No abstract

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5-10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.

No abstract

The proto-oncogene product pp60cs`c is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60C-cs in neurons and the existence of a neuronal pp6OCSrC variant, pp60c-srCN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We M. E. K. Mattson, U. Hammerling, E. Mohall, K. Hall, and S. Pahlman, Growth Factors, in press).Postmitotic neurons of rat brain and chicken retina show high expression of src protein and kinase activity (11,14,22,57), and the specific src kinase activity increases during in vitro differentiation of mouse embryo carcinoma cells (42) and rat striatal neurons (14). The src protein in rat brain neurons is modified in the N terminus (pp60-srcN) and has a specific tyrosine kinase activity higher than that of the nonneuronal form, pp60c-src (10, 11). However, pp6Oc-srcN isolated from established human neuroblastoma cell lines appeared not to have a specific kinase activity higher than that of pp6Oc-src prepared from fibroblasts or glioblastoma cells (48,66 (46). In this report, we show that the specific src kinase activity in SH-SY5Y cells increases threefold during neuronal differentiation induced by TPA. This increase does not involve a shift in the ratio of expressed c-src to c-srcN products.

The detection software and the ARP set needed for the analysis of a query protein reported here are properties of the Swedish National Food Agency and are available upon request. The protein sequence sets used in this work are publicly available on http://www.slv.se/templatesSLV/SLV_Page____9343.asp. Allergenicity assessment for specific protein sequences of interest is also possible via ulfh@slv.se