Whole-genome scan to detect quantitative trait loci associated with milk protein composition in 3 French dairy cattle breeds.

In the context of the PhénoFinLait project, a genome-wide analysis was performed to detect quantitative trait loci (QTL) that affect milk protein composition estimated using mid-infrared spectrometry in the Montbéliarde (MO), Normande (NO), and Holstein (HO) French dairy cattle breeds. The 6 main milk proteins (α-lactalbumin, ß-lactoglobulin, and αS1-, αS2-, ß-, and κ-caseins) expressed as grams per 100g of milk (% of milk) or as grams per 100g of protein (% of protein) were estimated in 848,068 test-day milk samples from 156,660 cows. Genotyping was performed for 2,773 MO, 2,673 NO, and 2,208 HO cows using the Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA). Individual test-day records were adjusted for environmental effects and then averaged per cow to define the phenotypes analyzed. Quantitative trait loci detection was performed within each breed using a linkage disequilibrium and linkage analysis approach. A total of 39 genomic regions distributed on 20 of the 29 Bos taurus autosomes (BTA) were significantly associated with milk protein composition at a genome-wide level of significance in at least 1 of the 3 breeds. The 9 most significant QTL were located on BTA2 (133 Mbp), BTA6 (38, 47, and 87 Mbp), BTA11 (103 Mbp), BTA14 (1.8 Mbp), BTA20 (32 and 58 Mbp), and BTA29 (8 Mbp). The BTA6 (87 Mbp), BTA11, and BTA20 (58 Mbp) QTL were found in all 3 breeds, and they had highly significant effects on κ-casein, ß-lactoglobulin, and α-lactalbumin, expressed as a percentage of protein, respectively. Each of these QTL explained between 13% (BTA14) and 51% (BTA11) of the genetic variance of the trait. Many other QTL regions were also identified in at least one breed. They were located on 14 additional chromosomes (1, 3, 4, 5, 7, 15, 17, 19, 21, 22, 24, 25, 26, and 27), and they explained 2 to 8% of the genetic variance of 1 or more protein composition traits. Concordance analyses, performed between QTL status and sequence-derived polymorphisms from 13 bulls, revealed previously known causal polymorphisms in LGB (BTA11) and GHR (BTA20 at 32 Mbp) and excluded some other previously described mutations. These results constitute a first step in identifying causal mutations and using routinely collected mid-infrared predictions in future genomic selection programs to improve bovine milk protein composition.

Detection of quantitative trait loci for resistance to gastrointestinal nematode infections in Creole goats.

This study aimed to identify regions of the genome affecting resistance to gastrointestinal nematodes in a Creole goat population naturally exposed to a mixed nematode infection (Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum) by grazing on irrigated pasture. A genome-wide quantitative trait loci (QTL) scan was performed on 383 offspring from 12 half-sib families. A total of 101 microsatellite markers were genotyped. Traits analysed were faecal egg count (FEC), packed cell volume (PCV), eosinophil count and bodyweight (BW) at 7 and 11 months of age. Levels of activity of immunoglobulin A (IgA) and activity of immunoglobulin E (IgE) anti-Haemonchus contortus L3 crude extracts and adult excretion/secretion products (ESPs) were also analysed. Using interval mapping, this study identified 13 QTL for parasite resistance. Two QTL linked with FEC were found on chromosomes 22 and 26. Three QTL were detected on chromosomes 7, 8 and 14 for eosinophil counts. Three QTL linked with PCV were identified on chromosomes 5, 9 and 21. A QTL for BW at 7 months of age was found on chromosome 6. Lastly, two QTL detected on chromosomes 3 and 10 were associated with IgE anti-L3, and IgE anti-ESP was linked with two QTL on chromosomes 1 and 26. This study is the first to have identified regions of the genome linked with nematode resistance in a goat population using a genome scan. These results provide useful tools for the understanding of parasite resistance in small ruminants.

Microsatellite mapping of quantitative trait loci affecting female reproductive tract characteristics in Meishan x Large White F(2) pigs.

A QTL analysis of female reproductive data from a 3-generation experimental cross between Meishan and Large White pig breeds is presented. Six F(1) boars and 23 F(1) sows, progeny of 6 Large White boars and 6 Meishan sows, produced 502 F(2) gilts whose reproductive tract was collected after slaughter at 30 d of gestation. Five traits [i.e., the total weight of the reproductive tract, of the empty uterine horns, of the ovaries (WOV), and of the embryos], as well as the length of uterine horns (LUH), were measured and analyzed with and without adjustment for litter size. Animals were genotyped for a total of 137 markers covering the entire porcine genome. Analyses were carried out based on interval mapping methods, using a line-cross regression and a half-full sib maximum likelihood test. A total of 18 genome-wide significant (P < 0.05) QTL were detected on 9 different chromosomes (i.e., SSC 1, 5, 6, 7, 9, 12, 13, 18, and X). Five genome-wide significant QTL were detected for LUH, 4 for weight of the empty uterine horns and WOV, 2 for total weight of the reproductive tract, and 1 for weight of the embryos. Twenty-two additional suggestive QTL were also detected. The largest effects were obtained for LUH and WOV on SSC13 (9.2 and 7.0% of trait phenotypic variance, respectively). Meishan alleles had both positive (e.g., on SSC7) and negative effects (e.g., on SSC13) on the traits investigated. Moreover, the QTL were generally not fixed in founder breeds, and opposite effects were in some cases obtained in different families. Although reproductive tract characteristics had only a moderate correlation with reproductive performances, most of the major QTL detected in this study were previously reported as affecting female reproduction, generally with reduced significance levels. This study thus shows that focusing on traits with high heritability might help to detect loci involved in low heritability major traits for breeding.

Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie.

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH(154)RQS, GIRQ(211)QS, GIRRK(222)S and GIRRQP(240)) derived from the wild-type allele (G(127)I(142)R(154)R(211)Q(222)S(240)) by a single-codon mutation, and two (S(127)IRRQP(240) and GM(142)RRQP(240)) by a double-codon mutation. A case-control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I(142)R(154)R(211)Q(222)/IRRQ goats, the odds of scrapie cases in IRQ(211)Q/IRRQ and IRRK(222)/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M(142)RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP(240) homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5-18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Detection of quantitative trait loci for teat number and female reproductive traits in Meishan × Large White F2 pigs.

A quantitative trait locus (QTL) analysis of female reproductive data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six F1 boars and 23 F1 sows, progeny of six LW boars and six MS sows, produced 573 F2 females and 530 F2 males. Six traits, i.e. teat number (TN), age at puberty (AP), ovulation rate (OR), weight at mating (WTM), number of viable embryos (NVE) and embryo survival (ES) at 30 days of gestation were analysed. Animals were genotyped for a total of 137 markers covering the entire porcine genome. Analyses were carried out based on interval mapping methods, using a line-cross (LC) regression and a half-full sib (HFS) maximum likelihood test. Genome-wide (GW) highly significant (P < 0.001) QTL were detected for WTM on SSC 7 and for AP on SSC 13. They explained, respectively, 14.5% and 8.9% of the trait phenotypic variance. Other GW significant (P < 0.05) QTL were detected for TN on SSC 3, 7, 8, 16 and 17, for OR on SSC 4 and 5, and for ES on SSC 9. Two additional chromosome-wide significant (P < 0.05) QTL were detected for TN, three for WTM, four for AP, three for OR, three for NVE and two for ES. With the exception of the two above-mentioned loci, the QTL explained from 1.2% to 4.6% of trait phenotypic variance. QTL alleles were in most cases not fixed in the grand-parental populations and Meishan alleles were not systematically associated with higher reproductive performance.

An international parentage and identification panel for the domestic cat (Felis catus).

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex 'core' panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs.

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.

Genetic diversity within and between European pig breeds using microsatellite markers.

An important prerequisite for a conservation programme is a comprehensive description of genetic diversity. The aim of this study was to use anonymous genetic markers to assess the between- and the within-population components of genetic diversity for European pig breeds at the scale of the whole continent using microsatellites. Fifty-eight European pig breeds and lines were analysed including local breeds, national varieties of international breeds and commercial lines. A sample of the Chinese Meishan breed was also included. Eleven additional breeds from a previous project were added for some analyses. Approximately 50 individuals per breed were genotyped for a maximum of 50 microsatellite loci. Substantial within-breed variability was observed, with the average expected heterozygosity and observed number of alleles per locus being 0.56 [range 0.43-0.68] and 4.5 respectively. Genotypic frequencies departed from Hardy-Weinberg expectations (P < 0.01) in 15 European populations, with an excess of homozygotes in 12 of them. The European breeds were on average genetically very distinct, with a Wright F(ST) index value of 0.21. The Neighbour-Joining tree drawn from the Reynolds distances among the breeds showed that the national varieties of major breeds and the commercial lines were mostly clustered around their breeds of reference (Duroc, Hampshire, Landrace, Large White and Piétrain). In contrast, local breeds, with the exception of the Iberian breeds, exhibited a star-like topology. The results are discussed in the light of various forces, which may have driven the recent evolution of European pig breeds. This study has consequences for the interpretation of biodiversity results and will be of importance for future conservation programmes.

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers.

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.

Detection of quantitative trait loci for fat androstenone levels in pigs.

A QTL analysis of fat androstenone levels from a three-generation experimental cross between Large White and Meishan pig breeds was carried out. A total of 485 F2 males grouped in 24 full-sib families, their 29 parents and 12 grandparents were typed for 137 markers distributed over the entire porcine genome. The F2 male population was measured for fat androstenone levels at 100, 120, 140, and 160 d of age and at slaughter around 80 kg liveweight. Statistical analyses were performed using two interval mapping methods: a line-cross (LC) regression method, which assumes alternative alleles are fixed in founder lines, and a half- full-sib (HFS) maximum likelihood method, where allele substitution effects were estimated within each half- and full-sib family. Both methods revealed genomewide significant gene effects on chromosomes 3, 7, and 14. The QTL explained, respectively, 7 to 11%, 11 to 15%, and 6 to 8% of phenotypic variance. Three additional significant QTL explaining 4 to 7% of variance were detected on chromosomes 4 and 9 using LC method and on chromosome 6 using HFS method. Suggestive QTL were also obtained on chromosomes 2, 10, 11, 13, and 18. Meishan alleles were associated with higher androstenone levels, except on chromosomes 7, 10, and 13, although 10 and 13 additive effects were near zero. The QTL had essentially additive effects, except on chromosomes 4, 10, and 13. No evidence of linked QTL or imprinting effects on androstenone concentration could be found across the entire porcine genome. The steroid chromosome P450 21-hydroxylase (CYP21) and cytochrome P450 cholesterol side chain cleavage subfamily XIA (CYP11A) loci were investigated as possible candidate genes for the chromosome 7 QTL. No mutation of coding sequence has been found for CYP21. Involvement of a candidate regulatory mutation of CYP11A gene proposed by others can be excluded in our animals.

Genetic linkage mapping of quantitative trait loci for behavioral and neuroendocrine stress response traits in pigs.

A QTL analysis of behavioral and neuroendocrine responses to a "novel environment" stress was conducted in a three-generation experimental cross between Meishan and Large White pig breeds. A total of 186 F2 males and 182 F2 females were studied for their behavioral and neuroendocrine reactivity to a novel environment test at 6 wk of age. Locomotion, vocalization, and defecation rate, as well as exploration time, were measured for 10 min. Blood samples were taken immediately before and after the test to measure plasma levels of ACTH, cortisol, and glucose. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: a line-cross regression method, where founder lines were assumed to be fixed for different QTL alleles, and a half-/full-sib maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Both methods revealed a highly significant gene effect for poststress cortisol level (P < 0.001) and a significant effect for basal cortisol level (P < 0.05) at the end of the q arm of chromosome 7, explaining, respectively, 20% and 7% of the phenotypic variance. Meishan alleles are associated with higher cortisol levels and are partially dominant (for poststress levels) over Large White alleles. Other significant gene effects on biological measures were detected on chromosomes 1 and 17 (ACTH response to stress), 3, 5, and 8 (glucose levels). The SSC 17 QTL explains 12% of the phenotypic variance of poststress ACTH levels, with a suggestive evidence of imprinting effects. Meishan alleles are associated with lower poststress ACTH levels. Gene effects of low amplitude only were found for behavioral reactivity traits. Considering the effects of stress neuroendocrine systems on energy fluxes and protein deposition, and the importance of stress reactivity for meat quality and animal welfare, these results open new perspectives for pig selection.

Detection of quantitative trait loci for growth and fatness in pigs.

A quantitative trait locus (QTL) analysis of growth and fatness data from a three-generation experimental cross between Meishan (MS) and Large White (LW) pig breeds is presented. Six boars and 23 F1 sows, the progeny of six LW boars and six MS sows, produced 530 F2 males and 573 F2 females. Nine growth traits, i.e. body weight at birth and at 3, 10, 13, 17 and 22 weeks of age, average daily gain from birth to 3 weeks, from 3 to 10 weeks and from 10 to 22 weeks of age, as well as backfat thickness at 13, 17 and 22 weeks of age and at 40 and 60 kg live weight were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using two interval mapping methods: a line-cross (LC) regression method where founder lines were assumed to be fixed for different QTL alleles and a half-/full-sib (HFS) maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Both methods revealed highly significant gene effects for growth on chromosomes 1, 4 and 7 and for backfat thickness on chromosomes 1, 4, 5, 7 and X, and significant gene effects on chromosome 6 for growth and backfat thickness. Suggestive QTLs were also revealed by both methods on chromosomes 2 and 3 for growth and 2 for backfat thickness. Significant gene effects were detected for growth on chromosomes 11, 13, 14, 16 and 18 and for backfat thickness on chromosome 8, 10, 13 and 14. LW alleles were associated with high growth rate and low backfat thickness, except for those of chromosome 7 and to a lesser extent early-growth alleles on chromosomes 1 and 2 and backfat thickness alleles on chromosome 6.

A first genotyping assay of French cattle breeds based on a new allele of the extension gene encoding the melanocortin-1 receptor (Mc1r).

The seven transmembrane domain melanocortin-1 receptor (Mc1r) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. Upon the binding of agonist (melanocortin hormone, alpha-MSH) or antagonist (Agouti protein) ligands, the melanosomal synthesis of eumelanin and/or phaeomelanin pigments is stimulated or inhibited, respectively. Different alleles of the extension gene were cloned from unrelated animals belonging to French cattle breeds and sequenced. The wild type E allele was mainly present in Normande cattle, the dominant E(D) allele in animals with black color (i.e. Holstein), whereas the recessive e allele was identified in homozygous animals exhibiting a more or less strong red coat color (Blonde d'Aquitaine, Charolaise, Limousine and Salers). A new allele, named E1, was found in either homozygous (E1/E1) or heterozygous (E1/E) individuals in Aubrac and Gasconne breeds. This allele displayed a 4 amino acid duplication (12 nucleotides) located within the third cytoplasmic loop of the receptor, a region known to interact with G proteins. A first genotyping assay of the main French cattle breeds is described based on these four extension alleles.

Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov.

Information from a scrapie epidemic in a closed INRA Romanov flock is presented. Performances, pedigree, histopathological diagnoses and PrP genotypes were recorded from the beginning of the outbreak (in 1993). Between 1st of April, 1993 and 1st of May, 1997, 1015 animals were exposed to scrapie, and 304 died from this disease. A major influence of the polymorphisms at codons 136, 154 and 171 is shown, A136H154Q171 allele carriers proving to be nearly as resistant as A136R154R171 carriers. A possible relationship between gastrointestinal parasitism and scrapie is discussed. There is evidence of maternal transmission, with a risk ratio for artificially fed lambs of 67 percent of the risk of lambs fed by their mother. Our results strongly suggest that resistant animals were not healthy carriers or at least were less infectious when comparing risk for lambs born to healthy dams either of resistant (risk = 0.431) or of susceptible (risk = 1.000) genotype.

Parentage testing of Day 10 equine embryos by amplified PCR analysis of microsatellites.

Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared to those of the dam to identify those of maternal origin. The paternal alleles were then searched for within the genotype of the 3 sires, and the stallion(s) that exhibited the particular allele was said to be compatible with the embryo for this microsatellite. In this way, the true sire was identified correctly for all 21 embryos.

A genetic linkage map of the male goat genome.

This paper presents a first genetic linkage map of the goat genome. Primers derived from the flanking sequences of 612 bovine, ovine and goat microsatellite markers were gathered and tested for amplification with goat DNA under standardized PCR conditions. This screen made it possible to choose a set of 55 polymorphic markers that can be used in the three species and to define a panel of 223 microsatellites suitable for the goat. Twelve half-sib paternal goat families were then used to build a linkage map of the goat genome. The linkage analysis made it possible to construct a meiotic map covering 2300 cM, i.e., > 80% of the total estimated length of the goat genome. Moreover, eight cosmids containing microsatellites were mapped by fluorescence in situ hybridization in goat and sheep. Together with 11 microsatellite-containing cosmids previously mapped in cattle (and supposing conservation of the banding pattern between this species and the goat) and data from the sheep map, these results made the orientation of 15 linkage groups possible. Furthermore, 12 coding sequences were mapped either genetically or physically, providing useful data for comparative mapping.

Sequence conservation of microsatellites between Bos taurus (cattle), Capra hircus (goat) and related species. Examples of use in parentage testing and phylogeny analysis.

A panel of 70 bovine microsatellites was tested for amplification from goat DNA. Forty-three could be successfully amplified by PCR, 20 of which were tested for polymorphism. Three were applied for parentage testing in goat families and their exclusion probability evaluated. Fourteen were cloned and sequenced from goat DNA, and goat and bovine sequences were compared to evaluate interspecific conservation. Correlation between the structure of the dinucleotide repeat and the number of alleles was studied and indicated that interruption(s) in the repeat could explain the difference in the levels of polymorphism between the two species. This study provides a valuable in vivo clue to the mechanism generating polymorphism in microsatellites. Sequence conservation was also observed for several microsatellites with two wild species of Bovidae, Nilgaï (Boselaphus tragocamelus) and Himalayan Tur (Capra cylindricornis), and with one species of Cervidae, the fallow deer (Cervus dama). This study showed that an estimated 40 per cent of the microsatellites isolated from cattle will prove useful to study the caprine genome and to characterize economically important genetic loci in this species. Moreover, bovine microsatellites were shown to constitute very useful tools for the study of genetic diversity of the Artiodactyla.

The effect of phosphate on the unfolding-refolding of phosphoglycerate kinase induced by guanidine hydrochloride.

Phosphate ions were found to stabilize the native structure of phosphoglycerate kinase without modifying the folding pathway. The transition curves obtained from different signals: enzyme activity, ellipticity at 220 nm and fluorescence intensity at 336 nm (excitation at 292 nm) are shifted to smaller guanidine hydrochloride cm values in the absence of phosphate. The kinetic characteristics are qualitatively similar, unfolding rate constants being slightly smaller in the presence of phosphate. The mechanism by which the native structure of phosphoglycerate kinase is stabilized by phosphate probably occurs upon specific phosphate binding to the nucleotide beta- or gamma-phosphate binding site of nucleotides.