Different evolutionary behavior of structurally related, repetitive sequences occurring in the same Balbiani ring gene in Chironomus tentans

Abstract

The Balbiani ring 2 (BR 2) gene in Chironomus tentans is highly internally repeated. Two Many eukaryotic genes display structural features suggesting that they have evolved through the amplification of intragenic sequences (1-7). The genes encoding structural proteins have often maintained a conspicuous repetitive structure that is a valuable asset in the analysis of the evolutionary process. In one gene family of this type-the Balbiani ring (BR) genes in the dipteran Chironomus tentans-the repetitive feature of the genes is particularly prominent, with a hierarchic arrangement of the repetitive sequences. The BR genes contain the genetic information for large salivary secretory proteins (8, 9), which are used to spin tubes housing the aquatic larvae. The principal structures of the BR 1 (10-12) and BR 2 (13-15) genes in C. tentans, the BR 2 gene in Chironomus pallidivittattus (16), and the BR b (17) and BR c (18) genes in Chironomus thummi have been revealed, and, in all cases, the genes consist of a large number of 150-to 300-base-pair (bp)-long repeat units that are tandemly repeated. All repeat units have one constant region (C region) and one subrepeat region (SR region),t the latter being formed by a number of subrepeats. Furthermore, all BR genes show a high degree of sequence similarity, suggesting that they may share a common origin. The characterization of the various BR genes therefore allows the study of the evolution of a set of different but closely related genes within and between species, each gene consisting of several hundred repeat units specific to that gene but still being the result of amplifications starting from a common ancestor sequence (13). In BR 2 in C. tentans, there exist two different but related types of repeat units-the a and P types-which form separate sequence blocks and probably together cover most of the BR 2 gene (13,14). In this study we investigate the homogeneity of the blocks and record striking structural differences between them. These findings suggest that the blocks evolved at different times and that both sequence correction mechanisms and selection at the protein level operate to various extents on the different repeat structures along the gene. MATERIALS AND METHODSIsolation of DNA. Total C. tentans DNA was extracted from crude nuclei preparations as described (13).Cloning Procedures. The construction and cloning of cDNA transcripts from salivary gland 75S RNA has been described by Sumegi et al. (13).Southern Blot Analysis. Total C. tentans DNA was cleaved completely with Hinfl (New England Biolabs) and separated in a 5% agarose gel containing 40% formamide according to Sun et al. (19). The DNA fragments were transferred to nitrocellulose filters (20) and hybridized with the 32P-labeled cDNA inserts according to Thomas (21). The hybridization signal was quantified by scanning densitometry using a Joyce-Loebl densitometer.DNA Sequence Determination. Hinfl fragments from the recombinant plasmids were treated with DNA polymerase I (New England Biolabs) and...