Skip to main content
. 1998 Feb;18(2):1115–1124. doi: 10.1128/mcb.18.2.1115

FIG. 5.

FIG. 5

Analysis of VLPs from wild-type and NLS mutant Ty1 strains. (A) Sucrose gradient fractionation profiles showing VLP peaks. The top of the gradient (20% sucrose) is presented at the left side of the x axis; the bottom of the gradient (70% sucrose) is at the right. The y axis represents absorbance at 254 nm. It is important to note that VLPs could be isolated from wild-type and NLS mutant Ty1 strains (small peaks under vertical arrows). The first peak at the top of the gradient is bulk protein, and the last peak at the right is the extreme bottom of the gradient. (B) Biochemical analysis of VLPs. The peak fraction from each VLP preparation was examined for RT and integration activities in vitro. All of the preparations showed normal RT activity. The activities of positive control avian myeloblastosis virus RT and the negative control, no enzyme (data not shown), were 93,500 and 465 cpm, respectively. VLPs isolated from the wild-type strain (pX3), or the NLS mutant strains (IN B1 and B2) supported normal levels of integration, whereas the negative control IN mutant strain (GM315) was 16-fold less active. OD280, optical density at 280 nm. (C) In vitro integration assay (25). The artificial transposon AT-2 serves as a substrate for Ty1 IN and is integrated into the target plasmid in vitro when VLPs are added. AT-2 carries the dhfr gene which confers trimethoprim resistance and thus serves as a selectable marker when the transposition reaction mixture is transformed into Escherichia coli. The target plasmid contains the bla gene conferring ampicillin resistance, while transposon recombinants carry both markers. The number of recombinants (Ampr Tmpr colonies) divided by the total number of targets recovered (Ampr colonies) provides the recombinant frequency. (D) Immunoblots of nine peak fractions from each gradient probed with MAb 8B11, showing similar yields of encapsidated IN protein in the four preparations. Molecular masses are indicated on the left in kilodaltons. WT, wild type; GM315, negative control IN mutant strain.