A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.     

Fibrinolysis in Cholestatic Jaundice

The fibrinolytic system was studied in primary biliary cirrhosis (16 patients) and large bile duct obstruction (10 patients, nine of whom had carcinoma). Plasma fibrinolysis (plasminogen activator activity) was decreased and fibrinogen increased in both groups of patients, particularly in those with large duct obstruction. These changes were related to the degree of cholestasis. Plasminogen activator activity was inversely related to serum triglyceride levels in patients with primary biliary cirrhosis. Urokinase inhibitors were decreased in both groups and antiplasmins increased in patients with large duct obstruction; fibrin/fibrinogen degradation products were normal in primary biliary cirrhosis and moderately increased in large duct obstruction. None of these fibrinolytic indices was related to the degree of cholestasis. Fibrinolytic activity and fibrinogen returned almost to normal levels after palliative surgery in the three patients with large duct obstruction who were studied. The decreased plasma fibrinolysis and increased fibrinogen may be due to altered lipid metabolism in cholestatic jaundice. In patients undergoing surgery for large duct obstruction there may be an increased risk of thrombosis.     

An inducible targeted tagging system for localized saturation mutagenesis in Arabidopsis

We describe a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis (Arabidopsis thaliana). In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock-inducible transposase fusion is utilized to control the levels of transposase gene expression, generating transpositions that can be subsequently stabilized without requiring crossing or segregation. We have mapped 40 single-copy lines by thermal asymmetric interlaced-PCR, which can be used as potential launch pads for heat shock mutagenesis. Using a starter line selected for detailed analysis, the efficiency of tagging over a 50-kb region in the genome was examined. Hits were obtained in the targeted genes with multiple alleles for most genes, with approximately equal numbers of hits detected in genes on either side of the T-DNA. These results establish the feasibility of our approach for localized saturation mutagenesis in Arabidopsis. This system is very efficient and much less laborious as compared to conventional crossing schemes and may be generally applicable to other plant species for which large-scale T-DNA tagging is not currently feasible.