Abstract
To study the regulation of expression of the chicken ovalbumin gene by steroid hormones, the entire ovalbumin gene and its flanking sequences were cloned together with the bacterial gene for xanthine-guanine phosphoribosyltransferase in plasmid pBR322. This recombinant plasmid was linearized and used to transform an estrogen-responsive breast carcinoma cell line (MCF-7) which was shown to possess estrogen receptors and to be estrogen responsive. Transformants were selected by their ability to grow in a medium containing mycophenolic acid and xanthine. The entire ovalbumin gene was integrated into high molecular weight DNA within all transformants analyzed and it retained its original sequence organization. Ovalbumin mRNA and protein were identified from these transformant cells and they were found to be indistinguishable from the authentic counterparts. An 8- to 10-fold increase in the amount of ovalbumin mRNA was observed to be present in cells cultured in 10(-8)M estradiol. We also constructed a hybrid gene containing the 5'-flanking sequence and the first exon of the ovalbumin gene which was linked to the xanthine-guanine phosphoribosyltransferase gene such that expression of this bacterial gene would be promoted and regulated by the chicken sequences. After introduction of this hybrid gene into MCF-7 cells, we observed that the survival of the transformed cells in our selection medium was highly dependent on the presence of estradiol. Our results indicated that the chicken ovalbumin sequence was expressed properly and was regulated to some extent by estradiol in this heterologous system.
Highlights
Together with the bacterial gene for xanthine-guanine 11).an attractivemodel of steroid hormone actionmay phosphoribosyltransferase in plasmid pBR322
Min gene which was linked to the xanthine-guanine reported that the expression of a fusion gene containing the phosphoribosyltransferase gene such that expression 5”flanking sequences of the chicken lysozyme gene and the of this bacterialgene would bepromoted and regulated SV40 T antigen gene could be regulated by steroid hormones by the chicken sequences
In order to study the steroid-controlledexpression of this gene, we have transferred the entire ovalbumin gene together with its flanking sequences intohuman estrogen-responsive breast Steroid hormones play an important role in regulating the carcinoma (MCF-7) cells which possess significant levels of expression of specific genes in eucaryotes
Summary
12-kb fragment of chicken DNA containing the entire oval- entire chicken DNA insertandabout 0.2 kb of plasmid bumin gene together with 3.5 and 0.5 kb of sequences flanking sequence (Fig. 1).SalI cleaves the plasmid sequence oncebut its 5' and 3' termini was isolated by partial HindIII digestion does not cut thechicken DNA and the XGPRTgenes. PSV2-gpt clones, our samples were analyzed for the presence of chicken was digested with BarnHI and PuuII so that a 2.1-kb DNA ovalbumin by the solid phase immunoassay described under fragment containing that bacterial XGPRT gene and the "Materials and Methods." The sensitivity of this assay was SV40 sequences required for its expression could be isolated within the picogram range. The entire chicken DNA insert can be excisedout of pOV12- more, chicken ovalbumin was detected in the growth gpt by HindIII and can be cleaved into three fragments of media of each of the clones (Fig.2).suggesting thatthe. This enzyme digests pOV12-gpt DNA into many small frag- semiquantitative, it allowed a rapid screening of the transments and one large fragment of12.2 kb consisting of the formed clones to identify those whichexpressed both the selection marker and the heterologous chicken gene
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