A two-phase approach for detecting recombination in nucleotide sequences

Genetic recombination can produce heterogeneous phylogenetic histories within a set of homologous genes. Delineating recombination events is important in the study of molecular evolution, as inference of such events provides a clearer picture of the phylogenetic relationships among different gene sequences or genomes. Nevertheless, detecting recombination events can be a daunting task, as the performance of different recombinationdetecting approaches can vary, depending on evolutionary events that take place after recombination. We recently evaluated the effects of postrecombination events on the prediction accuracy of recombination-detecting approaches using simulated nucleotide sequence data. The main conclusion, supported by other studies, is that one should not depend on a single method when searching for recombination events. In this paper, we introduce a two-phase strategy, applying three statistical measures to detect the occurrence of recombination events, and a Bayesian phylogenetic approach in delineating breakpoints of such events in nucleotide sequences. We evaluate the performance of these approaches using simulated data, and demonstrate the applicability of this strategy to empirical data. The two-phase strategy proves to be time-efficient when applied to large datasets, and yields high-confidence results.Comment: 5 pages, 3 figures. Chan CX, Beiko RG and Ragan MA (2007). A two-phase approach for detecting recombination in nucleotide sequences. In Hazelhurst S and Ramsay M (Eds) Proceedings of the First Southern African Bioinformatics Workshop, 28-30 January, Johannesburg, 9-1

Meeting Highlights: Genome Sequencing and Biology 2001

We bring you a report from the CSHL Genome Sequencing and Biology Meeting, which has a long and prestigious history. This year there were sessions on large-scale sequencing and analysis, polymorphisms (covering discovery and technologies and mapping and analysis), comparative genomics of mammalian and model organism genomes, functional genomics and bioinformatics

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus <i>Pneumocystis jirovecii</i>.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by &lt;i&gt;Pneumocystis jirovecii&lt;/i&gt; This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single &lt;i&gt;P. jirovecii&lt;/i&gt; strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the &lt;i&gt;P. jirovecii&lt;/i&gt; cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. &lt;b&gt;IMPORTANCE&lt;/b&gt; &lt;i&gt;Pneumocystis jirovecii&lt;/i&gt; is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens

Bacterial artificial chromosomes as analytical basis for gene transcriptional machineries

Bacterial Artificial Chromosomes (BACs) had been minimal components of various genome-sequencing projects, constituting perfect analytical basis for functional genomics. Here we describe an enhancer screening strategy in which BAC clones that cover any genomic segments of interest are modified to harbor a reporter cassette by transposon tagging, then processed to carry selected combinations of gene regulatory modules by homologous recombination mediated systematic deletions. Such engineered BAC-reporter constructs in bacterial cells are ready for efficient transgenesis in mice to evaluate activities of gene regulatory modules intact or absent in the constructs. By utilizing the strategy, we could speedily identify a critical genomic fragment for spatio-temporally regulated expression of a mouse cadherin gene whose structure is extraordinarily huge and intricate. This BAC-based methodology would hence provide a novel screening platform for gene transcriptional machineries that dynamically fluctuate during development, pathogenesis and/or evolution

High-throughput sequencing of the T-cell receptor repertoire: pitfalls and opportunities.

T-cell specificity is determined by the T-cell receptor, a heterodimeric protein coded for by an extremely diverse set of genes produced by imprecise somatic gene recombination. Massively parallel high-throughput sequencing allows millions of different T-cell receptor genes to be characterized from a single sample of blood or tissue. However, the extraordinary heterogeneity of the immune repertoire poses significant challenges for subsequent analysis of the data. We outline the major steps in processing of repertoire data, considering low-level processing of raw sequence files and high-level algorithms, which seek to extract biological or pathological information. The latest generation of bioinformatics tools allows millions of DNA sequences to be accurately and rapidly assigned to their respective variable V and J gene segments, and to reconstruct an almost error-free representation of the non-templated additions and deletions that occur. High-level processing can measure the diversity of the repertoire in different samples, quantify V and J usage and identify private and public T-cell receptors. Finally, we discuss the major challenge of linking T-cell receptor sequence to function, and specifically to antigen recognition. Sophisticated machine learning algorithms are being developed that can combine the paradoxical degeneracy and cross-reactivity of individual T-cell receptors with the specificity of the overall T-cell immune response. Computational analysis will provide the key to unlock the potential of the T-cell receptor repertoire to give insight into the fundamental biology of the adaptive immune system and to provide powerful biomarkers of disease

국내 콩 모자이크 바이러스 개체군의 진화 양상 분석

학위논문(석사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2023. 2. 김국형.콩 모자이크 바이러스 (SMV)는 전 세계적으로 발견되며, 콩 재배에 있어 만성적이고, 심각한 피해를 미치는 병원균으로 알려져 있다. 이번 연구에서는 한국의 41개 지역의 콩 재배 포장으로부터 12개의 새로운 SMV 분리주를 확보하였고, 기존에 보고된 분리주들과 비교함으로써 개체군 진화 양상을 분석하였다. 계통분석을 통해 전체 SMV 개체군 내에서 4 개의 주요 분기군을 발견하여 A에서 D 로 명명하였으며, 각 분기군이 지리적인 특징을 보이는 것을 확인하였다. 한국에서의 SMV 진화양상 파악을 위해 한국 분리주를 사용하여 개체군 분석이 진행되었다. 재조합 분석을 통해 12개의 새로운 분리주들 중 7개 분리주에서 재조합 신호를 발견하여, SMV 개체군에서 재조합이 매우 빈번하게 일어났음을 확인할 수 있었다. 특히, 3개의 분리주는 기존 서열에서는 발견되지 않았던 새로운 재조합 중단점을 가지고 있어 재조합이 최근까지도 지속적으로 SMV 진화에 영향을 미치고 있었음을 시사하였다. 개체군의 유전적 구조를 분석하기 위해 진행된 27개의 비 재조합 분리주를 사용한 분석에서 대부분의 분리주들은 주로 분기군 A와 B에 속했으며 각 분기군은 통계적으로 유의미한 유전적 차이를 보이는 것을 확인하였다. 유전적 다양성은 A보다 B에서 크게 나타났지만, 두 분기군에서 모두 11개 단백질 도메인 암호영역에서 공통적으로 음성 선택이 일어나는 것이 확인되었다. 2016년 이후 보고된 분리주는 대부분 B에서 발생한 것을 확인하였고 새로 발생한 분기군을 B1 이라 명명하였다. 아미노산 서열 유사도 분석을 통해 기존 B 분기군인 B2와 새로운 발생한 분기군인 B1의 주된 차이는 P1 단백질 부분에서 발생하였음을 확인할 수 있었는데, 이 결과는 최근 SMV 개체군에서 기주범위나 병원성에 변화가 나타났음을 시사한다.Soybean mosaic virus (SMV) is the most chronic and devastating soybean pathogen worldwide. In this study, 12 new isolates were detected from the 41 soybean-growing regions in Korea. Phylogenetic analysis revealed 4 main phylogroups (A to D) in the global SMV population and showed the geographic features of each phylogroup. To identify the population dynamics in Korea, we conducted an additional analysis using Korean isolates. Recombination analysis prevailed 7 of 12 new isolates were recombinants. In addition, 3 showed unique signals suggesting recombination constantly affected the evolution of SMV. In total 27 non-recombinant Korea SMV isolates, only 2 phylogroups, A and B, remained, and the statistically significant genetic difference was checked. Even though isolates of B showed higher genetic diversity than A, both phylogroups are under negative selection pressure in all 11 domains. In addition, phylogroup B, which contains G6 and G7 strains, seems to be preferred with the recently emerged subgroup, B1. In the sequence similarity plot, the novel subgroup (B1) showed the highest distinction in the P1 region, suggesting that host range or pathogenicity changes occur in the SMV population. These results provide information about the evolutionary flow of the SMV population in Korea.I. INTRODUCTION 1 II. MATERIALS AND METHODS 3 1. Sample collection and total RNA extraction 3 2. Virome analysis 3 3. SMV genome sequencing and assembly 4 4. Sequence alignment and phylogenetic analysis 6 5. Haplotype network analysis 6 6. Recombination analysis 6 7. Calculation of the population genetic parameter 7 III. RESULTS 9 1. Virus detection and SMV genome sequencing 9 2. Phylogenetic analysis of SMV population 11 3. Detection of recombination signal 13 4. Genetic variation in the Korean SMV population 16 5. Neutrality test and selection pressure analysis 16 6. Differentiation of Korean SMV populations 18 IV. DISCUSSION 21 V. LITERATURE CITED 25 VI. SUPPLEMENTARY TABLE 31 VII. ABSTRACT IN KOREAN 35석

A Stem Cell-Based Tool for Small Molecule Screening in Adipogenesis

Techniques for small molecule screening are widely used in biological mechanism study and drug discovery. Here, we reported a novel adipocyte differentiation assay for small molecule selection, based on human mesenchymal stem cells (hMSCs) transduced with fluorescence reporter gene driven by adipogenic specific promoter - adipocyte Protein 2 (aP2; also namely Fatty Acid Binding Protein 4, FABP4). During normal adipogenic induction as well as adipogenic inhibition by Ly294002, we confirmed that the intensity of green fluorescence protein corresponded well to the expression level of aP2 gene. Furthermore, this variation of green fluorescence protein intensity can be read simply through fluorescence spectrophotometer. By testing another two small molecules in adipogenesis –Troglitazone and CHIR99021, we proved that this is a simple and sensitive method, which could be applied in adipocyte biology, drug discovery and toxicological study in the future

Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line

<p>Abstract</p> <p>Background</p> <p>Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic β-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the <it>pdx1 </it>(pancreatic-duodenal homeobox 1) gene.</p> <p>Methods</p> <p>Single (hemizygous) transgenic mice of the <it>pdx1-cre</it>^Cre/0genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages.</p> <p>Results</p> <p>In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous <it>pdx1 </it>was not observed.</p> <p>Conclusions</p> <p>The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.</p

Sequence analysis of two alleles reveals that intra-and intergenic recombination played a role in the evolution of the radish fertility restorer (Rfo)

Background \ud Land plant genomes contain multiple members of a eukaryote-specific gene family encoding proteins with pentatricopeptide repeat (PPR) motifs. Some PPR proteins were shown to participate in post-transcriptional events involved in organellar gene expression, and this type of function is now thought to be their main biological role. Among PPR genes, restorers of fertility (Rf) of cytoplasmic male sterility systems constitute a peculiar subgroup that is thought to evolve in response to the presence of mitochondrial sterility-inducing genes. Rf genes encoding PPR proteins are associated with very close relatives on complex loci. \ud Results \ud We sequenced a non-restoring allele (L7rfo) of the Rfo radish locus whose restoring allele (D81Rfo) was previously described, and compared the two alleles and their PPR genes. We identified a ca 13 kb long fragment, likely originating from another part of the radish genome, inserted into the L7rfo sequence. The L7rfo allele carries two genes (PPR-1 and PPR-2) closely related to the three previously described PPR genes of the restorer D81Rfo allele (PPR-A, PPR-B, and PPR-C). Our results indicate that alleles of the Rfo locus have experienced complex evolutionary events, including recombination and insertion of extra-locus sequences, since they diverged. Our \ud analyses strongly suggest that present coding sequences of Rfo PPR genes result from intragenic recombination. We found that the 10 C-terminal PPR repeats in Rfo PPR gene encoded proteins result from the tandem duplication of a 5 PPR repeat block. \ud Conclusions \ud The Rfo locus appears to experience more complex evolution than its flanking \ud sequences. The Rfo locus and PPR genes therein are likely to evolve as a result of \ud intergenic and intragenic recombination. It is therefore not possible to determine which genes on the two alleles are direct orthologs. Our observations recall some \ud previously reported data on pathogen resistance complex loci. \u