Meiosis in Drosophila: seeing is believing.

Recently many exciting advances have been achieved in our understanding of Drosophila meiosis due to combined cytological and genetic approaches. New techniques have permitted the characterization of chromosome position and spindle formation in female meiosis I. The proteins encoded by the nod and ncd genes, two genes known to be needed for the proper partitioning of chromosomes lacking exchange events, have been identified and found to be kinesin-like motors. The effects of mutations in these genes on the spindle and chromosomes, together with the localization of the proteins, have yielded a model for the mechanism of female meiosis I. In male meiosis I, the chromosomal regions responsible for homolog pairing have been resolved to the level of specific DNA sequences. This provides a foundation for elucidating the molecular basis of meiotic pairing. The cytological techniques available in Drosophila also have permitted inroads into the regulation of sister-chromatid segregation. The products of two genes (mei-S332 and ord) essential for sister-chromatid cohesion have been identified recently. Additional advances in understanding Drosophila meiosis are the delineation of a functional centromere by using minichromosome derivatives and the identification of several regulatory genes for the meiotic cell cycle.

Introduction of the transposable element Minos into the germ line of Drosophila melanogaster.

A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Drosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp70-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp70-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three of the insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp70-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.

Identification of a retroviroid-like element from plants.

The biological nature of carnation small viroid-like RNA (CarSV RNA), a 275-nt circular molecule with self-cleaving hammerhead structures in its strands of both polarities, was investigated. The lack of infectivity observed in a series of transmission assays in carnation indicates that CarSV RNA, in spite of sharing structural similarities with viroid and viroid-like satellite RNAs from plants, does not belong to either of these two groups. Additional evidence in this direction comes from the observation that CarSV RNA also exists in carnation plants as DNA tandem repeats. In this respect, CarSV RNA is similar to a small transcript of a tandemly repeated DNA sequence of the newt genome. Moreover, CarSV and newt RNAs have similarities in their sequences as well as in some characteristics of their corresponding hammerhead structures. Further analyses have revealed that CarSV DNA is found directly fused to DNA sequences of carnation etched ring caulimovirus, a pararetrovirus, most likely in the form of an extrachromosomal element. The properties of the CarSV RNA/DNA system are those of a retroviroid-like element having some features in common with viroid and viroid-like satellite RNAs from plants and others with the newt transcript.

Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.

Efficient high-resolution genetic mapping of mouse interspersed repetitive sequence PCR products, toward integrated genetic and physical mapping of the mouse genome.

The ability to carry out high-resolution genetic mapping at high throughput in the mouse is a critical rate-limiting step in the generation of genetically anchored contigs in physical mapping projects and the mapping of genetic loci for complex traits. To address this need, we have developed an efficient, high-resolution, large-scale genome mapping system. This system is based on the identification of polymorphic DNA sites between mouse strains by using interspersed repetitive sequence (IRS) PCR. Individual cloned IRS PCR products are hybridized to a DNA array of IRS PCR products derived from the DNA of individual mice segregating DNA sequences from the two parent strains. Since gel electrophoresis is not required, large numbers of samples can be genotyped in parallel. By using this approach, we have mapped > 450 polymorphic probes with filters containing the DNA of up to 517 backcross mice, potentially allowing resolution of 0.14 centimorgan. This approach also carries the potential for a high degree of efficiency in the integration of physical and genetic maps, since pooled DNAs representing libraries of yeast artificial chromosomes or other physical representations of the mouse genome can be addressed by hybridization of filter representations of the IRS PCR products of such libraries.

Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes.

Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species. However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations. Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH. Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture. BAC clones as small as 40 kb were successfully mapped. To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis. The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis.

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms.

Of the approximately 380 families of angiosperms, representatives of only 10 are known to form symbiotic associations with nitrogen-fixing bacteria in root nodules. The morphologically based classification schemes proposed by taxonomists suggest that many of these 10 families of plants are only distantly related, engendering the hypothesis that the capacity to fix nitrogen evolved independently several, if not many, times. This has in turn influenced attitudes toward the likelihood of transferring genes responsible for symbiotic nitrogen fixation to crop species lacking this ability. Phylogenetic analysis of DNA sequences for the chloroplast gene rbcL indicates, however, that representatives of all 10 families with nitrogen-fixing symbioses occur together, with several families lacking this association, in a single clade. This study therefore indicates that only one lineage of closely related taxa achieved the underlying genetic architecture necessary for symbiotic nitrogen fixation in root nodules.

Da patenteabilidade de genes humanos

São inegáveis o caráter universal e a importância dos avanços tecnológicos e científicos originados das pesquisas genéticas. O sequenciamento do genoma humano, a identificação das principais sequências de DNA contidas nos seus genes e suas respectivas funções biológicas, bem como suas possíveis aplicações biomédicas, são de incalculável importância. Os genes, muito embora possam ser biologicamente caracterizados como compostos químicos, possuem um conteúdo informacional que se revela indispensável ao desenvolvimento da engenharia genética, figurando como elemento básico e central de suporte às inovações biotecnológicas. Desta forma, importante analisar a relevância da aplicação de mecanismos jurídicos como forma de fomento à contínua evolução biotecnológica sob a ótica do desenvolvimento econômico e social do país, princípios constitucionais justificadores da proteção de referidos desenvolvimentos técnicos por meio do intelecto e intervenção humanos na natureza. Para tanto, deve-se levar em consideração que a inexistência de tutela jurídica específica pode gerar desincentivo aos investimentos capazes de possibilitar o desenvolvimento de tais tecnologias, ao passo que uma tutela jurídica muito ampla poderá ocasionar indevida restrição ao acesso a tais insumos biológicos, de modo a gerar um efeito adverso àquele buscado. Assim, deve-se compatibilizar a proteção dos resultados obtidos através do desenvolvimento biotecnológico em relação à potencial dificuldade originada de uma eventual restrição ao acesso a tais elementos fundamentais à pesquisa e desenvolvimento genéticos. É neste contexto que se procura um balizamento entre os diferentes interesses e posicionamentos a respeito da patenteabilidade dos genes humanos, visando solução jurídica que permita um ambiente seguro e propício ao desenvolvimento da engenharia genética, e dos inúmeros benefícios que poderão daí se originar. O presente estudo se voltará, portanto, à análise da necessidade, condições, suficiência e extensão da tutela jurídica a ser conferida pela outorga de direitos patentários aos genes humanos.

Transient Activation of Meox1 Is an Early Component of the Gene Regulatory Network Downstream of Hoxa2

Hox genes encode transcription factors that regulate morphogenesis in all animals with bilateral symmetry. Although Hox genes have been extensively studied, their molecular function is not clear in vertebrates, and only a limited number of genes regulated by Hox transcription factors have been identified. Hoxa2 is required for correct development of the second branchial arch, its major domain of expression. We now show that Meox1 is genetically downstream from Hoxa2 and is a direct target. Meox1 expression is downregulated in the second arch of Hoxa2 mouse mutant embryos. In chromatin immunoprecipitation (ChIP), Hoxa2 binds to the Meox1 proximal promoter. Two highly conserved binding sites contained in this sequence are required for Hoxa2-dependent activation of the Meox1 promoter. Remarkably, in the absence of Meox1 and its close homolog Meox2, the second branchial arch develops abnormally and two of the three skeletal elements patterned by Hoxa2 are malformed. Finally, we show that Meox1 can specifically bind the DNA sequences recognized by Hoxa2 on its functional target genes. These results provide new insight into the Hoxa2 regulatory network that controls branchial arch identity.

Partitioned likelihood support and the evaluation of data set conflict

In simultaneous analyses of multiple data partitions, the trees relevant when measuring support for a clade are the optimal tree, and the best tree lacking the clade (i.e., the most reasonable alternative). The parsimony-based method of partitioned branch support (PBS) forces each data set to arbitrate between the two relevant trees. This value is the amount each data set contributes to clade support in the combined analysis, and can be very different to support apparent in separate analyses. The approach used in PBS can also be employed in likelihood: a simultaneous analysis of all data retrieves the maximum likelihood tree, and the best tree without the clade of interest is also found. Each data set is fitted to the two trees and the log-likelihood difference calculated, giving partitioned likelihood support (PLS) for each data set. These calculations can be performed regardless of the complexity of the ML model adopted. The significance of PLS can be evaluated using a variety of resampling methods, such as the Kishino-Hasegawa test, the Shimodiara-Hasegawa test, or likelihood weights, although the appropriateness and assumptions of these tests remains debated.

Presence of activatable Shiga toxin genotype (stx2d) in Shiga toxigenic Escherichia coli from livestock sources

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c) (vha), stx(2c) (vhb), or stx(2d) (EH250). Subsequently, the stx(2c) (vha) and stx(2c) (vhb) operons were screened for the absence of a PstI site in the stx(2a) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable stx(2d) Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

Searching oligo sets of human chromosome 12 using evolutionary strategies

DNA Microarray is a powerful tool to measure the level of a mixed population of nucleic acids at one time, which has great impact in many aspects of life sciences research. In order to distinguish nucleic acids with very similar composition by hybridization, it is necessary to design microarray probes with high specificities and sensitivities. Highly specific probes correspond to probes having unique DNA sequences; whereas highly sensitive probes correspond to those with melting temperature within a desired range and having no secondary structure. The selection of these probes from a set of functional DNA sequences (exons) constitutes a computationally expensive discrete non-linear search problem. We delegate the search task to a simple yet effective Evolution Strategy algorithm. The computational efficiency is also greatly improved by making use of an available bioinformatics tool.

Systematics and evolution of ticks with a list of valid genus and species names

In recent years there has been much progress in our understanding of the phylogeny and evolution of ticks, in particular the hard ticks (Ixodidae). Indeed, a consensus about the phylogeny of the hard ticks has emerged which is quite different to the working hypothesis of 10 years ago. So that the classification reflects our knowledge of ticks, several changes to the nomenclature of ticks are imminent or have been made. One subfamily, the Hyalomminae, should be sunk, while another, the Bothriocrotoninae, has been created (Klompen, Dobson & Barker, 2002). Bothriocrotoninae, and its sole genus Bothriocroton, have been created to house an early-diverging ('basal') lineage of endemic Australian ticks that used to be in the genus Aponomma. The remaining species of the genus Aponomma have been moved to the genus Amblyomma. Thus, the name Aponomma is no longer a valid genus name. The genus Rhipicephalus is paraphyletic with respect to the genus Boophilus. Thus, the genus Boophilus has become a subgenus of the genus Rhipicephalus (Murrell & Barker, 2003). Knowledge of the phylogenetic relationships of ticks has also provided new insights into the evolution of ornateness and of their life cycles, and has allowed the historical zoogeography of ticks to be studied. Finally, we present a list of the 899 valid genus and species names of ticks as of February 2004.

Sociality and the rate of molecular evolution

The molecular clock does not tick at a uniform rate in all taxa but maybe influenced by species characteristics. Eusocial species (those with reproductive division of labor) have been predicted to have faster rates of molecular evolution than their nonsocial relatives because of greatly reduced effective population size; if most individuals in a population are nonreproductive and only one or few queens produce all the offspring, then eusocial animals could have much lower effective population sizes than their solitary relatives, which should increase the rate of substitution of nearly neutral mutations. An earlier study reported faster rates in eusocial honeybees and vespid wasps but failed to correct for phylogenetic nonindependence or to distinguish between potential causes of rate variation. Because sociality has evolved independently in many different lineages, it is possible to conduct a more wide-ranging study to test the generality of the relationship. We have conducted a comparative analysis of 25 phylogenetically independent pairs of social lineages and their nonsocial relatives, including bees, wasps, ants, termites, shrimps, and mole rats, using a range of available DNA sequences (mitochondrial and nuclear DNA coding for proteins and RNAs, and nontranslated sequences). By including a wide range of social taxa, we were able to test whether there is a general influence of sociality on rates of molecular evolution and to test specific predictions of the hypothesis: (1) that social species have faster rates because they have reduced effective population sizes; (2) that mitochondrial genes would show a greater effect of sociality than nuclear genes; and (3) that rates of molecular evolution should be correlated with the degree of sociality. We find no consistent pattern in rates of molecular evolution between social and nonsocial lineages and no evidence that mitochondrial genes show faster rates in social taxa. However, we show that the most highly eusocial Hymenoptera do have faster rates than their nonsocial relatives. We also find that social parasites (that utilize the workers from related species to produce their own offspring) have faster rates than their social relatives, which is consistent with an effect of lower effective population size on rate of molecular evolution. Our results illustrate the importance of allowing for phylogenetic nonindependence when conducting investigations of determinants of variation in rate of molecular evolution.