Specific Genomic DNA Sequences Research Articles

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries.

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.

Flow cytometry assay for the detection of single-copy DNA in human lymphocytes.

Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.

Isolation and application of P genome-specific DNA sequences of Agropyron Gaertn. in Triticeae.

Different types of P genome sequences and markers were developed, which could be used to analyze the evolution of P genome in Triticeae and identify precisely wheat- A. cristatum introgression lines. P genome of Agropyron Gaertn. plays an important role in Triticeae and could provide many desirable genes conferring high yield, disease resistance, and stress tolerance for wheat genetic improvement. Therefore, it is significant to develop specific sequences and functional markers of P genome. In this study, 126 sequences were isolated from the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) products of microdissected chromosome 6PS. Forty-eight sequences were identified as P genome-specific sequences by dot-blot hybridization and DNA sequences analysis. Among these sequences, 22 displayed the characteristics of retrotransposons, nine and one displayed the characteristics of DNA transposons and tandem repetitive sequence, respectively. Fourteen of 48 sequences were determined to distribute on different regions of P genome chromosomes by fluorescence in situ hybridization, and the distributing regions were as following: all over P genome chromosomes, centromeres, pericentromeric regions, distal regions, and terminal regions. We compared the P genome sequences with other genome sequences of Triticeae and found that the similar sequences of the P genome sequences were widespread in Triticeae, but differentiation occurred to various extents. Additionally, thirty-four molecular markers were developed from the P genome sequences, which could be used for analyzing the evolutionary relationship among 16 genomes of 18 species in Triticeae and identifying P genome chromatin in wheat-A. cristatum introgression lines. These results will not only facilitate the study of structure and evolution of P genome chromosomes, but also provide a rapid detecting tool for effective utilization of desirable genes of P genome in wheat improvement.

Fabrication of nanopores with ultrashort single-walled carbon nanotubes inserted in a lipid bilayer.

We describe a protocol for the insertion of ultrashort single-walled carbon nanotubes (SWCNTs) to form nanopores in a Montal-Mueller lipid bilayer. The SWCNTs are designed to bind to a specific analyte of interest; binding will result in the reduction of current in single-channel recording experiments. The first stage of the PROCEDURE is to cut and separate the SWCNTs. We cut long, purified SWCNTs with sonication in concentrated sulfuric acid/nitric acid (3/1). Isolation of ultrashort SWCNTs is carried out by size-exclusion HPLC separation. The second stage is to insert these short SWCNTs into the lipid bilayer. This step requires a microinjection probe made from a glass capillary. The setup for protein nanopore research can be adopted for the single-channel recording experiments without any special treatment. The obtained current traces are of very high quality, showing stable baselines and little background noise. Example procedures are shown for investigating ion transport and DNA translocation through these SWCNT nanopores. This nanopore has potential applications in molecular sensing, nanopore DNA sequencing and early disease diagnosis. For example, we have selectively detected modified 5-hydroxymethylcytosine in single-stranded DNA (ssDNA), which may have implications in screening specific genomic DNA sequences. The protocol takes ∼15 d, including SWCNT purification, cutting and separation, as well as the formation of SWCNT nanopores for DNA analyses.

Ultrashort single-walled carbon nanotubes in a lipid bilayer as a new nanopore sensor

An important issue in nanopore sensing is to construct stable and versatile sensors that can discriminate analytes with minute differences. Here we report a means of creating nanopores that comprise ultrashort single-walled carbon nanotubes inserted into a lipid bilayer. We investigate the ion transport and DNA translocation through single-walled carbon nanotube nanopores and find that our results are fundamentally different from previous studies using much longer single-walled carbon nanotubes. Furthermore, we utilize the new single-walled carbon nanotube nanopores to selectively detect modified 5-hydroxymethylcytosine in single-stranded DNA, which may have implications in screening specific genomic DNA sequences. This new nanopore platform can be integrated with many unique properties of carbon nanotubes and might be useful in molecular sensing such as DNA-damage detection, nanopore DNA sequencing and other nanopore-based applications.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.

Identification of Aurora-A as a Direct Target of E2F3 during G2/M Cell Cycle Progression

Aurora-A is a centrosome kinase and plays a pivotal role in G(2)/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G(1)/S, accumulate during G(2)/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G(2)/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus, the E2F3-Aurora-A axis could be an important target for cancer intervention.

Functional Characteristics of a Highly Specific Integrase Encoded by an LTR-Retrotransposon

BackgroundThe retroviral Integrase protein catalyzes the insertion of linear viral DNA into host cell DNA. Although different retroviruses have been shown to target distinctive chromosomal regions, few of them display a site-specific integration. ZAM, a retroelement from Drosophila melanogaster very similar in structure and replication cycle to mammalian retroviruses is highly site-specific. Indeed, ZAM copies target the genomic 5′-CGCGCg-3′ consensus-sequences. To enlighten the determinants of this high integration specificity, we investigated the functional properties of its integrase protein denoted ZAM-IN.Principal FindingsHere we show that ZAM-IN displays the property to nick DNA molecules in vitro. This endonuclease activity targets specific sequences that are present in a 388 bp fragment taken from the white locus and known to be a genomic ZAM integration site in vivo. Furthermore, ZAM-IN displays the unusual property to directly bind specific genomic DNA sequences. Two specific and independent sites are recognized within the 388 bp fragment of the white locus: the CGCGCg sequence and a closely apposed site different in sequence.ConclusionThis study strongly argues that the intrinsic properties of ZAM-IN, ie its binding properties and its endonuclease activity, play an important part in ZAM integration specificity. Its ability to select two binding sites and to nick the DNA molecule reminds the strategy used by some site-specific recombination enzymes and forms the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.

Identifying the genome of wood barley Hordelymus europaeus (Poaceae: Triticeae)

Wood barley, Hordelymus europaeus, was compared with other Triticeae species by Southern and fluorescence in situ hybridisation using total genomic DNA and repetitive sequences as probes. On Southern blots, the total genomic probe from H. europaeus hybridised strongly to DNA of its own species and to Leymus and Psathyrostachys, indicating the presence of Ns genome in H. europaeus. Furthermore, the total genomic probe from P. fragilis hybridised to DNA of H. europaeus as much as to all of the Psathyrostachys and Leymus species examined. Ns genome-specific DNA sequences isolated from L. mollis (pLmIs1, pLmIs44 and pLmIs53) hybridised essentially to H. europaeus and all of the species of Leymus and Psathyrostachys. Chromosomal localization of these clones on H. europaeus confirmed the presence of Ns genome-specific DNA on all chromosomes, indiscriminately. Under moderate hybridisation stringency the Ns genome-specific probes, together with repetitive sequences pTa71 and pAesKB7, produced species-specific RFLP banding profiles on Southern blots. A phenetic tree based on these profiles revealed a distinct Ns species cluster within the Triticeae, represented by Leymus and Psathyrostachys species. Hordelymus europaeus belonged to this Ns cluster. Chromosomal mapping of the 18S-25S and the 5S ribosomal genes, together with the repetitive sequence pLrTaiI, corroborated that H. europaeus was most probably related to Leymus, especially the European/Eurasian members of sect. Leymus. In an attempt to identify the genome of H. europaeus, different approaches were employed; the results clearly showed that wood barley had the Ns basic genome and nothing else.

Characterization of Dasypyrum Villosum (L.) Candargy Chromosomes by Fluorescent in situ Hybridization

Fluorescent in situ hybridization (FISH) was utilized to study disomic wheat (Triticum aestivum)/Dasypyrum villosum chromosome addition lines to characterize the V genome chromosomes of D. villosum L. Candargy. The V genome specific DNA sequence, pHv62; the rye sequence, pSc119.2, the 18S-5.8S-26S rDNA genes, pTa71, and the 5S rDNA sequence, cP5S, were used as probes. Six of the seven added D. villosum chromosomes hybridized with pHv62. One of these, 3V, was shown to be a Robertsonian translocation with a wheat chromosome. The presence of the seventh addition line chromosome was verified by FISH using total genomic D. villosum DNA as probe. FISH with cP5S detected a site on 5V, whilst pTa71 revealed a single site on the short arm of IV, but pSc119.2 detected sites on all the chromosomes of D. villosum. The pHv62 probe was also used in the application of FISH to a number of other species in the Triticeae, including five species of Aegilops, barley and the tetraploid Dasypyrum breviaristatum. Sites were detected only in Thinopyrum bessarabicum and Secale cereale, and this together with the above evidence for pTa71 and pSc119.2 suggests that D. villosum is phylogenetically closest to Th. bessarabicum and S. cereale, and also is not related to Dasypyrum breviaristatum.

Variation in the distribution of a genome-specific DNA sequence on chromosomes reveals evolutionary relationships in the Triticum and Aegilops complex

The present study analyzed the distribution pattern of the Ae. speltoides–derived repetitive clone pGc1R-1 in the Triticum/Aegilops complex. Fluorescence in situ hybridization analysis showed that clone pGc1R-1 is a S-genome-specific repetitive sequence that hybridized to the S-genome of three species in the section Sitopsis, Aegilops speltoides (S), Ae. longissima (Sl), and Ae. sharonensis (Ssh), but not to Ae. bicornis (Sb) and Ae. searsii (Ss), nor to any other diploid Aegilops species. This clone also hybridized to the very closely related G-genome of T. timopheevii subsp. armeniacum and T. timopheevii ssp. timopheevii, but not to the B-genome of T. turgidum and T. aestivum. Hybridization also was observed in the polyploid Aegilops species, Ae. kotschyi (UkSk), Ae. peregrina (UpSp), and Ae. vavilovii (XvaDvaSva). Large inter- and intraspecific variations were observed. Our results confirm that the S genome is related more to the Sl and Ssh genomes than to the Sb and Ss genomes; there is a greater affinity between the G and S genomes than between the B and S genomes. Mechanisms to account for the variation in the FISH pattern with different genomes include sequence amplification and deletion. Variation in the distribution of this genome-specific DNA sequence, pGc1R-1, on chromosomes can be used to reveal evolutionary relationships in the Triticum and Aegilops complex.

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.

Genetic Analysis of DNA Excreted in Urine: A New Approach for Detecting Specific Genomic DNA Sequences from Cells Dying in an Organism

Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool. Mice received subcutaneous injections of either human Raji cells or purified (32)P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA. In the experiments on laboratory animals, we found that approximately 0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific ALU: sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K-ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas. The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.

Triple helix-forming oligonucleotides target psoralen adducts to specific chromosomal sequences in human cells.

The ability to target photochemical adducts to specific genomic DNA sequences in cells is useful for studying DNA repair and mutagenesis in intact cells, and also as a potential mode of gene-specific therapy. Triple helix-forming DNA oligonucleotides linked to psoralen (psoTFOs) were designed to deliver UVA-induced psoralen photoadducts to two distinct sequences within the human interstitial collagenase gene. A primer extension assay demonstrated that the appropriate psoTFO selectively damages a collagenase cDNA target. Site-specific genomic psoTFO DNA adducts were detected by a single-strand ligation PCR assay. The adduct, formed at a single site by a psoTFO in purified genomic DNA, contrasted with the multiple sites that were damaged within the observed segment of the collagenase gene upon treatment with free psoralen and subsequent photoactivation. When treated with psoTFOs, both repair-deficient fibroblasts from xero- derma pigmentosum complementation group A and HT1080 fibrosarcoma cells exhibited site-specific DNA adducts following UVA irradiation. Addition of phorbol ester, a transcriptional activator of the collagenase gene, to xeroderma pigmentosum cells did not detectably alter the initial levels of damage produced by psoTFOs, suggesting that further stimulation of transcription neither improves accessibility of psoTFOs to their targets nor enhances removal of non-covalently bound psoTFOs.

Visual verification of close disposition between a rice A genome-specific DNA sequence (TrsA) and the telomere sequence.

A rice A genome-specific tandem repeat sequence (TrsA) and telomeric nucleotide sequences, (TTTAGGG)n, were simultaneously detected by multicolor fluorescence in situ hybridization (McFISH) using rice prometaphase chromosomes. Six pairs of TrsA sites visualized by fluorescence signals were all localized on the long arms close to the telomeric regions. Differences in the copy number of TrsA at the different sites were visualized both by the size of the telomeric condensation block stained with Giemsa solution and the signal intensity after FISH with TrsA. McFISH analyses using interphase nuclei could resolve close disposition of TrsA and telomere and also gave rough estimation of the distance between them. The functional significance of the close disposition of TrsA and telomere is discussed.

Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza.

Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r = 0.939) and the total chromosomal area (r = 0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.

The use of double fluorescence in situ hybridization to physically map the positions of 5S rDNA genes in relation to the chromosomal location of 18S–5.8S–26S rDNA and a C genome specific DNA sequence in the genusAvena

A physical map of the locations of the 5S rDNA genes and their relative positions with respect to 18S-5.8S-26S rDNA genes and a C genome specific repetitive DNA sequence was produced for the chromosomes of diploid, tetraploid, and hexaploid oat species using in situ hybridization. The A genome diploid species showed two pairs of rDNA loci and two pairs of 5S loci located on both arms of one pair of satellited chromosomes. The C genome diploid species showed two major pairs and one minor pair of rDNA loci. One pair of subtelocentric chromosomes carried rDNA and 5S loci physically separated on the long arm. The tetraploid species (AACC genomes) arising from these diploid ancestors showed two pairs of rDNA loci and three pairs of 5S loci. Two pairs of rDNA loci and 2 pairs of 5S loci were arranged as in the A genome diploid species. The third pair of 5S loci was located on one pair of A-C translocated chromosomes using simultaneous in situ hybridization with 5S rDNA genes and a C genome specific repetitive DNA sequence. The hexaploid species (AACCDD genomes) showed three pairs of rDNA loci and six pairs of 5S loci. One pair of 5S loci was located on each of two pairs of C-A/D translocated chromosomes. Comparative studies of the physical arrangement of rDNA and 5S loci in polyploid oats and the putative A and C genome progenitor species suggests that A genome diploid species could be the donor of both A and D genomes of polyploid oats. Key words : oats, 5S rDNA genes, 18S-5.8S-26S rDNA genes, C genome specific repetitive DNA sequence, in situ hybridization, genome evolution.

Molecular evidence forTriticum speltoidesas a B-genome progenitor of wheat (Triticum aestivum)

In polyploid wheat, the origin of the B-genome donor has remained relatively unknown in spite of a number of investigations attempting to identify the parental species. A project was designed to isolate and clone a genome-specific DNA sequence from Triticum speltoides L. to determine if that species could be the B-genome donor. A cloning scheme involving the prescreening of 1-kb fragments followed by colony, dot blot, and Southern blot hybridization screenings was used to isolate a speltoides-specific sequence (pSp89.XI). The methods used allowed for rapid isolation of a genome-specific sequence when screened against total DNA from closely related species. Subsequent analyses showed that the sequence was barely detected in any of the other genomes of the annual Sitopsis section. The results of dot blot and Southern blot analyses established that (i) the sequence pSP89.XI, specific to T. speltoides relative to the other species of the Sitopsis section, was present in the genomes of tetraploid and hexaploid wheat, (ii) the relative abundance of pSp89.XI seemed to decrease from the diploid to the polyploid wheats, and (iii) the existence of a related, but modified B genome in polyploid wheat compared with that in modern T. speltoides was probable. Key words : genome-specific, DNA.

Absence of genomic imprinting at the DiGeorge locus.

Fluorescence in situ hybridization (FISH) has been used to visualize specific genomic DNA sequences in interphase nuclei. Timing of replication can be measured by FISH to interphase nuclei: nuclei with a sequence that has not replicated reveal two single signals (G1), whereas those in which the sequence has replicated show two signal doublets (G2). Asynchronous nuclei show a single signal on one allele and a double hybridization dot on the other homologue. In general, most sequences replicate synchronously on the two homologues, with only 10% of nuclei showing an asynchronous hybridization pattern. However, for the sequences known about to be imprinted, approximately 30% of nuclei reveal asynchronous replication. Little is known whether or not the proximal region of chromosome 22, involved in the DiGeorge syndrome [1], is imprinted. We have, therefore, examined the replication timing pattern of the DiGeorge critical region (DGCR).

Inhibition of RNA Polymerase II Transcription Causes Chromatin Decondensation, Loss of Nucleolar Structure, and Dispersion of Chromosomal Domains

Fluorescencein situhybridization and immunofluorescence have been used to visualize specific genomic DNA sequences and proteins in interphase nuclei treated with transcriptional inhibitors. The adenosine analog 5,6-dichloro-β-d-ribofuranosylbenzimidazole (DRB) and α-amanitin selectively inhibit transcription by RNA polymerase II at low doses. Upon exposure to DRB or α-amanitin the fibrillar components of the normally compact nucleolus unravel into necklace-like structures which represent highly extended linear arrays of ribosomal (r)RNA genes. Similarly, blocks of tandemly repeated satellite DNAs dissociate into extended beaded strands. Localized (euchromatic) chromosome domains and even whole chromosome territories disperse throughout the nuclear interior. Treatment of cells with actinomycin D (AMD) at doses that block rRNA synthesis does not cause significant decondensation of nucleolar, heterochromatic, and interphase chromosome domains. Interestingly, both α-amanitin and AMD cause coilin to associate with the nucleolar domain. In AMD-treated cells, coilin is enriched in nucleolar caps abutting upon the residual nucleolus. After α-amanitin treatment, coilin is concentrated in numerous beads closely associated with individual rDNA transcription units within nucleolar necklaces. The changes in higher-order nuclear structure are reversible in cell cultures exposed to nontoxic doses of transcriptional inhibitors. It therefore may be concluded that nuclear topographic organization is dependent on a continued transcription of nuclear genes, but not of the rRNA genes.