Inhibitor Alpha-amanitin Research Articles

Involvement of SMAD Signaling in Bovine Early Embryonic Development.

We have previously demonstrated a positive functional role for maternal (oocyte-derived) follistatin in control of time to first cleavage of bovine embryos, subsequent development to the blastocyst stage and in blastocyst cell allocation to the trophectoderm lineage. Follistatin binds to and inhibits activity of members of the transforming growth factor superfamily, whose signals are mediated intracellularly primarily through activation of receptor associated SMADs (rSMADs) including SMAD 2/3 (activin, TGF-beta) and SMAD 1/5/8 (BMPs). Thus, we hypothesized that follistatin's positive actions on early embryogenesis may be mediated via inhibition of rSMAD signaling. To address this hypothesis we determined the temporal expression and effects on bovine early embryogenesis of siRNA mediated knockdown of SMAD 4, a common component of above signaling pathways which complexes with respective rSMADs. Temporal changes in SMAD 4 mRNA during early embryogenesis were determined by real time PCR analysis of embryos collected at pronuclear, 2-, 4-, 8- and 16-cell and morula and blastocyst stages. SMAD 4 mRNA abundance was highest in 2-cell embryos and decreased dramatically to very low levels between the 8- to 16-cell stages, suggesting that SMAD 4 transcripts in early embryos are oocyte-derived. The maternal origin of SMAD 4 transcripts was confirmed by real time PCR analysis of embryos cultured to the 8-cell stage in the presence of the transcription inhibitor alpha-amanitin. Furthermore, SMAD 4 mRNA was higher in alpha-amanitin treated embryos, suggesting potential transcriptional dependence of observed decline in maternal SMAD 4 mRNA. To determine the functional role of SMAD 4 in bovine early embryogenesis, multiple siRNA species targeting bovine SMAD 4 were synthesized and evaluated for efficacy in reducing SMAD 4 mRNA levels (in 4-cell embryos) following microinjection into presumptive zygotes. Two of the validated SMAD 4 siRNA species with > 90 % efficiency in targeting SMAD 4 mRNA were combined and the efficacy of this siRNA cocktail (utilized in all subsequent studies) in reducing SMAD 4 mRNA and protein in early embryos verified. Presumptive zygotes were then injected with SMAD 4 siRNA, a negative control siRNA or served as uninjected controls. Effects of SMAD 4 knockdown on proportion of embryos cleaving early (within 30 h post insemination; hpi), total cleavage rates (determined 48 hpi), proportion of embryos developing to 8-16 cell (72 hpi) and blastocyst stages (d 7) and on blastocyst CDX2 mRNA abundance (index of trophectoderm cell allocation) were determined. SMAD 4 knockdown significantly reduced the proportion of embryos that cleaved early and embryonic development to 8- to 16-cell and blastocyst stages relative to uninjected and negative control siRNA injected embryos. Such results support a functional role for SMAD signaling in control of time to first cleavage and subsequent development to the blastocyst stage. However, effects of SMAD 4 knockdown on abundance of CDX2 mRNA in resulting blastocysts were not observed. Collectively, results support a requirement for the common SMAD (SMAD 4) of maternal origin in promoting bovine early embryogenesis, but do not suggest that previously observed stimulatory effects of follistatin on embryonic development are likely mediated via blocking stimulation of classical SMAD 4 dependent rSMAD signaling via endogenous TGF-beta superfamily members. (poster)

Isolation of aDrosophilaamplification origin developmentally activated by transcription

We exploited the Drosophila Amplicon in Follicle Cells, DAFC-62D, to identify a new metazoan amplification origin, ori62. In addition to the origin, DAFC-62D contains two other developmental stage-specific binding regions for the Origin Recognition Complex (ORC) and the replicative helicase MCM2-7. All three of these regions are required for proper amplification. There are two rounds of amplification initiation at ori62, and the second round is preceded by transcription across ori62. We show by alpha-amanitin inhibition that RNA polymerase II (RNAPII) transcription is required to localize MCM2-7 (but not ORC) to permit the second round of origin firing. This role for transcription appears unique to DAFC-62D, because neither other DAFCs nor ectopic transposons with the DAFC-62D replication elements bounded by functional chromatin insulators are affected by alpha-amanitin. By sequential chromatin immunoprecipitation, we show that the MCM complex and RNAPII are bound to the same 100-500 bp pieces of chromatin during late origin firing. These results raise the possibility that RNAPII may recruit MCM2-7 at some metazoan replication origins.

Histone Hyperacetylation during SV40 Transcription Is Regulated by p300 and RNA Polymerase II Translocation

The effects of the RNA polymerase II (RNAPII) translocation inhibitors alpha amanitin and 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) and an siRNA targeting p300 on the presence of RNAPII, p300, hyperacetylated H4 and H3 and unmodified H4 and H3 in transcribing simian virus 40 (SV40) minichromosomes were determined. Following treatment with alpha amanitin we observed a profound reduction in the occupancy of the promoter by RNAPII, the loss of p300 from chromatin fragments containing RNAPII, and an increase in the amount of unmodified H4 and H3 associated with the RNAPII. Treatment with DRB had little effect on the presence of RNAPII or p300 but also resulted in a significant increase in the amount of unmodified H4 and H3 present in chromatin fragments associated with RNAPII. Following treatment with a p300 small interfering RNA (siRNA), we observed a significant decrease in late transcription and a corresponding reduction in the amounts of p300 and hyperacetylated histones associated with the transcribing SV40 minichromosomes. We conclude that in transcribing SV40 minichromosomes histone hyperacetylation and deacetylation is dependent upon the presence of p300 and an as yet unknown histone deacetylase associated with the RNAPII complex that occurs coordinately as the RNAPII complex moves through a nucleosome.

Transcription of mammalian messenger RNAs by a nuclear RNA polymerase of mitochondrial origin

Transcription of eukaryotic genes is performed by three nuclear RNA polymerases, of which RNA polymerase II is thought to be solely responsible for the synthesis of messenger RNAs. Here we show that transcription of some mRNAs in humans and rodents is mediated by a previously unknown single-polypeptide nuclear RNA polymerase (spRNAP-IV). spRNAP-IV is expressed from an alternative transcript of the mitochondrial RNA polymerase gene (POLRMT). The spRNAP-IV lacks 262 amino-terminal amino acids of mitochondrial RNA polymerase, including the mitochondrial-targeting signal, and localizes to the nucleus. Transcription by spRNAP-IV is resistant to the RNA polymease II inhibitor alpha-amanitin but is sensitive to short interfering RNA specific for the POLRMT gene. The promoters for spRNAP-IV differ substantially from those used by RNA polymerase II, do not respond to transcriptional enhancers and contain a common functional sequence motif.

Transcription-Dependent Polyubiquitination of RNA Polymerase II Requires Lysine 63 of Ubiquitin

Lysine-63-linked polyubiquitin chains are not thought to signal protein degradation but instead signal for a variety of cellular processes including some types of DNA repair. RNA polymerase (Pol) II is polyubiquitinated following DNA damage or upon treatment of nuclear extracts with the transcription inhibitor alpha-amanitin. Here, we report, using a reaction in vitro, that transcription-dependent polyubiquitination of RNA Pol II consists of lysine-63-linked chains. This modification is specific for RNA Pol II engaged in active transcription and arrested by alpha-amanitin.

Expression pattern of the maternal factor zygote arrest 1 (Zar1) in bovine tissues, oocytes, and embryos.

Zygote arrest 1 (Zar1) is an ovary-specific maternal factor that plays an essential role during the oocyte-to-embryo transition in mouse. In this species, Zar1 expression is strictly limited to the oocyte, the zygote and, at a lower level, the 2-cell embryo. Aim of the present study was to analyze the presence and the expression pattern of the Zar1 ortholog in bovine tissues and embryos. Reverse transcription (RT)-polymerase chain reaction (PCR) analysis was performed in a panel of bovine tissues, in oocytes and pre-implantation in vitro produced embryos. The results demonstrated that a Zar1 ortholog is present in cattle. In the adult, the gene is expressed in ovary, testis, muscle, and myocardium. The gene is also expressed in the oocyte, the zygote, and in all the stages of embryonic development until blastocyst formation. A semi-quantitative RT-PCR analysis revealed that Zar1 levels are constant through in vitro development with the exception of the 4-cell stage, when a significant increase is observed. The exposure of fertilized oocytes to the RNA polymerase II inhibitor alpha-amanitin was able to suppress this Zar1 increase indicating that transcription of this gene occurs at the 4-cell stage. Zar1 is conserved in cattle but has an expression pattern different from the mouse. In particular, Zar1 expression in the adult is not limited to the ovary and in the embryo is expressed well beyond the oocyte to embryo transition. Moreover, the identification of Zar1 transcription at the 4-cell stage represents the first characterization of one of the genes expressed in cattle embryos before the major onset of embryonic transcription.

Effect of intrauterine insemination with spermatozoa or foreign protein on the mechanism of action of oestradiol in the rat oviduct.

Previously, we showed that oestradiol accelerates oviductal egg transport through a non-genomic action involving oviductal protein phosphorylation in non-mated rats, and through a genomic action in mated rats. Thus, sensory stimulation, seminal fluid or sperm cells may be the source of signals that switch the mechanism of action of oestradiol in the oviduct to a genomic pathway. The present study examined the ability of spermatozoa to switch the mode of action of oestradiol in the absence of the sensory stimulation and seminal fluid provided by mating. Pro-oestrous rats were inseminated in each uterine horn with epididymal spermatozoa and 12 h later were injected subcutaneously with oestradiol and intrabursally with the mRNA synthesis inhibitor alpha-amanitin. The number of eggs in the oviduct, assessed 24 h later, showed that alpha-amanitin blocked the oestradiol-induced egg transport acceleration, indicating that the interaction of spermatozoa with the genital tract shifts the action of oestradiol from non-genomic to genomic. Other rats were inseminated with live or dead spermatozoa and then treated with the protein kinase inhibitor H-89, and oestradiol. Treatment with H-89 did not block the oestradiol-induced acceleration of egg transport in these rats, although dead spermatozoa did not enter the oviduct, indicating that the mere presence of spermatozoa in the uterus abrogated the non-genomic action of oestradiol in the oviduct. Treatment with H-89 also failed to prevent the acceleration of oviductal egg transport induced by oestradiol in rats inseminated with hamster spermatozoa or with BSA, whereas in rats inseminated with their own serum (autologous proteins), H-89 was able to prevent the effect of oestradiol. This finding reveals that the effect of insemination on the mode of action of oestradiol is neither species-nor sperm-specific and it is produced by foreign organic material. It can be concluded that the presence of spermatozoa or foreign protein in the uterus is one of the components of mating that is capable of switching the action of oestradiol in the oviduct from a non-genomic to a genomic mode.

Effect of intrauterine insemination with spermatozoa or foreign protein on the mechanism of action of oestradiol in the rat oviduct

Previously, we showed that oestradiol accelerates oviductal egg transport through a non-genomic action involving oviductal protein phosphorylation in non-mated rats, and through a genomic action in mated rats. Thus, sensory stimulation, seminal fluid or sperm cells may be the source of signals that switch the mechanism of action of oestradiol in the oviduct to a genomic pathway. The present study examined the ability of spermatozoa to switch the mode of action of oestradiol in the absence of the sensory stimulation and seminal fluid provided by mating. Pro-oestrous rats were inseminated in each uterine horn with epididymal spermatozoa and 12 h later were injected subcutaneously with oestradiol and intrabursally with the mRNA synthesis inhibitor alpha-amanitin. The number of eggs in the oviduct, assessed 24 h later, showed that alpha-amanitin blocked the oestradiol-induced egg transport acceleration, indicating that the interaction of spermatozoa with the genital tract shifts the action of oestradiol from non-genomic to genomic. Other rats were inseminated with live or dead spermatozoa and then treated with the protein kinase inhibitor H-89, and oestradiol. Treatment with H-89 did not block the oestradiol-induced acceleration of egg transport in these rats, although dead spermatozoa did not enter the oviduct, indicating that the mere presence of spermatozoa in the uterus abrogated the non-genomic action of oestradiol in the oviduct. Treatment with H-89 also failed to prevent the acceleration of oviductal egg transport induced by oestradiol in rats inseminated with hamster spermatozoa or with BSA, whereas in rats inseminated with their own serum (autologous proteins), H-89 was able to prevent the effect of oestradiol. This finding reveals that the effect of insemination on the mode of action of oestradiol is neither species-nor sperm-specific and it is produced by foreign organic material. It can be concluded that the presence of spermatozoa or foreign protein in the uterus is one of the components of mating that is capable of switching the action of oestradiol in the oviduct from a non-genomic to a genomic mode.

Cyclin B1 levels in the porcine 4-cell stage embryo.

The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor alpha-amanitin. Cyclin B1 protein was not detectable in embryos cultured in medium without alpha-amanitin for 5, 10, 18 or 25 h P4CC followed by culture in medium with alpha-amanitin to 33 P4CC. These results suggest that the maternal to zygotic transition of mRNA production that occurs at the 4-cell stage of the pig embryo does not result in an increase in cyclin B1 production. In addition, cyclin B1 protein levels remained constant in the absence of embryonic genome activation at the 4-cell stage.

Structural basis of transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution.

The structure of RNA polymerase II in a complex with the inhibitor alpha-amanitin has been determined by x-ray crystallography. The structure of the complex indicates the likely basis of inhibition and gives unexpected insight into the transcription mechanism.

Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.

Transient expression of a translation initiation factor is conservatively associated with embryonic gene activation in murine and bovine embryos.

In the present study the abundance of mRNAs for eukaryotic translation initiation factors eIF-1A (formerly known as eIF-4C), -2alpha, -4A, -4E, and -5 was examined in in vivo-derived mouse embryos throughout preimplantation development using a semiquantitative reverse transcription-polymerase chain reaction assay. Although the mRNA profile for each gene is unique, only mRNA for eIF-1A transiently increases during embryonic gene activation (EGA) at the 2-cell stage, and this was confirmed by an independent hybridization-based assay. In in vitro-developed bovine embryos, mRNA for eIF-1A was transiently detected at the 8-cell stage, when the major activation of the genome occurs in this species. As in the mouse, detection in 8-cell bovine embryos was sensitive to the transcriptional inhibitor alpha-amanitin. It was also observed at the same time relative to cleavage in embryos cultured in defined medium under a reduced oxygen environment, and in medium supplemented with serum and somatic cells in 5% CO2 in air. Neither the chronology of early cleavage divisions nor the yield of bovine blastocysts differed in these culture media. Our results suggest that transient expression of eIF-1A in the mouse and cow is a conserved pattern of gene expression associated with EGA in mammals.

Disruption of downstream chromatin directed by a transcriptional activator.

Promoter-proximal pausing during transcriptional elongation is an important way of regulating many diverse loci, including the human hsp70 gene. Pausing of RNA polymerase can be enhanced by chromatin structure. We demonstrate that activation of hsp70 leads to disruption of transcribed chromatin in front of RNA polymerase. In vivo, disruption of chromatin in the first 400 bp of the transcribed region of hsp70 following heat shock is resistant to the transcriptional inhibitor alpha-amanitin. Disruption of chromatin farther downstream also occurs following activation but is sensitive to alpha-amanitin, suggesting that polymerase movement is needed to disrupt distal portions of the hsp70 gene. In vitro, disruption of transcribed chromatin is dependent on the presence of the human heat shock factor 1 (HSF1) activation domains. These experiments demonstrate that HSF1 can direct disruption of chromatin in transcribed regions. We suggest that this is one of the mechanisms used by HSF1 to facilitate transcriptional elongation.

Effects of amino acids and alpha-amanitin on bovine embryo development in a simple protein-free medium.

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor alpha-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O2:5% CO2:90% N2 at 39 degrees C for the first 40-44 hr in modified KSOM, and embryos with > or = 4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1-4, and with the addition of alpha-amanitin in experiment 5. In experiment 1, addition of 0.5x of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1x to 5x, did not stimulate embryo development. In experiments 2-4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, alpha-amanitin (20 microM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst.

Enrichment of middle repetitive element Bm-1 transcripts in translationally active RNA fractions of the silkmoth, Bombyx mori.

The Bm-1 repetitive element family represents a group of transcribed repetitive sequences in the genome of the silkmoth Bombyx mori. In the Bm-5 and BmN permanent cell lines studied here, alpha-amanitin inhibition and nuclear 'run-on' experiments demonstrated that approximately 80% of the Bm-1 transcripts are produced by RNA polymerase II. Bm-1 transcripts are dramatically enriched in poly A+ and polysomal RNA fractions compared to total RNA in these two cell lines. In the Bm-5 cell line, from total to poly A+ and polysomal RNA fractions, Bm-1 transcripts are enriched approximately 4 and 2 times, respectively, while in the BmN cell line these same fractions are enriched about 2 and 19 times compared to total RNA. This suggests that the Bm-1 transcripts may be involved in post-transcriptional processes or control of translation. Our data also revealed less size heterogeneity of Bm-1 transcripts in polysomal as compared to nuclear fractions. In the Bm-5 and BmN cell lines, the size of most transcripts containing Bm-1 sequences increases from approximately 1700 nt in the nucleus to 3000 nt in the polysomal fraction, both fractions with RNA much larger than the Bm-1 consensus sequence (250 bp). This raises the possibility that some Bm-1 elements are transcribed as part of larger transcripts containing mRNA by way of 'read-through', and may be involved in post-transcriptional regulation of gene expression as cis and/or trans acting elements.

Transcriptional competition and homeosis in Drosophila.

Interference between different classes of RNA polymerase II alleles causes a mutant phenotype called the "Ubx effect" that resembles one seen in flies haploinsufficient for the transcription factor, Ultrabithorax (Ubx). Flies carrying the mutation in the largest subunit of Drosophila RNA polymerase II, RpII215(4), display the Ubx effect when heterozygous as in RpII215(4)/+ but not when homozygous mutant or wild type. In this report we demonstrate that the interaction between alleles in different classes of polymerase occurs even in the absence of transcription by the wild-type polymerase. We utilized the resistance to the transcriptional inhibitor alpha-amanitin conferred by RpII215(4) to show that RpII215(4)/+ flies raised on alpha-amanitin-containing food still show the Ubx effect and are indistinguishable from flies raised on normal food. We demonstrate using HPLC that the intracellular concentration of alpha-amanitin in the developing larvae is sufficient to inhibit transcription by alpha-amanitin-sensitive polymerase. Furthermore, fluorescein-labeled alpha-amanitin accumulates in imaginal discs, which are the precursor cells for the tissue showing the homeotic transformation in adults. We conclude that the interaction between different classes of RNA polymerase II alleles resulting in the Ubx effect occurs prior to the block in transcription caused by alpha-amanitin.

Initiator-dependent transcription in vitro by a wheat germ chromatin extract

The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.

RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.

Transcription of U2 small nuclear RNA (snRNA) genes in eukaryotes is executed by RNA polymerase II and is dependent on extragenic cis-acting regulatory sequences which are not found in other genes. Here we have mapped promoter elements of the Trypanosoma brucei U2 snRNA gene by transient DNA expression of mutant constructs in insect form trypanosomes. Unlike other eukaryotic U2 snRNA genes, the T. brucei homolog is transcribed by an RNA polymerase III-like enzyme on the basis of its sensitivity to the inhibitors alpha-amanitin and tagetitoxin. Thus, the trypanosome U2 snRNA provides a unique example of an RNA polymerase III transcript carrying a trimethylated cap structure. The promoter of this gene consists of three distinct elements: an intragenic sequence close to the 5' end of the coding region, which is probably required to position the polymerase at the correct transcription start site; and two extragenic elements, located 110 and 160 nucleotides upstream, which are essential for U2 snRNA gene expression. These two elements closely resemble both in sequence and in distance from each other the A and B box consensus sequences of the internal control regions of tRNA genes.

RNA Polymerase III-Mediated Transcription of the Trypanosome U2 Small Nuclear RNA Gene Is Controlled by both Intragenic and Extragenic Regulatory Elements

Transcription of U2 small nuclear RNA (snRNA) genes in eukaryotes is executed by RNA polymerase II and is dependent on extragenic cis-acting regulatory sequences which are not found in other genes. Here we have mapped promoter elements of the Trypanosoma brucei U2 snRNA gene by transient DNA expression of mutant constructs in insect form trypanosomes. Unlike other eukaryotic U2 snRNA genes, the T. brucei homolog is transcribed by an RNA polymerase III-like enzyme on the basis of its sensitivity to the inhibitors alpha-amanitin and tagetitoxin. Thus, the trypanosome U2 snRNA provides a unique example of an RNA polymerase III transcript carrying a trimethylated cap structure. The promoter of this gene consists of three distinct elements: an intragenic sequence close to the 5' end of the coding region, which is probably required to position the polymerase at the correct transcription start site; and two extragenic elements, located 110 and 160 nucleotides upstream, which are essential for U2 snRNA gene expression. These two elements closely resemble both in sequence and in distance from each other the A and B box consensus sequences of the internal control regions of tRNA genes.

Regulation of Expression of Cholinergic Neuronal Phenotypic Markers in Neuroblastoma LA‐N‐2

Cholinergic neurons in PNS and CNS are identified by the presence of choline acetyltransferase and the accumulation of choline by a high-affinity, sodium-coupled choline transporter to be used for acetylcholine synthesis. It appears that expression of choline acetyltransferase can be altered by several physiological conditions, including hormones and trophic factors, but little is known about control of expression of the sodium-coupled choline carrier or whether these two phenotypic markers are regulated similarly. In the present study, the cholinergic human neuroblastoma LA-N-2 was used to investigate regulation of expression of choline acetyltransferase and choline uptake activity associated with differentiation and neurite extension. Cells grown in serum-containing basal medium maintained a relatively undifferentiated morphology, expressed low levels of choline acetyltransferase activity, and accumulated choline by a sodium-dependent process followed by conversion to acetylcholine. Transfer of cells to an enriched, serum-free defined medium resulted in morphological and neurochemical differentiation, with an enhancement of cholinergic phenotype. Hemicholinium-sensitive choline uptake activity was increased about sixfold over a 4-day period, with no change in choline acetyltransferase or acetylcholinesterase specific activity. Acetylcholine synthesis was increased in parallel with the changes in choline accumulation; choline metabolism in the differentiated cells differed significantly from that observed in the undifferentiated cells, with proportionally less converted to phosphorylcholine and proportionally more remaining as unmetabolized choline and converted to acetylcholine. The enhanced choline accumulation appeared to be mediated by an increased number of choline carriers, demonstrated by increased binding of the affinity ligand [3H]-choline mustard to the transporter and by an increased Vmax for the uptake process. The increased expression of the transport function appeared to be under transcriptional control, as the enhancement of uptake was blocked by the RNA polymerase II inhibitor alpha-amanitin as well as by the protein synthesis inhibitor cycloheximide. These results show that expression of sodium-coupled choline carriers and choline acetyltransferase may be regulated separately in the differentiating neuroblastoma LA-N-2 and that neurotransmitter synthesis is controlled by provision of precursor rather than at the level of the biosynthetic enzyme.