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HZE particle effects in space.

Among the various particulate components of ionizing radiation in space, heavy ions (the so-called HZE particles) have been of special concern to radiobiologists. To understand the ways by which HZE particles of cosmic radiation interact with biological systems, methods have been developed to precisely localize the trajectory of an HZE particle relative to the biological object and to correlate the physical data of the particle with the biological effects observed along its path. In a variety of test systems, injuries were traced back to the traversal of a single HZE particle, such as somatic mutations, and chromosomal aberrations in plant seeds, development disturbances and malformations in insect and salt shrimp embryos, or cell death in bacterial spores. In the latter case, a long-ranging killing effect around the particle's track was observed. Whereas, from spaceflight experiments, substantial infomation has been accumulated on single HZE particle effects in resting systems and in a few embryonic systems, there is a paucity of data on cosmic radiation effects in whole tissues or animals, especially mammalians.

Animals↗

Balbiani ring 3 in Chironomus tentans encodes a 185-kDa secretory protein which is synthesized throughout the fourth larval instar.

We have continued to map and identify genes encoding a family of secretory proteins. These proteins are synthesized in larval salivary glands of the midge, Chironomus tentans, and assemble in vivo into insoluble silk-like threads. The genes for several secretory proteins exist in Balbiani rings (BRs) on salivary-gland polytene chromosomes. A randomly primed cDNA clone, designated pCt185, hybridized in situ to BR3 and was shown on Northern blots to originate from a salivary gland-specific 6-kb poly(A) + RNA. The partial cDNA sequence contained 483 nucleotides including one open reading frame (ORF) encoding 160 amino acids (aa). A striking feature of the ORF was the periodic distribution of cysteine residues (Cys-X-Cys-X-Cys-X6-Cys) which occurred approximately every 22 aa. A cDNA-encoded 18-aa sequence was selected for chemical peptide synthesis. When affinity-purified antipeptide antibodies were incubated with a Western blot containing salivary-gland proteins they reacted specifically with a 185-kDa secretory protein (sp185). Developmental studies showed that sp185 and its mRNA were present in salivary glands throughout the fourth larval instar. Thus sp185 and a family of 1000-kDa secretory proteins are encoded by a class of genes that are expressed throughout the fourth instar. This contrasts with the developmentally regulated expression of the sp140 and sp195 genes whose expression is maximal during the prepupal stages of larval development.

Amino Acid Sequence↗

The cDNA and deduced protein sequence of house fly NADPH-cytochrome P450 reductase.

Antisera to purified house fly NADPH-cytochrome P450 reductase were used to select cDNA clones from an expression library of abdomens of phenobarbital-treated house flies. A partial cDNA of 1841 bp containing a TAG termination codon, a consensus polyadenylation site and 269 bp of 3' untranslated sequence was obtained. Sequencing of a genomic clone coupled with mRNA sequencing yielded the complete coding sequence including the starting ATG. The resulting open reading frame of 2013 nucleotides codes for a protein of 671 residues. The native reductase apoprotein has a molecular weight of 76,366 and the deduced molecular weight of the holoenzyme (i.e. with 1 mol of FAD and FMN) is 77,608. The sequence of the house fly P450 reductase protein is highly similar to that of rabbit liver, the overall amino acid positional identity is 54.5% and the overall identity among eukaryotic P450 reductases is about 25%. The P450 reductase gene of 19-23 kb was located on chromosome III, as shown by comparison of RFLP-patterns of the P450 reductase gene in two house fly strains and their hybrids.

Amino Acid Sequence↗

Isolation of white gene orthologue of the sawfly, Athalia rosae (Hymenoptera) and its functional analysis using RNA interference.

We isolated and characterized the white gene orthologue of the sawfly, Athalia rosae (Hymenoptera). The A. rosae white (Ar white) cDNA cloned was 2058-bp long encoding 685 amino acids in a single open reading frame (ORF). Comparison of the cDNA sequence with the genomic DNA sequence revealed that the ORF was derived from 11 exons. Ar white was a single copy gene as evidenced by genomic Southern blotting and its cytological localization on the metaphase chromosomes. The deduced amino acid sequence aligned well with known insect white orthologous gene products sharing conserved regions such as the ATP-binding motif and the six transmembrane-spanning segments. Expression of Ar white was detected at embryonic and pupal stages by Northern blotting. In situ hybridization detected the embryonic expression in a pair of the lateral tips of protocephalic placodes from where optic organs are formed. Ar white function was examined using double-stranded RNA (dsRNA)-mediated interference. The synthesized dsRNA targeting Ar white transcripts caused a decrease in the level of the original mRNAs, and resulted in the white phenocopy in the embryonic eye pigmentation when microinjected into eggs from wild-type females. The effects occurred in a dose-dependent manner.

ATP-Binding Cassette Transporters↗

One-hundred and five new potential Drosophila melanogaster genes revealed through STS analysis.

Complementation analysis had suggested that the Drosophila melanogaster genome contains approximately 5000 genes, but it is now generally accepted that the actual number is several times as high. We report here an analysis of 1788 anonymous sequence tagged sites (STSs) from the European Drosophila Genome Project (EDGP), totalling 463 kb. The data reveal a substantial number of previously undescribed potential genes, amounting to 6.1% of the number of Drosophila genes already in the sequence databases.

Amino Acid Sequence↗

Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere.

Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis [1,2]. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region [3]. By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives [4], we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote. The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast [5], the requirements for cohesion are separable from those for kinetochore assembly.

Animals↗

The defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes.

A recombinant Anopheles gambiae defensin peptide was used to define the antimicrobial activity spectrum against bacteria, filamentous fungi and yeast. Results showed that most of the Gram-positive bacterial species tested were sensitive to the recombinant peptide in a range of concentrations from 0.1 to 0.75 microM. No activity was detected against Gram-negative bacteria, with the exception of some E. coli strains. Growth inhibitory activity was detected against some species of filamentous fungi. Defensin was not active against yeast. The kinetics of bactericidal and fungicidal effects were determined for Micrococcus luteus and Neurospora crassa, respectively. Differential mass spectrometry analysis was used to demonstrate induction of defensin in the hemolymph of bacteria-infected adult female mosquitoes. Native peptide levels were quantitated in both hemolymph and midgut tissues. The polytene chromosome position of the defensin locus was mapped by in situ hybridization.

Animals↗

Chromosomal evolution trends of the genus Panstrongylus (Hemiptera, Reduviidae), vectors of Chagas disease.

The genus Panstrongylus includes 14 species widely distributed from Mexico to Argentina, some of them with great epidemiological significance as vectors of Chagas disease. We study the karyotype and the male meiotic process of Panstrongylus chinai, P. geniculatus, P. herreri, P. lignarius, P. megistus, P. rufotuberculatus and P. tupynambai. All species present the same sex mechanism (X(1)X(2)Y in males and X(1)X(1)X(2)X(2) in females) and they also have 20 autosomes, with the exception of P. megistus that only presents 18 autosomes. The analysis of C-banding patterns and meiotic chromosome behaviour show a great level of variability allowing the identification of three clearly differentiated groups. In the first group, we only include P. megistus because of its unusual number of autosomes. The second group includes P. chinai, P. herreri, P. lignarius and P. rufotuberculatus. Their autosomes present terminal heterochromatic regions that appear scattered throughout the nucleus and associated with the sex chromosomes. Actually, P. herreri and P. lignarius can be considered cytogenetically identical. Our results are in agreement with morphological, ecological and molecular data indicating that they should be regarded as the same species. The third group only includes P. tupynambai that shows autosomes without C-positive regions. Panstrongylus geniculatus shares characters will all the three groups. Its karyotypic features are extremely polymorphic depending on their geographic origin. Some populations do not show any heterochromatic regions, while others exhibit few or several heterochromatic blocks. The chromosomal variability observed, together with its wide distribution and phenetic variability, suggest that P. geniculatus is a species complex comprising at least two distinct species. Considering the entire subfamily, the level of cytogenetic variation in Panstrongylus is lower than that observed in Triatoma but considerably more than that of Rhodnius, which is a very homogenous genus in terms of chromosome appearance and behaviour. This would endorse the closer relationship between Panstrongylus and Triatoma, and their divergence from Rhodnius, in accordance with current tribal classification.

Animals↗

Genetic analysis of Drosophila virilis sex pheromone: genetic mapping of the locus producing Z-(11)-pentacosene.

Z-(11)-pentacosene, Drosophila virilis sex pheromone, is predominant among the female cuticular hydrocarbons and can elicit male courtship behaviours. To evaluate the genetic basis of its production, interspecific crosses between D. novamexicana and genetically marked D. virilis were made and hydrocarbon profiles of their backcross progeny were analysed. The production of Z-(11)-pentacosene was autosomally controlled and was recessive. Of the six D. virilis chromosomes only the second and the third chromosomes showed significant contributions to sex pheromone production, and acted additively. Analysis of recombinant females indicated that the locus on the second chromosome mapped to the proximity of position 2-218.

Alkenes↗

The rate of poleward chromosome motion is attenuated in Drosophila zw10 and rod mutants.

Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force responsible for poleward chromosome motion.

Animals↗

Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico.

RAPD-PCR polymorphisms at 57 presumptive loci were used to examine the breeding structure of the mosquito Aedes aegypti in Puerto Rico. Mosquitoes were sampled from 16 locations in six cities and samples were located in a nested spatial design to examine local patterns of gene flow. Allele frequencies were estimated assuming (1) that genomic regions amplified by RAPD-PCR segregate as dominant alleles, (2) that genotypes at RAPD loci are in Hardy-Weinberg proportions, (3) identity in state (iis) among dominant amplified alleles and (4) iis among null alleles. The average genic heterozygosity was 0.354, more than twice the level detected in earlier allozyme surveys. Nested analysis of variance indicated extensive genetic differentiation among locations within cities. Effective migration rates (Nm) among cities were estimated from FST assuming an island model of migration. Estimates of Nm ranged from 9.7 to 12.2 indicating a high dispersal rate. The large number of polymorphisms revealed by RAPD-PCR allowed the distribution of FST and linkage disequilibrium to be examined among loci and demonstrated that small samples inflate FST and linkage disequilibrium. No linkage disequilibrium maintained through epistasis was detected among alleles at the 57 loci.

Aedes↗

Chromosomal distribution of the 412 retrotransposon in natural populations of Drosophila simulans.

The insertion site localization of the 412 retrotransposable element was analysed by in situ hybridization to the polytene chromosomes of 57 individual genomes from 25 natural populations of Drosophila simulans. The 412 insertion sites along the chromosomes show a tendency to aggregate in the distal and proximal ends of the 2R arm, and in several local regions along the 3R arm. The distribution of the 412 insertion sites, weighted by DNA content, along the chromosome arms reveals an overall tendency for the site number to increase from the middle of the arm to the base and tip, with a decrease at the tips, especially pronounced for the X chromosome. Such a distribution differs slightly from that of D. melanogaster, which globally shows an increase of the 412 site number from base to tip of the chromosome arms, indicating differing behaviour of the 412 element in the two species. These results are discussed in connection with the recombination rate along the chromosome arms.

Animals↗

Chromosomal inversions and the reproductive isolation of species.

Recent genetic studies have suggested that many genes contribute to differences between closely related species that prevent gene exchange, particularly hybrid male sterility and female species preferences. We have examined the genetic basis of hybrid sterility and female species preferences in Drosophila pseudoobscura and Drosophila persimilis, two occasionally hybridizing North American species. Contrary to findings in other species groups, very few regions of the genome were associated with these characters, and these regions are associated also with fixed arrangement differences (inversions) between these species. From our results, we propose a preliminary genic model whereby inversions may contribute to the speciation process, thereby explaining the abundance of arrangement differences between closely related species that co-occur geographically. We suggest that inversions create linkage groups that cause sterility to persist between hybridizing taxa. The maintenance of this sterility allows the species to persist in the face of gene flow longer than without such inversions, and natural selection will have a greater opportunity to decrease the frequency of interspecies matings.

Animals↗