Temporally regulated expression of Cre recombinase in neural stem cells

The use of mouse gene targeting to study molecules important in neural development is oftentimes impaired by early embryonic lethality. In order to address later roles for such molecules, specifically in neural stem cells, we generated transgenic mice that express both the tetracycline-inducible molecule rtTA-M2 and GFP under the control of the neural precursor specific form of nestin. Developmental analysis of these mice demonstrates that GFP expression is exclusive to the neural tube. Adult expression of GFP is seen only in known areas of adult neurogenesis, namely, the subventricular zone and the dentate gyrus. When crossed with a second transgenic mouse (TetOp-Cre) that expresses the Cre recombinase under the control of the tetracycline responsive promotor, we demonstrate temporal induction of Cre in bigenic animals exposed to doxycycline. We further demonstrate the feasibility of this approach by using the ROSA-26 reporter mouse to mediate recombination in neural precursor cells.     

Separation and detection of vitellogenin in fish plasma by capillary zone electrophoresis

A method for coupling capillary zone electrophoresis (CZE) with rapid membrane chromatography purification (RMCP) was established for the analysis of vitellogenin (VTG) in male fish plasma induced with 17ss-estrodiol. CZE analyses of purified VTG were performed in a buffer containing 25 mM sodium borate (pH 8.4). A 50 microm i.d. fused-silica capillary was used for separation and the detection was carried out by UV-diode array at 214 nm. Inter- and intra-assay variabilities of the proposed method were less than 10.06 and 1.95%, respectively. The method has good linear relationship over the scope of 15-2250 microg/ml with a correlation coefficient of R2 = 0.9965 and a detection limit of 7.0 microg/ml. The established CZE method was also applied to directly separate and identify VTG from fish plasma. The results indicated this method could minimize interferences from plasma proteins, allowing the detection of at least 62.5 microg/ml of VTG proteins in total proteins. This is a rapid and easy method to determine the quantity and purity of VTG compared to Bradford method and SDS-PAGE.     

Determination of protein charge by capillary zone electrophoresis

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a "charge ladder" approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.     

Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone

Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada.     

Chloroplast DNA variation and population structure in the widespread forest tree, Eucalyptus grandis

Recognition of genetic structure of populations and the ability to identify vulnerable populations is useful for the formation of conservation management strategies for plants. Eucalyptus grandis is a tall forest tree that has a major area of occurrence in subtropical eastern Australia, with smaller populations located in the east coast tropics. Many widespread forest species exhibit population differentiation that corresponds to geographic regions. However, Eucalyptus grandis appears to be an exception based on isozyme and morphological data. This is intriguing given a large discontinuity between northern populations and those in the southern part of the species range. In this study, the distribution of a maternally inherited chloroplast locus was examined because it was more likely to reveal genetic structure due to the slower evolution of the chloroplast genome and limited dispersal of seed in eucalypts. As expected, the G _ST for chloroplast DNA was higher than that for nuclear DNA but indicated low population differentiation for a forest tree species. Phylogeographic analysis indicated that the 15 populations grouped into three broad geographical regions; however, overall population structure was weak suggesting that the large geographical disjunction in the distribution of E. grandis may be relatively recent. A paradigm for conservation management of E. grandis based on chloroplast DNA haplotype distribution would take into account the low differentiation among populations.     

Sequence Representation and Prediction of Protein Secondary Structure for Structural Motifs in Twilight Zone Proteins

Characterizing and classifying regularities in protein structure is an important element in uncovering the mechanisms that regulate protein structure, function and evolution. Recent research concentrates on analysis of structural motifs that can be used to describe larger, fold-sized structures based on homologous primary sequences. At the same time, accuracy of secondary protein structure prediction based on multiple sequence alignment drops significantly when low homology (twilight zone) sequences are considered. To this end, this paper addresses a problem of providing an alternative sequences representation that would improve ability to distinguish secondary structures for the twilight zone sequences without using alignment. We consider a novel classification problem, in which, structural motifs, referred to as structural fragments (SFs) are defined as uniform strand, helix and coil fragments. Classification of SFs allows to design novel sequence representations, and to investigate which other factors and prediction algorithms may result in the improved discrimination. Comprehensive experimental results show that statistically significant improvement in classification accuracy can be achieved by: (1) improving sequence representations, and (2) removing possible noise on the terminal residues in the SFs. Combining these two approaches reduces the error rate on average by 15% when compared to classification using standard representation and noisy information on the terminal residues, bringing the classification accuracy to over 70%. Finally, we show that certain prediction algorithms, such as neural networks and boosted decision trees, are superior to other algorithms.This research was supported in part by the Natural Sciences and Engineering Research Council of Canada (NSERC).     

A gradient analysis based on the buffer zones of urban landscape pattern of the constructed area in Guigang City, Guangxi, China

Over the last century urbanization on the landscape has increased and intensified. Urban development has a great impact on the environment at the local, regional and even global levels. As a driving force in global change, the need to understand the dynamics of urban pattern and its change in an accurate and efficient manner is ever more pressing. Based on aerial color infrared photography in 1985 and QuickBird satellite imagery in 2004 and according to the standard for plan and construction of city land use, the landscape of Guigang City was divided into 11 types. In the landscape classification maps, 31 buffer zones, each being 200 m wide, were divided. With the aid of GIS software ArcView 3.2 and landscape pattern analysis software FRAGSTATS 3.3, the landscape spatial patterns of each buffer zone were analyzed at the landscape level and class level. The landscape indices, such as patch size, patch fractal dimension, diversity index and evenness index, were calculated. The results indicated the following: The total area of the residential land and the communal land in 2004 covered 46.3% of the entire constructed area of Guigang City and the sum of the patch number of the two patch types occupied about 39.7% of the total patch number, while the percentages were respectively 48.2% and 45.4% in 1985. This showed that the mosaic landscape with the residential land and communal facilities land became increasingly unclear following urban development and landscape diversity. Based on the gradient analysis with the landscape-level index and the class-level index, there were two business and finance centers in the constructed area. One was located in the urban center with a range of 0.8 km; the other was from the 10^th to the 16^th zone. In each buffer zone, the mean patch size was larger and the landscape shape was more regular in 2004 than in 1985. Furthermore, the Shannon diversity index of each buffer zone rose in 2004 with patch richness and evenness increasing. The landscape index computed for the main landscape types (communal facilities land, industrial estate land, residential land and farmland) in the middle of the buffer zones clearly indicated the changes taking place in urbanization. The patch size and the patch number of industrial estate land and farmland also pointed to these changes, while communal facilities land and residential land assumed another similar tendency towards changes. In each gradient zone, industrial estate land had the most complex shape, the lowest area percentage of each zone and the biggest mean patch size, whereas communal facilities land and residential land were opposite to industrial estate land. Farmland had a steady percentage along the buffer zone and its continuous distribution had an important impact on the urban eco-environment. The question of how to relate the pattern of changing characteristics along the buffer zone to the urban ecological process and urban ecological planning remains to be studied further in the future.     

FEMALE HETEROGAMETY AND SPECIATION: REDUCED INTROGRESSION OF THE Z CHROMOSOME BETWEEN TWO SPECIES OF NIGHTINGALES

Several lines of evidence suggest that the X chromosome plays a large role in intrinsic postzygotic isolation. The role of the Z chromosome in speciation is much less understood. To explore the role of the Z chromosome in reproductive isolation, we studied nucleotide variation in two closely related bird species, the Thrush Nightingale ( Luscinia luscinia ) and the Common Nightingale ( L. megarhynchos ). These species are isolated by incomplete prezygotic isolation and female hybrid sterility. We sequenced introns of four Z-linked and eight autosomal loci and analyzed patterns of polymorphism and divergence using a divergence-with-gene flow framework. Our results suggest that the nightingale species diverged approximately 1.8 Mya. We found strong evidence of gene flow after divergence in both directions, although more introgression occurred from L. megarhynchos into L. luscinia . Gene flow was significantly higher on the autosomes than on the Z chromosome. Our results support the idea that the Z chromosome plays an important role in intrinsic postzygotic isolation in birds, although it may also contribute to the evolution of prezygotic isolation through sexual selection. This highlights the similarities in the genetic basis of reproductive isolation between organisms with heterogametic males and organisms with heterogametic females during the early stages of speciation.