A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.     

Genetic variation in caribou and reindeer (Rangifer tarandus)

Genetic variation at seven microsatellite DNA loci was quantified in 19 herds of wild caribou and domestic reindeer (Rangifer tarandus) from North America, Scandinavia and Russia. There is an average of 2.0-6.6 alleles per locus and observed individual heterozygosity of 0.33-0.50 in most herds. A herd on Svalbard Island, Scandinavia, is an exception, with relatively few alleles and low heterozygosity. The Central Arctic, Western Arctic and Porcupine River caribou herds in Alaska have similar allele frequencies and comprise one breeding population. Domestic reindeer in Alaska originated from transplants from Siberia, Russia, more than 100 years ago. Reindeer in Alaska and Siberia have different allele frequencies at several loci, but a relatively low level of genetic differentiation. Wild caribou and domestic reindeer in Alaska have significantly different allele frequencies at the seven loci, indicating that gene flow between reindeer and caribou in Alaska has been limited.     

Regulation of alternative splicing by SRrp86 and its interacting proteins

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.     

Simian virus 40 large T antigen binds a novel Bcl-2 homology domain 3-containing proapoptosis protein in the cytoplasm

A 193-kDa SV40 large T antigen (T-Ag)-binding protein, designated p193, was identified and cloned. Inspection of the deduced amino acid sequence revealed the presence of a short motif similar to the Bcl-2 homology (BH) domain 3, suggesting that p193 may be a member of a family of apoptosis promoting proteins containing only BH3 motifs. In support of this, p193 expression promoted apoptosis in NIH-3T3 cells. Deletion of the BH3 motif abolished p193 apoptosis activity. p193-induced apoptosis was antagonized by co-expression of Bcl-X(L). Immune cytologic analysis indicated that p193 is localized to the cytoplasm of transfected cells. p193-induced apoptosis was also antagonized by co-expression of T-Ag, which resulted in the cytoplasmic localization of both proteins. The p193 binding site was mapped to an N-terminal region of T-Ag previously implicated in transforming activity. These results suggest that T-Ag possesses an antiapoptosis activity, independent of p53 sequestration, which is actuated by T-Ag/p193 binding in the cytoplasm.     

Identification and characterization of a novel serine-arginine-rich splicing regulatory protein

We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.     

gp120-induced alterations of human astrocyte function: Na(+)/H(+) exchange, K(+) conductance, and glutamate flux

Many human immunodeficiency virus(HIV)-infected patients suffer from impaired neurological function anddementia. This facet of the disease has been termed acquiredimmunodeficiency syndrome (AIDS)-associated dementia complex (ADC).Several cell types, including astrocytes and neurons, are notproductively infected by virus but are involved in ADC pathophysiology.Previous studies of rat astrocytes showed that an HIV coat protein(gp120) accelerated astrocyte Na⁺/H⁺ exchangeand that the resultant intracellular alkalinization activated apH-sensitive K⁺ conductance. The present experiments wereconducted to determine whether gp120 affected human astrocytes in thesame fashion. It was found that primary human astrocytes express apH-sensitive K⁺ conductance that was activated onintracellular alkalinization. Also, gp120 treatment of whole cellclamped human astrocytes activated this conductance specifically.Furthermore, gp120 inhibited glutamate uptake by primary humanastrocytes. These altered physiological processes could contribute topathophysiological changes in HIV-infected brains. Because thegp120-induced cell physiological changes were partially inhibited bydimethylamiloride (an inhibitor of Na⁺/H⁺exchange), our findings suggest that modification of human astrocyte Na⁺/H⁺ exchange activity may provide a means ofaddressing some of the neurological complications of HIV infection.