Biodiversity of 52 chicken populations assessed by microsatellite typing of DNA pools

In a project on the biodiversity of chickens funded by the European Commission (EC), eight laboratories collaborated to assess the genetic variation within and between 52 populations from a wide range of chicken types. Twenty-two di-nucleotide microsatellite markers were used to genotype DNA pools of 50 birds from each population. The polymorphism measures for the average, the least polymorphic population (inbred C line) and the most polymorphic population (Gallus gallus spadiceus) were, respectively, as follows: number of alleles per locus, per population: 3.5, 1.3 and 5.2; average gene diversity across markers: 0.47, 0.05 and 0.64; and proportion of polymorphic markers: 0.91, 0.25 and 1.0. These were in good agreement with the breeding history of the populations. For instance, unselected populations were found to be more polymorphic than selected breeds such as layers. Thus DNA pools are effective in the preliminary assessment of genetic variation of populations and markers. Mean genetic distance indicates the extent to which a given population shares its genetic diversity with that of the whole tested gene pool and is a useful criterion for conservation of diversity. The distribution of population-specific (private) alleles and the amount of genetic variation shared among populations supports the hypothesis that the red jungle fowl is the main progenitor of the domesticated chicken.     

Design and structural analysis of an engineered thermostable chicken lysozyme.

A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (delta Tm approximately +10.5 degrees C) and chemical (delta Cm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 degrees C) to 4.0 (differential scanning calorimetry, 74 degrees C) kcal mol-1 greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with delta Tm = 3.3 degrees C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.     

Novel function of guanidine hydrochloride in reverse micellar extraction of lysozyme from chicken egg white

The efficiency of guanidine hydrochloride (GuHCl) addition in the suppression of gel formation and the extraction of lysozyme during reverse micellar extraction from chicken egg white was investigated. A low concentration of GuHCl in the feed permitted the successful extraction of lysozyme in its native form without gel formation, which is perceived as a novel function of GuHCl. The highest recovery and specific activity of lysozyme were obtained at a GuHCl concentration of 0.06 M in 25 mM AOT reverse micellar extraction from 20-fold-diluted natural chicken egg white. Lysozyme and ovalbumin CD spectra in the corresponding GuHCl aqueous solutions revealed no changes in the higher order structures of the proteins. Furthermore, the specific activity of lysozyme in the feed was well preserved in the GuHCl system. (c) 1995 John Wiley & Sons, Inc.     

Autosomal genetic control of the activity of a new variant ornithine transcarbamylase in chicken kidney

The mode of inheritance of the gene for chick kidney ornithine transcarbamylase (OTC), found previously as a genetic variant, was investigated. White Leghorn B line males homozygous for the allele for the variant OTC gene were selected using the California Gray breed, having a near-absolute deficiency of the enzyme. Then further crosses of the two breeds were made. The mean value of the OTC level of F₁ progeny was about 170 units. Chicks from the backcross generation were divided into two groups, of high activity and low activity, in a ratio of 1:1. F₂ chicks were divided into three groups: one-fourth of the chicks were classified as a super high group, one-half were high, and the remaining one-fourth were low the mean values for OTC level were 356.7, 196.4, and 15.6 units, respectively. From these results, it was suggested that the variant OTC represents a simple autosomal incompletely dominant trait.This work was supported in part by a grant-in-aid (No. 012207) for the scientific research from the Ministry of Education, Science, and Culture of Japan.     

中国家鸡的起源探讨

测定了6个家鸡品种30个个体的线粒体D0环区539bp的碱基序列,并与GenBank中的红原鸡、灰原鸡、绿原鸡、黑尾原鸡及鹌鹑的相应序列作比较分析,构建了分析系统树。结果表明,原鸡属4个种间差异较大,其中家鸡与泰国及邻近地区红原鸡关系最近,与印尼红原鸡、黑尾原鸡、灰原鸡、绿原鸡及鹌鹑的关系依次变远。提示中国家鸡可能起源于泰国及邻近地区的红原鸡。结果还显示,该原鸡的两个亚种（G.g.gallus和G.g.spadiceus)间的遗传分化程度显著低于家鸡亚种内的分化程度,这两个亚种似乎可以归为同一亚种。     

Comparison of SNPs and microsatellites for assessing the genetic structure of chicken populations

Many studies in human genetics compare informativeness of single‐nucleotide polymorphisms (SNPs) and microsatellites (single sequence repeats; SSR) in genome scans, but it is difficult to transfer the results directly to livestock because of different population structures. The aim of this study was to determine the number of SNPs needed to obtain the same differentiation power as with a given standard set of microsatellites. Eight chicken breeds were genotyped for 29 SSRs and 9216 SNPs. After filtering, only 2931 SNPs remained. The differentiation power was evaluated using two methods: partitioning of the Euclidean distance matrix based on a principal component analysis (PCA) and a Bayesian model‐based clustering approach. Generally, with PCA‐based partitioning, 70 SNPs provide a comparable resolution to 29 SSRs. In model‐based clustering, the similarity coefficient showed significantly higher values between repeated runs for SNPs compared to SSRs. For the membership coefficients, reflecting the proportion to which a fraction segment of the genome belongs to the ith cluster, the highest values were obtained for 29 SSRs and 100 SNPs respectively. With a low number of loci (29 SSRs or ≤100 SNPs), neither marker types could detect the admixture in the Gödöllö Nhx population. Using more than 250 SNPs allowed a more detailed insight into the genetic architecture. Thus, the admixed population could be detected. It is concluded that breed differentiation studies will substantially gain power even with moderate numbers of SNPs.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

Kinetic epitope mapping of the chicken lysozyme.HyHEL-10 Fab complex: delineation of docking trajectories.

The rate constants, k(on), for the formation of hen (chicken) lysozyme (HEWL). Fab-10 complexes have been determined for wild-type (WT) and epitope-mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow 10-fold range [(0.18 to 1.92) x 10(6) M(-1)s(-l)]. The affinity constants, K(D), cover a broader, 440-fold, range from 0.075 to 33 nM. Values of K(D) as high as 7 microM were obtained for the complexes prepared from some mutations at HEWL positions 96 and 97, but the associated kinetic constants could not be determined. The values of k(on) are negatively correlated with side-chain volume at position 101HEWL, but are essentially independent of this parameter for position 21HEWL substitutions. The multiple mutations made at positions 21HEWL and 101HEWL provide sufficient experimental data on complex formation to evaluate phi values [phi = (deltadeltaGon)/(deltadeltaG(D))] at these two positions to begin to define trajectories for protein-protein association. The data, when interpreted within the concept of a two-step association sequence embracing a metastable encounter complex intermediate, argue that the rate determining step at position 21HEWL (phiavg = 0.2) is encounter complex formation, but the larger phi(avg) value of 0.36 experienced for most position 101HEWL mutations indicates a larger contribution from the post-encounter annealing process at this site for these replacements.     

Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules.

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 degrees C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9 A) and I55L/S91T/D101S (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to delta delta G298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.     

溶菌酶的研究进展

溶菌酶普遍存在于动物、植物和微生物中。溶菌酶是一种小分子碱性蛋白,长期以来一直被作为一种“模型”体系．用于研究蛋白质的空间构象、酶动力学及其与分子进化、分子免疫间的关系。介绍了溶菌酶的来源、结构、性质、作用机制。并对近年来其在食品工业、医学和酶工程中的应用进行了综述;分析了溶菌酶应用中存在的主要问题,并对其应用前景进行了展望。     

C-terminus of TRAP in Staphylococcus can enhance the activity of lysozyme and lysostaphin

In Staphylococcus aureus , the target of RNAIII activating protein (TRAP) is a membrane-associated protein whose C-terminus can be used as a vaccine to provide protection against staphylococcal infection. Here, we show for the first time by surface plasmon resonance and enzyme-linked immunosorbent assay that TRAP can specifically bind lysozyme and lysostaphin through its C-terminus (amino acids 155–167) and enhance lysozomal activities in vitro . It was also found that the traP mutant strain is more resistant to lysostaphin than wild-type. Our previous data showed that the C-terminus of TRAP might be extracellular. So our results suggested that the C-terminus of TRAP could act as the specific targeting protein of the lysozyme/lysostaphin on the S. aureus cell wall and the biological significance of the interaction might be to facilitate lysozyme/lysostaphin-mediated cell lysis.     

中国红原鸡和泰国红原鸡遗传多样性分析

利用29个微卫星DNA标记对来自中国的红原鸡Gallus gallus spadiceus亚种和来自泰国的红原鸡Gallus gallus gallus亚种进行遗传多样性分析, 评估亚种内的遗传变异和亚种间的遗传分化, 结果表明: 共检测到168个等位基因, 每个位点的等位基因数从2到13不等, 所有位点平均的期望杂合度和PIC值分别为0.5780和0.53。中国和泰国红原鸡29个微卫星位点平均有效等位基因数分别为3.79和4.79, 平均基因杂合度为0.5379和0.6385, 两个红原鸡亚种均表现出较高的群体杂合度和丰富的遗传多样性。群体分化系数为19.4%(P<0.01), 两个红原鸡亚种间的Reynolds’遗传距离和Nm值分别为0.157和1.040。由此可见, Gallus gallus spadiceus亚种和Gallus gallus gallus亚种群体具有不同的群体遗传结构, 群体之间存在明显的遗传分化, 并不能将其认定为是同一亚种, 这也为中国家鸡具有独立的起源提供了一定的佐证。     

Environmental robustness and the adaptability of populations

Recent work has shown that genetic robustness can either facilitate or impede adaptation. But the impact of environmental robustness on adaptation remains unclear. Environmental robustness helps ensure that organisms consistently develop the same phenotype in the face of "environmental noise" during development. Under purifying selection, those genotypes that express the optimal phenotype most reliably will be selectively favored. The resulting reduction in genetic variation tends to slow adaptation when the population is faced with a novel target phenotype. However, environmental noise sometimes induces the expression of an alternative advantageous phenotype, which may speed adaptation by genetic assimilation. Here, we use a population-genetic model to explore how these two opposing effects of environmental noise influence the capacity of a population to adapt. We analyze how the rate of adaptation depends on the frequency of environmental noise, the degree of environmental robustness in the population, the distribution of environmental robustness across genotypes, the population size, and the strength of selection for a newly adaptive phenotype. Over a broad regime, we find that environmental noise can either facilitate or impede adaptation. Our analysis uncovers several surprising insights about the relationship between environmental noise and adaptation, and it provides a general framework for interpreting empirical studies of both genetic and environmental robustness.     

Surprisingly diverged populations of Saccharomyces cerevisiae in natural environments remote from human activity

The budding yeast, Saccharomyces cerevisiae, is a leading system in genetics, genomics and molecular biology and is becoming a powerful tool to illuminate ecological and evolutionary principles. However, little is known of the ecology and population structure of this species in nature. Here, we present a field survey of this yeast at an unprecedented scale and have performed population genetics analysis of Chinese wild isolates with different ecological and geographical origins. We also included a set of worldwide isolates that represent the maximum genetic variation of S. cerevisiae documented so far. We clearly show that S. cerevisiae is a ubiquitous species in nature, occurring in highly diversified substrates from human‐associated environments as well as habitats remote from human activity. Chinese isolates of S. cerevisiae exhibited strong population structure with nearly double the combined genetic variation of isolates from the rest of the world. We identified eight new distinct wild lineages (CHN I–VIII) from a set of 99 characterized Chinese isolates. Isolates from primeval forests occur in ancient and significantly diverged basal lineages, while those from human‐associated environments generally cluster in less differentiated domestic or mosaic groups. Basal lineages from primeval forests are usually inbred, exhibit lineage‐specific karyotypes and are partially reproductively isolated. Our results suggest that greatly diverged populations of wild S. cerevisiae exist independently of and predate domesticated isolates. We find that China harbours a reservoir of natural genetic variation of S. cerevisiae and perhaps gives an indication of the origin of the species.     

Co-segregation of alleles at a microsatellite locus within the macrophage expressed lysozyme gene and levels of serum lysozyme activity in two half-sib families of Polish Black and White Lowland cattle

Alleles at a microsatellite locus within the macrophage expressed lysozyme gene were shown to co-segregate with lysozyme activity in two half-sib families of Polish Black and White Lowland cattle. The bimodal distribution of lysozyme activities in both progeny groups is concordant with the occurrence of the alternative paternal alleles. The microsatellite is linked to a locus for high lysozyme activity that accounts for 70–95% of the phenotypic variation of both offspring groups considering the lysozyme activities of animals being older than 1 month.     

Subordination stress in Nile tilapia and its effect on plasma lysozyme activity

Dominant Nile tilapia had significantly higher lysozyme activity than did subordinate fishes (α=0·05). Plasma lysozyme level was not correlated with sex, parental origin, rearing length, weight, condition factor or rearing density.     

Microsatellite loci in Japanese quail and cross-species amplification in chicken and guinea fowl

In line with the Gifu University''s initiative to map the Japanese quail genome, a total of 100 Japanese quail microsatellite markers isolated in our laboratory were evaluated in a population of 20 unrelated quails randomly sampled from a colony of wild quail origin. Ninety-eight markers were polymorphic with an average of 3.7 alleles per locus and a mean heterozygosity of 0.423. To determine the utility of these markers for comparative genome mapping in Phasianidae, cross-species amplification of all the markers was tested with chicken and guinea fowl DNA. Amplification products similar in size to the orthologous loci in quail were observed in 42 loci in chicken and 20 loci in guinea fowl. Of the cross-reactive markers, 57.1% in chicken and 55.0% in guinea fowl were polymorphic when tested in 20 birds from their respective populations. Five of 15 markers that could cross-amplify Japanese quail, chicken, and guinea fowl DNA were polymorphic in all three species. Amplification of orthologous loci was confirmed by sequencing 10 loci each from chicken and guinea fowl and comparing with them the corresponding quail sequence. The microsatellite markers reported would serve as a useful resource base for genetic mapping in quail and comparative mapping in Phasianidae.     

Interspecific microsatellite markers for the study of pinniped populations

Microsatellites have rapidly become the marker of choice for a wide variety of population genetic studies. Here we describe 20 pinniped microsatellite markers which have been tested across 18 pinniped species. The majority of these markers have broad utility in all pinnipeds and provide a strong base for detailed population genetic studies in the Pinnipedia.     

Poststress levels of lysozyme and cortisol in adult rainbow trout: heritabilities and genetic correlations

Lysozyme response in stressed rainbow trout was compared with measurements of poststress cortisol activity. Estimates of heritability of lysozyme and cortisol were both moderate-to-high and both traits displayed positive genetic correlations in pair-wise comparisons of stress exposures. Genetic correlations between lysozyme and cortisol in stressed rainbow trout tended to be negative, although insignificant. Neither lysozyme, nor cortisol exhibited significant correlations with serum haemolytic activity. It is concluded that the data do not confirm earlier suggestions that lysozyme should be superior to cortisol in consistency of stress response in rainbow trout.