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The mammalian ME1 gene encodes a non-tissue-specific, helix-loop-helix transcription factor that is enriched in morphogenetically active regions during development. Regulation of mouse ME1 gene expression is controlled by a novel initiator (ME1 Inr) that promotes transcription from the center of a 13 bp poly(dA) tract. We show here that the ME1 Inr autonomously directs initiation from the poly(dA) tract both in vitro and in vivo. This transcription was dependent upon two protein complexes; MBPalpha, which associated directly with the poly(dA) tract, and MBPbeta, which introduced an approximately 60 degrees bend immediately downstream of the poly(dA) tract. The MBPalpha and MBPbeta binding sites were strikingly conserved in homologous DNA from several mammalian species and the frog Xenopus laevis. These results suggest that the ME1 Inr constitutes a robust nucleation site that promotes transcription initiation in the absence of conventional promoter elements.

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The murine E-protein gene ME1 encodes a non-tissue-specific, helix-loop-helix transcription factor that is associated with morphological development. ME1 gene expression is regulated by a TATA-less promoter that contains multiple Sp1 consensus elements, E-boxes, and a novel transcription initiation site. In this study, we compared DNA homologous to the ME1 promoter from vertebrate species ranging from frog to human. A region of striking sequence similarity was identified in a region corresponding to the ME1 transcription initiation site (ME1 Inr). Within this region, a poly d(A) tract and a 9-bp inverted repeat (5'-GTCCGCCTG) were highly conserved in all species that were examined. Protein complexes that recognized these DNA elements were present among distant vertebrates (frog, chick, monkey and human), and were able to bend the ME1 Inr to a similar extent (approximately 60 degrees) as the previously described murine MBP alpha and MBP beta proteins. Collectively, these results suggest that an ME1 Inr-like element and its associated proteins functioned in an ancestral vertebrate more than 350 million years ago.

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E-proteins comprise a subfamily of helix-loop-helix transcription factors that have been identified in arthropods and several chordate taxa. In mammals, there are three classes of E-protein genes (E2A, E2-2, and HEB) that encode related, and often interchangeable, gene products. We have determined that the clawed frog Xenopus laevis contains twice the number of transcriptionally active E-protein genes when compared with other vertebrate species. Based upon genomic Southern blots and nucleotide sequence comparisons, it is likely that the additional X. laevis genes arose from tetraploidization. During embryogenesis, XE2A (homologue of mammalian E2A) transcripts were broadly expressed in anterior and posterior regions of the embryo while homologues of E2-2 (XE2.2) and HEB (XE1.2) appeared in vertebrate-specific structures including the pineal gland, olfactory bulb, and brachial arches. A phylogenetic analysis of these genes and other known metazoan E-proteins suggests that there were two periods of marked E-protein gene expansion; one that predated the radiation of vertebrates, and the other that coincided with Xenopus tetraploidization. Both of these periods were characterized by the rapid evolution of E2-2 and HEB-class genes, but not of E2A. We propose that the former genes acquired new or specialized roles during early chordate evolution and also more recently in Xenopus, as reflected by the stereotypic expression patterns of these genes during X. laevis development.

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The GAP-43 promoter region contains seven E-boxes (E1 to E7) that are organized in two clusters, a distal cluster (E3 to E7) and a proximal cluster (E1 and E2). Deletion analysis and site-directed mutagenesis of the GAP-43 promoter region showed that only the most proximal E1 E-box significantly modulates GAP-43 promoter activity. This E-box is conserved between the rat and human GAP-43 promoter sequences in terms of flanking sequence, core sequence (CAGTTG), and position. We found that endogenous E-box-binding proteins present in neuronal N18 cells recognize the E1 E-box and activate the GAP-43 promoter. The transcriptional activity of the GAP-43 promoter was repressed not only by the negative regulator Id2 protein, but also by two class A basic helix-loop-helix proteins, E12 and ME1a. In vitro analyses showed that both ME1a and E12 bind to the E1 E-box as homodimers. By Northern analyses, we established an inverse correlation between the level of E12 and ME1a mRNAs and GAP-43 mRNA in various neuronal cell lines as well as in ME1a-overexpressing PC12 cells. Therefore, we have identified a cis-acting element, the E1 E-box, located in the GAP-43 promoter region that modulates either positively or negatively the expression of the GAP-43 gene depending on which E-box-binding proteins occupy this site. Together, these data indicate that basic helix-loop-helix transcription factors regulate the expression of the GAP-43 gene and that the class A ME1a and E12 proteins act as down-regulators of GAP-43 expression.

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We describe a streamlined whole-mount in situ hybridization protocol that utilizes high concentrations of the detergent sodium dodecyl sulfate (SDS). Our results suggest that SDS is an effective blocking agent in Xenopus laevis embryos which permeabilizes membranes without disrupting morphology. Consequently, riboprobes appeared to disperse uniformly within the embryo and several arduous and/or laborious steps of conventional procedures could be eliminated without compromising the technique.

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We have cloned a minisatellite tandem array (XTA) from Xenopus laevis that contains approximately 200 copies of the 20-bp repeat 5'-CCAACAGCCTGCCCATCCAT-3'. The XTA sequence is present only once per haploid genome and is polymorphic with respect to repeat number and location of flanking restriction endonuclease sites. Although the 20-bp repeat has not previously been described, flanking sequences suggest that it lies proximal to coding regions in the Xenopus genome.

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We have isolated four non-tissue-specific helix-loop-helix (HLH) transcription factors from Xenopus laevis (Xl). While some are clearly orthologous to known mammalian HLH proteins, others have dramatic amino-acid differences in otherwise highly conserved protein domains. We propose that these changes arose following the tetraploidization of Xl approx. 30 Myr ago.

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Xenopus laevis GAP-43 (XGAP-43) is highly related to other vertebrate GAP-43 proteins in its N-terminal region which contains a membrane-targeting sequence, serine phosphorylation site, and calmodulin binding domain. Unlike other species examined, however, there appear to be two GAP-43-class genes in X. laevis which resulted from the genome duplication in Xenopus approximately 30 million years ago. During embryogenesis, XGAP-43 is expressed in a complex spatiotemporal pattern that is consistent with its putative role in neuronal growth and development.

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The mouse ME1 gene (HEB, REB and GE1, homologues in human, rat and chick, respectively) is a member of the nontissue-specific helix-loop-helix (HLH) gene family that includes E2A, E2-2 and Drosophila daughterless. We have examined the factors that control ME1 gene expression. ME1 is a single copy gene that spans > or = 150 kb of DNA and contains > 10 exons. Transcription was directed by an unusual initiator element that contained a 13 bp poly d(A) tract flanked by palindromic and inverted repeat sequences. Both RNase protection and primer extension analyses mapped the ME1 transcriptional start site to the center of the 13 bp poly d(A) tract. The ME1 initiator and its proximal sequences were required for promoter activity, supported basal levels of transcription, and contributed to cell type-specific gene expression. Other cis-elements utilized by the TATA-less ME1 promoter included a cluster of Sp1 response elements, E-boxes and a strong repressor. Collectively, our results suggest that the ME1 initiator and other cis-elements in the proximal promoter play an important role in regulating ME1 gene expression.

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Class A basic-helix-loop-helix (bHLH) proteins have been referred to as ubiquitous and are believed to have redundant functions. They are involved in the control of several developmental pathways, such as neurogenesis and myogenesis. To rationalize the existence of multiple class A bHLH proteins, we evaluated the differences and similarities between ME1a and ME2, two class A bHLH proteins, highly expressed in differentiating neuronal cells. In situ hybridization analyses reveal that ME1a and ME2 are characterized by distinguishable patterns of expression in areas of the adult mouse brain where neuronal plasticity occurs. Also, DNA-binding assays show that both proteins bind to E-boxes as homodimers and heterodimers, and show differences in their DNA-binding specificities, which suggest selective interactions with different binding sites of target genes. In addition, in vitro DNA-binding assays demonstrate that Id2 forms heterodimers with ME1a and ME2. As a result of these interactions, their DNA-binding activity is abolished. Furthermore, overexpression of Id2 in neuronal cells suppresses ME1a and ME2 transcriptional activity. Based on our data, we hypothesize that ME1a and ME2 may activate gene expression of different target genes and therefore are likely to be differently involved during neurogenesis.

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We report the isolation of a cDNA encoding the mouse class A bHLH transcription factor ME2 and the analysis of its expression. ME2 is expressed in the cerebral cortex, Purkinje and granule cell layers of the cerebellum, olfactory neuroepithelium, pyramidal cells of hippocampal layers CA1-CA4, and in the granular cells of the dentate gyrus. The specific expression of ME2 during development and in the regions of neuronal plasticity in the adult brain suggest that ME2 may have a regulatory function in developmental processes as well as during neuronal plasticity.

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Id-like helix-loop-helix (HLH) proteins, which lack a basic DNA binding domain, have been suggested to serve as general inhibitors of differentiation. We present data that Id2 is expressed in specific cell types during neurogenesis and in the adult. At early stages of neurogenesis, Id2 is expressed in the ventricular zone of neuroepithelium. After the first neuronal populations are born, the expression of Id2 is down regulated in neuroepithelial cells and continues to be high in Purkinje cells of the cerebellum, in mitral cells of the olfactory bulb, and in layers 2, 3, and 5 of the cerebral cortex. In neuronally differentiating cell lines, the Id2 expression is up regulated (PCC7), down regulated (NG108), or unchanged (N18) during differentiation. Analyses of promoter sequences of the Id2 gene identify the region which is responsible for the down regulation of transcription during neuronal differentiation. Our data indicate that Id2 has different functions in different cell types during neurogenesis.

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Several class A basic helix-loop-helix (bHLH) transcription factors have been cloned from the developing mouse and chick nervous system. The cloned cDNAs (ME1, ME2, ME3, ME4, in the mouse and GE1, GE2 in the chick) have HLH coding regions highly homologous to other known class A bHLH genes. The genes corresponding to ME1 and GE1 are abundantly expressed during development of the central nervous system. ME1 and GE1 are expressed in proliferating neuroblasts and in cells at the initial stages of differentiation (for example in the external granule cell layer of the cerebellum and in the lateral region of the ventricular zone in the developing neural tube and cortex). They are also expressed at high levels in morphogenetically active regions such as limb buds, somites and mesonephric tubules. The expression of ME1 and GE1 decreases once cellular differentiation is over. Based on the expression of ME1 and GE1 in regions of active cellular proliferation and differentiation and on the known role of other bHLH factors in development, we suggest that ME1 and GE1 play important roles during development of the nervous system as well as in other organ systems.

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The neuronal growth cone plays a crucial role in forming the complex brain architecture achieved during development, and similar nerve terminal mechanisms may operate to modify synaptic structure during adulthood. The growth cone leads the elongating axon towards appropriate synaptic targets by altering motility in response to a variety of extracellular signals. Independently of extrinsic clues, neurons manifest intrinsic control of their growth and form (Banker and Cowan, 1979). Hence, there must be intracellular proteins which control nerve cell shape, so-called 'plasticity' or 'growth' genes. GAP-43 may be such a molecule (Skene and Willard, 1981; Benowitz and Lewis, 1983). For example, GAP-43 is localized to the growth cone membrane (Meiri et al. 1986; Skene et al. 1986) and can enhance filopodial formation even in non-neuronal cells (Zuber et al. 1989a). It includes a small region at the amino terminus for membrane association and perhaps growth cone targeting (Zuber et al. 1989b, Liu et al. 1991). We have found that Go, a member of the G protein family that links receptors and second messengers, is the major non-cytoskeletal protein in the growth cone membrane (Strittmatter et al. 1990). Double staining immunohistochemistry for GAP-43 and Go shows that the distributions of the two proteins are quite similar. Purified GAP-43 regulates the activity of purified Go (Strittmatter et al. 1990), a surprising observation since GAP-43 is an intracellular protein. We have compared the mechanism of GAP-43 activation of Go with that of G protein-linked receptors.2+ interactions between Go and GAP-43 suggest that Go plays a pivotal role in growth cone function, coordinating the effects of both extracellular signals and intracellular growth proteins.

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Neurons and other cells, such as those of epithelia, accumulate particular proteins in spatially discrete domains of the plasma membrane. This enrichment is probably important for localization of function, but it is not clear how it is accomplished. One proposal for epithelial cells is that proteins contain targeting signals which guide preferential accumulation in basal or apical membranes. The growth-cone membrane of a neuron serves as a specialized transduction system, which helps to convert cues from its environment into regulated growth. Because it can be physically separated from the cell soma, it has been possible to show that the growth-cone membrane contains a restricted set of total cellular proteins, although, to our knowledge, no proteins are limited to that structure. One of the most prominent proteins in the growth-cone membrane is GAP-43. Basi et al. have suggested that the N-terminus of GAP-43 might be important for the binding of GAP-43 to the growth-cone membrane. Skene and Virag recently found that the cysteines in the N-terminus are fatty-acylated and that this post-translational modification correlates with membrane-binding ability. We investigated the binding of GAP-43 to the growth-cone membrane by mutational analysis and by laser-scanning confocal microscopy of fusion proteins that included regions of GAP-43 and chloramphenicol acetyltransferase (CAT). We found that a short stretch of the GAP-43 N-terminus suffices to direct accumulation in growth-cone membranes, especially in the filopodia. This supports a previous proposal for the importance of this region of GAP-43 in determining the membrane distribution of GAP-43.

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The complete exonic and partial intronic sequence of the bovine CYP17 (P45017 alpha) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when approximately 500 bp of the 5'-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated.

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The neuron-specific protein GAP-43 is associated with the membrane of the nerve growth cone and thus may be important to the activity of this distinctive neuronal structure. Transient transfection of COS and NIH 3T3 cells with appropriate vectors resulted in expression of GAP-43 in these non-neuronal cells; as in neurons, transfected GAP-43 associated with the membrane. In addition, many long fine filopodial processes extended from the periphery of such transfected cells. Stable CHO cell lines expressing GAP-43 also exhibited processes that were more numerous, far longer, and more complex than those of CHO cell lines not transfected or transfected with control plasmids. Thus GAP-43 may directly contribute to growth cone activity by regulating cell membrane structure and enhancing extension of filopodial processes.

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Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.

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To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.

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