Fine mapping of the chicken salmonellosis resistance locus (SAL1)

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium that has a significant impact on both human and animal health. It is one of the most common food-borne pathogens responsible for a self-limiting gastroenteritis in humans and a similar disease in pigs, cattle and chickens. In contrast, intravenous challenge with S. Typhimurium provides a valuable model for systemic infection, often causing a typhoid-like infection, with bacterial replication resulting in the destruction of the spleen and liver of infected animals. Resistance to systemic salmonellosis in chickens is partly genetically determined, with bacterial numbers at systemic sites in resistant lines being up to 1000-fold fewer than in susceptible lines. Identification of genes contributing to disease resistance will enable genetic selection of resistant lines that will reduce Salmonella levels in poultry flocks. We previously identified a novel resistance locus on Chromosome 5, designated SAL1 . Through the availability of high-density SNP panels in the chicken, combined with advanced back-crossing of the resistant and susceptible lines, we sought to refine the SAL1 locus and identify potential positional candidate genes. Using a 6^th generation backcross mapping population, we have confirmed and refined the SAL1 locus as lying between 54.0 and 54.8 Mb on the long arm of Chromosome 5 ( F  = 8.72, P  = 0.00475). This region spans 14 genes, including two very striking functional candidates; CD27-binding protein ( Siva ) and the RAC -alpha serine/threonine protein kinase homolog , AKT1 ( protein kinase B , PKB ).     

A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L.

The effects of three different general purification protocols have been assessed quantitatively using mesophyll protoplasts of Brassica napus. Within the initial sample two distinct sub-populations were determined. The methods used influenced the ratio of the vacuolated to chloroplastic type protoplast sub-populations. Overall recovery rates of the initial sample varied according to the method used from 38% to 27%, but the relative recovery of the sub-populations varied considerably with a purified ratio of between 1.0:0.78 to 1.0:7.0. Size distribution profiles of the initial and purified populations are also presented.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

The Distribution and Identity of Assimilates in Tomato with Special Reference to Stem Reserves

Much of the work on the distribution of ¹⁴C-labelled assimilatesin tomato has been done in winter under low light intensities,and consequently the reported distribution patterns of ¹⁴C maynot be representative of plants growing in high light. Further,there are several somewhat conflicting reports on patterns ofdistribution of ¹⁴C-assimilates in young tomato plants. We soughtto clarify the situation by studying the distribution of ¹⁴C-assimilatesin tomato plants of various ages grown in summer when the lightintensity was high. In addition, the role of the stem as a storageorgan for carbon was assessed by (a) identifying the chemicalfractions in the stem internode below a fed leaf and monitoring¹⁴ C activity in these fractions over a period of 49 d, and(b) measuring concentrations of unlabelled carbohydrates inthe stem over the life of the plant. The patterns of distribution of ¹⁴C-assimilates we found fortomato grown under high light intensity confirmed some of thosedescribed for plants grown under low light, but export of ¹⁴Cby fed leaves was generally higher than reported for much ofthe earlier work. Lower leaves of young plants exported over50% of the ¹⁴C they fixed, although export fell sharply as theplants aged. Initially, the roots and apical tuft were strongsinks for assimilates, but they had declined in importance bythe time plants reached the nine-leaf stage. On the other hand,the stem became progressively more important as a sink for ¹⁴C-assimilates.Older, lower leaves exported more of their ¹⁴C-assimilates tothe upper part of the plant than to the roots, whereas youngleaves near the top of the plant exported more of their assimilatesto the roots. The stem internode immediately below a fed leafhad about twice the ¹⁴C activity of the internode above theleaf. Mature leaves above and below a fed leaf rarely importedmuch ¹⁴C, even when in the correct phyllotactic relationshipto the fed leaf. In the first 3 d after feeding leaf 5 of nine-leaf plants, theorganic and amino acid pools and the neutral fraction of theinternode below the fed leaf had most of the ¹⁴C activity, butby 49 d after feeding, the ethanolic-insoluble, starch and lipidfractions had most of the ¹⁴C activity. Glucose, fructose andsucrose were the main sugars in the stem. Although concentrationsof these sugars and starch declined in the stem as the plantsmatured, there was little evidence to indicate their use infruit production. Stems of plants defoliated at the 44-leafstage had lower concentrations of sugars and starch at maturity,and produced less fruit than the controls. It was concludedthat tomato is sink rather than source limited with respectto carbon assimilates, and that the storage of carbon in thestem for a long period is possibly a residual perennial traitin tomato.Copyright 1994, 1999 Academic Press Lycopersicon esculentum, tomato, assimilate distribution, ¹⁴C, internode storage, sink-source relationships, starch, stem reserves, sugars     

Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.