An Improved Enrichment Broth for Isolation of Escherichia coli O157, with Specific Reference to Starved Cells, from Radish Sprouts

An enrichment broth was developed for the efficient isolation of Escherichia coli O157 from radish sprouts. The broth was buffered peptone water containing 0.5% sodium thioglycolate (STG-BPW), which was designed to allow growth of E. coli O157 in starved and unstarved states. However, this medium suppressed the growth of non-carbohydrate-fermenting obligate aerobes whose colonial appearance on sorbitol MacConkey agar containing cefixime and tellurite (CT-SMAC) resembled that of E. coli O157. Both starved and unstarved cells of E. coli O157 experimentally inoculated into radish sprouts were successfully recovered with STG-BPW enrichment in all cases, most of which showed marked disappearance of E. coli O157-like colonies on CT-SMAC.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Freemartinism among singleton bovine females born from multiple embryo transfer

Heterosexual chimerism among singleton females produced by multiple nonsexed embryo transfer (MNET singleton females) was investigated using chromosome typing and PCR (polymerase chain reaction)-amplification of male-specific DNA (msDNA). Of the 22 animals tested, 21 were classified as normal by both methods (i.e., showing no male cells among 100 metaphase spreads in chromosome typing and being msDNA negative in PCR). No morphological abnormalities of the genital organs were observed among 19 MNET single females. One MNET singleton female was, however, classified as a freemartin by PCR (male-specific DNA positive), but it was classified as normal cytogenetically. This individual probably had a low degree of heterosexual chimerism, and it seems that the chimerism derived from MNET was difficult to diagnose by chromosome typing, although it was detectable by PCR. The genital organs of this individual (15-mo-old Aberdeen Angus) were normal in form (both external and internal) and size. However, a very small structure, resembling seminiferous tubule, was found in the left ovary. It may be concluded that most MNET singleton females are expected to have normal reproductive function.     

Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium

Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDC _ST on the chromosome and samAB on a 60-MDa cryptic plasmid. The roles of theumuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S. typhimurium were investigated. Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,-8DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e. 2-nitrofluorene (2NF) and 2-amino- 3-methyldipyrido[1,2-a:3 ,2-d]imi-dazole (Glu-P-1). Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined. Deletion of umuDC _STSubstantially lowered the reversion rate induced by l-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF). Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens. DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052. These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by l-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.     

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Cells of Nitella flexilis were made inexcitable by treatmentwith 10 mM KCl for more than 24 h. A Ca²⁺-sensitive photoproteinaequorin was injected into the cytoplasm of such cells. Forvacuolar per fusion, the central part of an aequorin-loadedcell was immersed in silicone oil, and both cell ends bathedin the perfusion medium were cut off. A large light emissionfrom aequorin was observed when the vacuole was perfused witha hypotonic medium whose osmotic pressure was adjusted to halfof the osmotic pressure of the cell sap. This shows that hydrationof the cytoplasm triggers release of Ca²⁺ from internal stores,since influx of Ca²⁺ from silicone oil is excluded. Hydration of cells was induced in another way. Cells were firstdehydrated by transferring them from 10 mM KCl solution to thatwith 250 mM sorbitol added. This procedure did not affect thecytoplasmic streaming. When cells were rehydrated by transferringthem to 10 mM KCl solution, cytoplasmic streaming was eitherstopped or slowed down in a few seconds. A quick light emissionfrom aequorin was observed in the rehydration, evidence thatcytoplasmic streaming was inhibited by an increase in the cytoplasmicCa²⁺ concentration. Both streaming cessation and aequorin lightemission were observed even in KCl-treated cells which werefurther treated with 5 mM EGTA. Thus, the increase in Ca²⁺ isconcluded to be caused by the release of Ca²⁺ from internalstores. These results support our previous hypothesis [Tazawa et al.(1994) Plant Cell Physiol. 35:63] that, in Nitella flexilis,the increase in the concentration of Ca²⁺ in the cytoplasm whichoccurs on the endoosmotic side of the cell during transcellularosmosis is caused by hydration of the cytoplasm. (Received June 6, 1994; Accepted December 26, 1994)     

Water Soluble Pigments Containing Xylose and Glucose in Gametangia of the Green Alga, Bryopsis maxima

Several water-soluble pigments were purified from gametangiaof Bryopsis maxima by liquid chromatography and characterizedby pyridylamination and high-performance anion-exchange chromatography.The structure of the main red pigment is proposed based on thedata of infrared spectrum, Mass spectrum, ¹H and ¹³C NMR spectraand pyridylamino analysis. As a consequence, this pigment containeda tetrapyrrole with phytol and a sugar chain comprised of xyloseand glucose. The sequence of the sugars in the chain was determinedbased on its Mass spectrum. The pigment was similar to chlorophyll-originpigments observed in other plants. No aldehyde group, however,was present at C5 in the open tetrapyrrole chain. (Received August 3, 1994; Accepted November 10, 1994)     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni