A dwarf transgenic mouse (DTM) line has been established in which mice express relatively high levels of a mutated bovine (b) GH gene. This bGH analog binds to mouse liver membrane preparations with an affinity similar to that of wild-type bGH. The mean growth ratio of these mice is approximately 0.7 relative to that of their nontransgenic littermates. Serum insulin-like growth factor-I (IGF-I) levels of DTM were found to be approximately half those in nontransgenic littermates. Liver GH receptor levels were up-regulated in DTM or wild-type bGH transgenic mice. Pituitary GH levels were negatively correlated with serum IGF-I concentrations. Wild-type bGH transgenic mice contain relatively high serum IGF-I and low pituitary GH levels, whereas DTM possess low serum IGF-I and high pituitary GH levels. The decrease in serum IGF-I resulting from the interaction between the bGH analog, the endogenous mouse GH, and GH receptor(s) apparently leads to a dwarf phenotype. These data suggest that this bGH analog has uncoupled GH ligand-receptor binding from IGF-I production and acts as a functional antagonist to the action of endogenous mGH.

To determine the importance of the third a-helix in bovine growth hormone (bGH) relative to growthrelated biological activities, the following experimental approach was used: (i) mutagenesis ofhelix III of bGH to generate an idealized amphiphilic helix; (ii) in vitro expression analyses of the mutated bGH gene in cultured mouse L cells; (iii) mouse liver membrane binding studies ofwild-type and mutated bGH; and (iv) expression ofthe mutated gene in the transgenic mouse. An altered bGH gene (pBGHlOA6-M8) was generated that encodes the following changes: glutamate-117 to leucine, glycine-119 to arginine, and alanine-122 to aspartate. The plasmid pBGHlOA6-M8 was shown to be expressed in, and its protein product secreted by, mouse L cells. The altered hormone possessed the same binding affinity to mouse liver membrane preparations as wild-type bGH. Transgenic mice containing the mutated bGH gene, however, showed a significant growthsuppressed phenotype. The degree of suppression was directly related to serum levels of the altered bGH molecule. >90% amino acid sequence identity between bGH and pGH, it is likely that bGH has a similar three-dimensional structure. By aligning these a-helical structures into a two-dimensional Edmundson wheel projection (24), it is clear that an amphiphilic a-helical segment exists between amino acid residues 109 and 126 in the third a-helical region (ref. 25; Fig. 1). However, amino acids 117, 119, and 122 are positioned so that an idealized amphiphilic a-helix is not generated.In this study, we have used the following approach to determine whether this amphiphilic a-helix is important in the growth-related properties ofbGH: (i) mutagenesis ofthe third a-helix of bGH so as to generate an idealized amphiphilic a-helix (Fig. 1); (ii) expression analyses of the wild-type and mutated bGH genes in cultured mouse L cells; (iii) mouse liver membrane binding studies comparing wild-type and mutated bGH; and (iv) production of transgenic mice containing wild-type and mutated bGH genes.

Bovine GH (bGH) analogs with single amino acid substitutions at positions 117 (bGH-E117L), 119 (bGH-G119R), and 122 (bGH-A122D) were generated. These analogs bind to mouse liver membrane preparations with affinities similar to native bGH. However, transgenic mice which express the analogs demonstrate different phenotypes ranging from dwarfism to gigantism. For example, expression of bGH or bGH-E117L result in large transgenic mice. In contrast, transgenic mice with a growth phenotype similar to nontransgenic animals result from expression of bGH-A122D. Surprisingly, transgenic mice with relatively high serum levels of bGH-G119R possessed a dwarf phenotype. Together these results suggest that Gly 119 and Ala 122 are involved in growth-promoting activity of GH.

Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states.