三倍体湘云鲫线粒体DNA序列变异性分析

对26尾三倍体湘云鲫的线粒体tRNA-Thr基因、tRNA-Pro基因和部分控制区的核苷酸序列进行了测定,获得26条长度为837—839 bp的同源基因序列,共发现65个多态性核苷酸变异位点,多态位点比例为0.077,定义了8种单元型。在湘云鲫8种单元型中确认了DNA复制终止相关的序列TAS、中央保守区序列(CSB-F、CSB-E和CSB-D)和保守序列CSB1,8种单元型含有3—5个TAS序列。在65个变异位点中,大部分序列变异为转换,8种单元型之间的序列差异在0.1%—6.3%之间。该研究为三倍体湘云鲫的繁殖和遗传改良提供了一些有价值的信息。     

EXMOTIF: efficient structured motif extraction

Background  

Extracting motifs from sequences is a mainstay of bioinformatics. We look at the problem of mining structured motifs, which allow variable length gaps between simple motif components. We propose an efficient algorithm, called EXMOTIF, that given some sequence(s), and a structured motif template, extracts all frequent structured motifs that have quorum q. Potential applications of our method include the extraction of single/composite regulatory binding sites in DNA sequences.     

Targeted single molecule sequencing methodology for ovarian hyperstimulation syndrome

Background

One of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility.

Results

Target enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing.

Conclusions

Combining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1451-2) contains supplementary material, which is available to authorized users.     

Heterochromatin and highly repeated DNA sequences in rye (Secale cereale)

Secale cereale DNA, of mean fragment length 500 bp, was fractionated by hydroxylapatite chromatography to allow recovery of a very rapidly renaturing fraction (C₀t 0–0.02). This DNA fraction was shown to contain several families of highly repeated sequence DNA. Two highly repeated families were purified; (1) a fraction which renatured to a density of 1.701 g/ cc and comprised 2–4% of the total genome, and (2) polypyrimidine tract DNA which comprised 0.1% of the total genome. The 1.701 g/cc DNA consisted of short sequence repeat units (5–50 bp long) tandemly repeated in blocks 30 kb long, while a portion of the polypyrimidine tract DNA behaved as part of a much larger block of tandemly repeated sequences. The chromosomal location of these sequences was determined by the in situ hybridisation of radioactive, complementary RNA to root tip mitotic chromosomes and showed the 1.701 g/cc sequences to be largely limited to the telomeric blocks of heterochromatin, accounting for 25–50% of the DNA present in these parts of the chromosomes. The polypyrimidine tracts were distributed at interstitial locations with 20–30% of the sequences at three well defined sites. The combined distributions of the 1.701 g/cc DNA sequences and polypyrimidine tracts effectively individualised each rye chromosome thus providing a sensitive means of identifying these chromosomes. The B chromosomes present in Secale cereale cv. Unevita, did not show defined locations for the sequences analysed. — The data are discussed in terms of the structure of the rye genome and the generality of the observed genomic arrangement of highly repeated sequence DNA.     

Impacts of low coverage depths and post-mortem DNA damage on variant calling: a simulation study

Background

Massively parallel sequencing platforms, featuring high throughput and relatively short read lengths, are well suited to ancient DNA (aDNA) studies. Variant identification from short-read alignment could be hindered, however, by low DNA concentrations common to historic samples, which constrain sequencing depths, and post-mortem DNA damage patterns.

Results

We simulated pairs of sequences to act as reference and sample genomes at varied GC contents and divergence levels. Short-read sequence pools were generated from sample sequences, and subjected to varying levels of “post-mortem” damage by adjusting levels of fragmentation and fragmentation biases, transition rates at sequence ends, and sequencing depths. Mapping of sample read pools to reference sequences revealed several trends, including decreased alignment success with increased read length and decreased variant recovery with increased divergence. Variants were generally called with high accuracy, however identification of SNPs (single-nucleotide polymorphisms) was less accurate for high damage/low divergence samples. Modest increases in sequencing depth resulted in rapid gains in total variant recovery, and limited improvements to recovery of heterozygous variants.

Conclusions

This in silico study suggests aDNA-associated damage patterns minimally impact variant call accuracy and recovery from short-read alignment, while modest increases in sequencing depth can greatly improve variant recovery.

     

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides

Summary DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5 terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3 terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.     

Human telomeric DNA sequences are a major target for the antitumour drug bleomycin

The DNA sequence specificity of the cancer chemotherapeutic agent bleomycin was examined in a human telomeric DNA sequence and compared with that of non-telomeric sequences. The target DNA sequence contained 17 repeats of the human telomeric sequence and other primary sites of bleomycin cleavage. The 377-base-pair target DNA was fluorescently labelled at the 3′-end, damaged with bleomycin and electrophoresed in an ABI 3730 automated capillary sequencer to determine the intensity and sequence specificity of bleomycin damage. The results revealed that bleomycin cleaved primarily at 5′-GT in the telomeric sequence 5′-GGGTTA. Maxam–Gilbert chemical sequencing reactions were utilised as DNA size markers to determine the precise sites of bleomycin cleavage. The telomeric region contained strong sites of bleomycin cleavage and constituted 57% of the 30 most intense bleomycin damage sites in the DNA sequence examined. These data indicated that telomeric DNA sequences are a major site for bleomycin damage.     

Identifying Cis-Regulatory Sequences by Word Profile Similarity

Background

Recognizing regulatory sequences in genomes is a continuing challenge, despite a wealth of available genomic data and a growing number of experimentally validated examples.

Methodology/Principal Findings

We discuss here a simple approach to search for regulatory sequences based on the compositional similarity of genomic regions and known cis-regulatory sequences. This method, which is not limited to searching for predefined motifs, recovers sequences known to be under similar regulatory control. The words shared by the recovered sequences often correspond to known binding sites. Furthermore, we show that although local word profile clustering is predictive for the regulatory sequences involved in blastoderm segmentation, local dissimilarity is a more universal feature of known regulatory sequences in Drosophila.

Conclusions/Significance

Our method leverages sequence motifs within a known regulatory sequence to identify co-regulated sequences without explicitly defining binding sites. We also show that regulatory sequences can be distinguished from surrounding sequences by local sequence dissimilarity, a novel feature in identifying regulatory sequences across a genome. Source code for WPH-finder is available for download at http://rana.lbl.gov/downloads/wph.tar.gz.     

Effect of Methanol on the Nucleotide Binding to High-Affinity Sites on Chloroplast Coupling Factor 1

The effects of methanol on the nucleotide binding to isolatedchloroplast coupling factor 1 (CF₁) were investigated. IsolatedCF₁ has four kinds of nucleotide binding sites; a barely dissociableADP-binding site (site A), two slowly exchangeable high-affinitysites with different affinities for ADP (sites B and C) whichare not catalytic sites, and several low-affinity sites (Hisaboriand Sakurai 1984). Methanol at 20% (v/v) slightly acceleratedthe binding of ADP to CF₁ but did not influence the number ofbinding sites. Methanol at 10–24% (v/v) affected neitherthe total amounts of bound adenine nucleotides (2.5 mol/molCF₁) nor the incorporation of labeled ADP from the medium (1.5mol/mol CF₁ into the slowly exchangeable sites (sites A, B,C). These results indicate that no appreciable exchange of ADPoccurred at site A at 10–24% (v/v) methanol and excludethe possibility of direct participation of nucleotide bindingat this site in the regulation of ATPase. In 32% methanol, theamount of the labeled ADP bound increased, suggesting some exchangeat site A. Methanol at 20% (v/v) greatly increased the affinitiesof sites B and C for ADP, CDP, GDP, UDP and PP_i. Conformational change of CF₁ induced by the binding of nucleotidesto site(s) B (and C) increased the resistance of CF₁ to inactivationby methanol at high concentrations or by cold treatment. (Received August 16, 1984; Accepted January 23, 1985)     

The organization of DNA segments undergoing repair synthesis during pachytene

DNA regions undergoing programmed repair synthesis during pachytene were isolated and used as a probe for analyzing the organization of these regions. Segments that are the sites of nick-repair activity are referred to as PsnDNA. These segments are distributed at intervals ranging from 30–350 kilobase pairs (kbp) within about half the genome. The other half of the genome, which consists of DNA molecules longer than 350 kbp under defined conditions of extraction, lacks these segments. PsnDNA sequences range in length from about 150–300 base pairs (bp) and are arranged in larger P.DNA units measuring 0.8–3.0 kbp. P.DNA units have three identifiable regions. Each end region consists of a PsnDNA sequence and the middle region contains sequences that do not share homology with PsnDNA and have a much lower repeat number. Pachytene nicking of PsnDNA sequences is polar with respect to the orientation of individual DNA strands. Most of the PsnDNA sequences are present at the 5 ends of the single strands generated in vivo by endonuclease action. Nicking is probably repeated at each PsnDNA site during early and midpachytene, and both members of a duplex are nicked within any single P.DNA region.     

EFFECTS OF TEMPERATURE ON CHRONIC HYPOXIA TOLERANCE IN THE NON-INDIGENOUS BROWN MUSSEL, PERNA PERNA (BIVALVIA: MYTILIDAE) FROM THE TEXAS GULF OF MEXICO

Effects of temperature (15°, 20° and 25°C), O₂ partialpressure (P_O2=0, 1, 2, 4, and 6 kPa), and individual size(12–79 mm shell length; SL) on survivorship of specimensof the non-indigenous, marine, brown mussel, Perna perna, fromTexas were investigated to assess its potential distributionin North America. Its hypoxia tolerance was temperature-dependent,survivorship being significantly extended at lower temperaturesunder all tested lethal P_O2. Incipient tolerated P_O2 was 4 and6 kPa at 15 and 20°C, respectively, with >50% mortalityoccurring at 25°C at all tested levels of hypoxia. P_O2 hadless of an effect on survival of hypoxia than temperature. At25°C, survivorship was not different over a P_O2 range of0–2 kPa and increased only at 4 and 6 kPa. Survivorshipwas size-dependent. Median survival times increased with increasingSL in anoxia and P_O2=1 kPa, but at 2, 4 and 6 kPa,smaller individuals survived longer than larger individuals.With tolerance levels similar to other estuarine bivalve species,P. perna should withstand hypoxia encountered in estuarine environments.Thus, its restriction to intertidal rocky shores may be dueto other parameters, particularly its relatively low temperaturetolerance. (Received 26 January 2004; accepted 31 March 2005)     

Lack of association of colonic epithelium telomere length and oxidative DNA damage in Type 2 diabetes under good metabolic control

Background

Telomeres are DNA repeat sequences necessary for DNA replication which shorten at cell division at a rate directly related to levels of oxidative stress. Critical telomere shortening predisposes to cell senescence and to epithelial malignancies. Type 2 diabetes is characterised by increased oxidative DNA damage, telomere attrition, and an increased risk of colonic malignancy. We hypothesised that the colonic mucosa in Type 2 diabetes would be characterised by increased DNA damage and telomere shortening.

Methods

We examined telomere length (by flow fluorescent in situ hybridization) and oxidative DNA damage (flow cytometry of 8 – oxoguanosine) in the colonic mucosal cells of subjects with type 2 diabetes (n = 10; mean age 62.2 years, mean HbA1c 6.9%) and 22 matched control subjects. No colonic pathology was apparent in these subjects at routine gastrointestinal investigations.

Results

Mean colonic epithelial telomere length in the diabetes group was not significantly different from controls (10.6 [3.6] vs. 12.1 [3.4] Molecular Equivalent of Soluble Fluorochrome Units [MESF]; P = 0.5). Levels of oxidative DNA damage were similar in both T2DM and control groups (2.6 [0.6] vs. 2.5 [0.6] Mean Fluorescent Intensity [MFI]; P = 0.7). There was no significant relationship between oxidative DNA damage and telomere length in either group (both p > 0.1).

Conclusion

Colonic epithelium in Type 2 diabetes does not differ significantly from control colonic epithelium in oxidative DNA damage or telomere length. There is no evidence in this study for increased oxidative DNA damage or significant telomere attrition in colonic mucosa as a carcinogenic mechanism.     

Genome structure of Tetrahymena pyriformiis

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0×10⁸ base pairs (or 1.2×10¹¹ daltons). About 90% of the macronuclear DNA fragments 200–300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7–9% at a rate corresponding to moderate repetitive sequences; 3–4% of such DNA fragments reassociate at C₀t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with Sl-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200–300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called zero time binding or foldback DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to Sl-nuclease treatment. The Sl-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.     

The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T (n 7) mononucleotide repeats. Each SSR sequence motif, along with ±200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber (Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.Communicated by C. Möllers