Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+ o exchanges for bath Na+ i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+ i affinities and current–voltage relationships, significant differences were observed in their Na+ i- and Ca2+ i-dependent regulatory properties. Both isoforms underwent Na+ i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+ i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+ i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+ i, but their responses to regulatory Ca2+ i differed markedly. For both isoforms, the application of cytoplasmic Ca2+ i led to a decrease in outward exchange currents. This negative regulation by Ca2+ i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+ i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca2+ i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca2+ i occurred by a direct mechanism and indirectly through effects on Na+ i-induced inactivation. Our results show that regulatory Ca2+ i decreases Na+ i-induced inactivation of CALX1.2, whereas it stabilizes the Na+ i-induced inactive state of CALX1.1. These effects of Ca2+ i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

Cytoplasmic Na

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About one-half of the ribosomal repeat unit of two isolates of Pythium ultimum was amplified by means of the polymerase chain reaction using one primer pair. The amplified region includes a small part of the large subunit ribosomal RNA gene, about half of the small subunit ribosomal RNA gene, and the entire intergenic region. The intergenic region of both isolates of P. ultimum has length heterogeneity due to the presence of subrepeat arrays (Klassen and Buchko 1990). PCR amplification of the heterogeneous target DNA resulted in sets of fragments which accurately reflect the heterogeneity in the target DNA, although there is a preferential amplification of the smaller targets. PCR product sizes ranged from 4.6 to 5.8 kb.

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The 5S ribosomal RNA (rRNA) genes in eukaryotes may occur either interspersed with the other rRNA genes in the ribosomal DNA (rDNA) repeat, or in separate tandem arrays, or dispersed throughout the genome. In Pythium species and in several related Oomycetes, polymerase chain reaction (PCR) amplification of the nontranscribed spacer (NTS) region with one primer specific for the 5S gene revealed, with several exceptions, that the 5S rRNA gene was present in the rDNA repeat of those species with filamentous sporangia and was absent from the rDNA repeat of those with globose or unknown sporangia. When present, the gene was located approximately 1 kb downstream of the large-subunit rRNA gene and on the strand opposite that on which the other rRNA genes were located. This was confirmed in P. torulosum by sequencing of the gene and its flanking regions. The 5S rRNA genes of P. ultimum were found in tandem arrays outside the rDNA repeat. Evidence of dispersed 5S rRNA sequences was also observed. For many of the species of Pythium having the 5S gene in the NTS, the region between the large-subunit rRNA gene and the 5S gene was found to have length heterogeneity. Oomycetes related to Pythium were also found to have the 5S gene in the NTS, although sometimes in the opposite orientation. This may mean that the presence of the gene in the NTS is ancestral for the Oomycetes and that the absence of the gene in the NTS in those Pythiums with globose sporangia is due to loss of the gene from the rDNA repeat in an ancestor of this group of species. These results favor the view that 5S rRNA gene linkage to the rRNA cistron existed prior to the unlinked arrangement seen in most plants and animals.