MATERIALES TERMOPLASTICOS CON ESTRUCTURA CRISTALINA
5.7.6 Sistema de refrigeración [8]
In the pioneering work by Endo et al ( 1987 ) on rat 28S rRNA modification by ricin A chain, the site of action was deduced from direct RNA sequencing of a naturally occurring 550 nucleotide 3' terminal fragment. Furthermore, the authors showed that depurination of the rRNA at this position, A4 3 2 4, by ricin A chain could be assayed by the appearance of an approx. 390 nucleotide fragment produced b y amine-catalysed cleavage of the 28S rRNA at this site. This elucidation of the mode of action of ricin A chain presented the opportunity to study the site of action of ricin A chain on other eukaryotic ribosomes and to investigate whether other ribosomes were also inactivated by the removal of the same adenine in the highly conserved sequence identified in rat 28S rRNA. However the approach used by Endo and coworkers of RNA sequencing on small naturally occurring fragments of the rRNA was not directly applicable to other ribosomes where comparable fragments were not known. Instead primer extension coupled with dideoxynucleotide sequencing was used. The later approach has been used extensively in structural probing of E.coli rRNA ( Moazed et al., 1988, S t e m et al., 1986 ). The rational for its use is based on the observation of Hagenbuchle et al. ( 1978) and Youvan and Hearst ( 1979 ) that reverse transcriptase is unable to read certain chemically modified bases in an RNA template. Pauses or stops give rise to bands corresponding to the length of the cDNA from the 5' end of the primer to the nucleotide immediately preceding the modified position. In the case of ricin A chain catalysed depurination of the rRNA it was envisaged that the removal of the
base would cause an additional band not present when untreated rRNA was used as the template. This approach has the advantage that it is less time consuming than direct RNA sequencing but it does rely on prior sequence knowledge to be able to design the primer.
Initially yeast ribosomes were targeted for study. The reasons for this were three fold; firstly the sequence of the LSU- rRNA was known both for Saccharomyces cerevisiae ( Georgiev et a l . , 1981 ) and Saccharomyces carlsbergensis ( Veldman et al., 1981 ). Secondly, more is known about the structure and function of the yeast cytoplasmic ribosome than that of any other eukaryote. Part of the reason for this progress is the well-defined genetics in yeast and the availability of mutants with altered ribosomal proteins (reviewed in Warner, 1982 ). Thirdly, other work in progress in our laboratory was aimed at obtaining yeast mutants with ricin-resistant ribosomes. The biochemical characterisation of the altered component(s) in such mutants could shed light on the action of ricin A chain. It was thought that all these would be of help if further investigation into the site of action of ricin A chain was carried out. On a practical note the isolation of intact ribosomes from yeast proved to be relatively unproblematic and little degradation of the rRNA is apparent in these ribosomes.
S.cerevisiae 26S rRNA is 3392 nucleotides in length, considerably shorter than rat 28S rRNA and it is almost identical to the corresponding LSU-rRNA from S . carlsbergensis. This is especially true in the 3' terminal region encompassing the highly conserved sequence identified by Endo - over the last 400 nucleotides b oth LSU- rRNAs are identical except for the addition of a single base in
carlsbergensis. This has the consequence that although the base in the conserved loop analogous to A4 3 2 4 in rat 28S rRNA and
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1 1 1 E.coli 23S rRNA in both yeasts is designated A3Q2 4, it is 368 nucleotides and 369 nucleotides from the 3' end in S.cerevisiae and S .carlsbergensis respectively.The site of action of ricin A chain was also investigated on plant ribosomes using wheat germ ribosomes as the system. Although no sequence for the entire 25S rRNA has been published, part of the 3' terminus has been deduced from sequence comparisons of the cloned intergenic region of wheat rDNA with other LSU-rRNAs ( Barker et al ., 1988 ). This region covers the ricin A chain / <x -sarcin loop but because the entire sequence of the 25S rRNA is not known it is not possible to designate numbers to the nucleotides.
In these and subsequent experiments described in other chapters recombinant ricin A chain was used. The source of this is discussed in section 2.1.1. It differs from the native ricin A chain only in the presence of an additional residue ( methionine ) at it s N-terminus and the fact that it is non-glycosylated. The results of the following chapter show the native and recombinant proteins to be identical in activity and specificity.
SECTION 3.2 RESULTS AND DISCUSSION.
3.2.1 Effects of Ricin A Chain on Yeast Ribosomes.
Ricin A chain has been reported to inhibit protein synthesis in a number of eukaryotic cell-free translation systems, including
the yeast lysate ( May, 1989 ). To test whether yeast ribosomes are modified by ricin A chain in a way analogous to that reported for rat 28S rRNA, ribosomes were purified from S.cerevisiae and incubated with and without ricin A chain for 1 hour. The rRNA was extracted and where indicated incubated with aniline to cleave at the site of depurination and the rRNA fractionated on 1.2% (w/v) agarose 50% formamide gels as described in section 2.6.1. ( Fig. 3.2.1 ). The treatment of S.cerevisiae ribosomes with ricin A chain ( tracks 3 and 4 ) or the incubation of extracted, untreated rRNA with aniline (tracks 5 and 6 ) does not cause the release of any additional fragments of rRNA when compared to the rRNA from control ribosomes (tracks 1 and 2 ). However when the yeast ribosomes are first incubated with ricin A chain and the extracted rRNA incubated with aniline a fragment is released ( arrowed in tracks 7 and 8 ). To confirm that the fragment represents the 3' end of the 26S rRNA a probe to this sequence was generated from the EcoRl fragment E of S. carlsbergensis rDNA ( Veldman et a l ., 1981 ).
3.2.2 Generation of a Probe for the 3' end of 26S rRNA.
The EcoRl fragment E of S . calsbergensis rDNA was a gift from Rudi Plants and contains 496 bp of the 3* terminus of the 26S rRNA gene arid extends a further 100 bp into the spacer region ( Fig. 3.2.2 ). Since this also contains approx. 125 bases upstream of the adenine analogous to the site of depurination in rat 28S rRNA a smaller probe was generated from the EcoRl fragment E. The strategy for the cloning of this fragment is shown in Fig. 3.2.3. Briefly, the
Fig. 3.2.1 Gel electrophoresis of rRNA from S. cerevisiae ribosomes treated with ricin A chain.
Yeast ribosomes ( 50 ug ) were incubated with 50 ng ricin A chain for 1 hour at 30°C in a final volume of 100 ul Endo buffer. A control, without ricin A chain was similarly incubated. The rRNA was extracted and where indicated treated with aniline as described in sections 2.4.1 and 2.5.1. The rRNA was fractionated on a 1.2% (w/v) agarose/ forraamide gel and stained with EtBr ( section 2.6.1 ). 1. control rRNA.
2. control rRNA.
3. rRNA from ribosomes incubated with ricin A chain. 4. rRNA from ribosomes incubated with ricin A chain. 5. aniline treated rRNA from control ribosomes.
6. aniline treated rRNA from control ribosomes.
7. aniline treated rRNA from ricin A chain treated ribosomes. 8. aniline treated rRNA from ricin A chain treated ribosomes.