ISSN:
1617-4623
Keywords:
GUS transient expression
;
Mobile DNA
;
Zea mays
;
Petunia hybrida
;
Cotransfection
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Summary The excision of a Ds-like transposable element (AcΔ) is mediated in trans by the transposable element Ac or its derivatives in Petunia protoplasts cotransfected with two plasmid DNAs. Excision restores the activity of the β-glucuronidase (GUS) gene that is otherwise shut off by the presence of AcΔ in its leader sequence. A transient expression assay (histochemical test) is used to detect the β-glucuronidase activity at the protoplast level. The number of blue-stained protoplasts is a measure of the excision frequency. With AcΔ alone a near-zero background of GUS activity is detected, which is weakly enhanced by the presence, in trans, of either the wild-type Ac or the coding region (ORFa) transcribed from the 2′ promoter of Agrobacterium tumefaciens TR-DNA. A strong enhancement is observed when a truncated Ac coding region, also under the control of the 2′ promoter, is supplied in trans. The truncated version has ATG10 at codon 103 in frame with ORFa and is preceded by 7 out-of-frame ATGs. The assay is quick and well suited for detection of excision frequencies above the value obtained with the wild-type Ac. The presence of empty donor sites following excision can be demonstrated by PCR amplification and direct sequencing of the appropriate DNA fragment.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00259446
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