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718 BIOCHEMICAL SOCIETY TRANSACTIONS Hunter, I. & Skerrow, D. (1982) Biochim. Biophys. Acia 714, 164169 Matoltsy, A. G. (1964) Nature (London) 201, 113&1131 McGuire, J., Osber, M. & Lightfoot, L. (1984) Br. J . Dermaiol. 111 (Suppl. 27), 27-37 Expression of two aldolase A mRNA species in different human and rat tissues F. SCHWEIGHOFFER, F. MENNECIER, P. MAIRE, L. BACHNER, and A. KAHN Inserm U 129, 24 rue du Fg St Jacques, 75674 Paris Cedex 14, France The development stage- and tissue-specific expression of multiple mRNAs encoding a same protein is now a welldocumented phenomenon (Schibler et al., 1983; MacGrogan et al., 1985). Recently, it has been shown in rat (Mukai et al., 1984; Tsutsumi et al., 1984) that aldolase A is translated from two mRNAs, of 1650 and 1550 bases; the latter was detected in adult muscle, whereas the former was present in brain and re-expressed in hepatomas. Fig. 1 shows a ‘Northern’ blot analysis of the two aldolase A mRNAs in different rat and human tissues. In rat, the heavier species is the only one detected in non-muscular organs, while the lighter species is the single detectable form in adult gastrocnemius. In foetus, by contrast, only the heavier form was seen before the 19th day of foetal life; then the 1550 base-long mRNA progressively takes over the 1650 one. The lighter aldolase A mRNA species appears to be specific to fast twitch muscle since, by contrast, adult soleus and heart muscle exhibit only the heavier mRNA species, like the non-muscular tissues. In man, the lighter mRNA form is already present in a 3-month-old foetus muscle, in an equal amount to the heavier species. Also in contrast to the rat, this 1650 base mRNA is still detectable as a minor species in the adult muscle. The peculiar occurrence of slow twitch fibres in human skeletal muscle compared with rat muscle might explain this result. The whole content of aldolase A mRNA is roughly the same in human adult and foetal skeletal muscle, and this is in contrast with the aldolase A protein level, which increases during muscle development. Fig. 2 shows primer extension experiments performed with rat brain, heart and gastrocnemius muscle mRNAs as templates. Adult gastrocnemius allowed a 370 base extension of our primer, while heart and brain mRNA used as templates gave longer extensions, of 420,490 and 560 bases. Therefore, the aldolase A mRNAs present in these tissues differ by their 5’ ends. An heterogeneity of the heavier species can be observed in brain and heart. Discussion The products of extension using the same primer upon the two types of aldolase A mRNA were different in size; this size difference is of the same range as the size difference between the two mRNAs. Therefore, the 5’ end differences can in themselves account for the total difference between those two messengers. It has been previously demonstrated that a single aldolase A isoenzyme exists in different tissues (Gracy et al., 1970; Ikehara et a]., 1970) and that both heavier and lighter mRNA species have a very homologous, probably identical, 3’ non-coding extension (Mukai e t al., 1984). Fig. 1. Northern blot experimenl Total mRNA was deposited on to each slot. Man: (1) adult liver, 1Opg of mRNA; (2) 3-month-old foetus liver, 10 pg; (3) adult lung, 5 pg; (4) adult muscle, 2 pg; (5) 3-monthold foetus muscle, 4pg; (6) testis, 5 pg; (7) pineal gland, 6pg; (8) adult muscle, 2pg. Rat: (1) 17-day-old foetus muscle, 30 pg; (2) and (10) adult brain, 20pg; (3) 19-day-old foetus muscle, 30pg; (4) and (8) adult gastrocnemius muscle, 3pg; (5) newborn rat muscle, 25 pg; (6) adult soleus muscle, 15 pg; (7) 3-day-old muscle, 18 pg; (9) adult heart, 20 pg; (1 1) 6-day-old muscle, 15 pg. 1986 616th MEETING, LONDON 719 3 70 Fig. 2. Primer extension experiment Templates used: (1) rat brain poly (A’) RNA, 28 pg; (2) rat heart poly (A’) RNA, 22pg; (3) rat gastrocnemius poly (A’) RNA, 5pg; (4) rat liver poly (A’) RNA, 5pg; ( 5 ) short exposure of lane (3). The primer was a 80 base-pair fragment Hinfl-Hue111 of the aldolase A cDNA, labelled with [a-”P]dATP by filling the 3’ recessed end at the Hinfl site. The differences between the aldolase A mRNA forms seem therefore to occur through their 5‘ non-coding stretches. Several possible mechanisms leading to the expression of such different messengers could then be proposed. Two promoters, one ubiquitous and the other specific to adult fast twitch muscle, could exist. Otherwise, the same pre- mRNA transcript might be differentially spliced, or even both mechanisms might occur, as has been demonstrated for myosin light chains (Periasmy et al., 1984). Nevertheless, one cannot totally exclude that the different aldolase A mRNAs could be specified by different genes. It has been shown that in man, at least two genes carried by different chromosomes (Hagenauer et a[., 1985), and in rat at least five genes (P.Maire, unpublished work) are present. There is no information until now on how many of these genes are transcribed. In fast twitch fibres, the rise of the aldolase A mRNA lighter species level is parallel to that of other specific muscular protein mRNAs such as glycogen phosphorylase M, creatine kinase M (F. Schweighhoffer, unpublished work) cr-actin (Minty et al., 1982) and adult myosin heavy chain (Wild et al., 1984). In conclusion, the expression of aldolase A mRNAs is qualitatively and quantitatively related to muscle development. The lighter mRNA species is absolutely specific to the differentiated fast twitch muscle fibres, while the heavier species accounts for foetal, ubiquitous aldolase A expression. Gracy, R. W., Lacko, A. G., Brox, L. W., Adelman, A. C. & Horecker, B. L. (1970) Arch. Biochem. Biophys. 136, 4 8 M 9 0 Hagenauer, O., N’guyen Van-Long, Mennecier, F., Kahn, A. & Frezal, J. (1985) Cyrogenef. Cell Genet. 40,605409 Ikehara, Y., Endo, H. & Okada, Y. (1970) Arch. Biochem. Biophys. 136, 491497 MacGrogan, M., Simonsen, C. C., Smouse, D. T., Farnham, P. J. & Schimke, R. T. (1985) J. B i d . Chem. 260, 2307-2314 Minty, A. J., Alonso, S., Caravatti, M. & Buckingham, M. E. (1982) Cell 30, 185-192 Mukai, T., Joh, K., Miyahara, H., Sakakibara, M., Arai, Y. & Hori, K. (1984) Biochem. Biophys. Res. Commun. 119, 575-581 Periasamy, M., Strehler, E. E., Garfinkel, L. I., Gubits, R. M., RutzOpazo, N. & Nadal-Ginard, B. (1984) J. B i d . Chem. 259, 13595-13604 Schibler, U., Hagenbiichle, O., Wellauer, P. K. & Pittet, A. C. (1983) Cell 33, 501-508 Tsutsumi, R., Tsutsumi, K., Numazaki, M. & Ishikawa, K. (1984) Eur. J. Biochem. 142, 161-164 Wild, 1. J. F., Boyd, C. D., Bester, A. J. & Van Helden, P.D. (1984) Nucleic Acids Res. 12, 2717-2729 Expression of brain-specific ID sequence is not restricted to the brain, and a novel complementary cID sequence is found in L-type pyruvate kinase mRNA (a liver-specific messenger) YU-CHUN LONE, MARIE-PIERRE SIMON, AXEL KAHN and JOBLLE MARIE Inserm U 129, Chu Cochin. 24 rue du Fg St Jacques, 75014 Paris Cedex 14, France We have recently shown that the pyruvate kinase (PK) gene is transcribed in the liver in three distinct translatable mRNA species of 3.2 2.2 and 2 k b (Simon et al., 1983). These three different pyruvate kinase mRNAs only differ by their 3’ untranslated extension (J. Marie, M. P. Simon, Y . C. Lone, M. Cognet & A. Kahn, unpublished work). In order to study the role of these messengers and the regulation of their expression in the liver, we have sequenced both the whole coding region and the 3’ untranslated region of the 3.2 kb PK mRNA. Abbreviations used: kb, kilobases; PK, pyruvate kinase. Vol. 14 Fig. 1 shows the nucleotide sequence of 3PK cDNA clones (llC6, 12H2, 2B8) which covers the whole 3’ noncoding region of the 3.2 kb PK mRNA. We have found that the 3.2 kb mRNA possesses in its 3’ non-coding region a sequence complementary to the brain-specific identifier sequence (ID) described by Sutcliffe and co-workers (Sutcliffe et al., 1982, 1984a, b; Milner et al., 1984); however, this sequence which is surrounded by two A h family elements is absent in the two other PK mRNAs (Fig. I). This cID sequence present in the PK mRNA was subcloned in both orientations in M 13 single-stranded phage, both strands being used as probes to analysis the expression of transcripts containing I D and cID sequences in various tissues. Fig. 2 shows the Northern blot and dot blot analysis in various tissues, ID sequence probe hybridized, as expected, with the two small brain-specific RNAs, BCI and BC2