PRNP gene variation in Pakistani cattle and buffaloes

Bovine spongiform encephalopathy (BSE) is a neurodegenerative prion protein misfolding disorder of cattle. BSE is of two types, classical BSE and atypical BSE which in turn is of two types, H-type BSE and L-type BSE. Both H-type BSE and L-type BSE are primarily sporadic prion disorders. However, one case of H-type BSE has recently been associated with E211K polymorphism in the prion protein gene (PRNP). Two polymorphisms in the bovine PRNP are also associated with susceptibility to classical BSE: a 23 bp insertion/deletion (indel) in the PRNP promoter region and a 12 bp indel in the first intron. No information regarding BSE susceptibility in Pakistani cattle is available. The present study aimed at achieving this information. A total of 236 cattle from 7 breeds and 281 buffaloes from 5 breeds were screened for E211K polymorphism and 23 bp and 12 bp indels employing triplex PCR. The E211K polymorphism was not detected in any of the animals studied. The 23 bp insertion allele was underrepresented in studied cattle breeds while the 12 bp insertion allele was overrepresented. Both 23 bp and 12 bp insertion alleles were overrepresented in studied buffalo breeds. Almost 90% of alleles were insertion alleles across all studied buffalo breeds. The average frequency of 23 bp and 12 bp insertion alleles across all studied cattle breeds was found to be 0.1822 and 0.9407, respectively. There were significant differences between Pakistani and worldwide cattle in terms of allele, genotype and haplotype frequencies of 23 bp and 12 bp indels. The higher observed frequency of 12 bp insertion allele suggests that Pakistani cattle are relatively more resistant to classical BSE than European cattle. However, the key risk factor for classical BSE is the dietary exposure of cattle to contaminated feedstuffs.     

Genomic aspects of age-related macular degeneration

Age-related macular degeneration (AMD) is a major late-onset posterior eye disease that causes central vision to deteriorate among elderly populations. The predominant lesion of AMD is the macula, at the interface between the outer retina and the inner choroid. Recent advances in genetics have revealed that inflammatory and angiogenic pathways play critical roles in the pathophysiology of AMD. Genome-wide association studies have identified ARMS2/HTRA1 and CFH as major AMD susceptibility genes. Genetic studies for AMD will contribute to the prevention of central vision loss, the development of new treatment, and the maintenance of quality of vision for productive aging.     

Comparison of different procedures of DNA analysis for sex identification in the endangered bearded vulture (<Emphasis Type="Italic">Gypaetus barbatus</Emphasis>)

During the last century, bearded vulture populations have declined and are threatened by extinction in Europe. Conservation efforts such as captive-bird breeding programs require the knowledge of the sex of individuals. The bearded vulture is difficult to sex morphologically because it is sexually monomorphic. Until now, there were no published genetic methods to sex this species. In our study, we tested different methods based on polymerase chain reaction analysis of the chromobox-helicase-DNA binding protein gene. This gene is located on both sex chromosomes, but the two copies differ in size depending on chromosomal location. Differences can be detected by digestion with restriction enzymes or with the amplification refractory mutation system technique. These methods are quick, accurate, and inexpensive and allow a large scale sex typing of bearded vultures.     

四引物扩增受阻突变体系PCR快速测定绵羊 BMPR-IB基因型方法的建立

四引物ARMS PCR是检测SNP有效、快速、简便的方法.绵羊BMPR-lB基因是控制Booroola绵羊多胎性状的主效基因,此研究目的在于建立一种对BMPR-IB基因四引物ARMS PCR检测方法.根据四引物ARMS PCR技术原理,在绵羊BMPR-IB基因突变位点(A746G)设计一对特异性引物,并在突变点两侧设计一对参照引物,用来扩增含有突变点的DNA片段,可在一步PCR反应中根据电泳图谱准确判断绵羊个体的BMPR-IB基因型,对比PCR-RFLP检测结果表明,所建立的方法简单,操作简便,大大提高了检测效率.     

Developmental and activity-dependent regulation of ARMS/Kidins220 in cultured rat hippocampal neurons

Neurotrophin activation of Trk receptors elicits diverse effects on neuronal survival, differentiation, and synaptic plasticity. One of the central questions is how specificity is encoded in neurotrophin receptor signaling and actions. A unique downstream protein is the Ankyrin-Repeat Rich Membrane Spanning (ARMS)/Kinase D-interacting substrate-220 kDa (Kidins220), a very abundant scaffold protein in the hippocampus. To determine the roles of ARMS/Kidins220 in hippocampal neurons, we have analyzed the effects of synaptic activity upon the regulation and distribution of ARMS/Kidins220. At early times in vitro (<7 DIV), synaptic activity was low and ARMS/Kidins220 levels were high. As synaptic activity and markers for synapse maturation, such as PSD-95, increased, ARMS/Kidins220 significantly decreased to a plateau by later times in vitro (>12 DIV). Immunocytochemistry showed ARMS/Kidins220 to be concentrated at the tips of growing processes in immature cultures, and more diffusely distributed in older cultures. To examine the apparent inverse relationship between activity and ARMS/Kidins220 levels, neuronal firing was manipulated pharmacologically. Chronic exposure to TTX increased ARMS/Kidins220 levels, whereas bicuculline caused the opposite effect. Moreover, using shRNA to decrease ARMS/Kidins220 levels produced a corresponding increase in synaptic activity. We find that ARMS/Kidins220 may function in neuronal development as an indicator and potentially as a homeostatic regulator of overall synaptic strength in hippocampal neurons.     

Identification of the endangered Australian orchid <Emphasis Type="Italic">Microtis angusii</Emphasis> using an allele-specific PCR assay

The endangered orchid, Microtis angusii, was recently described from a single population consisting of approximately 100 plants. This species is morphologically very similar to close relatives, and taxonomic difficulties have hindered attempts to identify further populations for protection. Here we present a rapid, economical, PCR-based assay for the effective identification of this species based on rDNA sequence variation. Using two single nucleotide substitutions in the internal transcribed spacer (ITS) region of the ribosomal DNA that are diagnostic for M. angusii, we developed an allele-specific PCR that can be easily visualized on a standard agarose gel, avoiding the use of expensive restriction enzymes and DNA sequencing reagents and equipment. Using PCR primer pairs for both the M. angusii, and the alternate allele, we also detected an individual heterozygous for the two alleles, indicating a need for further detailed genetic study. We performed a ‘blind trial’ to confirm the utility of this assay. Microtis angusii samples were successfully discriminated from amongst several congeners, and a further, previously unknown, population of the species was identified.     

Disentangling the complex microbial community of coral reefs using standardized Autonomous Reef Monitoring Structures (ARMS)

Autonomous Reef Monitoring Structures (ARMS) have been applied worldwide to describe eukaryotic cryptic reef fauna. Conversely, bacterial communities, which are critical components of coral reef ecosystem functioning, remain largely overlooked. Here we deployed 56 ARMS across the 2,000‐km spread of the Red Sea to assay biodiversity, composition and inferred underlying functions of coral reef‐associated bacterial communities via 16S rRNA gene sequencing. We found that bacterial community structure and diversity aligned with environmental differences. Indeed, sea surface temperature and macroalgae cover were key in explaining bacterial relative abundance. Importantly, taxonomic and functional alpha diversity decreased under more extreme environmental conditions (e.g., higher temperatures) in the southern Red Sea. This may imply a link between bacterial community diversity and functional capabilities, with implications for conservation management. Our study demonstrates the utility of ARMS to investigate the response of coral reef‐associated bacterial communities to environmental change.     

Kidins220/ARMS Modulates the Activity of Microtubule-regulating Proteins and Controls Neuronal Polarity and Development

In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubule-regulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.