41 results on '"Tsai, M-J."'
Search Results
2. Two distinct class A helix-loop-helix transcription factors, E2A and BETA1, form separate DNA binding complexes on the insulin gene E box.
- Author
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Peyton, M, primary, Moss, L G, additional, and Tsai, M J, additional
- Published
- 1994
- Full Text
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3. Synergism between steroid response and promoter elements during cell-free transcription.
- Author
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Allan, G F, primary, Ing, N H, additional, Tsai, S Y, additional, Srinivasan, G, additional, Weigel, N L, additional, Thompson, E B, additional, Tsai, M J, additional, and O'Malley, B W, additional
- Published
- 1991
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4. Recombinant human glucocorticoid receptor induces transcription of hormone response genes in vitro.
- Author
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Tsai, S Y, primary, Srinivasan, G, additional, Allan, G F, additional, Thompson, E B, additional, O'Malley, B W, additional, and Tsai, M J, additional
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- 1990
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5. Superactive estrogen receptors. Potent activators of gene expression.
- Author
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Elliston, J F, primary, Tsai, S Y, additional, O'Malley, B W, additional, and Tsai, M J, additional
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- 1990
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6. Regulation of in vitro transcription by progesterone receptor. Characterization and kinetic studies.
- Author
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Bagchi, M K, primary, Tsai, S Y, additional, Weigel, N L, additional, Tsai, M J, additional, and O'Malley, B W, additional
- Published
- 1990
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7. Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells.
- Author
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Liou, J Y, Shyue, S K, Tsai, M J, Chung, C L, Chu, K Y, and Wu, K K
- Abstract
The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.
- Published
- 2000
8. Molecular characterization of the rat insulin enhancer-binding complex 3b2. Cloning of a binding factor with putative helicase motifs.
- Author
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Shieh, S Y, Stellrecht, C M, and Tsai, M J
- Abstract
Cell-specific expression of the rat insulin II gene is in part mediated through an element located in the 5'-flanking region. The element, termed RIPE3b (-126 to -101), confers beta-cell-specific expression in conjunction with an adjacent element RIPE3a (-110 to -86). Here we report the characterization of one of the RIPE3b-binding complexes, 3b2. UV cross-linking analysis demonstrated that it is composed of at least three polypeptides: p58, p62, and p110. Furthermore, a cDNA was isolated via expression screening for binding to RIPE3b. Sequence analysis reveals that the encoded protein, designated Rip-1, possessed putative helicase motifs and a potential transcription activation domain. Overexpression of Rip-1 in cells greatly enhances the 3b2 binding complex, suggesting that Rip-1 is involved in the binding of 3b2.
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- 1995
9. A far upstream ovalbumin enhancer binds nuclear factor-1-like factor.
- Author
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Bradshaw, M S, Tsai, M J, and O'Malley, B W
- Abstract
Band-shifting and DNase I footprinting analyses detected a specific DNA binding protein extracted from oviduct nuclei that binds to the ovalbumin gene 5' sequence between -1094 and -1125. This “-1100” fragment, when inserted upstream of the SV40 or ovalbumin promoters fused to a chloramphenicol acetyltransferase reporter gene, enhances chloramphenicol acetyltransferase activity 5-10-fold following transfection into CV1 cells. The sequence to which the oviduct factor binds contains a nuclear factor-1 (NF-1) half-site (GCCAA). An oligonucleotide matching the sequence of the adenovirus NF-1 binding site competed for binding to the -1100 footprinted region with a higher affinity than an oligonucleotide for the -1100 region itself. Similarly, the -1100 region oligonucleotide also competes for binding of the factor to the NF-1 oligonucleotide. These data suggest that the oviduct factor which binds to the -1100 region is an NF-1-like protein that serves as a steroid hormone-independent enhancer of the ovalbumin gene transcription.
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- 1988
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10. The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors.
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Onate, S A, Boonyaratanakornkit, V, Spencer, T E, Tsai, S Y, Tsai, M J, Edwards, D P, and O'Malley, B W
- Abstract
Steroid receptors are ligand-inducible transcription factors, and their association with steroid receptor coactivators (SRCs) upon binding to DNA is necessary for them to achieve full transcriptional potential. To understand the mechanism of SRC-1 action, its ability to interact and enhance the transcriptional activity of steroid receptors was analyzed. First, we show that SRC-1 is a modular coactivator that possesses intrinsic transcriptional activity when tethered to DNA and that it harbors two distinct activation domains, AD1 and AD2, needed for the maximum coactivation function of steroid receptors. We also demonstrate that SRC-1 interacts with both the amino-terminal A/B or AF1-containing domain and the carboxyl-terminal D/E or AF2-containing domain of the steroid receptors. These interactions are carried out by multiple regions of SRC-1, and they are relevant for transactivation. In addition to the inherent histone acetyltransferase activity of SRC-1, the presence of multiple receptor-coactivator interaction sites in SRC-1 and its ability to interact with components of the basic transcriptional machinery appears to be, at least in part, the mechanism by which the individual activation functions of the steroid receptors act cooperatively to achieve full transcriptional activity.
- Published
- 1998
11. The mouse bone morphogenetic protein-4 gene. Analysis of promoter utilization in fetal rat calvarial osteoblasts and regulation by COUP-TFI orphan receptor.
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Feng, J Q, Chen, D, Cooney, A J, Tsai, M J, Harris, M A, Tsai, S Y, Feng, M, Mundy, G R, and Harris, S E
- Abstract
Bone morphogenetic protein-4 (BMP-4) is one of a member of related polypeptides that are important in bone formation and other developmental processes. We isolated the BMP-4 gene from a mouse genomic library and characterized the exon-intron structure and one of the candidate promoters. Two alternative 5'-noncoding exons, 1A and 1B, were identified by reverse transcription polymerase chain reaction assays. Quantitative competitive polymerase chain reaction using Exon 1A, Exon 1B, and Exon 3 primers indicate the 1A-containing transcript is the primary BMP-4 mRNA expressed in bone cell cultures. Primer extension analysis supports that 1A is the major promoter utilized in bone cell cultures as well as in 9.5-day mouse embryos. 1A promoter activity indicate selective DNA regions functional in bone cells. We found potential regulatory response regions in the 1A 5'-flanking region of the BMP-4 gene for the chicken ovalbumin upstream-transcription factor I (COUP-TFI). Specific binding to the COUP-TFI response regions in the BMP-4 1A promoter was demonstrated. By co-transfection of a COUP-TFI expression plasmid with the BMP-4 1A promoter in fetal rat calvarial osteoblasts, we demonstrated that COUP-TFI inhibits the BMP-4 promoter activity. This suggests that COUP-TFI could act as a silencer for BMP-4 transcription in vivo.
- Published
- 1995
12. Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription.
- Author
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Zarucki-Schulz, T, Tsai, S Y, Itakura, K, Soberon, X, Wallace, R B, Tsai, M J, Woo, S L, and O'Malley, B W
- Abstract
The role of the eucaryotic T-A-T-A box (Hogness box) homology sequence located approximately 30 base pairs upstream from the RNA initiation site has been further examined by oligodeoxynucleotide-directed site-specific mutagenesis. A method was employed which allows insertion of nucleotide-specific mutations into virtually any double-stranded, recombinant plasmid DNA. A synthetic mixed oligonucleotide, bearing defined multiple nucleotide substitutions at a single site, was used both as a specific mutagen during primed DNA repair synthesis in vitro, as well as a highly sensitive hybridization probe for the identification of the mutated cloned DNA. Using this methodology, an A leads to G transition mutation was introduced into the second position of the ovalbumin gene T-[A]-T-A box, and the effect of modifying this highly conserved nucleotide on the expression of the mutant DNA was analyzed in a cell-free transcription system. Comparison of two allelic ovalbumin genes, slightly divergent upstream from the TATA box, resulted in identical in vitro transcription efficiencies. While correct initiation of ovalbumin RNA transcripts was not affected, the efficiency of gene expression using the mutant template, compared to either the corresponding wild-type sequence or the allelic gene, was markedly reduced. These results suggest that it is the T-A-T-A box sequence which plays a role in the efficient initiation of RNA transcription in vitro and further supports the implication that this region may serve a promoter-related function in eucaryotic transcription.
- Published
- 1982
- Full Text
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13. Mutational Analysis of the Chicken Progesterone Receptor
- Author
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Dobson, A D, Conneely, O M, Beattie, W, Maxwell, B L, Mak, P, Tsai, M J, Schrader, W T, and O'Malley, B W
- Abstract
Oligonucleotide-directed site mutagenesis was used to prepare a series of chicken progesterone receptor deletion mutants in an attempt to elucidate structure-function relationships of the receptor. These mutants spanned the entire 659-amino acid coding region of the A form of the receptor. The ability of these mutants to bind progesterone was analyzed following in vitrotranscription and translation. Results obtained indicate that a large portion of the protein ranging from amino acid 420 to the extreme carboxyl terminus is necessary to maintain the protein in a conformation which is capable of binding hormone. Following transient cotransfection of mutant receptor proteins into CV-1 cells along with a reporter gene containing an authentic GRE/PRE (PRE-TK-CAT), our results indicated that any deletion throughout the entire molecule results in a decrease in transcriptional activation. Most of these decreases result from an inability of the mutant receptor proteins to bind DNA or hormone. However, two areas of the receptor have been identified which are unrelated to either DNA or hormone binding but markedly affect the ability of the receptor to transactivate target genes.
- Published
- 1989
- Full Text
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14. Multiple origins of transcription for the human placental lactogen genes.
- Author
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Selvanayagam, C S, Tsai, S Y, Tsai, M J, Selvanayagam, P, and Saunders, G F
- Abstract
Transcriptional activity of the three human placental lactogen (hPL) genes was compared in vitro and their relation to the hPL RNA species obtained from term placenta was analyzed. We used in vitro transcription system containing the HeLa cell crude extract as the source for RNA polymerase II and initiation factors. Gene fragments of identical length of 0.96 kilobase pair made from hPL1, hPL3, and hPL4 each contained the 5‘ flanking sequence of 497 base pairs, the first exon, the first intron, and a portion of the second exon. More than 90% transcripts of the hPL1 template were 470 nucleotides long, indicating that transcription was initiated at the proposed cap site. hPL3 and hPL4 genes generated heterogeneous RNA products of about 430, 470, 520, and 680 nucleotides suggesting that multiple start points were recognized for RNA synthesis in vitro. alpha-Amanitin sensitivity of transcription indicated that the DNA-dependent RNA synthesis was carried out by RNA polymerase II. These results show that hPL1, hPL3, and hPL4 genes have functional promoters and multiple initiation sites for transcription. Primer extension analysis of the 5‘ termini of hPL RNA isolated from term placenta shows that 82-83% of the transcripts are initiated at a region 29 base pairs downstream from a “TATA” sequence. This origin is observed in vitro for the transcript 470 nucleotides long. An additional upstream initiation region (-53) accounts for 8% of transcripts in term placenta and corresponds to the origin for the in vitro transcript of 520 nucleotides. At least three other sites 15, 23, and 39 base pairs downstream from the major cap site are functional in vivo. The initiation site at +40 is utilized preferentially for transcription from hPL3 and hPL4 genes in vitro. We have mapped the different transcription origins on hPL genes.
- Published
- 1984
- Full Text
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15. Steroid hormone regulation of the gene encoding the chicken heat shock protein hsp 108.
- Author
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Baez, M, Sargan, D R, Elbrecht, A, Kulomaa, M S, Zarucki-Schulz, T, Tsai, M J, and O'Malley, B W
- Abstract
We have previously isolated a partial cDNA clone encoding a heat shock protein which has been termed hsp 108 (Zarucki-Schulz, T., Kulomaa, M. S., Headon, D. R., Weigel, N. L., Baez, M., Edwards, D. O., McGuire, W. L., Schrader, W. T., and O'Malley, B. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6358-6362; Sargan, D. R., Tsai, M.-J., and O'Malley, B. W. (1985) Biochemistry 25, 6252-6258). Here we examine the expression of the hsp 108 gene in steroid-stimulated chick oviducts. After 16 h of secondary stimulation with estrogen or progesterone, a 20-50-fold increase in hsp 108 mRNA is detected above unstimulated levels. RNA quantitation by Rot analysis shows that in these oviducts there are 75 molecules of hsp 108 mRNA/oviduct cell. Nuclear “run-off” assays indicate only a 2-4-fold increase in the rate of transcription of the gene in response to either sex steroid, suggesting that the gene is regulated both at the transcriptional level and by mRNA stabilization. On hormone withdrawal, the concentration of hsp 108 mRNA in the oviduct falls to unstimulated control levels within 4 days. Chronic stimulation of the chicks with estrogen (or high acute doses of estrogen) attenuates specifically the inductive response of the hsp 108 gene, but not of ovalbumin, under these conditions. This is not due to a significant reduction of the transcription rate of the gene. We have previously shown that hsp 108 is expressed constitutively in many tissues of the chick (Sargan, D. R., Tsai, M.-J., and O'Malley, B. W. (1985) Biochemistry 25, 6252-6258). In tissues which are not responsive to hormones, no short-term effects of hormone administration on the gene were observed. In the spleen there is a reproducible slow activation of the gene, but the kinetics of this response suggest that it is not a primary response to the hormone. Thus, this hsp 108 gene codes for an interesting new eucaryotic heat shock protein which is regulated also by steroid hormones in a tissue-specific manner.
- Published
- 1987
- Full Text
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16. Purification and characterization of chicken ovalbumin upstream promoter transcription factor from HeLa cells.
- Author
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Wang, L H, Tsai, S Y, Sagami, I, Tsai, M J, and O'Malley, B W
- Abstract
A transcription factor which binds to the chicken ovalbumin upstream promoter (COUP) sequence spanning between −70 and −90 is required for efficient transcription of the ovalbumin gene. This COUP transcription factor has been purified approximately 200,000-fold by a combination of conventional column and sequence-specific DNA affinity column chromatography. A few polypeptides were identified in the purified preparation on sodium dodecyl sulfate gel. Upon renaturation, all the major polypeptides in the molecular size range between 43 and 53 kDa bound specifically to the COUP sequence. Furthermore, at least one of the renatured polypeptides in the region of 43-45 kDa retained transcriptional activity. The binding of the COUP transcription factor to the ovalbumin promoter cannot be competed by DNA fragments which contain the CCAAT box promoter sequence. Since the COUP and CCAAT binding proteins can be separated on an S300 column, they are distinct molecules. Using band-shifting assays and 3' and 5' deletion mutants and oligonucleotide mutants, the sequence important for binding was mapped.
- Published
- 1987
- Full Text
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17. Tissue-specific and developmental regulation of the rat insulin II gene enhancer, RIPE3, in transgenic mice.
- Author
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Stellrecht, C M, DeMayo, F J, Finegold, M J, and Tsai, M J
- Abstract
The rat insulin II gene enhancer, RIPE3 (-126 to -86), mediates beta-islet cell-specific activity in transfection assays. To investigate the in vivo activity of RIPE3, we generated mice carrying a transgene consisting of three copies of RIPE3 linked to a minimal chicken ovalbumin promoter in conjunction with sequences encoding the human growth hormone gene. 13 transgenic mice were obtained, 11 of which expressed the transgene, as determined by serum radioimmunoassay for human growth hormone. Expression of the transgene was assessed for cell specificity by immunocytochemistry. The pancreatic islet cells invariably stained for growth hormone, while the acinar and ductal cells did not. Staining of adjacent sections for insulin, glucagon, and somatostatin revealed that growth hormone was expressed in the beta-cell in all of the mice analyzed, but in some mice alpha-cells also contained growth hormone. RNase protection analysis revealed that the tissues that consistently express the transgene in these animals are the pancreas and brain. Developmental analysis revealed that the transgene was expressed in the pancreatic bud at embryonic day 9.5, corresponding to the temporal expression pattern of the insulin gene. These results signify that an element as small as 41 base pairs is capable of regulating pancreatic temporal and spatial gene expression in vivo.
- Published
- 1997
18. Differential binding of the chicken ovalbumin upstream promoter (COUP) transcription factor to two different promoters.
- Author
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Hwung, Y P, Wang, L H, Tsai, S Y, and Tsai, M J
- Abstract
The COUP (chicken ovalbumin upstream promoter) transcription factor binds to the upstream promoters of both the chicken ovalbumin and the rat insulin II genes, even though the two binding sites have no obvious sequence similarity (Hwung, Y.-P., Crowe, D. T., Wang, L.-H., Tsai, S. Y., and Tsai, M.-J. (1988) Mol. Cell. Biol. 8, 2070-2077). Comparison of the contact points and important nucleotides in the two binding sites suggests that the COUP transcription factor binds to them in different ways. The purine contacts in the two promoters are limited to one face of the DNA helix; however, studies on phosphate contacts suggest that the COUP transcription factor wraps around the ovalbumin promoter, while it binds to only one face of the DNA helix in the insulin promoter. In addition, the binding factor makes a more extended contact on the insulin promoter as compared to the ovalbumin promoter. The potential significance of this novel finding is discussed.
- Published
- 1988
- Full Text
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19. Generation of monoclonal antibodies to RNA polymerase II for the identification of transcriptional factors.
- Author
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Tsai, S Y, Dicker, P, Fang, P, Tsai, M J, and O'Malley, B W
- Abstract
Monoclonal antibodies were raised against a partially purified RNA polymerase II preparation from hen oviduct. Hybridomas were screened using enzyme-linked immunosorbent assays, and those producing antibodies against the RNA polymerase II preparation were cloned twice. The specificity of monoclonal antibodies for RNA polymerase II was determined by three types of assays: first, by immunoblot assay; second, by removal of enzyme activity using indirect immunoprecipitation; and third, by affinity column chromatography. Several monoclonal antibodies were identified. One of them, 4C3-21 was characterized in detail here. This monoclonal antibody recognized the native form of RNA polymerase II from chicken oviduct, calf thymus, and HeLa cells, but it did not recognize Escherichia coli RNA polymerase. In addition, after interacting with RNA polymerase, this antibody did not inhibit total RNA synthesis nor specific initiation of transcription on cloned ovalbumin fragments in vitro. Finally, since the antibody did not react with polymerase subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we deduced that the antibody interacted with the native form of the enzyme at a site which is not involved in initiation or elongation of RNA synthesis. This allowed us to undertake an unique approach utilizing antibody affinity column chromatography for purification of transcriptional factors. Using this approach, we demonstrated that certain transcription factor(s) were associated with RNA polymerase II and could be absorbed to an affinity column as an RNA polymerase-transcriptional factor complex. Differential elution can be carried out subsequently. Therefore, this antibody column should prove to be very useful for the purification of transcriptional regulatory factor(s) which bind to RNA polymerase.
- Published
- 1984
- Full Text
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20. Effect of estrogen on gene expression in the chick oviduct. Kinetics of initiation of in vitro transcription on chromatin.
- Author
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Hirose, M, primary, Tsai, M J, additional, and O'Malley, B W, additional
- Published
- 1976
- Full Text
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21. Effects of estrogen on gene expression in the chick oviduct. Control of ovalbumin gene expression by non-histone proteins.
- Author
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Tsai, S Y, primary, Tsai, M J, additional, Harris, S E, additional, and O'Malley, B W, additional
- Published
- 1976
- Full Text
- View/download PDF
22. Effect of estrogen on gene expression in the chick oviduct. Studies on the initiation of RNA synthesis on chromatin in vitro.
- Author
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Tsai, M J, primary, Tsai, S Y, additional, Towle, H C, additional, and O'Malley, B W, additional
- Published
- 1976
- Full Text
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23. The Chicken Progesterone Receptor A and B Isoforms Are Products of an Alternate Translation Initiation Event
- Author
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Conneely, O M, primary, Kettelberger, D M, additional, Tsai, M J, additional, Schrader, W T, additional, and O'Malley, B W, additional
- Published
- 1989
- Full Text
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24. Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene in chromatin.
- Author
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Harris, S E, primary, Schwartz, R J, additional, Tsai, M J, additional, O'Malley, B W, additional, and Roy, A K, additional
- Published
- 1976
- Full Text
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25. Effects of estrogen on gene expression in the chick oviduct. IV. Initiation of RNA synthesis on DNA and chromatin.
- Author
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Tsai, M-J, primary, Schwartz, R J, additional, Tsai, S Y, additional, and O'Malley, B W, additional
- Published
- 1975
- Full Text
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26. Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin.
- Author
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Schwartz, R J, primary, Tsai, M-J, additional, Tsai, S Y, additional, and O'Malley, B W, additional
- Published
- 1975
- Full Text
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27. Effects of estrogen on gene expression in chick oviduct. The role of chromatin proteins in regulating transcription of the ovalbumin gene.
- Author
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Tsai, S Y, primary, Harris, S E, additional, Tsai, M J, additional, and O'Malley, B W, additional
- Published
- 1976
- Full Text
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28. Effect of estrogen on gene expression in the chick oviduct. Correlation between nuclear-bound estrogen receptor and chromatin initiation site for transcription.
- Author
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Kalimi, M, primary, Tsai, S Y, additional, Tsai, M J, additional, Clark, J H, additional, and O'Malley, B W, additional
- Published
- 1976
- Full Text
- View/download PDF
29. Effect of estrogen on gene expression in the chick oviduct.
- Author
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Towle, H C, primary, Tsai, M J, additional, Tsai, S Y, additional, and O'Malley, B W, additional
- Published
- 1977
- Full Text
- View/download PDF
30. Repression of estrogen-dependent stimulation of the oxytocin gene by chicken ovalbumin upstream promoter transcription factor I.
- Author
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Burbach JP, Lopes da Silva S, Cox JJ, Adan RA, Cooney AJ, Tsai MJ, and Tsai SY
- Subjects
- Animals, Base Sequence, Binding, Competitive, COUP Transcription Factor I, Chickens, Gene Expression Regulation, Molecular Sequence Data, Mutation, Oligodeoxyribonucleotides, Rats, DNA-Binding Proteins metabolism, Estrogens pharmacology, Ovalbumin genetics, Oxytocin genetics, Promoter Regions, Genetic, Transcription Factors metabolism
- Abstract
The orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TF I) fully prevented not only the activation of the oxytocin gene by retinoic acid and thyroid hormone but also completely repressed the estrogen-dependent stimulation in transfected P19 EC cells. DNase I footprinting showed that the COUP-TF I protein bound to the 5'-flanking region of the oxytocin gene at the site of the distal composite hormone response element, which mediates the responses to estrogen, retinoic acid, and thyroid hormone. Electrophoretic mobility shift assay using this composite hormone response element as probe showed that COUP-TF I and the estrogen receptor competed for binding but did not form a heterodimer. The binding by COUP-TF I was stronger than the binding of the estrogen receptor. Thus, the mechanism of repression involves occupancy of integrated binding sites. By mutagenesis of the composite hormone response element, the COUP-TF I binding site and the estrogen response element could be separated, resulting in functional dissociation of the repressive action of COUP-TF I and the induction by estrogen. The results show that repression of gene expression by COUP-TF I is not limited to receptors that act through heterodimerization but also extends to the homodimer-forming estrogen receptor in a context-dependent manner. This interaction between COUP-TF I and the estrogen receptor may provide a physiological mechanism of selective antagonism of gene regulation by estrogens.
- Published
- 1994
31. Expression of the trans-active factors that stimulate insulin control element-mediated activity appear to precede insulin gene transcription.
- Author
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Robinson GL, Peshavaria M, Henderson E, Shieh SY, Tsai MJ, Teitelman G, and Stein R
- Subjects
- Animals, Base Sequence, Binding Sites, Cell Line, DNA-Binding Proteins metabolism, Glucagon biosynthesis, HeLa Cells, Humans, Islets of Langerhans cytology, Mice, Molecular Sequence Data, Neurons cytology, Neurons metabolism, Oligodeoxyribonucleotides, Rats, Transfection, DNA metabolism, Enhancer Elements, Genetic, Gene Expression, Insulin biosynthesis, Insulin genetics, Islets of Langerhans metabolism, Promoter Regions, Genetic, Transcription, Genetic
- Abstract
Cell type-specific expression of the major differentiated products of alpha (glucagon) and beta (insulin) cells are regulated by sequences found within their 5'-flanking region. Specific transcription of the insulin gene appears to be principally controlled by a single cis-acting DNA element, termed the insulin control element (ICE). The ICE activator acts in combination with other positive regulatory factors that interact within this region to generate the correct, cell type-specific expression. In the present study, we show that the ICE activator is not only present but is functionally active in the islet glucagon-producing alpha cell line, alpha TC6. Analysis of the expression of various transfected insulin enhancer expression plasmids demonstrated that the insulin enhancer is active in alpha TC6 cells, although at a lower level than in beta cells. The reduced transcription from these constructs appears to be a consequence of the lack of other essential positive regulator(s). The alpha TC6 cells were also shown to display neuronal-like properties. Since islet cells appear to evolve from an alpha-like precursor cell that transiently expresses neuronal cell markers, these results would indicate that the ICE activator factor is induced before transcription of the insulin gene in the developing islet.
- Published
- 1994
32. Multiple mechanisms of chicken ovalbumin upstream promoter transcription factor-dependent repression of transactivation by the vitamin D, thyroid hormone, and retinoic acid receptors.
- Author
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Cooney AJ, Leng X, Tsai SY, O'Malley BW, and Tsai MJ
- Subjects
- Animals, Base Sequence, Binding, Competitive, COUP Transcription Factor I, Calcitriol metabolism, Carrier Proteins physiology, Chickens, DNA metabolism, Molecular Sequence Data, Receptors, Calcitriol, Receptors, Retinoic Acid, Receptors, Steroid physiology, Receptors, Thyroid Hormone physiology, DNA-Binding Proteins metabolism, Ovalbumin genetics, Promoter Regions, Genetic, Receptors, Cell Surface physiology, Transcription Factors metabolism, Transcriptional Activation
- Abstract
The chicken ovalbumin upstream promoter transcription factor (COUP-TF) is a member of the steroid/thyroid hormone receptor superfamily about which little is known of its functional role in the cell. However, it is able to repress hormonal induction of target genes by vitamin D3 receptor (VDR), thyroid hormone receptor (TR), and retinoic acid receptor (RAR). We have shown previously that COUP-TF can bind a wide variety of A/GGGTCA repeats. This promiscuous recognition of response elements correlates with the ability of COUP-TF I to repress other receptors that bind to A/GGGTCA repeats with different spacings between the half-sites. Here we show that repression of transactivation by these receptors is a general phenomenon for the COUP-TF subfamily, as inhibition is also observed with COUP-TF II. This repression is also dose-dependent on COUP-TF. Inhibition of VDR, TR, and RAR activities also occurs through natural physiological response elements found in the osteocalcin, myosin heavy chain, and beta RAR promoters, respectively. In search of the mechanisms of repression by COUP-TF we show that it does not involve the formation of detectable functionally inactive heterodimers between COUP-TF and VDR, TR, and RAR. Instead, we show that the mechanism of repression could occur at three different levels: (a) active silencing of transcription and dual competition for; (b) occupancy of DNA binding sites; and (c), heterodimer formation with retinoid X receptor, the coregulator of VDR, TR, and RAR. The silencing activity was localized to the putative ligand binding domain of COUP-TF. We postulate that COUP-TF may play a master role in regulating transactivation by VDR, TR, and RAR.
- Published
- 1993
33. Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation.
- Author
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Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ, and O'Malley BW
- Subjects
- Base Sequence, Carrier Proteins ultrastructure, Heat-Shock Proteins metabolism, Humans, In Vitro Techniques, Macromolecular Substances, Molecular Sequence Data, Oligodeoxyribonucleotides chemistry, Protein Conformation drug effects, Receptors, Progesterone drug effects, Receptors, Progesterone ultrastructure, Receptors, Retinoic Acid, Recombinant Proteins metabolism, Recombinant Proteins ultrastructure, Carrier Proteins metabolism, Progesterone pharmacology, Progestins antagonists & inhibitors, Receptors, Progesterone metabolism, Tretinoin pharmacology
- Abstract
Antihormones are potent antagonists of hormone action in vivo, but the mechanism underlying this antagonism is not understood. Several steroid hormones transform (activate) their receptors from a cytosolic, non-DNA binding 8 S sedimentation form to a nuclear, DNA binding 4 S form. Transformation is accompanied by the loss of associated heat shock proteins. We have previously demonstrated that an additional hormone-dependent step, separate from heat shock protein removal, is required for activation of the human progesterone receptor. We have devised an assay in which the human progesterone receptor translated in vitro binds to its specific response element in a hormone-dependent manner. As assessed by limited proteolytic digestion, hormone treatment of the nascent receptor induces a dramatic conformational change within the protein. The conformational change occurs in the absence of DNA and renders the entire ligand binding domain resistant to digestion by proteases. A number of antiprogestins, including RU486, induce an equally dramatic, but distinct, structural alteration of the ligand binding domain. The distinction centers upon the final 30 to 40 amino acids at the carboxyl terminus. The conformational change can be induced by ligand prior to dissociation of the 8 S complex and is not induced by heat shock protein removal in the absence of hormone. Remarkably, virtually identical hormone-induced conformational changes were detected following proteolytic analysis of in vitro translated retinoic acid receptors. Our data indicate that the sole necessary event in the activation of steroid receptors is conformational modification by the ligand. Furthermore, we conclude that transcriptional inactivation of steroid receptors by antihormones involves the induction of an inappropriate structural conformation at the extreme carboxyl terminus of the ligand binding domain.
- Published
- 1992
34. Members of the steroid hormone receptor superfamily interact with TFIIB (S300-II).
- Author
-
Ing NH, Beekman JM, Tsai SY, Tsai MJ, and O'Malley BW
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Nucleus metabolism, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Glutathione Transferase genetics, Glutathione Transferase metabolism, HeLa Cells, Humans, Models, Structural, Molecular Sequence Data, Oligodeoxyribonucleotides, Polymerase Chain Reaction, Receptors, Steroid isolation & purification, Recombinant Fusion Proteins metabolism, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Restriction Mapping, Transcription Factor TFIIB, Transcription Factors isolation & purification, Transcription, Genetic, Transcriptional Activation, Receptors, Estrogen metabolism, Receptors, Steroid metabolism, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
The S300-II factor was discovered as an activator of ovalbumin gene transcription with the chicken ovalbumin upstream promoter-transcription factor (COUP-TF, Sagami, I., Tsai, S. Y., Wang, H., Tsai, M.-J., and O'Malley, B. W. (1986) Mol. Cell. Biol. 6, 4259-4267). Although S300-II does not bind DNA selectively, it stabilizes the binding of COUP-TF to its ciselement (Tsai, S. Y., Sagami, I., Wang, H., Tsai, M.-J., and O'Malley, B. W. (1987) Cell 50, 701-709). Purified S300-II is also required for steroid receptor-activated transcription. Cloning and sequencing of S300-II showed that it is the general transcription factor TFIIB. Specific protein-protein interactions between recombinant S300-II/TFIIB and three members of the steroid hormone receptor superfamily, COUP-TF, estrogen receptor, and progesterone receptor, indicate that S300-II/TFIIB is one of the targets of these transactivators. Interestingly, a truncated estrogen receptor construct containing only the N-terminal transcription activation function 1 did not interact with S300-II/TFIIB in our assay, revealing that individual transcription activation functions of a single steroid hormone receptor may contact different targets. Demonstration of a direct association of S300-II/TFIIB and COUP-TF, independent of additional "adaptor" proteins, suggests that members of the steroid hromone receptor superfamily facilitate the transcription of activated genes at least in part via protein-protein interactions with the general transcription factor TFIIB.
- Published
- 1992
35. Hormone activation of baculovirus expressed progesterone receptors.
- Author
-
Elliston JF, Beekman JM, Tsai SY, O'Malley BW, and Tsai MJ
- Subjects
- Animals, Cell Line, Chickens, Cloning, Molecular, Genetic Vectors, Humans, Insecta, Kinetics, Progesterone metabolism, Reading Frames, Receptors, Progesterone genetics, Transfection, Baculoviridae genetics, Progesterone pharmacology, Receptors, Progesterone metabolism, Transcription, Genetic drug effects
- Abstract
Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression.
- Published
- 1992
36. Identification of cis- and trans-acting factors regulating the expression of the human insulin receptor gene.
- Author
-
Lee JK, Tam JW, Tsai MJ, and Tsai SY
- Subjects
- Animals, Base Sequence, Carcinoma, Hepatocellular metabolism, Cells, Cultured, Chloramphenicol O-Acetyltransferase genetics, Chloramphenicol O-Acetyltransferase metabolism, DNA Fingerprinting, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Fibroblasts metabolism, Humans, Liver Neoplasms metabolism, Methylation, Molecular Sequence Data, Plasmids, Promoter Regions, Genetic, Rats, Receptor, Insulin metabolism, Transcription, Genetic, Transfection, Tumor Cells, Cultured, Gene Expression Regulation, Receptor, Insulin genetics, Trans-Activators metabolism
- Abstract
The functional organization of the human insulin receptor (hIR) promoter was analyzed by deletion mutagenesis and protein-DNA interaction studies. A series of deletion mutants was expressed transiently in two human hepatocytes, HepG2 and PLC. The results revealed that the promoter region between -692 and -345 is essential for efficient transcription of the hIR gene. Multiple trans-acting factors were identified by band shift and footprinting analyses. Sp1 binds to a cluster of GC boxes and two GGGAGG hexamers locating at -637 to -594. Adjacent to GC boxes, there are two regions, from -550 to -530 and from -522 to -503, which bind to two novel factors, IRNF-I and IRNF-II. These two factors are distributed differentially in different cell lines. Linker scanning mutations on GC, GA boxes, or the IRNF-I binding site significantly decreased the transcriptional activity, indicating that IRNF-I and Sp1 are important for hIR promoter activity. In addition, we demonstrated that glucocorticoid-dependent transcriptional induction of hIR mRNA in vivo is conferred by a glucocorticoid response element in the hIR promoter. Taken together, these results imply that transcription of the human insulin receptor gene is regulated by multiple protein-DNA interactions occurring within the defined promoter region.
- Published
- 1992
37. Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene.
- Author
-
Shieh SY and Tsai MJ
- Subjects
- Animals, Base Sequence, Binding Sites, Binding, Competitive, Cell Line, Cricetinae, DNA, HeLa Cells, Islets of Langerhans cytology, Islets of Langerhans metabolism, Molecular Sequence Data, Mutation, Organ Specificity genetics, Rats, Tumor Cells, Cultured, DNA-Binding Proteins metabolism, Enhancer Elements, Genetic, Insulin genetics
- Abstract
Pancreatic beta-cell-specific expression of the insulin gene is mediated, at least in part, by an enhancer element termed the rat insulin promoter element 3 (RIPES) found within the rat insulin II gene between positions -126 and -86. Here we identify three distinct factors interacting with RIPE3, namely 3a1, 3a2, and 3b1, which bind to the sequences between -100 to -90, -108 to -99, and -115 to -107, respectively. Factors 3a1 and 3b1 are beta-cell specific whereas 3a2 is ubiquitously distributed. The 3a1 site contains the consensus binding sequence (CANNTG) for a group of DNA-binding proteins called basic-helix-loop-helix proteins. We showed in this study that the 3a1 binding activity contains E12/E47, a member of the basic-helix-loop-helix protein family, or an E12/E47-like protein. Sequence comparison of the 3a2 and 3b1 binding sites suggest that they are unique and may bind to novel transcription factors. Mutation analysis of each individual binding site in transient expression experiments indicates that all of the three binding sites contribute to the enhancer activity of the RIPE3 in beta-cells. Mutation in any one of the three binding sites not only disrupts binding of the corresponding factor but decreases RIPE3 enhancer activity by 4-7-fold. The results suggest that interactions among the 3a1, 3a2, and 3b1 factors are required for maximum enhancer activity of the RIPE3 in insulin-producing cells.
- Published
- 1991
38. Studies on the mechanism of functional cooperativity between progesterone and estrogen receptors.
- Author
-
Bradshaw MS, Tsai SY, Leng XH, Dobson AD, Conneely OM, O'Malley BW, and Tsai MJ
- Subjects
- Base Sequence, Binding Sites, Blotting, Western, Cell Line, DNA, Molecular Sequence Data, Transfection, Progesterone metabolism, Receptors, Estrogen metabolism
- Abstract
Steroid response elements (SREs) cooperate with many different cis-acting elements including NF-1 sites, CACCC boxes, and other SREs to induce target gene expression (Schule, R., Muller, M., Otsuka-Murakami, H., and Renkawitz, R. (1988) Nature 332, 87-90; Strahle, U., Schmid, W., and Schutz, G. (1988) EMBO J. 7, 3389-3395). Induction of gene expression can be additive or synergistic with respect to the level of activation by either transactivators. Two mechanisms have been proposed for how synergism occurs: 1) cooperative binding of transcriptional activators to DNA or 2) simultaneous interaction of individually bound activators with a common target protein. We have shown previously that cooperative binding of receptors is important for synergism between two progesterone response elements (PREs). Here we showed that an estrogen response element (ERE) and a PRE can also functionally cooperate and this synergism between an ERE and a PRE is not contributed by cooperative DNA binding. Furthermore, we have demonstrated that the activation domains of the progesterone receptor (PR) (C1Act) are required for synergism between two PREs and sufficient for confirming cooperative binding. However these two activation domains of PR are not sufficient for synergism between an ERE and a PRE. Additional regions within the NH2-terminal and COOH-terminal domains are also required for synergistic interaction between two heterologous SREs.
- Published
- 1991
39. Definition of 5' and 3' structural boundaries of the chromatin domain containing the ovalbumin multigene family.
- Author
-
Lawson GM, Knoll BJ, March CJ, Woo SL, Tsai MJ, and O'Malley BW
- Subjects
- Animals, Base Sequence, Chickens, DNA Restriction Enzymes metabolism, Deoxyribonuclease I, Deoxyribonucleases metabolism, Endonucleases metabolism, Female, Kinetics, Repetitive Sequences, Nucleic Acid, Transcription, Genetic, Chromatin metabolism, Cloning, Molecular, Genes, Ovalbumin genetics, Oviducts metabolism
- Abstract
Hen oviduct nuclei were subjected to pancreatic DNase I treatment under conditions known to preferentially degrade transcriptionally active genes (Weintraub, H., and Groudine, M. (1976) Science (Wash. D. C.) 93, 848-856). The ovalbumin gene, its structurally related genes, X and Y, and the spacer and flanking DNA were all found to exist in a DNase I-sensitive configuration. The DNase I-sensitive region was extended more than 20 kilobases beyond the 5' end of the X gene and approximately an equal distance beyond the 3' end of the ovalbumin gene before it became DNase I-resistant. The transition from a DNase I-sensitive to a -resistant conformation in oviduct chromatin occurred in a gradient fashion with 10 kilobases of DNA. Thus, ovalbumin and its related genes, X and Y, exist in a 100-kilobase DNase-sensitive domain in the oviduct tissue. In contrast, the entire domain was resistant to DNase I in spleen, liver, and erythrocyte nuclei. When the transcription of ovalbumin, X, and Y genes was eliminated by the withdrawal of hormone from estrogen-stimulated chicks, the entire domain remained in a DNase I-sensitive configuration. We conclude that DNase I-sensitive domains may provide the structural capability for gene expression and appear to be a result of the differentiation process since they are cell-specific and contain potentially expressible genes of that cell type. Repetitive sequences within this domain have been mapped and the possible relationship of these repetitive sequences to the DNase I-sensitive structure is discussed.
- Published
- 1982
40. Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene.
- Author
-
Scott MJ, Huckaby CS, Kato I, Kohr WJ, Laskowski M Jr, Tsai MJ, and O'Malley BW
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chickens, Cloning, Molecular, Cosmids, DNA isolation & purification, Exons, Introns, Nucleic Acid Hybridization, Egg Proteins genetics, Egg Proteins, Dietary, Genes, Genetic Linkage, Ovomucin genetics, Protease Inhibitors genetics
- Abstract
We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.
- Published
- 1987
41. Transcription factors from oviduct and HeLa cells are similar.
- Author
-
Tsai SY, Tsai MJ, Kops LE, Minghetti PP, and O'Malley BW
- Subjects
- Animals, Carrier Proteins metabolism, Chickens, DNA-Binding Proteins, Female, HeLa Cells metabolism, Humans, Ovalbumin genetics, Species Specificity, Genes, Oviducts metabolism, Transcription Factors
- Abstract
Total HeLa cell extract was separated into multiple components using first DEAE-Sephadex and then phosphocellulose column chromatography. Four major fractions, DE175, DE500, P100, and P1000, from HeLa cells are found to be essential for accurate and efficient transcription of cloned ovalbumin DNA fragments in the reconstituted system. DE175 serves as the source for RNA polymerase II and DE500 minimizes the synthesis of small molecular size RNA. P100 enhances the levels of specific transcription, while P1000 is absolutely required for correct initiation. Chick oviduct crude extract was also fractionated into multiple components according to the same procedure. Similar efficiency of specific initiation could be obtained when an individual fraction (DE175, DE500, P100, or P1000) from oviduct cells was exchanged for a fraction from HeLa cells. These results indicate that chick oviduct tissue also contains general transcription factors like that of HeLa cells and these factors can be fractionated according to the same procedure. In this fractionation scheme, we were able to separate the bulk of RNase activity into the P350 fraction which was not required for initiation of ovalbumin RNA transcription. Thus, this reconstituted system is suitable for studying in vitro transcription in a homologous system derived from tissue extracts, even though a substantial amount of cellular ribonuclease may be associated with it.
- Published
- 1981
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