Olga O. Zaytseva

Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs

Much of the research on insulators in Drosophila has been done with transgenic constructs using the white gene (mini-white) as reporter. Hereby we report that the sequence between the white and CG32795 genes in Drosophila melanogaster contains an insulator of a novel kind. Its functional core is within a 368 bp segment almost contiguous to the white 3′UTR, hence we name it as Wari (white-abutting resident insulator). Though Wari contains no binding sites for known insulator proteins and does not require Su(Hw) or Mod(mdg4) for its activity, it can equally well interact with another copy of Wari and with unrelated Su(Hw)-dependent insulators, gypsy or 1A2. In its natural downstream position, Wari reinforces enhancer blocking by any of the three insulators placed between the enhancer and the promoter; again, Wari–Wari, Wari–gypsy or 1A2–Wari pairing results in mutual neutralization (insulator bypass) when they precede the promoter. The distressing issue is that this element hides in all mini-white constructs employed worldwide to study various insulators and other regulatory elements as well as long-range genomic interactions, and its versatile effects could have seriously influenced the results and conclusions of many works.

New data on three molecular markers from different cellular genomes in Mediterranean accessions reveal new insights into phylogeography of Pisum sativum L. subsp. elatius (Bieb.) Schmalh.

Twelve accessions classified as Pisum sativum subsp. elatius, mostly from West and Central Mediterranean, were analysed for three markers from different cellular genomes: rbcL (plastid genome), coxI (mitochondrial genome) and SCA (nuclear genome). Based on geographical distribution of their allele combinations analysed in this and the earlier study, we suggest a putative history of wild representatives of P. sativum. The ancestor of this species belonged to lineage A (coxI+, rbcL+, SCAf); it appeared in East Mediterranean, then spread westward most probably during one of the Pleistocene coolings when the sea was smaller, so that representatives of lineage A remained in the Eastern Mediterranean and on the islands of Sicily and Menorca. Mutation leading to the loss of the restriction site for PsiI in coxI−, gave rise to lineage C (coxI−, rbcL+, SCAf) which spread widely in the Mediterranean and is now found in France, Greece and Ethiopia. Mutation leading to rbcL− gave rise to lineage D (coxI−, rbcL−, SCAf), now found in Egypt (P. sativum subsp. jomardii) and Spain. Mutational transition of SCAf to SCAs most probably took place in North-Eastern Mediterranean since the resulting lineage B (coxI−, rbcL−, SCAs) now occupies the Tauro-Caucasian area. In Asia Minor and North Israel, line B met the ancestral line A so that both lines coexist there presently. The lineage B gave rise to the cultivated P. sativum subsp. sativum.

Phylogenetic reconstruction at the species and intraspecies levels in the genus Pisum (L.) (peas) using a histone H1 gene.

A phylogenetic analysis of the genus Pisum (peas), embracing diverse wild and cultivated forms, which evoke problems with species delimitation, was carried out based on a gene coding for histone H1, a protein that has a long and variable functional C-terminal domain. Phylogenetic trees were reconstructed on the basis of the coding sequence of the gene His5 of H1 subtype 5 in 65 pea accessions. Early separation of a clear-cut wild species Pisum fulvum is well supported, while cultivated species Pisum abyssinicum appears as a small branch within Pisum sativum. Another robust branch within P. sativum includes some wild and almost all cultivated representatives of P. sativum. Other wild representatives form diverse but rather subtle branches. In a subset of accessions, PsbA-trnH chloroplast intergenic spacer was also analysed and found less informative than His5. A number of accessions of cultivated peas from remote regions have a His5 allele of identical sequence, encoding an electrophoretically slow protein product, which earlier attracted attention as likely positively selected in harsh climate conditions. In PsbA-trnH, a 8bp deletion was found, which marks cultivated representatives of P. sativum.

Divergence and population traits in evolution of the genus Pisum L. as reconstructed using genes of two histone H1 subtypes showing different phylogenetic resolution.

Two histone H1 subtype genes, His7 and His5, were sequenced in a set of 56 pea accessions. Phylogenetic reconstruction based on concatenated His5 and His7 sequences had three main clades. First clade corresponded to Pisum fulvum, the next divergence separated a clade inside Pisum sativum in the broad sense that did not correspond strictly to any proposed taxonomical subdivisions. According to our estimations, the earliest divergence separating P. fulvum occurred 1.7±0.4MYA. The other divergence with high bootstrap support that separated two P. sativum groups took place approximately 1.3±0.3MYA. Thus, the main divergences in the genus took place either in late Pliocene or in early Pleistocene, the time of onset of the profound climate cooling in the northern hemisphere. The ω=K(a)/K(s) ratio was 2.5 times higher for His5 sequences than for His7. Thus, His7 gene, coding for a unique subtype specific for actively growing tissues, might have evolved under stricter evolutionary constraints than His5, that codes for a minor H1 subtype with less specific expression pattern. For this reason phylogenetic reconstructions separately obtained from His5 sequences resolved tree topology much better than those obtained from His7 sequences. Computational estimation of population dynamic parameters in the genus Pisum L. from His5-His7 sequences using IMa2 software revealed a decrease of effective population size on the early stage of Pisum evolution.

Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

Refinement of the collection of wild peas (Pisum L.) and search for the area of pea domestication with a deletion in the plastidic psbA-trnH spacer

The plastidic psbA-trnH spacer was sequenced in 78 accessions representing the genus Pisum L. A nucleotide substitution C64T in the spacer was found to be specific for Pisum abyssinicum A. Br.; a substitution T75G occurred in five accessions of the wild pea [Pisum sativum L. subsp. elatius (Bieb.) Schmalh.], one from France, three from Greece and one from Turkey, and also in a primitive landrace from Afghanistan with signs of contamination. A 7-bp tandem duplication was found in two P. sativum subsp. elatius accessions, from Turkey and Georgia. All (except for the probably contaminated Afghan accession) of the cultivated subspecies P. sativum L. subsp. sativum had a deletion of one copy of a tandem 8-bp repeat. The same deletion was found in two wild pea (P. sativum subsp. elatius) accessions, from Bulgaria and Georgia, belonging to the same earlier defined lineage B as the cultivated pea. They are supposed to represent the ancestral evolutionary lineage of the cultivated pea. It is noteworthy that accessions from the proposed Core Area of the Near East founder crop domestication in south-eastern Turkey do not have the deletion. Most of wild representatives of lineage B have a scarcely pigmented flower with almost non-opening standard, but those from Transcaucasia have a normal flower. The revealed area of co-existence of the earlier defined wild pea lineages A and B (differing in alleles of three molecular markers from different cellular genomes) was extended from Turkey to include Georgia and probably North Africa. Accessions claimed to represent wild peas were tested for spontaneous pod dehiscing and 14 of them were disproved as such. They are enriched with ‘recombinant’ marker combinations and most probably resulted from hybridisation of wild and cultivated peas, either in nature or while reproducing in germplasm collections.

Profiling and genetic control of the murine immunoglobulin G glycome

AbstractImmunoglobulin G (IgG) glycosylation is essential for function of the immune system, but the genetic and environmental factors that underlie its inter-individual variability are not well defined. The Collaborative Cross (CC) genetic resource harnesses over 90% of the common genetic variation of the mouse. By analyzing the IgG glycome composition of 95 CC strains, we made several important observations: (i) glycome variation between mouse strains was higher than between individual humans, despite all mice having the same environmental influences; (ii) five genetic loci were found to be associated with murine IgG glycosylation; (iii) variants outside traditional glycosylation site motifs affected glycome variation; (iv) bisecting N-acetylglucosamine (GlcNAc) was produced by several strains although most previous studies have reported the absence of glycans containing the bisecting GlcNAc on murine IgGs; and (v) common laboratory mouse strains are not optimal animal models for studying effects of glycosylation on IgG function.Comprehensive glycome profiling of immunoglobulin G (IgG) in 95 strains of mice from the Collaborative Cross genetics resource reveals the extent and variability of IgG glycosylation in vivo.

MIgGGly (mouse IgG glycosylation analysis) - a high-throughput method for studying Fc-linked IgG N-glycosylation in mice with nanoUPLC-ESI-MS

Immunoglobulin G (IgG) N-glycosylation is crucial for its effector functions. It is a complex trait, and large sample sets are needed to discover multiple genetic factors that underlie it. While in humans such high-throughput studies of IgG N-glycans became usual, only one has been carried out in mice. Here we describe and validate a method for the relative quantification of IgG Fc-linked N-glycans in a subclass-specific manner using nano-reverse phase liquid chromatography coupled with mass-spectrometry (nanoRP-LC-MS) applied to murine IgG. High-throughput data processing is ensured by the LaCyTools software. We have shown that IgG isolation procedure is the main source of technical variation in the current protocol. The major glycoforms were quantified reliably with coefficients of variation below 6% for all the analytes with relative abundances above 5%. We have applied our method to a sample set of 3 inbred strains: BALB/c, C57BL/6 and C3H and observed differences in subclass-specific and strain-specific N-glycosylation of IgG, suggesting a significant genetic component in the regulation of Fc-linked IgG N-glycosylation.

Coat color allele and mtDNA haplotype distributions in Russian cat populations: a citizen-science research

This work has started as a project for the summer school in molecular and theoretical biology. Efficient teaching and learning modern methods of molecular biology and population genetics in a two-week study course for high school students needs an attractive subject. We chose phylogeography of the domestic cat as the subject of our project because 1) everybody likes cats; 2) cats are polymorphic for several coat color mutations, which can be easily detected by street survey or in the photographs; 3) samples for DNA extraction are easy to collect without posing ethical and biosafety problems; 4) rich background information of geographical distribution of coat color alleles and variation in mtDNA is available; 5) phylogeography of cat population in Russia is poorly studied and therefore new data collected in this large territory may shed a light on the global cat distribution and on the origin of the fancy breeds. During the project students studied coat color and mitotype distribution across Russian random bred cats. The basics of field (observations of natural cat populations, hair collection), formal (inheritance of coat colors), mathematical population (allele frequency distributions and their comparisons, building phylogenetic trees, multidimensional scaling), and molecular (DNA extraction and amplification, quality control of DNA sequencing data) genetics were covered. We scored coat color phenotypes in 1182 cats and sequenced mtDNA control region from hair samples of 38 cats from 18 geographical sites. Analysis of coat color alleles frequencies and mitotype distribution confirmed relative homogeneity of gene pools of Russian cat populations, indicating their recent origin. We found several unique mitotypes and demonstrated that OL1 mitotype, previously found only in Siberian fancy breed, was present in random bred cats from several Russian cities. This contributes to the discussion on the origin of the Siberian breed of cats, and supports the view that Siberians is a recent breed created in the 1980s by breeding of selected representatives of the random bred population.

Network based conditional genome wide association analysis of human metabolomics

Background Genome-wide association studies (GWAS) have identified hundreds of loci influencing complex human traits, however, their biological mechanism of action remains mostly unknown. Recent accumulation of functional genomics (‘omics’) including metabolomics data opens up opportunities to provide a new insight into the functional role of specific changes in the genome. Functional genomic data are characterized by high dimensionality, presence of (strong) statistical dependencies between traits, and, potentially, complex genetic control. Therefore, analysis of such data asks for development of specific statistical genetic methods. Results We propose a network-based, conditional approach to evaluate the impact of genetic variants on omics phenotypes (conditional GWAS, cGWAS). For each trait of interest, based on biological network, we select a set of other traits to be used as covariates in GWAS. The network could be reconstructed either from biological pathway databases or directly from the data. We evaluated our approach using data from a population-based KORA study (n=1,784, 1.7 M SNPs) with measured metabolomics data (151 metabolites) and demonstrated that our approach allows for identification of up to five additional loci not detected by conventional GWAS. We show that this gain in power is achieved through increased precision of genetic effect estimates, and in presence of specific ‘contra-intuitive’ pleiotropic scenarios (when genetic and environmental sources of covariance are acting in opposite manner). We justify existence of such scenarios, and discuss possible applications of our method beyond metabolomics. Conclusions We demonstrate that in context of metabolomics network-based, conditional genome-wide association analysis is able to dramatically increase power of identification of loci with specific ‘contra-intuitive’ pleiotropic architecture. Our method has modest computational costs, can utilize summary level GWAS data, and is applicable to other omics data types. We anticipate that application of our method to new and existing data sets will facilitate progress in understanding genetic bases of control of molecular and complex phenotypes. Short abstract We propose a network-based, conditional approach for genome-wide analysis of multivariate omics phenotypes. Our methods can incorporate prior biological knowledge about biological pathways from external sources. We evaluated our approach using metabolomics data and demonstrated that our approach has bigger power and allows for identification of additional loci. We show that gain in power is achieved through increased precision of genetic effect estimates, and in presence of specific ‘contra-intuitive’ pleiotropic scenarios (when genetic and environmental sources of covariance are acting in opposite manner). We justify existence of such scenarios, and discuss possible applications of our method beyond metabolomics.