Retrotranspositions in orthologous regions of closely related grass species

Background  

Retrotransposons are commonly occurring eukaryotic transposable elements (TEs). Among these, long terminal repeat (LTR) retrotransposons are the most abundant TEs and can comprise 50–90% of the genome in higher plants. By comparing the orthologous chromosomal regions of closely related species, the effects of TEs on the evolution of plant genomes can be studied in detail.     

Divergent protein coding regions in otherwise closely related androgen-regulated mRNAs

Rat seminal vesicles serve as a model system for studying androgen action. We have sequenced and compared full length cDNAs for two major proteins (S and F) synthesised and secreted under hormonal control. Overall, mRNAS and mRNAF share 57% nucleotide sequence homology suggesting that their genes arose by duplication of a common ancestor. However, the mRNAs display a highly unusual regional distribution of sequence homology, with the untranslated regions (UTRs) being substantially more homologous than the protein-coding regions (PCRs). Detailed analysis of nucleotide substitutions at synonymous and replacement sites shows that the PCRs have evolved very rapidly. Evolutionary conservation of the UTRs is no higher than that of UTRs generally and thus provides no evidence of a specific regulatory role for the UTRs in androgen action. The primary sequences of proteins S and F have diverged so rapidly that they are the best examples of neutrally evolving proteins for which comparative nucleotide sequence data are available. However, despite their rapid divergence, the predicted higher order structures for both proteins consist largely of non-regular conformation. This is discussed in terms of their roles as structural components of the rodent copulatory plug.     

Identifying quantitative trait loci and determining closely related stalk traits for rind penetrometer resistance in a high-oil maize population

Stalk lodging in maize causes annual yield losses between 5 and 20% worldwide. Many studies have indicated that maize stalk strength significantly negatively correlates with lodging observed in the field. Rind penetrometer resistance (RPR) measurements can be used to effectively evaluate maize stalk strength, but little is known about the genetic basis of this parameter. The objective of this study was to explore a genetic model and detect quantitative trait loci (QTL) of RPR and determine relationships between RPR and other stalk traits, especially cell wall chemical components. RPR is quantitative trait in nature, and both additive and non-additive effects may be important to consider for the improvement of RPR. Nine additive-effect QTLs covering nine chromosomes, except chromosome 5, and one pair of epistatic QTLs were detected for RPR. CeSA11 involved in cellulose synthesis and colorless2 involved in lignin synthesis were identified as possible candidate genes for RPR. Internode diameter (InD), fresh weight of internode (FreW), dry weight of internode (DryW), fresh weight and dry weight as well as cell wall components per unit volume significantly positively correlated with RPR. The internode water content (InW) significantly negatively correlated with RPR. Notably, these traits significantly correlated with RPR, and the QTLs of these traits co-localized with those of RPR. The corresponding results obtained from correlation analysis and QTL mapping suggested the presence of pleitropism or linkage between genes and indicated that these different approaches may be used for cross authentication of relationships between different traits.     

Aspartate isomerization in the complementarity-determining regions of two closely related monoclonal antibodies

The aspartic acid residues (Asp) present in the complementarity-determining regions (CDRs) of the light chains of two recombinant monoclonal antibodies (MAbs), MAb I and MAb II, are highly susceptible to isomerization due to the presence of glycine residues (Gly) on their C-terminal ends. Asp isomerization in these MAbs leads to formation of the isoaspartate (IsoAsp) and the cyclic imide (Asu) variants of these MAbs. Both MAb I and MAb II, employed in this study, elicit their pharmacological responses through binding human IgE. The formation of the MAb variants as a result of Asp isomerization significantly reduces the binding affinities of these antibodies to IgE, thereby reducing their potencies. Here we report on significant differences in the susceptibility of the MAb I and the MAb II to Asp isomerization. The molecular basis for these differences in rates of Asp isomerization was elucidated. The effect of primary sequence on Asp isomerization was evaluated using pentapeptide models of the MAbs, which included the labile Asp residues and their neighboring amino acid residues. The separation of the parent MAbs and pentapeptides from their isomerization products was achieved using hydrophobic interaction chromatography (HIC) and rp-HPLC, respectively. Structural characterization of the MAbs was performed using differential scanning calorimetry (DSC), circular dichroism (CD), and X-ray crystallography. Our investigations demonstrate that the differences in the Asp isomerization rates between MAb I and MAb II can be attributed to structural factors including the conformational flexibility and the extent of solvent exposure of the labile Asp residue.     

Application of a new method for GWAS in a related case/control sample with known pedigree structure: identification of new loci for nephrolithiasis

In contrast to large GWA studies based on thousands of individuals and large meta-analyses combining GWAS results, we analyzed a small case/control sample for uric acid nephrolithiasis. Our cohort of closely related individuals is derived from a small, genetically isolated village in Sardinia, with well-characterized genealogical data linking the extant population up to the 16(th) century. It is expected that the number of risk alleles involved in complex disorders is smaller in isolated founder populations than in more diverse populations, and the power to detect association with complex traits may be increased when related, homogeneous affected individuals are selected, as they are more likely to be enriched with and share specific risk variants than are unrelated, affected individuals from the general population. When related individuals are included in an association study, correlations among relatives must be accurately taken into account to ensure validity of the results. A recently proposed association method uses an empirical genotypic covariance matrix estimated from genome-screen data to allow for additional population structure and cryptic relatedness that may not be captured by the genealogical data. We apply the method to our data, and we also investigate the properties of the method, as well as other association methods, in our highly inbred population, as previous applications were to outbred samples. The more promising regions identified in our initial study in the genetic isolate were then further investigated in an independent sample collected from the Italian population. Among the loci that showed association in this study, we observed evidence of a possible involvement of the region encompassing the gene LRRC16A, already associated to serum uric acid levels in a large meta-analysis of 14 GWAS, suggesting that this locus might lead a pathway for uric acid metabolism that may be involved in gout as well as in nephrolithiasis.     

Use of closely related affected individuals for the genetic study of complex diseases in founder populations

We propose a method, the maximum identity length contrast (MILC) statistic, to locate genetic risk factors for complex diseases in founder populations. The MILC approach compares the identity length of parental haplotypes that are transmitted to affected offspring with the identity length of those that are not transmitted to affected offspring. Initially, the statistical properties of the method were assessed using randomly selected affected individuals with unknown relationship. Because both nuclear families with multiple affected sibs and large pedigrees are often available in founder populations, we performed simulations to investigate the properties of the MILC statistic in the presence of closely related affected individuals. The simulation showed that the use of closely related affected individuals greatly enhances the power of the statistic. For a given sample size and type I error, the use of affected sib pairs, instead of affected individuals randomly selected from the population, could increase the power by a factor of two. This increase was related to an increase of kinship-coefficient contrast between haplotype groups when closely related individuals were considered. The MILC approach allows the simultaneous use of affected individuals from a founder population and affected individuals with any kind of relationship, close or remote. We used the MILC approach to analyze the role of HLA in celiac disease and showed that the effect of HLA may be detected with the MILC approach by typing only 11 affected individuals, who were part of a single large Finnish pedigree.     

The meaning of genetic research results: reflections from individuals with and without a known genetic disorder

In the debate about whether to return individual genetic results to research participants, consideration of the nature of results has taken precedence over contextual factors associated with different study designs and populations. We conducted in-depth interviews with 24 individuals who participated in a genotype-driven study of cystic fibrosis: 9 of the individuals had cystic fibrosis, 15 had participated as healthy volunteers, and all had gene variants of interest to the researchers. These interviews revealed that the two groups had different ideas about the meaningfulness of genetic results. Our findings point to the importance of understanding research context, such as participants' relationship with the researcher and whether they have the disease condition under study, when considering whether to return individual results.     

Identifying individuals at risk for fracture in Guatemala

Introduction

The FRAX calculator combines a set of clinical risk factors with country-specific incidence rates to determine the ten-year absolute risk of major osteoporotic fracture. However, regional or country-specific databases from Central American countries are not available. We compared the use of various FRAX databases and the Pluijm algorithm in determining risk of fracture.

Methods

We collected clinical risk factor data needed for the FRAX calculator and Pluijm algorithm of Hispanic women in Guatemala and calculated the FRAX absolute risk measures of major osteoporotic fracture and hip fracture. Subjects were postmenopausal women greater than age 40 with no history of using medication that affect bone. A random sample of 204 women in 34 different regions women in Guatemala City was visited in their homes to complete the surveys. The Pluijm risk score and FRAX risk score using the US Hispanic, Spain, and Mexican databases were calculated.

Results

We used the US NOF guidelines for treatment which suggest a treatment threshold for patients with a 10-year hip fracture probability ≥3% or a 10-year major osteoporotic fracture risk ≥20%. The number of patients meeting the suggested threshold limits for treatment using the Spain and Mexico calculators were identical. There was 100% conformity in threshold limits for both hip and major osteoporotic fracture risk. The mean conformity for any fracture risk between US Hispanic and the other two databases was 97.5%. Conformity was 99.0% based on major osteoporotic fracture and 97.5% based on risk of hip fracture. The Pluijm evaluation shows conformity of 87.2% and 83.3%, respectively, when compared to the US Hispanic and Spain/Mexico FRAX thresholds for risk of fracture.

Discussion

Although the different FRAX databases provide variations in the absolute risk of fracture, the overall conformity to treatment thresholds amongst the US Hispanic, Spain, and Mexico databases show the database used would have little effect as to the decision to treat. The Pluijm tool conforms to the FRAX thresholds and can be used as well. It does not matter which country-specific calculator or assessment tool is used, as there are a similar number of patients that would meet the intervention threshold.     

FT-IR spectroscopy for identification of closely related lactobacilli

Fourier Transform Infrared (FT-IR) spectroscopy was used to analyse 56 strains from four closely related species of Lactobacillus, L. sakei, L. plantarum, L. curvatus and L. paracasei. Hierarchical Cluster Analysis (HCA) was used to study the clusters in the data, but in the dendrogram, the spectra were not differentiated into four separate clusters corresponding to species. When the data were analysed with Partial Least Squares Regression (PLSR), the strains were differentiated into four clusters according to species. It was also possible to recognise strains that were incorrectly identified by conventional methods prior to the FT-IR analysis. PLSR was used to identify strains from three of the species, and the results were compared to two other multivariate methods, Soft Independent Modelling of Class Analogy (SIMCA) and K-Nearest Neighbour (KNN). The three methods gave equally good identification results. The results show that FT-IR spectroscopy in combination with PLSR, or other multivariate methods, is well suited for identification of Lactobacillus at the species level, even in quite large data sets.     

Testing for ancient admixture between closely related populations

One enduring question in evolutionary biology is the extent of archaic admixture in the genomes of present-day populations. In this paper, we present a test for ancient admixture that exploits the asymmetry in the frequencies of the two nonconcordant gene trees in a three-population tree. This test was first applied to detect interbreeding between Neandertals and modern humans. We derive the analytic expectation of a test statistic, called the D statistic, which is sensitive to asymmetry under alternative demographic scenarios. We show that the D statistic is insensitive to some demographic assumptions such as ancestral population sizes and requires only the assumption that the ancestral populations were randomly mating. An important aspect of D statistics is that they can be used to detect archaic admixture even when no archaic sample is available. We explore the effect of sequencing error on the false-positive rate of the test for admixture, and we show how to estimate the proportion of archaic ancestry in the genomes of present-day populations. We also investigate a model of subdivision in ancestral populations that can result in D statistics that indicate recent admixture.     

Pedstrip: extracting a maximal subset of available,unrelated individuals from a pedigree

SUMMARY: Certain types of genetic analysis are simplified by assembling a collection of unrelated individuals, e.g. case-control experiments. If a family study is being performed then it will be necessary to extract subsets of unrelated, available individuals from pedigress. Our program provides an optimal method for performing this task. AVAILABILITY: The software is available, free of charge, on request from Oxagen Ltd. SUPPLEMENTARY INFORMATION: http://www.oxagen.co.uk     

Identifying critical regions for spike propagation in axon segments

Morphological reconstructions of axon segments reveal the abundance of geometrical ultrastructures that can dramatically affect the propagation of Action Potentials (AP). Moreover, deformations and swellings in axons resulting from brain traumas are associated to many neural dysfunctions and disorders. Our aim is to develop a computational framework to distinguish between geometrical enlargements that lead to minor changes in propagation from those that result in critical phenomenon such as reflection or blockage of the original traveling spike. We use a few geometrical parameters to model a prototypical shaft enlargement and explore the parameter space characterizing all possible propagation regimes and dynamics in an unmylienated AP model. Contrary to earlier notions that large diameter increases mostly lead to blocking, we demonstrate transmission is stable provided the geometrical changes occur in a slow manner. Our method also identifies a narrow range of parameters leading to a reflection regime. The distinction between these three regimes can be evaluated by a simple function of the geometrical parameters inferred through numerical simulations. We suggest that evaluating this function along axon segments can detect regions most susceptible to (i) transmission failure due to perturbations, (ii) structural plasticity, (iii) critical swellings caused by brain traumas and/or (iv) neurological disorders associated with the break down of spike train propagation.     

DRG represents a family of two closely related GTP-binding proteins

In a previous publication we identified a novel human GTP-binding protein that was related to DRG, a developmentally regulated GTP-binding protein from the central nervous system of mouse. Here we demonstrate that both the human and the mouse genome possess two closely related drg genes, termed drg1 and drg2. The two genes share 62% sequence identity at the nucleotide and 58% identity at the protein level. The corresponding proteins appear to constitute a separate family within the superfamily of the GTP-binding proteins. The DRG1 and the DRG2 mRNA are widely expressed in human and mouse tissues and show a very similar distribution pattern. The human drg1 gene is located on chromosome 22q12, the human drg2 gene on chromosome 17p12. Distantly related species including Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae also possess two drg genes. In contrast, the genomes of archaebacteria (Halobium, Methanococcus, Thermoplasma) harbor only one drg gene, while eubacteria do not seem to contain any. The high conservation of the polypeptide sequences between distantly related organisms indicates an important role for DRG1 and DRG2 in a fundamental pathway.     

Identifying chromosomal fragile sites from individuals: a multinomial statistical model

The inability to identify fragile sites from data for single individuals remains the major obstacle to determining whether these chromosomal loci are predisposed to cancer-causing and evolutionary rearrangements. We describe a novel statistical model that is amenable to data from single individuals and that establishes site-specific chromosomal breakage as nonrandom with respect to the distribution of total breakage. Our method tests incrementally smaller subsets of the data for homogeneity under a multinomial model that assigns equal probabilites to a maximal set of nonfragile sites and unrestricted probabilities to the remaining fragile sites with significantly higher numbers of breaks. We show how standardized Pearson's chi-square (X²) and likelihood-ratio (G²) statistics can be appropriately used to measure goodness-of-fit for sparse contingency (individual-based) data in this model. A sample application of this approach indicates extensive variation in fragile sites among individuals and marked differences in fragile-site inferences from pooled as opposed to per-individual data.     

Evidence for extensive recent intron transposition in closely related fungi

Though spliceosomal introns are a major structural component of most eukaryotic genes and intron density varies by more than three orders of magnitude among eukaryotes [1-3], the origins of introns are poorly understood, and only a few cases of unambiguous intron gain are known [4-8]. We utilized population genomic comparisons of three closely related fungi to identify crucial transitory phases of intron gain and loss. We found 74 intron positions showing intraspecific presence-absence polymorphisms (PAPs) for the entire intron. Population genetic analyses identified intron PAPs at different stages of fixation and showed that intron gain or loss was very recent. We found direct support for extensive intron transposition among unrelated genes. A substantial proportion of highly similar introns in the genome either were recently gained or showed a transient phase of intron PAP. We also identified an intron transfer among paralogous genes that created a new intron. Intron loss was due mainly to homologous recombination involving reverse-transcribed mRNA. The large number of intron positions in transient phases of either intron gain or loss shows that intron evolution is much faster than previously thought and provides an excellent model to study molecular mechanisms of intron gain.     

An enzyme-linked immunosorbent assay for quassin and closely related metabolites

A microtitration plate enzyme-linked immunosorbent assay has been developed for quassin and closely related seco-triterpenes. The method is much more sensitive than conventional chromatographic techniques and enables the routine analysis of large numbers of samples with a detection limit of 5 ng quassin. Antibody production was elicited by injecting a conjugate of iso-quassinic acid linked to bovine serum albumin into rabbits. Important features of the technique are (a) the use of a double-antibody, noncompetitive enzyme-linked immunosorbent assay for a low-molecular-weight, nonantigenic compound using microtitration plates; and (b) that the initial incubation of samples with primary antiserum can be carried out in buffer containing up to 20% ($\text{v}\text{v}$) methanol with minimal loss of sensitivity. The structural requirements for recognition of the hapten by the antiserum are considered based on the levels of cross-reaction found with a wide range of other quassinoids.     

16S-23S ribosomal DNA intergenic spacer regions in cellulolytic myxobacteria and differentiation of closely related strains

The diversity of 16S-23S rDNA intergenic spacer regions (ISR) among cellulolytic myxobacterial strains was assayed. Agarose gel electrophoresis of PCR amplification products from ten strains shows that there are at least four copies of rRNA operons in the genus Sorangium, based on their size and restriction enzymatic digest maps. There are two sequence organization patterns: tRNA(Ile)-tRNA(Ala)-containing ISR and tRNA-lacking ISR. The tRNA-containing ISRs are highly similar among strains and within a strain (more than 98% similarity) and contain the essential functional regions, such as a ribonuclease III recognition site and an antiterminator recognition site boxA. The tRNA-lacking ISR has no such functional sites that are important for yielding mature rRNA, which suggests that this type of rRNA operons might be degenerate. The tRNA-lacking ISR is divided into two types based on their sizes and sequences, which exhibits about 90% similarity within each type. Thus, the tRNA-lacking ISR polymorphisms can be used to discriminate among different strains of sorangial species.