Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression.

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding alpha subunits, and the other is a structural beta subunit. The existence of at least one more nAChR gene, probably encoding a beta subunit, is indicated. Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. localpha1, localpha3, and locbeta1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Loc

Cloning of the human NCNF gene.

We have cloned from a cDNA library of human testis tissue the human homologue to the mouse nuclear orphan receptor NCNF (neuronal cell nuclear factor). The open reading frame encodes a protein of 480 amino acids, the sequence of which (EMBL accession no. X99975) is 98.3% identical to the mouse homologue. Northern blot analysis of adult human tissues revealed a broad pattern of tissue expression. Similar to NCNF expression in mouse testis, two transcript forms of the single copy gene are expressed in human tissues. The two transcript forms which differ only in their 3'UTR, result in human from differential polyadenylation, in mouse from alternative splicing. Based on the high level of sequence identity of human and murine NCNF, it is likely that also the human nuclear receptor is involved in the control of neurogenesis and gametogenesis.

Neuronal cell nuclear factor--a nuclear receptor possibly involved in the control of neurogenesis and neuronal differentiation.

We have cloned from a cDNA library of neuronal derivatives of retinoic-acid-induced embryonic carcinoma cells a nuclear receptor that may be involved in the control of late neurogenesis and early neuronal differentiation. The receptor which is practically identical in sequence with germ cell nuclear factor, has been designated neuronal cell nuclear factor (NCNF). NCNF is exclusively expressed in the neuronal derivatives of PCC7-Mz1 cells, with the expression beginning within hours of exposure to retinoic acid. In the developing mouse brain, NCNF is expressed in the marginal zones of the neuroepithelium which are known to contain young postmitotic neurons. NCNF binds to the DR0 sequence thereby silencing transcription. Because NCNF does not recognize hormone response elements of other nuclear receptors tested and does not heterodimerize with these, it probably binds exclusively as a homodimer. NCNF may induce neuronal differentiation by repressing the activity of genes that permit cell fates other than the neuronal one.

Cloning of several genes coding for retinoic acid nuclear receptors in the mouse embryonal carcinoma cell line PCC7-MZ1.

Mouse embryonal carcinoma cell line PCC7-Mz1 can be induced by retinoic acid (RA) to differentiate into several well defined phenotypes of neuroectodermal origin (Lang, E. et al. (1989) J. Cell. Biol. 109, 2481-2493). Several subclones of the cell line (clonal variants) differ from each other in their developmental potential. To test whether these differences in cellular fate are due to somatic mutations in specific genes of these cells, we have cloned full length cDNAs coding for the alpha 1 and beta 2 isoforms, and partial length cDNAs coding for the alpha 2, beta 1 and beta 3 isoforms of the retinoic acid nuclear receptor (RAR). The cloned cDNAs did not differ in sequence from those of normal mouse cells. Using as probe the beta 2-RAR promoter region from mouse liver, we also checked for restriction fragment length polymorphism in the promoter regions of RA-inducible and RA-resistant cell variants. No alterations in this region of RAR genes was found in the clonal variants tested. The different patterns of derivatives produced by the variants upon exposure to RA therefore cannot be caused by somatic mutations in RAR genes of the tumor cell lines.

Expression of retinoic acid nuclear receptors in the mouse embryonal carcinoma cell line PCC7-Mz1.

Mouse embryonal carcinoma cell line PCC7-Mz1 can serve as a model of mammalian neural development [1989, J. Cell. Biol. 109, 2481-2493]. Upon exposure to all-trans retinoic acid (RA), Mz1 cells differentiate into a stable pattern of neurons, astroglia and fibroblasts whereas variants of the parental cell line either are restricted in their patterns of derivatives or do not respond at all to RA. Using gene probes specific for the alpha 1, alpha 2 and beta 2 isoforms of the retinoic acid nuclear receptor, we have studied by Northern blot analysis the expression of these transcription factors in uninduced and induced cells of clone Mz1 and in variants with different developmental potential. alpha 1-RAR is expressed constitutively in all variants independent of whether RA is present or not. Soon after addition of 10(-7) M RA, alpha 2-RAR is induced in RA-responsive cells reaching within a few hours a plateau level that remains unchanged throughout the developmental process. In contrast, the beta 2 isoform is expressed only transiently after RA-induction despite the continuous presence of RA. Other RAR isoforms are expressed only in trace amounts.

In vitro transcription with extracts of nuclei of Drosophila embryos.

An in vitro transcription system has been developed from 0.3M NaCl extracts of nuclei of Drosophila embryos. Optimal transcription in the Drosophila embryo extract (DEX) was at 5mM MgCl2, 70mM KCl, 25 degrees C and with promoter concentrations of 0,75-1.0 pmol/assay. In vitro transcription from the Adenovirus-2 major late and the Drosophila histone gene promoters was studied in particular. S1-nuclease protection experiments showed that in vitro transcription from these promoters was accurate. In vitro transcription from the Adenovirus-2 major late promoter was less efficient than from histone gene H3 and H4 promoters in DEX. Vicecersa, in vitro transcription from Adenovirus-2 major late promoter was more efficient in HeLa whole cell extracts. The efficiencies of transcription from histone gene promoters decreased in DEX in the order H4 greater than or equal to H3 greater than H2a. Transcription from H2b and H1 promoters was not detected in DEX. The transcription from the Adenovirus-2 major late promoter was completely inhibited by histone H3 and H4 promoters. Preincubation of DEX with the adenoviral template, however, did not inhibit transcription from histone H3 and H4 promoters. The transcription start sites of histone genes H3 and H4 are separated by 160 base pairs. The H3 and H4 transcription start sites were subcloned separately. Now, a competition of transcription from the H3/H4 promoters with the Adenovirus-2 major late promoter was observed. The competition studies suggest that preincubation of DEX with the adenoviral template inhibited transcription from the H3 promoter more strongly than from the H4 promoter.

Transcription of cloned tRNA and 5S RNA genes in a Drosophila cell free extract.

We describe the preparation of a cell-free extract from Drosophila Kc cells which allows transcription of a variety of cloned eukaryotic RNA polymerase III genes. The extract has low RNA-processing nuclease activity and thus the major products obtained are primary transcripts.