Clifford D. Carpenter

Detection of Estrogen Receptor-β Messenger Ribonucleic Acid and 125I-Estrogen Binding Sites in Luteinizing Hormone-Releasing Hormone Neurons of the Rat Brain

Luteinizing hormone-releasing hormone (LHRH) neurons of the forebrain play a pivotal role in the neuroendocrine control of reproduction. Although serum estrogen levels influence many aspects of LHRH neuronal activity in the female, earlier studies were unable to detect estrogen receptors (ERs) within LHRH neurons, thus shaping a consensus view that the effects of estradiol on the LHRH neuronal system are mediated by interneurons and/or the glial matrix. The present studies used dual-label in situ hybridization histochemistry (ISHH) and combined LHRH-immunocytochemistry/125I-estrogen binding to readdress the estrogen-receptivity of LHRH neurons in the female rat. In ISHH experiments we found that the majority of LHRH neurons exhibited hybridization signal for the “β” form of ER (ER-β). The degree of colocalization was similar in topographically distinct populations of LHRH neurons and was not significantly altered by estradiol (67.2±1.8 % in ovariectomized and 73.8±4.2 % in ovariectomized and estradiol-tre...

Genes Encoding Glycine-Rich Arabidopsis thaliana Proteins with RNA-Binding Motifs Are Influenced by Cold Treatment and an Endogenous Circadian Rhythm

We have characterized the expression of two members of a class of Arabidopsis thaliana glycine-rich, putative RNA-binding proteins that we denote Ccr1 and Ccr2. Southern blot analysis indicates that Ccr1 and Ccr2 are members of a small gene family. Both Ccr1 and Ccr2 mRNA levels were influenced by a circadian rhythm that has an unusual phase for plants, with maximal accumulation at 6:00 PM and minimal accumulation at 10:00 AM. The level of CCR1 protein, however, remained relatively constant throughout the cycle. The transcript accumulation patterns of the Ccr1 and Ccr2 genes differed considerably from conditions that affect the expression of similar genes from maize, sorghum, and carrot. Levels of Ccr1 and Ccr2 mRNAs were unchanged in wounded plants, increased at least 4-fold in cold-stressed plants, and decreased 2- to 3-fold in abscisic acid-treated plants. Ccr1 transcript levels decreased in response to drought, whereas Ccr2 transcript levels increased under the same conditions. Based on the presence of additional Ccr transcripts in dark-grown plants, we propose that Ccr transcripts may be subjected to a light- or dark-mediated regulation.

Direct and Indirect Regulation of Gonadotropin-Releasing Hormone Neurons by Estradiol

Abstract Estrogen signaling to GnRH neurons is critical for coordinating the preovulatory surge release of LH with follicular maturation. Until recently it was thought that estrogen signaled GnRH neurons only indirectly through numerous afferent systems. This minireview presents new evidence indicating that GnRH neurons are directly regulated by estradiol (E2), primarily through estrogen receptor (ER)-β, and indirectly through E2-sensitive neurons in the anteroventral periventricular (AVPV) region. The data described suggest that E2 generally represses GnRH gene expression but that this repression is transiently overcome by indirect E2-dependent signals relayed by AVPV neurons. We also present evidence that the AVPV neurons responsible for relaying E2 signals to GnRH neurons are multifunctional gamma aminobutyric acid-ergic/glutamatergic/neuropeptidergic neurons.

Recombination between satellite RNAs of turnip crinkle virus

Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat‐RNA D, sat‐RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat‐RNAs and DI RNAs (sat‐RNA C). When plants were inoculated with TCV, sat‐RNA D and in vitro sat‐RNA C transcripts containing non‐viable mutations in the 5′ domain, recombinant sat‐RNAs were recovered. These recombinants were composed of sat‐RNA D at the 5′ end and sat‐RNA C sequences at the 3′ end. Analysis of 20 independent recombination junctions revealed that unequal crossing‐over had occurred in planta in a region of sequence similarity between the two sat‐RNAs which resulted in the duplication of 3‐16 nucleotides. Thirty percent of the sat‐RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat‐RNA C or sat‐RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat‐RNA C. Based on the similarity between this sequence of sat‐RNA C, the right side junction of DI RNA G and the 5′ end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat‐RNA C and the 5′ end of the sat‐RNAs, a replicase‐driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator‐2 in the rat brain and brainstem

Dioxin exposure alters a variety of neural functions, most likely through activation of the arylhydrocarbon receptor (AhR) pathway. Many of the adverse effects, including disruption of circadian changes in hormone release and depressed appetite, seem to be mediated by hypothalamic and/or brainstem neurons. However, it is unclear whether these effects are direct or indirect, because there have been no comprehensive studies mapping the expression of components of the AhR pathway in the brain. Therefore, we used a sensitive in situ hybridization histochemical (ISHH) method to map the neural expression of AhR mRNA, as well as those of the mRNAs encoding the AhR dimerization partners, arylhydrocarbon receptor nuclear translocator (ARNT) and ARNT2. We found that AhR, ARNT, and ARNT2 mRNAs were widely distributed throughout the brain and brainstem. There was no neuroanatomic evidence that AhR is preferentially colocalized with ARNT or ARNT2. However, ARNT2, unlike ARNT expression, was relatively high in most regions. The most noteworthy regions in which we found AhR, ARNT, and ARNT2 mRNA were several hypothalamic and brainstem regions involved in the regulation of appetite and circadian rhythms, functions that are disrupted by dioxin exposure. These regions included the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), nucleus of the solitary tract (NTS), and the dorsal and median raphe nuclei. This neuroanatomic information provides important clues as to the sites and mechanisms underlying the previously unexplained effects of dioxins in the central nervous system. J. Comp. Neurol. 427:428–439, 2000.

Open reading frames of turnip crinkle virus involved in satellite symptom expression and incompatibility with Arabidopsis thaliana ecotype Dijon.

Carmoviruses are single-stranded, single component RNA viruses that include turnip crinkle virus (TCV) and the recently discovered cardamine chlorotic fleck virus (CCFV). Full-length, biologically active cDNAs were constructed for the TCV-M isolate and the Blue Lake isolate of CCFV. Using chimeric viruses constructed between isolates of TCV that produce mild or severe symptoms when coinoculated with a virulent satellite RNA, a Glu residue at position 1,144 in the polymerase open reading frame was identified as being involved in satellite-mediated symptom expression. To analyze viral determinants involved in resistance, chimeric viruses with precisely exchanged open reading frames were produced between TCV, which does not infect the Arabidopsis thaliana ecotype Dijon (Di-0), and CCFV, which can infect Di-0, TCV with the coat protein of CCFV was able to systemically infect Di-0 although whole plant hybridizations revealed that the hybrid virus spread more slowly than either of the two parental viruses. These results indicate that the two parental viruses. These results indicate that the coat protein is an important viral determinant in the resistance of Di-0 to TCV.

Novel proteins belonging to the troponin C superfamily are encoded by a set of mRNAs in sea urchin embryos.

The properties of several cDNA clones representing a family of mRNAs found in the embryonic ectoderm of Strongylocentrotus purpuratus are described. We have previously shown that these mRNAs (termed Spec for Strongylocentrotus purpuratus ectoderm) accumulate in the presumptive dorsal ectoderm of post-cleavage stage embryos and code for a group of 10 to 12 low molecular weight acidic proteins. We demonstrate here, using antibodies raised against the major Spec proteins, that the proteins are localized in the cytoplasm of dorsal ectoderm cells. Hybridization analysis and DNA sequencing show that the mRNAs coding for these proteins, although all related, can be divided into two subfamilies. Comparison of the translational reading frames of the Spec mRNAs with known protein sequences shows a significant homology with troponin C-related proteins, especially in the calcium-binding domains. We suggest that the Spec proteins are previously uncharacterized members of the troponin C superfamily.

A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos

Abstract Xenopus laevis eggs and gastrula stage embryos were fractionated into three equal sections normal to the animal-vegetal axis, and poly(A) + RNA was isolated from each section. Hybridization of these poly(A) + RNAs with [ 32 P]cDNA synthesized using animal or vegetal poly(A) + RNAs showed no detectable differences in the extents or rates of reaction. Thus, the vast majority of poly(A) + RNAs are not segregated along the animal-vegetal axis. To increase the sensitivity of these experiments, [ 32 P]cDNAs were prepared which had reduced levels of RNA sequences from the animal region of the gastrula stage embryo or spawned unfertilized egg. Hybridization reactions with these probes showed that 3 to 5% of the input cDNA represents poly(A) + RNA sequences enriched 2- to 20-fold in the vegetal region of the egg or gastrula stage embryo.

The gad2 Promoter Is a Transcriptional Target of Estrogen Receptor (ER) α and ERβ: A Unifying Hypothesis to Explain Diverse Effects of Estradiol

Estradiol (E2) regulates a wide range of neural functions, many of which require activation of estrogen receptor α (ERα) and/or ERβ, ligand-gated transcriptional regulators. Surprisingly, very few neural gene targets of ERs have been identified, and these cannot easily explain the myriad effects of E2. GABA regulates most of the same neural functions as E2, and GABAergic neurons throughout the brain contain ER. Therefore, we examined whether E2 directly regulates expression of glutamic acid decarboxylase 2 (gad2), the enzyme primarily responsible for GABA synthesis for synaptic release. Using dual luciferase assays, we found that E2, but not other gonadal steroids, stimulated the activity of a 2691 bp rat gad2 promoter reporter construct. Activation required either ERα or ERβ, and ERβ did not repress ERα-mediated transactivation. Site-directed mutagenesis studies identified three estrogen response elements (EREs) with cell-specific functions. An ERE at −711 upstream of the gad2 translational start site was essential for transactivation in both MCF-7 breast cancer cells and SN56.B5.G4 neural cells, but an ERE at −546 enhanced transcription only in neural cells. A third ERE at −1958 was inactive in neural cells but exerted potent transcriptional repression in E2-treated MCF-7 cells. Chromatin immunoprecipitation assays in mouse GABAergic N42 cells confirmed that E2 induced ERα binding to a DNA fragment containing sequences corresponding to the −546 and −711 EREs of the rat promoter. Based on these data, we propose that direct transcriptional regulation of gad2 may explain, at least in part, the ability of E2 to impact such a diverse array of neural functions.

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.