The identification of the precise structural features of yeast sterol molecules required for the essential "sparking" function has been a controversial area of research. Recent cloning and gene disruption studies in Saccharomyces cerevisiae have shown that C-24 methylation (ERG6), C-5 desaturation (ERG3) and delta 8-delta 7 isomerization (ERG2) are not required, while C-14 demethylation (ERG11) and C-14 reduction (ERG24) are each required for aerobic viability. Earlier observations had indicated that C-14 demethylase deficient strains could be restored to aerobic growth by suppressor mutations that caused a deficiency in C-5 desaturase. These strains were reported to synthesize some ergosterol, indicating that they contained leaky mutations in both ERG11 and ERG3, thereby making it impossible to determine whether the removal of the C-14 methyl group was required for aerobic viability. The availability of the ERG11 and ERG3 genes has been used in this study to construct strains that contain null mutants in both ERG11 and ERG3. Results show that these double disruption strains are viable and that spontaneously arising suppressors of the ERG11 disruption are erg3 mutants. The erg11 mutants of S. cerevisiae are compared to similar mutants of Candida albicans that are viable in the absence of the erg3 lesion.

We have sequenced the structural gene and flanking regions for lanosterol 14 alpha-demethylase (14DM) from Saccharomyces cerevisiae. An open reading frame of 530 codons encodes a 60.7-kDa protein. When this gene is disrupted by integrative transformation, the resulting strain requires ergosterol and, as expected, grows only in the absence of oxygen. The deduced amino acid sequence of 14DM includes a hydrophobic segment near the amino terminus which may be a transmembrane domain. The deduced sequence has been compared with those of eight other eukaryotic P450s, each from a different family within the P450 superfamily. These comparisons indicate that this yeast gene is the first member of a new P450 family, P450LI. The P450, designated P450LIA1, is more closely related to mammalian P450s than to the bacterial P450cam. In fact, both the yeast P450 and several mammalian P450s have equivalent alignment scores when each is compared with the bovine P450scc. Matrix comparisons of the amino acid sequence of this P450 with those of mammalian P450s reveal three conserved regions. The DNA region 5' to the structural 14DM gene includes poly(dA:dT) sequences and a repeating hexamer sequence.

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Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity. This region begins 275-310 amino acids from the amino terminus of each P-450, continues for =170 residues, and ends 35-50 amino acids before the carboxyl terminus. The region can be divided into four domains of sequence similarity, each possessing its own pattern of invariant, conserved, and variable amino acids. The four domains are 56,20, 59, and 28 residues long and are connected by three shorter segments of limited sequence similarity. The number of residues in these short segments varies with the P-450 protein but ranges from 0 to 20 residues. Consensus sequences based on these similarities can be used to determine whether the sequence of an unidentified peptide resembles that expected for a P-450. Sequence similarities between proteins sometimes reflect constraints imposed by the requirements of a common function. The fourth domain of the P-450s, for example, contains an invariant cysteine that provides the axial thiolate ligand to the heme iron. Other relationships between the four domains and P-450 function can be examined by in vitro mutagenic procedures that alter the conserved amino acids or modify the distance between domains.The P-450 cytochromes are components in monooxygenase systems that catalyze a wide variety of oxidative reactions in prokaryotes and eukaryotes. These reactions include steps in the synthesis and degradation of such compounds as cholesterol, steroid hormones, and prostaglandins. P-450 proteins are also involved in the metabolism of drugs and in the activation and inactivation of carcinogens. A recent analysis of >60 P-450s from eukaryotes and one prokaryote led to the organization of the known P-450s into 10 different families with a total of 15 subfamilies. Amino acid sequences within a P-450 family are >36% similar, while sequences within a subfamily are :70% similar (1). Although sequence relatedness between P-450 families is low, these families are still grouped into one P-450 superfamily according to standard criteria (2).The eukaryotic branch of the cytochrome P-450 superfamily arose >1000 million years ago (1, 3) and sequence similarities among these ancient families might identify conserved domains of structure or function. Since complete amino acid sequences had been published for proteins from eight eukaryotic P-450 families, we used a representative sequence from each family in a series of sequence comparisons. A multiple alignment of the eight sequences revealed one region of similarity shared by all. Sequence similarities in this region reside in four domains connected by short segments of variable length. The region will provide a focus for experiments that probe the relationship of amino acid sequence to structure and function in the P-450 superfamily. METHODS Amino Acid Sequences. The source for each eukaryotic P-450 sequence used in these comparisons was as follows: LAw, rat liver (4); c, rat liver (5); 17a, bovine adrenal cortex (6); scc,...

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In our most recent update of P450 gene superfamily nomenclature , at least one reader has expressed confusion about the Fig. 1 abscissa labeled "Evolutionary Distance" (d). The erroneous assumption was that the numbers represented millions or billions of years before the present. This is not the case. The scale does not represent linear time. The branchings of distinct genes in this figure represent a combination of orthologous genes in different species and gene duplication events within the same species. Analyses of less complicated P450 phylogenetic trees are presented elsewhere ."Cytochrome P450" is a deceptive name. This term is misleading because it connotes that all P450 gene products are the "same protein" in different tissues and species; this is clearly not the case. Cytochrome P450 is not like, for example, cytochrome c. The functions of different P450 enzymes vary widely (Nebert and Gonzalez, 1987;. Therefore, we expect that the evolutionary constraints on these proteins will also vary widely. One consequence of this is a variation in the evolutionary rate among different P450 genes. This variation is most apparent when branch points representing the same historical divergence event are found at very different evolutionary distances. Two examples are provided to emphasize this point. First, the bacterial P450 sequences diverged from eukaryotic P450 sequences presumably at a fixed point in time, yet there are three prokaryote/eukaryote branch points on the tree, all at very large distances from one another: i.e., d = 1.7 for the CYP55 vs. (CYP105, CYP107) branch point; d = 3.4 for the bacterial cluster plus fungal CYP55 vs. other eukaryotes plus CYP102; and d = 2.1 for the CYP52 and CYP102 families. The second example can be seen with the chicken P450 genes. The CYP2H subfamily and the CYP17 family each branch at d = 0.7-0.9 from other P450 genes. However, the chicken CYP19 branches at d = 0.4, i.e., about one-half that of the other values. Since birds diverged from land animals at a fixed point in time i.e., approximately 320 million years before the present the difference in d suggests that CYP19 is about two times more conserved than the other chicken P450 genes in the tree (Nebert et ai, 1989).In addition to the variation in rate within a single species, there is the further complication of variation between lineages (cf. p. 587 of Nelson and Strobel, 1987). For example, mammals appear to have evolved more rapidly than birds or reptiles. For those who insist on a time scale for P450 evolution, the time scale must therefore be kept

Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.