Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus.

High mutation rates have driven RNA viruses to shorten their genomes to the minimum possible size. Mammalian (+)-strand RNA viruses and retroviruses have responded by reducing the number of cis-acting regulatory elements, a constraint that has led to the emergence of the polyprotein. Poliovirus is a (+)-stranded picornavirus whose polyprotein, encoded by an open reading frame spanning most of the viral RNA, is processed by virus-encoded proteinases. Despite their genetic austerity, picornaviruses have retained long 5' untranslated regions, which harbour cis-acting elements that promote initiation of translation independently of the uncapped 5' end of the viral messenger RNA. These elements are termed 'internal ribosomal entry sites' and are formed from highly structured RNA segments of at least 400 nucleotides. How these elements function is not known, but special RNA-binding proteins may be involved. The ribosome or its 40S subunit probably binds at or near a YnXmAUG motif (where Y is a pyrimidine and X is a purine) at the 3' border of the internal ribosomal entry site, which either provides the initiating codon or enables the ribosome to translocate to one downstream (E.W. et al., submitted). Initiation from most eukaryotic messenger RNAs usually occurs by ribosomal recognition of the 5' and subsequent scanning to the AUG codon. Here we describe a genetic strategy for the dissection of polyproteins which proves that an internal ribosomal entry site element can initiate translation independently of the 5' end.     

Characterization of single-nucleotide polymorphisms in coding regions of human genes.

A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.     

Tyro-3 family receptors are essential regulators of mammalian spermatogenesis

We have generated and analysed null mutations in the mouse genes encoding three structurally related receptors with tyrosine kinase activity: Tyro 3, Axl, and Mer. Mice lacking any single receptor, or any combination of two receptors, are viable and fertile, but male animals that lack all three receptors produce no mature sperm, owing to the progressive death of differentiating germ cells. This degenerative phenotype appears to result from a failure of the tropic support that is normally provided by Sertoli cells of the seminiferous tubules, whose function depends on testosterone and additional factors produced by Leydig cells. Tyro 3, Axl and Mer are all normally expressed by Sertoli cells during postnatal development, whereas their ligands, Gas6 and protein S, are produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter. Here we show that the concerted activation of Tyro 3, Axl and Mer in Sertoli cells is critical to the role that these cells play as nurturers of developing germ cells. Additional observations indicate that these receptors may also be essential for the tropic maintenance of diverse cell types in the mature nervous, immune and reproductive systems.     

Diverse behavioural defects caused by mutations in Caenorhabditis elegans unc-43 CaM kinase II

Calcium/calmodulin-dependent serine/threonine kinase type II (CaMKII) is one of the most abundant proteins in the mammalian brain, where it is thought to regulate synaptic plasticity and other processes. Activation of the multisubunit kinase by calcium is effectively cooperative and can persist long after transient calcium rises. Despite extensive biochemical characterization of CaMKII and identification of numerous in vitro kinase targets, little is known about its function in vivo. Here we report that unc-43 encodes the only Caenorhabditis elegans CaMKII. A gain-of-function unc-43 mutation reduces locomotory activity, alters excitation of three muscle types and lengthens the period of the motor output of a behavioural clock. Null unc-43 mutations cause phenotypes generally opposite to those of the gain-of-function mutation. Mutations in the unc-103 potassium channel gene suppress a gain-of-function phenotype of unc-43 in one tissue without affecting other tissues; thus, UNC-103 may be a tissue-specific target of CaMKII in vivo.