Grating metrology for X-ray and V-UV synchrotron beamlines at SOLEIL.

Diffraction gratings are key elements of soft X-ray synchrotron beamlines. Besides wavelength dispersion, specific parameters can be tailored to adjust the energy dependent efficiency and focusing, and to correct wavefront aberrations. As key elements of a beamline, any departure from the design values can severely reduce the overall performance. On the other hand, known non-conformities can often be corrected by slight adjustment of the alignment parameters. A careful and accurate metrology is therefore required before installation on the beamline. After presenting what variable line spacing gratings, variable groove depth gratings, and alternate multilayer gratings are, the use of the SOLEIL long trace profiler for the measurement of groove density variation along the surface and of the atomic force microscope for the groove geometry and roughness characterizations will be discussed. A few examples of grating metrology will be presented and analyzed with the help of optical simulations.

Surface shape determination with a stitching Michelson interferometer and accuracy evaluation.

Stitching methods are increasingly used for determining the surface shape of large and high precision optical elements used in synchrotron beamlines. They consist in reconstructing the surface topography from multiple measurements on overlapping parts of the optics aperture by various algorithms. This paper is an attempt to investigate how true and accurate such a reconstruction can be. Error sources are identified and evaluated throughout the acquisition and processing steps. The analysis is based on the example SOLEIL Michelson interferometer for nano-topography, a dedicated measurement bench for stitching interferometry. We propose a method for determining the error made on the estimate of the interferometric reference surface from the stitching dataset. This determination is made before and independently of the stitching procedure itself.

Characterization of Carbon-Contaminated B4C-Coated Optics after Chemically Selective Cleaning with Low-Pressure RF Plasma.

Boron carbide (B4C) is one of the few materials that is expected to be most resilient with respect to the extremely high brilliance of the photon beam generated by free electron lasers (FELs) and is thus of considerable interest for optical applications in this field. However, as in the case of many other optics operated at light source facilities, B4C-coated optics are subject to ubiquitous carbon contaminations. Carbon contaminations represent a serious issue for the operation of FEL beamlines due to severe reduction of photon flux, beam coherence, creation of destructive interference, and scattering losses. A variety of B4C cleaning technologies were developed at different laboratories with varying success. We present a study regarding the low-pressure RF plasma cleaning of carbon contaminated B4C test samples via inductively coupled O2/Ar, H2/Ar, and pure O2 RF plasma produced following previous studies using the same ibss GV10x downstream plasma source. Results regarding the chemistry, morphology as well as other aspects of the B4C optical coating before and after the plasma cleaning are reported. We conclude that among the above plasma processes only plasma based on pure O2 feedstock gas exhibits the required chemical selectivity for maintaining the integrity of the B4C optical coatings.

Thank you for not flowering: conservation genetics and gene flow analysis of native and non-native populations of Fraxinus (Oleaceae) in Ireland.

The risks of gene flow between interfertile native and introduced plant populations are greatest when there is no spatial isolation of pollen clouds and phenological patterns overlap completely. Moreover, invasion probabilities are further increased if introduced populations are capable of producing seeds by selfing. Here we investigated the mating system and patterns of pollen-mediated gene flow among populations of native ash (Fraxinus excelsior) and mixed plantations of non-native ash (F. angustifolia and F. excelsior) as well as hybrid ash (F. excelsior × F. angustifolia) in Ireland. We analysed the flowering phenology of the mother trees and genotyped with six microsatellite loci in progeny arrays from 132 native and plantation trees (1493 seeds) and 444 potential parents. Paternity analyses suggested that plantation and native trees were pollinated by both native and introduced trees. No signs of significant selfing in the introduced trees were observed and no evidence of higher male reproductive success was found for introduced trees compared with native ones either. A small but significant genetic structure was found (φft=0.05) and did not correspond to an isolation-by-distance pattern. However, we observed a significant temporal genetic structure related to the different phenological groups, especially with early and late flowering native trees; each phenological group was pollinated with distinctive pollen sources. Implications of these results are discussed in relation to the conservation and invasiveness of ash and the spread of resistance genes against pathogens such as the fungus Chalara fraxinea that is destroying common ash forests in Europe.

Carbon contamination of soft X-ray beamlines: dramatic anti-reflection coating effects observed in the 1 keV photon energy region.

Carbon contamination is a general problem of under-vacuum optics submitted to high fluence. In soft X-ray beamlines carbon deposit on optics is known to absorb and scatter radiation close to the C K-edge (280 eV), forbidding effective measurements in this spectral region. Here the observation of strong reflectivity losses is reported related to carbon deposition at much higher energies around 1000 eV, where carbon absorptivity is small. It is shown that the observed effect can be modelled as a destructive interference from a homogeneous carbon thin film.

Transgenic analysis of the response of the rat calbindin-D 9k gene to vitamin D.

The promoter of the calbindin-D 9k (CaBP9k) gene, previously analyzed in transgenic mice, contains all of the information necessary for expression of a transgene similar to the endogenous gene and also for an appropriate response to vitamin D. In the present study we first investigated the role of a putative vitamin D-responsive element (9k/VDRE), located at nucleotides -489 to -445 on the rat CaBP9k promoter gene, using transgenic mice. As expected, the pattern of transgene expression in mice carrying this putative VDRE mutated in its whole promoter context was similar to that in mice bearing the wild-type sequence. These transgenic mice also responded to 1,25-dihydroxyvitamin D3 in the same way as those bearing the wild-type transgene and as those carrying a transgene with a large deletion (from -2894 to -117) eliminating the putative 9k/VDRE. Thus, the putative 9k/VDRE is not required for the control of rat CaBP9k gene expression by vitamin D in vivo. We also found that responsiveness to 1,25-dihydroxyvitamin D3 depends on the site at which the transgene is integrated into the host genome, in a tissue-specific manner. These data together with the fact that vitamin D-responsive sequences are present in a two-module region (from -3731 to -2894 and/or -117 to +365) and that this region does not contain any classical VDRE show that the CaBP9k gene is submitted to a non-conventional control by vitamin D.

Intestinal expression of the calbindin-D9K gene in transgenic mice. Requirement for a Cdx2-binding site in a distal activator region.

The calbindin-D9K gene encodes a vitamin D-induced calcium-binding protein that is expressed as a marker of small intestine differentiation. We have shown that 4580 base pairs of its 5' DNA regulatory region can target reporter transgene expression in the intestine and cause this transgene to respond like the endogenous gene to vitamin D active metabolite and that the homeoprotein Cdx2 is bound to the TATA box in the intestine. We now show that the 4580 base pairs construct confers a differentiated pattern of reporter transgene expression in the intestine and that cooperation between the proximal promoter and a distal element located in an opened chromatin structure is responsible for the intestinal expression and vitamin D responsiveness of the transgene. Gel shift and footprinting assays using duodenal nuclear extracts indicate that this distal element contains a Cdx2-binding site. Finally, a mutation in this distal Cdx2-binding site dramatically decreases intestinal expression in transgenic mice. This report, using an in vivo approach, demonstrates the crucial role of Cdx2 for the transcription of an intestinal gene.

Upregulation of Calbindin-D-28k immunoreactivity by excitatory amino acids.

Excessive or prolonged exposure to excitatory amino acids (EAA) are thought to be neurotoxic by altering calcium homeostasis. A protective role of Calbindin-D-28 k (Calbindin) has been postulated due to its capacity to buffer calcium. Calbindin is highly expressed in the Purkinje cells (PCs), of the cerebellar cortex. Changes of the Calbindin immunoreactivity (IR) by the EAA has been here investigated in cerebellar slices maintained in vitro. It was found that at low temperature, PCs are very slightly immunoreactive and therefore the experiments were done at 22 degrees C. The results show that Calbindin-IR increases in PCs exposed to the neurotoxic agonists, Kainic acid (KA) and AMPA as well as to glutamate (Glu), the endogenous EAA. The increase is very rapid and slowly reversible; is induced by excitatory and excitotoxic concentrations of the agonists; is independent of the calcium influx. While KA- and AMPA-induced Calbindin-IR is blocked by CNQX, the KA/AMPA receptor antagonist, Glu-induced Calbindin-IR is only slightly decreased by CNQX and AP5, the NMDA receptor antagonist. It is concluded that Calbindin-containing neurons can increase their calcium buffering capacity in response to EAA binding to specific receptors, the response being independent of, but concomitant to calcium influx.

Control of nuclear transcription of vitamin D-dependent genes by vitamin D.

Vitamin D acts on the genome via its active metabolite, calcitriol, which is bound to its nuclear receptor (vitamin D receptor) and a DNA response element. The characterization of the DNA target of the vitamin D receptor in vitamin D-activated or -repressed genes and structure-function analysis of the vitamin D receptor have led to several advances. These include a better understanding of the mechanisms of transactivation via the vitamin D receptor by the description of direct and indirect interactions of the vitamin D receptor with the basal transcriptional machinery. Physiological evidence for heterodimerization of the vitamin D receptor with the retinoid X receptor, and with other liganded or unliganded nuclear receptors, has indicated how the genetic response to vitamin D can be modulated. This modulation can also be brought about by cooperation between vitamin D receptor and transcription factors, by the action of a dominant negative isoform of vitamin D receptor, and by cross-talk between the signalling pathways for vitamin D and growth factors. These new concepts, plus the development of analogues of calcitriol, all indicate considerable progress towards vitamin D therapy for several disorders, including renal diseases.

Estradiol-dependent uterine leiomyomas in transgenic mice.

Uterine leiomyomas are a major health problem for women of reproductive age. The molecular biology of these tumors is poorly understood partly because of the lack of relevant animal models. We have produced transgenic mice expressing the simian virus 40 T antigen driven by the promoter of the Calbindin-D9K (CaBP9K) gene and either -1,000 or -117 bp of regulatory sequences so as to establish in vivo, uterine smooth muscle tumor models. Six transgenic mouse lines were obtained. Leiomyomas developed in all of them, with an almost complete penetrance of the phenotype. The smooth muscle tumors arose in different parts of the female reproductive tract. Leiomyomas usually developed in the corpus of the uterus, but one mouse line developed leiomyomas in the horn of the uterus, and another in the vagina. The CaBP9K regulatory sequences directing the expression of the Tag gene possess an estradiol responsive element, and accordingly, development of the tumors was strictly under the control of estrogen. Expression of the Tag gene is not only necessary for the initiation of the tumor but also for its development and maintenance. These transgenic mouse models should be useful for studying the pathobiology of uterine leiomyomas and could be instrumental in designing new therapeutic approaches to this disease.

Functional and growth properties of a myometrial cell line derived from transgenic mice: effects of estradiol and antiestrogens.

This study describes the properties of a myometrial cell line, m-M116, that was derived from a leiomyoma developed in an adult female transgenic mouse harboring the simian virus 40 large T antigen (Tag) under the control of the 5'-regulatory sequence of the calbindin D9k (CaBP9k) gene. As the expression of this transgene is governed by the CaBP9k estrogen-responsive element, m-M116 cells were grown in medium supplemented with 17 beta-estradiol. The cells were long lived, had Tag-positive nuclei, and were nontumorigenic when injected into nude mice. They formed irregular layers of elongated cells with typical features of uterine, smooth muscle cells, as assessed by the presence of alpha-smooth muscle actin and desmin filaments, estradiol and progesterone receptors, and expression of the CaBP9k gene. The rate of cell doublings and the expression of the Tag gene in early passaged cells depended on the presence of 17 beta-estradiol. Tamoxifen, a mixed estrogen agonist-antagonist, also stimulated the growth of m-M116 cells, whereas ICI 182 780, a pure antiestrogen, blocked cell growth. Later passages of m-M116 cells still had a smooth muscle phenotype, but proliferated even in the absence of 17 beta-estradiol. These mouse uterine smooth muscle cells obtained by targeted oncogenesis provide a useful model for studies of the progression of steroid-independent carcinomas.

Calbindin-D9k and calbindin-D28k expression in rat mineralized tissues in vivo.

Following their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium-binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D9k and calbindin-D28k in rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin-D28k accounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100-fold more concentrated than calbindin-D9k in both tissue types. Western blot analysis confirmed that the anticalbindin-D28k reactive species corresponded to the well characterized renal calbindin-D28k in the ectomesenchyme. In this tissue, calbindin-D28k was ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin-D28k appeared to be associated with rough endoplasmic reticulum. Calbindin-D9k antisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (approximately 10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins-D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D-dependent calcium-binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tissues, enamel, dentine, and bone.

cis-Acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box.

The calbindin-D9K (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the molecular mechanisms of intestine-specific transcription. The gene has been cloned, two major DNase-I-hypersensitive sites in the duodenum have been described, and a vitamin-D-response element has been identified. We have now analysed the transcription factors and regulatory sequences involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intestinal (CaCo-2) and non-intestinal (HeLa) cell lines defined two regions in the 5'-flanking sequences of the rat CaBP9k gene. A minimal proximal region (-117 to +20) promoted transcription in both intestinal expressing and non-expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream, which are responsible for complete repression in the non-intestinal cells. Intestinal transcription was specified by the proximal region, containing a specialized TATA box, and a distal region, which contains a previously described intestinal DNase-I-hypersensitive site. In vitro DNase I footprinting, electrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (-800 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Some of them appear to be binding sites for ubiquitous (nuclear factor 1) or hepatic-enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein alpha and beta factors. DNA binding studies and transfection experiments indicated that an intestine-specific transcription factor, caudal homeobox-2, binds to the TATA box of the rat CaBP9k gene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that several trans-acting factors, other than the vitamin D receptor, may be factors for intestine-specific CaBP9k gene expression.

Relationship between calbindin-D28K levels in the A and B cells of the rat endocrine pancreas and the secretion of insulin and glucagon: influence of vitamin D3 deficiency and 1,25-dihydroxyvitamin D3.

The pancreatic B cell is equipped with specific receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and contains vitamin D-dependent calcium binding proteins (calbindin-D). Insulin secretion is impaired by vitamin D deficiency and is restored by 1,25-(OH)2D3 (concomitantly with an improved calcium handling within B cells) but the effect of 1,25-(OH)2D3 on the pancreatic B cell via calbindin-D is unclear. Therefore we examined the relationship between calbindin-D28K or calbindin-D9K and the activity of the endocrine pancreas in normal (N), four week vitamin D-deficient (-D) and one week 1,25-(OH)2D3-replete (+D) rats. Calbindin-D9K was not found in the pancreas, neither in the islets nor in the exocrine part, of any of the groups of rats (N, -D, or+D). Surprisingly, total islet calbindin-D28K content was increased by vitamin D deficiency and partly restored by 1,25-(OH)2D3. Calbindin-D28K immunostaining was observed only on A and B cells in the endocrine part of the pancreas, the greatest staining being found in A cells. This difference in staining density was increased by vitamin D deficiency and decreased by 1,25-(OH)2D3 treatment. In vitro, 1,25-(OH)2D3 also produced a negative influence on calbindin-D28K staining in A cells, as demonstrated using pieces of pancreas incubated with the steroid for 2 h. No significant influence on labeling intensity of B cell calbindin-D28K could be shown. Plasma insulin and islet insulin release in response to 10 mM arginine stimulation were decreased in -D rats and enhanced in +D rats towards N values. In contrast, plasma glucagon and the amount of glucagon secretion, stimulated in vitro by 10 mM arginine or by low (1.7 mM) glucose concentration, was increased in -D rats and attenuated by 1,25-(OH)2D3. Thus there appears to be no relationship between the steady state level of B cell calbindin-D28K and the regulation of insulin secretion by 1,25-(OH)2D3 in vitamin D-deficient rats. However there is a correlation between A cell calbindin-D28K and glucagon secretion, which are both negatively regulated by 1,25-(OH)2D3. The predominance of calbindin-D28K in A cells raises the question as to how A and B cells interact and the role of calbindin-D28K in calcium handling.

Contrasting effects of tamoxifen and ICI 182 780 on estrogen-induced calbindin-D 9k gene expression in the uterus and in primary culture of myometrial cells.

Antiestrogens have a large range of tissue- and promoter-specific actions, many of which still remain unclear, particularly in the uterus. Thus, we have analyzed the effects of two antiestrogens, tamoxifen (TAM) and ICI 182 780 (ICI) on the uterine estrogen-responsive gene calbindin-D9k (CaBP9k), in the ovariectomized rat uterus, and in primary cultures of myometrial cells. In the ovariectomized rat uterus, estradiol (E2) or E2 plus TAM induced CaBP9k mRNA to the same levels in 6h. Rats given TAM alone had the same mRNA concentration, but maximal induction was obtained later, 12h after injection. ICI alone did not induce CaBP9k gene expression. Rats given E2 plus ICI had low uterine CaBP9k mRNA levels at 6-12h that became undetectable at 24h. Thus ICI has a full antagonistic effect on E2-induced CaBP9k gene. Estradiol receptor (ER) assays showed that TAM had a partial antagonist effect, while ICI had a full antagonist effect on the ER. We also analyzed the effect of TAM and ICI on CaBP9k gene expression in primary cultures of myometrial cells. The effects were similar to those observed in whole uterus. Thus, TAM has mixed effects, being an agonist for CaBP9k gene induction, and an antagonist for ER. ICI antagonizes the effects of E2 on the CaBP9k gene in myometrial cells and in the intact uterus, but in a way that does not involve a decrease in the cellular content of ER. Instead, it interferes with at least one of the events leading to transcriptional activation.

Identification of DNA sequences that bind retinoid X receptor-1,25(OH)2D3-receptor heterodimers with high affinity.

Vitamin D3 receptors (VDR) bind as heterodimers with retinoid X receptors (RXR) to vitamin D response elements (VDRE) and transactivate gene expression in a 1,25(OH)2D3-dependent manner. These elements are tandem direct repeats (DRs) of the hexamer RGGTCA separated by three nucleotides (DR3). We determined whether this DR3 was the optimal and/or only recognition sequence, by PCR-mediated binding site selection with reticulocyte lysate-expressed hVDR and mRXRalpha, and a pool of random sequences. We derived a consensus binding site for RXR-VDR heterodimers, RGGTCANN RRGTTCAB, and analyzed 10 of the 45 sequences slected by EMSA, methylation interference and transfection experiments: all the sequences were specific and acted as positive VDREs; the underlined purine of the spacer interacted with the heterodimer; the mutation of the third T in the second motif to a G did not influence VDRE activity. Thus, the selectivity of vitamin D pathway involving heterodimerization rather than VDR-homodimerization is not due to internal sequence variations. Except for mouse osteopontin VDRE, the natural VDREs would be efficient, only when helped by adjacent sequences and/or transactivators other than VDR and RXR.

Induction of calbindin-D 28K gene and protein expression by physiological stimuli but not in calcium-mediated degeneration in rat PC12 pheochromocytoma cells.

To understand the role of calbindin-D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin-D 28K is expressed in PC12 cells. The amounts of calbindin-D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D3. Calbindin-D 28K expression was also significantly up-regulated by stimuli (depolarization, low concentrations of Ca2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin-D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin-D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.

Influence of exogenous porcine growth hormone on vitamin D metabolism and calcium and phosphorus absorption in intact pigs.

The influence of growth hormone (GH) on vitamin D metabolism and calcium and phosphorus absorption in vivo is not clear. We, therefore, measured calcium and phosphorus balance, plasma 1,25-dihydroxyvitamin D (1,25(OH)2D), and intestinal vitamin D-dependent calcium-binding protein (CaBP 9k) in intact growing pigs given exogenous GH. Six 10-week-old pigs were given two daily subcutaneous injections of 50 micrograms porcine GH/kg body weight for 2 months; six control pigs were given vehicle. They were all fed a diet containing 1.1% Ca, 0.6% P, and 1000 IU vitamin D3/kg. Apparent Ca and P absorption and retention were measured in a 10-day balance trial at the end of the 2 months. The plasma levels of Ca, P, 1,25(OH)2D, IGF-I, and GH were determined, and the duodenal and jejunal mucosal CaBP 9k content was measured at slaughter. The plasma Ca and P of GH-treated pigs were unchanged, but all aspects of mineral metabolism, including the plasma 1,25(OH)2D concentration (40%), Ca absorption and retention (70%), P absorption (33%) and retention (45%), and jejunal CaBP 9k (40%), were stimulated, in addition to an increase in the circulating IGF-I concentration.

[Vitamin D and the immune system]. / Vitamine D et système immunitaire.

There is now increasing evidence that the hormonal form of vitamin D, 1,25(OH)2D3, is involved in the regulation of the immune system. Local production of the hormone in various infectious diseases can benefit the immune environment. 1,25(OH)2D3 exerts most of its actions only after it has bound to its specific nuclear receptor. These receptors are present in monocytes and activated lymphocytes. The hormone inhibits lymphocyte proliferation and immunoglobulin production in a dose-dependent fashion. It also blocks the accumulation of the mRNAs for IL-2, IFN-gamma and GM-CSF. It interferes with T helper cell (Th) function, reducing Th-induction of immunoglobulin production by B-cells and inhibits the passive transfer of cellular immunity by Th in vivo. The steroid hormone promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. The expression of Class II antigen by lymphocytes and monocytes is also affected. In vivo, 1,25(OH)2D3 is particularly effective in preventing auto-immune diseases such as experimental auto-immune encephalomyelitis, murine lupus, and diabetes in NOD mice. Synthetic analogues of vitamin D3 that bind to receptors but have no hypercalcemic effect in vivo have recently been developed for therapeutic use.

Calbindin-D9k gene expression in the uterus: study of the two messenger ribonucleic acid species and analysis of an imperfect estrogen-responsive element.

The calbindin D9k (CaBP9k) gene is under strict estrogen control in the rat uterus. This tissue contains two CaBP9k messenger RNA (mRNA) species. We have used primer extension analysis, reverse transcriptase associated with polymerase chain reaction, and RNase H digestion to show that these two mRNA species have the same structural features, including 5'- and 3'-ends, and poly(A) tail length. Our results suggest that the difference in electrophoretic mobilities of the two mRNA species might be due to interaction with another factor. We also analyzed the imperfect estrogen-responsive element (ERE) present on the first 5'-splice site of the rat CaBP9k gene. The oligonucleotide corresponding to the CaBP9k ERE was cloned in the plasmid pBLCAT2 (where the thymidine kinase promoter governs the expression of the chloramphenicol acetyl transferase gene) and transfected into MCF7 cells. This CaBP9k ERE was found to be a hormone-inducible enhancer that worked in an orientation-independent manner on a heterologous promoter and was functional at physiological hormone concentrations. One CaBP9k ERE conferred only weak (about 2-fold) estrogen induction, but two EREs cloned in tandem were strongly synergistic (14- to 16-fold). The CaBP9k ERE also bound to the partially purified estrogen receptor (ER) and to ER expressed in COS cells by gel shift assay. Methylation interference showed that all the guanine residues in both half-sites of the CaBP9k ERE were protected by ER binding. Thus, ER binds to the CaBP9k ERE in a way similar to other EREs. The gel shift assay results indicate that the strong synergistic effect of two EREs cloned in tandem is not due to cooperative binding between the two elements. As the CaBP9k gene is under strong estrogenic control in the uterus in vivo, the imperfect CaBP9k ERE may cooperate with another trans-acting factor to become fully efficient.