Yoshiaki Fujii-Kuriyama

The nucleotide sequence of human fibroblast interferon cDNA

DNA synthesized by in vitro reverse transcription of the interferon mRNA has been cloned and amplified as recombinant DNA, TpIF319-13 (Taniguchi et al., 1979). The nucleotide sequence of this IF cDNA which consists of 770 bp (excluding the A:T tails) has been determined. The data reported predict the hitherto unknown amino acid sequence of human fibroblast interferon and its putative signal peptide.

Determination of nucleotide sequence of cDNA coding rat glutathione peroxidase and diminished expression of the mRNA in selenium deficient rat liver

The cDNA for rat glutathione peroxidase mRNA was isolated from liver cDNA library in lambda gt11 by cross-hybridization using the mouse cDNA, and its nucleotide sequence was determined. The selenocysteine which constitutes an active center of this enzyme was encoded by TGA, a nonsense codon in general, as was the cases with mouse and human glutathione peroxidase. Northern blot analysis elucidated that the mRNA for glutathione peroxidase was markedly diminished in selenium deficient rat liver as compared with that of normal rat livers. The result suggested that the de novo synthesis of the mRNA would be regulated by selenium.

Expression of cytochrome P-450d by Saccharomyces cerevisiae

Rat liver microsomal cytochrome P‐450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast‐Escherichia coli shuttle vector consisting of rat liver P‐450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P‐450d was immunologically crossed with rat liver P‐450d. The hydroxylase activity of estra‐1,3,5(10)‐triene‐3,17β‐diol was 11 nmol/min per nmol P‐450d, which is comparable to that reported previously for rat liver P‐450d. The expressed P‐450d content was nearly 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorption of the reduced CO form.

Hypomethylation and expression of pepsinogen a genes in the fundic mucosa of human stomach

We have examined the correlation between the extents of methylation and expression of pepsinogen A genes in normal human tissues. Expression of pepsinogen A mRNA was detected only in the fundic mucosa of the stomach and both CCGG and GCGC sites in the genes region were less methylated in the fundic mucosa than in other non-expressing tissues. Thus, there was an inverse correlation between the extents of methylation and expression of pepsinogen A genes and the role of DNA methylation in the regulation of pepsinogen A genes expression during normal differentiation was suggested.

Close linkage of human chromosomal pepsinogen A genes

We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al.

Isolation of the chick myosin alkali light chain gene expressed in embryonic gizzard muscle and transitional expression of the light chain gene family in vivo.

A chick embryonic myosin alkali light chain L23 gene that is expressed transiently at embryonic stages in chick skeletal, cardiac and smooth muscles and in brain continuously from embryo to adult stages, was isolated and characterized. Sequence analysis showed that the exonic sequence of this gene was identical with that of embryonic myosin light chain mRNA except for one base replacement. This gene is a single gene of 5200 bases, which is divided into seven exons by six introns, and the positions of inserts of all the introns are well-conserved as in the skeletal and cardiac muscle myosin alkali light chain genes. Therefore, this embryonic myosin light chain gene can be classified as a member of the myosin alkali light chain gene family, and these three genes may have originated from a common ancestral gene. Transcription of the embryonic light chain gene starts from the same initiation site 33 bases upstream from ATG in embryonic muscle tissues and brain. Comparison of the nucleotide sequence around the promotor region of the embryonic myosin light chain gene with the corresponding regions of the skeletal and cardiac myosin light chain genes showed that the 11-base consensus sequence (TCCTATTTATAG) is present about 100 bases upstream from the transcription initiation site in each gene.

Single chicken cardiac myosin alkali light-chain gene generates two different mRNAs by alternative splicing of a complex exon

We have isolated and characterized two kinds of cDNA for the chicken cardiac myosin alkali light chain. The sequences of the two cDNAs are identical, except for a notable divergence in part of the 3 untranslated sequence. By analysis of isolated genomic clones, it was shown that the genomic sequences corresponding to the different sequences in the 3 untranslated regions of the two mRNAs were arranged within a limited part of a single stretch of DNA; also the two distinct 3 untranslated regions of the two mRNAs shared part of the last exon, which was 0.6 x 10(3) base-pairs long. There are two canonical acceptor sites available for RNA splicing in the last exon, the first being located at the 5 end of the exon, and the second at 370 base-pairs downstream from this end. Together with analysis by S1 nuclease mapping, the foregoing results lead us to conclude that, by the differential use of these two acceptor sites, a single gene generates two distinct mRNAs of 1.45 x 10(3) base-pairs and 1.1 x 10(3) base-pairs with or without the 5 half of the last exon. The two mRNAs appear to utilize the same modified poly(A) signal, AGTAAA, rather than the authentic AATAAA sequence present about 30 base-pairs downstream from the poly(A) attachment sites. This is probably because another consensus G + T-rich sequence is present at an appropriate distance from the AGTAAA sequence, but not from the AATAAA sequence. The gene for the cardiac myosin alkali light chain has proved to be expressed in ventricular muscle and in atrial and anterior latissimus dorsi muscles, the last of these being characteristic of slow skeletal muscle. In these muscles, two kinds of mRNA for the cardiac myosin alkali light chain, identical with those in ventricular muscle, were expressed and their relative amount in each tissue was almost the same as that in ventricular muscle.

Possible steroid binding site common to adrenal cytochrome P-450scc and prostatic steroid binding protein

By searching the entire PIR‐protein‐sequence data base, we have found that a dodecapeptide sequence in bovine adrenal cytochrome P‐450scc is closely related to that in rat prostatic steroid binding protein. The two proteins belong to unrelated protein families, but both have steroids as substrates or ligands. Thus, the dodecapeptides may be important for substrate/ligand recognition in the individual proteins.

Gene Structure and Regulation of Cytochrome P-450

Cytochrome P-450 is a collective term for a group of hemoproteins which catalyze monooxygenase reactions of steroids, fatty acids, prostaglandins and an almost infinite number of lipophilic xenobiotics. Generally speaking, most of the constituent members of the P-450 superfamily, if not all, are inducible enzymes. Their specific forms are induced in response to either endogenous or exogenous inducers.

Multiplicity of cytochrome P-450 and its gene structure

Cytochrome P-450 is widely distributed in nature from microorganisms to higher animals and plays an important role in the oxidative metabolism of a great variety of endogenous as well as exogenous lipophilic compounds (Sato & Omura, 1978; Lu & West, 1980). Recent studies involving immunological chemistry and protein chemistry have shown that multiple forms of cytochrome P-450 are present in rat liver microsomes and that their synthesis could be induced in different ways by the administration of various kinds of drugs (Sato & Omura, 1978; Lu & West, 1980). However, the molecular multiplicity and drug induction mechanism of cytochrome P-450 could best be understood by investigation at the gene (DNA) level using recombinant DNA technology.