Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

A PCR test for detecting Escherichia coli O157:H7 based on the identification of the small inserted locus (SILO157)

AIMS: This paper provides identification of a DNA sequence derived from Shiga toxin-producing Escherichia coli (STEC) O157:H7 and information on its utilization for detecting STEC O157 by PCR. METHODS AND RESULTS: Random Amplified Polymorphic DNA and DNA library were used to identify in STEC O157:H7 (strain EDL 933) a 2634-bp Small Inserted Locus, designated SILO157. Analysis of 211 bacterial strains showed that the PCR assays amplifying the SILO157 region could be used to detect STEC O157 with a good specificity. CONCLUSIONS: Characterization of a novel locus in STEC O157 is attractive since the serotype O157:H7 of STEC is still by far the most important serotype associated with more serious diseases. This island encodes putative proteins and especially one that is predicted to be an outer membrane protein designated IHP1. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations could now be developed to appreciate the role of the SILO157 in pathogenicity.     

HPLC analysis of plant DNA methylation: a study of critical methodological factors.

HPLC analysis of nucleosides is important for determining total DNA methylation in plants and can be used to help characterise epigenetic changes during stress, growth and development. This is of particular interest for in vitro plant cultures as they are highly susceptible to genetic change. HPLC methodologies have been optimised for mammalian and microbial DNA, but not for plants. This study examines critical methodological factors in the HPLC analysis of plant DNA methylation using in vitro cultures of Ribes ciliatum. HPLC revealed that complete removal of RNA from plant DNA extractions is difficult using RNase (A and T1) digestions and LiCl precipitation. This suggests that base analysis should be avoided when using these RNA removal techniques, as bases from residual RNA fragments will inflate peak areas for DNA-derived bases. Nucleoside or nucleotide analysis is therefore recommended as a more suitable option as RNA and DNA constituents can be readily separated. DNA digestion was also a critical factor as methylation was under-estimated following incomplete nuclease digestion and over-estimated following incomplete phosphatase digestion. The units of enzyme required for complete DNA digestion was optimised and found to be 20-200 times less for nuclease P1 and 15 times less for alkaline phosphatase as compared with previous protocols. Digestion performance was conveniently monitored using marker peaks that indicate incomplete digestion products. This study identifies critical components of HPLC analysis and offers a comprehensive guide for the stringent analysis of DNA methylation in plants.     

Detection of Escherichia coli serogroup O103 by real-time polymerase chain reaction

AIMS: The aims of the study were to identify the specific genes of O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O103 and to provide the basis for a specific real-time PCR test for rapid detection of E. coli O103. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species, were used to amplify the 12-kbp O103 O-antigen biosynthesis locus of STEC O103. A DNA library representative of this cluster allowed two O103-specific probes to be identified in the flippase (wzx) and UDP-galactose-4-epimerase (galE) genes. Two specific O103 serotyping real-time PCR tests based on these two genes were successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen real-time PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of real-time PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.     

Dynamics of a memory trace: effects of sleep on consolidation

BACKGROUND: There is evidence that sleep is important for memory consolidation, but the underlying neuronal changes are not well understood. We studied the effect of sleep modulation on memory and on neuronal activity in a memory system of the domestic chick brain after the learning process of imprinting. Neurons in this system become, through imprinting, selectively responsive to a training (imprinting) stimulus and so possess the properties of a memory trace. RESULTS: The proportion of neurons responsive to the training stimulus reaches a maximum the day after training. We demonstrate that sleep is necessary for this maximum to be achieved, that sleep stabilizes the initially unstable, selective responses of neurons to the imprinting stimulus, and that for sleep to be effective, it must occur during a particular period of time after training. During this period, there is a time-dependent increase in EEG activity in the 5-6 Hz band, that is, in the lower range of the theta bandwidth. The effects of sleep disturbance on consolidation cannot be attributed to fatigue or to stress. CONCLUSIONS: We establish that long-term trace consolidation requires sleep within a restricted period shortly after learning. Undisturbed sleep is necessary for the stabilization of long-term memory, measured at the behavioral and neuronal levels, and of long-term but not short-term neuronal responsiveness to the training stimulus.     

Cold susceptibility and disinfestation of Bactrocera invadens (Diptera: Tephritidae) in oranges

To develop a cold disinfestation treatment for the fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) that is rapidly spreading across Africa, research was conducted in Nairobi, Kenya, using flies from a laboratory culture and 'Valencia' orange (Citrus sinensis L. Osbeck) as the host. The developmental rate of B. invadens in Valencia oranges was determined at 28 degrees C, and the third instar was found to be the least susceptible of the egg and larval life stages to cold treatment at 1.1 degrees C in oranges. When 22,449 B. invadens third instars were exposed in oranges to a cold treatment with an approximate midpoint of 1.1 +/- 0.5 degrees C, the results suggested that a period of 16 d would be worthwhile verifying on a larger scale in oranges. Results from the first replicate of 16,617 larvae showed no survivors, but the second replicate of 23,536 larvae had three survivors. Because a longer cold treatment based on a mean temperature of 1.1 degrees C would create logistical difficulties for some export markets, further replicates were conducted at an approximate midpoint of 0.5 degrees C and at mean hourly maximum of 0.9 +/- 0.5 degrees C, for 16 d. After three replicates, in which 65,752 B. invadens third instars in total were treated with no survivors, the Japanese requirement of 99.99% mortality at the 95% confidence level was surpassed. The following treatment protocol for B. invadens larvae in oranges can therefore be recommended: fruit pulp to be maintained at temperatures of 0.9 degrees C or lower for 16 consecutive days.     

Significance of nucleotide sequence alignments: a method for random sequence permutation that preserves dinucleotide and codon usage

The similarity of two nucleotide sequences is often expressed in terms of evolutionary distance, a measure of the amount of change needed to transform one sequence into the other. Given two sequences with a small distance between them, can their similarity be explained by their base composition alone? The nucleotide order of these sequences contributes to their similarity if the distance is much smaller than their average permutation distance, which is obtained by calculating the distances for many random permutations of these sequences. To determine whether their similarity can be explained by their dinucleotide and codon usage, random sequences must be chosen from the set of permuted sequences that preserve dinucleotide and codon usage. The problem of choosing random dinucleotide and codon-preserving permutations can be expressed in the language of graph theory as the problem of generating random Eulerian walks on a directed multigraph. An efficient algorithm for generating such walks is described. This algorithm can be used to choose random sequence permutations that preserve (1) dinucleotide usage, (2) dinucleotide and trinucleotide usage, or (3) dinucleotide and codon usage. For example, the similarity of two 60-nucleotide DNA segments from the human beta-1 interferon gene (nucleotides 196-255 and 499-558) is not just the result of their nonrandom dinucleotide and codon usage.