Genotypes of sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase II gene in substrains of spontaneously hypertensive rats.

OBJECTIVE: To search for a genetic marker of the sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (SERCA) II gene in spontaneously hypertensive rats (SHRs) and to investigate differences in blood pressure and intracellular Ca2+ among some substrains of SHRs and Wistar-Kyoto (WKY) rats related to their SERCA II genotypes. DESIGN AND METHODS: The coding region of the SERCA II gene was sequenced in SHRs. Blood pressure and intracellular Ca2+ concentration ([Ca2+]i) in platelets were measured in substrains of SHRs and WKY rats with different SERCA II genotypes. RESULTS: A point mutation that provided restriction fragment length polymorphisms (RFLPs) by HindIII or Saul was found in the SERCA II gene. The polymerase chain reaction (PCR) products were digested by HindIII in SHR substrains and WKY-Kyoto rats, whereas they were digested by Saul in normotensive strains and SHR-Toho. Among SHR-Kyoto, SHR-Toho, WKY-Kyoto and WKY-Charles River, the substrains with the HindIII-digested SERCA II genotype showed slightly but significantly higher systolic blood pressure and augmented agonist-stimulated [Ca2+]i than those with the Saul-digested genotype. CONCLUSIONS: RFLPs were found in the SERCA II gene. In the substrain analysis of SHRs and WKY rats, higher blood pressure and increased [Ca2+]i were associated with the SERCA II genotype digested by HindIII. The SERCA II gene locus has the potential to contribute to the development of hypertension and abnormal intracellular Ca2+ metabolism in SHRs. These RFLPs in the SERCA II gene should be a useful genetic marker.

Expression of a MADS box gene, MEF2D, in neurons of the mouse central nervous system: implication of its binary function in myogenic and neurogenic cell lineages.

MEF2D, a member of myocyte-specific enhancer binding factor 2 (MEF2) gene family, was shown by Northern blot hybridization to be strongly expressed in the head portion of mouse embryos at later stages of ontogenesis, in the cerebellum and the cerebrum of adult mice, in cultured cell lines of neuronal origin, and in skeletal and cardiac muscles. During ontogenesis, MEF2D transcripts were detected by in situ hybridization in the olfactory bulb, entorhinal cortex, pyriform cortex, and hippocampus, in Purkinje and granule cells, and in large neurons in both the ventral and dorsal horns of spinal cord. Adult mice continued to express MEF2D in these limited areas of the central nervous system. Thus, MEF2D seems to be involved in either the differentiation process or the function of these neurons.

Spatial and temporal regulation of the rat calmodulin gene III directed by a 877-base promoter and 103-base leader segment in the mature and embryonal central nervous system of transgenic mice.

Three non-allelic rat calmodulin (CaM) genes CaMI, CaMII and CaMIII, which share no homology in their 5'-upstream regions, are coordinately expressed in neurons of the central nervous system (CNS). Deletion analysis of the CaMIII promoter showed that the upstream segments longer than 700 bases functioned as efficient promoters, and that the sequence from -133 to -65 was required for the activity of house-keeping type promoter in transient expression assays on a mouse glioma cell line C6. However, the transient expression seemed not to be cell type specific. To determine the temporal and spatial specificity of the promoter function, we produced transgenic mice carrying a fusion gene of the CaMIII segment from -877 to +103 and the lacZ reporter gene. In CNS of the adult transgenic mice, the localization of transgene expression was similar to that of endogenous CaMIII transcripts analyzed by in situ hybridization. The transgene was expressed prominently in pyramidal cells of the cerebral neocortex and the hippocampal regions CA1 to CA3, in Purkinje cells of the cerebellar cortex, and in neurons of the spinal cord, and moderately in granule cells of the dentate gyrus and the cerebellar cortex. In the developing CNS, the overall profiles of neuron-specific expression were also similar for both transgene and endogenous CaMIII that were expressed in the mantle layer and the dorsal root ganglia of the embryonal spinal cord. These results indicated that the neuron-specific expression of rat CaMIII was directed by this 877-base promoter sequence. The CaMIII segment used for the promoter of transgene contained a 29-bp sequence at -410, namely H3, which was conserved in the upstream regions of vertebrate CaMII and CaMIII. H3 seemed to play a pivotal role in the temporal and spatial expression of transgene in CNS, although the deletion of H3 did not decrease CAT activity in the transient expression. The transgene expression was not observed in the external granular cells of the developing cerebellum and in some neurons of the embryonic sensory ganglia in which the endogenous CaMIII was obviously expressed. Therefore, the other cis-acting element(s) located outside of this 877-bp segment seemed to be required for the temporal regulation of CaMIII in certain rudimentary neurons.

Spermatocyte-specific transcription by calmodulin gene II promoter in transgenic mice.

Transgenic mice carrying a fused gene of the 294-base upstream and 68-base leader sequences of a rat calmodulin gene, CaMII, and beta-galactosidase gene were made. Only spermatocytes expressed the transgene mRNA in the testes of four independent transgenic lines. The localization of transgene mRNA was consistent with that of the mouse endogenous CaMII analyzed by in situ hybridization with the probe of 3'-noncoding region of mouse CaMII. Thus, this short promoter of CaMII evidently conferred the expression of transgene only on spermatocytes but not on spermatogonia nor on spermatids of the testis. The rat CaMII promoter up to -294 contained no sequences that corresponded to any of the reported sequence features of genes expressed in the testis. Therefore, this short promoter region of CaMII seemed to carry a certain novel machinery for the spermatocyte-specific gene expression.

Expression of the rat calmodulin gene II in the central nervous system: a 294-base promoter and 68-base leader segment mediates neuron-specific gene expression in transgenic mice.

Deletion analysis of the rat CaMII promoter demonstrated that the segment from -294 to +68 bases of CaMII was efficient as a promoter in NIH3T3 by transient assay. We developed transgenic mice carrying a fusion gene of this promoter segment and a beta-galactosidase reporter gene. This short CaMII promoter mediated the transgene expression in pyramidal cells of the cerebral neocortex, the pyriformcortex and the hippocampal regions CA1 to CA3, in granule cells of the dentate gyrus, in Purkinje cells of the cerebellum, and in neurons of the lateral vestibular nucleus of pons and the spinal cord of adult transgenic mice. The expression of endogenous CaMII was precisely analyzed by in situ hybridization in the nervous tissues. The localization of transgene expression was consistent with those of the endogenous CaMII in the adult transgenic mice. In the embryos at 13.5-15.5 days of gestation, the transgene was expressed in various neurons similarly to the endogenous CaMII but certain subtle differences were observed in the localization of expression. This short promoter of rat CaMII carried two sequence stretches highly conserved in the mouse, dog, chicken and Xenopus CaMII promoters. These conserved stretches may be involved in the observed neuron-specific expression of rat CaMII gene.

Expression of three nonallelic genes coding calmodulin exhibits similar localization on the central nervous system of adult rats.

By Northern blot analysis with the digoxigenin-labeled antisense RNA probes of the noncoding regions, the transcripts of three calmodulin (CaM) genes, CaMI, CaMII, and CaMIII, were separately detected in 12 different tissues of adult Wistar albino rats, without any cross-hybridization. The mRNAs of all three CaM genes were abundant in the central nervous system (CNS) as well as in the testis, although ubiquitous expression was detected at low levels in the other tissues. There were subtle but significant differences in the tissue-specific distribution of the three CaM gene RNAs. By in situ hybridization, strong hybridization of the three CaM gene probes was observed in common in large projection neurons of the CNS: the hippocampal pyramidal cells, the cerebellar Purkinje cells, and the large neurons of the cerebral neocortex, the pyriform cortex, the mesencephalon, the pons, and the spinal cord. The expression of the three CaM genes was at lower levels in small interneurons of the CNS. These profiles of expression were almost the same among the three CaM genes. Thus, all three CaM genes were coordinately expressed in neurons of the adult rat CNS. Certain regulatory mechanisms of the three CaM genes seemed to mediate similar tissue- and cell type-specific expression in the CNS.

Four synonymous genes encode calmodulin in the teleost fish, medaka (Oryzias latipes): conservation of the multigene one-protein principle.

We cloned four distinct calmodulin (CaM)-encoding cDNAs from a small teleost fish, medaka (Oryzias latipes). The deduced amino acid (aa) sequences were exactly the same in these four genes and identical to the aa sequence of mammalian CaM, because of synonymous codon usages. The four cDNAs from medaka, termed CaM-A, -B, -C and -D, corresponded to mRNAs of 1.8, 1.4, 2.5 and 1.8 kb, respectively, in Northern blot analysis. Our results demonstrated that the 'multigene one-protein' principle of CaM synthesis is applicable to medaka, as well as to mammals whose CaM is encoded by at least three different genes.