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At least 1,189 records · Page 66Linked to original sources
PubMed1999

Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-kappa B transcription factors.

Interleukin-6 (IL-6) is a multifunctional cytokine expressed by angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs) that functions as an autocrine growth factor. In this study, we analyze the mechanism for Ang II-inducible IL-6 expression in quiescent rat VSMCs. Stimulation with the Ang II agonist Sar1 Ang II (100 nmol/L) induced transcriptional expression of IL-6 mRNA transcripts of 1.8 and 2.4 kb. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, Sar1 Ang II treatment induced IL-6 transcription in a manner completely dependent on the nuclear factor-kappaB (NF-kappaB) motif. Sar1 Ang II induced cytoplasmic-to-nuclear translocation of the NF-kappaB subunits Rel A and NF-kappaB1 with parallel changes in DNA-binding activity in a biphasic manner, which produced an early peak at 15 minutes followed by a nadir 1 to 6 hours later and a later peak at 24 hours. The early phase of NF-kappaB translocation was dependent on weak simultaneous proteolysis of the IkappaBalpha and beta inhibitors, whereas later translocation was associated with enhanced processing of the p105 precursor into the mature 50-kDa NF-kappaB1 form. Pretreatment with a potent inhibitor of IkappaBalpha proteolysis, TPCK, completely blocked Sar1 Ang IIAng II-induced NF-kappaB activation and induction of endogenous IL-6 gene expression, which indicated the essential role of NF-kappaB in mediating IL-6 expression. We conclude that Ang II is a pleiotropic regulator of the NF-kappaB transcription factor family and may be responsible for activating the expression of cytokine gene networks in VSMCs.

Angiotensin II↗
PubMed2006

FOG-1 represses GATA-1-dependent FcepsilonRI beta-chain transcription: transcriptional mechanism of mast-cell-specific gene expression in mice.

Cell-type-specific transcription of mouse high-affinity IgE receptor (FcepsilonRI) beta-chain is positively regulated by the transcription factor GATA-1. Although GATA-1 is expressed in erythroid cells, megakaryocytes, and mast cells, the expression of mouse FcepsilonRI beta-chain is restricted to mast cells. In the present study, we characterized the role of GATA-associated cofactor FOG-1 in the regulation of the FcepsilonRI beta-chain promoter. The expression levels of FOG-1, GATA-1, and beta-chain in each hematopoietic cell line were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FOG-1 expression was higher in the beta-chain-negative hematopoietic progenitor cell line Ba/F3 than in the beta-chain-positive mast cell line PT18. By contrast, GATA-1 expression was similar when comparing the 2 cell lines. A transient reporter assay demonstrated that the beta-chain promoter functioned in PT18 but not in Ba/F3 and that the transcription activity of the beta-chain promoter in PT18 was markedly suppressed by overexpression of FOG-1. Although the activity of the beta-chain promoter, which was upregulated by coexpression of GATA-1, was significantly suppressed by coexpression of FOG-1 in the simian kidney CV-1 cells (beta-chain(-), GATA-1(-), and FOG-1(-)), the transactivation of the beta-chain promoter by the GATA-1 mutant V205G, which cannot bind FOG-1, was not affected by coexpression of FOG-1. Further, overexpression of FOG-1 in PT18 resulted in decreases in cell surface expression of FcepsilonRI and beta-chain transcription. Finally, suppression of FOG-1 expression using an siRNA approach resulted in increased beta-chain promoter activity in Ba/F3. These results suggest that FOG-1 expression level regulates the GATA-1-dependent FcepsilonRI beta-chain promoter.

Animals↗
PubMed2000

The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB.

The human vitamin D receptor (hVDR) is a ligand-regulated transcription factor that mediates the actions of the 1,25-dihydroxyvitamin D3 hormone to effect bone mineral homeostasis. Employing mutational analysis, we characterized Arg-18/Arg-22, hVDR residues immediately N-terminal of the first DNA binding zinc finger, as vital for contact with human basal transcription factor IIB (TFIIB). Alteration of either of these basic amino acids to alanine also compromised hVDR transcriptional activity. In contrast, an artificial hVDR truncation devoid of the first 12 residues displayed both enhanced interaction with TFIIB and transactivation. Similarly, a natural polymorphic variant of hVDR, termed F/M4 (missing a FokI restriction site), which lacks only the first three amino acids (including Glu-2), interacted more efficiently with TFIIB and also possessed elevated transcriptional activity compared with the full-length (f/M1) receptor. It is concluded that the functioning of positively charged Arg-18/Arg-22 as part of an hVDR docking site for TFIIB is influenced by the composition of the adjacent polymorphic N terminus. Increased transactivation by the F/M4 neomorphic hVDR is hypothesized to result from its demonstrated enhanced association with TFIIB. This proposal is supported by the observed conversion of f/M1 hVDR activity to that of F/M4 hVDR, either by overexpression of TFIIB or neutralization of the acidic Glu-2 by replacement with alanine in f/M1 hVDR. Because the f VDR genotype has been associated with lower bone mineral density in diverse populations, one factor contributing to a genetic predisposition to osteoporosis may be the F/f polymorphism that dictates VDR isoforms with differential TFIIB interaction.

Amino Acid Sequence↗
PubMed1997

HSF1 transcription factor concentrates in nuclear foci during heat shock: relationship with transcription sites.

In this paper, we show that upon heat shock, HSF1 concentrates in the nucleus of diploid human fibroblasts in two large foci. The relative distribution of HSF1 nuclear foci and active heat shock protein (hsp) genes was investigated by combining fluorescence in situ hybridization (FISH) for the detection of hsp nuclear transcripts and immunofluorescence for the detection of HSF1. We show that the HSF1 foci are distinct from the sites of hsp70 and hsp90 genes transcription. This is the second report of ploidy-dependent foci of transcription factors that are independent of their specific transcription sites. However, the correlation between the number of HSF1 foci and the ploidy of the cells strongly supports the existence of a specific chromosomal target for HSF1 foci.

Apoptosis↗
PubMed2002

Activating transcription factor-2 mediates transcriptional regulation of gluconeogenic gene PEPCK by retinoic acid.

All-trans-retinoic acid (RA) is known to increase the rate of transcription of the PEPCK gene upon engagement of the RA receptor (RAR). RA also mediates induction of specific gene transcription via several signaling pathways as a nongenomic effect. Here we show that RA upregulation of PEPCK promoter activity requires the cAMP response element (CRE)-1 in addition to the RA-response element and that activating transcription factor-2 (ATF-2) binds the CRE element to mediate this effect. Furthermore, we show that RA treatment potentiates ATF-2-dependent transactivation by inducing specific phosphorylation of ATF-2 by p38beta kinase. ATF-2 activation by RA blocked the inhibitory intramolecular interaction of ATF-2 amino and carboxyl terminal domains in a p38beta kinase-dependent manner. Consistent with these results, RA treatment increased the DNA binding activity of ATF-2 on the PEPCK CRE-1 sequence. Taken together, the data suggest that RA activates the p38beta kinase pathway leading to phosphorylation and activation of ATF-2, thereby enhancing PEPCK gene transcription and glucose production.

Activating Transcription Factor 2↗
PubMed2002

The forkhead transcription factor FoxO regulates transcription of p27Kip1 and Bim in response to IL-2.

The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. We and others have shown that PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). In this study we have investigated the role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival. We show that IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27(Kip1) and the proapoptotic Bcl-2 family member Bim. Furthermore, we show that IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, we propose that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival.

Animals↗
PubMed2002

A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene.

Expression of the norepinephrine transporter (NET), which mediates the reuptake of norepinephrine into presynaptic nerve terminals, is restricted to noradrenergic (NA) neurons. We have demonstrated previously that the 9.0 kb upstream sequences and the first intron residing in the 5' untranslated area are critical for high-level and NA cell-specific transcription. Here, using transient transfection assays, we show that 4.0 kb of the 5' upstream sequences contains sufficient genetic information to drive reporter gene expression in an NA cell type-specific manner. Three functional domains appear to be potentially important for the regulation of human NET (hNET) gene transcription: an upstream enhancer region at -4.0 to -3.1 kb, a proximal domain at -133 to -75 bp, and a middle silencer region between these two domains. DNase I footprinting analysis of the proximal promoter region shows that a subdomain at -128 to -80 bp is protected in a cell-specific manner. We provide evidence that multiple protein factors interact with the proximal promoter domain to critically regulate the transcriptional activity of the hNET gene. In the middle of this proximal subdomain resides a homeodomain (HD)-binding core motif, which interacts with HD factors, including Phox2a and HoxA5, in an NA-specific manner. Cotransfection analyses suggest that HoxA5 and Phox2a may transactivate the hNET gene promoter. Together with previous studies indicating direct activation of dopamine beta-hydroxylase transcription by Phox2a/2b, the present results support a model whereby Phox2 proteins may coordinately regulate the phenotypic specification of NA neurons by activating both NA biosynthetic and reuptake genes.

5' Untranslated Regions↗
PubMed1992

Mechanism of assembly of the RNA polymerase II preinitiation complex. Transcription factors delta and epsilon promote stable binding of the transcription apparatus to the initiator element.

Assembly of RNA polymerase II with the core region of TATA box-containing promoters requires the action of the TATA factor and four transcription factors designated alpha, beta gamma, delta, and epsilon, which have each been purified to near homogeneity from rat liver. Evidence from previous studies argues that alpha and beta gamma play a crucial role in delivering RNA polymerase II to the promoter (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6205-6209). Here we describe the interaction of transcription factor delta with preinitiation intermediates assembled in the presence of either recombinant yeast TFIID or the high molecular mass, endogenous TATA factor tau from rat liver (Conaway, J. W., Hanley, J. P., Garrett, K. P., and Conaway, R. C. (1991) J. Biol. Chem. 266, 7804-7811). Results of template challenge experiments argue that delta enters the preinitiation complex through interactions with multiple components of the transcription apparatus. We observe that, in the presence of recombinant TFIID, delta interacts stably with the preinitiation complex only in the presence of RNA polymerase II, alpha, and beta gamma, whereas, in the presence of tau, delta is capable of interacting stably with the Initial Complex independently of RNA polymerase II. Results of restriction site protection experiments reveal that delta and epsilon promote binding of the transcription apparatus to the Initiator element and support the model that RNA polymerase II assembles at the core promoter in at least two discrete steps, first "touching down" near the TATA element and finally extending its interaction downstream to encompass the cap site.

Animals↗
PubMed1994

The transcription factor BSAP (NF-HB) is essential for immunoglobulin germ-line epsilon transcription.

Treatment of murine splenic B lymphocytes and certain B-lineage cell lines with mitogen (LPS) and the lymphokine IL-4 has been shown to induce expression of germ-line epsilon transcripts (l epsilon transcripts) and class switching to the C epsilon gene. Three protein complexes, one of which (complex 3) is constitutively expressed, have been shown to bind to a 179-base pair LPS/IL-4-responsive l epsilon promoter (Rothman, P., S. C. Li, B. Gorham, L. Glimcher, F. W. Alt, and M. Boothby. 1991. Mol. Cell. Biol. 11:5551). Complex 3 is indispensable for this inducible promoter activity. In this report, we have used electrophoretic mobility shift assays (EMSA) to demonstrate that the early B cell-specific transcription factor (BSAP) is involved in the formation of complex 3. In addition, BSAP is implicated functionally in l epsilon transcription because a BSAP binding site either from a sea urchin histone promoter (H2A-2.2) or from 5' of murine immunoglobulin S gamma 2a can substitute for the epsilon-associated site (epsilon(foot), as assayed by transient transfection assays of the l epsilon:CAT reporter constructs into the M12.4.1 B cell line. Like the sea urchin histone BSAP site, the complex 3 binding site (epsilon(foot)) functions as an upstream promoter element when assayed in the OVEC vector. These results indicate that BSAP is an essential protein required for LPS/IL-4 induction of the l epsilon promoter. In addition, experiments showing that a BSAP binding site from 5' of S gamma 2a also functions as an upstream promoter element in OVEC suggest a potential role for BSAP in regulation of the IgG2a isotype.

Animals↗
PubMed1994

Positive and negative regulation of retinoid X receptor gene expression by thyroid hormone in the rat. Transcriptional and post-transcriptional controls by thyroid hormone.

The 9-cis-retinoic acid receptors (RXRs), belonging to the members of the steroid/thyroid hormone receptor superfamily, act as auxiliary proteins, heterodimerizing with other nuclear receptors such as retinoic acid receptors (RARs), vitamin D receptor, thyroid hormone receptors, and peroxisome-proliferator activated receptor, thereby transactivating target genes in a ligand-dependent manner. We have previously reported that in the rat, thyroid hormone (TH) positively and negatively regulates the hepatic mRNA levels of RXR beta and RXR gamma, respectively. In the present study, we have tried to elucidate the level at which TH regulates the gene expression of RXR beta and RXR gamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis inhibitor (cycloheximide), blocked the induction of RXR beta mRNA by TH. On the other hand, none of these drugs inhibited the decrease of RXR gamma mRNA levels caused by TH. Nuclear run-on assays showed that the transcription rate of the RXR beta gene was positively regulated by TH, whereas the transcription of RXR gamma gene was not controlled by TH. Taken together, these results indicate that the gene expression of RXR beta is positively regulated by TH at transcriptional level, while the negative regulation of the RXR gamma gene expression by TH may occur at a post-transcriptional level in intact rat. Thus, the RXR-mediated signal transductions may be modulated in part through TH control of the levels of RXR beta and RXR gamma.

Animals↗
PubMed1993

Activating transcription factor-1 can mediate Ca(2+)- and cAMP-inducible transcriptional activation.

Increased intracellular cAMP and Ca2+ levels can activate transcription of a number of eukaryotic genes through a common promoter element, the cAMP/Ca2+ response element. This element is the binding site for activating transcription factor (ATF)/CREB proteins, an extensive transcription factor family. Here we report that one member of this family, ATF-1, can mediate both Ca2+ and cAMP transcriptional responses, but that the responses to the two pathways differ in magnitude. In contrast, another family member, CREB, has been shown to mediate Ca2+ and cAMP responses to similar levels. Taken together, these results suggest a mechanism that allows cells to integrate and differentiate gene regulation by cAMP and Ca2+.

Activating Transcription Factor 1↗
PubMed1996

Differential regulation of transcription by the NURR1/NUR77 subfamily of nuclear transcription factors.

NURR1 is an orphan member of the nuclear receptor superfamily of transcription factors that shares close sequence homology to the orphan nuclear receptor and immediate early gene product NUR77(NGF1 beta). The physiological role of NURR1 has not been established in mammalian cells. However, the observation that NURR1 and NUR77 interact with at least one common enhancer element (AAAAGGTCA), together with their partly overlapping but differential expression patterns in mammalian tissues, suggests that these proteins may have both shared and independent transcription regulatory functions. To identify potential target genes that may be regulated by NURR1, we analyzed its DNA binding properties to potential cis-acting enhancer elements. Using point mutagenesis of the AAAAGGTCA motif, we have identified three additional sequences that bind specifically to both NURR1 and NUR77, one of which serves as a functional enhancer element. Comparative analysis of the transcription regulatory properties of NURR1 and NUR77 indicates that the proteins can display opposing transregulatory activities that are influenced by the specific cis-acting sequences to which they bind. Our results indicate that the transcriptional responses of specific target genes to the NURR1/NUR77 subfamily may be differentially regulated by the relative cellular levels of NURR1 and NUR77 and influenced by the specific enhancer sequences that mediate their activity. Finally, we have identified several potential target genes of neuronal and neuroendocrine origin whose promoters contain this element.

Base Sequence↗
PubMed2007

Using the histone H2a transcript as an endogenous standard to study relative transcript abundance during bovine early development.

The requirement for sample standardization is basic to any relative RNA abundance assessment. In 2002, we published on the RNA abundance profiling of several housekeeping targets during early bovine embryonic development. It was then concluded that histone H2a was the most stable transcript across the studied developmental period. Since that time, several teams have applied this information, yet neglected to use the published set of primers. Here, we show that these other primer sets do not target the same histone H2a variant. Within the present report, the RNA abundance profiles of their respective targets, for example, histone H2a.1, H2a.z, and H2a.o were measured in developmental series spanning the immature oocyte to the blastocyst stage embryo. In order to more clearly define the conditions that impact the RNA abundance level measurement of these candidates, the state of polyadenylation and the origin of the transcript either from de novo transcription or from maternal stocks were taken into consideration. The histone H2a.z transcript is principally regulated by de novo transcription following embryonic genome activation, whereas the levels of H2a.1 and H2a.o variants are largely of maternal origin, conferring a more stable profile throughout the studied developmental window. The histone H2a.z was found solely in a polyadenylated state whereas the H2a.1 and H2a.o were found to be more abundant in a nonadenylated form. The presence of serum in the in vitro embryo production system also had some impact on the histone H2a.1 RNA level at the blastocyst stage.

Animals↗
PubMed1999

Transcription mapping in a medulloblastoma breakpoint interval and Smith-Magenis syndrome candidate region: identification of 53 transcriptional units and new candidate genes.

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith-Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith-Magenis syndrome.

Brain Neoplasms↗
PubMed1994

The loss of E-cadherin mRNA transcripts in rat prostatic tumors is accompanied by increased expression of mRNA transcripts encoding fibronectin and its receptor.

We compared the levels of mRNA transcripts encoding E-cadherin, N-cadherin, beta 1 integrin subunit, alpha 5 integrin subunit and fibronectin in the normal rat prostate gland, as well as in tumors derived from three invasive sublines (G, MatLyLu, AT-2) of the Dunning R-3227 rat prostatic adenocarcinoma. E-cadherin mRNA transcripts were only detectable in total RNA extracts prepared from normal rat prostates, whereas N-cadherin mRNA transcripts were only found in normal rat brains. In contrast, the mRNA transcripts encoding the beta 1 integrin subunit, alpha 5 integrin subunit and fibronectin were all elevated in the tumors, as compared to the levels of these transcripts in normal tissues. Our results suggest that there is an inverse correlation between cadherin and integrin mRNA levels in rat prostatic tumors.

Adenocarcinoma↗
PubMed2006

Mitochondrial disease activates transcripts of the unfolded protein response and cell cycle and inhibits vesicular secretion and oligodendrocyte-specific transcripts.

Mutations in gene products expressed in the mitochondrion cause a nuclear transcriptional response that leads to neurological disease. To examine the extent to which the transcriptional profile was shared among 5 mitochondrial diseases (LHON, FRDA, MELAS, KSS, and NARP), we microarrayed mutant and control groups in N-tera2, SH-SY5Y, lymphoblasts, fibroblasts, myoblasts, muscle, and osteosarcoma cybrids. Many more transcripts were observed to be significantly altered and shared among these 5 mitochondrial diseases and cell types than expected on the basis of random chance, and these genes are significantly clustered with respect to biochemical pathways. Mitochondrial disease activated multiple transcripts of the unfolded protein response (UPR), and of the cell cycle pathway, and low doses of the mitochondrial inhibitor rotenone induced UPR transcripts in the absence of cell death. By contrast, functional clusters inhibited by mitochondrial disease included: vesicular secretion, protein synthesis, and oligodendrogenesis. As it is known that UPR activation specifically inhibits vesicular secretion and protein synthesis, these data support the view that mitochondrial disease and dysfunction triggers the UPR, which in turn causes secretory defects which inhibit cellular migratory, synaptic, and oligodendrocytic functions, providing a testable hypothesis for how mitochondrial dysfunction causes disease. Since ischemic hypoxia, chemical hypoxia, and mitochondrial genetic disease (which could be considered 'genetic hypoxia') produce an overlapping induction of UPR and cell cycle genes which appears to have negative consequences, the modulation of these responses might be of benefit to patients with mitochondrial disease.

Cell Cycle Proteins↗
PubMed2001

Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.

Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flärdh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

Bacterial Proteins↗
PubMed1999

Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells--detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction.

In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca2+ channels have been amplified by reverse transcription-polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro, fragments of the three major transcripts alpha1C, alpha1A, and alpha1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca2+ channels. These data support the correlation between alpha1A and P-type Ca2+ channels (G1) and between alpha1E and R-type channels (G2 and G3). By including specific primer pairs for alpha1E the complimentary DNA fragments of indicative regions of alpha1E isoforms are amplified corresponding to the three most variable regions of alpha1E, the 5'-end, the II/III-loop, and the central part of the 3'-end. Although the complementary DNA fragments of the 5'-end of rat alpha1E yield a uniform reverse transcription-polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat alpha1E complementary DNA. It corresponds to the alpha1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of alpha1E. While fragments of a new rat alpha1E isoform are amplified from the 5'-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3'-coding region are detected, which in the last case are discriminated by a 129 base pair insertion. The shift of the alpha1E expression from a pattern seen in cerebellum (alpha1Ee) to a pattern identified in other regions of the brain (alpha1E-3) is discussed. These data show that: (i) alpha1E is expressed in rat brain as a structural homologue to the mouse and human alpha1E; and (ii) rat cerebellar granule cells in primary culture express a set of alpha1E isoforms, containing two different sized carboxy termini. Since no new transcripts of high-voltage-activated Ca2+ channels genes are identified using degenerate oligonucleotide primer pairs, the two isoforms differentiated by the 129 base pair insertion might correspond to the two R-type channels, G2 and G3, characterized in these neurons. Functional studies including recombinant cells with the different proposed isoforms should provide more evidence for this conclusion.

Amino Acid Sequence↗