Elena Drosopoulou

Identification of several cytoplasmic HSP70 genes from the Mediterranean mussel (Mytilus galloprovincialis) and their long-term evolution in Mollusca and Metazoa.

The HSP70 protein family consists one of the most conserved and important systems for cellular homeostasis under both stress and physiological conditions. The genes of this family are poorly studied in Mollusca, which is the second largest metazoan phylum. To study these genes in Mollusca, we have isolated and identified five HSP70 genes from Mytilus galloprovincialis (Mediterranean mussel) and investigated their short-term evolution within Mollusca and their long-term evolution within Metazoa. Both sequence and phylogenetic analyses suggested that the isolated genes belong to the cytoplasmic (CYT) group of the HSP70 genes. Two of these genes probably represent cognates, whereas the remaining probably represent heat-inducible genes. Phylogenetic analysis including several molluscan CYT HSP70s reveals that the cognate genes in two species have very similar sequences and form intraspecies phylogenetic clades, differently from most metazoan cognate genes studied thus far, implying either recent gene duplications or concerted evolution. The M. galloprovincialis heat-inducible genes show intraspecies phylogenetic clustering, which in combination with the higher amino acid than nucleotide identity suggests that both gene conversion and purifying selection should be responsible for their sequence homogenization. Phylogenetic analysis including several metazoan HSP70s suggests that at least two types of CYT genes were present in the common ancestor of vertebrates and invertebrates, the first giving birth to the heat-inducible genes of invertebrates, whereas the other to both the heat-inducible genes of vertebrates and the cognate genes of all metazoans. These analyses also suggest that inducible and cognate genes seem to undergo divergent evolution.

A Drosophila hsp70 gene contains long, antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci

A clone isolated from a Drosophila auraria heat-shock cDNA library presents two long, antiparallel, coupled (LAC) open reading frames (ORFs). One strand ORF is 1,929 nucleotides long and exhibits great identity (87.5% at the nucleotide level and 94% at the amino acid level) with the hsp70 gene copies of D. melanogaster, while the second strand ORF, in antiparallel in-frame register arrangement, is 1,839 nucleotides long and exhibits 32% identity with a putative, recently identified, NAD+-dependent glutamate dehydrogenase (NAD+-GDH). The overlap of the two ORFs is 1,824 nucleotides long. Computational analysis shows that this LAC ORF arrangement is conserved in other hsp70 loci in a wide range of organisms, raising questions about possible evolutionary benefits of such a peculiar genomic organization.

The ?-tubulin gene family evolution in the Drosophila montium subgroup of the melanogaster species group

The β1-, β2-, and β3-tubulin genes have been mapped by in situ hybridization on the polytene chromosomes of 11 selected species (15 strains) belonging to the Drosophila montium subgroup. Although the hybridization pattern among the strains of the same species does not differ, this pattern is significantly different among the species. The β-tubulin genes in the montium subgroup seem to be organized in a cluster, or in a semi-cluster, or are completely dispersed. The clustered arrangement is found in the North-Oriental sibling species D. auraria, D. triauraria, and D. quadraria. The semi-clustered arrangement, wherein the β1 and β2 genes are located at the same locus while β3 is at a different one, appears in the South-Oriental species D. bicomuta, D. serrata, and D. birchii, as well as in the Afrotropical species D. diplacantha and D. seguyi. The complete separation of the genes is observed in the Indian species D. kikkawai and D. jambulina and in the Afrotropical species D. vulcana. Based on the above results, a possible mode of evolution of the β-tubulin genes in the montium subgroup is attempted. In addition, phylogenetic relationships among the montium species are discussed.

The heat shock genes in the Drosophila montium subgroup: chromosomal localization and evolutionary implications

Thehsp70, hsp83, hsrω, and thesmall heat shock protein genes were mapped on the polytene chromosomes of six species, representative of the geographical distribution of theDrosophila montium subgroup of themelanogaster species group. In addition, based on hybridization conditions, the putative locus of thehsp68 gene is given. In contrast to the situation in themelanogaster subgroup species, thehsp70 locus is single in themontium species. Thehsp83, hsrω and thesmall hsp loci are also single in themontium genomes studied here, a common feature of allDrosophila species. Among thehsp genes studied, thesmall hsp genes and thehsrω-homologous sequences exhibit a higher degree of divergence between themelanogaster and themontium subgroups. Our results support the idea that themontium subgroup species has a genome organization closer to that of the common ancestor compared with themelanogaster subgroup species.

Isolation, annotation and applications of expressed sequence tags from the olive fly, Bactrocera oleae

The olive fruit fly, Bactrocera oleae, is the major pest of the olive tree. Despite its importance, very little genetic and molecular knowledge is available. The present study is a first attempt to identify and characterize B. oleae expressed sequence tags (ESTs). One hundred and ninety-five randomly selected cDNA clones were isolated and the obtained sequences were annotated through BLASTX similarity searches. A set of 159 unique putative transcripts were functionally assigned using Gene Ontology terms in broad categories of biological process, molecular function and cellular component based on D. melanogaster matches. Moreover, the cytogenetic location of 35 ESTs was determined by in situ hybridization to B. oleae polytene chromosomes. The resulting low-resolution EST map more than doubles the available entry points to the insect’s genome and can assist syntenic comparisons with other distant species. The deduced codon usage of the isolated ESTs suggested a conserved pattern of B. oleae with its closest relatives. Additionally, the comparative analysis of B. oleae ESTs with the homologous D. melanogaster genes led to the development of 17 nuclear EPIC-PCR markers for the amplification of intron sequences of 11 Tephritidae species. Sequencing analysis of several cross-amplified intron sequences revealed a high degree of conservation among Bactrocera species and a varying transferability of the generated markers across the examined genera, suggesting that this method can provide a useful tool for the clarification of phylogenetic relationships among different species, particularly in cases of species complexes.

Cytogenetic analysis of the Ethiopian fruit fly Dacus ciliatus (Diptera: Tephritidae)

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.

Genetic diversity, structure, and size of an endangered brown bear population threatened by highway construction in the Pindos Mountains, Greece

One of the major negative effects of roads is the creation of barriers to the movement of wildlife, ultimately disconnecting populations and increasing extinction risk. We collected genetic data from a threatened brown bear population in the central part of the Pindos mountain range in northwestern Greece to provide information about this, as yet genetically undescribed, population and to evaluate its status prior to the construction of a major highway. We used noninvasive genetic sampling methods and microsatellite analysis to investigate nuclear genetic diversity, population genetic structure, demographic history, relatedness within the population and estimated effective and total population size. Brown bears in the study area were found to possess a relatively high level of nuclear genetic diversity and low levels of inbreeding; the population did not show any signs of substructuring but seems to have gone through a genetic bottleneck in the recent past. The estimated effective population size was 29, and the total population size estimate obtained by two different methods was 33 and 51 individuals, respectively. Our results indicate a good conservation status of this bear population and provide baseline genetic data for the future evaluation of the effects on bears from the construction of a major highway, for monitoring the genetic status of this and other bear populations in Greece and for assessing gene flow in bear populations in southern Europe.

Genetic and cytogenetic analysis of the American cherry fruit fly, Rhagoletis cingulata (Diptera: Tephritidae)

The American eastern cherry fruit fly, Rhagoletis cingulata, a pest of cherries in the western hemisphere, invaded Europe in 1983, and since then dispersed to several European countries. Information on the genetics and cytogenetics of this pest is very scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of R. cingulata are presented here. The mitotic metaphase complement consists of six pairs of chromosomes with the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei and an extrachromosomal heterochromatic mass, which corresponds to the sex chromosomes. The banding patterns and the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between R. cingulata, R. completa and R. cerasi are also proposed, based on the comparison of chromosome banding patterns. Furthermore, the detection and characterization of Wolbachia pipientis in the R. cingulata population studied is presented and the potential correlation with the asynaptic phenomena found in its polytene complement is discussed. In addition, 10 out of 24 microsatellite markers developed for other Rhagoletis species are cross-amplified, evaluated and proposed as useful markers for population and genetic studies in R. cingulata.

Genetic and Cytogenetic Analysis of the Walnut-Husk Fly (Diptera: Tephritidae)

ABSTRACT Genetic and cytogenetic information is an essential basis for understanding the biology of insect pests, as well as for designing modern control strategies. The walnut husk fly, Rhagoletis completa (Cresson) (Diptera: Tephritidae), is an important pest of walnuts (Juglans spp.) in North America and has invaded Europe in the early 1990s. Studies on the genetics and cytogenetics of R. completa are scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of six pairs of chromosomes, the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The banding pattern as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed.

The complete mitochondrial genome of the loggerhead turtle Caretta caretta (Testudines: Cheloniidae): Genome description and phylogenetic considerations

The marine turtle Caretta caretta is a widely distributed species that is facing critical population decline, especially in the Mediterranean rookeries. Molecular markers, such as mitochondrial DNA (mtDNA) sequences, are of great importance for the description and monitoring of turtle migratory populations. The complete sequence of the C. caretta mitochondrial genome is presented here. The genome comprises 16,440 base pairs, containing 37 genes (13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes), and a control region, all organized similar to the majority of vertebrate mitogenomes. MtDNA length polymorphism and heteroplasmy were observed among, and within, individuals due to the variable size of a microsatellite repeat residing at the 3′ end of the control region. The use of the above repeat as a marker for individual fingerprinting is discussed. Furthermore, phylogenetic analyses among Testudines based on complete mitogenomes, as well as among marine turtles based on partial mtDNA sequences, are considered.