PRNP and SPRN genes polymorphism in atypical bovine spongiform encephalopathy cases diagnosed in Polish cattle

Polymorphisms in the coding region of the prion protein gene (PRNP) have been associated with the susceptibility and incubation period of prion diseases in humans and sheep. However, polymorphisms in this part of the bovine PRNP gene do not affect the classical bovine spongiform encephalopathy (BSE) susceptibility in cattle. Studies carried out in Germany have shown that insertion/deletion-type polymorphisms located in the promoter region of the bovine prion gene are possible genetic factors modulating BSE susceptibility by changing the level of PRNP expression. No such association was observed for atypical BSE cases; however, due to the rare nature of the disease, these results should be confirmed. Additionally, a single nonsynonymous mutation in PRNP codon 211 (E211K) was described in one H-type BSE case in the USA; however, it was not found in any other cases. Here, we performed genetic characterization of PRNP promoter indel variations and determined the polymorphism of open reading frames (ORFs) of PRNP and bovine prion-like Shadoo (SPRN) genes in six Polish atypical BSE cases and compared these results to the population of clinically healthy Polish Holstein cattle. No potentially pathogenic mutations were found in the PRNP ORF in atypical BSE-affected cattle, but our study showed a high frequency of deletions at the indel loci of PRNP promoter in these animals. Additionally, a rare sequence variation in the SPRN protein-coding sequence was found in one L-type atypical BSE-affected animal.     

Sporadic—but Not Variant—Creutzfeldt-Jakob Disease Is Associated with Polymorphisms Upstream of PRNP Exon 1

Human prion diseases have inherited, sporadic, and acquired etiologies. The appearance of the novel acquired prion disease, variant Creutzfeldt-Jakob disease (vCJD), and the demonstration that it is caused by the same prion strain as that causing bovine spongiform encephalopathy, has led to fears of a major human epidemic. The etiology of classical (sporadic) CJD, which has a worldwide incidence, remains obscure. A common human prion-protein-gene (PRNP) polymorphism (encoding either methionine or valine at codon 129) is a strong susceptibility factor for sporadic and acquired prion disease. However, a quantitative-trait-locus study of prion incubation periods in mice has demonstrated an important factor that is close to Prnp but is independent of its coding sequence or that of the nearby prion-like doppel gene (Prnd). We have analyzed the PRNP locus for such tightly linked susceptibility factors. Fifty-six polymorphic sites have been identified within 25 kb of the PRNP open reading frame, including sites within the PRNP promoter and the PRNP 3' untranslated region. These have been characterized in 61 Centre d'Etude du Polymorphisme Humain (CEPH) families, demonstrating extensive linkage disequilibrium around PRNP and the existence of 11 major European PRNP haplotypes. Haplotype frequencies estimated in healthy U.K. control individuals were very similar to those deduced in the CEPH families. A common haplotype was overrepresented in patients with sporadic CJD (sCJD). Through use of a log-linear modeling approach to simultaneously model Hardy-Weinberg and linkage disequilibria, a significant independent association was found between sCJD and a polymorphism upstream of PRNP exon 1 (P=.005), in addition to the strong susceptibility conferred by codon 129 (P=2x10(-8)). However, although our sample size was necessarily small, no association was found between these polymorphisms and vCJD or iatrogenic CJD, in keeping with their having distinct disease mechanisms. In addition, there was no evidence of a PRNP founder effect in the first reported geographical cluster of vCJD.     

Comparative genomic analysis of the whale (Pseudorca crassidens) PRNP locus.

There have been many studies of the morphology, behavioral audiograms, and population structure of the false killer whale (Pseudorca crassidens), but sequencing, mapping, and functional and comparative genomics studies are still largely unknown. In this paper, we sequenced three novel BAC clones corresponding to a total length of 308 kb and spanning the PRNP, PRND, and RASSF2 loci, and conducted comparative genomic analysis to examine the genomic structure of the false killer whale PRNP locus. We determined that the three genes show a high degree of conservation in their syntenic regions with respect to gene order, gene orientation, and the predicted coding sequence (CDS) between human and whale, whereas PRNT was not detected in whale. Interestingly, the predicted CDS in whale PRNP contained a novel type of 4-copy octarepeat resulting from a 24 bp deletion when compared with the human sequence. In addition, we identified a novel 1869 bp repeat unit in a region that is non-syntenic to human and cow sequences and is therefore considered to be whale-specific sequence. Our results will provide novel insights into the genomic changes that have occurred during evolution of mammalian PRNP loci, and may also have implications for research into prion disease.     

金黄色葡萄球菌A蛋白基因的克隆及序列分析

 我们构建了金黄色葡萄球菌Cowan1株(CMCC26111)的染色体文库,转化大肠杆菌后筛选出一株protein A的阳性克隆。SDS-PAGE及Western-blot结果显示该克隆株表达的重组protein A的分子量为30 000,较天然protein A的小。该克隆中的protein A基因片段的序列分析表明,它含有天然protein A基因的启动子、信号肽以及至少四个与IgGFc段结合的结构域,而不含天然protein A的胞壁结合区,并发现其中有24个碱基对与Uhlen报告的protein A基因不同,由此导致三个编码的氨基酸发生变化,但这些差异并不影响该重组protein A与IgG Fc段的特异结合。     

Comparative genomic organization of the human and bovine PRNP locus

We sequenced a 208-kb BAC clone spanning the bovine prion protein (PRNP) locus, and compared the genomic structure with that of human. As a result, we determined the precise breakpoint between the two syntenic genomes, located on the 5' UTR of the PRNP gene, and discovered two highly repetitive sequences near the breakpoint. Further analysis demonstrated that the genomic structure of three genes, PRNP, PRND, and RASSF2, within the syntenic region of the bovine genome is highly conserved in order and orientation. The PRNT locus was not found in bovine but is conserved in several primates, including human. Moreover, we confirmed that the bovine RASSF2 is composed of 10 exons, as is the human gene, showing some difference from a previous report. Our findings may provide useful clues for understanding the evolutional process in the PRNP locus and also the mechanism that allows TSE from cattle to infect humans.     

Conservation of the RD-BF-C2 organization in the pig MHC class-III region: mapping and cloning of the pig RD gene

The RD gene, named after the arginine (R) and aspartic acid (D) repeat in the central part of its protein, was initially mapped in the mouse H-2S subregion between C4 and BF. It was later mapped in the same position in the human MHC and here we show it is also conserved in the pig MHC class III region, close to the complement BF gene. A pig RD genomic clone was isolated from a γ-phage library. Hybridizations on genomic DNA separated with pulsed field gel electrophoresis identified common 220kb Nrul, 130 kb EagI and 200 kb Mlul bands for RD, BF and C2. The RD gene has also a 17 kb Kpnl and 11 kb Sad fragment in common with BFbut not with C2. The close linkage of the RD and BF genes was further established by hybridization of BF to a genomic γ-phage clone also containing the RD gene. This genomic RD clone overlaps with a γ -phage clone previously isolated and containing the complete BF gene and the 3' part of C2. The distance between RD and BF is about 6 kb. The junction between the two complement genes BF and C2 was sequenced and the BF 5' promoter region, overlapping the 3' noncoding region of C2, was compared with that of the human BF promoter. The overall homology was about 80% and all but one identified promoter elements were found in the same position in both genes. The results obtained demonstrate the RD-BF-C2 organization is strongly conserved between human, mouse and pig. No polymorphisms were detected in either the RD gene or in the BF promoter region using polymerase chain reaction and restriction fragment polymorphism analysis.     

Analysis of Arabidopsis PsbQ A Gene Expression in Transgenic Tobacco Reveals Differential Role of Its Promoter and Transcribed Region in Organ-specific and Light-mediated Regulation

Arabidopsis PsbQ, encoding a 16 kDa protein of the oxygen-evolving complex, is regulated by light and is expressed preferentially in leaf tissues. To analyze the components required for light-regulated and organ-specific expression of PsbQA, several promoter constructs were generated and expressed in tobacco. The 2.2 kb promoter could confer organ-specific expression of the reporter gene, whereas regulatory elements for light-dependent induction could not be located within this promoter and the transcribed region extending up to a second exon, represented by a genomic fragment encompassing the gene. The genomic fragment representing the transcribed region, however, could confer light regulation even on a constitutive promoter, as observed by steady-state mRNA analysis in T0 and T1 tobacco plants. The results obtained have led to the conclusion that regulatory elements for organ-specificity mainly reside in the promoter region whereas the transcribed region of the gene has an important role in light regulation.     

Detection of a new tissue-specific enhancer in the ct6 region of the regulatory region of the drosophila cut locus]

The cut locus of Drosophila is an interesting example of a complex eukaryotic locus responsible for the development of many tissues and organs. Most of this locus is regulatory. The entire locus was cloned by Tchurikov et al. in 1986 and Blochlinger et al. in 1988. The wing ctn enhancer located 80 kb upstream of the promoter was earlier found in a 2.7 kb EcoRI-BamHI DNA fragment. The locus region 65-80 kb remote from the promoter was assumed to control the development of wings and vibrissae. We have found a new enhancer region in the ct6 region of the locus, which was in a 5 kb BamHI-EcoRI DNA fragment adjacent to the ctn enhancer. This region is responsible for the expression of the reporter lacZ gene in many tissues and organs at all stages of Drosophila development (at least in the intestine, Malpighian tubules, thoracic and abdominal sensory organs, thoracic ganglia and in ring glands). Thus, the region located 75 kb upstream of the promoter has some properties of the locus control region (LCR).     

Analysis of polymorphic microsatellites within the bovine and ovine prion protein (PRNP) genes

Twenty-four microsatellite sites with at least three repeats were found in the bovine prion protein gene (PRNP) and 23 in the ovine PRNP gene. Eight microsatellite sites were polymorphic in cattle and six in sheep with up to 10 alleles per site. In many cases allelic DNA fragments had variants in microsatellite sites and in flanking regions. Distances between microsatellite sites in eight genes from cattle and sheep occurred on average every 0.9 kb. The numerous polymorphic microsatellite sites will improve analysis of phylogenetic origin of different PRNP alleles and trait association studies for bovine spongiform encephalopathy (BSE) and scrapie.     

The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3): promoter characterization and genomic structure

A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.