Localization of <Emphasis Type="Italic">Dras1</Emphasis> gene in polytene chromosomes of sibling species of <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

The Dras1 gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and their hybrids. A 1037-bp fragment of Dras1 gene from the D. virilis genome was used as the probe. The gene sequence was localized in the region of a 25 A-B disk in chromosome 2 (in accordance with the D. virilis polytene chromosome map (Gubenko and Evgen’ev, 1984).     

Structural comparison of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in the <Emphasis Type="Italic">Drosophila virilis</Emphasis> species group

One of the Drosophila montana hsp70 genes was cloned and sequenced. Its 3′-flanking sequence proved to harbor a fragment of the SGM mobile element. The element was also found in the hsp70 3′-flanking region of and other species of the species group. A reorganization of the cluster with the involvement of full-length SGM was found in a strain. It was assumed that the presence of SGM in the cluster is conserved among species of the group and that SGM played a role in evolutionarily rearrangements of the cluster.     

The relationships among the species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group inferred from the gene <Emphasis Type="Italic">Ras1</Emphasis> sequences

Comparative analysis of a group of closely related Drosophila species (D. virilis, D. lummei, D. novamexicana, D. americana texana, D. flavomontana, D. montana, D. borealis, D. lacicola, D. littoralis, D. kanekoi, and D. ezoana) was conducted based on an incomplete sequence of gene Ras1. The pattern of the relationships among the species corresponded to that expected from analysis of morphological and cytogenetic characters. Statistical data favoring neutrality of the substitutions examined in the Ras1 gene are presented. This character of the gene Ras1 evolution confers more reliability to reconstruction of phylogenetic relationships among closely related species. The resultant tree for main phylads of the group is as follows: D. virilis (D. lummei, D. montana, D. ezoana).     

Evolution and arrangement of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in two closely related species of the <Emphasis Type="Italic">virilis</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

To investigate the genetic basis of differing thermotolerance in the closely related species Drosophila virilis and Drosophila lummei, which replace one another along a latitudinal cline, we characterized the hsp70 gene cluster in multiple strains of both species. In both species, all hsp70 copies cluster in a single chromosomal locus, 29C1, and each cluster includes two hsp70 genes arranged as an inverted pair, the ancestral condition. The total number of hsp70 copies is maximally seven in the more thermotolerant D. virilis and five in the less tolerant D. lummei, with some strains of each species exhibiting lower copy numbers. Thus, maximum hsp70 copy number corresponds to hsp70 mRNA and Hsp70 protein levels reported previously and the size of heat-induced puffs at 29C1. The nucleotide sequence and spacing of the hsp70 copies are consistent with tandem duplication of the hsp70 genes in a common ancestor of D. virilis and D. lummei followed by loss of hsp70 genes in D. lummei. These and other data for hsp70 in Drosophila suggest that evolutionary adaptation has repeatedly modified hsp70 copy number by several different genetic mechanisms.     

RAPD and rep-PCR fingerprinting for characterization of <Emphasis Type="Italic">Bifidobacterium</Emphasis> species

DNA fingerprinting methods, RAPD with 7 random primers, and rep-PCR using both BOXA1R and (GTG)(5) ones, were used for the discrimination of 16 type and collection Bifidobacterium strains of 9 species of human origin, B. animalis ssp. animalis and B. animalis ssp. lactis and 7 Bifidobacterium strains collected in the Culture Collection of Dairy Microorganisms (CCDM). Both RAPD and rep-PCR methods provided similar results. The strains were identified as B. animalis ssp. lactis (6 strains) and B. adolescentis (1 strain). The reclassification of the collection strain CCM 3761 as B. pseudocatenulatum species (previously classified as B. adolescentis) was confirmed.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Chromosomal differentiation of the sibling species <Emphasis Type="Italic">Pichia membranifaciens</Emphasis> and <Emphasis Type="Italic">Pichia manshurica</Emphasis>

The molecular karyotyping analysis of 21 strains within the taxonomic complex Pichia membranifaciens allowed the sibling species P. membranifaciens and P. manshurica, as well as P. deserticola and P. punctispora, to be differentiated. Heterogeneity of the species P. membranifaciens at the variety level is discussed.     

A new <Emphasis Type="Italic">Dactylella</Emphasis> species from <Emphasis Type="Italic">Orbilia alba</Emphasis>

A new Dactylella species, Dactylella alba was isolated from the ascospores of Orbilia alba collected in Wenshan County, Yunnan Province, China. Conidiophores were either not branched or occasionally branched, bearing divergent sterigmata on the tip with single conidium on each. Conidia were elongated ellipsoids, 1–2 septate, mostly 1 septate. By combining the ITS sequence with morphological characteristics, a new anamorphic species is described and illustrated together with its teleomorph.     

Patterns of <Emphasis Type="Italic"> Hermes </Emphasis>transposition in <Emphasis Type="Italic"> Drosophila melanogaster</Emphasis>

Transposable elements are being developed as tools for genomics and for the manipulation of insect genotypes for the purposes of biological control. An understanding of their transposition behavior will facilitate the use of these elements. The behavior of an autonomous Hermes transposable element from Musca domestica in the soma and germ-line of Drosophila melanogaster was investigated using the method of transposon display. In the germ-line, Hermes transposed at a rate of approximately 0.03 jumps per element per generation. Within the soma Hermes exhibited markedly non-random patterns of integration. Certain regions of the genome were distinctly preferred over others as integration targets, while other regions were underrepresented among the integration sites used. One particular site accounted for 4.4% of the transpositions recovered in this experiment, all of which were located within a 2.5-kb region of the actin5C promoter. This region was also present within the Hermes element itself, suggesting that this clustering is an example of transposable element "homing". Clusters of integration sites were also observed near the original donor sites; these represent examples of local hopping. The information content (sequence specificity) of the 8-bp target site was low, and the consensus target site resembles that determined from plasmid-based integration assays.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

Transposon display supports transpositional activity of <Emphasis Type="Italic">P</Emphasis> elements in species of the <Emphasis Type="Italic">saltans</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> gene <Emphasis Type="Italic">Merlin</Emphasis> interacts with the clathrin adaptor protein gene <Emphasis Type="Italic">lap</Emphasis>

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer ^DBB together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

<Emphasis Type="Italic">Elaeocarpus</Emphasis> for <Emphasis Type="Italic">Flora Malesiana</Emphasis>: new taxa and understanding in the <Emphasis Type="Italic">Ganitrus</Emphasis> group

The Ganitrus group of Elaeocarpus L. (incl. Elaeocarpus Sect. Ptilanthus Schltr.) as found in Malesia is examined and some understanding reached. The group is more complex and speciose in eastern Malesia, especially in Papuasia (with the Philippines, Sulawesi and Maluku somewhat less rich) than in western and central Malesia. E. angustifolius Blume (syn. E. ganitrus & E. sphaericus auctt. mult.) — the source of the ornamental beads known as rudraksha of eastern commerce, from which malas or rosaries are made — is considered to be very variable in Malesia and probably varies further in the extremes of its range; outside Malesia the Ganitrus group is represented by ‘E. angustifolius s.l.’ only. The following species are recognised: E. altisectus Schltr. with a new subsp. carrii Coode from New Guinea; E. angustifolius Blume; E. avium Coode sp. nov. from New Guinea; E. buderi Coode; E. dolichostylus Schltr. with E. chloranthus treated as var. chloranthus (A. C. Sm.) Coode from New Guinea, with a new var. elliptifolius Coode from the Talaud/Sangihe Is. and a new var. hentyi Coode (replacing and re-interpreting subsp. collinus Coode) from New Guinea; E. fulvus Elmer; E. kaniensis Schltr.; E. ornatus Coode sp. nov. from New Guinea; E. osiae Coode sp. nov. from Bougainville I.; E. ptilanthus Schltr.; E. ramiflorus Merr. and E. trichopetalus Merr. & Quisumb. — a total of 12 species although the last two are perhaps not truly distinct from E. angustifolius. The stone sculpturing is discussed and some useful characters revealed. Attention is drawn to a collection from mid-altitude in Papua New Guinea, inadequately known but probably a new species in this group.