On-off switching of gene expression regulated with carbohydrate-lectin interaction.
A novel strategy for artificial regulation system of gene expression applying the specific molecular recognition between carbohydrate and lectin is proposed. Plasmid-lactose conjugates (pActin-lactose and pGFP-lactose) prepared via diazocoupling maintained the transcription activity with T7 RNA polymerase. Gel-shift assay showed that the pActin-lactose conjugates were specifically complexed with galactose-specific lectin RCA(120) with a strong binding affinity (K(a) = 7.6 x 10(5) M(-1) per Lac-unit). The complexes were observed to form aggregates of sub-several micrometer size by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). The activities of transcription and expression of the conjugates were evaluated, respectively, on the basis of the amount of transcript of pActin and the fluorescent intensity of the expressed GFP. These activities were repressed in the presence of an increasing concentration of RCA120, and then recovered by adding lactose, lactosylceramide-containing liposomes, and lactose-carrying polymers to the conjugate-RCA120 complex. Gel-shift assay and TEM observation revealed that the aggregation form of the complex was relaxed partially in the presence of the lactose derivatives, which increased the accessibility of T7 RNA polymerase to result in the recovery of transcription activity.
Matsuura
Kazunori
K
Department of Molecular Design, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan. mal4tcm@mbox.nc.kyushu-u.ac.jp
Hayashi
Katsuhiro
K
Kobayashi
Kazukiyo
K
D000199:Actins / Q000737:chemistry; D000818:Animals; D002241:Carbohydrates / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D015870:Gene Expression; D005786:Gene Expression Regulation; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D007785:Lactose / Q000737:chemistry; D037102:Lectins / Q000737:chemistry*; D008024:Ligands; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D008956:Models, Chemical; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D013696:Temperature; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry
D000199:Actins; D002241:Carbohydrates; D006027:Glycosides; D037102:Lectins; D008024:Ligands; D008081:Liposomes; D011108:Polymers; D014764:Viral Proteins; C008245:lactosides; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D007785:Lactose