Evolution of murine alpha 1-proteinase inhibitors: gene amplification and reactive center divergence

Abstract

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

Fig. 1

Southern blot analysis of α₁PI genes in Mus species. Liver DNA (10 µg) from each of several species was digested with EcoRI, and resulting fragments were fractionated on agarose gels, transferred to a nylon membrane, and hybridized to ³²P-labeled α₁-PI-specific probe. (See Materials and Methods.) The lanes are M. domesticus strain C57BL/6J (DOM); M. caroli (CAR); M. saxicola (SAX). Each lane contains DNA from a single animal; all were run on the same blot. Size markers are HindIII fragments of λ DNA, and are indicated to the left.

Fig. 2

Sequences of α₁PI cDNAs in Mus species. The sequence of the M. saxicola (SAX) α₁PI cDNA is shown aligned with those of M. caroli (CAR; Latimer et al. 1990) and M. domesticus (DOM; Sifers et al. 1990). Numbering is based on the M. saxicola sequence. Differences relative to M. saxicola are indicated; identities are depicted by dots (.) and deletions by dashes (−). Asterisks (*) mark the translational initiation codon at nucleotides 37–39 and the termination codon at nucleotides 1276–1278.

Fig. 3

Amino acid sequences for mammalian α₁PIs. The sequence of the α₁PI of M. saxicola (SAX) is shown aligned with those of M. domesticus (DOM; Sifers et al. 1990), M. caroli (CAR; Latimer et al. 1990), rat (R; Chao et al. 1990), sheep (SH; Brown et al. 1989), baboon (BA; Kurachi et al. 1981), and human (H; Rosenberg et al. 1984). Numbering is based on M. saxicola. Identities are indicated by dots (.) and deletions by dashes (−). The reactive center is overlined. An asterisk (*) marks the P₁ amino acid at residue 377.

Fig. 4

Sequences of the reactive centers of M. saxicola α₁PIs. The four reactive center regions deduced from analysis of 87 cDNA clones (see text) are aligned in the upper panel, with asterisks (*) indicating the codon that corresponds to the P₁ amino acid. Amino acid sequences for the reactive centers are aligned in the lower panel, with the P₁ amino acid indicated by an asterisk. Sequence differences are highlighted; identities are indicated by dots (.).

Fig. 5

Phylogeny of mammalian α₁PI mRNAs. The evolutionary relationships among mammalian species are shown in the upper panel, along with estimated times of divergence as determined primarily from the fossil record (Li et al. 1985; Tseng-Crank and Berger 1987; Britten 1986; Jouvin-Marche et al. 1988). BL/6 and BALB/C represent inbred strains C57BL/6J and BALB/cJ, respectively, of M. domesticus. The phylogeny of α₁PI mRNAs is shown in the lower panel, and is based upon the synonymous substitution rates in Table 1; branch lengths (in changes/100 sites) are indicated. The thick line highlights the relationships among mRNAs of Mus species. (See text for details.)