A novel gene trap method using terminator-REMI and 3' rapid amplification of cDNA ends (RACE) in Dictyostelium.
We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.
Takeda
Kosuke
K
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810, Hokkaido, Japan.
Saito
Tamao
T
Tanaka
Tomoyuki
T
Morio
Takahiro
T
Maeda
Mineko
M
Tanaka
Yoshimasa
Y
Ochiai
Hiroshi
H
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D017384:Sequence Deletion
D020413:3' Untranslated Regions; D016054:DNA, Protozoan; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases