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Isolation and Structure of the Gene for the Progesterone-Inducible Protein Uteroglobin

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Isolation and Structure of the Gene for the Progesterone-Inducible Protein Uteroglobin Proc. Nati Acad. Sci. USA Vol. 79, pp. 4853-4857, August 1982 Biochemistry Isolation and structure of the gene for the progesterone-inducible protein uteroglobin (DNA sequence/gene structure/steroid hormone action/uteroglobin cDNA/blastokinin gene) C. MENNE*, G. SUSKE, J. ARNEMANN, M. WENZ, A. C. B. CATO, AND M. BEATOt Institut fir Physiologische Chemie I, Philipps-Universitdt, Deutschhausstrasse 1-2, D-3550 Marburg, Federal Republic of Germany Communicated by Ervin Bfinning, April 29, 1982 ABSTRACT Uteroglobin is a small steroid-binding protein cific activity 50 Ci/mmol; 1 Ci = 3.7 X 10'° becquerels) was that is differentially regulated by steroid hormones in several tis- used as the radioactive label. Afteralkaline hydrolysis the cDNA sues of the rabbit. In endometrium, the levels of uteroglobin was isolated and a second strand was synthesized with reverse mRNA increase after progesterone administration due to an en- transcriptase and nonradioactive deoxyribonucleotide triphos- hanced rate oftranscription ofthe uteroglobin gene. As a prereq- phates at 1 mM each. The double-stranded cDNA was isolated uisite for understanding the molecular mechanisms that modulate and the 3' ends were elongated with short tracts of dCMP by uteroglobin gene expression, we have isolated and characterized incubation with [a-32P]dCTP and terminal deoxyribonucleo- the uteroglobin gene. We first synthesized, cloned, and sequenced tidyl transferase from calf thymus (4). Approximately 15-30 a uteroglobin cDNA that was used to screen a rabbit gene library dCMP residues were added and to show that the uteroglobin gene is not reiterated in the rabbit to each end. The tailed cDNA was genome. We obtained three recombinant phages containing uter- centrifuged through a neutral sucrose density gradient, and the oglobin gene sequences and covering 35 kilobases of the rabbit material that sedimented at around 7 S was pooled for further genome. The uteroglobin gene is 3 kilobases long and is composed experiments. of three short exons separated by a long and a short intron. The Circular plasmid pBR322 was linearized with Pst I and elon- complete coding sequence, the short intron, part of the large in- gated with terminal transferase and [3H]dGTP until 10-20 tron, and the flanking sequences have been subjected to sequence dGMP residues were added to each end. Twenty nanograms analysis. The salientfeatures ofthe nucleotide sequence, including ofelongated cDNA was annealed with 250 ng oftailed pBR322 the absence of a canonical "T-A-T-A box," are discussed. A pos- in 0.1 M NaCVl10 mM Tris HCI, pH 7.5/1 mM Na2EDTA at sible relationship is considered between the exon-intron structure 63°C for 10 min followed by 3 hr at 43°C. The samples were of the gene and the known structure and function of uteroglobin. slowly cooled to room temperature and used to transform com- petent Escherichia coli K-12 X1776 cells (5). Transformants The induction ofuteroglobin synthesis by steroid hormones in were selected by antibiotic sensitivity and in situ colony hy- the rabbit offers a suitable system for studying the modulation bridization by using [32P]cDNA as probe (3, 6). Clones that ap- ofgene expression in mammals (for a review, see ref. 1). In the peared positive after colony hybridization were grown in a small endometrium progesterone is the main steroid inducer, whereas volume and plasmid DNA was isolated by a rapid lysate pro- in the oviduct estrogens are effective inducers, and in the lung cedure (7). After digestion with Pst I the size ofthe inserts was glucocorticoids cause a moderate induction ofuteroglobin syn- analyzed on polyacrylamide gels. Preparative isolation ofplas- thesis. We have shown in a cell-free transcription system that mid DNA was performed according to published procedures the accumulation of uteroglobin mRNA in endometrial cells (8). after administration of ovarian hormones is mainly due to an Screening of the Rabbit Gene Library. The rabbit gene li- increased rate of transcription of the uteroglobin gene (2). brary (9) was screened by the in situ plaque-hybridization tech- A more detailed understanding ofthe molecular mechanisms nique (10). Sixteen square plates (22 x 22 cm), each containing underlying the hormonal control ofuteroglobin gene expression 7 X 10' phage plaques, were screened with 0.7% agarose as top depends on our knowledge ofthe structural organization ofthe layer (9) and clone pcUG-G8 labeled with 32P by nick-transla- gene and its putative regulatory sequences. An elucidation of tion (11) as hybridization probe. Positive plaques were purified the interaction between the hormone receptor and the regu- by replating three times and the phage DNA was isolated (9). latory sequences in chromatin may eventually provide answers Analysis of Uteroglobin Gene Sequences in Genomic DNA to long-standing questions on the mechanism ofaction ofsteroid and Recombinant Phages. High molecular weight DNA was hormones. prepared from different tissues offemale rabbits (12). Genomic In this paper we report the synthesis, cloning, and sequence DNA and DNA from recombinant phages were digested with analysis ofa uteroglobin cDNA and its use for the titration and the appropriate restriction enzymes, and the restriction frag- isolation of the uteroglobin gene. The primary structure of the ments were separated on 1% agarose gels. After transfer ofthe gene-coding sequences and the flanking regions is reported and DNA fragments to nitrocellulose filters by a modification ofthe discussed. Southern technique (13, 14), the filters were hybridized to either pcUG-G8 or uteroglobin mRNA labeled with 32p (11, MATERIALS AND METHODS 15). DNA Sequence Analysis. The sequence strategy is shown in Synthesis and Cloning of Double-Stranded Uteroglobin the corresponding figures. In general, the 5' ends of purified cDNA. The first cDNA strand was synthesized as previously restriction fragments were labeled with [y-32P]ATP and phage described by using partially purified preuteroglobin mRNA as T4 polynucleotide kinase (16). After secondary restriction the template (3). Actinomycin D was omitted and [3H]dCTP (spe- Abbreviations: bp, base pair(s); kb, kilobase(s). The publication costs ofthis article were defrayed in part by page charge * Present address: Institut fur Biologie, Universitat Tubingen, D-7400 payment. This article must therefore be hereby marked "advertise- Tubingen, Federal Republic of Germany. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 4853 Downloaded by guest on September 29, 2021 4854 Biochemistry: Menne et al. Proc. Nad Acad. Sci. USA 79 (1982) radioactive fragments were isolated on polyacrylamide gels and dons for the amino acids 13-70 of mature uteroglobin (17) and subjected to sequence analysis (16). In a few cases the 3' ends the 3' untranslated region of the mRNA. The previously pub- were labeled with the Klenow fragment E. coli DNA polymer- lished amino acid sequence (17) is confirmed by the nucleotide ase I and the corresponding [a-32P]dNTP. The BamHI fragment sequence ofthe cDNA, with the exception ofposition 61 where that contains the 5' end ofthe mRNA and the putative promoter we reported Gln and the nucleotide sequence indicates Glu sequences was labeled both at the 5' ends and at the 3' ends (17, 18). (in two separate experiments), and the two strands were sep- Determination of Uteroglobin Gene Copy Number. To de- arated in a5% polyacrylamide gel before sequence analysis (16). termine the number ofcopies ofthe uteroglobin gene per hap- Containments. Initial work with cDNA cloning and the loid rabbit genome, we hybridized trace amounts ofpcUG-G8 screening of the rabbit gene library were carried out under L3 insert, labeled with 32P, to rabbit genomic DNA and to salmon level of physical containment and B2 level of biological con- sperm DNA to which different amounts ofpcUG-G8 had been tainment as specified by the "Richtlinien zum Schutz vor Ge- added (19). In this type of experiment a standard straight line fahren durch in vitro rekombinierte Nukleinsiuren" ofthe Bun- is obtained relating the rate ofcDNA hybridization to the num- desministerium fur Forschung und Technologie ofthe Federal ber ofgene copies added (Fig. 2). From these results it is clear Republic of Germany. that the uteroglobin gene belongs to the class of unique genes and is only present in one to three copies per haploid genome. Experiments performed under less stringent hybridization con- RESULTS ditions gave no indication for the existence in the rabbit genome Synthesis and Cloning of a Uteroglobin cDNA Probe. Uter- of sequences related to the uteroglobin gene (data not shown). oglobin is composed of two identical subunits-each 70 amino Results similar to those depicted in Fig. 2 were obtained with acids long-that are synthesized as a precursor containing a sig- liver DNA and with DNA prepared from the endometrium of nal peptide of 21 amino acids at the NH2 terminus (1). Pre- control estrous rabbits, thus demonstrating that gene amplifi- uteroglobin mRNA prepared from membrane-bound endo- cation is not responsible for the increased levels ofuteroglobin metrial polysomes ofpseudopregnant rabbits is >70% pure (3). mRNA observed in endometrial cells of pseudopregnant ani- mals (20). This material was used for the synthesis of double-stranded Isolation and Structural Analysis of the Uteroglobin Gene. cDNA that was introduced into the Pst I site ofpBR322 by the For the isolation ofthe uteroglobin gene(s) a rabbit gene library oligo(G)oligo(C) tailing procedure and cloned in E. coli (see was screened with labeled pcUG-G8 (9, 10). Of one million Materials and Methods). Thirty clones gave positive hybridiza- phage plaques tested, three independent isolates were found tion signals with uteroglobin cDNA as probe. After digestion after three plaque purification steps. The inserts in these three with Pst I, 10 of them showed inserts of around 400 base pairs recombinant Charon 4A phages overlap partially and cover a (bp) and a similar restriction map with Alu I, Hpa II, Hinfl, and region extending over 35 kilobases (kb) of genomic DNA that Hae III.
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