MARCO NORMATIVO
1: Ambiente de control
The translation products of all of the 2A mutation-containing constructs derived from
pMD2 appeared to be similar (Figure 3.4.2). The lanes resulting from reactions in rabbit reticulocyte lysate all displayed a lar ge quantity of uncleaved [CAT2AGUS] and a diffuse
band co-migrating with CAT2A. Very faint bands co-migrating with GUS were seen in some cases. In the majority of lanes this band migrated slightly further than the genuine GUS cleavage product and was attributed to internal initiation at the methionine start codon
for GUS. Internal initiation was more pronounced in the wheat germ system. In wheat
germ extract there was also some uncleaved material for each of the mutant polyproteins. However, there appeared to be more of the CAT2A cleavage products in the wheat germ system. The band co-migrating with GUS was again much fainter than that for CAT2A. In all of these cases "2A-mediated proteolytic cleavage" as such does not appear to be occurring. Rather an interruption in translation after the 2A region seemed to have taken place. Plasmid pMD2.4.28 which encodes the mutations E->N and N->Q shows notably more cleavage products than the other mutants. Plasmids pMD2.3.8 and pMD2.3.7 which encode a PT mutation of P->S and I, respectively, also appeared to be cleaved to CAT2A
and GUS to a small extent. Immunoprécipitation of pMD2.3.8 with anti-CAT and anti- GUS polyclonal antibodies confirmed the product migrating with CAT2A to be derived
from the CAT protein and that migrating with GUS to be GUS specific (Figure 3.3.4). The translation products of all the 2A mutant constructs derived from pCAT2AGUS are more consistent with what one would expect from a proteolysis reaction. The efficiency of 2A cleavage is reduced in all of the mutated forms, however all mutants produce both CAT2A and GUS to some extent. The most efficient cleaving mutant 2A regions are those with E->Q, N->H , S->I and S->F mutations. The polyproteins containing the mutations E->D and N>E, and to a lesser extent the mutation of N->Q, produced small quantities of CAT2A and GUS proteins. The distinctive feature of these plasmids compared to those
derived from pMD2 is that the GUS gene commences immediately after the first proline residue of FMDV 2B (see Table 3.4.3). This might suggest that the sequence immediately
following 2A is important for translation of the second cleavage product.
The mutations encoding S->F and KLAG->KLAAG were placed in the context of both
that this might clarify the differences in the activities of 2A in the two systems. The translation products of these plasmids from both rabbit reticulocyte lysate and wheat germ extract reactions are seen in Figure 3.4.5. In both systems the 2A region containing mutation of serine to phenylalanine cleaves well. However, there is a marked increase in the ratio of CAT2A : GUS for pMD6.7 especially in the wheat germ extract system. The insertion of an alanine residue to give the motif KLAAG inactivates the proteolytic activity
of the 2A region. Once again however there was some CAT2A protein produced in the
reactions programmed with pMD31/10(5). This substantiated the hypothesis that the sequence following 2A was important for the translation of the second cleavage product. The products of transcription and translation of pMD4, encoding [CAT-'2A'-GUS] with a +1 frameshift over the 2A region, were somewhat unanticipated (Figure 3.4.3); if 2A were mediating a proteolytic cleavage dependent upon its primary sequence then the product of this reaction would surely be an uncleaved [CAT-'2A'-GUS] poly protein. In both rabbit reticulocyte and wheat germ systems there were bands, as expected, co-migrating with uncleaved [CAT2AGUS] polyprotein, and surprisingly a doublet of bands migrating similarly to CAT2A. Immunoprécipitation reactions using anti-CAT and anti-GUS polyclonal antibodies identified the doublet of bands as CAT specific. Thus, it appeared that no 2A-mediated cleavage had occurred. Instead an interruption in translation seemed to occur in a small percentage of cases, although it produced two products of the approximate size of CAT2A rather than one. One could postulate, therefore, that it was the RNA sequence of 2A which was important for the aiTest of translation.
3.4.4 Conclusions
• All of the [CAT2AGUS] polyproteins synthesised with mutated 2A regions displayed similar profiles. All had large amounts of uncleaved [CAT2AGUS] polyprotein, relatively lai'ge quantities for CAT2A and low levels of GUS, Thus the CAT2A : GUS
ratio was very large, and much greater than for the wild type sequence. This is in contrast with a proteolytic cleavage hypothesis.
• Translation of CAT'2A'GUS, in which the 2A region is in the +1 reading frame, appeared to result in two CAT specific products which were of the approximate size of CAT2A, This indicated that only the RNA sequence of 2A and / or the short stretch of 2B linker sequence encoding PFFF was important for the intermption in translation.