RESUMO
Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.
Assuntos
Escherichia coli/genética , Técnicas de Transferência de Genes , Genes Bacterianos , Engenharia Genética , Vetores Genéticos , Íntrons , Sequência de Bases , Southern Blotting , Cromossomos/genética , Códon de Terminação , Biblioteca Gênica , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Mutação Puntual , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.
Assuntos
DNA/genética , Marcação de Genes , Íntrons , RNA Catalítico/genética , Pareamento de Bases , Sequência de Bases , Linhagem Celular , DNA Viral/genética , Escherichia coli/genética , Genes pol , Terapia Genética , HIV-1/genética , Humanos , Lactococcus lactis/genética , Dados de Sequência Molecular , Provírus/genética , Receptores CCR5/genética , Recombinação Genética , TransfecçãoRESUMO
Functional foods are gaining more and more interest from health scientists. One way to improve the nutritional properties of foods may be to modify the starch component, often included to stabilize the food product. In this study two chemically modified starches-a 1-2% acetylated potato starch and a starch enriched with 2% beta-cyclodextrin-and a native, unmodified potato starch (control) were investigated with regard to 6-h energy expenditure, substrate metabolism, hormone concentrations, and subjective appetite sensations. Subjects were 11 healthy, normal-weight, young men. The starch (50 g) was prepared as a pudding with fruit sauce and whipped cream (3180 kJ, 49% of energy from carbohydrate, and 40% of energy from fat). The meal was given in the morning after a 2-d carbohydrate-rich, weight-maintenance diet. After the modified-starch meals, response patterns for plasma glucose (P < 0.01), insulin (P < 0.05); gastric inhibitory polypeptide (P < 0.05), subjective satiety (P < 0.05), and fullness (P = 0.06) were significantly different from response patterns after the meal with the control starch. Thus, a flattening of the glucose curve, a lower insulin and gastric inhibitory polypeptide response, and higher fullness ratings were observed after the meal with the beta-cyclodextrin starch. Satiety ratings were higher after both meals with modified starch than after the meal with the control starch. In conclusion, a minor modification insulinemic (1-2%) of native potato starch improved the glycemia, insulinemic, and satiating properties of a meal. This was especially true for the beta-cyclodextrin-enriched starch. Slower gastric-emptying rate or delayed intestinal absorption of the modified starch may explain the observed differences.
