Organization of proteins into complexes is crucial for many cellular functions. However, most proteomic approaches primarily detect protein interactions for soluble proteins but are less suitable for membrane-associated complexes. Here we describe a matingbased split ubiquitin system (mbSUS) for systematic identification of interactions between membrane proteins as well as between membrane and soluble proteins. mbSUS allows in vivo cloning of PCR products into a vector set, detection of interactions via mating, regulated expression of baits, and improved selection of interacting proteins. Cloning is simplified by introduction of attachment sites for GATEWAY. Homo-and heteromeric interactions between Arabidopsis K ؉ channels KAT1, AKT1, and AKT2 were identified.Tests with deletion mutants demonstrate that the C terminus of KAT1 and AKT1 is necessary for physical assembly of complexes. Screening of a sorted collection of 84 plant proteins with K ؉ channels as bait revealed differences in oligomerization between KAT1, AKT1, and AtKC1, and allowed detection of putative interacting partners of KAT1 and AtKC1. These results show that mbSUS is suited for systematic analysis of membrane protein interactions.split ubiquitin ͉ proteomics ͉ KAT1 ͉ Arabidopsis ͉ GATEWAY

In countries with a low prevalence of human T-lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T-lymphotropic viruses (STLV), which give an HTLV-indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T-cell lymphotropic virus strains (PTLV), including the two new types STLV-PH969 and STLV-PP1664, we developed generic and type-specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross-reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross-reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV-1 PCR positive and 1 was HTLV-II PCR positive. Sequencing of one of the HTLV-I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV-I. The majority of the seroindeterminate samples (91%) were however HTLV-PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV-I or HTLV-II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples.

No abstract

In countries with a low prevalence of human T-lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T-lymphotropic viruses (STLV), which give an HTLV-indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T-cell lymphotropic virus strains (PTLV), including the two new types STLV-PH969 and STLV-PP1664, we developed generic and type-specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross-reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross-reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV-1 PCR positive and 1 was HTLV-II PCR positive. Sequencing of one of the HTLV-I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV-I. The majority of the seroindeterminate samples (91%) were however HTLV-PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV-I or HTLV-II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples.