Pairing Of Homologous Sequences Research Articles

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Measuring functional similarity of lncRNAs based on variable K-mer profiles of nucleotide sequences.

Long non-coding RNAs are a class of essential non-coding RNAs with a length of more than 200 nts. Recent studies have indicated that lncRNAs have various complex regulatory functions, which play great impacts on many fundamental biological processes. However, measuring the functional similarity between lncRNAs by traditional wet-experiments is time-consuming and labor intensive, computational-based approaches have been an effective choice to tackle this problem. Meanwhile, most sequences-based computation methods measure the functional similarity of lncRNAs with their fixed length vector representations, which could not capture the features on larger k-mers. Therefore, it is urgent to improve the predict performance of the potential regulatory functions of lncRNAs. In this study, we propose a novel approach called MFSLNC to comprehensively measure functional similarity of lncRNAs based on variable k-mer profiles of nucleotide sequences. MFSLNC employs the dictionary tree storage, which could comprehensively represent lncRNAs with long k-mers. The functional similarity between lncRNAs is evaluated by the Jaccard similarity. MFSLNC verified the similarity between two lncRNAs with the same mechanism, detecting homologous sequence pairs between human and mouse. Besides, MFSLNC is also applied to lncRNA-disease associations, combined with the association prediction model WKNKN. Moreover, we also proved that our method can more effectively calculate the similarity of lncRNAs by comparing with the classical methods based on the lncRNA-mRNA association data. The detected AUC value of prediction is 0.867, which achieves good performance in the comparison of similar models.

Comparative analysis of the plastid and mitochondrial genomes of Artemisia giraldii Pamp.

Artemisia giraldii Pamp. is an herbaceous plant distributed only in some areas in China. To understand the evolutionary relationship between plastid and mitochondria in A. giraldii, we sequenced and analysed the plastome and mitogenome of A. giraldii on the basis of Illumina and Nanopore DNA sequencing data. The mitogenome was 194,298 bp long, and the plastome was 151,072 bp long. The mitogenome encoded 56 genes, and the overall GC content was 45.66%. Phylogenetic analysis of the two organelle genomes revealed that A. giraldii is located in the same branching position. We found 13 pairs of homologous sequences between the plastome and mitogenome, and only one of them might have transferred from the plastid to the mitochondria. Gene selection pressure analysis in the mitogenome showed that ccmFc, nad1, nad6, atp9, atp1 and rps12 may undergo positive selection. According to the 18 available plastome sequences, we found 17 variant sites in two hypervariable regions that can be used in completely distinguishing 18 Artemisia species. The most interesting discovery was that the mitogenome of A. giraldii was only 43,226 bp larger than the plastome. To the best of our knowledge, this study represented one of the smallest differences between all sequenced mitogenomes and plastomes from vascular plants. The above results can provide a reference for future taxonomic and molecular evolution studies of Asteraceae species.

Recombination in yeast based on six base pairs of homologous sequences: Structural instability in two sets of isomeric model expression plasmids.

Multicopy episomal yeast (Saccharomyces cerevisiae) plasmids are frequently employed in research and industrial production despite their known limited structural and segregational stability. Employing a set of six yEGFP3 model expression plasmids (identical in size but differing in the arrangement of their functional sequences, joined via six base pair SacI sequences), we used back transformation of total DNA extracted from yeast transformants into Escherichia coli to detect potential plasmid rearrangements. This approach revealed deletions, translocations, duplications, and flippings of functional sequences in our plasmids based on homologous recombination between the SacI sequences. To extend our findings, we assembled and analysed in the same way a corresponding plasmid set of six isoforms expressing the antibacterial insect peptide defensin A. In 833 individual ampR clones (both sets combined), we traced 28 cases (3.4%) with precise structural changes. However, the frequency in one isoform in the pIFC4.13X series, pIFC4.131, was particularly high with 18.5% (15 out of 81 clones), indicating that the architecture of this plasmid is unfavourable to the host. With an increased sensitivity, a Polymerase Chain Reaction (PCR) approach revealed further structural changes in at least half of the isoforms of each set. The changes are considered the consequence of homologous recombination events involving the SacI sequences in a random fashion. The frequency of plasmid alterations is the product of selection and counterselection seemingly favouring or disfavouring certain structures. Although no sole architectural arrangement stuck out as being particularly stable, we were able to determine with our approach unfavourable sequence associations that should be avoided.

Meiotic drive of female-inherited supernumerary chromosomes in a pathogenic fungus.

Meiosis is a key cellular process of sexual reproduction that includes pairing of homologous sequences. In many species however, meiosis can also involve the segregation of supernumerary chromosomes, which can lack a homolog. How these unpaired chromosomes undergo meiosis is largely unknown. In this study we investigated chromosome segregation during meiosis in the haploid fungus Zymoseptoria tritici that possesses a large complement of supernumerary chromosomes. We used isogenic whole chromosome deletion strains to compare meiotic transmission of chromosomes when paired and unpaired. Unpaired chromosomes inherited from the male parent as well as paired supernumerary chromosomes in general showed Mendelian inheritance. In contrast, unpaired chromosomes inherited from the female parent showed non-Mendelian inheritance but were amplified and transmitted to all meiotic products. We concluded that the supernumerary chromosomes of Z. tritici show a meiotic drive and propose an additional feedback mechanism during meiosis, which initiates amplification of unpaired female-inherited chromosomes.

Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba).

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F2 mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F2 plants were divided into three subpopulations according to the original F1 plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.

Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator‐like effector nucleases in medaka

Genome editing using engineered nucleases such as transcription activator-like effector nucleases (TALENs) has become a powerful technology for reverse genetics. In this study, we have described efficient detection methods for TALEN-induced mutations at endogenous loci and presented guidelines of TALEN design for efficient targeted mutagenesis in medaka, Oryzias latipes. We performed a heteroduplex mobility assay (HMA) using an automated microchip electrophoresis system, which is a simple and high-throughput method for evaluation of in vivo activity of TALENs and for genotyping mutant fish of F1 or later generations. We found that a specific pattern of mutations is dominant for TALENs harboring several base pairs of homologous sequences in target sequence. Furthermore, we found that a 5' T, upstream of each TALEN-binding sequence, is not essential for genomic DNA cleavage. Our findings provide information that expands the potential of TALENs and other engineered nucleases as tools for targeted genome editing in a wide range of organisms, including medaka.

Autoimmune Thyroid Disease Elicited by NY-ESO-1 Vaccination

Immunotherapies and targeted therapies are frequently associated with thyroid dysfunction, which is in contrast with the rare thyroid abnormalities induced by cytotoxic agents. Immunotherapy with NY-ESO-1, a tumor-associated antigen expressed by a number of malignancies, was reported to trigger hyperthyroidism or hypothyroidism in two HLA-A2 patients with ovarian cancer. We describe now a case of Graves' disease triggered by NY-ESO-1 in a HLA-A2-negative woman. A 32-year-old woman with a synovial sarcoma received radiotherapy, chemotherapy, and finally NY-ESO-1 vaccine. The patient was found to have HLA A11/A33(19), B13/B56(22), Cw3/-. One month after the beginning of immunotherapy, thyroid dysfunction was clinically suspected and Graves' disease was biochemically confirmed. Fearful of the antithyroid drugs' side effects, the patient was treated with a beta-blocker (propranolol, 80-20 mg/day). As hyperthyroidism progressively worsened, the patient underwent total thyroidectomy. We hypothesized that NY-ESO-1 shared partial homology with thyroid autoantigens (the so-called molecular mimicry mechanism) and that at least one pair of homologous sequences contained amino acid sequence binding motifs to a restricted number of HLA molecules. We used BLAST software to search amino acid sequence homologies between NY-ESO-1 and thyroid autoantigens (thyrotropin receptor [TSH-R], thyroperoxidase, and thyroglobulin), and the HLA ligand/motif database to look for HLA/T-cell receptor binding motifs in the regions of NY-ESO-1 and thyroid autoantigens that were homologous. We found 15 epitopic regions of NY-ESO-1 homologous to 15 regions of thyroid autoantigens, some of which epitopic: 5 of TSH-R, 8 of thyroglobulin, and 2 of thyroperoxidase. These homologous sequences contain binding motifs belonging to several HLA class I antigens, including HLA A2 and the patient's A11 and A33. Genetically predisposed patients who receive NY-ESO-1 vaccination are at risk to develop thyroid dysfunction. Considering the increasing use of NY-ESO-1, thyroid dysfunctions induced by NY-ESO-1 are expected to increase in cancer patients over the next years.

Comparative Analysis of Barophily-Related Amino Acid Content in Protein Domains ofPyrococcus abyssiandPyrococcus furiosus

Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

Chromosome Pairing: A Hidden Treasure No More

Chromosome Pairing: A Hidden Treasure No More

Sequence Alignment with an Appropriate Substitution Matrix

A widely used algorithm for computing an optimal local alignment between two sequences requires a parameter set with a substitution matrix and gap penalties. It is recognized that a proper parameter set should be selected to suit the level of conservation between sequences. We describe an algorithm for selecting an appropriate substitution matrix at given gap penalties for computing an optimal local alignment between two sequences. In the algorithm, a substitution matrix that leads to the maximum alignment similarity score is selected among substitution matrices at various evolutionary distances. The evolutionary distance of the selected substitution matrix is defined as the distance of the computed alignment. To show the effects of gap penalties on alignments and their distances and help select appropriate gap penalties, alignments and their distances are computed at various gap penalties. The algorithm has been implemented as a computer program named SimDist. The SimDist program was compared with an existing local alignment program named SIM for finding reciprocally best-matching pairs (RBPs) of sequences in each of 100 protein families, where RBPs are commonly used as an operational definition of orthologous sequences. SimDist produced more accurate results than SIM on 50 of the 100 families, whereas both programs produced the same results on the other 50 families. SimDist was also used to compare three types of substitution matrices in scoring 444,461 pairs of homologous sequences from the 100 families.

Sister chromatid junctions in the hyperthermophilic archaeon Sulfolobus solfataricus

Although the Archaea exhibit an intriguing combination of bacterial- and eukaryotic-like features, it is not known how these prokaryotic cells segregate their chromosomes before the process of cell division. In the course of our analysis of the third replication origin in the archaeon Sulfolobus solfataricus, we identify and characterise sister chromatid junctions in this prokaryote. This pairing appears to be mediated by hemicatenane-like structures, and we provide evidence that these junctions persist in both replicating and postreplicative cells. These data, in conjunction with fluorescent in situ hybridisation analyses, suggest that Sulfolobus chromosomes have a significant period of postreplicative sister chromatid synapsis, a situation that is more reminiscent of eukaryotic than bacterial chromosome segregation mechanisms.

Dynamic use of multiple parameter sets in sequence alignment

The level of conservation between two homologous sequences often varies among sequence regions; functionally important domains are more conserved than the remaining regions. Thus, multiple parameter sets should be used in alignment of homologous sequences with a stringent parameter set for highly conserved regions and a moderate parameter set for weakly conserved regions. We describe an alignment algorithm to allow dynamic use of multiple parameter sets with different levels of stringency in computation of an optimal alignment of two sequences. The algorithm dynamically considers various candidate alignments, partitions each candidate alignment into sections, and determines the most appropriate set of parameter values for each section of the alignment. The algorithm and its local alignment version are implemented in a computer program named GAP4. The local alignment algorithm in GAP4, that in its predecessor GAP3, and an ordinary local alignment program SIM were evaluated on 257 716 pairs of homologous sequences from 100 protein families. On 168 475 of the 257 716 pairs (a rate of 65.4%), alignments from GAP4 were more statistically significant than alignments from GAP3 and SIM.

GeneAlign: a coding exon prediction tool based on phylogenetical comparisons

GeneAlign is a coding exon prediction tool for predicting protein coding genes by measuring the homologies between a sequence of a genome and related sequences, which have been annotated, of other genomes. Identifying protein coding genes is one of most important tasks in newly sequenced genomes. With increasing numbers of gene annotations verified by experiments, it is feasible to identify genes in the newly sequenced genomes by comparing to annotated genes of phylogenetically close organisms. GeneAlign applies CORAL, a heuristic linear time alignment tool, to determine if regions flanked by the candidate signals (initiation codon-GT, AG-GT and AG-STOP codon) are similar to annotated coding exons. Employing the conservation of gene structures and sequence homologies between protein coding regions increases the prediction accuracy. GeneAlign was tested on Projector dataset of 491 human–mouse homologous sequence pairs. At the gene level, both the average sensitivity and the average specificity of GeneAlign are 81%, and they are larger than 96% at the exon level. The rates of missing exons and wrong exons are smaller than 1%. GeneAlign is a free tool available at .

SLAM web server for comparative gene finding and alignment.

SLAM is a program that simultaneously aligns and annotates pairs of homologous sequences. The SLAM web server integrates SLAM with repeat masking tools and the AVID alignment program to allow for rapid alignment and gene prediction in user submitted sequences. Along with annotations and alignments for the submitted sequences, users obtain a list of predicted conserved non-coding sequences (and their associated alignments). The web site also links to whole genome annotations of the human, mouse and rat genomes produced with the SLAM program. The server can be accessed at http://bio.math.berkeley.edu/slam.

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences.

Heterogeneity in Rates of Recombination in the 6-Mb Region Telomeric to the Human Major Histocompatibility Complex

Analysis of 784 informative meioses in the CEPH pedigrees revealed a total of 22 recombination events having occurred in the 6-Mb region between D6S265 (70 kb centromeric of HLA-A) and D6S276. These 22 breakpoints were localized with respect to anonymous polymorphic markers, leading to a detailed genetic map of the region telomeric to the human major histocompatibility complex. A nonrandom pattern of recombination was observed throughout this region: the low recombination rate of 0.19% within the 4-Mb interval centromeric to the HLA class I-like candidate gene for hemochromatosis indeed contrasts with the approximate 1% rate observed within the most telomeric two megabases. This reduced rate of recombination may be due to selective constraints depending on environmental factors related to immunity and iron status or to structural variations hampering proper meiotic pairing of homologous sequences. Population data from other human genome segments are now needed to determine whether linkage disequilibrium extending over 4 Mb is unique to this region.

Analysis of Amino Acid Substitution during Divergent Evolution: The 400 by 400 Dipeptide Substitution Matrix

Most formal methods for analyzing the divergent evolution of protein sequences assume a Markov model where position i in a polypeptide chain undergoes amino acid substitution independently from position i + 1. The large number of aligned homologous sequence pairs available from the exhaustive matching of the protein sequence database makes it possible to examine this assumption empirically. We have constructed a 400 by 400 matrix that reports empirical probabilities for the interconversion of all pairs of dipeptides in proteins undergoing divergent evolution. Comparison of these probabilities with those expected if substitution at adjacent positions in a protein sequence were independent reveals interesting patterns that arise through the breakdown of this assumption. Several of these are useful in extracting conformational information from patterns of conservation and variation in homologous protein sequences.

Molecular perspectives of chromosome pairing at meiosis

Ideas about the mechanisms that regulate chromosome pairing, recombination, and segregation during meiosis have gained in molecular detail over the last few years. The purpose of this article is to survey briefly the shifts in paradigms and experiments that have generated new perspectives. It has never been very clear what it is that brings together the homologous chromosomes at meiotic prophase. For a while it appeared that the synaptonemal complex might be the nuclear organelle responsible for synapsis, but the supporting evidence has not been entirely convincing. Whatever the mechanism, it has always been assumed that homologous synapsis creates the opportunity for homologous DNA sequences to initiate recombination. At present, alternative ideas are developing. Attractive is the concept that double strand DNA repair mechanisms, that find and use the undamaged homologue for repair, have evolved into a meiotic mechanism for the recognition and pairing of homologous sequences. Subsequent intimate synapsis of homologous chromosomes in the context of the synaptonemal complex may serve later functions in the regulation of interference and segregation at first anaphase. A number of areas that are being tested at present and some that may be investigated in the future are discussed at the end of the review.

Balbiani ring 1 gene in Chironomus tentans: Sequence organization and dynamics of a coding minisatellite

Balbiani ring (BR) genes in diptera encode large secretory proteins and are classical model systems for studies of gene expression. In Chironomus tentans, four closely related BR genes, BR 1, BR 2.1, BR 2.2 and BR 6 form a gene family. The BR genes have been partially characterized and are known to contain long arrays of tandemly arranged repeat units with an hierarchical repeat organization. Here, we report the sequence organization of the complete transcribed part of the BR 1 gene in C. tentans. The gene contains five exons and four introns. Three of the introns are located at the 5′ end and the fourth at the 3′ end of the gene. Exon 4 is approximately 35,000 bases long and is built completely from tandemly organized repeats. We show that this long repeat block contains two types of related repeat units, β and γ. Each type forms a large uninterrupted array, a 5′ β array and a 3′ γ array with a sharp border between them. In the hierarchical repeat structure in each repeat array, all repeats are virtually identical at one level of repetition, but shown differences at the next level. The whole repeat block in the BR 1 gene fluctuates in size between different alleles, but not by more than 10%. In contrast, within the block, the β and γ arrays vary in length between 8000 and 29,000 bases in an inverse fashion, together keeping the overall length requirement. We propose that the length of exon 4 is conserved by selection of cross-over products of a given length, and that the internal hierarchical sequence organization in the BR 1 gene is a consequence of the combined action of several different sequence turnover mechanisms, all dependent on the unequal pairing of homologous sequences at different, competing levels of repetition.