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Cold-sensitive mutations in the SPB genes (spb1-spb7) of Saccharomyces cerevisiae suppress the inhibition of translation initiation resulting from deletion of the poly(A)-binding protein gene (PAB1). The SPB4 protein belongs to a family of adenosine triphosphate (ATP)-dependent RNA helicases. The aberrant production of 25S ribosomal RNA (rRNA) occurring in spb4-1 mutants or the deletion of SPB2 (RPL46) permits the deletion of PAB1. These data suggest that mutations affecting different steps of 60S subunit formation can allow PAB-independent translation, and they indicate that further characterization of the spb mutations could lend insight into the biogenesis of the ribosome.

Publisher Summary Polyadenylate-binding protein (PAB) is specifically bound to the poly(A) tail of mRNA in eukaryotic cells.l-6 PAB is similar in aspects of its primary structure to a large family of eukaryotic RNA-binding proteins. A homologous domain has been identified in many other RNA-binding proteins, where it is often repeated as well. Deletion analysis of yeast PAB revealed that only the first half of a single domain is required for cell growth and division. The techniques presented here for the identification, purification, and characterization of PAB should be applicable to other RNA-binding proteins. The availability of large quantities of PAB for biochemical studies, together with the ease of genetic analysis in yeast, make PAB an excellent choice for further investigation of a large and growing family of RNAbinding proteins. The similar binding properties of nuclear and cytoplasmic PAB make it difficult to determine the relative abundance of each species in a crude extract.

The poly(A-binding protein (PAB) is required for translation initiation and poly(A) tail shortening in Saccharomyces cerevisiae (Sachs and Davis, 1989). Extragenic suppressors of a null mutation in PAB1 also affect the 60S ribosomal subunit. One of these spb genes (suppressor of P A B 1) encodes ribosomal protein L46 (SPB2), and another encodes a putative rRNA helicase (SPB4) (Sachs and Davis, 1990). Mutagenesis of an spb4-l strain revealed that many lethal mutations in genes other than PAB1 are suppressed by the spb4-l mutation. Furthermore, approximately 75% of these were also suppressed by deleting RPL46. These data suggest that some essential genes in yeast are made dispensable by altering the translational apparatus.

A new and convenient spectroscopic method for measuring monovalent cation flux in cells is described. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-dicarboxylic acid) by Cs+. A conventional fluorescence microscope can be used to measure the Cs+ flux. The usefulness of the technique is illustrated by measurement of acetylcholine receptor-mediated Cs+ flux in PC-12 cells, a sympathetic neuronal cell line. The results are the same as those obtained when radioactive tracer ions were used. The technique is applicable to any transmembrane process in which Cs+ can substitute for either Na+ or K+. The method has been developed to identify the different neurotransmitter receptors that control the translocation of monovalent cations and to locate the cells in central nervous system cell preparations that contain these receptors. The advantage of an optical method over tracer ion methods for biochemical and pharmacological studies of transmembrane processes in cells is described.

The site of action of phencyclidine, a powerful and increasingly abused drug, in sympathetic nerve cells has not previously been identified. Here it is demonstrated that phencyclidine is a powerful, noncompetitive inhibitor of the nicotinic acetylcholine receptor in a sympathetic nerve cell line, PC-12. In the presence of 1 mM carbamoylcholine the rate of the receptor-controlled influx of 22Na+ is reduced by a factor of 2 by 0.7 microM phencyclidine. Increasing concentrations of carbamoylcholine cannot reverse the inhibitory effect of the drug. Both the transmission of electrical signals between nerve cells and the secretion of catecholamines in the PC-12 cell line depend on the receptor-controlled ion flux. Thus phencyclidine interferes with at least two specific, physiologically important functions of these nerve cells. A new spectroscopic method has been developed to measure cation flux in cells. It is shown that this method can replace measurements of tracer ion flux.

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

The development of a spectrophotometric stopped-flow method to measure ion flux in membrane vesicles in the millisecond to minute time region is described in detail. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-disulfonic acid) by Cs+. The method has been applied to the measurement of acetylcholine-receptor-mediated ion flux in membrane vesicles prepared from the electric organs of both Electrophorus electricus and Torpedo californica. The method is applicable to any vesicle system in which Cs+ can substitute for either Na+ or K+. Loading of vesicles with the fluorescent dye is accomplished using the routine procedure for making the vesicles. The dye-loaded vesicles can be stored in liquid nitrogen before use. Neither the dye-loading procedure nor the presence of Cs+ changes the permeability of the membrane to ions, allowing ion-translocation measurements to be made in the millisecond to minute time region. The stopped-flow design presented allows two sequential mixings of solutions. The relationship between fluorescence quenching and ion flux as well as the interpretation of the ion flux data is described. It is shown that the data obtained with stopped-flow and Cs+ is identical to data obtained previously using a quench-flow technique and 86Rb+. The advantages of the present method over the quench-flow technique and a similar stopped-flow technique developed previously based on T1+ are described in detail.

Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value

Depletion of the essential poly(A) binding protein (PAB) in S. cerevisiae by promoter inactivation or by the utilization of a temperature-sensitive mutation (pab1-F364L) results in the inhibition of translation initiation and poly(A) tail shortening. Reversion analysis of pab1-F364L yielded seven independent, extragenic cold-sensitive mutations (spb1-spb7) that also suppress a PAB1 deletion. These mutations allow translation initiation without significantly changing poly(A) tail lengths in the absence of PAB, and they affect the amount of 60S ribosomal subunit. Consistent with this, SPB2 encodes the ribosomal protein L46. These data suggest that the 60S subunit mediates the PAB requirement of translation initiation, thereby ensuring that only intact poly(A)+ mRNA will be translated efficiently in vivo.

Summary The effect of suberyldicholine concentration (0.5 to 30 μM) on acetylcholine receptor-controlled ion flux was measured in membrane vesicles prepared from E. electricus . Both a quench flow apparatus and a stopped flow technique were used. The integrated rate equation, based on a minimum model that relates ligand binding to ion translocation, accounts for the effects of acetylcholine, carbamoylcholine, and suberyldicholine over a wide range of concentrations. The different maximum influx rates obtained with these 3 ligands are accounted for by their effect on the equilibrium between receptor: ligand complexes in the open and closed channel forms. The constants for these equilibria have been determined.

Nuclear and cytoplasmic poly(A)-binding proteins have been purified from Saccharomyces cerevisiae, and antisera have been used to isolate a gene that encodes them. The gene occurs in a single copy on chromosome 5 and gives rise to a unique, unspliced 2.1 kb transcript. The nuclear protein appears to be derived from the cytoplasmic one by proteolytic cleavage into 53 and 17 kd polypeptides that remain associated during isolation. DNA sequence determination reveals four tandemly arrayed 90 amino acid regions of homology that probably represent poly(A)-binding domains. A 55 residue A-rich region upstream of the initiator methionine codon in the mRNA shows an affinity for poly(A)-binding protein comparable to that of poly(A) 180–220 , raising the possibility of feedback regulation of translation.