SHORT COMMUNICATION Julius LukesÏ á Dmitri A. Maslov Unexpectedly high variability of the histone H4 gene in Leishmania Received: 16 August 1999 / Accepted: 10 September 1999 Abstract The sequence of the cDNA of the histone H4 gene of Leishmania tarentolae is reported herein. The predicted 100-amino-acid-long protein has the highest degree of identity with the histone H4 gene of L. in- fantum and shares with it a 5¢ region that shows a very low degree of identity with the corresponding region of histone H4 genes from other organisms. However, be- tween these two genes is a 7.7% nucleotide dierence that results in seven dierent amino acids, located in the 5¢, central, and 3¢ regions of the coding sequence. Such a divergence in the H4 gene, which is considered to be one of the most highly conserved genes, between closely re- lated members of the genus Leishmania is unexpected and may re¯ect some unusual features of these impor- tant proteins in kinetoplastid ¯agellates. Introduction Kinetoplastid protozoa of the genus Leishmania are the causative agents of a wide spectrum of human diseases occurring in most tropical and subtropical regions. Probably due to their ancient character, kinetoplastid ¯agellates retain many unique features, including un- usual gene organization and expression (Donelson 1999). Therefore, the characterization of their histone proteins is of considerable interest. Histones are small basic proteins that are omnipresent in eukaryotic cell nuclei, where they form nucleosomes, which consist of DNA wound around a histone octamer containing two molecules each of the core histones H2A, H2B, H3, and H4. Histone H4 is an extremely slowly evolving histone protein (Thatcher and Gorowski 1994). In the course of a study of nuclear-encoded proteins associated with the respiratory complexes we accidentally obtained the cDNA of histone H4 from the species L. tarentolae, and the results are reported herein. Promastigotes of L. tarentolae were cultivated as described elsewhere (Simpson and Braly 1971). On the basis of a partial N-terminal amino-acid sequence (DLPGKIVSV) derived from an as yet unidenti®ed kinetoplast protein, a highly degenerate primer (CTRRANGGNCCNTTYTADCANWSNCA) was designed and, with the total poly(A) mRNA serving as a template, was used for cDNA synthesis. The subsequent polymerase chain reaction was performed with the above-mentioned primer and the mini-exon primer (Campbell et al. 1984). After 30 cycles performed at 95 °C for 2 min, at 50 °C for 1 min, and at 72 °C for 3 min, one major band and several minor bands were ampli®ed. The most abundant 0.6-kb band was cloned into the pT7blue vector (Novagen), and both strands were sequenced (Fig. 1). Comparison of the sequence obtained with the Gen- Bank data base revealed similarity with several histone H4 genes, particularly with the H4 gene from L. in- fantum (Soto et al. 1997). The cDNA molecule was 585 bp long and contained in the 5¢ end the 39-bp spliced leader sequence that is part of all mRNA mole- cules in trypanosomatids. The region between the spliced leader and the start codon of the histone H4 gene was 85 bp long and had no sequence similarity with the corresponding region of histone H4 of L. infantum or with other sequences. The histone H4 gene is 300 bp long, exactly the same length as the L. infantum H4 gene. Surprisingly, there are 23 dierences between the 2 genes, located mainly in the 5¢ and 3¢ coding regions. Most of these nucleotide dierences are not silent and result in seven dierences in predicted amino-acid se- quences, localized in three clusters in the 3¢,5¢, and Parasitol Res (2000) 86: 259±261 Ó Springer-Verlag 2000 J. LukesÏ á D.A. Maslov Department of Biology, University of California, Riverside, CA 92521, USA J. LukesÏ (&) Institute of Parasitology, Czech Academy of Sciences, BranisÏovska 31, 37005 C Ï eske BudeÏjovice, Czech Republic e-mail: jula@paru.cas.cz Tel.: +420-38-7775416; Fax: +420-38-5300388 The sequence data reported herein have been submitted to Gen- Bank and assigned the accession number AF175386