An unbiased estimator of gene diversity in samples containing related individuals

Gene diversity is sometimes estimated from samples that contain inbred or related individuals. If inbred or related individuals are included in a sample, then the standard estimator for gene diversity produces a downward bias caused by an inflation of the variance of estimated allele frequencies. We develop an unbiased estimator for gene diversity that relies on kinship coefficients for pairs of individuals with known relationship and that reduces to the standard estimator when all individuals are noninbred and unrelated. Applying our estimator to data simulated based on allele frequencies observed for microsatellite loci in human populations, we find that the new estimator performs favorably compared with the standard estimator in terms of bias and similarly in terms of mean squared error. For human population-genetic data, we find that a close linear relationship previously seen between gene diversity and distance from East Africa is preserved when adjusting for the inclusion of close relatives.     

BchY-Based Degenerate Primers Target All Types of Anoxygenic Photosynthetic Bacteria in a Single PCR

To detect anoxygenic bacteria containing either type 1 or type 2 photosynthetic reaction centers in a single PCR, we designed a degenerate primer set based on the bchY gene. The new primers were validated in silico using the GenBank nucleotide database as well as by PCR on pure strains and environmental DNA.Anoxygenic photosynthetic bacteria are diverse and important members of microbial communities (11, 13, 17, 20). There are five bacterial phyla containing anoxygenic phototrophs: Proteobacteria (purple bacteria), Chlorobi (green sulfur bacteria), Chloroflexi (green nonsulfur bacteria), Acidobacteria (“Candidatus Chloracidobacterium thermophilum” [7]), and Firmicutes (heliobacteria). While Heliobacterium modesticaldum, Chlorobi, and “Ca. Chloracidobacterium thermophilum” have a type 1 reaction center (RC1) similar to photosystem I in Cyanobacteria and higher plants, Chloroflexi and Proteobacteria possess a type 2 reaction center (RC2) similar to photosystem II of oxygenic phototrophs (7, 16).Primers based on pufM, the gene encoding the M subunit of RC2, have been widely used to detect phototrophic purple bacteria (1, 4, 12, 19). However, phototrophic bacteria that do not possess RC2 are not retrieved when pufM is used as the target. Achenbach and coworkers (1) developed primers targeting rRNA genes of Chlorobi, Chloroflexi, and heliobacteria, while Alexander and coworkers (2) have developed primers to specifically detect green sulfur bacteria (Chlorobi) by using 16S rRNA and fmoA as gene targets and applied these primers in environmental studies (3). No currently available primer set can simultaneously target phototrophs containing either RC1 or RC2.Since it is well established that both RC1- and RC2-containing anoxygenic phototrophs synthesize bacteriochlorophylls (BChls), we searched for a universal anoxygenic photosynthesis gene marker among all enzymes involved in BChl biosynthetic pathways. All known pathways for chlorophyll and BChl biosynthesis branch from the heme biosynthesis pathway at protoporphyrin IX and continue to chlorophyllide a (Chlide a) through the same intermediates (9). Chlide a is the branching point that separates chlorophyll and BChl biosynthetic pathways. Moreover, pathways for the synthesis of different BChls are also split at this stage: chlorophyllide oxidoreductase converts Chlide a to 3-vinyl-bacteriophyllide a, which is the precursor for BChls a, b, and g, while a yet unknown enzyme reduces Chlide a to 3-vinyl-bacteriophyllide d, a precursor for antenna BChls c, d, and e in Chlorobium spp. (9). Since 3-vinyl-bacteriophyllide a is the last common intermediate in the synthesis of BChl a and BChl g, and the latter is the only BChl in heliobacteria (14, 15), chlorophyllide oxidoreductase is the only enzyme that is (i) present in anoxygenic phototrophic bacteria and not in oxygenic phototrophs and (ii) common to all known anoxygenic phototrophic bacterial species (with the exception of “Ca. Chloracidobacterium thermophilum,” where the pathway for BChl synthesis is not yet known). Analyzing multiple alignments of the subunits of chlorophyllide oxidoreductase, we found that only the Y subunit (encoded by the BchY gene) had two conserved regions distinguishing this protein from its closest homologs; therefore, the bchY gene was chosen as a universal marker for anoxygenic photosynthesis.Due to likely codon variations coding identical amino acid sequences in different genomes (19), degenerate BchY primers were designed by reverse translation of two conserved regions of the BchY alignment (Fig. ​(Fig.1):1): bchY_fwd (5′-CCNCARACNATGTGYCCNGCNTTYGG-3′ [26 bases; 2,048 variants; corresponding amino acid sequence, PQTMCPAFG]) and bchY_rev (5′-GGRTCNRCNGGRAANATYTCNCC-3′ [23 bases; 4,096 variants; corresponding amino acid sequence, GE{I/M}FP{A/ V}DP]). Each primer had no more than two bases deviating from known bchY sequences in the GenBank nr database (except for H. modesticaldum) as well as to environmental BchY variants in the GenBank env_nr database. None of these deviations were located in the 3′ ends of the primers (see Tables S2 and S3 in the supplemental material). These primers, therefore, were predicted to amplify a wide diversity of bchY genes under nonstringent PCR conditions (50 to 52°C annealing temperature). The lengths of the expected PCR products were either 480 bp (for green sulfur, green nonsulfur bacteria, and heliobacteria) or 510 bp (for purple bacteria).Open in a separate windowFIG. 1.Multiple-amino-acid alignment of BchY proteins. Sequence abbreviations: R.den, Roseobacter denitrificans (gi|110677524); R.gel, Rubrivivax gelatinosus (gi|29893484); R.cap, Rhodobacter capsulatus (gi|114868); C.lit, Congregibacter litoralis KT 71 (gi|88706663); H.hal, Halorhodospira halophila (gi|121998388); C.aur, Chloroflexus aurantiacus (gi|163849328); C.tep, Chlorobium tepidum (gi|66576270); and H.mod, Heliobacterium modesticaldum (gi|167629410).In order to check primer specificity in silico, a screening procedure was developed. Putative primer sites (tags) for both the bchY_fwd and the bchY_rev primers were gathered from the GenBank nucleotide collection (nt) by BLAST with relaxed search conditions; the tags having mismatches at the 3′ end or more than five overall mismatches from their primer were filtered out, and the remaining tags were mapped to their sequences mimicking PCR primer annealing. Fragments ranging from 300 to 700 bp (virtual “PCR products”) were retrieved from GenBank and annotated (see Table S4 in the supplemental material). All bchY genes present in the GenBank nt database were virtually “amplified,” pointing to the robustness of the primers and our in silico PCR analysis. On the other hand, all nonspecific “amplicons” have major deviations from the primer sequences and would likely not be amplified by a real PCR. The same screening procedure was performed against the GenBank environmental nucleotide collection (env_nt) (see Table S5 in the supplemental material), and as in the case with the nt database, only bchY fragments were virtually “amplified.”The BchY primer set was validated using five key control organisms, including the RC2-containing the purple sulfur bacterium Allochromatium vinosum and the purple nonsulfur bacterium Rhodobacter capsulatus as well as the RC1-containing green sulfur bacterium Chlorobium limicola, green nonsulfur bacterium Chloroflexus aurantiacus, and the heliobacterium H. modesticaldum. Amplifications yielded the predicted products of 510 bp from the purple bacteria and 480 bp from the green sulfur and nonsulfur bacteria and H. modesticaldum. Negative-control Escherichia coli and Synechocystis sp. strain PCC 6803 did not yield amplification products when the bchY primers were used.The designed BchY primer set successfully amplified bchY genes from DNA obtained from both marine (East Mediterranean Sea) and freshwater (Lake Kinneret) environments (see Table S6 in the supplemental material for best BLASTX hits for selected sequenced fragments). These habitats were chosen for testing due to the previously reported wide diversity of their anoxygenic phototrophs (8, 10, 18, 19). A phylogenetic tree of bchY gene fragments amplified from both freshwater and marine DNA samples is shown in Fig. ​Fig.22.Open in a separate windowFIG. 2.BchY phylogenetic tree based on a maximum likelihood tree to which short sequences were added by ARB parsimony. The branches that appeared on the original maximum likelihood tree are shown with thicker lines. Bootstrap values greater than 50% are indicated next to the branches. Sequences obtained in this study are shown in bold. For reasons of clarity, not all BchY sequences retrieved are shown in the tree. For cases in which a BchY fragment was found in more than three clones, the numbers of clones are given in parentheses. Clones m21_2 and m21_3 are identical to the bchY gene of Hoeflea phototrophica strain DFL-43 (6); the m20_2 clone was identical to the bchY gene of Dinoroseobacter shibae (5).Our study underlines the utility of the bchY gene as a molecular marker for revealing genetic heterogeneity in phototrophic microbial populations. Using both wide-scale bioinformatic analysis and PCR on control strains and naturally occurring microbial community DNA, we have confirmed the specificity and coverage of the proposed degenerate BchY primers.     

ARF Induces Autophagy by Virtue of Interaction with Bcl-xl

The ARF tumor suppressor controls a well-described p53/Mdm2-dependent oncogenic stress checkpoint. In addition, ARF has recently been shown to localize to mitochondria, and to induce autophagy; however, this has never before been demonstrated for endogenous ARF, and the molecular basis for this activity of ARF has not been elucidated. Using an unbiased mass spectrometry-based approach, we show that mitochondrial ARF interacts with the Bcl2 family member Bcl-xl, which normally protects cells from autophagy by inhibiting the Beclin-1/Vps34 complex, which is essential for autophagy. We find that increased expression of ARF decreases Beclin-1/Bcl-xl complexes in cells, thereby providing a basis for ARF-induced autophagy. Our data also indicate that silencing p53 leads to high levels of ARF and increased autophagy, thereby providing a possible basis for the finding by others that p53 inhibits autophagy. The combined data support the premise that ARF induces autophagy in a p53-independent manner in part by virtue of its interaction with Bcl-xl.The ARF tumor suppressor, p14^ARF in humans and p19^ARF in mouse, is a critical growth suppressor that is up-regulated by chronic mitogenic signals and localizes predominantly to the nucleolus. At the nucleolus and in the nucleoplasm, ARF can exert both p53-dependent and -independent growth suppressive function, by virtue of interaction with and inhibition of MDM2, nucleophosmin, E2F-1, CtBP, c-Myc, as well as others (see Ref. 1 for review). Recently, a small molecular weight variant of ARF, generated by translation from an internal methionine, has been discovered to localize primarily to mitochondria and to induce autophagy (2). More recently, another group has shown that full-length ARF, in addition to the small molecular weight variant, can likewise induce autophagy (3). However, neither of these studies revealed a mechanism whereby ARF induces autophagy.Autophagy is an evolutionarily conserved homeostatic process whereby cytosolic components are targeted for removal or turnover in membrane-bound compartments (autophagosomes) that fuse with the lysosome (for review see Ref. 4). This process regulates the turnover of damaged organelles and long-lived proteins that are too large to be delivered to the proteasome. Autophagy occurs constitutively at low levels and is greatly induced during period of metabolic stress, where lysosome-mediated digestion of sequestered molecules serves to release free amino acids and ATP to fuel the continued survival of the cell.Several genes are implicated in the control of autophagy. Perhaps most notable of these is Beclin-1, which is an evolutionarily-conserved mediator of autophagy, with structural similarity to the yeast autophagy gene Apg6/Vps30. Beclin-1 is a component of the class III PI3 kinase complex that includes Vps34; this complex regulates the formation and nucleation of autophagosomes, and the regulation of the activity of this complex is tightly regulated. For example, Beclin-1 possesses a BH3 domain that interacts with the BH3 binding groove of certain members of the Bcl-2 family, including Bcl-2, Bcl-xl, Bcl-w, and to a lesser extent, Mcl-1 (5–9). Binding of Bcl-2 family members to Beclin-1 inhibits autophagy, possibly by decreasing the kinase activity of the Beclin/Bcl-2/Vps34 complex (5) or by negatively regulating Beclin-1 oligomerization (10). The interaction between Beclin-1 and Bcl-2 family members is also regulated; for example, BH3-only proteins can bind directly to Bcl-2 family members and disrupt complex formation with Beclin-1 (11). Additionally, phosphorylation of Bcl-2 by Jun-N-terminal kinase (JNK) can interfere with its ability to bind to Beclin-1 (12). In all cases, dissociation of the Beclin-1/Bcl-2 complex is associated with induction of autophagy.In this report we confirm the findings of others that a fraction of ARF protein localizes to mitochondria and can induce autophagy. We show for the first time that endogenous ARF, up-regulated in non-transformed cells by oncogenes, is capable of inducing autophagy, and further that silencing of p53 is sufficient to de-repress ARF and induce autophagy. We report the identification of Bcl-xl as a mitochondrial ARF-binding protein, and show that ARF-mediated autophagy is enhanced in cells with Bcl-xl silenced. Finally, we show that ARF can reduce complex formation between Bcl-xl and Beclin-1. These data offer the first mechanistic insights into ARF-mediated autophagy. They also point to ARF as a novel regulator of Beclin/Bcl-xl complex formation.     

Leucyl-tRNA Synthetase-dependent and -independent Activation of a Group I Intron

Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.Although the aminoacyl-tRNA synthetases (aaRSs)⁶ are best known for their role in protein synthesis, many have functionally expanded and are essential to a wide range of other cellular activities that are unrelated to tRNA aminoacylation (1). The class I aaRSs, leucyl- (LeuRS or NAM2) and tyrosyl-tRNA synthetase (TyrRS or CYT-18) are required for RNA splicing of cognate group I introns in the mitochondria of certain lower eukaryotes (2). In yeast, processing of two related group I introns called bI4 and aI4α (Fig. 1) from the cob and cox1α genes, respectively, require yeast mitochondrial LeuRS (3, 4). Likewise, expression of Neurospora crassa mitochondrial genes, such as those for the large ribosomal RNA, is dependent on TyrRS for excising group I introns (5).Open in a separate windowFIGURE 1.Predicted secondary structures of the bI4 and aI4α group I introns. The secondary structure of the canonical core was based on previous predictions (19). Solid bold lines indicate linear connectivities of the nucleic acid strand with arrowheads oriented in the 5′ to 3′ direction. The dashed lines represent putative tertiary interactions. Dotted lines with numbers identify insertions where secondary structures were ambiguous. Arrows in the P1 and P9 domain show splice sites, whereas boxed nucleotides are paired regions.LeuRS facilitates RNA splicing in concert with a bI4 maturase that is encoded within the bI4 intron. Genetic investigations showed that an inactivated bI4 maturase resulting in deficient splicing activity of the bI4 and aI4α group I introns can be rescued by a suppressor mutation of LeuRS to restore mitochondrial respiration (4, 6). In addition, the splicing defect can be compensated by a mutant aI4α DNA endonuclease that is closely related to the bI4 maturase (7, 8).Previously, we used intracellular three-hybrid assays to demonstrate that LeuRS and bI4 maturase can independently bind to the bI4 intron and stimulate RNA splicing activity in the non-physiological yeast nucleus compartment (9). RNA-dependent two-hybrid assays also supported that the bI4 intron could simultaneously bind both the bI4 maturase and LeuRS. In this case, the RNA was co-expressed with LeuRS and bI4 maturase that was fused to either LexA or B42 to generate a two-hybrid response. This suggested that the bI4 intron was bridging these two protein splicing factors. In either the RNA-dependent two-hybrid or three-hybrid assays, bI4 intron splicing occurred only in the presence of LeuRS or bI4 maturase or both.We hypothesized that the bI4 maturase and LeuRS bind to distinct sites of the bI4 intron to form a ternary complex and promote efficient splicing activity. However, the functional basis of the collaboration between these two splicing cofactors or how either of them promotes RNA splicing remains unclear.We sought to characterize the respective splicing roles of the bI4 maturase and LeuRS via biochemical investigations. Previous attempts to develop an in vitro splicing assay for the bI4 intron or its closely related aI4α intron have failed (10, 11). It was hypothesized that the long length of the bI4 intron (∼1600 nucleotides) and its highly A:U-rich content (∼80%) hindered RNA folding in vitro as well as stabilization of its competent structure.Efforts to produce an active form of the bI4 intron have relied on building chimeric group I introns by interchanging RNA domains with the more stable Tetrahymena thermophila group I intron (11). Based on these results, it was proposed that the catalytic core of the bI4 group I intron was inherently defective (11). In this case, the group I intron would be expected to be completely dependent on its protein splicing factors similar to the bI3 intron that relies on the bI3 maturase and Mrs1 for activity (12). Thus, it was hypothesized that the bI4 maturase and/or LeuRS splicing factors aided the bI4 group I intron by targeting its core region to compensate for these deficiencies.We focused our efforts on re-designing the bI4 intron to develop a minimized molecule that might be competent for splicing. Because both the bI4 and aI4α group I introns rely on the bI4 maturase and LeuRS for their splicing activity, we compared their secondary structures to identify and eliminate peripheral regions outside of their catalytic cores. A small active derivative of the bI4 intron, comprised of just 380 nucleotides primarily from the canonical core, was generated. Thus, we show that, in and of itself, the canonical core of this group I intron is competent for splicing. Both the bI4 maturase and LeuRS enhance the splicing activity of the minimized bI4 intron. However, it is possible that protein-dependent splicing of the bI4 intron represents an intermediate evolutionary step in which the RNA activity is becoming increasingly dependent on its protein splicing factors.     

Peptide inhibitors of HIV-1 integrase: From mechanistic studies to improved lead compounds

The HIV-1 integrase enzyme (IN) catalyzes integration of viral DNA into the host genome. We previously developed peptides that inhibit IN in vitro and HIV-1 replication in cells. Here we present the design, synthesis and evaluation of several derivatives of one of these inhibitory peptides, the 20-mer IN1. The peptide corresponding to the N-terminal half of IN1 (IN1 1–10) was easier to synthesize and much more soluble than the 20-mer IN1. IN1 1–10 bound IN with improved affinity and inhibited IN activity as well as HIV replication and integration in infected cells. While IN1 bound the IN tetramer, its shorter derivatives bound dimeric IN. Mapping the peptide binding sites in IN provided a model that explains this difference. We conclude that IN1 1–10 is an improved lead compound for further development of IN inhibitors.     

Plant antigens cross-react with rat polyclonal antibodies against KLH-conjugated peptides

Keyhole limpet hemocyanin (KLH)-conjugated peptides are routinely used to raise polyclonal antibodies for biochemical or immunolocalization studies. Rats are suitable for producing antisera against plant antigens as they often lack non-specific response towards plant materials. We attempted to obtain rat antisera against peptides derived from several plant proteins. However, most antisera recognized the same background KLH-related plant antigen (KRAP) in Arabidopsis and tobacco. We characterized KRAP with respect to size and cellular localization and examined possible antigen-specific reasons for the failure of most immunizations. We also found no reports of successful use of rat anti-KLH-peptide antibodies in plant studies. We thus believe that the rat-KLH:peptide system is poorly suited for production of antibodies, especially against plant antigens, and should be used with caution, if at all.     

The Relationship between Imputation Error and Statistical Power in Genetic Association Studies in Diverse Populations

Genotype-imputation methods provide an essential technique for high-resolution genome-wide association (GWA) studies with millions of single-nucleotide polymorphisms. For optimal design and interpretation of imputation-based GWA studies, it is important to understand the connection between imputation error and power to detect associations at imputed markers. Here, using a 2 × 3 chi-square test, we describe a relationship between genotype-imputation error rates and the sample-size inflation required for achieving statistical power at an imputed marker equal to that obtained if genotypes at the marker were known with certainty. Surprisingly, typical imputation error rates (∼2%–6%) lead to a large increase in the required sample size (∼10%–60%), and in some African populations whose genotypes are particularly difficult to impute, the required sample-size increase is as high as ∼30%–150%. In most populations, each 1% increase in imputation error leads to an increase of ∼5%–13% in the sample size required for maintaining power. These results imply that in GWA sample-size calculations investigators will need to account for a potentially considerable loss of power from even low levels of imputation error and that development of additional genomic resources that decrease imputation error will translate into substantial reduction in the sample sizes needed for imputation-based detection of the variants that underlie complex human diseases.