RESUMEN
A genomic DNA library of Anopheles aquasalis Curry was screened for clones that hybridized more intensely to DNA from A. aquasalis than to DNA from A. benarrochi Gabaldon, Cova Garcia, and Lopez, A. konderi Galvao and Damasceno, A. nuneztovari Gabaldon cytotypes A, B, and C, A. oswaldoi (Peryassu), A. rangeli Gabaldon, Cova Garcia, and Lopez, or A. trinkae Faran. Two specific clones (2.5 kilobasepairs [kbp] and 3.0 kbp) from A. aquasalis were isolated. Both A. aquasalis-specific clones were from the intergenic spacer region of the ribosomal RNA (rRNA) cistron. Upon digestion with Rsa I, a 900-bp fragment from the clone AA-1 hybridized specifically to A. aquasalis DNA. Analysis of the DNA sequence of this fragment revealed four tandemly repeated 36-bp units. Three of these repeat units were identical, and the fourth was 94% identical to the others. The DNA sequence of a highly conserved region of these repeats was used to synthesize an oligonucleotide probe specific to A. aquasalis.
Asunto(s)
Anopheles/genética , ARN Ribosómico/genética , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Anopheles/clasificación , Anopheles/parasitología , Secuencia de Bases , Sondas de ADN/genética , ADN Ribosómico/genética , Femenino , Genes/genética , Biblioteca Genómica , Insectos Vectores/clasificación , Insectos Vectores/genética , Insectos Vectores/parasitología , Malaria/transmisión , Datos de Secuencia Molecular , ARN Ribosómico 28S/genética , Homología de Secuencia de Ácido Nucleico , América del Sur , Especificidad de la EspecieRESUMEN
We analyzed variation in mitochondrial DNA (mtDNA) of two neotropical mosquitoes, Anopheles rangeli (n = 181) and A. trinkae (n = 45), with very different distribution patterns in Latin America, to assess species boundaries for these putative sister taxa and to examine population genetic structure. Phylogenetic analyses revealed (1) support for the monophyletic origin of each species; (2) diagnostic restriction site differences between the species; (3) geographic partitioning of haplotypes by country in A. rangeli from Bolivia, Ecuador, and Venezuela compared with considerable overlap in haplotypes of A. trinkae from Bolivia and Ecuador; and (4) similar levels of mean haplotype and nucleotide diversity in both species, but lower levels of mean nucleotide divergence in A. trinkae compared with A. rangeli. We hypothesize that higher maternal gene flow and lower divergence in A. trinkae are most likely due either to a distinctive matrilineal history or to a smaller effective population size, which may have been influenced by a smaller, essentially linear geographic range along the eastern flank of the Andes. In the cladistic analysis of A. rangell, the Bolivian haplotypes appear to be more derived than those from Ecuador or Venezuela, yet there is no evidence to support the hypothesis of a recent range expansion from Ecuador into Bolivia.
Asunto(s)
Anopheles/genética , ADN Mitocondrial , Genes de Insecto , Variación Genética , Animales , Anopheles/clasificación , Haplotipos , FilogeniaRESUMEN
We compared rates of feeding on human hosts for blood-engorged female Anopheles quadrimaculatus species A, B and C1 collected from daytime resting sites in Manatee Springs State Park, Levy County, Florida during 1992-1993. Quick-blot DNA probes were used to identify mosquito taxa and also the presence of human blood in the mosquito gut. In collections from a campground area, human blood-feeding rates differed significantly among mosquito species (10.7% [19 of 177], 0%, [0 of 62], and 1.2%, [4 of 327]), respectively for species A, B and C1). In collections from a woodland site (1 km from the campground), 1.5% (2 of 129) of the species B females had fed on humans, whereas none of 19 species A or 159 species C1 females had done so. Of the three species in this study area, species A appears the most likely to be a biting pest of humans and a vector of human malaria.
Asunto(s)
Anopheles/fisiología , Animales , Anopheles/clasificación , Conducta Apetitiva , Sondas de ADN , Conducta Alimentaria/fisiología , Femenino , Florida , Humanos , Especificidad de la EspecieRESUMEN
Two Hsp82 genes were isolated from the malaria vector Anopheles albimanus in a single lambda phage clone. The two genes are in a head-to-head arrangement separated by approx. 0.9 kbp. Northern hybridizations and 5' RACE demonstrate that both genes are transcribed, have moderate levels of constitutive transcription, and are also heat-inducible with maximum transcript accumulation occurring after 40 degrees C heat shocks. Both genes have typical heat-shock promoters and conserved intron boundaries in the untranslated leaders. The open reading frames are 99.6% identical differing in only nine silent nucleotide positions in the 2166 bp ORFs. However, precisely outside the ORFs, the flanking DNA of the two genes shows no evidence of common derivation. The high degree of identity between the two ORFs appears to be a result of gene conversion occurring by a process similar to that previously suspected in the A. albimanus Hsp70 genes and several D. melanogaster genes arranged as palindromes. This process probably involves a stem-loop intermediate and is restricted in extent by flanking sequence divergence. These Hsp82 genes clearly demonstrate the extreme precision with which gene conversion can lead to protein-coding-region homogeneity yet allow flanking DNA divergence.
Asunto(s)
Anopheles/genética , Proteínas de Choque Térmico/genética , Sistemas de Lectura Abierta , Secuencia de Aminoácidos , Animales , Anopheles/química , Secuencia de Bases , Evolución Biológica , Northern Blotting , Clonación Molecular , ADN Complementario , Proteínas HSP90 de Choque Térmico , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiaeRESUMEN
The bacterial parathion hydrolase gene (opd) was expressed in transformed D. melanogaster under the control of an hsp70 promoter. Transformed lines carrying chimaeric genes designed for either cytoplasmic or secretory expression exhibited high- or low-level heat-shock-inducible transient resistance to paraoxon respectively. Greatest levels of resistance occurred approximately 12-16 h after heat shock and well after periods of maximal transcription. Insecticide resistance conferred by the cytoplasmic form of opd is expressed as a semidominant trait.
Asunto(s)
Drosophila melanogaster/genética , Esterasas/genética , Resistencia a los Insecticidas/genética , Paraoxon/toxicidad , Animales , Arildialquilfosfatasa , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Esterasas/biosíntesis , Esterasas/metabolismo , Expresión Génica , Genotipo , Calor , Datos de Secuencia Molecular , Mortalidad , Paratión/toxicidad , Regiones Promotoras Genéticas , Transformación GenéticaRESUMEN
The complete sequence (15,455 bp) of the mitochondrial DNA of the mosquito Anopheles quadrimaculatus species A is reported. This genome is compact and very A+T rich (77.4% A+T). It contains genes for 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and 13 subunits of the mitochondrial inner membrane respiratory complexes. The gene arrangement is the same as in Drosophila yakuba, except that the positions of two contiguous tRNAs are reversed and a third tRNA is transcribed from the complementary strand. Protein-coding genes, rRNAs, and most tRNAs were similar to D. yakuba. Two tRNAs had nonstandard secondary structures comparable with those of nematode mitochondrial tRNAs. The very small putative control region (625 bp) contains no sequence motifs similar to those used in vertebrates and other insects for initiation of transcription and replication.
Asunto(s)
Anopheles/genética , ADN Mitocondrial/genética , Genes de Insecto , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Codón/genética , Drosophila/genética , Genoma , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Proteínas/genética , ARN Ribosómico/genética , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The nucleotide sequence was determined for a portion of a 12-kb genomic DNA clone specific for Anopheles quadrimaculatus species A. Four short, internally repeated sequences were identified. Synthetic oligonucleotide probes were prepared based on these four repeats. The oligonucleotides are highly specific and can be reliably used to separate individuals of An. quadrimaculatus species A from members of other species of the complex.
Asunto(s)
Anopheles/genética , Sondas de ADN/química , Animales , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/química , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
Populations of the malaria vector Anopheles aquasalis from Venezuela, Trinidad, and Brazil were analyzed using restriction enzyme digestion of mitochondrial DNA (mtDNA). The five enzymes surveyed yielded 12 mtDNA haplotypes. Three restriction endonuclease profiles (Bcl I, Eco RI, and Hind III) differentiated the Venezuelan and Trinidadian A. aquasalis from two Brazilian populations. Estimates of mtDNA sequence divergence between the Venezuelan/Trinidadian populations and each Brazilian population (0.023-0.032), as well as divergence between the two Brazilian populations (0.030), were within the range of interspecific distances calculated for members of anopheline species complexes. Ribosomal DNA (rDNA) restriction profiles of the A. aquasalis populations examined were identical.
Asunto(s)
Anopheles/genética , Insectos Vectores/genética , Malaria , Animales , Brasil , ADN Mitocondrial/genética , Femenino , Variación Genética , Haplotipos , Humanos , Malaria/transmisión , Polimorfismo de Longitud del Fragmento de Restricción , Especificidad de la Especie , Trinidad y Tobago , VenezuelaRESUMEN
Four Hsp70 genes of the malaria vector Anopheles albimanus were isolated from a genomic DNA library as two non-overlapping clones each containing a pair of divergently transcribed genes having 75% DNA sequence similarity to the protein-coding regions of the Drosophila melanogaster Hsp70 genes. The clones were assigned to two loci on chromosome 2R by in situ hybridization. These clones hybridize strongly to heat-shock but only weakly to non-shocked mosquito RNA. The Hsp70 gene family of A. albimanus is undergoing concerted evolution probably by gene conversion. The general arrangement of the genes suggests that divergently transcribed pairs of genes at two loci is an ancient Dipteran arrangement predating the Nematocera/Cyclorrapha divergence.
Asunto(s)
Anopheles/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Insectos/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Genes de Insecto , Proteínas HSP70 de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , ARN/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The extent of intra- and inter-specific variation in mitochondrial DNA and nuclear ribosomal RNA gene restriction sites was determined for the four sibling species of the Anopheles quadrimaculatus complex. Individual mosquitoes were identified by allozyme analysis according to previously published keys, and the total genomic DNA of these same individuals was then cleaved with restriction enzymes. Restriction maps of mitochondrial DNA, including the positions of variable sites, were constructed for each species. No evidence for interspecific hybridization was found in the populations surveyed. There was little variation in restriction patterns within any given species, but differences occurred among the four. Three restriction enzymes (AvaI, HindIII, and PvuII) yielded species-specific DNA restriction patterns for the mitochondrial DNA, while AvaI and HindIII produced diagnostic patterns for the ribosomal DNA. Thus, restriction patterns were very useful for detecting cryptic species but less appropriate than isozymes for studying genetic structure of populations within species.
Asunto(s)
Anopheles/genética , ADN Mitocondrial/genética , ADN Ribosómico/genética , Animales , Sondas de ADN , Variación Genética , Filogenia , Polimorfismo Genético , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
The quick blot protocol is an improved technique for preparing crude insect homogenates for hybridization to nucleic acid probes. Individual insects are ground in wells of a microtiter plate and transferred to a dot blot manifold. This allows preparation of multiple filters and provides uniformity and an orderly arrangement of samples. The high background detection resulting from use of crude insect homogenates with nonradioactive detection systems was eliminated by incubating quick blot filters in a laundry stain remover containing proteases. We used mosquito species-specific DNA probes to demonstrate the effectiveness of nonradioactive DNA labeling systems with quick blots.
Asunto(s)
Culicidae/genética , Sondas de ADN/análisis , Mapeo Nucleótido/métodos , Animales , Hibridación de Ácido Nucleico , Trazadores Radiactivos , Especificidad de la EspecieRESUMEN
Maize (Zea mays, cv 'Black Mexican Sweet') (BMS) and tobacco (Nicotiana tabacum, cv 'Xanthi') tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells. DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA. Maize cells were treated with a plasmid carrying both the BAR gene, whose product confers resistance to the herbicide BASTA, and a gene encoding ß-glucuronidase (GUS). Tobacco cells were treated with two plasmids to co-transfer genes encoding neomycin phosphotransferase (NPTII) and GUS from the respective plasmids. Thirty-four BASTA-resistant BMS colonies and 23 kanamycin-resistant tobacco colonies recovered following selection contained intact copies of the BAR gene and NPTII genes, respectively, as determined by Southern blot analysis. Sixty-five percent of the resistant BMS colonies and 50% of the resistant tobacco colonies also expressed GUS activity. Intact copies of the GUS gene were observed in Southern blots of all resistant BMS and tobacco colonies that expressed GUS activity. These results indicate that a simple, inexpensive DNA delivery procedure employing silicon carbide fibers can be used to reproducibly transform cells of both monocotyledonous and dicotyledonous plant species.Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the University of Minnesota or the USDA, and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
RESUMEN
Temperature effects on Anopheles albimanus larval survival were investigated. Larvae were exposed to 30 min heat shocks at various temperatures. Almost no mortality was observed at 40 degrees C, but was complete at 43 degrees C. Increased larval thermotolerance could be induced by higher rearing temperature or by a 30 min exposure to 37 degrees C.
Asunto(s)
Anopheles/fisiología , Calor , Adaptación Fisiológica/fisiología , Animales , Ecología , Proteínas de Choque Térmico/biosíntesis , Larva/fisiología , Estaciones del AñoRESUMEN
The entire 15 kilobase (kb) Anopheles quadrimaculatus mitochondrial DNA (mtDNA) was cloned as three EcoRI fragments in a bacteriophage vector and then subcloned into plasmid vectors. The cloned DNA was physically mapped with restriction endonucleases, and the maps were compared to the restriction patterns of native A. quadrimaculatus mtDNA. Several genes were mapped by sequencing the ends of A. quadrimaculatus mtDNA subclones and by hybridization with the previously characterized Aedes albopictus mtDNA clones. These portions of the genetic map were identical in gene order to those of Drosophila yakuba. The predicted amino acid sequence of the protein coding regions that were sequenced were between 72% and 98% homologous to D. yakuba. The cloned mtDNA will be useful as a probe for population genetic analysis of mosquitoes.
Asunto(s)
Anopheles/genética , Clonación Molecular , ADN Mitocondrial/genética , Secuencia de Aminoácidos , Animales , Bacteriófagos , Secuencia de Bases , Sondas de ADN , ADN Mitocondrial/química , Desoxirribonucleasa EcoRI , Biblioteca de Genes , Vectores Genéticos , Hibridación de Ácido Nucleico , Plásmidos , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
A general method for obtaining species-specific repetitive DNA sequences is described. The method is based on the detection of recombinant DNA clones containing repetitive sequences using labeled total genomic DNA. These repetitive DNA sequences can be used to identify individual mosquito adults, pupae, and larvae squashed on filter membranes (squash blots). This technique was used to distinguish individuals of the four sibling species of the Anopheles quadrimaculatus complex. Repetitive DNA sequences and squash blots can be of use for rapid identification of other insect species in field collections.
Asunto(s)
Anopheles/aislamiento & purificación , ADN/análisis , Animales , Anopheles/genética , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Especificidad de la EspecieRESUMEN
Silicon carbide fiber-mediated delivery of DNA into intact plant cells was investigated. Black Mexican Sweet (BMS) maize (Zea mays) and tobacco (Nicotiana tabacum) suspension culture cells were vortexed in the presence of liquid medium, plasmid DNA encoding ß-glucuronidase (GUS), and silicon carbide fibers. Penetration of BMS cells by the silicon carbide fibers was observed by scanning electron microscopy of vortexed cells. Following fiber and DNA treatment, BMS cells transiently expressed GUS activity at a mean frequency of 139.5 units (one unit = one blue cell or one colony of blue cells) per sample. Treated tobacco cells expressed an average of 373 GUS units per sample. Untreated controls did not exhibit GUS activity. These results indicate that the silicon carbide fibers-vortex procedure can be used to rapidly and inexpensively deliver foreign DNA into intact plant cells for investigations of transient gene expression.
RESUMEN
Methods are described for the isolation of mitochondrial and total cellular DNA from mosquitoes. The mitochondrial and ribosomal DNA restriction patterns could be detected in the total DNA of an individual mosquito by the use of cloned probes. DNA restriction analysis may prove to be a useful alternative to isozyme electrophoresis for the study of insect population genetics.