Cloning and Sequencing of Cytochrome P450 1A Complementary DNA in Eel (Anguilla japonica)

Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3′ untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%–56%) than to CYP1A2 (49%–52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel. Received December 19, 1998; accepted February 18, 1999     

Cloning, Sequencing, and Phylogenetic Analysis of Complementary DNA of Novel Cytochrome P-450 CYP1A in Japanese Eel (Anguilla japonica)

Complementary DNA of cytochrome P-450 CYP1A, in addition to CYP1A1, has been isolated from Japanese eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 66 bp, an open reading frame of 1554 bp coding for 517 amino acids and a stop codon, and a 3′ untranslated region of 1166 bp. The predicted molecular weight of the Japanese eel CYP1A was approximately 58.5 kDa. The nucleotide sequence exhibited identities with the reported CYP1A1 sequences of 77% for Japanese eel, 75% for rainbow trout, 72% for scup, plaice, and butterfly fish, and 71% for toadfish. The deduced amino acid sequence exhibited identities with the reported CYP1A1 sequences of 78% for Japanese eel, 77% for rainbow trout, 75% for scup, 74% for toadfish, 73% for plaice, and 72% for butterfly fish. The novel eel CYP1A obtained had less similarity to the other teleost CYP1A1 proteins (72%–78%) than that of the eel CYP1A1 (74%–80%). When compared with mammalian CYP proteins, the novel eel CYP1A was more similar to the CYP1A1 proteins (54%–56%) than to the CYP1A2 proteins (50%–53%). The phylogenetic tree of the teleost CYP1A genes constructed using the maximum likelihood method suggested that the novel eel CYP1A is ubiquitous among the Anguilliformes. Received August 25, 2000; accepted November 30, 2000     

Induction of Two Forms of Eel Cytochrome P450 1A Genes by 3-Methylcholanthrene

In eel (Anguilla japonica), exposure to polyaromatic hydrocarbons such as 3-methylcholanthrene leads to induction of two CYP1A enzymes, CYP1A1 and CYP1A6. We studied the time course and tissue specificity of induction of messenger RNAs for CYP1A1 and CYP1A6 in eel by administering 3-methylcholanthrene intraperitoneally. In both cases, the drug induced a rapid increase of mRNAs and biphasic expression. In the liver, mRNA levels of CYP1A1 and CYP1A6 increased 22-fold at 3 hours and 27-fold at 6 hours after the administration, respectively, showing initial peaks in the induction. After the initial inductions, mRNA levels decreased unexpectedly. Following these temporary decreases, the mRNA levels again increased and reached levels that were 35 and 41 times the basal levels at 24 hours after administration, respectively. CYP1A1 and CYP1A6 resembled each other also in the tissue specificity of gene expression; the expression levels were liver ≫ gill > intestine > kidney. The rapid induction, the biphasic expression, and the tissue-specific expression were common features of gene expression in CYP1A1 and CYP1A6 and may come from common structures of the regulatory regions of the two genes. Received December 7, 1998; accepted February 15, 1999     

A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer

We have identified an enhancer responsible for induction by 3-methylcholanthrene in the upstream region of the CYP1A2 gene. The enhancer does not contain the invariant core sequence of XREs that are binding sites for the Ah receptor (AhR) and Arnt heterodimer. The enhancer did not show any inducible expression in Hepa-1-derived cell lines, C4 and C12, deficient of Arnt and AhR, respectively. On the other hand, bacterially expressed AhR-Arnt heterodimer could not bind to the enhancer. Mutational analysis of the enhancer revealed that a repeated sequence separated by six nucleotides is important for expression. A factor binding specifically to the enhancer was found by using gel shift assays. Bacterially expressed AhR-Arnt heterodimer interacted with the factor. A dominant negative mutant of the AhR to XRE activated the enhancer. Collectively, these results demonstrate that a novel induction mechanism is present in which the AhR-Arnt heterodimer functions as a coactivator.     

Analysis of the essential DNA region for OsEBP-89 promoter in response to methyl jasmonic acid

In rice, the characterization of OsEBP-89 is inducible by various stress-or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible by MeJA in tobacco leaves. To further determine the crucial sequences responsible for MeJA induction, we generated a series of deletion promoters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene driven by these mutant promoters show that the essential region for MeJA induction is positioned in the region between −1200 and −800 in OsEBP-89 promoter containing a G-box (−1127), which is distinct from the essential region containing ERE (−562) for ACC induction. In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli. OsEBP-89, essential DNA region, methyl jasmonic acid, transient assay, promoter, tobacco leaves Contributed equally to this work Supported by the National Basic Research Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation of China (Grant Nos. 30671135, 30525034 and 30730060)     

Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter

SBgLR (Solanum tuberosum genomic lysine-rich) is a pollen-specific gene cloned from potato (Solanum tuberosum L.). The region from −269 to −9 (The A of translation start site “ATG” as +1) of the SBgLR promoter was identified as critical for gene specific expression in pollen grains. Sequence analysis indicates a palindromic sequence “TTTCTATTATAATAGAAA” in the −227 to −209 region, in which two pollen-specific motifs TTTCT and AGAAA surround a unique putative TATA box. Moreover, nine putative pollen-specific motifs are located in the region between the TATA box and ATG. We placed the −227 to −9 region (reserving the palindrome) and the −222 to −9 region (breaking the palindrome) downstream of the CaMV35S enhancer, respectively, to construct two fusion promoters. Histochemical assays in transgenic plants demonstrated that the region from −222 to −9 is necessary and sufficient for pollen-specific expression of the uidA gene. However, the region of −227 to −9 is incapable of driving GUS expression in pollen grains and parts of vegetative tissues. A series of 5′ deletions from −269 to −9 of SBgLR promoter were constructed. A transient expression assay indicated that the region from the −227 to −9 suppressed gfp gene expression in pollen, and a positive regulatory element was present in the region of −253 to −227. The function of the palindromic sequence as a repressor inhibiting gene expression in pollen was further confirmed by the mutated promoter, breaking the palindrome by substituting its 3′-flanking five base pairs, which resumes the reporter gene expression in mature pollen.     

Sudan III dye strongly induces CYP1A1 mRNA expression in HepG2 cells

Sudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 μM Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20-fold after 16 and 24 h. The transactivation was due to the activation of a region located at -1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III--treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III.     

Characterization of Upstream Sequences of the <Emphasis Type="Italic">LOJ</Emphasis> Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.     

A novel nuclear factor, SREB, binds to a cis-acting element, SRE, required for inducible expression of the Aspergillus oryzae Taka-amylase A gene in A. nidulans

The Taka-amylase A gene (taaG2) of Aspergillus oryzae is inducibly expressed in A. nidulans upon exposure to inducing carbon sources, such as starch and maltose. In order to identify nuclear factor(s) possibly involved in the induction of the taaG2 gene, gel mobility shift assays and DNase I footprinting analyses were carried out, and revealed a novel nuclear factor in A. nidulans extracts, which specifically bound to two sites in the taaG2 promoter region, −204 to −189 and −182 to −168, which share the common sequence GGAAATT. The nuclear factor was detected in nuclei from both induced and uninduced mycelia. Mutational analysis within and around the binding sequences demonstrated that only the upstream binding sequence, designated SRE (starch responsive element), was required for the inducible expression of the taaG2 gene, and thus we designated the nuclear factor SREB (SRE binding factor). The downstream binding site contained an inverted SRE (ISRE) and played no role in the induction of taaG2 expression. SREB was shown by gel retardation assays to have higher affinity for SRE than for ISRE. Received: 26 January 1999 / Accepted: 10 November 1999