Hypoglycaemia, liver necrosis and perinatal death in mice lacking all isoforms of phosphoinositide 3-kinase p85 alpha

Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.     

Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.     

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.     

Myoglobin-like aerotaxis transducers in Archaea and Bacteria

Haem-containing proteins such as haemoglobin and myoglobin play an essential role in oxygen transport and storage. Comparison of the amino-acid sequences of globins from Bacteria and Eukarya suggests that they share an early common ancestor, even though the proteins perform different functions in these two kingdoms. Until now, no members of the globin family have been found in the third kingdom, Archaea. Recent studies of biological signalling in the Bacteria and Eukarya have revealed a new class of haem-containing proteins that serve as sensors. Until now, no haem-based sensor has been described in the Archaea. Here we report the first myoglobin-like, haem-containing protein in the Archaea, and the first haem-based aerotactic transducer in the Bacteria (termed HemAT-Hs for the archaeon Halobacterium salinarum, and HemAT-Bs for Bacillus subtilis). These proteins exhibit spectral properties similar to those of myoglobin and trigger aerotactic responses.