Detection of protein-protein interactions in plants using bimolecular fluorescence complementation

Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.     

Movement of cynomolgus and rhesus monkey spermatozoa collected from the lower female reproductive tract

Postcoital (pc) cervical mucus was collected in 73 menstrual cycles of cynomolgus monkeys and in 43 cycles of rhesus monkeys at 2,6,10,30 hr pc. Videomicrography was used to analyze sperm numbers and movement in the mucus. Both cynomolgus and rhesus monkeys had comparable populations of motile sperm in the mucus at 2 hr pc. However, by 6 hr pc, cervical mucus from cynomolgus monkeys contained twice as many total sperm and motile sperm as mucus from rhesus monkeys (P <.05). Mean swimming speeds of the free-swimming cervical sperm were similar for the two species at this time. No motile sperm were recovered in mucus from rhesus monkeys at 30 hr pc. In cynomolgus monkeys, however, 14 of the 26 animals examined at 30 hr pc had motile sperm in their mucus. These sperm exhibited lower percent molility, percent free-swimming sperm, and swimming speed than those sperm observed at 6 hr pc. Uterine sperm were collected by transcervical or transuterine aspiration from cynomolgus monkeys. In the transcervical technique, sperm were successfully obtained in four of nine animals examined at 6 hr and in four of five animals at 30 hr pc. The percentage of motile sperm in the uterine fluid was high, 82% ± 4%, and the swimming speeds (86 ± 2μm/sec) were higher than those observed in cervical mucus. Approximately 5–10% of the uterine sperm exhibited swimming motions similar to the hyperactivated motility seen in most mammals. These findings indicate that the sperm cervical mucus interaction in vivo in cynomolgus monkeys has more similarities to the human situation than does the interaction in rhesus monkeys.     

Experimental design for estimating integrals by numerical quadrature, with applications to pharmacokinetic studies

Many experimental situations, including bioavailability studies, require the estimation of integrals by numerical quadrature applied to dependent variable observations with measurement error. A strategy is described for selecting values for the independent variable (e.g. time). The strategy minimizes the expectation of the square of the difference between the exact integral and the quadrature approximation. This approach was applied to simulated pharmacokinetic problems, including the estimation of bioavailability. Results indicate that the procedure is potentially useful in reducing the variance of resulting estimates and that it appears to be robust with respect to prior assumptions about model parameter values.     

Watching Every Step of the Way: Junín Virus Attenuation Markers in the Vaccine Lineage

The Arenaviridae family includes several hemorrhagic fever viruses which are important emerging pathogens. Junín virus, a member of this family, is the etiological agent of Argentine Hemorrhagic Fever (AHF). A collaboration between the Governments of Argentina and the USA rendered the attenuated Junín virus vaccine strain Candid#1. Arenaviruses are enveloped viruses with genomes consisting of two single-stranded RNA species (L and S), each carrying two coding regions separated by a stably structured, non-coding intergenic region. Molecular characterization of the vaccine strain and of its more virulent ancestors, XJ13 (prototype) and XJ#44, allows a systematic approach for the discovery of key elements in virulence attenuation. We show comparisons of sequence information for the S RNA of the strains XJ13, XJ#44 and Candid#1 of Junín virus, along with other strains from the vaccine lineage and a set of Junín virus field strains collected at the AHF endemic area. Comparisons of nucleotide and amino acid sequences revealed different point mutations which might be linked to the attenuated phenotype. The majority of changes are consistent with a progressive attenuation of virulence between XJ13, XJ#44 and Candid#1. We propose that changes found in genomic regions with low natural variation frequencies are more likely to be associated with the virulence attenuation process. We partially sequenced field strains to analyze the genomic variability naturally occurring for Junín virus. This information, together with the sequence analysis of strains with intermediate virulence, will serve as a starting point to study the molecular bases for viral attenuation.     

Organization of the Hamster Cumulus Extracellular Matrix: A Hyaluronate-Glycoprotein Gel which Modulates Sperm Access to the Oocyte

The mammalian oocyte-cumulus complex contains an extracellular matrix rich in hyaluronate. Recently, the microstructure of the hamster cumulus extracellular matrix was described (52). In the present work, we investigated the organization of this matrix. We employed freeze-substitution methodologies to investigate ultrastructural effects of various treatments, including sperm enzymes, on the matrix. Protease treatment resulted in disruption with a loss of the fibrillar structures and some expansion; in contrast, hyaluronidase treatment completely solubilized the matrix. EDTA extraction revealed that the fibrils are composed of fine filaments. A discrete region of the matrix immediately surrounding the oocyte, the corona radiata, was resistant to EDTA disruption. We found that hyaluronate is an ubiquitous constituent of the microstructural elements of this extracellular matrix. The matrix exhibits a carbohydrate:protein ratio of approximately 2:1. SDS-PAGE revealed that glycosylated polypeptides are bound to the matrix. The lectins LCA and WGA had differing affinities for these polypeptides, and bound ubiquitously to the intact matrix. The present data suggest that glycoprotein-hyaluronate interaction is critical for maintenance of the cumulus extracellular matrix microstructure and for its physical properties.     

Analysis and design of RNA sequencing experiments for identifying isoform regulation

Through alternative splicing, most human genes express multiple isoforms that often differ in function. To infer isoform regulation from high-throughput sequencing of cDNA fragments (RNA-seq), we developed the mixture-of-isoforms (MISO) model, a statistical model that estimates expression of alternatively spliced exons and isoforms and assesses confidence in these estimates. Incorporation of mRNA fragment length distribution in paired-end RNA-seq greatly improved estimation of alternative-splicing levels. MISO also detects differentially regulated exons or isoforms. Application of MISO implicated the RNA splicing factor hnRNP H1 in the regulation of alternative cleavage and polyadenylation, a role that was supported by UV cross-linking-immunoprecipitation sequencing (CLIP-seq) analysis in human cells. Our results provide a probabilistic framework for RNA-seq analysis, give functional insights into pre-mRNA processing and yield guidelines for the optimal design of RNA-seq experiments for studies of gene and isoform expression.