RESUMO
In 2000, transgenes were detected in local maize varieties (landraces) in the mountains of Oaxaca, Mexico [Quist, D. & Chapela, I. H. (2001) Nature 414, 541-543]. This region is part of the Mesoamerican center of origin for maize (Zea mays L.), and the genetic diversity that is maintained in open-pollinated landraces is recognized as an important genetic resource of great cultural value. The presence of transgenes in landraces was significant because transgenic maize has never been approved for cultivation in Mexico. Here we provide a systematic survey of the frequency of transgenes in currently grown landraces. We sampled maize seeds from 870 plants in 125 fields and 18 localities in the state of Oaxaca during 2003 and 2004. We then screened 153,746 sampled seeds for the presence of two transgene elements from the 35S promoter of the cauliflower mosaic virus and the nopaline synthase gene (nopaline synthase terminator) from Agrobacterium tumefaciens. One or both of these transgene elements are present in all transgenic commercial varieties of maize. No transgenic sequences were detected with highly sensitive PCR-based markers, appropriate positive and negative controls, and duplicate samples for DNA extraction. We conclude that transgenic maize seeds were absent or extremely rare in the sampled fields. This study provides a much-needed preliminary baseline for understanding the biological, socioeconomic, and ethical implications of the inadvertent dispersal of transgenes from the United States and elsewhere to local landraces of maize in Mexico.
Assuntos
Plantas Geneticamente Modificadas/genética , Zea mays/genética , Agrobacterium tumefaciens/genética , Aminoácido Oxirredutases/genética , Caulimovirus/genética , DNA Recombinante/genética , DNA Recombinante/isolamento & purificação , Alimentos Geneticamente Modificados/efeitos adversos , Engenharia Genética/efeitos adversos , México , Plantas Geneticamente Modificadas/efeitos adversos , Regiões Promotoras Genéticas , Sementes/genéticaAssuntos
Alimentos Geneticamente Modificados , Transferência Genética Horizontal , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Caulimovirus/genética , DNA Recombinante/isolamento & purificação , DNA Viral/isolamento & purificação , Ecologia , Meio Ambiente , União Europeia , Genes de Plantas , México , Publicações Periódicas como Assunto , Política , Editoração , Pesquisa/normas , Estados UnidosAssuntos
DNA Bacteriano/isolamento & purificação , Plasmídeos/isolamento & purificação , Cloreto de Potássio/química , Dodecilsulfato de Sódio/química , DNA Bacteriano/análise , DNA Recombinante/análise , DNA Recombinante/isolamento & purificação , Escherichia coli/genética , Plasmídeos/análise , Plasmídeos/genéticaAssuntos
Baculoviridae/genética , DNA Recombinante/análise , Transfecção , Animais , Células Cultivadas , DNA Recombinante/isolamento & purificação , Desoxirribonuclease EcoRI , Desoxirribonucleases de Sítio Específico do Tipo II , Vetores Genéticos , Reação em Cadeia da Polimerase , Spodoptera/metabolismoRESUMO
We describe conditions for optimal recovery of recombinant plasmids after blunt-end ligation. It was found that one of the most critical parameters of the blunt-end ligation reaction is total DNA concentration (vector plus incoming DNA). This concentration was optimal in the range of 1-5 micrograms/ml of reaction mixture. Concentrations larger than 10 micrograms/ml result in strong inhibition. The optimal molar relationship between incoming DNA and vector was found to be 1 or less. Under these conditions, using dephosphorylated vector, recombinants are generated at a frequency of 10(6) colonies per microgram of insert, provided that transforming efficiency is about 5 x 10(7) colonies per microgram of plasmid DNA.
Assuntos
Clonagem Molecular/métodos , DNA Ligases/metabolismo , DNA Recombinante/isolamento & purificação , Plasmídeos , Fenômenos Químicos , Físico-Química , DNA Recombinante/metabolismoRESUMO
1. The analysis of total protoscolex DNA and some rDNA recombinants of Echinococcus granulosus by restriction endonuclease mapping and hybridization to rDNA probes indicated the complex organization of the ribosomal RNA genes and that some repeat units are larger than 15 kb. The non-transcribed spacer can be up to 13 kb in length in some repeat units. 2. Restriction site polymorphism was detected mainly in the nontranscribed spacer regions although some polymorphism was also observed in the 28S rRNA coding region. 3. On the basis of Southern blot hybridization using EcoRI-digested genomic DNA, we conclude that the repeat units containing an extra EcoRI site are present almost in the same proportion as the repeat units without the extra EcoRI site in the 28S rRNA coding region.
Assuntos
DNA Ribossômico/isolamento & purificação , Echinococcus/genética , RNA Ribossômico/isolamento & purificação , Animais , Southern Blotting , Clonagem Molecular , Sondas de DNA , DNA Recombinante/isolamento & purificação , Eletroforese em Gel de Ágar , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Mapeamento por RestriçãoRESUMO
The analysis of total protoscolex DNA and some rDNA recombinats of Echinococcus granulosus by restriction endonuclease mapping and hybridization to rDNA probes indicated the complex organization of the ribosomal RNA genes and that some repeat units are larger than 15 kb. The nontranscribed spacer can be up to 13 kb in length in some repeat units. Restriction site polymorphisms was detected mainly in the nontranscribed spacer regions although some polymorphisms was also observed in the 28S rRNA coding region. On the basis of Southern blot hybridization using EcoRi-digested genomic DNA, we conclude that the repeat units containing an extra EcoRI site are present almost in the same proportion as the repeat units without the extra EcoRi site in the 28S rRNA coding region
Assuntos
Animais , Clonagem Molecular , Echinococcus/genética , RNA Ribossômico/isolamento & purificação , Sondas de DNA , DNA Recombinante/isolamento & purificação , DNA Ribossômico/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Biblioteca Genômica , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição , RNA Ribossômico 18S/isolamento & purificação , RNA Ribossômico 28S/isolamento & purificaçãoRESUMO
Two recombinants between the phage M13 and the plasmid pBR322 were isolated, analyzing the plasmid content of over one hundred colonies obtained by transduction. The study of the structure of both recombinants indicates that a fragment of the M13 genome has been integrated to pBR322. In both cases, the fragment contains a part of the phage replication region inserted either in the vicinity or within the pBR322 replicon. The fact that the phage and plasmid replicons seem to be involved in the recombination event suggests that it is helpful when the replication begins. So far it has not been possible to isolate a recombinant taking the whole genomes of pBR322 and M13. This is, undoubtedly, due to the instability of the recombinant molecule.
Assuntos
Bacteriófagos/genética , DNA Recombinante/isolamento & purificação , DNA Viral/isolamento & purificação , Plasmídeos/genética , Transdução GenéticaRESUMO
The human insulin gene is flanked by a polymorphic locus that is located approximately 500 base pairs (bp) from the 5' end of the point where transcription begins (Bell et al. 1981; Bell et al, 1982). Its occurrence is due to an insertion-deletion region which gives rise to two major classes of alleles: those containing small insertions of 0-600 bp and those containing larger insertions of 1,600-2,200 bp (Owerbach and Nerup, 1982). Insertions of 600-1,600 bp are rare (Rotwein et al., 1983). The larger insertions have previously been reported to be associated with type 2 diabetes (Owerbach and Nerup, 1982). We have conducted studies on a Mexican-American population in Starr County, Texas (98% Mexican-American) and a Tunisian population in Tunis, Tunisia, to determine if the frequency distribution of these classes of insulin gene alleles are similar to the previously reported frequency distributions and if any of the classes of alleles are associated with type 2 diabetes in these populations. We conclude that none of the classes of insulin gene alleles are associated with type II diabetes among Mexican-Americans or Tunisians, and that the frequency distributions of the insulin gene alleles do not vary significantly between the Tunisians, Mexican-Americans, or the aggregate data resulting from combining the insulin gene frequencies of several of the populations described thus far (Bell et al., 1984).