Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared 'hollow' because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate-eosinophilic-clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub-adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose 'Hollow Sperm Syndrome (HSS)' to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub-adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through permanova, also indicated significant differences in bacterial communities based on the season.

Histone Deacetylases Regulate Multicellular Development in the Social Amoeba Dictyostelium discoideum

Epigenetic modifications of histones regulate gene expression and lead to the establishment and maintenance of cellular phenotypes during development. Histone acetylation depends on a balance between the activities of histone acetyltransferases and histone deacetylases (HDACs) and influences transcriptional regulation. In this study, we analyse the roles of HDACs during growth and development of one of the cellular slime moulds, the social amoeba Dictyostelium discoideum. The inhibition of HDAC activity by trichostatin A results in histone hyperacetylation and a delay in cell aggregation and differentiation. Cyclic AMP oscillations are normal in starved amoebae treated with trichostatin A but the expression of a subset of cAMP-regulated genes is delayed. Bioinformatic analysis indicates that there are four genes encoding putative HDACs in D. discoideum. Using biochemical, genetic and developmental approaches, we demonstrate that one of these four genes, hdaB, is dispensable for growth and development under laboratory conditions. A knockout of the hdaB gene results in a social context-dependent phenotype: hdaB- cells develop normally but sporulate less efficiently than the wild type in chimeras. We infer that HDAC activity is important for regulating the timing of gene expression during the development of D. discoideum and for defining aspects of the phenotype that mediate social behaviour in genetically heterogeneous groups.

Marking one-summer old whitefish with fluorescent pigment spraying method and results of whitefish stockings in the Gulf of Bothnia

Anadromous whitefish is one of the most important fish species in the Finnish coastal fisheries in the Gulf fo Bothnia. To compensate the lost reproduction due to river damming and to support the fisheries, several million one-summer old whitefish are released yearly into the Gulf of Bothnia. Since there are naturally reproducing whitefish in the Gulf as well, and the wild and stocked fish can not be separated in the catch, stocking impact can only be estimated by marking the stocked fish. Due to the small size and large number of released whitefish, the scattered fishery and large area where the whitefish migrate, most of the traditionally used fish marking methods were either unsuitable (e.g. Carlin-tags) or proved to be too expensive (e.g. coded wire tags). Fluorescent pigment spraying method offers a fast and cost-effective method to mass-mark young fish. However, the results are not always satisfactory due to low long-time retention of the marks in some species. The method has to be tested and proper marking conditions and methods determined for each species. This thesis is based on work that was accomplished while developing the fluorescent pigment spraying method for marking one-summer old whitefish fingerlings, and it draws together the results of mass-marking whitefish fingerlings that were released in the Gulf of Bothnia. Fluorescent pigment spraying method is suitable for one-summer old whitefish larger than 8 cm total length. The water temperature during the marking should not exceed 10o C. Suitable spraying pressure is 6 bars measured in the compressor outlet, and the distance of the spraying gun nozzle should be ca 20 cm from the fish. Under such conditions, the marking results in long-term retention of the mark with low or no mortality. The stress level of the fish (measured as muscle water content) rises during the marking procedure, but if the fish are allowed to recover after marking, the overall stress level remains within the limits observed in normal fish handling during the capture-loading-transport-stocking procedure. The marked whitefish fingerlings are released into the sea at larger size and later in the season than the wild whitefish. However, the stocked individuals migrate to the southern feeding grounds in a similar pattern to the wild ones. The catch produced by whitefish stocking in the Gulf of Bothnia varied between released fingerling groups, but was within the limits reported elsewhere in Finland. The releases in the southern Bothnian Bay resulted in a larger catch than those made in the northern Bothnian Bay. The size of the released fingerlings seemed to have some effect on survival of the fish during the first winter in the sea. However, when the different marking groups were compared, the mean fingerling size was not related to stocking success.

Development of salinity tolerance in rice by constitutive-overexpression of genes involved in the regulation of programmed cell death

Environmental factors contribute to over 70% of crop yield losses worldwide. Of these drought and salinity are the most significant causes of crop yield reduction. Rice is an important staple crop that feeds more than half of the world’s population. However among the agronomically important cereals rice is the most sensitive to salinity. In the present study we show that exogenous expression of anti-apoptotic genes from diverse origins, AtBAG4 (Arabidopsis), Hsp70 (Citrus tristeza virus) and p35 (Baculovirus), significantly improves salinity tolerance in rice at the whole plant level. Physiological, biochemical and agronomical analyses of transgenic rice expressing each of the anti-apoptotic genes subjected to salinity treatment demonstrated traits associated with tolerant varieties including, improved photosynthesis, membrane integrity, ion and ROS maintenance systems, growth rate, and yield components. Moreover, FTIR analysis showed that the chemical composition of salinity-treated transgenic plants is reminiscent of non-treated, unstressed controls. In contrast, wild type and vector control plants displayed hallmark features of stress, including pectin degradation upon subjection to salinity treatment. Interestingly, despite their diverse origins, transgenic plants expressing the anti-apoptotic genes assessed in this study displayed similar physiological and biochemical characteristics during salinity treatment thus providing further evidence that cell death pathways are conserved across broad evolutionary kingdoms. Our results reveal that anti-apoptotic genes facilitate maintenance of metabolic activity at the whole plant level to create favorable conditions for cellular survival. It is these conditions that are crucial and conducive to the plants ability to tolerate/adapt to extreme environments.

Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

The Emerging Phenotype: Ecological Genetics of Color, Form and Sex in the Common Frog

One of the main aims of evolutionary biology is to explain why organisms vary phenotypically as they do. Proximately, this variation arises from genetic differences and from environmental influences, the latter of which is referred to as phenotypic plasticity. Phenotypic plasticity is thus a central concept in evolutionary biology, and understanding its relative importance in causing the phenotypic variation and differentiation is important, for instance in anticipating the consequences of human induced environmental changes. The aim of this thesis was to study geographic variation and local adaptation, as well as sex ratios and environmental sex reversal, in the common frog (Rana temporaria). These themes cover three different aspects of phenotypic plasticity, which emerges as the central concept for the thesis. The first two chapters address geographic variation and local adaptation in two potentially thermally adaptive traits, namely the degree of melanism and the relative leg length. The results show that although there is an increasing latitudinal trend in the degree of melanism in wild populations across Scandinavian Peninsula, this cline has no direct genetic basis and is thus environmentally induced. The second chapter demonstrates that although there is no linear, latitudinally ordered phenotypic trend in relative leg length that would be expected under Allen s rule an ecogeographical rule linking extremity length to climatic conditions there seems to be such a trend at the genetic level, hidden under environmental effects. The first two chapters thus view phenotypic plasticity through its ecological role and evolution, and demonstrate that it can both give rise to phenotypic variation and hide evolutionary patterns in studies that focus solely on phenotypes. The last three chapters relate to phenotypic plasticity through its ecological and evolutionary role in sex determination, and consequent effects on population sex ratio, genetic recombination and the evolution of sex chromosomes. The results show that while sex ratios are strongly female biased and there is evidence of environmental sex reversals, these reversals are unlikely to have caused the sex ratio skew, at least directly. The results demonstrate that environmental sex reversal can have an effect on the evolution of sex chromosomes, as the recombination patterns between them seem to be controlled by phenotypic, rather than genetic, sex. This potentially allows Y chromosomes to recombine, lending support for the recent hypothesis suggesting that sex-reversal may play an important role on the rejuvenation of Y chromosomes.

FGFR1 regulated gene-expression, cell proliferation and differentiation in the developing midbrain and hindbrain

The neuroectodermal tissue close to the midbrain hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) regulates cellular survival, patterning and proliferation in the midbrain (Mb) and rhombomere 1 (R1) of the hindbrain. Signaling molecules of the IsO, such as fibroblast growth factor 8 (FGF8) and WNT1 are expressed in distinct bands of cells around the MHB. It has been previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. In the present study, we have compared the gene expression profiles of wild-type and Fgfr1 mutant embryos. We show that the loss of Fgfr1 results in the downregulation of several genes expressed close to the MHB and in the disappearance of gene expression gradients in the midbrain and R1. Our microarray screen identified several previously uncharacterized genes which may participate in the development of midbrain R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1. As Wnt1 expression at the MHB region requires the FGF signaling pathway, WNT target genes, including Drapc1, were also identified in our screen. The microarray data analysis also suggested that the cells next to the midbrain hindbrain boundary express distinct cell cycle regulators. We showed that the cells close to the border appeared to have unique features. These cells proliferate less rapidly than the surrounding cells. Unlike the cells further away from the boundary, these cells express Fgfr1 but not the other FGF receptors. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.

Ultrastructural features of the early secretory pathway in Trichoderma reesei

We have systematically analysed the ultra structure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFPfusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

Deciphering the Distinct Role for the Metal Coordination Motif in the Catalytic Activity of Mycobacterium smegmatis Topoisomerase I

Mycobacterium smegmatis topoisomerase I (Mstopol) is distinct from typical type IA topoisomerases. The enzyme binds to both single- and double-stranded DNA with high affinity, making specific contacts. The enzyme comprises conserved regions similar to type IA topoisomerases from Escherichia coli and other eubacteria but lacks the typically found zinc fingers in the carboxy-terminal domain. The enzyme can perform DNA cleavage m the absence of Mg2+ but religation needs exogenously added Mg2+. One molecule of Mg2+ tightly bound to the enzyme has no role in DNA cleavage but is needed only for the religation reaction. The toprim. (topoisomerase-primase) domain in MstopoI comprising the Mg2+ binding pocket, conserved in both type IA and type II topoisomerases, was subjected to mutagenesis to understand the role of Mg2+, in different steps of the reaction. The residues D108, D110, and E112 of the enzyme, which form the acidic triad in the DXDXE motif, were changed to alanines. D108A mutation resulted in an enzyme that is Mg2+ dependent for DNA cleavage unlike Mstopol and exhibited enhanced DNA cleavage property and reduced religation activity. The mutant was toxic for cell growth, most likely due to the imbalance in cleavage-religation equilibrium. In contrast, the E112A mutant behaved like wild-type enzyme, cleaving DNA in a Mg2+-independent fashion, albeit to a reduced extent. Intra- and intermolecular religation assays indicated specific roles for D108 and E112 residues during the reaction. Together, these results indicate that the D108 residue has a major role during cleavage and religation, while E112 is important for enhancing the efficiency of cleavage. Thus, although architecturally and mechanistically similar to topoisomerase I from E. coli, the metal coordination pattern of the mycobacterial enzyme is distinct, opening up avenues to exploit the enzyme to develop inhibitors.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDPglucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-D-galactose to alpha-D-galactose and the hitter for epimerization of UDP-galactose to UDP-glucose. Absence of C albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevsiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose. (c) 2006 Elsevier Inc. All rights reserved.

Accumulation of point mutations and reassortment of genomic RNA segments are involved in the microevolution of Puumala hantavirus in a bank vole (Myodes glareolus) population

"The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5x2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2-4.9% for the S segment, 0.2-4.8% for the M segment and 0.2-9.7% for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, 'A' and 'B'. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. SAMALA or SBMBLB.. A substantial proportion (8/40, 20%) of the newly characterized PUUV strains possessed reassortant genomes such as SBMALA, SAMBLB or SBMALB. These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains."

Analysis of the beta-glucoside utilization (bgl) genes of Shigella sonnei: evolutionary implications for their maintenance in a cryptic state

The pattern of expression of the genes involved in the utilization of aryl beta-glucosides such as arbutin and salicin is different in the genus Shigella compared to Escherichia coli. The results presented here indicate that the homologue of the cryptic bgl operon of E. coli is conserved in Shigella sonnei and is the primary system involved in beta-glucoside utilization in the organism. The organization of the bgl genes in 5. sonnei is similar to that of E. coli; however there are three major differences in terms of their pattern of expression. (i) The bglB gene, encoding phospho-beta-glucosidase B, is insertionally inactivated in 5. sonnei. As a result, mutational activation of the silent bgl promoter confers an Arbutin-positive (Arb(+)) phenotype to the cells in a single step; however, acquiring a Salicin-positive (Sal(+)) phenotype requires the reversion or suppression of the bglB mutation in addition. (ii) Unlike in E. coli, a majority of the activating mutations (conferring the Arb(+) phenotype) map within the unlinked hns locus, whereas activation of the E. coli bgl operon under the same conditions is predominantly due to insertions within the bglR locus. (iii) Although the bgl promoter is silent in the wild-type strain of 5. sonnei (as in the case of E. coli), transcriptional and functional analyses indicated a higher basal level of transcription of the downstream genes. This was correlated with a 1 bp deletion within the putative Rho-independent terminator present in the leader sequence preceding the homologue of the bglG gene. The possible evolutionary implications of these differences for the maintenance of the genes in the cryptic state are discussed.