ELSEVIER Biochimica et Biophysica Acta 1306 (1996) 133-136 BB. Biochi~ic~a etBiophysica P~ta Short sequence-paper Genomic structure of carp mitogen-activated protein kinase kinase 1 gene Jiann-Horng Leu a, Ming-Shyue Lee b, Kuan-Tien Chen c, Geen-Dong Chang b, Chen-Kung Chou d, Chang-Jen Huang a,* a Institute of Biological Chemistry., Academia Sinica, Taipei, Taiwan b Graduate Institute of Biochemical Sciences, and National Taiwan Uniuersi~, Taipei, Taiwan c Department of Surgery, Universi~.' Hospital, National Taiwan University, Taipei, Taiwan o Department of Medical Research, Veterans General Hospital, Taipei, Taiwan Received 30 November 1995; accepted 16 January 1996 Abstract Carp mitogen-activated protein kinase kinase 1 (cMKKI) gene was isolated from a liver genomic library. The sequence around the exon-intron boundaries and 2 kb of the promoter region were determined. Our data indicate that this gene is composed of 11 exons and 10 introns spanning about 9 kb. Multiple potential transcription initiation sites were located by primer extension analysis. Examination of 2 kb of 5'-flanking sequence revealed potential binding sites for a variety of transcription factors such as E2F, Ets-1, GATA-1, Myb, NF-IL6, Spl, and NF-kB. Keywords: Genomic DNA sequence; Mitogen-activated protein kinase kinase; (Cyprinus carpio) Mitogen-activated protein (MAP) kinases, also known as extracellular signal-regulated kinases (ERKs), are a family of serine/threonme kinases that mediate intra- cellular protein phosphorylation events. They have been shown to participate in a variety of signal transduction pathways and are rapidly activated in response to various extracellular stimuli [1-4]. Several subgroups of MAP kinase have been identified in vertebrates including the ERK1 and ERK2 [5,6], c-jun amino-terminal kinase (JNK) [7,8], p38 [9-11], and ERK5 [12]. The activation of MAP kinase is triggered by an up- stream activator, called MAP kinase kinase (MKK) or MAPK/ERK kinase (MEK) [13]. MEK is a dual speci- ficity kinase that phosphorylates MAP kinase on both tyrosine and threonine re:ddues [14,15], whereas it is acti- vated and phosphorylated by a further upstream activator on its serine/threonine residues [16]. Recently, there are also a growing number of MEKs [12,17,18]. Specific MEKs have been shown to phosphorylate specific MAP kinases in a given pathway. For example, MEK1 and MEK2 phosphorylate ERK1 and ERK2 in the cellular * Corresponding author. Fax: + 886 2 3635038. 0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0167-478 1(96)00023- 1 processes such as proliferation, differentiation, and devel- opment [4,13]. MEK3 and p38 are involved in the cytokine response [18,19], whereas MEK4 and JNK are shown to participate in the stress response [18]. More recently, MEK5 and ERK5 have been identified to constitute a new signal transduction pathway [12]. As an initial step to study signal transduction in fish, we have cloned a full-length cDNA encoding the carp MAP kinase kinase 1 (cMKKI) [20]. In this study, we further determined the genomic structure and the promoter region of this gene. A commercially available carp liver lambda FIX II genomic library (Stratagene, La Jolla, CA, USA) was screened using the carp cDNA coding for MKK1 [20] as a probe. The probe was labeled using a DIG DNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany). Two genomic clones (M1 and M2) were isolated from the library. As shown in Fig. 1, the carp MKK1 gene spanned about 9 kb and was composed of 11 exons and 10 introns. The sequence around the exon-intron boundaries was de- termined and shown in Table 1. All exon/intron bound- aries identified conformed to the GT/AG splice donor/acceptor rule [23]. Some exons were relatively small (46 bp to 78 bp), whereas the first and the last exons