Department of Molecular and Structural Biology, University of Aarhus, Bldg. 130, DK- 8000/Y~rhus C, Denmark

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1 Vol. 43, No. 4, November 17 BOCEMSTRY and MOLECULAR BOLOGY NTERNATONAL Poges CARACTERSATON OF BOVNE STRUCTURE-SPECFC RECOGNTON PROTEN 1 (SSRP1) edna Peter Kresten Nielsen, Jesper aaning, and Lars Sottrup-Jensen* Department of Molecular and Structural Biology, University of Aarhus, Bldg. 130, DK- 8000/Y~rhus C, Denmark Received August 4, 17 SUMMARY: A partial bovine SSRP1 (structure-specific recognition protein 1) cdna has been isolated from a osteoblast cdna library and sequenced. The bovine SSRP1 cdna of 1870 bp encoding 460 amino acid residues showed 86% and 8% sequence identity with human SSRP1 at the DNA and protein level, respectively. Expression of SSRP1 mrna was detected in many human tissues, with a particularly high level in kidney. Key words: Bovine; structure-specific recognition protein 1; SSRP1; high mobility group; MG; osteoblast; edna sequence. NTRODUCTON Structure-Specific Recognition Protein (SSRP1) containing approx. 710 residues is a member of the MG superfamily of DNA-binding proteins. The number of proteins containing MG domains is rapidly growing, and the superfamily comprises, among others, MG1 and MG2, the nucleolar transcription factor UBF, the lymphoid transcription factors LEF-1 and TCF-la, the fungal mating type genes mat-mc and MATA1, the mammalian sex-determining gene SRY, and the mitochondrial transcription factor mttf1. For review on the MG superfamily see Ref. 1. The biological role of SSRP1 is unknown. owever, human SSRP1 binds to DNA modified by the anticancer drug cisplatin (2), and the murine homologue denoted T160 recognises DNA sequences that act as V(D)J recombination signal sequences (3). Deletion analysis of mouse T160 has revealed that the MG domain alone is sufficient for interaction with and binding to DNA (3). Besides man and mouse the cdna sequence of rat (4), chicken (partial edna) (4) and Drosophila (5) SSRP1 have been Abbreviations: SSRP1, structure-specific recognition protein 1; MG, high mobility group. *Corresponding author. Fax: (+45) lsj@mbio, aau. dk /7/ /0 Copyright 17 by Academic Press Australia. 781 All rights of reproduction in any form reserved.

2 Vol. 43, No. 4, 17 BOCEMSTRY and MOLECULAR BOLOGY NTERNATONAL reported. ere we report the cloning and sequencing of a 1.8 kb bovine SSRP1 cdna, and by using a human RNA master blot hybridised with the bovine cdna under stringent conditions we have observed expression of the SSRP1 gene in a wide range of human tissues. The level of expression is particularly high in kidney. MATERALS AND METODS The SSRP1 edna was isolated from a bovine osteoblast lambda-unizap-xr cdna library with a degenerated 01igonucleotide probe (5"-GC(CA)GTA(TC)CA- (AG)AA(TC)ACGA-3") in an attempt to clone chondroitin sulphate proteoglycan edna. The edna library was screened using the tetramethylammonium chloride method at 47 ~ as described (6). Phage inserts that hybridised to the degenerated probe were converted into pbluescript phagemid by in vivo excision (Stratagene). Screening of 5xl@ plague forming units resulted in identification of twelve clones hybridising to the probe. Sequencing revealed that all edna clones encoded SSRP1. Sequencing on both strands was performed using a DNA sequencing kit with AmpliTaq DNA polymerase FS (Perkin Elmer, Applied Biosystem Division) and an Applied Biosystem 373A DNA Sequencer~ The sequence data were analysed with Sequencher (Gene Codes Corporation, P. O., Box 336, Ann Arbor, M 48106, U.S.A.) and Blast Network Service at the National Centre for Biotechnology nformation on the nternet. The nucleotide sequence has been submitted to GenBank under accession No. U8413. A human RNA master blot (Clontech, catalog #7770-1) was hybridised with a nick translated 3zp-labelled bovine SSRP1 edna probe. After two final washes in 1.5xSSC, 0.5% SDS at 65~ for 30 min the filter was autoradiographed. RESULTS AND DSCUSSON Comparison of edna sequences at the DNA 1eve1 revealed that bovine SSRP1 is very similar to mouse T160 and to SSRP1 from human, rat, chicken, and Drosophila (Table 1). At the protein level (460 residues) 8% sequence identity between the bovine and human SSRP1 was observed. As shown in Figure 1 the sequences of the MG domains of bovine, rat, chicken and human SSRP1 are very similar, except for Drosophila where 55% sequence identity is found when comparing bovine and Drosophila SSRP1. n addition, other conserved regions, including the acidic, basic &, and the mixed charge domains (for definition see Refs. 2, 5) are present in the bovine sequence. Previous work has shown that a 2.8 kb SSRP1 mrna is conserved between humans and rodents (2, 7). The sequence identity of 86% between the 1.8 kb partial bovine 5' edna and the human SSRP1 edna indicates that bovine and human SSRP1 mrna have the same size. Northern blot analysis of tissues from baboon has indicated a similar level of expression of SSRP1 in heart, ileum, jejunum, kidney, liver, muscle and spleen. A relatively higher level of expression was observed in baboon brain (2). 782

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5 Vol, 43, No. 4, 17 BOCEMSTRYond MOLECULAR BOLOGY NTERNATONAL i B C D E F G Figure 2. Results of hybridisation of a human RNA master blot with bovine SSRP1 cdna. Tissues: 1A, whole brain; 1B, occipital lobe; 1C, heart; 1D, testis; 1E, kidney; 1F, appendix; 1G, fetal brain; 1, yeast total RNA (100 ng); 2A, amygdale; 2B, putamen; 2C, aorta; 2D ovary; 2E, liver; 2F, lung; 2G, fetal heart; 2, yeast trna (100 ng); 3A, caudate nucleus; 3B substantia nigra; 3C skeletal muscle; 3D, pancreas; 3E, small intestine; 3F, trachea; 3G, fetal kidney; 3 E.coli rrna (100 ng); 4A, cerebellum; 4B, temporal lobe; 4C colon; 4D pituitary gland; 4E spleen; 4F placenta; 4G fetal liver; 4, E. coli DNA (100 ng); 5A cerebral cortex; 5B thalamus; 5C bladder; 5D adrenal gland; 5E thymus; 5G fetal spleen; 5 poly r(a) (100 ng); 6A frontal lobe; 6B, subthalamic nucleus; 6C, uterus; 6D thyroid gland; 6E peripheral leukocyte; 6G fetal thymus; 6, human Cot 1 DNA (100 ng); 7A, hippocampus; 7B, spinal cord; 7C, prostate; 7D salivary gland; 7E lymph node; 7G fetal lung; 7 human DNA (100 ng); 8A, medulla oblongata; 8C, stomach; 8D, mammary gland; 8E, bone marrow; 8, human DNA (500 ng). Using a human RNA master blot hybridised with the bovine cdna under stringent conditions, expression of the SSRP1 gene was observed in a wide range of human tissues (Figure 2). nterestingly, a high level of SSRP1 mrna was observed in human kidney. The reason for the different expression patterns of human and baboon SSRP1 is not clear at present. ACKNOWLEDGEMENTS We thank Dick eineg~ird and Yngve Sommarin, University of Lund, for donating a bovine osteoblast lambda-unizap-xr cdna library. This work was supported by the Danish Research Academy (Ph.D. Training Grant to P.K.N.) and a Basic Research Grant from the University of Aarhus (L.S.-J.). REFERENCES 1. Laudet, V., Stehelin, D., and Clevers,. (13) Nucleic Acids Res. 21, Bruhn, S., Pil, P. M., Essigmann, J. M., ousman, D. E., and Lippard, S. (12) Proc. Natl. Acad. Sci. USA 8, o 785

6 . Vol. 43, No. 4, 17 BOCEMSTRYond MOLECULAR BOLOGY NTERNATONAL Shirakata, M., fippi, K., Usuda, S., Okazaki, K., Yoshida, K., and Sakano,. (11) Mol. Cell Biol. 11, Wang, L., Precht, P., Balakr, R., and orton, W. E. (13) Nucleic Acids Res. 21, 143. Bruhn, S. L., ousman, D. E., and Lippard, S. (13) Nucleic Acids Res. 21, onor6, B., Madsen, P., and Leffers,. (13) J. Biochem. Biophys. Meth. 27, Toney, J.., Donahue, B. A., Kellett, P. J., Bruhn, S. L., Essigmann, J. M., and Lippard, S. J. (18) Proc. Natl. Acad. Sci. USA 86,