Repeated large-scale retreat and advance of Totten Glacier indicated by inland bed erosion.

Climate variations cause ice sheets to retreat and advance, raising or lowering sea level by metres to decametres. The basic relationship is unambiguous, but the timing, magnitude and sources of sea-level change remain unclear; in particular, the contribution of the East Antarctic Ice Sheet (EAIS) is ill defined, restricting our appreciation of potential future change. Several lines of evidence suggest possible collapse of the Totten Glacier into interior basins during past warm periods, most notably the Pliocene epoch, causing several metres of sea-level rise. However, the structure and long-term evolution of the ice sheet in this region have been understood insufficiently to constrain past ice-sheet extents. Here we show that deep ice-sheet erosion-enough to expose basement rocks-has occurred in two regions: the head of the Totten Glacier, within 150 kilometres of today's grounding line; and deep within the Sabrina Subglacial Basin, 350-550 kilometres from this grounding line. Our results, based on ICECAP aerogeophysical data, demarcate the marginal zones of two distinct quasi-stable EAIS configurations, corresponding to the 'modern-scale' ice sheet (with a marginal zone near the present ice-sheet margin) and the retreated ice sheet (with the marginal zone located far inland). The transitional region of 200-250 kilometres in width is less eroded, suggesting shorter-lived exposure to eroding conditions during repeated retreat-advance events, which are probably driven by ocean-forced instabilities. Representative ice-sheet models indicate that the global sea-level increase resulting from retreat in this sector can be up to 0.9 metres in the modern-scale configuration, and exceeds 2 metres in the retreated configuration.

Developmental regulation of VDJ recombination by the core fragment of the T cell receptor alpha enhancer.

The role of T cell receptor alpha enhancer (E alpha) cis-acting elements in the developmental regulation of VDJ recombination at the TCR alpha/delta locus was examined in transgenic mice containing variants of a minilocus VDJ recombination substrate. We demonstrate that the 116-bp T alpha 1,2 core enhancer fragment of the 1.4-kb E alpha is sufficient to activate the enhancer-dependent step of minilocus rearrangement, and that within T alpha 1,2, intact binding sites for TCF/LEF and Ets family transcription factors are essential. Although minilocus rearrangement under the control of the 1.4-kb E alpha initiates at fetal day 16.5 and is strictly limited to alpha beta T cells, we find that rearrangement under the control of T alpha 1,2 initiates slightly earlier during ontogeny and occurs in both gamma delta and alpha beta T cells. We conclude that the core fragment of E alpha can establish accessibility to the recombinase in developing thymocytes in vivo in a fashion that is dependent on the binding of TCF/LEF and Ets family transcription factors, but that these and other factors that bind to the E alpha core cannot account for the precise developmental onset of accessibility that is provided by the intact E alpha. Rather, our data suggests a critical role for factors that bind E alpha outside of the core T alpha 1,2 region in establishing the precise developmental onset of TCR alpha rearrangement in vivo.

Clonal deletion and clonal anergy in the thymus induced by cellular elements with different radiation sensitivities.

The present study demonstrates that immune tolerance can be achieved in the thymus both by clonal deletion and by clonal inactivation, but that the two tolerant states are induced by cellular elements with different radiation sensitivities. TCR engagement of self antigens on bone marrow-derived, radiation-sensitive (presumably dendritic) cells induces clonal deletion of developing thymocytes, whereas TCR engagement of self antigens on radiation-resistant cellular elements, such as thymic epithelium, induces clonal anergy. The nondeleted, anergic thymocytes can express IL-2-Rs but are unable to proliferate in response to either specific antigen or anti-TCR antibodies, and do develop into phenotypically mature cells that emigrate out of the thymus and into the periphery.

Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one TCR alpha, one TCR beta, and two CD3 epsilon chains.

The stoichiometry of the subunits that comprise the T cell antigen receptor (TCR) complex is not completely known. In particular, it is uncertain whether TCR alpha and TCR beta proteins are present in the TCR complex as one or multiple heterodimeric pairs. In this study we have used mice transgenic for two different TCR alpha and two different TCR beta proteins to determine the number of TCR alpha and TCR beta chains in a single TCR complex. Individual thymocytes and splenic T cells from double TCR transgenic mice simultaneously expressed all four transgenic TCR proteins on their surfaces. Because the individual TCR alpha and individual TCR beta proteins were biochemically distinguishable, we were able to examine association among the transgenic TCR products. We found that each TCR alpha chain paired with each TCR beta chain, but that each TCR complex contained only one TCR alpha and one TCR beta protein. Furthermore, quantitative immunofluorescence revealed that T cells expressed twice as many CD3 epsilon as TCR beta proteins. These findings demonstrate that there are precisely one TCR alpha, one TCR beta, and two CD3 epsilon chains in each TCR/CD3 complex expressed on the surfaces of both thymocytes and mature T cells.

Regulation of T cell receptor delta gene rearrangement by CBF/PEBP2.

We have analyzed transgenic mice carrying versions of a human T cell receptor (TCR)-delta gene minilocus to study the developmental control of VDJ (variable/diversity/joining) recombination. Previous data indicated that a 1.4-kb DNA fragment carrying the TCR-delta enhancer (E(delta)) efficiently activates minilocus VDJ recombination in vivo. We tested whether the transcription factor CBF/PEBP2 plays an important role in the ability of E(delta) to activate VDJ recombination by analyzing VDJ recombination in mice carrying a minilocus in which the deltaE3 element of E(delta) includes a mutated CBF/PEBP2 binding site. The enhancer-dependent VD to J step of minilocus rearrangement was dramatically inhibited in three of four transgenic lines, arguing that the binding of CBF/PEBP2 plays a role in modulating local accessibility to the VDJ recombinase in vivo. Because mutation of the deltaE3 binding site for the transcription factor c-Myb had previously established a similar role for c-Myb, and because a 60-bp fragment of E(delta) carrying deltaE3 and deltaE4 binding sites for CBF/PEBP2, c-Myb, and GATA-3 displays significant enhancer activity in transient transfection experiments, we tested whether this fragment of E(delta) is sufficient to activate VDJ recombination in vivo. This fragment failed to efficiently activate the enhancer-dependent VD to J step of minilocus rearrangement in all three transgenic lines examined, indicating that the binding of CBF/PEBP2 and c-Myb to their cognate sites within E(delta), although necessary, is not sufficient for the activation of VDJ recombination by E(delta). These results imply that CBF/PEBP2 and c-Myb collaborate with additional factors that bind elsewhere within E(delta) to modulate local accessibility to the VDJ recombinase in vivo.

Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei.

A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.

Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development.

Males with X-linked severe combined immunodeficiency (XSCID) have defects in the common cytokine receptor gamma chain (gamma c) gene that encodes a shared, essential component of the receptors of interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15. The Janus family tyrosine kinase Jak3 is the only signaling molecule known to be associated with gamma c, so it was hypothesized that defects in Jak3 might cause an XSCID-like phenotype. A girl with immunological features indistinguishable from those of XSCID was therefore selected for analysis. An Epstein-Barr virus (EBV)-transformed cell line derived from her lymphocytes had normal gamma c expression but lacked Jak3 protein and had greatly diminished Jak3 messenger RNA. Sequencing revealed a different mutation on each allele: a single nucleotide insertion resulting in a frame shift and premature termination in the Jak3 JH4 domain and a nonsense mutation in the Jak3 JH2 domain. The lack of Jak3 expression correlated with impaired B cell signaling, as demonstrated by the inability of IL-4 to activate Stat6 in the EBV-transformed cell line from the patient. These observations indicate that the functions of gamma c are dependent on Jak3 and that Jak3 is essential for lymphoid development and signaling.

Subjective memory complaints and awareness of memory functioning in mild cognitive impairment: a systematic review.

OBJECTIVES: Subjective memory complaint (SMC) is central to the diagnosis of mild cognitive impairment (MCI). People with MCI are at a higher risk of progressing to dementia, and research on SMC is contradictory in terms of the accuracy of SMC and its predictive role for future dementia. One possible reason for these contradictory findings is that the level of awareness of memory function may vary among people with MCI. This review examines whether the level of awareness of memory functioning varies amongst people classified as having MCI and whether there is support for the suggestion that the level of awareness in MCI predicts future progression to dementia. METHOD: Sixteen studies were identified which evaluate the awareness level in people classified as having MCI in either a clinical or research setting. In addition to the outcome of each study, the conceptualization of awareness, 'object' of awareness and methodology were also considered. RESULTS: There is evidence to show that the level of awareness in MCI does vary, and this may have implications for future progression to dementia. CONCLUSIONS: Given the increased risk of progression to dementia for those identified as having MCI, the role of awareness should be explored further with due consideration given to the conceptualization of awareness and the methodology employed. The finding of variability in awareness has implications for the use of SMC in the diagnostic criteria for MCI.

Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis.

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.

Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

Cooperation among multiple transcription factors is required for access to minimal T-cell receptor alpha-enhancer chromatin in vivo.

To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor alpha enhancer (Ealpha), we analyzed enhancer occupancy in thymocytes of transgenic mice in vivo by genomic footprinting. We found that the formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5' of the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type Ealpha, all sites are unoccupied on versions of Ealpha with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB. Our data indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single Ealpha binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable binding site occupancy as well as the activation of transcription and V(D)J recombination.

Lack of functional immunoregulatory cells in a patient with mycosis fungoides and circulating Sézary cells.

Peripheral blood lymphocytes obtained at various intervals from normal individuals and from a patient with mycosis fungoides (MF) were cultured with phytohemagglutinin (PHA) for 3 days to activate suppressor cells. After being cultured, the PHA-treated cells were irradiated, washed, and then transferred to fresh medium with PHA. The PHA responsiveness of the cells from normal individuals was suppressed approximately 90% by autologous or normal allogeneic lymphocytes activated for 3 days with PHA, whereas the cells activated for 3 days with PHA from the patient with MF lacked the capacity to inhibit the mitogenic response of autologous or allogeneic lymphocytes. These data suggest that this patient lacked suppressor T-cells that have a specificity for helper T-cells.

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin.

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Identification of a major up-stream transcription start site for the human progonadotropin-releasing hormone gene used in reproductive tissues and cell lines.

Previous studies suggested that GnRH gene transcripts in human tissues may be derived from an upstream transcriptional start site in addition to the well characterized hypothalamic start site. To resolve this issue we characterized the transcriptional start sites of the human GnRH gene in a human placental tumor cell line (JEG) and a human breast tumor cell line (MDA). Using primer extension and reverse transcription-polymerase chain reaction (RT-PCR) assay, we identified a discrete upstream transcriptional start site 579 bases up-stream from the hypothalamic site in both JEG and MDA cell lines. The up-stream start site lacks the TATA and CAAT elements often present in RNA polymerase-II promoters, but contains the sequence GGTCTTGCT located 84 bases 5' to the up-stream start site similar to other genes that lack TATA/CAAT boxes. RT-PCR quantitation shows that the up-stream start site is the major transcriptional start site, representing 74% of the cytoplasmic transcripts in JEG cells and 67% in MDA cells. Supporting this observation, transfection assay using a human GnRH promoter/luciferase reporter gene construct containing only the up-stream transcription start site has a higher level of transcriptional activity than the human GnRH promoter/luciferase reporter construct containing only the down-stream start site. A high relative abundance (approximately 45%) of total GnRH mRNAs were also found in the nucleus of both cell lines, which did not appear to be a consequence of the nuclear/cytoplasmic fractionation procedure. To determine if this upstream start site was used in normal GnRH-expressing human tissues, we analyzed RNA from a variety of postmortem/surgical procedure tissue samples. RT-PCR analysis together with Southern blot analysis demonstrated the presence of GnRH mRNA in human pituitary, cerebral cortex, testes, ovary, and mammary gland for the first time as well as verified GnRH gene expression in hypothalamus and placenta. The up-stream transcriptional start site is used only in reproductive tissues, such as placenta, testes, ovary, and mammary gland, suggesting tissue-specific regulation at this site.

Analysis of proopiomelanocortin gene expression during prenatal development of the rat pituitary gland.

The POMC gene is transcribed in 5-10% of adult rat anterior pituitary cells and nearly all intermediate lobe cells, but the end product peptide hormones produced by these two cell populations are distinct. We have here used in situ hybridization with 3H-POMC cRNA probes to investigate the temporal and spatial distribution of POMC mRNA containing cells during development of both lobes of the adenohypophysis. These hybridization patterns were, in addition, compared to the immunocytochemical distribution of POMC-related peptides on the same or adjacent sections. In both POMC populations, POMC-mRNA detection preceded POMC-derived peptide accumulation by at least 0.5 days. POMC-mRNA was first detected in the ventral aspect of Rathke's pouch (which will give rise to the anterior lobe) at e 13, which was two days earlier than POMC transcripts were first detected in the intermediate lobe; this result provides strong evidence that factors eliciting POMC transcription in these two populations act at distinct times during development. Further, since POMC mRNA containing cells were restricted to the anterior lobe during stages when intermediate lobe-like POMC peptides have been characterized biochemically, these results confirm immunocytochemical results and demonstrate that developmentally regulated alterations in the extent of POMC posttranslational processing occur during prenatal differentiation of anterior lobe POMC cells. POMC expression in the intermediate lobe was first observed in the midcaudal region in the midsagittal plane and subsequently extended in a gradient-like pattern to the rest of the intermediate lobe. Finally, semiquantitation of in situ hybridization signals documented that increases in the relative levels of POMC mRNA/cell occur at specific ages in both anterior and intermediate lobe. POMC cell populations and demonstrated that adult POMC mRNA/cell levels are not achieved before birth in either the anterior or intermediate lobe POMC cell populations.

Corticotropin-releasing hormone stimulates proopiomelanocortin transcription by cFos-dependent and -independent pathways: characterization of an AP1 site in exon 1.

The POMC gene, encoding a hormonal precursor protein, is primarily expressed in the pituitary in a tissue-specific manner. The POMC gene is transcriptionally regulated by a variety of hormones and neuropeptides and the second messengers cAMP and Ca++. Using the corticotrope-derived AtT20 cell line, we have previously shown that overexpression of cFos stimulates POMC transcription. The aim of this work was to analyze whether cFos directly interacts with the POMC gene in basal and corticotropin-releasing hormone (CRH) stimulated cells. Using progressively deleted POMC promoter sequences or heterologous promoter constructs coupled to the chloramphenicol acetyl transferase reporter gene, we demonstrate the existence of a major cFos- responsive sequence within the first exon of the POMC gene. This sequence, TGACTAA, appears functionally indistinguishable from the canonical AP1 binding site. When fused to a minimal promoter, this sequence confers inducibility by cFos and CRH. Gel shift analyses with CRH-stimulated AtT20 nuclear extracts or in vitro synthesized proteins revealed that this sequence efficiently binds Fos and Jun. Expression of c-fos anti-sense mRNA reduced CRH-stimulated POMC transcription, thus indicating that, at least in part, cFos mediates the effect of CRH on POMC transcription. However, deletion of this major exonic AP1 site from the POMC constructs greatly reduced the effect of c-fos overexpression but did not suppress POMC stimulation by CRH, indicating that CRH stimulates POMC transcription by one or more cFos-independent mechanism(s).

Stimulation of pituitary luteinizing hormone secretion by gonadotropin-releasing hormone is not coupled to beta-luteinizing hormone gene transcription.

The physiological stimulation and inhibition of release of several peptide and protein hormones appears to be associated with transcription of the respective peptide- and protein hormone-encoding genes. In the current studies we investigated whether this was also true for the rat pituitary LH system. Using an anterior pituitary primary tissue culture system, we have analyzed the effects of 10(-9) M GnRH on beta LH gene transcription using a transcription run-on assay, and on nuclear beta LH RNA levels using a highly sensitive solution hybridization-S1-nuclease protection assay. GnRH-stimulated release of LH does not appear to be coupled to a significant change in the rate of beta LH gene transcription, but is associated with both a 2- to 3-fold increase in levels of the beta LH primary transcript and processing intermediates and a rapid decrease in the levels of fully processed beta LH mRNA in the nucleus. No significant change in beta LH cytoplasmic mRNA levels, however, was associated with GnRH-stimulated release of LH, in vitro. Our findings suggest that unlike several other peptide and protein hormone systems, stimulated release of beta LH by GnRH in vitro is not associated with an increase in beta LH gene transcription or cytoplasmic mRNA levels.

Gonadotropin regulation of the rat proopiomelanocortin promoter: characterization by transfection of primary ovarian granulosa cells.

To characterize the transcriptional effects of human (h)FSH and hCG on the POMC gene, primary rat granulosa cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid under the control of the POMC promoter and 5' region. POMC-CAT contains a fragment of the rat POMC gene, extending from nucleotide -704 to nucleotide +63, fused to the CAT gene. Treatment of POMC-CAT-transfected cells with either hFSH (20 ng/ml) or hCG (10 ng/ml) significantly increased CAT enzyme activity; however, neither hCG nor hFSH increased CAT enzyme activity in cells transfected with pSV2-CAT, a reporter plasmid under the control of the SV40 virus promoter and 5' region. The phosphodiesterase inhibitor isobutylmethylxanthine or the nonhydrolyzable cAMP analog cAMP-chlorothiophenyl significantly increased CAT activity in POMC-CAT-transfected granulosa cells. Human FSH stimulated transcription 10, 20, and 40 h after treatment, but FSH stimulation at the two earlier time points was 2.5- to 5.5-fold greater than that at 40 h. Gonadotropin-stimulated steroidogenesis was equivalent in POMC-CAT-transfected granulosa cells, untransfected, and mock-transfected cells. This indicates that transfection left the physiological hormone response intact. These data demonstrate the following. 1) 767 basepairs of the rat POMC gene are enough to confer gonadotropin stimulation on the CAT marker gene in granulosa cells. 2) Although the POMC promotor lacks a well conserved cAMP response element, either of two different pharmacological manipulations of granulosa cells that raise intracellular cAMP can also stimulate POMC-driven CAT expression. 3) Transfected primary cultures of granulosa cells provide a nontransformed, physiologically relevant model with which to study hormone-regulated gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein.

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.