Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii

Background

DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes.

Results

Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation.

Conclusion

In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Distribution of energy reserves in a viviparous skink: Does tail autotomy involve the loss of lipid stores?

Abstract Caudal autotomy is an effective defensive strategy used by many lizards to facilitate escape during predatory encounters. However, it has several potentially severe consequences, including a range of energetic costs that are believed to result from the depletion of caudal lipid reserves during tail loss. In this study we examined the possible effect of caudal autotomy on the energetic reserves of a small viviparous skink, Niveoscincus metallicus (O'Shaughnessy 1874). Animals of each sex were collected on three occasions to assess the distribution of lipid stores. In addition, the frequency and position of naturally occurring tail breaks were determined. Both abdominal and caudal lipid stores are present in N. metallicus; however, caudal fat bodies comprise the majority (55–78%) of these fat reserves. Temporal variation in fat body mass, both abdominal and caudal, was evident. There was a significant relationship between the two fat stores, which was distorted in pregnant females, when relatively more fat was stored in the tail. Examination of the distribution of caudal fat in the tail revealed that the majority (90–95%) occurs within the proximal third of the tail. The remainder is located in the middle portion of the tail, with no reserves in the most distal tail section. During late pregnancy, females store relatively more fat closer to the body. The frequency of tail loss in a natural population of N. metallicus was extremely high (78%). Tail breaks were normally distributed along the length of the tail (i.e. most near the middle and fewer distal and proximal breaks). Thus there was a relatively high chance of some lipid depletion as a result of tail loss, but because 76% of breaks occur in the middle and distal thirds of the tail, there is a high probability that tail loss results in only minimal (i.e. <10%) lipid depletion. This is the first instance where both the energetic ‘value’ of the tail and the likelihood of lipid depletion during tail loss have been determined in a lizard. Overall, the combination of the aggregation of caudal fat reserves and position of naturally occurring tail breaks may enable N. metallicus to combine caudal fat storage and tail autotomy with minimal conflict.     

Biogeographic barriers in south‐eastern Australia drive phylogeographic divergence in the garden skink,Lampropholis guichenoti

Aim To investigate the impact of climatic oscillations and recognized biogeographic barriers on the evolutionary history of the garden skink (Lampropholis guichenoti), a common and widespread vertebrate in south‐eastern Australia. Location South‐eastern Australia. Methods Sequence data were obtained from the ND4 mitochondrial gene for 123 individuals from 64 populations across the entire distribution of the garden skink. A range of phylogenetic (maximum likelihood, Bayesian) and phylogeographic analyses (genetic diversity, Tajima’s D, Φ_ST, mismatch distribution) were conducted to examine the evolutionary history and diversification of the garden skink. Results A deep phylogeographic break (c. 14%), estimated to have occurred in the mid–late Miocene, was found between ‘northern’ and ‘southern’ populations across the Hunter Valley in northern New South Wales. Divergences among the geographically structured clades within the ‘northern’ (five clades) and ‘southern’ (seven clades) lineages occurred during the Pliocene, with the location of the major breaks corresponding to the recognized biogeographic barriers in south‐eastern Australia. Main conclusions Climatic fluctuations and the presence of several elevational and habitat barriers in south‐eastern Australia appear to be responsible for the diversification of the garden skink over the last 10 Myr. Further molecular and morphological work will be required to determine whether the two genetic lineages represent distinct species.     

Decline in lizard species diversity,abundance and ectoparasite load across an elevational gradient in the Australian alps

The rapid changes in altitude, and associated habitat, of mountain ecosystems make them ideal natural laboratories for testing the effect of environmental heterogeneity on species assemblage. Our understanding of the sensitivity of Australian reptiles to elevational clines is limited. We examined lizard distribution across three elevation zones (montane, subalpine and alpine), spanning from 900 to 1840 m above sea level, in the Australian alps. We aimed to examine how elevation influences species diversity and abundance, and ectoparasite load, and whether species alter their habitat use amongst different elevational zones. Active searches were conducted across the elevation zones to identify lizard community structure (at least 16 species) across elevational zones, along with skink habitat preferences and the ectoparasite load. Skink diversity and abundance were negatively correlated with increased elevation. The alpine zone had significantly lower diversity and abundance of skinks. Habitat use differed amongst both elevations and species. Ectoparasite prevalence was also significantly diminished in the alpine zone. Ectoparasites only infected a subset of the skink community, with ectoparasite load increasing as the active season progressed. This study provides evidence of the complex interplay between elevation and species diversity, as well as the differences in ectoparasite pressure along elevational gradients in the Australian alps.     

Know when to run,know when to hide: can behavioral differences explain the divergent invasion success of two sympatric lizards?

Invasive species represent a select subset of organisms that have successfully transitioned through each stage of the introduction process (transportation, establishment, and spread). Although there is a growing realization that behavior plays a critical role in invasion success, few studies have focused on the initial stages of introduction. We examined whether differences in the grouping tendencies and exploratory behavior of two sympatric lizard species could contribute to their divergent invasion success. While the nondirected activity of the two species did not differ, the invasive delicate skink (Lampropholis delicata) was found to be more exploratory than the congeneric noninvasive garden skink (L. guichenoti), which enabled it to more effectively locate novel environments and basking site resources. The delicate skink also exhibited a greater tendency to hide, which may act to enhance its probability of ensnarement in freight and cargo and decrease its likelihood of detection during transit. The grouping tendencies of the two species did not differ. Together, our results suggest that while the two species have an equivalent "opportunity" for unintentional human-assisted transportation, several pre-existing behavioral traits may enhance the success of the delicate skink in negotiating the initial stages of the introduction process, and subsequent post-establishment spread.     

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin‐8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B‐gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal ‘9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.