Utility of low-copy nuclear gene sequences in plant phylogenetics

Low-copy nuclear genes in plants are a rich source of phylogenetic information. They hold a great potential to improve the robustness of phylogenetic reconstruction at all taxonomic levels, especially where universal markers such as cpDNA and nrDNA are unable to generate strong phylogenetic hypotheses. Low-copy nuclear genes, however, remain underused in plant phylogenetic studies due to practical and theoretical complications in unraveling the evolutionary dynamics of nuclear gene families. The lack of the universal markers or universal PCR primers of low-copy nuclear genes has also hampered their phylogenetic utility. It has recently become clear that low-copy nuclear genes are particularly helpful in resolving close interspecific relationships and in reconstructing allopolyploidization in plants. Gene markers that are widely, if not universally, useful have begun to emerge. Although utilizing low-copy nuclear genes usually requires extra lab work such as designing PCR primers, PCR-cloning, and/or Southern blotting, rapid accumulation of gene sequences in the databases and advances in cloning techniques have continued to make such studies more feasible. With the growing number of theoretical studies devoted to the gene tree and species tree problem, a solid foundation for reconstructing complex plant phylogenies based on multiple gene trees began to build. It is also realized increasingly that fast evolving introns of the low-copy nuclear genes will provide much needed phylogenetic information around the species boundary and allow us to address fundamental questions concerning processes of plant speciation. Phylogenetic and molecular evolutionary analyses of developmentally important genes will add a new dimension to systematic and evolutionary studies of plant diversity.     

Quantitative protein sequencing using mass spectrometry: Computer-aided assembly of protein sequences from N-terminal peptide sequences

A computer program has been developed that reconstructs partial or total amino acid sequences of proteins from the partial N-terminal sequences of selected peptides derived from specific cleavage of the protein by proteolytic and/or chemical methods.     

Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination.     

Towards resolving Lamiales relationships: insights from rapidly evolving chloroplast sequences

Background  

In the large angiosperm order Lamiales, a diverse array of highly specialized life strategies such as carnivory, parasitism, epiphytism, and desiccation tolerance occur, and some lineages possess drastically accelerated DNA substitutional rates or miniaturized genomes. However, understanding the evolution of these phenomena in the order, and clarifying borders of and relationships among lamialean families, has been hindered by largely unresolved trees in the past.     

The boundary of macaque rDNA is constituted by low-copy sequences conserved during evolution

In Macaca mulatta, the single rDNA array is flanked by a patchwork of sequences including subregions of human Yp11.2, 4q35.2, and 10p15.3. This composite DNA region is characterized by unique or low-copy sequences, resembling a potentially transcribed region. The analysis of Cercopithecus aethiops, Presbytis cristata, and Hylobates lar suggests that this complex sequence organization could be shared by Old World monkey and lesser ape species. After the lesser apes/great apes divergence, the unique or nonduplicated DNA region underwent amplification and spreading, preferentially marking the p arm of acrocentric chromosomes bearing the rDNA. The molecular analysis of human acrocentric chromosomes revealed some extent of remodeling of the rDNA boundary: near the human NOR, a large 4q35.2 duplication partially resembles that found in MMU; conversely, infrequently represented Yp11.2 sequences totally differed from those of the macaque, and 10p15.3 sequences were lacking. Thus, although evolutionary events modified the sequence organization of the MMU rDNA boundary, its overall sequence feature and the preferential location in vicinity to the NOR have been conserved.     

Systematics of Chaetognatha under the light of molecular data, using duplicated ribosomal 18S DNA sequences

While the phylogenetic position of Chaetognatha has became central to the question of early bilaterian evolution, the internal systematics of the phylum are still not clear. The phylogenetic relationships of the chaetognaths were investigated using newly obtained small subunit ribosomal RNA nuclear 18S (SSU rRNA) sequences from 16 species together with 3 sequences available in GenBank. As previously shown with the large subunit ribosomal RNA 28S gene, two classes of Chaetognatha SSU rRNA gene can be identified, suggesting a duplication of the whole ribosomal cluster; allowing the rooting of one class of genes by another in phylogenetic analyses. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the molecular data, and statistical tests showed (1) that there are three main monophyletic groups: Sagittidae/Krohnittidae, Spadellidae/Pterosagittidae, and Eukrohniidae/Heterokrohniidae, (2) that the group of Aphragmophora without Pterosagittidae (Sagittidae/Krohnittidae) is monophyletic, (3) the Spadellidae/Pterosagittidae and Eukrohniidae/Heterokrohniidae families are very likely clustered, (4) the Krohnittidae and Pterosagittidae groups should no longer be considered as families as they are included in other groups designated as families, (5) suborder Ctenodontina is not monophyletic and the Flabellodontina should no longer be considered as a suborder, and (6) the Syngonata/Chorismogonata and the Monophragmophora/Biphragmophora hypotheses are rejected. Such conclusions are considered in the light of morphological characters, several of which are shown to be prone to homoplasy.     

A program for template matching of protein sequences

The matching of a template to a protein sequence is simplifiedby treating it as a special case of sequence alignment. Restrictionof the distances between motifs in the template controls againstspurious matches within very long sequences. The program usingthis algorithm is fast enough to be used in scanning large databasesfor sequences matching a complex template. Received on August 17, 1987; accepted on January 11, 1988     

A rhesus monkey reference label atlas for template driven segmentation

Background We have acquired dual‐echo spin‐echo (DE SE) MRI data of the rhesus monkey brain since 1994 as part of an ongoing study of normal aging. To analyze these legacy data for regional volume changes, we have created a reference label atlas for the Template Driven Segmentation (TDS) algorithm. Methods The atlas was manually created from DE SE legacy MRI data of one behaviorally normal, young, male rhesus monkey and consisted of 14 regions of interest (ROI’s). We analyzed the reproducibility and validity of the TDS algorithm using the atlas relative to manual segmentation. Results ROI volumes were comparable between the two segmentation methodologies, except TDS overestimated the volume of basal ganglia regions. Both methodologies were highly reproducible, but TDS had lower sensitivity and comparable specificity. Conclusions TDS segmentation calculates accurate volumes for most ROI’s. Sensitivity will be improved in future studies through the acquisition of higher quality data.     

Targeted de novo phasing and long-range assembly by template mutagenesis

Short-read sequencers provide highly accurate reads at very low cost. Unfortunately, short reads are often inadequate for important applications such as assembly in complex regions or phasing across distant heterozygous sites. In this study, we describe novel bench protocols and algorithms to obtain haplotype-phased sequence assemblies with ultra-low error for regions 10 kb and longer using short reads only. We accomplish this by imprinting each template strand from a target region with a dense and unique mutation pattern. The mutation process randomly and independently converts ∼50% of cytosines to uracils. Sequencing libraries are made from both mutated and unmutated templates. Using de Bruijn graphs and paired-end read information, we assemble each mutated template and use the unmutated library to correct the mutated bases. Templates are partitioned into two or more haplotypes, and the final haplotypes are assembled and corrected for residual template mutations and PCR errors. With sufficient template coverage, the final assemblies have per-base error rates below 10^–9. We demonstrate this method on a four-member nuclear family, correctly assembling and phasing three genomic intervals, including the highly polymorphic HLA-B gene.     

Nucleic acid-independent retrovirus assembly can be driven by dimerization

The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.     

Optical resolution by crystallization from melt using enantiomer derivatives as resolving agents: nonlinear behavior of resolving agent mixtures

Substituted racemic 1-phenylethylamine derivatives were reacted in molten phase with chiral N-substituted 1-phenylethylamine dicarboxylic acid derivatives (4-9), and their mixtures (e.g., 4+6, 7+8, etc.) in different ratios, and the crystalline quasi-racemic diastereomers so formed were separated. Nonlinear behavior of the mixtures of resolving agents having related structure and considerable positive and negative chiral-chiral interactions were observed.     

A mitogenomic perspective on the basal teleostean phylogeny: resolving higher-level relationships with longer DNA sequences

A recent study demonstrated that mitochondrial genomic (mitogenomic) data comprising nucleotide sequences from the concatenated protein-coding (no 3rd codon positions) plus transfer RNA (stem regions only) genes reproduced the expected phylogeny of teleosts with high statistical support. We reexamined the interrelationships of the five major, basal teleostean lineages (Osteoglossomorpha, Elopomorpha, Clupeomorpha, Ostariophysi, and Protacanthopterygii; given various rankings) using mitogenomic data for which five alternative phylogenetic hypotheses have been previously proposed on the basis of both morphological and molecular analyses. In addition to previously determined complete mitochondrial DNA (mtDNA) sequences from eight basal teleosts and two outgroups, we determined the complete mtDNA sequences (excluding a portion of the control region) for two, purposefully chosen species of Osteoglossomorpha (Osteoglossum bicirrhosum and Pantodon buchholzi), and the data were subjected to maximumparsimony and maximum-likelihood analyses. The resultant tree topologies from the two methods were congruent, although they differed from any of the previously proposed hypotheses. Furthermore, the mitogenomic data confidently rejected all of these hypotheses with high statistical significance.     

Heterochromatic sequences in a Drosophila whole-genome shotgun assembly

Background

Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly.

Results

WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm.

Conclusions

Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes.     

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a P_ADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a P_HIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.