Mapping of the human IL10 gene and further characterization of the 5' flanking sequence

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two Class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 or Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

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Event-specific qualitative and quantitative detection of transgenic rice Kefeng-6 by characterization of the transgene flanking sequence

European Food Research and Technology ◽

10.1007/s00217-010-1389-1 ◽

2010 ◽

Vol 232 (2) ◽

pp. 297-305 ◽

Cited By ~ 23

Author(s):

Wei-Xia Wang ◽

Ting-Heng Zhu ◽

Feng-Xiang Lai ◽

Qiang Fu

Keyword(s):

Transgenic Rice ◽

Quantitative Detection ◽

Flanking Sequence ◽

Qualitative And Quantitative

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In-depth characterization of the cisplatin mutational signature in human cell lines and in esophageal and liver tumors

10.1101/189233 ◽

2017 ◽

Author(s):

Arnoud Boot ◽

Mi Ni Huang ◽

Alvin W.T. Ng ◽

Szu-Chi Ho ◽

Jing Quan Lim ◽

...

Keyword(s):

Liver Tumors ◽

Secondary Malignancies ◽

Flanking Sequence ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Non Invasive ◽

Mutational Signature ◽

Hepatocellular Carcinomas ◽

Non Negative Matrix Factorization

AbstractBackground and aimsCisplatin reacts with DNA, and thereby likely generates a characteristic pattern of somatic mutations, called a mutational signature. Despite widespread use of cisplatin in cancer treatment and its role in contributing to secondary malignancies, its mutational signature has not been delineated. We hypothesize that cisplatin’s mutational signature can serve as a biomarker to identify cisplatin mutagenesis in suspected secondary malignancies. Knowledge of which tissues are at risk of developing cisplatin-induced secondary malignancies could lead to guidelines for non-invasive monitoring for secondary malignancies after cisplatin chemotherapy.MethodsWe performed whole genome sequencing of 10 independent clones of cisplatin-exposed MCF-10A and HepG2 cells, and delineated the patterns of single- and dinucleotide mutations in terms of flanking sequence, transcription strand bias, and other characteristics. We used the mSigAct signature presence test and non-negative matrix factorization to search for cisplatin mutagenesis in hepatocellular carcinomas and esophageal adenocarcinomas.ResultsAll clones showed highly consistent patterns of single- and dinucleotide substitutions. The proportion of dinucleotide substitutions was high: 8.1% of single nucleotide substitutions were part of dinucleotide substitutions, presumably due to cisplatin’s propensity to form intra-and inter-strand crosslinks between purine bases in DNA. We identified likely cisplatin exposure in 9 hepatocellular carcinomas and 3 esophageal adenocarcinomas. All hepatocellular carcinomas for which clinical data were available and all esophageal cancers indeed had histories of cisplatin treatment.ConclusionsWe experimentally delineated the single- and dinucleotide mutational signature of cisplatin. This signature enabled us to detect previous cisplatin exposure in human hepatocellular carcinomas and esophageal adenocarcinomas with high confidence.

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Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.5043-5056.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 5043-5056

Author(s):

K Shirahige ◽

T Iwasaki ◽

M B Rashid ◽

N Ogasawara ◽

H Yoshikawa

Keyword(s):

Essential Element ◽

The Other ◽

Flanking Sequence ◽

Functional Elements ◽

Autonomously Replicating Sequence ◽

The Core ◽

Initiation Of Replication ◽

Flanking Region ◽

Delta G

We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI. The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4. Nine ARS fragments were identified, and their positions were mapped on the chromosome. Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations. The organization of functional elements composed of core and stimulative sequences was found to be variable. Single core sequences were identified in eight of nine ARSs. The remaining ARS (ARS603) essential element is composed of two core-like sequences. The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS. Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs. In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence. In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.

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