RESUMEN
Heat shock protein 70 (Hsp70) is the major player that underlies adaptive response to hyperthermia in all organisms studied to date. We investigated patterns of Hsp70 expression in larvae of dipteran species collected from natural populations of species belonging to four families from different evolutionary lineages of the order Diptera: Stratiomyidae, Tabanidae, Chironomidae and Ceratopogonidae. All investigated species showed a Hsp70 expression pattern that was different from the pattern in Drosophila. In contrast to Drosophila, all of the species in the families studied were characterized by high constitutive levels of Hsp70, which was more stable than that in Drosophila. When Stratiomyidae Hsp70 proteins were expressed in Drosophila cells, they became as short-lived as the endogenous Hsp70. Interestingly, three species of Ceratopogonidae and a cold-water species of Chironomidae exhibited high constitutive levels of Hsp70 mRNA and high basal levels of Hsp70. Furthermore, two species of Tabanidae were characterized by significant constitutive levels of Hsp70 and highly stable Hsp70 mRNA. In most cases, heat-resistant species were characterized by a higher basal level of Hsp70 than more thermosensitive species. These data suggest that different trends were realized during the evolution of the molecular mechanisms underlying the regulation of the responses of Hsp70 genes to temperature fluctuations in the studied families.
Asunto(s)
Dípteros/fisiología , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico , Animales , Evolución Biológica , Dípteros/genética , Dípteros/crecimiento & desarrollo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la EspecieRESUMEN
Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.
Asunto(s)
Lino/genética , Cromosomas de las Plantas/genética , Lino/ultraestructura , Marcadores Genéticos , Genotipo , Cariotipificación , Repeticiones de Microsatélite , Polimorfismo GenéticoRESUMEN
The functional characteristics of the DNA fragments responsible for chromosome attachment to the nuclear envelope during the interphase (neDNAs) have been studied. The neDNAs flanking the transgene have been found to promote a steadily high rate of its expression, irrespective of the site of its insertion into the host chromosomes. At the same time, neDNAs themselves have no transcription regulatory functions.
Asunto(s)
Cromosomas/fisiología , Membrana Nuclear/fisiología , Proteínas Nucleares/genética , Transportadoras de Casetes de Unión a ATP/genética , Animales , Animales Modificados Genéticamente , ADN/fisiología , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster , Color del Ojo/genética , Proteínas del Ojo/genética , Regulación de la Expresión Génica , Genes Reporteros , Interfase , Membrana Nuclear/genética , TransgenesAsunto(s)
Drosophila melanogaster/genética , Drosophila/genética , Mutación , Interferencia de ARN , Retroelementos , Animales , Genoma , Modelos Genéticos , ARN/genética , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Transcripción Genética , TransgenesRESUMEN
Various copies of mobile element Penelope previously described in Drosophila virilis have been investigated in different strains of D. virilis and transgenic strains of D. melanogaster transformed by P-based constructs containing full-size copy of Penelope. It has been demonstrated that most of Penelope copies in both species carry large terminal inverted repeats (TIR) and contain deletions of various size at their 5'end of ORF. Junctions between TIR and ORF usually carry microhomology of various length enabling to postulate a hypothesis explaining the molecular mechanism underlying the origin of such complex structures. Penelope copies usually carry 34 bp repeat located in direct orientation at both end of ORF and target site duplications of various length.
Asunto(s)
Regiones no Traducidas 5'/genética , Drosophila/genética , Evolución Molecular , Sistemas de Lectura Abierta/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Retroelementos/genética , Animales , Especificidad de la EspecieRESUMEN
C banding, Ag-NOR staining, FISH with pTa71 (45S rDNA) and pTa794 (5S rDNA), and RAPD-PCR analysis were used to study the genome and chromosome polymorphism in four varieties (Frisson, Sparkle, Rondo, and Finale) and two genetic lines (Sprint-2 and SGE) of pea Pisum sativum L. A comparison of the C-banding patterns did not reveal any polymorphism within the varieties. The most significant between-variety differences were observed for the size of C bands on satellite chromosomes 4 and 7. All grain pea varieties (Frisson, Sparkle, and Rondo) had a large C band in the satellite of chromosome 4 and a medium C band in the region adjacent to the satellite thread on chromosome 7. C bands were almost of the same size in the genetic lines and vegetable variety Finale. In all accessions, 45S rDNA mapped to the secondary constriction regions of chromosomes 1, 3, and 5. The signal from chromosome 5 in the lines was more intense than in the varieties. Ag-NOR staining showed that the transcriptional activity of the 45S rRNA genes on chromosome 7 was higher than on chromosome 4 in all accessions. No more than four Ag-NOR-positive nucleoli were observed in interphase nuclei. Statistical analysis of the total area of Ag-NOR-stained nucleoli did not detect any significant difference between the accessions examined. RAPD-PCR analysis revealed high between-variety and low within-variety genomic polymorphism. Chromosomal and molecular markers proved to be promising for genome identification in pea varieties and lines.
Asunto(s)
Cromosomas de las Plantas/genética , Genoma de Planta/fisiología , Pisum sativum/genética , Polimorfismo Genético , Nucléolo Celular/genética , Bandeo Cromosómico/métodos , ADN de Plantas/genética , ADN Ribosómico/genética , ARN Ribosómico/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Especificidad de la EspecieRESUMEN
Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18-2.5S-26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum. L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.
Asunto(s)
Mapeo Cromosómico , ADN Ribosómico/genética , Lino/genética , Hibridación Fluorescente in Situ , Cariotipificación , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Drosophila/genética , Retroelementos , Animales , Southern Blotting , Quimera , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Femenino , Amplificación de Genes , Regulación de la Expresión Génica , Vectores Genéticos , Genoma , Disgenesia Gonadal , Hibridación in Situ , Masculino , Especificidad de Órganos , Especificidad de la Especie , Transcripción Genética , Transformación GenéticaRESUMEN
To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.
Asunto(s)
Cromosomas , Sondas de ADN , Hordeum/genética , Ribosomas/genética , ADN Ribosómico , Humanos , Hibridación Fluorescente in Situ , ARN Ribosómico 18S/genética , ARN Ribosómico 28S/genéticaRESUMEN
RNA preparations synthesized in vitro were used to study the influence of RNA interference on the Kruppel gene activity in Drosophila embryos. RNA complementary in parallel orientation to the mRNA fragment proved to induce the development of Kruppel phenocopies. The data obtained indicate that mechanisms of specific regulation of gene activity exist in Drosophila cells, which are sensitive to the formation of both parallel and antiparallel RNA-RNA duplexes that include mRNA of the corresponding gene.
Asunto(s)
Drosophila/genética , Genes Homeobox , Fenotipo , ARN Complementario/genética , ARN Mensajero/genética , Animales , Drosophila/embriología , Embrión no Mamífero , MicroinyeccionesRESUMEN
The cut locus of Drosophila is an interesting example of a complex eukaryotic locus responsible for the development of many tissues and organs. Most of this locus is regulatory. The entire locus was cloned by Tchurikov et al. in 1986 and Blochlinger et al. in 1988. The wing ctn enhancer located 80 kb upstream of the promoter was earlier found in a 2.7 kb EcoRI-BamHI DNA fragment. The locus region 65-80 kb remote from the promoter was assumed to control the development of wings and vibrissae. We have found a new enhancer region in the ct6 region of the locus, which was in a 5 kb BamHI-EcoRI DNA fragment adjacent to the ctn enhancer. This region is responsible for the expression of the reporter lacZ gene in many tissues and organs at all stages of Drosophila development (at least in the intestine, Malpighian tubules, thoracic and abdominal sensory organs, thoracic ganglia and in ring glands). Thus, the region located 75 kb upstream of the promoter has some properties of the locus control region (LCR).
Asunto(s)
Drosophila/genética , Elementos de Facilitación Genéticos , Región de Control de Posición , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Animales , Proteínas de Drosophila , Genes Reporteros , Proteínas de Homeodominio , Operón Lac , Datos de Secuencia Molecular , Factores de TranscripciónAsunto(s)
Evolución Biológica , Elementos Transponibles de ADN , Drosophila/genética , Genoma , Animales , Genes de InsectoAsunto(s)
Drosophila/genética , Elementos de Facilitación Genéticos , Proteínas de Insectos/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , ADN , Proteínas de Drosophila , Proteínas de Homeodominio , Proteínas de Insectos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Mapeo Físico de Cromosoma , Unión Proteica , Factores de TranscripciónRESUMEN
Expression of the esterase S gene of Drosophila virilis was studied in transgenic experiments. Truncated genomic copy of this gene including 400 bp of 5' regulatory region was integrated into the genome of Drosophila melanogaster. The products of the transferred gene were detected. It was found that strict temporal and tissue specificity of the esterase S gene expression is conserved in transformed flies. The results suggest that this specificity is evidently determined by the regulatory region of the esterase S gene and controlled by cis mechanism.
Asunto(s)
Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/genética , Proteínas de Drosophila , Drosophila/enzimología , Expresión Génica , Genes de Insecto , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Animales Modificados Genéticamente , Carboxilesterasa , Hidrolasas de Éster Carboxílico/análisis , Mapeo Cromosómico , Sondas de ADN , Drosophila/genética , Drosophila melanogaster , Regulación Enzimológica de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Mapeo RestrictivoRESUMEN
Drosophila melanogaster was transformed with the esterase S gene from Drosophila virilis. This gene is strongly activated in ejaculatory bulbs of mature males of Drosophila virilis. The closely related gene from Drosophila melanogaster is activated in ejaculatory ducts. The tissue- and stage-specific expression of incomplete genomic copy of the esterase S gene integrated into the Drosophila melanogaster genome is the same as in Drosophila virilis. These data show that tissue and stage specificity is determined by relatively small 5' regulatory region of the esterase S gene. The comparison between deduced amino-acid sequences of the esterase S of Drosophila virilis and esterase 6 of Drosophila melanogaster was performed. These sequences revealed 50% homology.
Asunto(s)
Drosophila melanogaster/genética , Drosophila/genética , Esterasas/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Drosophila/enzimología , Femenino , Regulación Enzimológica de la Expresión Génica , Genes de Insecto , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Distribución Tisular , TransfecciónRESUMEN
Transformation of Drosophila melanogaster using P-element-based vectors yielded 129 sublines, which carried mini-white gene copies in the different genome regions. Dependence of mini-white gene expression on the location, gene dosage, and sex of the transformed individuals was analyzed. The mutation lzb was shown to suppress mini-white gene expression, the degree of suppression depending on the location and dosage of the mini-white gene.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Elementos Transponibles de ADN , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas del Ojo , Transformación Genética , Animales , Color del Ojo/genética , Femenino , Dosificación de Gen , Vectores Genéticos , Heterocigoto , Homocigoto , Hormonas de Insectos/genética , MasculinoRESUMEN
A previously described system of a Drosophila melanogaster mutative strain (MS), which originates from a stable strain (SS), is characterized by genetic instability caused by transposition of the retrotransposon gypsy. New unstable strains were obtained by microinjections of the gypsy transposable copy into SS embryos. In situ hybridization experiments revealed amplification and active transposition of gypsy in SS derivatives. At the same time, introduction of the gypsy transposable copy into another stable strain (208) did not lead to appearance of genetic instability. Genetic instability in the MS system is apparently induced by a combination of two factors: the presence of a gypsy transposable copy and mutation(s) in the gene(s) regulating its transpositions.