MODS is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends failing between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MODS, in contrast, produces mRNAs with 5' ends that anl fail upstream of both ATGs. To determine how MODS encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MODS and MODS-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MODS protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence. Numerous examples of chemical reactions catalyzed inboth the mitochondrion and some other cellular compartment have been documented (reviewed in reference 24). The majority of enzymes that catalyze reactions in two cellular compartments are probably the products of separate genes. However, some single genes encode isozymes, which we propose to call sorting isozymes, that are sorted to more than one cellular compartment (5,9,11,16,18, 40,41,47,49,52; reviewed in reference 46). These genes encode at least two translation initiation sites and multiple mRNAs with 5' ends falling upstream of as well as in between potential translation initiation sites (6,9,16, 40,41,49,52). Thus, the mRNAs have different coding capabilities with respect to amino-terminal sequences.Amino-terminal sequences play an important role in mitochondrial targeting (reviewed in reference 3), and in several cases the presence or absence of the peptide sequences encoded between the first and second ATGs has been shown to be essential for import of the mitochondrial isozyme (5, 9, 40). However, in at least one case, the amino-terminal extension is not essential for mitochondrial targeting and a single translation product is able to distribute to both mitochondrial and nuclear compartments (14, 36 teins, one having 11 more amino acids at the amino terminus than the other. By analogy with other genes that code sorting isozymes, the longer protein would be expected to, be ...

MODS, a nuclear gene ofSaccharomyces cerevisiae, encodes two isozymic forms of a tRNA-modification enzyme. These enzymes modify both cytoplasmic and mitochondrial tRNAs. Two inframe ATGs of the MODS gene are used for initiation of translation, and the form of the protein translated from the first AUG is imported into mitochondria. Protein translated from the second AUG functions in the cytoplasm. Since all transcripts contain both of these translational start sites and two proteins are made, the question arises as to the factors that influence the translation start-site choice. Extending the 5' ends of the MOD5 mRNA to include leader sequences of the ADH1 (alcohol dehydrogenase defective) transcript produces signficant changes in the choice of AUGs. This suggests that for wild-type MOD5 transcripts, the length or structure of the leader sequence plays a role in AUG choice. The nucleotides surrounding the first ATG ofMODS also have an effect on translation initiation. Altering these nucleotides changes initiation choice and suggests that ribosomal bypass of a suboptimal AUG is another mechanism controlling the alternate use of two initiation codons. Our data support the model that at least one MOD5 transcript is able to produce two proteins with different N-terminal sequences.

In 15 normal men, cimetidine taken orally in a dose of 300 mg twice a day for 3 days reduced to similar extents the rate constants for formation (ki) of the three principal metabolites of antipyrine (AP): 29.9% +/- 8.5% (mean +/- SD) for 4-hydroxyantipyrine (4-OH-AP); 28.3% +/- 6.3% for 3-hydroxymethylantipyrine (3-OHM-AP); and 22.4% +/- 5.6% for N-demethylantipyrine (NDM-AP). AP clearance declined 24.3%; AP salivary t 1/2 rose 33%; and corrected AP apparent volume of distribution was unchanged. In one apparently normal subject, however, kis for formation of 3-OHM-AP and NDM-AP rose after cimetidine even though AP clearance declined 19.7%. This surprising result, which suggests that cimetidine can exert an inductive effect on the hepatic mixed-function oxidases of some subjects, was checked by restudying the individual. Very similar values occurred on repetition. The average increase in kis for NDM-AP and 3-OHM-AP was 172.2% and 34.0%. These unusual results in this subject indicate that at least two distinguishable forms of cytochrome P-450 participate in AP metabolism in man. Cimetidine appeared to reduce the amount of AP absorbed from the gut in 10 of our 15 normal subjects.

To determine whether genetic mechanisms control large interindividual variations in theophylline elimination in normal uninduced human subjects, and, if so, to test the possibility that these genetic factors are transmitted as a simple Mendelian trait, theophylline was administered to 79 unrelated adults, six sets of monozygotic twins, six sets of dizygotic twins, and six two-generation families. Thereafter, in urine collected from each subject at regular intervals for 48 h, concentrations of theophylline and its three principal metabolites were measured and rate constants of formation of these metabolites calculated.The twin study, designed to determine the relative contributions of genetic and environmental factors to large interindividual variation in theophylline elimination, revealed predominantly genetic control. Values for this genetic component, designated heritability (HI2), of interindividual variation in rate constants of metabolite formation were 0.61, 0.84, and 0.95 for 3-methylxanthine, 1-methyluric acid, and 1,3-dimethyluric acid, respectively. HI2 for the overall theophylline elimination rate constant (kei) was lower (0.34).In the 79 unrelated adults, each distribution curve for rate constants of formation of each theophylline metabolite appeared to be trimodal. By contrast, the distribution curve for the overall theophylline elimination rate constant appeared to be either unimodal or bimodal. The extent of interindividual variation was fourfold for theophylline kei and 6-8-fold for the three principal metabolites. High correlations among the three rate constants in individual subjects suggested their regulation by a single shared factor.In six families carefully selected to be under near basal environmental conditions so that hepatic theophylline metabolism of each family member would be neither markedly induced nor inhibited, phenotypes for theophylline metabolite rate constants were assigned. This assignment of phenotype was made by the position of each family member's rate constant on the three distribution curves that were generated from the 79 unrelated subjects. In each family, pedigree analysis of the three phenotypes for each rate constant was consistent with their control by two alleles at a single genetic locus and with autosomal codominant transmission. Frequencies of the two alleles at each genetic locus controlling rate constants of formation of theophylline metabolites were similar (p = 0.49, 0.53, and 0.52). In the three families studied with antipyrine (AP) as well as with theophylline, AP