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The 5'-flanking region of the rat insulin II gene (-448 to +50) is sufficient for tissue-specific expression. To further determine the tissue-specific cis-acting element(s), important sequences defined by linker-scanning mutagenesis were placed upstream of a heterologous promoter and transfected into insulin-producing and -nonproducing cells. Rat insulin promoter element 3 (RIPE3), which spans from -125 to -86, was shown to confer beta-cell-specific expression in either orientation. However, two subregions of RIPE3, RIPE3a and RIPE3b (defined by linker-scanning mutations), displayed only marginal activities. These results suggest that the two subregions cooperate to confer tissue specificity, presumably via their cognate binding factors.

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.

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.

The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [(3)H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [(3)H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.

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.

A study was made of the function of the intervening sequences in the ovalbumin gene, Radioactively labeled DNA probes for the intervening sequences were prepared and RNA's were isolated from whole cells, nuclei, and polysomes of estrogen-stimulated chick oviducts. The concentrations of messenger RNA (mRNA) transcripts from ovalbumin structural sequences (mRNAov) and transcripts corresponding to intervening sequences were then estimated by hybridization to cloned DNA probes. Oviduct tissue contains approximately 58,000 molecules of mRNAov sequences per tubular gland cell and most of these sequences are present in the cytoplasm. In contrast, there are 200 to 300 molecules of RNA per cell which are transcribed from the intervening sequences of the natural ovalbumin gene and almost all of these are found in the nucleus. The difference in distribution of structural and intervening sequence transcripts suggests that, unlike mature mRNA, the intervening sequences are not preferentially transported to cytoplasmic polysomes.

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.

The [3H]estradiol exchange assay was used to characterize the nuclear estrogen receptor from the chick oviduct. After diethylstilibestrol (DES) treatment (14 days), the oviduct nuclei contained estrogen receptors that manifested high affinity (Kd = 2 X 10(-9)M) and low capacity binding (4000 to 5000 sites/cell) for estradiol. DES and estradiol competed significantly for [3H]estradiol binding, while testosterone and progesterone were ineffective. These binding sites were found in the oviduct and liver but not in the spleen, kidney, or muscle. Following salt extraction from nuclei, the receptor had a sedimentation coefficient of 4 S when analyzed by centrifugation in high and low salt sucrose density gradients. The [3H]estradiol exchange assay was used to examine the relationships between nuclear-bound receptor and RNA polymerase initiation sites on oviduct chromatin. Within 20 min after a single injection of 2.5 mg of DES to withdrawn chicks, a maximum number of estradiol receptor-binding sites was detected in oviduct nuclei. Within 30 min after DES injection, the total number of RNA initiation sites also increased, reaching 100% of control values. Daily injections of DES to unstimulated chicks resulted in a gradual increase in the nuclear content of estradiol receptor, which reached a maximum at 6 days and thereafter declined gradually up to 18 days of hormone treatment. A maximum number of initiation sites for RNA synthesis was also attained at 4 to 6 days of DES treatment and thereafter declined. When DES was withdrawn after 14 to 18 days of hormone stimulation, the numbers of nuclear estradiol receptor sites and initiation sites for RNA synthesis both declined gradually, reaching half-maximal values in 3 days and approached control values after 7 to 8 days of withdrawal. The increase in the concentration of nuclear estradiol receptor sites and the number of initiation sites for RNA synthesis also showed a close correlation with the dosage of DES administration. Both attained maximum levels at 1.25 mg of DES with a half-maximal value of 0.5 mg. The close correlation between the concentration of nuclear-bound estradiol receptors and the number of initiation sites for RNA synthesis in vivo is at present only a temporal correlation but does raise the possibility that gene transcription in chick oviduct may depend upon the amount of estradiol receptor bound to the target cell nuclei.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.

We have identified the 5' termini of ovomucoid RNA transcripts isolated from estrogen stimulated chick oviduct by S1-nuclease mapping. Most of the ovomucoid RNA molecules (88-89%) are initiated at a predominant region which is positioned at about 30 basepairs downstream from a "TATATAT" sequence. However, there is at least one additional initiation region which accounts for 9% of the ovomucoid RNA transcripts. It is located at approximately 80 basepairs further upstream (5') from the major initiation region. DNA sequence data reveal that an AT-rich sequence resembling the "TATA" box is also present 30 nucleotides before this minor region. Our finding provides additional insight into the molecular mechanism involved in the transcription initiation of eucaryotic genes, and indicates that in vivo synthesis of mRNA may not require an absolute site for initiation of transcription.

RNA was transcribed from chromatin isolated from chick oviduct and spleen by using RNA polymerase from Escherichia coli. RNA was also transcribed from whole chick DNA using E. coli RNA polymerase. DNA complementary to ovalbumin messenger RNA (cDNAov) was then used as a hybridization probe to estimate the concentration of ovalbumin messenger RNA sequences (mRNAov) in these in vitro transcripts. Although chromatin from unstimulated chick oviduct was capable of substantial RNA synthesis, no detectable mRNAov sequences could be found in the transcript. Likewise, mRNAov sequences could not be found in RNA synthesized from spleen chromatin using E. coli RNA polymerase. However, chromatin from estrogen-stimulated chick oviducts was capable of supporting synthesis of ovalbumin mRNA. We estimate that approximately 0.01% of the RNA synthesized from estrogen-stimulated chromatin was mRNAov sequences. When RNA synthesized from chick DNA was tested with the cDNAov probe, mRNAov sequences could be detected in a concentration of approximately 10% that found in the RNA transcript from estrogen-stimulated chromatin. This was as expected if the ovalbumin gene is considered to be in the "open or derepressed" region of the estrogen-stimulated oviduct chromatin. Chromatin isolated from chicks withdrawn from hormone for 12 days was only capable of supporting mRNAov synthesis in vitro at a level of 5 to 10% of that observed in chromatin prepared from estrogen-stimulated chicks, thus indicating the requirement for estrogen to maintain the ovalbumin gene in the available or "open" state in the majority of oviduct cells. These data militate against post-transcriptional control as the primary mechanism of steroid hormone regulation of specific mRNA synthesis in the chick oviduct system, and favor primary gene derepression as the most likely mechanism for estrogen induction of ovalbumin synthesis.

We present data which map the 5' and 3' ends of transcripts of the natural ovalbumin gene. First, hybridization of radiolabeled 5' and 3' fragments of the gene to oviduct nuclear RNA which had been electrophoresed in agarose gels and transferred to DBM paper localized the 5' and 3' ends of precursor molecules and demonstrated that the largest ovalbumin RNA molecules detectable by this method (approximately 7.8 kb) were similar in size to the ovalbumin natural gene. Second, hybridization of end-labeled probes to oviduct nuclear RNA followed by digestion with S1 nuclease and analysis on polyacrylamide gels mapped more precisely the 5' and 3' ends of the precursor molecules, and these termini were coincident with the beginning and end of the structural sequence of the natural gene. These results suggest that the 7.8 kb precursor is likely to be the primary transcript of the ovalbumin gene.

Two transcription factors, COUP and S300-II, were isolated and partially purified from HeLa cell nuclear extracts. Both factors are required for the efficient transcription in vitro of the ovalbumin gene but not the simian virus 40 early genes. COUP factor binds to the chicken ovalbumin upstream promoter (COUP) sequence which lies between -70 to -90 base pairs upstream from the cap site. A series of competition experiments with a band-shifting assay was carried out to determine the relative affinity of COUP box transcription factor for various promoters. We found that a promoter DNA fragment isolated from the ovalbumin gene competes better than those isolated from the ovomucoid, Y, and alpha-genes. In contrast, the the simian virus 40 early genes, the beta-globin gene, and the adenosine deaminase gene promoters do not compete well in this assay. The molecular weight of the COUP factor was estimated by S-300 column chromatography, glycerol gradient centrifugation to be 90,000. However, two bands were observed in sodium dodecyl sulfate gel electrophoresis of cross-linked COUP factor to a 32P-labeled oligonucleotide containing the COUP sequence. The protein moieties of the major and minor bands were estimated to be 85,000 to 90,000 and 40,000 to 45,000, respectively. The S300-II factor with an apparent molecular weight of 45,000 in an S-300 column is required for function in an in vitro reconstituted transcription system. In contrast to the COUP factor, the S300-II factor does not have apparent specificity for binding to the ovalbumin gene promoter. The S300-II factor may function by interacting with RNA polymerase or other DNA-binding transcription factors.

To define the functional domains of the chicken progesterone receptor (CPR) and to establish the origin of the receptor A and B proteins, we have cloned and sequenced the complete cDNA encoding the CPR. The receptor A and B proteins arise by alternate initiation of translation from a single mRNA transcript. Using deletion mutants in the receptor A cDNA, we have determined that the DNA binding and gene activator regions reside in the central portion of the protein, while the hormone binding domain is located in the C-terminal region. The target gene activator site of the receptor is modulated by interaction with the hormone binding domain.

Structural sequences that are extensively separated by nonstructural intervening sequences in the natural ovalbumin gene are coordinately expressed in target and nontarget tissue. The intervening sequences, which consist of unique sequences in the chick genome, are transcribed in their entirety. The amount of nuclear RNA corresponding to these sequences, however, is approximately 10 times less than that observed for structural sequences. The accumulation of RNA corresponding to structural and intervening sequences during acute estrogen stimulation suggests either that there are different rates of transcription for these regions of the ovalbumin gene or that RNA sequences corresponding to the intervening sequences are preferentially processed and degraded. Comparison of the in vitro expression of portions of the ovalbumin gene in nuclei isolated from chronically stimulated oviducts indicates that both structural and intervening sequences are preferentially transcribed in vitro at rates approximately 500 times greater than expected for random transcription of the haploid chick genome. In addition, electrophoresis of oviduct nuclear RNA on agarose gels containing methylmercury hydroxide reveals multiple species of RNA that are from 1.3 to over 4 times larger than ovalbumin mRNA and hybridize to both structural and intervening sequences of the ovalbumin gene. These results are consistent with transcription of the entire ovalbumin gene into a large precursor molecule followed by excision of the intervening sequences and appropriate ligation of the structural sequences to form the mature mRNA.

Three chicken genomic DNA clones containing the U1 RNA sequence were isolated from a chicken gene library and characterized. Two of these clones, CL64 and CL111, are overlapping clones which show several single-nucleotide changes in the U1 coding sequence, suggesting that they probably are alleles of the same sequence. The U1 sequence in the third clone, CL40, is more divergent. Flanking regions of these genes do not share any sequence homology between each other or with the previously isolated chicken genomic clone CL59. A short repeat CGGGG appears 28 times upstream of the U1 sequence in CL59. Another repeat, GCACC, is repeated 14 times upstream of the U1 region in CL40.

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.

An in vitro system [Weil, P. A., Luse, D. S., Segall, J. & Roeder, R. G. (1979) Cell 18, 469-484 and Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L., (1980) Proc. Natl. Acad. Sci USA 77, 3855-3859] was adapted for studying initiation of transcription of the ovalbumin gene. The DNA template was a cloned ovalbumin gene fragment that contained 5' flanking sequences, the first structural sequence region, and a portion of the first intervening sequence. A HeLa cell crude extract was used as the source of RNA polymerase and initiation factors. Correct initiation was judged by the sizes of the transcription products generated from ovalbumin templates truncated at various positions before the 3' end of the gene. Transcription of the specific product was carried out by RNA polymerase II, as judged from alpha-amanitin sensitivity. A series of deletion mutants was constructed by trimming 5' flanking sequences of the ovalbumin DNA template by using exonuclease III. The DNAs generated were then cloned in pBR322 and used as templates to determine which sequences were necessary for initiation of transcription. Specific initiation of the ovalbumin gene was unaffected by deletion of all but 61 nucleotides of the 5' flanking sequence but completely abolished by deletion of all but 26 nucleotides of 5' flanking sequence. Thus, a region between 61 and 26 nucleotides upstream from the cap site, which includes the Hogness box (T-A-T-A-T-A-T) at position 32-26, is essential for the correct initiation of the ovalbumin gene. Nevertheless, natural DNA fragments containing false Hogness boxes (T-A-T-A-A-A-A and T-A-T-A-T-A-T) not normally located in the immediate 5' flanking region of an authentic gene did not serve as promoters for initiation of transcription. These results suggest that the Hogness box is essential, but not sufficient, for specific initiation of RNA synthesis.

Problems involved in using the Hg-nucleotide technique for in vitro chromatin transcription are 2-fold. First, Escherichia coli RNA polymerase can utilize endogenous RNA as template and synthesize complementary sequences which remain base-paired to the template, thereby allowing it to bind to the SH-Sepharose column and copurify with the newly synthesized Hg-RNA. Second, non-mercurated endogenous RNA can bind to the SH-Sepharose through aggregation with Hg-RNA and thus be retained in the final RNA preparation. These two problems associated with the Hg-nucleotide technique can be minimized by modifying the conditions for RNA synthesis and SH-Sepharose chromatography. Using the modified procedure the Hg-nucleotide and SH-Sepharose technique can remove more than 90% of endogenous RNA contaminants. In order to directly demonstrate that the mRNAov sequences detected in vitro result from de novo transcription of oviduct chromatin, experiments were carried out which show that the hybridizable RNA sequences contain the Hg element and that the synthesis of these RNA sequences is sensitive to low concentrations of actinomycin D. These combined results strongly suggest that the majority of mRNAov sequences detected by hybridization to cDNAov is indeed due to DNA-dependent RNA synthesis by E. coli RNA polymerase and not due to an artifact of endogenous RNA contamination. This observation was further supported by data obtained using a filter hybridization method which measures directly the mRNAov sequences present in [3H]RNA synthesized from chromatin. The 3H-labeled ovalbumin messenger RNA was assayed by hybridization to cloned pOV230 DNA containing the ovalbumin structural gene sequence. With this modified Hg-nucleotide-SH-Sepharose technique and filter hybridization technique, we have restudied the in vitro transcription of the ovalbumin gene from chromatins isolated at different stages of hormone-induced oviduct development. The results are in agreement with our previous findings which suggest that the primary regulation of ovalbumin synthesis by steroid hormones occurs at the transcriptional level.

The location of CR1 middle repetitive sequences within or near the boundaries of the ovalbumin DNase I sensitive domain has suggested that CR1 sequences may play a role in defining transition regions of DNase I sensitivity in hen oviduct nuclei. We have examined this apparent relationship of CR1 sequences and transitions of chromatin structure by determining the DNase I sensitivity in oviduct nuclei of a 47-kilobase region that contains five CR1 sequences and the transcribed ovomucoid and ovoinhibitor genes. We find that three of the CR1 sequences occur within a broad transition region of decreasing DNase I sensitivity downstream of the ovomucoid gene. Another CR1 is in a region of decreased DNase I sensitivity within the ovoinhibitor gene. The fifth CR1 sequence is in a DNase I sensitive region between the two genes but which is less sensitive to DNase I digestion than the region immediately upstream from the ovomucoid gene. Thus, the CR1 sequences occur within regions of reduced relative DNase I sensitivity, suggesting that CR1s could facilitate the formation of a chromatin conformation that is less sensitive to DNase I digestion. Unexpectedly, the noncoding strand of sequences within and immediately adjacent to the 5' end of the actively transcribed ovomucoid and ovalbumin genes was less sensitive to DNase I digestion than their respective coding strands.

We have isolated and sequenced a genomic fragment containing sequences complementary to chicken U1 RNA. The sequence of this genomic U1 gene is completely homologous and colinear with that of chicken U1 RNA. This U1 gene is part of a multigene family (6--10 copies per haploid genome), and these loci do not appear to be closely clustered. Sequences complementary to other snRNAs are not present within the 2.5 kb genomic fragment containing the U1 gene. We have determined that U1 RNA is synthesized by polymerase II; however, a "Hogness box" is not present upstream from its cap site at the position usually observed for mRNA genes. The synthesis of U1 RNA in oviduct nuclei during different states of hormonal induction also appears to be constitutive.

By use of cloned DNA fragments as probes, low levels of ovalbumin RNA sequences (structural and intervening sequences) were detected in nuclear RNA extracts of nontarget tissues, such as liver, spleen, brain, and heart of chicks. The expression of the ovalbumin gene sequences was hormone dependent. In estrogen-stimulated chicks, a low level of ovalbumin RNA sequences, ranging from 0.2 to 0.7 molecule per cell, was present in nontarget tissues while less than 0.01 molecule per cell could be found in the same tissues of unstimulated chicks. A significant amount of the ovalbumin mRNA sequences was also found in polysomes of liver and brain. The ovalbumin mRNA sequences could be translated into proteins which were only localized in a few cells among the entire population of liver cells as determined by an immunocytochemical assay. These results suggest that there are some cells in liver, spleen, heart, and brain which can respond to hormone stimulation and produce ovalbumin mRNA and its translational product.

We describe the isolation of recombinant phages from a chicken gene library which contain two genes designated X and Y. These two genes are linked to the ovalbumin gene (OV) in the order 5'-X-Y-ovalbumin-3' [Royal, A., Garapin, A., Cami, B., Perrin, F., Mandel, J. L., LeMeur, M., Bregegegre, F., Gannon, F, LePennec, J. P., Chambon, P., & Kourilsky, P. (1979) Nature (London) 279, 125-132]. Both genes contain multiple intervening sequences and share limited sequence homology with the authentic ovalbumin gene but are expressed in oviduct cells at different levels. X and Y hybridization probes were prepared in order to study the expression and the relative hormonal responsiveness of these three genes in chicken oviduct. The sequence specificity of the probes was demonstrated by Southern hybridization assays. Northern hybridization studies using the X and Y gene probes indicated the presence of putative precursor molecules in stimulated oviduct ribonucleic acid preparations, which differ in size from those observed for ovalbumin. R0t analysis has demonstrated that, similar to the ovalbumin gene, the level of X and Y gene transcripts is increased by the steroid hormone estrogen, but to varying degrees. The extent of hormonal responsiveness of the three closely related genes is in the order (normalized) relative to ovalbumin of OV:Y:X congruent to 100:10:1. Pulse-labeling studies of these three closely linked genes suggest that in estrogen-stimulated oviduct, the markedly different steady-state levels of the X, Y, and ovalbumin gene transcripts reflect their differential transcription rates.

Synthesis of uteroglobin in rabbit uterine epithelial cells during early pregnancy is induced by progesterone and apparently suppressed by estrogen. To assess whether the steroid hormones exert regulatory effects on the expression of the uteroglobin gene, we performed transcriptional rate assays in endometrial nuclei prepared from ovariectomized rabbits subjected to different hormonal treatments. The isolated nuclei were incubated with [3H]UTP to assess the synthesis of newly elongated RNA transcripts by the incorporation of [3H]UMP. The percentage of labeled RNA represented by uteroglobin sequences was determined by hybridization with cloned uteroglobin DNA immobilized on nitrocellulose filters. Progesterone alone (3 mg/kg X day) for 5 days increased endometrial DNA content 16-fold, total cellular transcriptional activity 4-fold, and relative uteroglobin gene transcription about 6-fold. 17 beta-Estradiol alone (50 micrograms/kg X day) for 5 days had no significant effect on any of these three variables. Not until after 2 days of treatment did progesterone increase the relative rate of uteroglobin gene transcription, whereas total transcriptional activity per mg DNA increased rapidly on the first day of progesterone administration. Treatment of progesterone-stimulated rabbits with estradiol for 5 days reduced tissue DNA content by 80%, and a single injection of estradiol caused a small decrease in uteroglobin gene transcription. Chronic treatment with progesterone alone was sufficient to reduce uteroglobin gene transcription to control levels, without a decrease in tissue DNA content. Progesterone thus regulates uteroglobin synthesis in the uterus at least at three levels: 1) by exerting a mitogenic effect on endometrial cells, 2) by enhancing total cellular transcriptional activity, and 3) by preferential stimulation of uteroglobin gene transcription. Physiologically, the decline in uteroglobin synthesis after 5 days of pregnancy appears to be the result of a decrease in uteroglobin gene transcription in the face of the continued rise in blood progesterone, and this effect may be aided by antiprogestational actions of estradiol.

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.

To understand the molecular basis of the steroid hormone regulated expression of the ovalbumin gene, we sought to identify and isolate nuclear factors from chicken oviduct which interact specifically with the ovalbumin promoter. Using DNase I footprinting and mobility shift assays, we have defined at least four distinct protein binding sites, OV-150, OV-220, OV-250 and OV-330, in the promoter region between -100 to -400. Binding competition and protein fractionation studies revealed the existence of two distinct proteins, each recognizing two promoter sites: Both OV-330 and OV-250 are recognized by one protein factor which is distinct from the one binding to both OV-220 and OV-150. The location of the DNase I footprints coincides with those of in vivo chromatin hypersensitive sites. The OV-330 site is located in a sequence area required for the repression of the gene in the absence of hormone. The factor binding to OV-330 has been substantially purified and renaturation experiments indicate that the binding activity is associated with a polypeptide(s) of Mr 40K.

The transcription of DNA sequences flanking the 5' end and 3' end of the ovalbumin gene was examined. First, various restriction endonuclease fragments corresponding to the 5' and 3' regions of the gene isolated and used as hybridization probes to assay for the presence of transcripts corresponding to these different regions in the chick oviduct nuclear RNA. Very little, if any, of the transcripts corresponding to sequences flanking the 5' and 3' structural sequences of the ovalbumin gene was detected in the steady-state nuclear RNA. Second, RNA was pulse labeled either in isolated nuclei or in an oviduct tissue suspension system and hybridized to DNA filters containing purified fragments of various 5'- and 3'-flanking regions. Our results again demonstrated that RNA was not synthesized from the 5'- and 3'-flanking regions surrounding the gene. Taken together, these results are consistent with the postulate that flanking DNA sequences are not transcribed and that the largest RNA species detected in the nuclear RNA are the initial transcripts.

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.