Interferon α/β genes from a marsupial, Macropus eugenii

Abstract

This paper describes the cloning of full length marsupial type I interferon (IFN) genes and their flanking regions using a genome walking approach and PCR primers based on previously isolated partial DNA sequences. We confirm that the two major classes of Tammar Wallaby type I IFN genes are homologous with the eutherian IFN-α and IFN-β gene families. The wallaby IFN genes share a number of conserved features with their eutherian counterparts, including codons for cysteines at equivalent positions, implying similar secondary structures for the encoded proteins, and promoter regions with conserved putative regulatory motifs. Moreover, the wallaby genes have AT-rich elements in their flanking sequence corresponding to the mRNA 3′-untranslated regions, also implying that, as in eutherian mammals, rapid mRNA degradation plays a role in regulating expression of these genes. The complex nature of the type I IFN gene families in viviparous mammals (eutherians and marsupials) may reflect their recruitment into nonimmunological processes and this concept is discussed.

Introduction

The type I interferons (IFNs) are an important group of cytokines most recognised for their anti-viral action but which also possess activities related to immunomodulation, tumour suppression, cell growth and differentiation and reproduction [1], [2], [3], [4], [5]. In addition to functional differences, type I IFNs are distinguished from the type II IFN, IFN-γ, which also has anti-viral properties, based on a lack of similarity in primary amino acid structure as well as differences in gene organisation. Type I IFNs have been extensively studied in birds and, more particularly, in some eutherian mammals, at both the molecular and functional levels. In birds, although their homology relationship to mammalian type I IFNs is controversial, a relatively homogeneous assortment of genes has thus far been documented, consisting of probably no more than 10 loci in any avian species [6], [7], [8]. In contrast, a number of eutherians have a more complex gene family for the type I IFNs, encoding five structurally and/or functionally distinct subtypes - IFN-α, -β, -δ, -τ and -ω [9]. However, little direct evidence for how and why this increase in complexity came about in these eutherians during the course of evolution is available. One way of assessing this transition would be to investigate the type I IFN gene family repertoire in taxa phylogenetically intermediate to birds and eutherian mammals, namely, the ‘other’ mammals, marsupials and monotremes. A knowledge of type I IFN genes in these groups would thus provide a paradigm for the intervening steps in the evolutionary processes that have led to the high degree of multiplicity and form of these genes in eutherians.

Recently, we have investigated the type I IFN genes of a model marsupial species, the Tammar Wallaby (Macropus eugenii) by a PCR-based survey of partial DNA sequences [10]. This revealed that this species possesses a complement of type I IFN genes that parallels that of eutherian species in terms of gene family complexity. It was also shown that some of the Tammar Wallaby genes are more closely related to the eutherian genes encoding either the subtypes related to IFN-α (IFN-α, -δ, -τ and -ω) or IFN-β, than they are to each other. However, wallaby genes orthologous to the minor subtypes related to IFNA (IFND, IFNW and IFNT) were not present, indicating that at the time of the marsupial-eutherian split approximately 130 million years ago (MYA) [11], the common ancestor of these two groups of mammals probably possessed distinct IFN-α and IFN-β genes but that the genes for the minor subtypes related to IFN-α had not yet arisen and were subsequently only to do so in some lineages of eutherians. In this study, to further investigate the wallaby type I IFNs, we cloned the full-length genomic copies and flanking regions, by genomic-walking PCR [12], of representatives from the two major subtype gene families (IFNA and IFNB) in the Tammar Wallaby.