Studies in Cyperaceae in southern Africa 43: Scirpoides varius Browning — a new name for Scirpus varius Boeck. ex. C.B. Clarke

Scirpoides varius Browning is established to accommodate the southern African endemic sedge Scirpus varius Boeck. ex C.B. Clarke, an illegitimate name in Scirpus. Morphological considerations support the transfer of the taxon to Scirpoides Ség., and there is no hindrance to the use of the epithet ‘varius’ in Scirpoides.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Evidence that the requirements for ATP and wheat germ initiation factors 4A and 4F are affected by a region of satellite tobacco necrosis virus RNA that is 3' to the ribosomal binding site

A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP.     

Population Structure With Localized Haplotype Clusters

We propose a multilocus version of F_ST and a measure of haplotype diversity using localized haplotype clusters. Specifically, we use haplotype clusters identified with BEAGLE, which is a program implementing a hidden Markov model for localized haplotype clustering and performing several functions including inference of haplotype phase. We apply this methodology to HapMap phase 3 data. With this haplotype-cluster approach, African populations have highest diversity and lowest divergence from the ancestral population, East Asian populations have lowest diversity and highest divergence, and other populations (European, Indian, and Mexican) have intermediate levels of diversity and divergence. These relationships accord with expectation based on other studies and accepted models of human history. In contrast, the population-specific F_ST estimates obtained directly from single-nucleotide polymorphisms (SNPs) do not reflect such expected relationships. We show that ascertainment bias of SNPs has less impact on the proposed haplotype-cluster-based F_ST than on the SNP-based version, which provides a potential explanation for these results. Thus, these new measures of F_ST and haplotype-cluster diversity provide an important new tool for population genetic analysis of high-density SNP data.GENOME-WIDE data sets from worldwide panels of individuals provide an outstanding opportunity to investigate the genetic structure of human populations (Conrad et al. 2006; International Hapmap Consortium 2007; Jakobsson et al. 2008; Auton et al. 2009). Populations around the globe form a continuum rather than discrete units (Serre and Paabo 2004; Weiss and Long 2009). However, notions of discrete populations can be appropriate when, for example, ancestral populations were separated by geographic distance or barriers such that little gene flow occurred.F_ST (Wright 1951; Weir and Cockerham 1984; Holsinger and Weir 2009) is a measure of population divergence. It measures variation between populations vs. within populations. One can calculate a global measure, assuming that all populations are equally diverged from an ancestral population, or one can calculate F_ST for specific populations or for pairs of populations while utilizing data from all populations (Weir and Hill 2002). One use of F_ST is to test for signatures of selection (reviewed in Oleksyk et al. 2010).F_ST may be calculated for single genetic markers. For multiallelic markers, such as microsatellites, this is useful, but single-nucleotide polymorphisms (SNPs) contain much less information when taken one at a time, and thus it is advantageous to calculate averages over windows of markers (Weir et al. 2005) or even over the whole genome. The advantage of windowed F_ST is that it can be used to find regions of the genome that show different patterns of divergence, indicative of selective forces at work during human history.Another measure of human evolutionary history is haplotype diversity. Haplotype diversity may be measured using a count of the number of observed haplotypes in a region or by the expected haplotype heterozygosity based on haplotype frequencies in a region. Application of this regional measure to chromosomal data can be achieved by a haplotype block strategy (Patil et al. 2001) or by windowing (Conrad et al. 2006; Auton et al. 2009).One problem with the analysis of population structure based on genome-wide panels of SNPs is that a large proportion of the SNPs were ascertained in Caucasians, potentially biasing the results of the analyses. Analysis based on haplotypes is less susceptible to such bias (Conrad et al. 2006). This is because haplotypes can be represented by multiple patterns of SNPs; thus lack of ascertainment of a particular SNP does not usually prevent observation of the haplotype. On a chromosome-wide scale, one cannot directly use entire haplotypes, because all the haplotypes in the sample will almost certainly be unique, thus providing no information on population structure. Instead one can use haplotypes on a local basis, either by using windows of adjacent markers or by using localized haplotype clusters, for example those obtained from fastPHASE (Scheet and Stephens 2006) or BEAGLE (Browning 2006; Browning and Browning 2007a).Localized haplotype clusters are a clustering of haplotypes on a localized basis. At the position of each genetic marker, haplotypes are clustered according to their similarity in the vicinity of the position. Both fastPHASE and BEAGLE use hidden Markov modeling to perform the clustering, although the specific models used by the two programs differ.Localized haplotype clusters derived from fastPHASE have been used to investigate haplotype diversity, to create neighbor-joining trees of populations, and to create multidimensional scaling (MDS) plots (Jakobsson et al. 2008). It was found that haplotype clusters showed different patterns of diversity to SNPs, while the neighbor-joining and MDS plots were similar between haplotype clusters and SNPs.In this work, we apply windowed F_ST methods to localized haplotype clusters derived from the BEAGLE program (Browning and Browning 2007a,b, 2009). We consider population-average, population-specific, and pairwise F_ST estimates (Weir and Hill 2002). Population-average F_ST''s either assume that all the populations are equally diverged from a common ancestor, which is not realistic, or represent the average of a set of population-specific values. This can be convenient in that the results are summarized by a single statistic; however, information is lost. A common procedure is to calculate F_ST for each pair of populations, and these values reflect the degree of divergence between the two populations. Different levels of divergence are allowed for each pair of populations but each estimate uses data from only that pair of populations. On the other hand, population-specific F_ST''s allow unequal levels of divergence in a single analysis that makes use of all the data.We compare results from the localized haplotype clusters to those using SNPs directly. The results of applying localized haplotype clusters to population-specific F_ST estimation are very striking, showing better separation of populations and a more realistic pattern of divergence than for population-specific F_ST estimation using SNPs directly. We also use BEAGLE''s haplotype clusters in a haplotype diversity measure and investigate the relationship between this measure of haplotype-cluster diversity and the recombination rate.     

Eukaryotic elongation factor 1A interacts with the upstream pseudoknot domain in the 3' untranslated region of tobacco mosaic virus RNA

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3' untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.     

Constitutive activation of NF-kappa B and secretion of interleukin-8 induced by the G protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus involve G alpha(13) and RhoA

The Kaposi's sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor kappa B (NF-kappa B) and secretion of interleukin-8, and this response involves activation of G alpha(13) and RhoA. The induced expression of a NF-kappa B luciferase reporter was partially reduced by pertussis toxin and the G beta gamma scavenger transducin, and enhanced by co-expression of G alpha(13) and to a lesser extent, G alpha(q). These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-kappa B activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the G alpha(13)-RhoA pathway in KSHV-GPCR-mediated NF-kappa B activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA(T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-kappa B activation by KSHV-GPCR, and by a constitutively active G alpha(13)(Q226L). KSHV-GPCR-induced NF-kappa B activation is accompanied by increased secretion of IL-8, a function mimicked by the activated G alpha(13) but not by an activated G alpha(q)(Q209L). These results suggest coupling of KSHV-GPCR to the G alpha(13)-RhoA pathway in addition to other G proteins.     

Eukaryotic initiation factor 4B from wheat and Arabidopsis thaliana is a member of a multigene family

Clones of eukaryotic initiation factor (eIF) 4B from wheat and Arabidopsis thaliana were obtained from cDNA and genomic libraries. The exon/intron organization of the genes from wheat and A. thaliana is very similar. The deduced amino acid sequences for the wheat and Arabidopsis eIF4B proteins showed overall similarity to each other, but very little similarity to eIF4B from other eukaryotes. The recombinant form of eIF4B supports polypeptide synthesis in an in vitro translation system and reacts with antibodies to native wheat eIF4B. In contrast to mammalian eIF4B and eIF4A, the combination of wheat eIF4B and eIF4A does not stimulate RNA-dependent ATP hydrolysis activity; however, wheat eIF4B does stimulate eIF4F and eIF4A RNA-dependent ATP hydrolysis activity. Interestingly, eIF4B does not stimulate eIF(iso)4F and eIF4A hydrolysis activity. Gel filtration experiments indicate wheat eIF4B, like its mammalian counterpart, self-associates to form a homodimer.