Large-subunit ribosomal RNA-targeted probes forPseudo-nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole-cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996-1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole-cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor-binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis crossreacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole-cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells.

Pseudo-nitzschia austrulis Frenguelli is a marine pennatc diatom associated with the production of domoic acida neurocxcitatory amino acid linked to illness and mortality of humans and wildlife, Distinguishing P. austrulis from its co-occurring congeners is labor intensive and time consuming because of a requirement for scanning electron microscopy. Hcrc, we apply large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotides in wholecell and sandwich hybridization formats to identify and enumerate this species collected from pure cultures and natural populations. Whole-cell hybridization employed fluorescently labeled probes, filter-based sample processing, and epifluorescence microscopy to enumerate labeled cells. In contrast, sandwich hybridization was accomplished by homogenizing cells in a chaotropic solution and performing two hybridization reactions: capture of LSU rRNA using an oligonuclcotide coupled to a macroscopic solid support and binding of signal probe to a region of LSU rRNA near that of the capture site. Sandwich hybrids were detected calorimetrically; color intensity was proportional to the abundance of target species in the original sample. The sandwich hybridization assay was semiautomated with a robotic processor. Both whole-cell and sandwich hybridization are useful techniques for identifying P. australis as it occurs in nature. Sandwich hybridization potentially offers the most rapid and simple means to accomplish this task when screening large numbers of environmental samples.Fundamental challenges common to all studies of harmful algal blooms (HABs) are identifying, enumerating, and mapping the distributions of toxic species as they occur in nature. Recent workshops on HABs have recognized these problems and the need to develop novel techniques to speed and ease these tasks (Anderson et al. 1993;ECOHAB 1995). At present such operations are difficult because of the time and labor required to count potentially toxic species collected in discrete samples and the need to repeat such procedures hundreds of times in order to chart organisms' growth and movement. In this contribution we apply large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotide probes to detect and quantify Pseudo-nitzschia austrulis Frenguelli, a marine pennate diatom, collected from pure cultures and natural populations. This species is linked to the production of domoic acid (DA), a neuroexcitatory amino acid responsible for a human disorder known as amnesic shellfish poisoning (ASP: Per1 et al. 1990; Todd 1993).

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SynopsisA series of high-resolution melting curves were obtained by the continuous direct-derivative method [Blake, R. D. & Lefoley, S. G. (1978) Biochirn. Biophys. Acta 518,233-2461 on lambda DNA (~1~5 7 s~ strain) under varying conditions of "a+]. Examination of the denaturation patterns at close intervals of "a+] indicates that frequent changes in mechanism occur below 0.04M Na+, while almost none occurs above 0.1M Na+. Changes at low "a+] generally occur in an abrupt fashion, in most cases within a 3 mM change in "a+], and in at least one case within 0.6 mM, indicating the balance between alternative mechanisms is frequently quite delicate. These changes involve segments of between 900 and 1500 or more base pairs in length and are therefore not insignificant. Changes at low "a+] reflect a perturbation of the energetic balance between competing mechanisms by weakly screened long-range electrostatic forces. Some perturbation probably also arises from variations in the linear charge density of the double helix induced by the proximity of premelted loop segments; however, this contribution cannot be evaluated without a detailed denaturation map.At high "a+] the mechanism of melting is more conserved, permitting the dependence of subtransitional melting temperature t i ) on "a+] to be examined for almost all 34 f 2 subtransitions. The G + C composition of segments responsible for each subtransition was determined by a quantitative spectral method. relating AH,,, and d t i ) / d log [Na+] to the fraction of Na+ released during melting, appears to indicate almost 40% more Na+ is bound to the single-stranded G and/or C residues than to A and T residues. This is consistent with a much shorter mean axial spacing and higher charge density in the former, particularly single-stranded G residues, which have an extraordinary tendency to stack.

Filaggrin is an intermediate filament-associated protein that is involved in aggregation of keratin filaments in fully cornified cells of the mammalian epidermis, and is an important marker for epidermal differentiation. In this report, the sequence of a rat cDNA clone coding for a portion of the polymeric precursor, profilaggrin, is presented. The cDNA is 2,314 bp long with 1,875 bp of coding region ending with an A-T-rich 3' noncoding region. Genomic analysis indicates that the profilaggrin gene consists of 20 +/- 2 repeats of 1,218 bp of sequence coding for 406 amino acids, making the mRNA at least 25-27 kb in length. Each repeat consists of a filaggrin domain and a linker sequence with an estimated size of 380 and 26 amino acids, respectively. High levels of profilaggrin mRNA are found only in keratinizing epithelia. Comparison of the rat filaggrin sequence with that of mouse and human filaggrin and with the sequence of phosphorylated peptides from mouse profilaggrin indicates that the proteins share extensive amino acid sequence similarities, especially in the two phosphorylated regions. Proteolytic processing sites are also quite similar in rat and mouse. The three species show blocks of sequence that are similar in length and composition which alternate with sequences that are variable in length. This analysis suggests that the evolution of the present-day filaggrins has been constrained by maintenance of phosphorylation sites and overall amino acid composition. The cDNAs for the profilaggrins are similar in structure, reflecting genes that have simple repeating structures and lack introns within their coding regions. Mouse and rat profilaggrin terminate with a nonpolar sequence atypical of the rest of the coding region, and have similar 3' noncoding regions. To explain these observations, a novel evolutionary model is proposed.