Biomedical subjects

H Imataka

Publications and source records attributed to H Imataka.

28 records · Page 2Linked to original sources

PubMed1993

cDNA cloning and transcriptional properties of a novel GC box-binding protein, BTEB2.

We have cloned a cDNA for a novel GC box-binding protein designated BTEB2 from a human placenta cDNA library using rat BTEB cDNA (Imataka et al. (1992). EMBO J. 11,3663-3671. as a hybridization probe. BTEB2 consists of 219 amino acids and contains three contiguous zinc finger motifs at its C-terminus. The zinc finger domains showed 59% and 64% sequence similarity to those of Sp1 and BTEB, respectively. Adjacent to the N-terminal of the zinc finger motifs, a short sequence rich in basic amino acids is conserved between BTEB2 and Sp1. Furthermore, This basic sequence concurs with the N-terminal half of the consensus sequence for basic domains of the proteins containing both helix-loop-helix and leucine zipper motifs. The other region of BTEB2 is notably rich in proline, serine, threonine, and alanine residues. BTEB2 expressed in Escherichia coli showed DNA-binding activity whose specificity was closely similar to that of Sp1. Cotransfection experiments using Hepa-1 cells (a mouse hepatoma cell line) with a BTEB2 expression plasmid and GC box-containing reporter plasmids revealed that BTEB2 apparently activated the expression of the CAT activity. Moreover, when BTEB2 was fused to GAL4 DNA-binding domain, the chimeric protein could enhance the transcription through promoters containing GAL4-binding sites. Analysis of the BTEB2 mRNA by RNA blot analysis demonstrated that the mRNA was expressed specifically in testis and placenta with different sizes, 20S and 28S, respectively, among various organs examined.

Amino Acid Sequence↗

PubMed1993

Activation of the human immunodeficiency virus type 1 long terminal repeat by BTEB, a GC box-binding transcription factor.

BTEB, a GC box-binding transcription factor, was tested for its ability to activate the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). An electrophoretic mobility shift assay demonstrated specific binding of BTEB to GC boxes of the HIV-1 LTR. When a BTEB expression vector was cotransfected into A3.01 cells with a fusion gene of HIV-1 LTR and chloramphenicol acetyltransferase (CAT) structural gene, the CAT activity was increased. This increase was accompanied by an increase in the content of CAT mRNA. Transcriptional activity of the HIV-1 LTR, stimulated by Tat, was further enhanced by the expression of BTEB. BTEB also activated the LTR activity in cooperation with phorbol 12-myristate 13-acetate. Northern blot analysis showed that various T cell and macrophage/monocyte cell lines expressed the BTEB mRNA to a level comparable with that of Sp1, another GC box-binding transcription factor. These results suggest that BTEB, like Sp1, is involved in transcriptional activation of the HIV-1 LTR.

Base Sequence↗

PubMed1993

Comparison of DNA-binding properties between BTEB and Sp1.

We have expressed truncated forms of BTEB and Sp1 in Escherichia coli and investigated the DNA-binding properties of the two proteins. The two proteins as well as their chimeric proteins protected the same DNA region in the BTE sequence (a GC box in the P-4501A1 gene) as examined by ortho-phenanthroline-Cu footprinting. The region overlapped nearly perfectly with the GC box consensus sequence. Methylation interference footprinting revealed that all the guanines within the region and two other guanines in the close vicinity interacted with the proteins. Competitive gel mobility shift assay using various synthetic oligonucleotides of the GC box sequences as the competitors demonstrated that BTEB and Sp1 have similar sequence specificities for DNA binding. We have purified the bacterially expressed BTEB and measured the dissociation constant of the BTEB-BTE complex using gel mobility shift assay. The dissociation constant was (3.0 +/- 1.0) x 10(-10) M and was comparable to that of Sp1 binding to a GC box. Taken together, these findings indicate that the binding modes of BTEB and Sp1 to the GC box are similar to each other.

Base Sequence↗

PubMed1992

Regulation of CYP1A1 expression.

CYP1A1 is considered to be involved mainly in oxidative metabolism of exogenous chemicals and drugs. Synthesis of this hemoprotein is induced in livers, lungs, and other tissues of experimental animals by the administration of these chemicals. Regulatory mechanisms of the induction process of the protein have been investigated by the DNA transfer method using the isolated genomic DNA. At least two kinds of cis-acting regulatory DNA sequences are localized 5' upstream of the gene. One is distributed five times in a relatively wide range from -0.5 to -3.5 kb and functions as an inducible enhancer-designated xenobiotic responsive element or XRE. The other is localized just upstream of the TATA sequence and acts as a regulatory element for the constitutive expression. The two DNA elements are required for a high level of the inducible expression. Their cognate DNA binding factors are recognized in the nuclear extracts of Hepa-1 cells and rat liver cells which show the inducible expression of CYP1A1 in response to the inducer. This paper discusses the regulatory mechanisms of CYP1A1 gene expression by summarizing the present state of knowledge about properties of the DNA regulatory elements and their cognate DNA-binding factors.

Amino Acid Sequence↗

PubMed1992

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P-450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys-Cys/His-His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV-1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus-1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Transcriptional regulation of 3-methylcholanthrene-inducible P-450 gene responsible for metabolic activation of aromatic carcinogenes.

We have investigated the transcriptional regulation of 3-methylcholanthrene (3MC)-inducible P-450c gene which is involved in the metabolic activation of polycyclic aromatic carcinogens. Reverse genetic study using the fusion gene composed of the 5' upstream sequence of P-450c gene and the structure gene for bacterial chloramphenicol acetyltransferase (CAT) and a cultured cell line of Hepa-1 cells localized two kinds of cis-acting regulatory DNA elements. One is designated XRE or xenobiotic responsive element which is responsible for the inducibility of the gene and is distributed 5 times in the region from -3.0 to -0.5 kb. The other is BTE or basal transcription element whose deletion reduces a low level of the constitutive CAT expression to a background level, and which is localized immediately upstream of the TATA sequence. Both kinds of regulatory elements are necessary for a high level of inducible expression. Gel mobility shift assay strongly suggests that the binding protein to the XRE is an Ah receptor with a specific affinity for 3MC or 2,3,7,8,tetrachlorodibenzo-p-dioxin (TCDD). Without inducer treatment, cryptic form of the binding protein occurs only in the cytoplasm of the Hepa-1 cells. Upon treatment with the inducer, the cryptic form of the binding protein exhibits binding activity to XRE and, at the same time, translocates to the nuclei. The BTE-binding protein is an ubiquitous nuclear factor and its cDNA cloning reveals the DNA-binding feature with zinc finger motifs.

Animals↗

PubMed1989

Biosynthetic pathways of testosterone and estradiol-17 beta in slices of the embryonic ovary and testis of the chicken (Gallus domesticus).

To elucidate synthetic pathways of testosterone and estradiol-17 beta in embryonic gonads of the chicken, metabolism of various 14C-labeled steroids in slices of the left ovaries and paired testes of 15- and 9-day-old chicken embryos was examined. (1) Fifteen-day-old chicken embryos: From pregnenolone, more 17 alpha-hydroxypregnenolone was produced than progesterone in the ovary, while more progesterone was produced than 17 alpha-hydroxypregnenolone in the testis. From 17 alpha-hydroxypregnenolone, however, only dehydroepiandrosterone was detected as a product in both gonads. Dehydroepiandrosterone was converted mainly into androstenedione and its 5 beta-reduced derivatives by both gonads. Progesterone was converted into 5 beta-pregnane-3,20-dione more than into 17 alpha-hydroxyprogesterone by both gonads. Both gonads metabolized 17 alpha-hydroxyprogesterone, androstenedione, and testosterone predominantly into their corresponding 5 beta-reduced steroids, while production of androstenedione from 17 alpha-hydroxyprogesterone and of testosterone from androstenedione was limited. Estradiol-17 beta was produced from androstenedione and testosterone only by the ovary. (2) Nine-day-old chicken embryos: From pregnenolone, production of progesterone and 17 alpha-hydroxypregnenolone was similar in the ovary. On the other hand, in the testis, more progesterone was produced than 17 alpha-hydroxypregnenolone from pregnenolone. For delta 4-3-oxo steroids, strong activity of 5 beta-reductase was demonstrated in both gonads. From these results, both delta 4- and delta 5-pathways are involved in the formation of testosterone and then finally of estradiol-17 beta by the embryonic gonads of the chicken, and relative preference for the pathway seems to depend on sexes and embryonic ages. In addition, it is suggested that steroidogenesis in these embryonic gonads is characterized by marked activity of 5 beta-reductase, irrespective of sexes or ages.

17-alpha-Hydroxypregnenolone↗

PubMed1988

Sexual differences of steroidogenic enzymes in embryonic gonads of the chicken (Gallus domesticus).

The left ovary and testis of 15-day-old embryos of the chicken were compared in the enzyme activities related to steroidogenesis. The activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary was similar to that of the testis. Activities of 17 alpha-hydroxylase and C-17-C-20 lyase in the ovary were 2.5 and 2.6 times those in the testis. From the CO-induced difference spectrum, the content of cytochrome P-450 in the ovarian microsomes was estimated as 27.6 pmol/mg protein. However, no detectable amount of cytochrome P-450 was observed in the testicular microsomal fraction. The substrate (progesterone)-induced difference spectrum was appreciable only in the ovarian microsomes. The activity of microsomal NADPH-cytochrome c reductase in the ovary was significantly higher than that in the testis. The activities of 17 beta-hydroxysteroid dehydrogenase in both gonads were similar to each other, when androstenedione was used as substrate. However, its activity in the ovary was 1.4 and 3.1 times that in the testis, when dehydroepiandrosterone and estrone were used as substrate, respectively. Aromatase activity in the ovary was over 100 times that in the testis, as assessed by release of [3H]water from [1-3H]testosterone. Appreciable amounts of radioactive estradiol-17 beta and estrone were formed from [4-14C]testosterone and [7-3H]androstenedione, respectively, only by the ovarian tissue. 5 beta-Reductase activity in the ovary was 1.4 times that in the testis.

Aldehyde-Lyases↗

PubMed1988

Developmental changes of steroidogenic enzyme activities in the embryonic gonads of the chicken: the sexual difference.

Steroidogenic enzyme activities in the left ovary and the testes of 9- to 15-day-old chicken embryos were measured, and development of the activities was compared between sexes. Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase in the ovary and in the testis was comparable, and did not change throughout the period examined. Activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the ovary was similar to or higher than that in the testis, depending on substrates employed. In both gonads, 17 beta-HSD activity did not change or tended to decrease from 9 to 15 days of development. On the other hand, activities of 17 alpha-hydroxylase, C-17--C-20 lyase in the ovary were three to eight times those in the testis, and aromatase activity in the ovary was definitely higher than that in the testis at all stages examined. The activities of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase significantly increased from 9 to 11 days only in the ovary. From 13 to 15 days, the activities of 17 alpha-hydroxylase and C-17--C-20 lyase markedly increased only in the testis. These results suggest that, in the gonads of developing chicken embryos, there are sexual differences in the regulation of 17 alpha-hydroxylase, C-17--C-20 lyase, and aromatase activities.

17-Hydroxysteroid Dehydrogenases↗

PubMed1985

Effect of Fe2+ -induced lipid peroxidation upon microsomal steroidogenic enzyme activities of porcine adrenal cortex.

When porcine adrenocortical microsomes were treated with Fe2+, enhanced production of malondialdehyde was observed as a result of membrane lipid peroxidation. By treatment of the microsomes with Fe2+, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with delta 5-delta 4 isomerase, concentration of cytochrome P-450 and the activity of the cytochrome-involving enzyme systems such as 17 alpha- and 21-hydroxylases were significantly reduced. 17 alpha-Hydroxylase activity was more effectively decreased by Fe2+ than that of 21-hydroxylase. On the other hand, activity of NADH- and NADPH-cytochrome c reductases remained unchanged or somewhat increased. Both the induction of lipid peroxidation and the decrease of the enzyme activities were prevented by alpha-tocopherol and N,N' -diphenyl-rho-phenylenediamine.

3-Hydroxysteroid Dehydrogenases↗