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.     

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.     

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).     

<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.     

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.     

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.     

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.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

<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.     

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).     

Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

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.     

The role of acoustic signals in courtship behavior of <Emphasis Type="Italic">Drosophila virilis</Emphasis>

The role of the male acoustic signal in courtship behavior in Drosophila virilis was studied by a video-typing method. Three series of the experiments were performed: tests with intact flies, with wingless males and intact females, and with intact males and females with the aristas removed. It was demonstrated that touching and licking were the most prolonged elements of the male courtship. It was noted that removal of the wings in males or aristas in females resulted in an increase in the duration of almost all elements of the courtship behavior (following, touching, licking, singing, and circling) and in a significant decrease in the portion of successful copulations. It was demonstrated that the courtship structure in the experiments with females without aristas changed to a greater extent than in tests with wingless males.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.