A genetic analysis of DNA sequence requirements for Dissociation state I activity in tobacco.

Abstract

Our objective was to test whether the double Ds structure correlated with Dissociation state I activity @e., high frequency of chromosome breakage and low frequency of reversion) in maize exhibited similar properties in tobacco. A genetic assay was established to test double Ds and related structures for their ability to cause loss of the linked marker genes streptomycin phosphotransferase and P-glucuronidase in transgenic tobacco. An engineered double Ds element and a simple Ds element showed behavior consistent with that of state I and state II Ds elements, respectively, as described for maize. DNA structural rearrangements accompanied marker gene loss. Dissection of the double Ds structure showed that a left end and a right end of Ds in direct orientation were sufficient for the instability observed. This result suggested that left and right ends of Ds in direct orientation can participate in aberrant transposition events, consistent with two different models for double Ds-induced chromosome breakage proposed previously. Both models predict that the inversion of a half Ds element accompanies the aberrant transposition event. Such an inversion was detected by polymerase chain reaction experiments in tobacco and maize only when Activator activity was present in the genome.

Methylation pattern of Activator transposase binding sites in maize endosperm.

“…The abberant intrachromatid transposition or SIN model (Doring et al, 1984(Doring et al, , 1989English et al, 1993) proposes that two directly oriented Ds ends on the same chromatid undergo aberrant transposition. In contrast, the abberant interchromatid transposition or SIS model suggests that, after replication, a 5'end on one and a 3'end on the other daughter chromatid combine to form an aberrant transposition complex (Greenblatt, 1984;Fedoroff et al, 1989;English et al, 1993English et al, , 1995Weil and Wessler, 1993). English et al (1995) have consistently shown that the SIS mechanism predominates in tobacco.…”

Aberrant Transpositions of Maize Double Ds-Like Elements Usually Involve Ds Ends on Sister Chromatids.

“…In our previous work (English et al, 1993), we extended this idea by proposing that an excision product might be formed by ligation of the sequences that had originally flanked the excised Ds ends. The sister Chromatids would be fused at this position, forming an acentric or dicentric chromatid, and chromosome breakage would result.…”

“…Thus, the empty site sequences isolated by PCR experiments (English et al, 1993) probably represent the precise positions of sister chromatid fusion or "chromatid fusion products." Other models explaining the structures that were observed would be complicated enough to make them unlikely.…”

“…An engineered double Ds element in tobacco behaves like a chromosome-breaking Ds element with respect to marker gene loss and reversion, and a left end (LE) and a right end (FIE) of Ds in direct orientation (that is, half a double Ds) are sufficient for Ac-dependent marker gene loss (English et al, 1993). Examples of these different types of Ds structures are shown in Figure 1.…”

“…With this model as a reference point, two models have been proposed to show how aberrant transpositions involving the Ds LE and RE in direct rather than inverted orientation could result in chromosome breakage. In the sister chromatid substrate (SIS) model (Fedoroff, 1989;English et al, 1993), the Ds LE and RE from sister chromatids participate in an aberrant transposition event (Figures 2E to 2H). In contrast, in the single chromatid substrate (SIN) model (Doring and Starlinger, 1984;English et al, 1993), the aberrant transposition involves Ds ends on the same chromatid (Figures 21 to 2L).…”