Calcium-dependent inhibition of renin secretion: TMB-8 is a non-specific antagonist

Intracellular Ca (Cai) is an inhibitory second messenger in renin secretion, and it has been hypothesized that some first messengers--especially angiotensin II [A-II] and antidiuretic hormone [ADH], and possibly A1-adenosine receptor antagonists as well--increase Cai and thereby inhibit renin secretion by causing the release or mobilization of Ca from intracellular sites of sequestration. The present experiments were designed to test this hypothesis, by using 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8), a putative antagonist of Ca release from intracellular sequestration sites. The rat renal cortical slices preparation was used. Basal renin secretory rate was unaffected by 1 and 10 microM TMB-8, but more than doubled in response to 100 microM TMB-8. Basal renin secretory rate was inhibited by A-II (1 microM), by ADH (200 units/1), by an A1-adenosine receptor agonist (N6-cyclohexyladenosine, or CHA; 0.5 microM), and by an alpha-adrenergic agonist (methoxamine; 10 microM). Only the inhibitory effect of methoxamine was blocked by 1 and 10 microM TMB-8, but these concentrations had no effect on basal secretory rate. At 100 microM, TMB-8 blocked the inhibitory effects of ADH as well as of methoxamine, but failed to block the inhibitory effects of CHA and A-II. However, these observations cannot be taken as evidence that methoxamine and ADH, but not CHA and A-II, inhibit renin secretion by a mechanism involving release of Ca from intracellular sequestration sites, because 100 microM TMB-8 clearly had non-specific effects. Among them, it completely blocked the inhibitory effect of K-depolarization on renin secretion. Collectively, at least three separate actions of TMB-8 must be invoked to explain the present results. Likely candidates are an Na-channel blocking effect and a Ca channel blocking effect in addition to antagonism of the release of Cai.     

Slipped-strand mispairing: a major mechanism for DNA sequence evolution

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing- over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.      

Complete sequences of the rRNA genes of Drosophila melanogaster

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.      

Statistical aspects of genetic mapping in autopolyploids.

Many plant species of agriculture importance are polyploid, having more than two copies of each chromosome per cell. In this paper, we describe statistical methods for genetic map construction in autopolyploid species with particular reference to the use of molecular markers. The first step is to determine the dosage of each DNA fragment (electrophoretic band) from its segregation ratio. Fragments present in a single dose can be used to construct framework maps for individual chromosomes. Fragments present in multiple doses can often be used to link the single chromosome maps into homologous groups and provide additional ordering information. Marker phenotype probabilities were calculated for pairs of markers arranged in different configurations among the homologous chromosomes. These probabilities were used to compute a maximum likelihood estimator of the recombination fraction between pairs of markers. A likelihood ratio test for linkage of multidose markers was derived. The information provided by each configuration and power and sample size considerations are also discussed. A set of 294 RFLP markers scored on 90 plants of the species Saccharum spontaneum L. was used to illustrate the construction of an autopolyploid map. Previous studies conducted on the same data revealed that this species of sugar cane is an autooctaploid with 64 chromosomes arranged into eight homologous groups. The methodology described permitted consolidation of 54 linkage groups into ten homologous groups.