~I]~ECHNICAL~[~IPS observed in this fungusm: the association of two nuclear genes leads to an early arrest of growth a few days after ascospore germination. This p h e n o t y p e is correlated with a specific deletion of the mitochondrial DNA that parallels the situation observed in the case of m y o p a t h y described by Zeviani et al. 5 . The two genes triggering this site-specific event are well characterized genetically. The first one is very closely linked to the matingtype minus haplotyp'e but'its nature and function are not yet known. The s e c o n d is a mutant allele of the structural gene of the cytoplasmic ribosomal protein $12 n. The mutation increases translational accuracy and affects the mycelial growth of the fungus 12,13. What is the significance of the strong similarity between the Rig protein, which does not s e e m to be a ribosomal protein, and a ribosomal protein from a lower eukaryote? The former is highly.expressed in the nucleus of actively dividing cells and seems to be involved in the control of nuclear DNA replication while the latter might control, directly or indirectly, the integrity of mitochondrial DNA in a lower eukaryote. It is difficult to speculate about the biological implications and the significance of
this observation at present. Nevertheless, there is evidence for the oncogenic potential of translation. As mentioned above, overexpression of the elF-4E translation initiation factor has been shown to induce tumorigenic transformation-~. The authors suggest that this effect may be at the level of translational regulation of certain oncogenes or growth factors. In this regard, it is interesting to note that several p r o t o - o n c o g e n e s seem to be translationally regulated in vivo and in vitro 14A5. On the other hand, the roles played b y nuclear-encoded mitochondrial proteins in both neoplastic transformation and myopathies or cell death remain to be elucidated. The evolutionary conservation of a protein that can be either ribosomal (in bacteria and lower eukaryotes) or nucleoplasmic (in mammals) has to be considered in the context of this complex network.
Acknowledgements I thank C. Caries, F. Bouet and Y. Blouquit for peptide separation and microsequencing, and M. Picard for critical reading of the manuscript.
348, 334-336 3 Lazaris-Karatzas, A., Montinc, K.S. and Sonenberg, N. (1990) Nature 345, 544-547 4 Wallace, D.C. (1989) Trends Genet. 5, 9-13 5 Zeviani, M. et al. (1989) ;Vature339, 309-311 6 lnoue, C. etal. (1987) t'roc. ,~}tll Acad. Sci. USA 84, 66594~662 7 lnoue, C. et aL (1988) Biocbem Bioph3,£ Res. C~)mmun. 150, 1302-1308 8 Mankin, A.S. (1989) kE'BSLett. 24-(). 13-16 9 Ohkubo, S. et al. (1987) Mol. Getz. Genet. 210, 314-322 lO Belcour, I,., Begel, O. and Picard. MI Proc. NatlAcad. Sci. t3)4 (in press) 11 Dequard-Chablat. M., CoppinRaynal, E., Picard-Bennoun, M. and Madjar, J-J, (1986) J. Mol. BioL 190, 167-175 12 Picard-Bennoun, M. (1976) Mol. Gen. Genet. 147, 299-306 13 Picard-Bennoun, M. (1980)Mol. Gen. Genet. 183, 175-180 14 Marth, J.D. el al. (1988) ,\~lttov 332, 171-173 15 Acland, P., Dixon, M., Peters. G. and Dickson, C. (1988) ,Valttm 3i3. 662--665
MICHELLEDEQUARD-CHABLAT
References 1 Kruh, J. and Defer, N. (1989) Regard Biochim. 2, 32-38 2 Hockenbery, D. etal. (1990)Natuw
l*lsl 'lift de G(',letique el de 31 crab uh)~,e. I R, I CVRS DI ~54 Bdt 400 ['m~'ervit(,Pan~ ~'ml F91405 O~t O' Ced~:v. t:nmce.
Improved resolution of ra_diolabeiled proteins by blotting before autoradiography Autoradiography of 3~S-radiolabelled proteins that have been sepa~ted in polyacrylamide gels is commonly achieved by A B C the d~ing of the get under vacuum and subsequent exposure to X-~y film, O ~ of the disadvantages of ~ technique is that the amount of radioactivity detected is reduced be'cause of shielding by the gel itself. The .sensitivity can be improved by impregrTmting the gel with scintillants before dryingi,z, but this can lead to a reduction in resolution of the detected t4.3 kDabands, because the scintillant method relies upon the detection of scattered light radiation that results from the ~-particles impinging on the scintillant, In experiments wlaere it is necessary to measure molecular weights accurately or to resolve proteins of similar mobility it would be particularly desirable to improve the sensitivit7 of autoradiography without compromising resolution. We show here that this can be achieved by transferring radiolabelled proteins from the gel to a protein-binding membrane before exposure to X-ray film. (Quantitative comparison between different proteins is not possible with our method, because of variations in blotting efficiency between proteins, although such comparisons are difficult with any method because of variations in the content of labelled amino acids.) Radiolabelled protein was obtained by transcnption and translation in vitro of a linearized plasmid, pGB3b~, using SP6 RNA polymerase and wheatgerm extract in the presence of PSS]methionine. Equal amounts of proteins were separated on a 1 mm thick polyacD'lamide gel, after which individual tracks were treated in the following ways: track A, the gel was soaked in fixer (25% v/v propan-2-ol. 10% v/v acetic acid~ for 60 min and then dried under vacuum at 80°C for 90 min: track B. the gel was soaked in fixer for 30 min. then in the scintillant AmplifyTM (Amersham International) for 30 rain and then dried as above: track C. the gel was soaked in 48 m~l Tris. 39 m,a glycine, 20% v/v methanol, pH 9.2 for 20 min. after
TIG AtGUST 1 9 9 I VOL. 7 NO. 8
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this observation at present. Nevertheless, there is evidence for the oncogenic potential of translation. As mentioned above, overexpression of the elF-4E translation initiation factor has been shown to induce tumorigenic transformation-~. The authors suggest that this effect may be at the level of translational regulation of certain oncogenes or growth factors. In this regard, it is interesting to note that several p r o t o - o n c o g e n e s seem to be translationally regulated in vivo and in vitro 14A5. On the other hand, the roles played b y nuclear-encoded mitochondrial proteins in both neoplastic transformation and myopathies or cell death remain to be elucidated. The evolutionary conservation of a protein that can be either ribosomal (in bacteria and lower eukaryotes) or nucleoplasmic (in mammals) has to be considered in the context of this complex network.
Acknowledgements I thank C. Caries, F. Bouet and Y. Blouquit for peptide separation and microsequencing, and M. Picard for critical reading of the manuscript.
348, 334-336 3 Lazaris-Karatzas, A., Montinc, K.S. and Sonenberg, N. (1990) Nature 345, 544-547 4 Wallace, D.C. (1989) Trends Genet. 5, 9-13 5 Zeviani, M. et al. (1989) ;Vature339, 309-311 6 lnoue, C. etal. (1987) t'roc. ,~}tll Acad. Sci. USA 84, 66594~662 7 lnoue, C. et aL (1988) Biocbem Bioph3,£ Res. C~)mmun. 150, 1302-1308 8 Mankin, A.S. (1989) kE'BSLett. 24-(). 13-16 9 Ohkubo, S. et al. (1987) Mol. Getz. Genet. 210, 314-322 lO Belcour, I,., Begel, O. and Picard. MI Proc. NatlAcad. Sci. t3)4 (in press) 11 Dequard-Chablat. M., CoppinRaynal, E., Picard-Bennoun, M. and Madjar, J-J, (1986) J. Mol. BioL 190, 167-175 12 Picard-Bennoun, M. (1976) Mol. Gen. Genet. 147, 299-306 13 Picard-Bennoun, M. (1980)Mol. Gen. Genet. 183, 175-180 14 Marth, J.D. el al. (1988) ,\~lttov 332, 171-173 15 Acland, P., Dixon, M., Peters. G. and Dickson, C. (1988) ,Valttm 3i3. 662--665
MICHELLEDEQUARD-CHABLAT
References 1 Kruh, J. and Defer, N. (1989) Regard Biochim. 2, 32-38 2 Hockenbery, D. etal. (1990)Natuw
l*lsl 'lift de G(',letique el de 31 crab uh)~,e. I R, I CVRS DI ~54 Bdt 400 ['m~'ervit(,Pan~ ~'ml F91405 O~t O' Ced~:v. t:nmce.
Improved resolution of ra_diolabeiled proteins by blotting before autoradiography Autoradiography of 3~S-radiolabelled proteins that have been sepa~ted in polyacrylamide gels is commonly achieved by A B C the d~ing of the get under vacuum and subsequent exposure to X-~y film, O ~ of the disadvantages of ~ technique is that the amount of radioactivity detected is reduced be'cause of shielding by the gel itself. The .sensitivity can be improved by impregrTmting the gel with scintillants before dryingi,z, but this can lead to a reduction in resolution of the detected t4.3 kDabands, because the scintillant method relies upon the detection of scattered light radiation that results from the ~-particles impinging on the scintillant, In experiments wlaere it is necessary to measure molecular weights accurately or to resolve proteins of similar mobility it would be particularly desirable to improve the sensitivit7 of autoradiography without compromising resolution. We show here that this can be achieved by transferring radiolabelled proteins from the gel to a protein-binding membrane before exposure to X-ray film. (Quantitative comparison between different proteins is not possible with our method, because of variations in blotting efficiency between proteins, although such comparisons are difficult with any method because of variations in the content of labelled amino acids.) Radiolabelled protein was obtained by transcnption and translation in vitro of a linearized plasmid, pGB3b~, using SP6 RNA polymerase and wheatgerm extract in the presence of PSS]methionine. Equal amounts of proteins were separated on a 1 mm thick polyacD'lamide gel, after which individual tracks were treated in the following ways: track A, the gel was soaked in fixer (25% v/v propan-2-ol. 10% v/v acetic acid~ for 60 min and then dried under vacuum at 80°C for 90 min: track B. the gel was soaked in fixer for 30 min. then in the scintillant AmplifyTM (Amersham International) for 30 rain and then dried as above: track C. the gel was soaked in 48 m~l Tris. 39 m,a glycine, 20% v/v methanol, pH 9.2 for 20 min. after
TIG AtGUST 1 9 9 I VOL. 7 NO. 8
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