The organization of the alpha-tubulin gene family in the Drosophila montium subgroup of the melanogaster species group.

The alpha 1-, alpha 2-, alpha 3-, and alpha 4-tubulin genes have been mapped by in situ hybridization to the polytene chromosomes of five species representative of the Drosophila montium subgroup geographical distribution. A lambda phage clone containing alpha 1-tubulin specific sequences was isolated from a genomic DNA library of Drosophila auraria and its restriction endonuclease pattern is presented. Both well-characterized heterologous and homologous probes were used to assess orthogonality of gene members between species groups. The in situ hybridization pattern observed in all species studied is consistent with that of Drosophila melanogaster, since alpha 1-, alpha 2-, and alpha 3-tubulin genes are located on the same polytene arm, and the alpha 4-tubulin gene is found on a different arm. Cross-hybridization was observed among alpha 1-, alpha 2-, and alpha 3-tubulin specific sequences in all species studied, using either heterologous or homologous probes. However, unlike D. melanogaster, in all montium species studied, both alpha 1- and alpha 3-tubulin specific probes hybridize to the same polytene band, indicating a clustered organization of the above genes. The chromosomal organization of this gene family would suggest that taxa within the montium subgroup are closer to their common ancestors than are the taxa in the melanogaster species group. A mode of evolution for this gene family in Drosophila is proposed.

Induction of a subgroup of acute phase protein genes in mouse liver by hyperthermia.

We have demonstrated that two members of the acute phase reactant family of positively regulated genes, alpha 1-acid glycoprotein (AGP-1 and AGP-2) and C-reactive protein (CRP) are induced by hyperthermia, while two others, the serum amyloid A (SAA) and alpha 1-antitrypsin (AT) genes, are not. Albumin (ALB), a negative acute phase reactant gene, is also induced by hyperthermia. The AGP-1, AGP-2, and CRP genes require glucocorticoids, but not IL-6, IL-1 beta or TNF alpha in response to hyperthermia. As with LPS, the C/EBP beta mRNA levels increased, while the C/EBP alpha mRNA levels decreased in response to LPS. In contrast to the LPS response, C/EBP delta was unchanged. Protein pool levels and DNA-binding activities of the 35 and 20 kDa C/EBP beta isoforms increase, whereas protein pool levels of the 42 kDa C/EBP alpha decrease and the 30kDa remained high. These studies suggest that the synthesis of specific C/EBP alpha and C/EBP beta isoforms is induced by hyperthermia, and that the regulation of the AGP-1 and AGP-2 genes during heat stress may involve one of these isoforms. The difference between the responses to hyperthermia and LPS is that the former, may not involve the participation of cytokines. Furthermore, since cis-acting heat shock elements (HSE) are located in the promoter regions of the ALB, CRP, and C/EBP beta genes, these regulatory sequences may be involved in the in vivo activation of these genes by hyperthermia.

The heat-shock gene hsp83 of Drosophila auraria: genomic organization, nucleotide sequence, and long antiparallel coupled ORFs (LAC ORFs).

The genomic organization of the hsp83 gene of Drosophila auraria, a far-eastern endemic species belonging to the montium subgroup of the melanogaster species group, is presented here. Based on in situ hybridization on polytene chromosomes, cDNA and genomic clone mapping, nucleotide sequencing, and genomic Southern analysis, hsp83 is shown to be present as a single-copy gene at locus 64B on the 3L chromosome arm in D. auraria. This gene is organized into two exons separated by a 929-bp intron. The first exon represents the mRNA leader sequence and is not translated, while the coding region, having a length of 2,151 bp, is solely included in the second exon. Nucleotide sequence comparisons of D. auraria hsp83 with homologous sequences from other organisms show high conservation of the coding region (88-92% identity) in the genus Drosophila, in addition to the conserved genomic organization of two-exons-one-intron, of comparable size and arrangement. A phylogenetic tree based on the protein sequences of homologous genes from representative organisms is in accord with the accredited phylogenetic position of D. auraria. In the hsp83 gene region, a second case of long antiparallel coupled open reading frames (LAC ORFs) for this species was found. The antiparallel to the hsp83 gene ORF is 1,554 bases long, while the two ORFs overlap has a size of 1,548 bp. The anti-hsp83 ORF does not show significant homology to any known gene sequences. In addition, no similar LAC ORF structures were found in homologous gene regions of other organisms.

The hsp70 locus of Drosophila auraria (montium subgroup) is single and contains copies in a conserved arrangement.

The restriction endonuclease pattern of a number of hsp70-homologous clones isolated from a library of heat shock cDNA from Drosophila auraria, a species belonging to the montium subgroup of the melanogaster species group, reveals two types of clones, A and B, differing in a single restriction site. Both types, as well as hsp70-specific probes derived from both hsp70 loci of Drosophila melanogaster, hybridize in situ with a single band at region 32 A of the 2L polytene arm, indicating a clustered organization of the hsp70 gene copies in D. auraria. The longest type B clone was sequenced and it was found that one strand contains an open reading frame (ORF) exhibiting great identity with a previously described hsp70 gene of D. auraria (now denoted as type A) and with its counterparts of D. melanogaster, while its second strand, unlike the type A clone, does not contain a long antiparallel coupled ORF (LAC ORF) because of a base substitution resulting in a premature stop codon. After additional data had been derived from isolation and characterization of hsp70-homologous genomic clones, together with Southern analysis of genomic DNA, we found that two hsp70 gene copies are present at the above locus of D. auraria with an inverted tandem repeat organization, while the presence of a third hsp70 gene is not clearly evident. The above results are compared with those observed at the homologous loci of some melanogaster subgroup species (D. melanogaster and its sibling species), in which, however, the hsp70 locus is duplicated, and with the more distantly related Dipteran Anopheles albimanus.

The glutamate dehydrogenase, E74 and putative actin gene loci in the Drosophila montium subgroup. Chromosomal homologies among the montium species and D. melanogaster.

DNA-specific sequences from an enzyme-coding gene (glutamate dehydrogenase, gdh), a regulatory protein-coding gene (E74) and genes of the actin family were mapped by in situ hybridization on the polytene chromosomes of six species representative of the geographical distribution of the Drosophila montium subgroup of the melanogaster species group. In all species studied, one hybridization signal was detected for the gdh and E74 genes, and seven signals for the actin genes. The distribution of the actin-related loci in five montium species is similar to that of the other Drosophila species studied so far, although they present an extra signal. This distribution differs in the sixth montium species studied, D. kikkawai. Taking into account the present results, as well as previous data obtained mainly by in situ hybridizations, homologies among the polytene chromosomes of the montium subgroup species, as well as between these species and D. melanogaster, were also established.

Variations in the heat-induced protein pattern of several Drosophila montium subgroup species (Diptera:Drosophilidae).

After temperature elevation, the newly synthesized polypeptides from several Drosophila montium subgroup species, of the melanogaster species group, were analyzed in denaturing acrylamide gels. The pattern obtained is characteristic of the heat shock response already documented for many other Drosophila species, although the relative electrophoretic mobility of the "small" heat shock proteins exhibits a species-specific pattern. Based on the above pattern, the montium species are placed in three distinct groups. The present data is consistent with that previously used to propose a northeast to southwest evolutionary mode of expansion for the montium subgroup species.

Heat shock gene expression during recovery after transient cold shock in Drosophila auraria (Diptera: Drosophilidae).

Newly synthesized polypeptides during recovery from prolonged cold treatment (0 degree to -1 degree C) of Drosophila auraria, a montium subgroup species, of the melanogaster species group, were analysed in denaturing polyacrylamide gels. In addition, during the cold shock recovery period, Northern analysis of the hsp83 mRNA was performed. A significant induction of two polypeptides, which exhibited electrophoretic mobilities, with the heat inducible 83 and 70 kD hsp83 and hsp70 was detected, but no such induction was evident in the so-called 'small' hsp genes. These results are compared and discussed with those observed in D. melanogaster.

The Drosophila montium subgroup species. Phylogenetic relationships based on mitochondrial DNA analysis.

Mitochondrial DNA (mtDNA) restriction site maps for three Drosophila montium subgroup species of the melanogaster species group, inhabiting Indian and Afrotropical montium subgroup territories, were established. Taking into account previous mtDNA data concerning six oriental montium species, a phylogeny was established using distance-matrix and parsimony methods. Both genetic diversity and mtDNA size variations were found to be very narrow, suggesting close phylogenetic relationships among all montium species studied. The phylogenetic trees that were constructed revealed three main lineages for the montium subgroup species studied: one consisting of the Afrotropical species Drosophila seguyi, which is placed distantly from the other species, one comprising the north-oriental (Palearctic) species, and one comprising the southwestern (south-oriental, Australasian, Indian, and Afrotropical) species. The combination of the mtDNA data presented here with data from other species belonging to the melanogaster and obscura subgroups revealed two major clusters: melanogaster and obscura. The melanogaster cluster is further divided into two compact lineages, comprising the montium subgroup species and the melanogaster complex species; the species of the other complex of the melanogaster subgroup, yakuba, disperse among the obscura species. The above grouping is in agreement with the mtDNA size variations of the species. Overall, among all subgroups studied, the species of the montium subgroup seem to be the most closely related.

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

The hsp70, hsp83, hsromega, and the small heat shock protein genes were mapped on the polytene chromosomes of six species, representative of the geographical distribution of the Drosophila montium subgroup of the melanogaster species group. In addition, based on hybridization conditions, the putative locus of the hsp68 gene is given. In contrast to the situation in the melanogaster subgroup species, the hsp70 locus is single in the montium species. The hsp83, hsromega and the small hsp loci are also single in the montium genomes studied here, a common feature of all Drosophila species. Among the hsp genes studied, the small hsp genes and the hsromega-homologous sequences exhibit a higher degree of divergence between the melanogaster and the montium subgroups. Our results support the idea that the montium subgroup species has a genome organization closer to that of the common ancestor compared with the melanogaster subgroup species.

The Afrotropical Drosophila montium subgroup: Balbiani ring 1, polytene chromosomes, and heat shock response of Drosophila vulcana.

A detailed photographic map of the salivary gland polytene chromosomes of Drosophila vulcana, an Afrotropical species of the montium subgroup of the melanogaster group, is presented, along with chromosomal rearrangements, such as reverse tandem duplications and inversions, the well-formed Balbiani ring 1, and the most prominent puffs during normal larval and white prepupal development and after ecdysone treatment. In addition, the heat inducible protein and puffing pattern and the loci of the major heat shock genes, namely, hsp70, hsp83, the "small" hsps, and a putative hsp68, of this species were studied. In the light of the data revealed by the above studies, phylogenetic relationship among the montium subgroup species are attempted.

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 beta-tubulin gene family evolution in the Drosophila montium subgroup of the melanogaster species group.

The beta 1-, beta 2-, and beta 3-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 beta-tubulin genes in the montium subgroup seem to be organized in a cluster, or in a semicluster or are completely dispersed. The clustered arrangements is found in the North-Oriental sibling species D. auraria, D. triauraria, and D. quadraria. The semiclustered arrangement, wherein the beta 1 and beta 2 genes are located at the same locus while beta 3 is at a different one, appears in the South-Oriental species D. bicornuta, 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 beta-tubulin genes in the montium subgroup is attempted. In addition, phylogenetic relationships among the montium species are discussed.

Cytotaxonomic differentiation of the afrotropical Drosophila montium subgroup: D. diplacantha and D. seguyi. The major role of reverse tandem duplications.

Aiming to establish phylogenetic relationships among species of the montium subgroup, detailed polytene chromosome maps are given showing intraspecific polymorphism and ecdysone induced larval puffing pattern profiles of two Afrotropical members of this subgroup, Drosophila diplacantha and D. seguyi. Both species exhibit two unique characteristics that define the montium subgroup, namely, a large number of reverse tandem duplications and a progressive darkening of anterior spiracles of the late third instar larvae, which is accompanied by a definite temporal and spatial puffing pattern of the salivary gland chromosomes. In contrast with the well-formed Balbiani ring 1 (BR1) observed in Oriental and Indian montium species, BR1 exhibits a different developmental profile in D. diplacantha, while it is obscured in D. seguyi. Although phyletic comparisons of five species from five different complexes within the subgroup show some conservation in banding and puffing pattern homologies, an analysis to assign map sections by sequential rearrangements remains unresolved at this time. The evolution of the subgroup is discussed in relation with the sharing of reverse tandem duplications, especially those including the montium BRs.

The beta-tubulin genes of Drosophila auraria are arranged in a cluster.

When the beta 1-, beta 2- and beta 3-tubulin-specific DNAs from Drosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes of Drosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a lambda EMBL3 genomic library of D. auraria, and they all contain beta-tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.

Mitochondrial DNA evolution in the Montium-species subgroup of Drosophila.

Mitochondrial DNA (mtDNA) restriction-site maps for six species (10 strains) of the Drosophila montium subgroup were established. A total of 50 restriction sites were mapped, corresponding to 1.67% of the mtDNA genome. On the basis of differences in the restriction sites, nucleotide divergence (delta) was calculated for each pair of species (strains), and phylogenetic trees were constructed by using distance-matrix and parsimony methods. Comparison of the resultant phylogenetic trees shows that the sibling species D. auraria and D. quadraria are closely related. At the other extreme, considerable divergence was observed between the two strains of D. serrata and between D. serrata and D. birchii, a finding that contrasts with their grouping within the same species complex. Nevertheless, our data indicate that these six oriental montium species are rather closely related.

Rapid isolation of mitochondrial DNA. Mitochondrial DNA from Drosophila serrata.

A simple and rapid method for isolation of high quality mitochondrial DNA (mtDNA) is presented in this report. Using this method, isolation and restriction site maps for 10 enzymes of the mtDNA of Drosophila serrata were established.

Insecticidal effects of essential oils. A study of the effects of essential oils extracted from eleven Greek aromatic plants on Drosophila auraria.

Effects of the essential oils (EOs) extracted from eleven aromatic plants belonging to the Lamiaceae family (common in the Greek flora) were examined upon three different developmental stages of Drosophila auraria. All of the EOs examined exhibited insecticidal effects, either by preventing egg hatching, or by causing the death of larvae and adult flies. In several cases, malformation and/or prohibition of puparium formation was also observed.

The polytene chromosomes of Drosophila triauraria and D. quadraria, sibling species of D. auraria.

The polytene chromosomes of Drosophila triauraria and D. quadraria, two of the sibling species of D. auraria, were examined. The polytene chromosomes of all three species exhibit very clear homology. Unlike the stock of D. auraria that we studied, D. triauraria and D. quadraria carry heterozygous paracentric inversions. In both species, 2R and 3R are the arms where these inversions are concentrated. In addition, in D. quadraria, the 3L chromosome arm is very complicated because of heterozygous inversions. The mode of inheritance of these rearrangements was studied. A homozygous strain for all chromosome arms of D. triauraria was isolated, while a homozygous strain was obtained only for the arms X, 2L, 3L, and 4 of D. quadraria. Like D. auraria, both species show a large number of inverted tandem duplications in the paired condition, even in the chromosomes of their hybrids. Small deletions were also detected, one of which, in D. triauraria, is homozygous terminal. Hypotheses are discussed concerning the relationships of the species and the existence of inverted tandem duplications.

The Balbiani ring and the polytene chromosomes of Drosophila bicornuta.

A study of the BR1 and of the most prominent puffs during larval development and after in vitro ecdysterone treatment, as well as of the banding pattern and inverted tandem chromosomal duplications of the salivary gland chromosomes of Drosophila bicornuta, is presented in this report. These data are compared and discussed with those of D. auraria and D. serrata, two other montium species.

The prominent role of ectopic pairing in the toroidal conformation of Drosophila polytene chromosome bands.

An examination of the polytene chromosomes of some Drosophila species from the montium subgroup, namely, D. auraria, D. serrata, D. jambulina and D. bicornuta, revealed an intimate relationship between ectopic pairing and the toroidal conformation of the polytene chromosome bands. The possible significance of these events is discussed.