Sequence and Functional Conservation of the Intergenic Region Between the Head-to-Head Genes Encoding the Small Heat Shock Proteins aB-Crystallin and HspB2 in the Mammalian Lineage Linda Doerwald, 1, * Teun van Rheede, 1, * ,  Ron P. Dirks, 2 Ole Madsen, 1 Remco Rexwinkel, 1 Siebe T. van Genesen, 1 Gerard J. Martens, 2 Wilfried W. de Jong, 1 Nicolette H. Lubsen 1 1 Department of Biochemistry, Faculty of Science, University of Nijmegen, Nijmegen, The Netherlands 2 Department of Animal Physiology, Faculty of Science, University of Nijmegen, Nijmegen, The Netherlands Received: 7 January 2004 / Accepted: 7 June 2004 [Reviewing Editor: Dr. Manyuan Long] Abstract. An unexpected feature of the large mammalian genome is the frequent occurrence of closely linked head-to-head gene pairs. Close appo- sition of such gene pairs has been suggested to be due to sharing of regulatory elements. We show here that the head-to-head gene pair encoding two small heat shock proteins, aB-crystallin and HspB2, is closely linked in all major mammalian clades, suggesting that this close linkage is of selective advantage. Yet aB- crystallin is abundantly expressed in lens and muscle and in response to a heat shock, while HspB2 is abundant only in muscle and not upregulated by a heat shock. The intergenic distance between the genes for these two proteins in mammals ranges from 645 bp (platypus) to 1069 bp (opossum), with an average of about 900 bp; in chicken the distance was the same as in duck (1.6 kb). Phylogenetic foot- printing and sequence alignment identified a number of conserved sequence elements close to the HspB2 promoter and two farther upstream. All known reg- ulatory elements of the mouse aB-crystallin promoter are conserved, except in platypus and birds. The lens- specific region 1 (LSR1) and the heat shock elements (HSEs) lack in birds; in platypus the LSR1 is reduced to a Pax-6 site, while the Pax-6 site in LSR2 and a HSE are absent. Most likely the primordial mam- malian aB-crystallin promoter had two LSRs and two HSEs. In transfection experiments the platypus aB-crystallin promoter retained heat shock respon- siveness and lens expression. It also directed lens expression in Xenopus laevis transgenes, as did the HspB2 promoter of rat or blind mole rat. Deletion of the middle of the intergenic region including the up- stream enhancer affected the activity of both the rat aB-crystallin and the HspB2 promoters, suggesting sharing of the enhancer region by the two promoters. Key words: aB-Crystallin — HspB2 — Head-to- head genes — Small heat shock proteins — Heat shock — Phylogenetic footprinting Introduction Bidirectional gene pairs, located so close that pro- moter regions overlap, are surprisingly common in eukaryotes. For example, Adachi and Lieber (2002) recently found that almost 30% of the housekeeping genes in man were located in a bidirectional fashion less than 1 kb apart. As eukaryotic genomes are large relative to the number of genes, one would expect genes to drift apart unless there is a selective advan- tage to maintaining the gene pair. A recent analysis of divergent genes in human chromosomes 20, 21, and 22 showed a biphasic distribution of the intergenic distance between such genes, with most genes being *Both authors contributed equally.   Deceased May 21, 2003. Correspondence to: Nicolette H. Lubsen; email: nhl@sci.kun.nl J Mol Evol (2004) 59:674–686 DOI: 10.1007/s00239-004-2659-y