SUMMARYA viroid-like RNA was detected in nucleic acid preparations from two of the three commercial hop varieties grown in Spain. It had a size very close to that of avocado sunblotch viroid (ASBV), although dot-blot analysis revealed that it was very different in base sequence from ASBV, coconut cadang-cadang viroid, hop stunt viroid (HSV) and citrus exocortis viroid. In its physical and biological properties, the viroid-like RNA differed from the previously characterized HSV.Viroids are the only class of subviral agents endowed with autonomous replication whose molecular structure is well known. They comprise a single-stranded circular RNA which is highly self-complementary (Diener, 1979) and has a size range from 246 to 375 nucleotide residues (S~inger, 1984;Keese & Symons, 1985;Riesner & Gross, 1985). Viroids cause several diseases of higher plants (Riesner & Gross, 1985), although they can also replicate in other plants without detectable damage to the host (Owens et al., 1978). The last observation led some years ago to the proposal that an increasing number of viroid-like RNAs would be isolated from apparently healthy plants (Diener, 1979). This assumption has been confirmed recently in the case of grapevine (Sano et al., 1985(Sano et al., , 1986Flores et al., 1985). The detection of viroid-like RNAs has been greatly facilitated by the electrophoretic technique of Schumacher et aL (1983), which takes advantage of the very different mobilities of circular and linear RNA molecules of similar size in denaturing polyacrylamide gels. In the present communication we report that by applying this approach to RNA from hops, a new viroid-like RNA has been detected which is different from hop stunt viroid (HSV) described previously .Young leaves and flowers from individual hop plants (Humulus lupulus L.) were taken in the early summer of 1986 from different areas of Le6n (Spain). Samples were collected from the three hop varieties grown in Spain: H-3, H-7 and 'Fino de Alsacia'. Frozen tissue (20 g) was homogenized with a Willems Polytron (Kinematica, Kriens-Lucerne, Switzerland) in an extraction medium: 80 ml of water-saturated phenol, 16 ml 0.2 M-Tris-HC1 pH 8.9, 4 ml 0.1 M-EDTA pH 7, 4 ml 5 ~ SDS and 0.5 ml 2-mercaptoethanol (Semancik & Weathers, 1972). After centrifugation at 6000 g for 15 min the upper aqueous phases were re-extracted with 0.5 vol. of water-saturated phenol. The second aqueous phases were adjusted to a final volume of 40 ml with distilled water and the composition was adjusted to that of STE buffer (50 mM-Tris-HC1 pH 7.2, 100 mM-NaC1 and 1 mM-EDTA) and 35~ ethanol. Following the addition of 2 g of cellulose (CF-11, Whatman) (Franklin, 1966), the mixtures were gently shaken in centrifuge tubes for 10 to 15 min and centrifuged at 1000 g for 5 min. The cellulose pellets were washed three times with 60 ml of 35 ~ ethanol in STE and then with 20 ml of STE. The nucleic acids (and some other accompanying substances) eluted in the last wash were precipitated with 2.5 vol. of ethanol at -20 °C overnight,...

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.Identification of brewery bacterial isolates has traditionally been accomplished biochemically by determining the assimilation and fermentation patterns of a number of carbohydrates and nitrogen sources. Advances have been made in automating and improving detection times using biochemical methods. Both the Biolog system and API Rapid CH kits have proven to be useful for identifying beer spoilage lactic acid bacteria. However, biochemical identification is not accurate for determining the genotypic differences of microorganisms. A more accurate method for genotype determination is that of the molecular biological approach of ribotyping by comparing similarities in the rRNA gene sequences. A more recent ribotyping technique is the patented method called riboprinting. This method is based on restriction endonuclease digestion of bacterial chromosomal DNA, followed by Southern hybridization to probes for sequences in the regions of bacterial DNA coding for the 5S-16S-23S (the Escherichia coli rrnB rRNA operon) rRNA operon (1). The probes have been developed that are directed to highly conserved regions of the rRNA operon present in all eubacteria and can therefore be used for ribotyping most bacteria. Restriction fragments analyzed by probe hybridization range in size from approximately 1 to 50 kb, meaning that the fragments could potentially represen...

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The fermenting ability of Saccharomyces at low temperatures is crucial for the development of alcoholic beverages, but the key factors for the cold tolerance of yeast are not well known. In this report, we present the results of a screening for genes able to confer cold tolerance by overexpression in a laboratory yeast strain auxotrophic for tryptophan. We identified genes of tryptophan permeases (TAT1 and TAT2), suggesting that the first limiting factor in the growth of tryptophan auxotrophic yeast at low temperatures is tryptophan uptake. This fact is of little relevance to industrial strains which are prototrophic for tryptophan. Then, we screened for genes able to confer growth at low temperatures in tryptophan-rich media and found several genes related to phosphate uptake (PHO84, PHO87, PHO90 and GTR1). This suggests that without tryptophan limitation, uptake of inorganic phosphate becomes the limiting factor. We have found that overexpression of the previously uncharacterized ORF YCR015c/CTO1 increases the uptake of inorganic phosphate. Also, genes involved in ergosterol biosynthesis (NSG2) cause improvement of growth at 10°C, dependent on tryptophan uptake, while the gluconeogenesis gene PCK1 and the proline biosynthesis gene PRO2 cause an improvement in growth at 10°C, independent of tryptophan and phosphate uptake.