Quantitative model for prediction of hydrodynamic size of nonionic reverse micelles.

The sizes of nonionic reverse micelles were investigated as a function of the molecular structure of the surfactant, the type of oil, the total concentration of surfactant [NP], the ratio of surfactant to total surfactant (r), the water to surfactant molar ratio (omega), temperature, salt concentration, and polar phase. The basis of our investigation was a mixture of nonylphenol polyethoxylates--NP4 and NP7, various polar phases, and several oils. Micelle sizes were determined using dynamic light scattering (DLS). A central composite experimental design was used to quantitatively model micelle size as a function of omega, surfactant concentration, and r. The model has demonstrated the capability of predicting the mean diameter of micelles from 4 to 13 with a precision of +/-2 nm as measured by DLS. This quantitative correlation between the size of reverse micelles and the synthetic variables provides the foundation for choosing experimental conditions to control reverse micelle size. In turn, this allows control of the size of nanoparticles synthesized within them.

[The unusual mobile element Penelope and its behavior in distant Drosophila species]. / Neobychnyi mobil'nyi élement Penelope i ego povedenie v otdalennykh vidakh Drozofily.

The retroelement Penelope isolated from Drosophila virilis has a very unusual structure and codes for reverse transcriptase and an endonuclease belonging to the UvrC type. As shown previously, Penelope is a key element in induction of the hybrid dysgenesis syndrome described in D. virilis, which also involves mobilization of several unrelated mobile element families. Here we report a successful introduction of Penelope into the D. melanogaster genome by P element-mediated transformation. In the new host genome, Penelope is actively transcribed producing major transcript which coincides with that detected in dysgenic hybrids of D. virilis. In situ hybridization on D. melanogaster polytene chromosomes and Southern blotting revealed multiple transpositions of Penelope in the transformed D. melanogaster strains. We determined the structure of six Penelope copies inserted into D. melanogaster chromosomes. Some transformed D. melanogaster strains showed dysgenesis effects similar to those observed in hybrids from D. virilis dysgenic crosses.

Varicose veins possess greater quantities of MMP-1 than normal veins and demonstrate regional variation in MMP-1 and MMP-13.

BACKGROUND: Studies have reported that structural proteins such as elastin and collagen are decreased in varicose veins compared to normal controls. We hypothesized that the changes observed in varicose vein wall composition may be related to alterations in extracellular matrix remodeling proteins, such as the matrix metalloproteases and serine proteases. In addition we hypothesized that there may be regional variation in the expression of these enzymes within the leg. PATIENTS AND MATERIALS: One-centimeter segments of the proximal and distal greater saphenous vein (GSV) were obtained from patients undergoing ligation and stripping for venous insufficiency (vv) (n = 15) or GSV harvest in conjunction with coronary artery bypass grafting (CABG) (n = 7). All vv patients had incompetence of the GSV by color flow duplex. Vein specimens were examined for MMP-1, 3, and 13, tryptase, and GAPDH mRNA using semiquantitative RT-PCR analysis. Quantification of MMP-1 and 13 (active/latent forms) and tryptase was performed using Western blot analysis. Western blots were analyzed using scanning densitometry and standardized to normal controls and values expressed as the median densitometric index (D.I.). Nonparametric statistical methods (Wilcoxan signed rank test and Mann-Whitney U test) were used for analysis. RESULTS: We were able to amplify MMP-1, MMP-13, and tryptase mRNA from both proximal and distal segments of all greater saphenous veins studied. MMP-3 mRNA, however, was not found in either segment of any of the veins examined. A semiquantitative analysis of RT-PCR products comparing the ratio of MMP-1, MMP-13, or tryptase mRNA to GAPDH mRNA showed no difference between cases and controls nor proximal vs distal vein segments. Western blot analysis revealed larger quantities of MMP-1 in varicose veins than in nondiseased veins from CABG patients (48.0 +/- 36.7 D.I. vs 12.5 +/- 6.8 D.I., P = 0.036). Investigation into the regional variation of proteases revealed lower amounts of MMP-1 in distal than in proximal vein segments (37.9 +/- 35.0 D.I. vs 44.1 +/- 41.6 D.I., P = 0.01). Similarly, we found significantly less MMP-13 in distal segments of varicose veins than in proximal segments (152.8 +/- 130.0 D.I. vs 206.7 +/- 173.3 D.I., P = 0.006). CONCLUSIONS: This study found that MMP-1 protein is increased in varicose veins when compared to controls despite no differences in mRNA expression. In addition we found that there is regional variation of MMP-1 and MMP-13 in diseased varicose veins. Lower leg veins have significantly reduced amounts of these proteolytic enzymes when compared to veins of the upper thigh. These data suggest that posttranscriptional regulatory controls could be responsible for the observed differences.

Phylogeny of the subgenus sophophora (Diptera: drosophilidae) based on combined analysis of nuclear and mitochondrial sequences.

Sequences from the nuclear (nu) alcohol dehydrogenase gene, the nu 28S ribosomal RNA locus, and the mitochondrial cytochrome oxidase II gene were used both individually and in combined analyses to infer the phylogeny of the subgenus Sophophora (Diptera: Drosophilidae). We used several optimality criteria, including maximum likelihood, maximum parsimony, and minimum evolution, to analyze these partitions to test the monophyly of the subgenus Sophophora and its four largest species groups, melanogaster, obscura, saltans, and willistoni. Our results suggest that the melanogaster and obscura species groups are each monophyletic and form a closely related clade. The Neotropical clade, containing the saltans and willistoni species groups, is also recovered, as previous studies have suggested. While the saltans species group is strongly supported as monophyletic, the results of several analyses indicate that the willistoni species group may be paraphyletic with respect to the saltans species group.

[Structure and evolutionary role of the Penelope mobile element in Drosophila species of the virilis group]. / Struktura i évoliutsionnaia rol' mobil'nogo élementa Penelopa u vidov Drosophila gruppy virilis.

The mobile element Penelope is activated and mobilizes several other transposons in dysgenic crosses in Drosophila virilis. Its structure proved to be complex and to vary greatly in all examined species of the virilis group. Phylogenetic analysis of the reverse transcriptase (RT) domain assigned Penelope to a new branch, rather than to any known family, of LTR-lacking retroelements. Amino acid sequence analysis showed that the C-terminal domain of the Penelope polyprotein is an active endonuclease, which is related to intron-encoded endonucleases and to bacterial repair endonuclease UrvC, and may act as an integras. Retroelements coding for a putative endonuclease that differs from typical integrase have thus far not been known. The N-terminal domain of the Penelope polyprotein was shown to contain a protease with significant homology to HIV-1 protease. Phylogenetic analysis divided the Penelope copies from several virilis species into two subfamilies, one including virtually identical full-length copies, and the other comprising highly divergent defective copies. The results suggest both vertical and horizontal transfer of the element. Possibly, Penelope invasion recurred during evolution and contributed to genome rearrangement in the virilis species. Chromosome aberrations detected in D. virilis, which is now being invaded by Penelope, is direct evidence for this assumption.

The structure and evolution of Penelope in the virilis species group of Drosophila: an ancient lineage of retroelements.

The Penelope element is the key element responsible for mobilization of other transposable elements in the course of hybrid dysgenesis in Drosophila virilis. Penelope has an unusually complex, highly variable organization in all studied species of the virlis group. Thc BRIDGE1 element from the fish Fugu rubripes is homologous to Penelope, and database searches detected additional homologous sequences among Expressed Sequence Tags from the flatworm Schistosoma mansonii and the nematode Ancylostoma caninum. Phylogenetic analysis shows that the reverse transcriptase of the Penelope group does not belong to any of the characterized major retroelement lineages, but apparently represents a novel branch of non-LTR retroelements. Sequence profile analysis results in the prediction that the C-terminal domain of the Penelope polyprotein is an active endonuclease related to intron-encoded endonucleases and the bacterial repair endonuclease UvrC, which could function as an integrase. No retroelements containing a predicted endonuclease of this family have been described previously. Phylogenetic analysis of Penelope copies isolated from several species of the virilis group reveals two subfamilies of Penelope elements, one of which includes full-length copies whose nucleotide sequences are almost identical, whereas the other one consists of highly diverged defective copies. Phylogenetic analysis of Penelope suggests both vertical transmission of the element and probable horizontal transfers. These findings support the notion that Penelope invasions occurred repeatedly in the evolution of the virilis group.

Perspective: transposable elements, parasitic DNA, and genome evolution.

The nature of the role played by mobile elements in host genome evolution is reassessed considering numerous recent developments in many areas of biology. It is argued that easy popular appellations such as "selfish DNA" and "junk DNA" may be either inaccurate or misleading and that a more enlightened view of the transposable element-host relationship encompasses a continuum from extreme parasitism to mutualism. Transposable elements are potent, broad spectrum, endogenous mutators that are subject to the influence of chance as well as selection at several levels of biological organization. Of particular interest are transposable element traits that early evolve neutrally at the host level but at a later stage of evolution are co-opted for new host functions.

Drosophila mediopunctata P elements: a new example of horizontal transfer.

Sequences homologous to the P element of Drosophila melanogaster were previously identified in Drosophila mediopunctata, a member of the tripunctata group, subgenus Drosophila. We report here that the P element is present in about three to five copies in the D. mediopunctata genome. While one of the insertion sites appears to be fixed, others may be polymorphic, indicating relatively recent P element activity. Phylogenetic analysis revealed that the D. mediopunctata element belongs to the canonical subfamily of P elements and that divergence of the D. mediopunctata element from other members of this subfamily ranges from 2% to 5% at the nucleotide level. This is the first report of a canonical P element outside the subgenus Sophophora. Based primarily on the striking incongruence between P element and host species phylogenies, the presence of a canonical P element in D. mediopunctata is most likely explained by horizontal transfer between species.

Mobile elements and chromosomal evolution in the virilis group of Drosophila.

Species of the virilis group of Drosophila differ by multiple inversions and chromosome fusions that probably accompanied, or led to, speciation. Drosophila virilis has the primitive karyotype for the group, and natural populations are exceptional in having no chromosomal polymorphisms. We report that the genomic locations of Penelope and Ulysses transposons are nonrandomly distributed in 12 strains of D. virilis. Furthermore, Penelope and Ulysses insertion sites in D. virilis show a statistically significant association with the breakpoints of inversions found in other species of the virilis group. Sixteen newly induced chromosomal rearrangements were isolated from the progeny of D. virilis hybrid dysgenic crosses, including 12 inversions, 2 translocations, and 2 deletions. Penelope and Ulysses were associated with the breakpoints of over half of these new rearrangements. Many rearrangement breakpoints also coincide with the chromosomal locations of Penelope and Ulysses insertions in the parental strains and with breakpoints of inversions previously established for other species of the group. Analysis of homologous sequences from D. virilis and Drosophila lummei indicated that Penelope insertion sites were closely, but not identically, located at the nucleotide sequence level. Overall, these results indicate that Penelope and Ulysses insert in a limited number of genomic locations and are consistent with the possibility that these elements play an important role in the evolution of the virilis species group.

Horizontal transfer and selection in the evolution of P elements.

The roles of selection and horizontal transfer in the evolution of the canonical subfamily of P: elements were studied in the saltans and willistoni species groups of the genus Drosophila (subgenus Sophophora). We estimate that the common ancestor of the canonical P: subfamily dates back 2-3 Myr at the most, despite the much older age (more than 40 Myr) of the P: family as a whole. The evolution of the canonical P: subfamily is characterized by weak selection at nonsynonymous sites. These sites have evolved at three quarters the rate of synonymous sites, in which no selective constraints were detected. Their recent horizontal transfer best explains the high degree of similarity among canonical P: elements from the saltans and willistoni species groups. These results are consistent with a model of P:-element evolution in which selective constraints are imposed at the time of horizontal transfer. Furthermore, it is estimated that the spread and diversification of the canonical subfamily involved a minimum of 11 horizontal transfer events among the 18 species surveyed within the past 3 Myr. The presence of multiple P: subfamilies in the saltans and willistoni species groups is likely to be the result of multiple invasions that have previously swept through these taxa in a succession of horizontal transfer events. These results suggest that horizontal transfer among eukaryotes might be more common than anticipated.

Invasion of Drosophila virilis by the Penelope transposable element.

The Penelope family of transposable elements (TEs) is broadly distributed in most species of the virilis species group of Drosophila. This element plays a pivotal role in hybrid dysgenesis in Drosophila virilis, in which at least four additional TE families are also activated. Here we present evidence that the Penelope family of elements has recently invaded D. virilis. This evidence includes: (1) a patchy geographical distribution, (2) genomic locations mainly restricted to euchromatic chromosome arms in various geographical strains, and (3) a high level of nucleotide similarity among members of the family. Two samples from a Tashkent (Middle Asia) population of D. virilis provide further support for the invasion hypothesis. The 1968 Tashkent strain is free of Penelope sequences, but all individuals collected from a 1997 population carry at least five Penelope copies. Furthermore, a second TE, Ulysses, has amplified and spread in this population. These results provide evidence for the Penelope invasion of a D. virilis natural population and the mobilization of unrelated resident transposons following the invasion.

A P element-homologous sequence in the house fly, Musca domestica.

Sequences homologous to the P transposable element have been identified in Musca domestica. Sequence analysis of a genomic clone (Md-P1) indicates that, although the house fly P element has lost its coding capacity, the basic general structure of drosophilid P elements is present. The house fly P element sequence shares a number of structural features with that from the blow fly, Lucilia cuprina, including a large intron separating exons 1 and 2, two additional introns interrupting exon 2 and the apparent absence of inverted repeat termini. Within a relatively well-conserved central region, the house fly sequence shows 59% similarity to the D. melanogaster P element, but distal regions are more diverged. Southern blot analysis of several strains indicated the presence of at least four P element copies.

Distribution and evolution of mobile elements in the virilis species group of Drosophila.

The distributions of Penelope and Ulysses, two transposable elements that can induce hybrid dysgenesis, were studied in several species groups of Drosophila. No significant hybridization to Penelope and Ulysses probes was detected by Southern blot analyses of species outside the virilis group. In contrast, both element families have had a long residence in all species of the virilis species group, as indicated by their strong presence in the heterochromatic chromocenter. Except for D. kanekoi, D. lummei, and some strains of D. virilis, species of the group carry full-sized, and at least potentially functional, copies of both element families. Consistent with the occurrence of recent transposition, Penelope and Ulysses elements are located at different chromosomal sites in different geographical strains of the same species. A total of 79 Penelope and 47 Ulysses euchromatic insertion sites were localized to chromosomal subsections in species of the virilis group. Highly significant deviations from independence of the distributions of Penelope and Ulysses and previously established inversion breakpoints were documented, suggesting that these transposable elements may have played an important role in genomic reorganization and evolution of the virilis species group, which is especially rich in karyotypic variation.

How valuable are model organisms for transposable element studies?

Model organisms have proved to be highly informative for many types of genetic studies involving 'conventional' genes. The results have often been successfully generalized to other closely related organisms and also, perhaps surprisingly frequently, to more distantly related organisms. Because of the wealth of previous knowledge and their availability and convenience, model organisms were often the species of choice for many of the earlier studies of transposable elements. The question arises whether the results of genetic studies of transposable elements in model organisms can be extrapolated in the same ways as those of conventional genes? A number of observations suggest that special care needs to be taken in generalizing the results from model organisms to other species. A hallmark of many transposable elements is their ability to amplify rapidly in species genomes. Rapid spread of a newly invaded element throughout a species range has also been demonstrated. The types and genomic copy numbers of transposable elements have been shown to differ greatly between some closely related species. Horizontal transfer of transposable elements appears to be more frequent than for nonmobile genes. Furthermore, the population structure of some model organisms has been subject to drastic recent changes that may have some bearing on their transposable element genomic complements. In order to initiate discussion of this question, several case studies of transposable elements in well-studied Drosophila species are presented.

Molecular evolution of P transposable elements in the Genus drosophila. II. The obscura species group.

A phylogenetic analysis of P transposable elements in the Drosophila obscura species group is described. Multiple P sequences from each of 10 species were obtained using PCR primers that flank a conserved region of exon 2 of the transposase gene. In general, the P element phylogeny is congruent with the species phylogeny, indicating that the dominant mode of transmission has been vertical, from generation to generation. One manifestation of this is the distinction of P elements from the Old World obscura and subobscura subgroups from those of the New World affinis subgroup. However, the overall distribution of elements within the obscura species group is not congruent with the phylogenetic relationships of the species themselves. There are at least four distinct subfamilies of P elements, which differ in sequence from each other by as much as 34%, and some individual species carry sequences belonging to different subfamilies. P sequences from D. bifasciata are particularly interesting. These sequences belong to two subfamilies and both are distinct from all other P elements identified in this survey. Several mechanisms are postulated to be involved in determining phylogenetic relationships among P elements in the obscura group. In addition to vertical transmission, these include retention of ancestral polymorphisms and horizontal transfer by an unknown mating-independent mechanism.

Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences.

Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group.

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.