Joseph P. Stein

Ovomucoid intervening sequences specify functional domains and generate protein polymorphism

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/exon junctions. The junction sequences surrounding four of the introns are redundant; however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. The ovomucoid gene sequence around intron F can be used to predict the cause of an internal deletion polymorphism in the ovomucoid protein, which is an apparent error in the processing of the ovomucoid pre-mRNA. We also compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordial ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.

The evolution of a complex eucaryotic gene.

Two thirds of the natural chicken ovomucoid gene has been sequenced, including all exons and the intron sequences surrounding all fourteen intron/exon junctions. The junctions sequences surrounding four of the introns are redundant: however, the sequences surrounding the other three introns contain no redundancies and thus the splicing sites at either end of these three introns are unambiguous. The splicing in all cases conforms to the GT-AG rule. We compare the structural organization of the ovomucoid gene with the ovomucoid protein sequence to examine theories of the evolution of ovomucoids as well as the origin of intervening sequences. This analysis suggests that the present ovomucoid gene evolved from a primordia ovomucoid gene by two separate intragenic duplications. Furthermore, sequence analyses suggest that introns were present in the primordial ovomucoid gene before birds and mammals diverged, about 300 million years ago. Finally, the positions of the introns within the ovomucoid gene support the theory that introns separate gene segments that code for functional domains of proteins and provide insight on the manner by which eucaryotic genes were constructed during the process of evolution.

The Structure and Regulation of the Natural Chicken Ovomucoid Gene

The molecular mechanism by which steroid hormones regulate specific gene expression has been an area of acute interest during the past several years. One particularly attractive model system for studying this hormonal regulation has been the hen oviduct (O’Malley et al. 1969). A number of laboratories, in addition to our own, have utilized this model system for investigations of eucaryotic molecular biology (Oka and Schimke 1969; Palmiter and Schimke 1973; Palmiter et al. 1976; Cox 1977; Hynes et al. 1977; Garapin et al. 1978b; Mandel et al. 1978). Administration of estrogen to the newborn chick stimulates oviduct growth and differentiation and results in the appearance of a number of new specific intracellular proteins (O’Malley et al. 1969; Hynes et al. 1977; Palmiter 1973; Chan et al. 1973; Harris et al. 1973, 1975; Sullivan et al. 1973; O’Malley and Means 1974). The synthesis of one of these proteins, ovalbumin, has been studied extensively. Ovalbumin mRNA has been purified (Rosen et al. 1975), and a full-length dsDNA copy synthesized (Monahan et al. 1976b) and cloned in a bacterial plasmid (McReynolds et al. 1977). More recently, ovalbumin genomic DNA sequences have been isolated from restriction enzyme digests of hen DNA and cloned (Woo et al. 1978). The other three major proteins under estrogenic control in the oviduct tubular gland cell, ovomucoid, conalbumin and lysozyme, have been less extensively studied (Palmiter 1972; Hynes et al. 1977).

THE OVOMUCOID GENE ORGANIZATION, STRUCTURE AND REGULATION

Publisher Summary This chapter focuses on the ovomucoid gene organization, structure, and regulation. The molecular mechanism by which steroid hormones regulate specific gene expression has been an area of acute interest. One attractive model system for studying this hormonal regulation has been the hen oviduct. Administration of estrogen to the newborn chick stimulates oviduct growth and differentiation and results in the appearance of a number of new specific intracellular proteins. The synthesis of one of these proteins, ovalbumin, has been studied. Ovalbumin mRNA has been purified and a full-length dsDNA copy synthesized and cloned in a bacterial plasmid. Moreover, ovalbumin genomic DNA sequences have been isolated from restriction enzyme digests of hen DNA and cloned. The chapter discusses the cloning of the ovomucoid structural gene sequence. It also describes the synthesis and cloning of a complementary DNA copy and identification of an ovomucoid structural gene clone And discusses a partial restriction map of pOM100.

Gene Structure and Evolution

The organization of eukaryotic genes has been an area of acute interest to us for several years. One particularly attractive model system for studying the structural organization of functionally related genes in a single tissue has been the hen oviduct (O’Malley et al. 1969). The chicken ovalbumin gene has been cloned and sequenced (Rosen et al. 1975; Monahan et al. 1976; McReyn-olds et al. 1977; Woo et al. 1978; Woo et al. 1981). More recently, the chicken ovomucoid gene was cloned and partially sequenced (Stein et al. 1978; Lai et al. 1979; Catterall et al. 1979; Stein et al. 1980; Catterall et al. 1980). We discovered that both of these oviduct genes exhibited a surprisingly complex structure; each gene contained seven nontranslated regions of DNA sequence (intervening sequences or introns) interspersed among eight genomic DNA regions (exons) that code for the mature polypeptide chains. A number of laboratories in addition to our own have identified the existence of these intervening sequences in diverse eukaryotic genes (Breathnach et al. 1977; Weinstock et al. 1978; Garapin et al. 1978; Mandel et al. 1978; Lindenmaier et al. 1979; Cochet et al. 1979; Brack and Tonegawa 1977; Maki et al. 1980; Early et al. 1980; Jeffreys and Flavell 1977; Tilghman et al. 1978; Valenzuela et al. 1978; Lomedico et al. 1979; Bell et al. 1980; Nunberg et al. 1980; Gorin and Tilgham 1980; Fyrberg et al. 1980).