Exogenous artificial DNA forms chromatin structure with active transcription in yeast

AbstractYeast artificial chromosomes (YACs) are important tools for sequencing, gene cloning, and transferring large quantities of genetic information. However, the structure and activity of YAC chromatin, as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events, have not been widely explored. Here, we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported data-carrying chromosome (dChr). In addition, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional, and proteomic characteristics of the exogenous dChr. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. The dChr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs. The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids. A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.

Download Full-text

Characterization of four dispersed repetitive DNA sequences from Zea mays and their use in constructing contiguous DNA fragments using YAC clones

Genome ◽

10.1139/g96-102 ◽

1996 ◽

Vol 39 (4) ◽

pp. 811-817 ◽

Cited By ~ 12

Author(s):

Keith J. Edwards ◽

Jacky Veuskens ◽

Heather Rawles ◽

Allan Daly ◽

Jeffrey L. Bennetzen

Keyword(s):

Repetitive Dna ◽

Dna Sequences ◽

Local Level ◽

Repetitive Sequences ◽

Maize Genome ◽

Dna Fragments ◽

Mitotic Chromosomes ◽

Artificial Chromosomes ◽

Key Words Maize ◽

Yeast Artificial Chromosomes

We have isolated four repetitive DNA fragments from maize DNA. Only one of these sequences showed homology to sequences within the EMBL database, despite each having an estimated copy number of between 3 × 104 and 5 × 104 per haploid genome. Hybridization of the four repeats to maize mitotic chromosomes showed that the sequences are evenly dispersed throughout most, but not all, of the maize genome, whereas hybridization to yeast colonies containing random maize DNA fragments inserted into yeast artificial chromosomes (YACs) indicated that there was considerable clustering of the repeats at a local level. We have exploited the distribution of the repeats to produce repetitive sequence fingerprints of individual YAC clones. These fingerprints not only provide information about the occurrence and organization of the repetitive sequences within the maize genome, but they can also be used to determine the organization of overlapping maize YAC clones within a contiguous fragment (contigs). Key words : maize, repetitive DNA, YACs.

Download Full-text

Deoxyribonucleic Acid as a Tool for Digital Information Storage: An Overview

THE INDIAN JOURNAL OF VETERINARY SCIENCES AND BIOTECHNOLOGY ◽

10.21887/ijvsbt.15.1.1 ◽

2019 ◽

Vol 15 (01) ◽

pp. 1-8

Author(s):

Ashish C Patel ◽

C G Joshi

Keyword(s):

Data Storage ◽

Dna Sequences ◽

Consensus Sequence ◽

Random Access ◽

Information Storage ◽

Digital Data ◽

Digital Information ◽

Multiple Sequence ◽

Digital World ◽

Digital File

Current data storage technologies cannot keep pace longer with exponentially growing amounts of data through the extensive use of social networking photos and media, etc. The "digital world” with 4.4 zettabytes in 2013 has predicted it to reach 44 zettabytes by 2020. From the past 30 years, scientists and researchers have been trying to develop a robust way of storing data on a medium which is dense and ever-lasting and found DNA as the most promising storage medium. Unlike existing storage devices, DNA requires no maintenance, except the need to store at a cool and dark place. DNA has a small size with high density; just 1 gram of dry DNA can store about 455 exabytes of data. DNA stores the informations using four bases, viz., A, T, G, and C, while CDs, hard disks and other devices stores the information using 0’s and 1’s on the spiral tracks. In the DNA based storage, after binarization of digital file into the binary codes, encoding and decoding are important steps in DNA based storage system. Once the digital file is encoded, the next step is to synthesize arbitrary single-strand DNA sequences and that can be stored in the deep freeze until use.When there is a need for information to be recovered, it can be done using DNA sequencing. New generation sequencing (NGS) capable of producing sequences with very high throughput at a much lower cost about less than 0.1 USD for one MB of data than the first sequencing technologies. Post-sequencing processing includes alignment of all reads using multiple sequence alignment (MSA) algorithms to obtain different consensus sequences. The consensus sequence is decoded as the reversal of the encoding process. Most prior DNA data storage efforts sequenced and decoded the entire amount of stored digital information with no random access, but nowadays it has become possible to extract selective files (e.g., retrieving only required image from a collection) from a DNA pool using PCR-based random access. Various scientists successfully stored up to 110 zettabytes data in one gram of DNA. In the future, with an efficient encoding, error corrections, cheaper DNA synthesis,and sequencing, DNA based storage will become a practical solution for storage of exponentially growing digital data.

Download Full-text

Murine Albino-Deletion Complex: High-Resolution Microsatellite Map and Genetically Anchored YAC Framework Map

Genetics ◽

10.1093/genetics/147.2.787 ◽

1997 ◽

Vol 147 (2) ◽

pp. 787-799

Author(s):

Brad A Rikke ◽

Dabney K Johnson ◽

Thomas E Johnson

Keyword(s):

Positional Cloning ◽

Interspecific Backcross ◽

Genetic Distances ◽

Sequence Length ◽

Recombination Rates ◽

Oak Ridge ◽

Artificial Chromosomes ◽

Microsatellite Map ◽

Specific Locus ◽

Yeast Artificial Chromosomes

The murine albino-deletion complex developed as part of the Oak Ridge specific-locus test covers 6–11 cM of chromosome 7. This complex has proven to be a valuable resource for localizing traits to a small target region suitable for positional cloning. In this study, we mapped the endpoints of deletions in this complex using all of the available Mit simple-sequence length polymorphism (SSLP) markers. Concurrently, this mapping has determined the map order of nearly all of the SSLP markers, most of which were previously unresolved. The SSLP-based deletion map was confirmed and genetic distances were determined using the European Collaborative Interspecific Backcross panel of nearly a thousand mice. The average SSLP marker resolution is 0.3–0.4 cM, comparable to the cloning capacity of yeast artificial chromosomes (YACs). The SSLP markers were then used to construct a genetically anchored YAC framework map that further confirms the deletion map. We find that the largest deleted region distal to Tyr is about two to three times larger than the largest proximal deleted region, and the original C3H/101 regions flanking the deletions (moved to an St2A cch/cch background) are smaller than anticipated, which we suggest may result from increased recombination rates immediately flanking the deleted regions.

Download Full-text

A Novel Ty1-Mediated Fragmentation Method for Native and Artificial Yeast Chromosomes Reveals That the Mouse Steel Gene is a Hotspot for Ty1 Integration

Genetics ◽

10.1093/genetics/143.2.673 ◽

1996 ◽

Vol 143 (2) ◽

pp. 673-683

Author(s):

Jacob Z Dalgaard ◽

Mukti Banerjee ◽

M Joan Curcio

Keyword(s):

Transcription Unit ◽

Yeast Genome ◽

Physical Analysis ◽

Single Chromosome ◽

Artificial Chromosomes ◽

Unique Site ◽

High Fraction ◽

A Site ◽

Yeast Artificial Chromosomes ◽

Random Insertion

Abstract We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24 retrotransposon insertions into two different mousederived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into aYAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.

Download Full-text

Pancreatic cancer prognosis is predicted by an ATAC-array technology for assessing chromatin accessibility

Nature Communications ◽

10.1038/s41467-021-23237-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

S. Dhara ◽

S. Chhangawala ◽

H. Chintalapudi ◽

G. Askan ◽

V. Aveson ◽

...

Keyword(s):

Low Cost ◽

Disease Free Survival ◽

Chromatin Accessibility ◽

Cancer Prognosis ◽

Ductal Adenocarcinoma ◽

Binding Motifs ◽

Free Survival ◽

Array Technology ◽

Treatment Naïve ◽

Generation Sequencing

AbstractUnlike other malignancies, therapeutic options in pancreatic ductal adenocarcinoma (PDAC) are largely limited to cytotoxic chemotherapy without the benefit of molecular markers predicting response. Here we report tumor-cell-intrinsic chromatin accessibility patterns of treatment-naïve surgically resected PDAC tumors that were subsequently treated with (Gem)/Abraxane adjuvant chemotherapy. By ATAC-seq analyses of EpCAM+ PDAC malignant epithelial cells sorted from 54 freshly resected human tumors, we show here the discovery of a signature of 1092 chromatin loci displaying differential accessibility between patients with disease free survival (DFS) < 1 year and patients with DFS > 1 year. Analyzing transcription factor (TF) binding motifs within these loci, we identify two TFs (ZKSCAN1 and HNF1b) displaying differential nuclear localization between patients with short vs. long DFS. We further develop a chromatin accessibility microarray methodology termed “ATAC-array”, an easy-to-use platform obviating the time and cost of next generation sequencing. Applying this methodology to the original ATAC-seq libraries as well as independent libraries generated from patient-derived organoids, we validate ATAC-array technology in both the original ATAC-seq cohort as well as in an independent validation cohort. We conclude that PDAC prognosis can be predicted by ATAC-array, which represents a low-cost, clinically feasible technology for assessing chromatin accessibility profiles.

Download Full-text

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes

Human Molecular Genetics ◽

10.1093/hmg/6.1.59 ◽

1997 ◽

Vol 6 (1) ◽

pp. 59-68 ◽

Cited By ~ 9

Author(s):

P. Mogayzel

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary Cell ◽

Chinese Hamster Ovary ◽

Chinese Hamster ◽

Ovary Cell ◽

Artificial Chromosomes ◽

Yeast Artificial Chromosomes

Download Full-text

Variability of the Sheep Lung Microbiota

Applied and Environmental Microbiology ◽

10.1128/aem.00540-16 ◽

2016 ◽

Vol 82 (11) ◽

pp. 3225-3238 ◽

Cited By ~ 27

Author(s):

Laura Glendinning ◽

Steven Wright ◽

Jolinda Pollock ◽

Peter Tennant ◽

David Collie ◽

...

Keyword(s):

Spatial Variability ◽

Dna Sequences ◽

Bacterial Communities ◽

16S Rrna Genes ◽

Rrna Genes ◽

Large Animal ◽

Local Factors ◽

Adult Sheep ◽

Sequencing Technologies ◽

Host Influence

ABSTRACTSequencing technologies have recently facilitated the characterization of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system. Protected specimen brush samples were collected from three spatially disparate segmental bronchi of six adult sheep (age, 20 months) on three occasions (day 0, 1 month, and 3 months). To further explore the spatial variability of the microbiotas, more-extensive brushing samples (n= 16) and a throat swab were taken from a separate sheep. The V2 and V3 hypervariable regions of the bacterial 16S rRNA genes were amplified and sequenced via Illumina MiSeq. DNA sequences were analyzed using the mothur software package. Quantitative PCR was performed to quantify total bacterial DNA. Some sheep lungs contained dramatically different bacterial communities at different sampling sites, whereas in others, airway microbiotas appeared similar across the lung. In our spatial variability study, we observed clustering related to the depth within the lung from which samples were taken. Lung depth refers to increasing distance from the glottis, progressing in a caudal direction. We conclude that both host influence and local factors have impacts on the composition of the sheep lung microbiota.IMPORTANCEUntil recently, it was assumed that the lungs were a sterile environment which was colonized by microbes only during disease. However, recent studies using sequencing technologies have found that there is a small population of bacteria which exists in the lung during health, referred to as the “lung microbiota.” In this study, we characterize the variability of the lung microbiotas of healthy sheep. Sheep not only are economically important animals but also are often used as large animal models of human respiratory disease. We conclude that, while host influence does play a role in dictating the types of microbes which colonize the airways, it is clear that local factors also play an important role in this regard. Understanding the nature and influence of these factors will be key to understanding the variability in, and functional relevance of, the lung microbiota.

Download Full-text