We determined the coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 mRNA of the rat by analysis of three cloned cDNAs and by primer extension methods. The deduced amino acid sequence of the cytochrome is composed of 491 amino acids, and its predicted molecular weight and amino acid composition concur with those determined with the purified protein. The sequence of one of the three cloned cDNAs is not identical with that of the other two. In their 922 overlapping nucleotides, 14 nucleotide substitutions occur, and 7 of them result in 6 amino acid changes, therefore indicating the presence of at least two similar but distinct mRNAs for phenobarbital-inducible cytochrome P-450. These substitutions occur in a limited portion of the sequence, apparently forming some sort of a "variable region."

The gene structure of a phenobarbital-inducible rat liver cytochrome P-450 was elucidated by sequence analysis of two isolated genomic clones. The total length of the gene was approximately 14 kilobases and was separated into nine exons by eight intervening sequences. Nucleotide sequences of all exon/intron boundaries follow the G-T/A-G rule. A putative transcription initiation site was assigned to an A, 30 base pairs upstream from the initiation codon by SI nuclease protection mapping. A possible "TATA" equivalent sequence, C-A-T-A-A-A, was found 27 base pairs further upstream from this initiation site. A poly(A) attachment site was determined to be 386 or 387 base pairs downstream from the termination codon by comparison with the cDNA sequence. Detailed comparison with two cDNA sequences determined previously showed the coding nucleotide sequence of the genomic clones to concur with that of the pcP-450pb2 cDNA clone coding for cytochrome P-450e except for three neutral base substitutions. Therefore, we conclude that the gene sequence determined here is for the cytochrome P-450e gene or a similar gene. On the other hand, 40 base substitutions were found in about 1,900 base pairs compared between the sequences of the genomic clone and the other cDNA clones (pcP-450pbl and 4) coding for cytochrome P-450b, and 15 of them result in 14 amino acid replacements in the total 491 amino acid residues. These base substitutions occur in relatively limited regions of the sequences. Most of them are found in exons 6, 7, 8, and 9; most frequently in exon 7.Cytochrome P-450 is a component of the microsomal monooxygenase system which metabolizes endogenous substrates such as steroids and fatty acids as well as exogenous substrates such as many kinds of drugs and other lipophilic xenobiotics. This unusual metabolic versatility of the monooxygenase system results from the participation of multiple forms of cytochrome P-450, each of which exhibits a different but, in some cases, overlapping substrate specificity (1-3). It is known, in addition, that most of these species of cytochrome P-450 are inducible enzymes; administration of drugs and other xenobiotics to animals induces the synthesis of specific forms of cytochrome P-450 (2, 3). The molecular multiplicity and mechanisms of induction of cytochrome P-450 can be best understood by using recombinant DNA technology to perform studies at the gene or DNA level.We previously reported deduced primary amino acid sequences of phenobarbital-inducible cytochrome P-450 from sequence analysis of the cDNA clones (4). By using these cDNAs as probes, we have isolated several genomic clones of the cvtochrome P-450 which are independent of one another on the basis of their restriction analyses (5).Here we report the structural analysis of one of the isolated genes for phenobarbital-inducible rat liver cytochrome P-450 and a comparison of the nucleotide sequences of the genomic and cDNA clones. MATERIALS AND METHODS.Restriction endonucleases were obtained from Takara Shuzo (Kyoto, Japa...

SUMMARY: The productive structure and productivity of a Sargassum macrocarpum C. Agardh population were studied from June 1993 to July 1994 in Fukawa Bay facing the Sea of Japan, Yamaguchi Prefecture. S. macrocarpum formed a dense population at a depth of 8 m in the study area. Using the stratified clip technique, monthly changes in the productive structure from 1993 to 1994 were clarified. The dry weight of leaves and main branches increased with the elongation of branches. Thalli in the middle to high stratum began to bear receptacles from March 1994 and the dry weight of receptacles was approximately one‐third of the standing crop in June. The loss of leaves increased from April to June 1994, and the loss of main branches and receptacles from June to July 1994. Productivity of branches and receptacles reached maxima of 4.67 g dry wt/m2 per day from February to March and 5.33 g dry wt/m2 per day from April to May, respectively. Productivity of the leaves, however, was almost constant at approximately 2 g dry wt/m2 per day from July 1993 to March 1994. Therefore, maximum productivity of the S. macrocarpum population of 7.17 g dry wt/m2 per day occurred from February to March. Annual net production of newly sprouting branches in June 1993 was 1600.1 g dry wt/m2 per year based on the summation method, which was calculated from the monthly changes in the productive structure.

We previously identified cDNA clones for rat cytochrome P-450 of the phenobarbital-inducible type by sequence analysis [Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., & Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797]. With these cloned cDNAs as probe, the multiplicity of phenobarbital-inducible cytochrome P-450 gene in rat genome was investigated by three approaches. The first approach was the Cot analysis of the total rat liver DNA under conditions of DNA excess. With internal and external markers used as gene-number standards, the reassociation kinetics were studied, which suggested the presence of approximately six genes or gene-like sequences hybridizable to phenobarbital-inducible cytochrome P-450 cDNA per rat haploid genome. The second was the isolation of the cytochrome P-450 genes from a rat genomic library. From a screening of about 1 X 10(6) plaques, nine clones with an approximately 15-kb insert were isolated. Restriction maps and Southern blot analysis of the cloned DNAs showed that six out of nine isolated clones contained DNA inserts independent of one another. The third was Southern blot analysis of rat genomic DNA with restriction enzyme EcoRI. Approximately 12 positive bands were demonstrated with the cDNA probe, seven to eight of which showed the same mobilities as the fragments in the isolated six genomic clones, suggesting that some other genes or gene-like DNA sequences remained to be cloned.

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