The genetics of scrapie in sheep and goats

Scrapie, an invariably fatal disease of sheep and goats, is a transmissible spongiform encephalopathy (TSE). The putative infectious agent is the host-encoded prion protein, PrP. The development of scrapie is closely linked to polymorphisms in the host PrP gene. The pathogenesis of most TSEs involves conversion of normal, cellular PrP into a protease-resistant, pathogenic isoform called PrPSc. The conversion to PrPSc involves change in secondary structure; it is impacts on these structural changes that may link polymorphisms to disease. Within the structured C-terminal part of PrP polymorphisms have been reported at 15 and 10 codons of the sheep and goat PrP genes respectively. Three polymorphisms in sheep are acutely linked to the occurrence of scrapie: A136V, R154H and Q171R/H. These generate five commonly observed alleles: ARQ, ARR, AHQ, ARH and VRQ. ARR and AHQ are associated with resistance; ARQ, ARH and VRQ are associated with susceptibility. There are subtle effects of specific allele pairings (genotypes). Generally, more susceptible genotypes have younger ages at death from scrapie. Different strains of scrapie occur which may attack genotypes differently. Different sheep breeds vary in the assortment of the five alleles that they predominantly encode. The reason for this variation is not known. Furthermore, certain genotypes may be susceptible to scrapie in some breeds and resistant in others. The explanation is not known, but may relate to different scrapie strains circulating in different breeds, or there may be effects of other genes which modulate the effect of PrP.     

Prion protein gene (PrP) polymorphisms in healthy sheep in Turkey

Scrapie, a transmissible spongiform encephalopathy (TSE) or prion disease, is a fatal, neurodegenerative disease in sheep and goats. This disease has been known in Europe for more than 250 years. Susceptibility to scrapie is associated with polymorphisms in the sheep prion protein gene (PrP) gene. In sheep, polymorphism in the PrP gene has been identified at a number of codons, and polymorphisms at codons 136, 154 and 171 have reported linkage with susceptibility to scrapie. Polymorphisms at the PrP locus were studied in 413 animals representing three native sheep breeds (Imroz, Chios and Kıvırcık) in Turkey. Genomic DNA was obtained from blood, and genotypes were screened using PCR and direct DNA sequencing. We report 17 genotypes derived from seven different alleles. The most frequent genotype in the Kıvırcık sheep is ARQ/ARQ, whereas the ARR/ARQ genotype is predominant in the Chios and Imroz breeds. In general, the ARQ haplotype was the predominant haplotype. ARQ haplotype was also predominant in the Kıvırcık and Chios sheep breeds, whereas the Imroz sheep predominantly had the ARR haplotype. The susceptibility-associated VRQ haplotype was found in 2.38%, 0.35% and 0.81% of the Imroz, Kıvırcık and Chios sheep, respectively. Moreover, seven additional polymorphisms have been detected at codons G127S, G127V, H143R, G145S, Y172D, N174Y and Q189L. Among these polymorphisms, the N174Y allele is a novel polymorphism, and the G145S allele is a novel allele for a known polymorphic locus.     

Frequencies of PrP genotypes and their implication for breeding against scrapie susceptibility in nine Pakistani sheep breeds

Prion protein (PrP) gene of 308 sheep was genotyped to investigate polymorphisms at scrapie-associated codons 136, 154 and 171 to assess the resistance of nine different Pakistani sheep breeds to natural/typical scrapie. As a result six genotypes were established on the basis of polymorphic codons 154 and 171. The most scrapie-susceptible codon 136 (A/V) was monomorphic (A) in all breeds. Wild-type genotype ARQ/ARQ was detected with maximum prevalence ranging from 63.2% in crossbred Pak-karakul to 100% in native Buchi, Kachi and Thalli breeds. The most frequent of typical scrapie-associated genotypes was ARQ/ARR as indicated by five of nine breeds. The coding region of PrP gene of 49 animals from the total sampled was also sequenced to ascertain additional polymorphisms. Polymorphism was found in 13 animals of the six breeds in codons 101(Q/R), 112(M/T), 146(N/S) and 189(Q/L) and ten genotypes were established on the basis of these polymorphic codons. Only Hissardale possessed five of the ten genotypes. The most frequent genotype was M¹¹²ARQ/T¹¹²ARQ detected in Hissardale, Pak-karakul and Awassi, whereas genotypes ARQr²³¹/ARQr²³¹ and ARQR²³¹/ARQr²³¹ (established on the basis of silent polymorphism agg/cgg-R/R) were detected in all breeds. Some animals consisted of three polymorphisms at different PrP codons that are not common in European breeds. An infrequent double heterozygosity (c/c a/g g/t) for codon 171 resulting in a genotype R/H was also detected in three animals each one from Kajli, Hissardale and Pak-karakul. This study concludes that all native sheep breeds are poor in scrapie-resistant PrP genotypes and could contract scrapie if exposed to prions.     

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.     

Prion protein gene polymorphisms in four goat breeds of Pakistan

Four different goat breeds (Pak-Angora, Dera Din Panah, Naachi and Teddy) of Pakistan were selected to investigate polymorphism in the prion protein gene (PrP gene) responsible for scrapie disease resistance in goats. Initially, genotyping of 187 animals of these four breeds by restriction fragment length polymorphism (RFLP) was done to see the genotype for codon 136 and 154. All the animals were monomorphic with a genotype of AARR except one animal of Teddy breed having the genotype of AARH. Sequencing of PrP gene of twenty animals representing these four goat breeds revealed two genotypes PPSSSS and PPSSPS with haplotypes PSS and PSP of PrP gene at the codon numbers 42, 138, and 240. All four breeds showed both wild type monomorphic sequence and mutant polymorphic sequences of these codons. The mutants of 42 and 138 codons translate the same amino acids as with the wild type sequences, while the mutant of codon 240 is responsible for a different amino acid translation i.e., serine to proline. In short, this study provides preliminary information about alleles and genotypes of PrP gene in four goat breeds of Pakistan.     

Nucleotide variants in prion-related protein (testis-specific) gene (PRNT) and effects on Chinese and Mongolian sheep phenotypes

ABSTRACT

Studies of the ovine prion-related protein (testis-specific) gene (PRNT), including studies of genetic diversity, have highlighted its potential relationship to scrapie infection and economically important ovine traits. PRNT was previously reported to be highly polymorphic in Portuguese sheep. To characterize genetic polymorphisms in this gene in Asian sheep, a direct sequencing method was used to detect polymorphic loci in PRNT in 285 individual sheep from four Chinese and one Mongolian breeds. Seven SNP variants in PRNT were identified, including three novel variants (g.93G>A, g.162G>T, and g.190A>G) and four previously reported variants (g.17 C>T, g.112G>C, g.129C>T, and g.144A>G). In the five breeds that we analyzed, the mutation frequencies of g.190A>G in Lanzhou Fat-tail sheep (LFTS) and g.129C>T in the other four varieties were high (F>0.5). Moreover, thirteen different haplotypes that had a comparable distribution in the tested breeds were also identified; ‘C-G-G-C-A-G-A’ occurred at the highest frequency in the five sheep breeds. Additionally, we previously explored the significance of relationships between polymorphisms in PRNP or PRND and ovine growth performance. Here, we also performed correlation analysis in all tested loci. These loci polymorphisms were significantly associated with ten different growth traits (P<0.05), except for g.93G>A. Meanwhile, in contrast to a previous study, there was no significant association between the seven SNP loci analyzed and our previously reported sheep PRND or PRNP insertion/deletion mutations. Our findings may provide new insights into polymorphic variation in ovine PRNT, which may contribute to genetic improvements in economic traits that are important for sheep breeding.     

Molecular cloning and polymorphism analysis of the prion protein gene in Tan sheep of Ningxia, China

The resistance or susceptibility of sheep to scrapie is associated with polymorphisms of the prion protein gene (PRNP), particularly, single nucleotide polymorphisms (SNPs) in amino acid positions 136, 154 and 171. The prion protein (PrP) gene sequence and the deduced amino acid alignment of prion protein in Tan sheep, a local Chinese sheep breed traditionally raised in Ningxia, northwestern China, were determined and variability of the PrP amino acids sequence was analyzed in this study. The PrP nucleic acids and amino acids sequences of 112 Tan sheep were highly homogenous, although polymorphism of the PrP gene was detected at several sites, particularly codons 106, 154, and 171. The analysis of both sequences revealed that the most predominant allele at codons 136, 154 and 171 in Tan sheep was ARQ, which was known to be associated with high susceptibility to scrapie in sheep. The result suggests that Tan sheep is potentially susceptible to scrapie. Our findings provide valuable information for future breeding projects to scrapie resistance in Tan sheep.     

Prion Protein Gene Variability in Spanish Goats. Inference through Susceptibility to Classical Scrapie Strains and Pathogenic Distribution of Peripheral PrPsc

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrP^sc) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrP^sc distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.     

Genetic variability of the PRNP gene in goat breeds from Northern and Southern Italy

Aims:  To determine the variability of the prion protein gene ( PRNP) in goats from Northern and Southern Italy.
Methods and results:  Genomic DNA isolated from goat blood was polymerase chain reaction (PCR)-amplified for the coding region of the PRNP gene and then sequenced. In total, 13 polymorphic sites were identified: G37V, T110P, G127S, M137I, I142M, I142T, H143R, R154H, P168Q, T194P, R211Q, Q222K and S240P (substitutions I142T and T194P are novel) giving rise to 14 haplotypes. Clear frequency differences between Northern and Southern breeds were found and confirmed by genetic distance analysis.
Conclusions:  Differences in allele distribution were found between Northern and Southern goats, in particular regarding the M142 and K222 alleles, possibly associated to scrapie resistance; philogeographical analysis supported the idea that Northern and Southern breeds may be considered as separate clusters.
Significance and impact of the study:  In Italy only limited studies have been carried out on caprine PRNP genotype distribution; this study is important to fill this lack of information. Moreover the finding of significant differences among allele distributions in Northern and Southern goats, especially if involved in modulating resistance/susceptibility, need to be carefully considered for the feasibility of selection plans for resistance to scrapie.     

Association between expression of reproductive seasonality and alleles of melatonin receptor 1A in goats

To determine whether a link exists between reproductive seasonality and the structure of the melatonin receptor 1A (MTNR1A) gene, the latter was studied in year-round estrous breeds (Jining Grey and Boer goats) and seasonal estrous breeds (Liaoning Cashmere, Inner Mongolia Cashmere, Wendeng milk and Beijing native goats). A large fragment of exon 2 of MTNR1A gene was amplified by PCR using sheep sense and antisense primers in 260 does of six breeds. The uniform 824 bp PCR product was digested with restriction endonucleases MnII and RsaI, and checked for the presence of restriction sites. No polymorphism at the MnII cleavage sites was detected in all six goat breeds and no relationship could be established between the MnII cleavage sites of MTNR1A gene and reproductive seasonality in goats. For polymorphic RsaI cleavage site at base position 53, only genotype RR (267 bp/267 bp) was detected in Jining Grey goats, both genotype RR and genotype Rr (267 bp/320 bp) were found in all other goat breeds, no genotype rr (320 bp/320 bp) was detected in all six goat breeds. Frequency of genotype RR was obviously higher, and frequency of genotype Rr was obviously lower in year-round estrous goat breeds than in seasonal estrous goat breeds. Sequencing revealed one mutation (G52A) in genotype Rr compared with genotype RR. For polymorphic RsaI cleavage site, the differences of genotype distributions were significant (P<0.05) between year-round estrous goat breeds and seasonal estrous goat breeds. These results preliminarily showed an association between genotype RR and year-round estrus in goats, and an association between genotype Rr and seasonal estrus in goats.     

青藏高原藏羚羊朊蛋白基因及氨基酸序列分析

羊瘙痒病是一种自然发生的传染性海绵状脑病,它可以在绵羊和山羊羊群之中传播,其疾病的易感性、潜伏期和跨物种传播的能力主要受宿主朊蛋白基因(prion protein gene,PRNP)的影响。研究藏羚羊PRNP有助于明确藏羚羊和羊瘙痒病之间的关系,从而更好地保护藏羚羊。参照GenBank上已经发表的绵羊PRNP序列设计引物,然后对藏羚羊的PRNP序列进行扩增、测序和分析。测序结果显示藏羚羊PRNP序列由771个核苷酸构成,编码256个氨基酸。序列分析结果显示21只藏羚羊PrP(prion protein,PrP)氨基酸序列完全同源,并且与野生型绵羊PrP氨基酸序列完全一致。此外,藏羚羊PrP氨基酸序列与山羊(99.2%)、汤氏瞪羚(99.2%)、印度羚(99.2%)、驯鹿(98%)、马鹿(98%)、家牛(97.7%)和水牛(96.1%)也具有较高的同源性。在对PrP氨基酸序列136、154、171位多态性分析后发现本次收集的21只藏羚羊样本可能和野生型绵羊一样对羊瘙痒病易感。本研究为羊瘙痒病在藏羚羊羊群中可能出现的风险提供了理论依据,提示要加强藏羚羊羊群中羊瘙痒病的监测。     

Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.     

Prion protein (PrP) gene polymorphism in Red Maasai and Black Head Persian sheep breeds in Tanzania: Consistent profile regardless of locations

The status of scrapie in Africa is largely unknown. The susceptibility to scrapie and its pathology is determined by amino acid polymorphisms at positions 136 (A/V), 154 (R/H) and 171 (Q/R/H) of the ovine PrP gene (PrP genotype) of the animals. Despite the widely studied PrP gene polymorphisms worldwide, limited data is available on PrP genotypes of sheep from the African continent. Previously, we have reported six PrP genotypes derived from four different alleles in Red Maasai and Black Herd Persian (BHP), the ARQ/ARQ genotype being more frequent in the Red Maasai than in the BHP sheep. The highly susceptible VRQ allele was not found in any of the sheep breeds; the ARR allele was absent in the Red Maasai or occurred at a low frequency in the BHP. The lack of the highly susceptible VRQ alleles among Tanzanian sheep examined, necessitated further examinations on genetic susceptibility in sheep of the same breeds, but originating from an entirely different part of Tanzania. Consistently, ARQ/ARQ genotype was observed in 88% of Red Maasai and 64% of BHP sheep, ARQ/AHQ genotype was present in 12% in Red Maasai and 36% in BHP. Neither the highly resistant (ARR/ARR), nor the highly susceptible (VRQ/VRQ) genotypes were found. The ARQ and AHQ were the only alleles observed. The ARQ allele constituted 94%, 82% and 67% in the Red Maasai, BHP and cross-bred sheep, respectively. The AHQ constituted 6%, 18% and 33%, respectively. Data reported here, provide additional information on genetic susceptibility of the Red Maasai, BHP and their crosses; they may be helpful in policy formulation for future prevention of scrapie.     

Mouse-adapted sporadic human Creutzfeldt-Jakob disease prions propagate in cell culture

Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt-Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human prions.     

GnRHR gene polymorphisms and their effects on reproductive performance in Chinese goats

In this study, polymorphisms in the goat GnRHR gene exon 1 were detected by PCR-SSCP and DNA sequencing methods in 786 individuals from two different goat breeds. Two haplotypes (A and B), two observed genotypes (AA and AB), and two single nucleotide polymorphisms (SNPs) were detected, which resulted in five amino acid substitutions. The frequencies of haplotypes A and B in the two goat breeds were 0.78–0.83 and 0.17–0.22, respectively. The SNP locus was in Hardy–Weinberg disequilibrium in the two goat breeds (P < 0.05). Polymorphisms of the GnRHR gene were shown to be associated with litter size in the two goat breeds. The SNPs in the goat GnRHR gene had significant effects on litter size (P < 0.05). Therefore, these results suggest that the GnRHR gene is a strong candidate gene that affects litter size in goat.     

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay

The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.     

Genetic Variability of PRNP in Chinese Indigenous Goats

Polymorphism of the prion protein gene (PRNP) is usually associated with scrapie susceptibility or resistance. To determine the variability of PRNP in Chinese indigenous goat breeds, we isolated genomic DNA from goat blood and amplified and sequenced the coding region of the gene. We identified 10 polymorphic sites that gave rise to 28 haplotypes. Clear frequency differences were found between northern and southern breeds and confirmed by genetic distance analysis, except for the Tangshan dairy goat. Phylogeographic analysis supported the idea that northern and southern breeds might be considered separate clusters, except for the Tangshan dairy goat. The finding of significant differences in allele distribution in northern and southern goats, especially if involved in modulating resistance/susceptibility, needs to be carefully considered for the feasibility of selection plans for resistance to scrapie.     

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.     

Analysis on DNA sequence of goat RFRP gene and its possible association with average daily sunshine duration

Goat RFRP gene was cloned and its mutations were detected in thirteen goat breeds whose reproductive seasonality and litter size were different. Then sequence characteristics were analyzed and association analyses were performed to reveal the relationships between mutations of RFRP gene and average daily sunshine duration, reproductive seasonality as well as litter size in goats. A 4,862 bp DNA fragment of goat RFRP gene was obtained and the complete CDS of 591 bp encodes 196 amino acids, having high homology with that of other mammals. The protein was predicted to be a secreted protein with a signal peptide of 21 amino acids. Moreover, two mutations (A712G, T1493C) in 5' regulatory region and one mutation (A3438T) in exon 2 were detected. The test of genotype distribution in six selective goat breeds showed that there was no uniform significant association between the three polymorphisms and seasonal reproduction. The association just existed in some goat breeds for each locus. Interestingly, however, there was a strong positive correlation (r = 0.830, P = 0.003) between the G allele frequency of the A712G locus and average daily sunshine duration in ten local goat breeds, suggesting that RFRP gene has undergone a selective pressure in sunshine duration and may have indirect relationship with reproductive seasonality in goats. Additionally, no significant difference was found in litter size between genotypes in prolific Jining Grey goats.     

Human cellular prion protein hPrPC is sorted to the apical membrane of epithelial cells

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells.