Determination of RET Sequence Variation in an MEN2 Unaffected Cohort Using Multiple-Sample Pooling and Next-Generation Sequencing.

Multisample, nonindexed pooling combined with next-generation sequencing (NGS) was used to discover RET proto-oncogene sequence variation within a cohort known to be unaffected by multiple endocrine neoplasia type 2 (MEN2). DNA samples (113 Caucasians, 23 persons of other ethnicities) were amplified for RET intron 9 to intron 16 and then divided into 5 pools of <30 samples each before library prep and NGS. Two controls were included in this study, a single sample and a pool of 50 samples that had been previously sequenced by the same NGS methods. All 59 variants previously detected in the 50-pool control were present. Of the 61 variants detected in the unaffected cohort, 20 variants were novel changes. Several variants were validated by high-resolution melting analysis and Sanger sequencing, and their allelic frequencies correlated well with those determined by NGS. The results from this unaffected cohort will be added to the RET MEN2 database.

Genomic organization, chromosomal localization, and transcriptional variants of the murine Pactolus gene.

The Pactolus gene encodes a cell surface protein similar to that of the beta integrin subunit family. The murine Pactolus gene is comprised of 16 exons that encompass 24 kb of the genome. The genomic organization of the Pactolus gene is very similar to that described for the human beta2 integrin gene (and deduced for murine beta2 integrin), including a separate exon containing only 5' untranslated sequences. The Pactolus gene was mapped to a terminal region of murine Chromosome (Chr) 16, distinct from the previously mapped site of the beta2 integrin gene on murine Chr 10. The Pactolus gene encodes three distinct transcripts via alternative splicing. The Pactolus A transcript encodes the full-length protein including transmembrane and cytoplasmic domains, while the Pactolus B transcript truncates translation before reaching these membrane-anchoring sequences. A newly discovered form, Pactolus C, is found in neonatal samples (along with Pactolus A) and would also encode a prematurely terminated protein. This form is derived from an alternative splicing event that skips part of exon 11, deletes exon 12, and uses an alternative acceptor site upstream of exon 13. The formation of the Pactolus B and C forms is thus governed by a complex alternative splicing mechanism that is affected by the developmental status of the animal.

Localization of a C-terminal region of lambda2 protein in reovirus cores.

The 144-kDa lambda2 protein is a structural component of mammalian reovirus particles and contains the guanylyltransferase activity involved in adding 5' caps to reovirus mRNAs. After incubation of reovirus T3D core particles at 52 degrees C, the lambda2 protein became sensitive to partial protease degradation. Sequential treatments with heat and chymotrypsin caused degradation of a C-terminal portion of lambda2, leaving a 120K core-associated fragment. The four other proteins in cores--lambda1, lambda3, mu2, and sigma2--were not affected by the treatment. Purified cores with cleaved lambda2 were subjected to transmission cryoelectron microscopy and image reconstruction. Reconstruction analysis demonstrated that a distinctive outer region of lambda2 was missing from the modified cores. The degraded region of lambda2 corresponded to the one that contacts the base of the sigma1 protein fiber in reovirus virions and infectious subvirion particles, suggesting that the sigma1-binding region of lambda2 is near its C terminus. Cores with cleaved lambda2 were shown to retain all activities required to transcribe and cap reovirus mRNAs, indicating that the C-terminal region of lambda2 is dispensable for those functions.

Nonrandom segregation of parental alleles in reovirus reassortants.

To test for nonrandom segregations among their 10 genomic RNA segments, we examined a set of 83 reassortants derived from mammalian reovirus type 1 Lang and type 3 Dearing. After confirming the genotypes of the reassortants, we performed statistical analyses on the distributions of parental alleles for each of the 10 gene segments, as well as for the 45 possible pairings of the 10 segments. The analyses revealed nonrandom associations of parental alleles in the L1-L2, L1-M1, L1-S1, and L3-S1 segment pairs, at levels indicating high statistical significance (P < 0.005). Such associations may reflect specific interactions between viral components (protein-protein, protein-RNA, or RNA-RNA) and may influence both the evolution of reoviruses in nature and their genetic analysis in the laboratory. The data may also support an hypothesis that reovirus reassortants commonly contain mutations that improve their fitness for independent replication.

The deduced Vibrio cholerae RecA amino-acid sequence.

The nucleotide sequence of the recA gene of Vibrio cholerae (Vc) has been determined. The amino acid (aa) sequence of the protein product is very similar to other known RecA aa sequences. However, this sequence does not agree with a previously reported Vc RecA aa sequence [Ghosh et al., Nucleic Acids Res. 20 (1992) 372].