in human cell cultures after long-term passage

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8–12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.     

New BAR tools for mining expression data and exploring Cis‐elements in Arabidopsis thaliana

Identifying sets of genes that are specifically expressed in certain tissues or in response to an environmental stimulus is useful for designing reporter constructs, generating gene expression markers, or for understanding gene regulatory networks. We have developed an easy‐to‐use online tool for defining a desired expression profile (a modification of our Expression Angler program), which can then be used to identify genes exhibiting patterns of expression that match this profile as closely as possible. Further, we have developed another online tool, Cistome, for predicting or exploring cis‐elements in the promoters of sets of co‐expressed genes identified by such a method, or by other methods. We present two use cases for these tools, which are freely available on the Bio‐Analytic Resource at http://BAR.utoronto.ca .     

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures.     

The Distribution of Some Hydrolytic Enzymes in the Wheat Seed

A histochemical study of dry and germinated wheat embryos andseeds has been made to identify the sites of activity of 3'-and 5'-nucleotidases, phosphatases, lipase, and esterase. Theenzymes all showed the same distribution, with the coleorhizaas the most prominent site of activity in the dry embryo, especiallywhen compared with the adjacent roots with very low activity.Investigations of the whole seed after 24 h germination showeda very high activity in the aleurone cells and high activitiesin the scutellum and coleorhiza. The epithelium and provasculartissues of the scutellum showed moderate to low activity whilethe endosperm had little or none. Hydrolase activity also wasfound in the testa. The function of the coleorhiza. as firstfood reserve for the roots and its location at the most importantsite of water entry, is discussed.     

Genetic Analysis of Giardia from Hoofed Farm Animals Reveals Artiodactyl-Specific and Potentially Zoonotic Genotypes

ABSTRACT. Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture or growth in suckling mice, were compared genetically by analysis of DNA amplified from loci encoding variant surface proteins or the enzyme glutamate dehydrogenase and by allozyme analysis. The isolates were heterogeneous, but all showed affinity with genetic Assemblage A-one of two major assemblages defined previously by analysis of Giardia from humans. Three distinct genotypes were evident. Ten isolates (eight axenic and two established in suckling mice) from an alpaca, pig, horse, cattle and sheep were indistinguishable from human-derived G. intestinalis belonging to a previously designated genetic group (Group I). This genotype seems to have broad host specificity, including a zoonotic potential for humans. Five isolates (two axenic and three established in suckling mice) from an alpaca, a horse and sheep had close affinity with human-derived Group I and Group I1 G. inresrinalis genotypes. The other 16 isolates (comprising both axenic and suckling mouse-propagated cultures derived from cattle, sheep, alpaca, a goat and pigs in Australia and Europe) differed from all other Giardia with "duodenalis" morphology that have been examined by these methods and they segregated as a highly distinct sublineage (referred to herein as 'Novel livestock') within genetic Assemblage A. The predominance of 'Novel livestock' genotypes in the test panel and their apparent exclusive association with artiodactyl hosts indicates that they may be confined to this group of mammals. Assemblage B genotypes, which are prevalent in humans and some other animal species, were not detected.