The Ac transposase consists of several, functionally distinct
--Ute Behrens-Jung, Reinhard Kunze and Sandra Kuehn
By means of a transient Ds-excision assay we have recently investigated the transpositional activities of several Ac transposase (TPase) mutants, and their effects upon coexpression with the active TPase derivative TPase(103-807) (Kunze et al., Proc. Natl. Acad. Sci. USA 90, 7094-7098, 1993). Some of these mutant TPase derivatives act as dominant inhibitors of transposition, leading to the conclusion that the active TPase is an oligomeric protein.
Since a fraction of the mutant, inactive TPase expression vectors used in that study gave rise to much lower protein levels in the transfected protoplasts than the "wild-type" TPase vector, it could not be determined whether they influence the Ds excision frequency upon coexpression with the "wild-type" TPase. Therefore, we have constructed modified plasmids which express the mutant TPases with similar levels as the "wild-type" TPase vector. The results of these experiments - performed under the same conditions as described in the above-mentioned publication - are summarized in Figure 1. Extending our previous results, we found four additional dominant mutants (369TR, 390RV, 445TR and 462TR), and a number of inactive, recessive mutants. The weird transposition frequency boost caused by coexpression of small amounts of mutants
The distribution of dominant and recessive mutations along the polypeptide chain indicates functionally distinct regions of the TPase. With only one exception (mutant 388CR) all mutants between the N-terminus of the active TPase derivative (amino acid [aa] 103) and aa 585 are transpositionally inactive. The C-terminal about 180 aa's seem to be more tolerant against (presumably small) structural disturbances, as four out of five two-aa-insertions in this region are transpositionally active. Nonetheless, the C-terminal about 100 aa's are required for the transposition reaction, as their deletion inactivates the protein.
The inactive TPase mutants fall into two groups - dominant and recessive - which seem to be clustered along the polypeptide chain. Except for mutant 249(S)AD which possibly exhibits an intermediate effect, all inactive mutants between the N-terminus and aa 270, and between aa's 369 and 524 are dominant, whereas two-aa-insertion mutants between aa's 297 and 341, and between aa's 577 and 709 are recessive, respectively. The dominant mutants are most likely still capable of interacting with the "wild-type" TPase. The recessive phenotype could have different causes: (a) the disturbance of protein structure is severe and not locally restricted, preventing any functional interaction with the "wild-type" TPase; (b) the mutants have specifically lost the ability to interact with the "wild-type" TPase; or (c) the mutants can specifically interact with the "wild-type" TPase, but the affected functions are not required in every subunit of the active TPase oligomer.
In order to distinguish between these three possibilities, we have begun to do complementation experiments with pairs of inactive, recessive mutants. We obtained preliminary results indicating that coexpression of mutants 297PR and 709RV leads to the formation of active TPase. Accordingly, these two mutants can specifically interact and fall into two different complementation groups. Presumably, the Ac TPase consists of several distinct domains with independent functions.
1. Distribution and relative activities of mutations along the N-terminally
truncated TPase(103-807). Certain segments of the protein are highlighted
in column "TPase(103-807)": "PQ" = P109QPQPQPQPEPQPQPQPEPE128. "DNA" =
DNA binding domain. Protein regions with more than 30% sequence identity
between the Ac TPase and the putative Tam3 TPase are indicated
as "a", "b1", "b2", "c" and "d". Column "Mutant" shows the approximate
locations of individual mutations. For the nomenclature of mutations the
single amino acid letter code is used: D(n-m)
= deletion of ORFa residues n to m. HnA = substitution of Hisn
with Ala. nPR = insertion of Pro and Arg behind ORFa residue n.
249(S)AD = substitution of Tyr250 with Ser and insertion of
Ala and Asp. Protoplasts were co-transfected with 10 mg
reporter plasmid and, either 10 mg
(mutant) TPase plasmid alone (Column "mutant alone"), or 3 mg
"wild type" TPase plasmid and 15 mg
mutant TPase plasmid (column "mutant + WT"). The number of blue-stained,
i.e. GUS-positive protoplasts obtained with "wild type" plasmid alone was
taken as 100%. The values shown in columns "mutant alone" and "mutant
+ WT" are the averages of three to six independent co-transfections and
two platings per transfection.
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