chromosomal locus
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2021 ◽  
Author(s):  
King L. Hung ◽  
Jens Luebeck ◽  
Siavash R. Dehkordi ◽  
Ceyda Coruh ◽  
Julie A. Law ◽  
...  

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we present a method for targeted purification of megabase-sized ecDNA by combining in-vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA (CRISPR-CATCH). We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells. Targeted purification of ecDNA versus chromosomal DNA enabled phasing of genetic variants and provided definitive proof of an EGFRvIII mutation on ecDNA and wild-type EGFR on chromosomal DNA in a glioblastoma neurosphere model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNA compared to the native chromosomal locus in the same cells. Finally, separation of ecDNA species by size and sequencing allowed accurate reconstruction of megabase-sized ecDNA structures with base-pair resolution. CRISPR-CATCH is a new addition to the toolkit for studying focal amplifications in cancer and will accelerate studies aiming to explore the genetic and epigenetic landscapes of ecDNA.


2021 ◽  
Author(s):  
Jillella Mallikarjun ◽  
J Gowrishankar

In Escherichia coli, three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB. The isoforms have earlier been suggested to additionally participate in DNA damage repair and replication restart. It is also known that the proteins RecA and RecBCD are needed for repair of DNA double-strand breaks (DSBs) in E. coli. Here we show that strains lacking IF2-1 are profoundly sensitive to two-ended DSBs in DNA generated by radiomimetic agents phleomycin or bleomycin, or by endonuclease I-SceI. However, these strains remained tolerant to other DSB-generating genotoxic agents or perturbations to which recA and recBC mutants remained sensitive, such as to mitomycin C, type-2 DNA topoisomerase inhibitors, or DSB caused by palindrome cleavage behind a replication fork. Data from genome-wide copy number analyses following I-SceI cleavage at a single chromosomal locus suggested that, in a strain lacking IF2-1, the magnitude of break induced replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in absence of IF2-1 it is the annealing of a RecA nucleoprotein filament to its homologous target that is weakened, which in turn leads to a specific failure in assembly of Ter-to-oriC directed replisomes needed for consummation of two-ended DSB repair.


2021 ◽  
Vol 23 (5) ◽  
pp. 715-722
Author(s):  
Yu. H. Antypkin ◽  
L. H. Kyrylova ◽  
O. O. Miroshnykov ◽  
O. O. Yuzva ◽  
L. Yu. Silaieva ◽  
...  

Aim. To analyze current data of scientific literature on the etiology, pathogenesis, approaches to the diagnosis and treatment of rare orphan disease – pantothenate kinase-associated neurodegeneration and to describe the clinical case of this disease. Pantothenate kinase-associated neurodegeneration (PKAN) is a rare inherited autosomal recessive disorder caused by mutations in the PANK2 gene, which is located at the chromosomal locus: 20p13-p12.3. The article presents a literature review and a case report of the diagnosis and treatment of 8-year-old girl with pantothenate kinase-associated neurodegeneration who suffered from progressive motor impairment. Among the dominant clinical manifestations is a growing extrapyramidal motor disorder, the so-called “dystonic storm”, which can range from severe sensorimotor motor deficits in infants and young children to mild parkinsonism in adults. MRI signs in the form of specific changes in the T2-weighted mode, which detects typical areas of hyperintensive signal in globus pallidus, surrounded by a border of hypointensive signal (“tiger’s eye”) are particular important. Neuroimaging data most likely make possible to suspect a correct diagnosis, reducing the time and cost of additional examinations. There is no pathogenetic treatment of this disease now. The main directions of currently available symptomatic drug therapy are described. Clinical trials of detoxifying drugs that reduce iron levels in the body (chelation) and surgical treatments are currently underway. Conclusions. The presented clinical observation once again confirms the complexity of the diagnostic search for orphan diseases of the nervous system. A key place in the diagnosis of pantothenate kinase-associated neurodegeneration belongs to the neuroimaging methods and molecular genetic testing data.


Author(s):  
Michelle Naidoo ◽  
Fayola Levine ◽  
Tamara Gillot ◽  
Akintunde T. Orunmuyi ◽  
E. Oluwabunmi Olapade-Olaopa ◽  
...  

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (CRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). The 8q24 chromosomal locus is a region of very high PCa susceptibility that carries genetic variants associated with high risk of PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes a cluster of microRNAs including, microRNA-1205 (miR-1205), which are largely understudied. Herein, we demonstrate that miR-1205 is underexpressed in PCa cells and tissues and suppresses CRPC tumors in vivo. To characterize the molecular pathway, we identified and validated fry-like (FRYL) as a direct molecular target of miR-1205 and observed its overexpression in PCa cells and tissues. FRYL is predicted to regulate dendritic branching, which led to the investigation of FRYL in neuroendocrine PCa (NEPC). Resistance toward ADT leads to the progression of treatment related NEPC often characterized by PCa neuroendocrine differentiation (NED), however, this mechanism is poorly understood. Underexpression of miR-1205 is observed when NED is induced in vitro and inhibition of miR-1205 leads to increased expression of NED markers. However, while FRYL is overexpressed during NED, FRYL knockdown did not reduce NED, therefore revealing that miR-1205 induces NED independently of FRYL.


PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0252624
Author(s):  
Zhirong Fu ◽  
Srinivas Akula ◽  
Chang Qiao ◽  
Jinhye Ryu ◽  
Gurdeep Chahal ◽  
...  

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.


2021 ◽  
Vol 12 ◽  
Author(s):  
Krishan K. Vishnolia ◽  
Aleksandar Rakovic ◽  
Celine Hoene ◽  
Karim Tarhbalouti ◽  
Zouhair Aherrahrou ◽  
...  

Soluble guanylyl cyclase (sGC) protein is a heterodimer formed by two subunits encoded by GUCY1A1 and GUCY1B1 genes. The chromosomal locus 4q32.1 harbors both of these genes, which has been previously significantly associated with coronary artery disease, myocardial infarction, and high blood pressure. Blood pressure is influenced by both the environment and genetics and is complemented by several biological pathways. The underlying mechanisms associated with this locus and its genes still need to be investigated. In the current study, we aimed to establish the zebrafish as a model organism to investigate the mechanisms surrounding sGC activity and blood pressure. A zebrafish mutant gucy1a1 line was generated using the CRISPR-Cas9 system by inducing a 4-bp deletion frameshift mutation. This mutation resulted in a reduction of gucy1a1 expression in both heterozygote and homozygote zebrafish. Blood flow parameters (blood flow, arterial pulse, linear velocity, and vessel diameter) investigated in the gucy1a1 mutants showed a significant increase in blood flow and linear velocity, which was augmented in the homozygotes. No significant differences were observed for the blood flow parameters measured from larvae with individual morpholino downregulation of gucy1a1 and gucy1b1, but an increase in blood flow and linear velocity was observed after co-morpholino downregulation of both genes. In addition, the pharmacological sGC stimulator BAY41-2272 rescued the impaired cGMP production in the zebrafish gucy1a1± mutant larvae. Downregulation of cct7 gene did not show any significant difference on the blood flow parameters in both wild-type and gucy1a1± background larvae. In summary, we successfully established a zebrafish platform for investigating sGC-associated pathways and underlying mechanisms in depth. This model system will have further applications, including for potential drug screening experiments.


Author(s):  
Soichirou Satoh ◽  
Takayuki Hata ◽  
Naoto Takada ◽  
Makoto Tachikawa ◽  
Mitsuhiro Matsuo ◽  
...  

ABSTRACTHorizontal gene transfer can occur between phylogenetically distant organisms, such as prokaryotes and eukaryotes. In these cases, how do the translocated genes acquire transcriptional competency in the alien genome environment? According to the conventional view, specific loci of the eukaryotic genome are thought to provide transcriptional competency to the incoming coding sequences. To examine this possibility, we randomly introduced the promoterless luciferase (LUC)-coding sequences into the genome of Arabidopsis thaliana cultured cells and performed a genome-wide “transgene location vs. expression” scan. We found that one-third of the 4,504 mapped LUC genes were transcribed. However, only 10% of them were explained by conventional transcriptional fusions with the annotated genes, and the remainder of the genes exhibited novel transcription that occurred independently of the chromatin configuration or transcriptional activity inherent to the given chromosomal locus; rather, their transcriptional activation occurred stochastically at about 30% of each insertion event, but independent of the integration sites. We termed this activation phenomenon as an integration-dependent stochastic transcriptional activation, a new type of response of the plant genome to incoming coding sequences. We discuss the possible roles of this phenomenon in the evolution of eukaryotic genomes.


eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julien G Roth ◽  
Kristin L Muench ◽  
Aditya Asokan ◽  
Victoria M Mallett ◽  
Hui Gai ◽  
...  

Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of ‘footprint’-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information—available with the cell lines by request from the Simons Foundation Autism Research Initiative—with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Alex Avallone ◽  
Kerry L. Bartie ◽  
Sarah-Louise C. Selly ◽  
Khanam Taslima ◽  
Antonio Campos Mendoza ◽  
...  

Abstract Tilapia is one of the most commercially valuable species in aquaculture with over 5 million tonnes of Nile tilapia, Oreochromis niloticus, produced worldwide every year. It has become increasingly important to keep track of the inheritance of the selected traits under continuous improvement (e.g. growth rate, size at maturity or genetic gender), as selective breeding has also resulted in genes that can hitchhike as part of the process. The goal of this study was to generate a Local Ancestry Interence workflow that harnessed existing tilapia genotyping-by-sequencing studies, such as Double Digest RAD-seq derived Single-Nucleotide Polymorphism markers. We developed a workflow and implemented a suite of tools to resolve the local ancestry of each chromosomal locus based on reference panels of tilapia species of known origin. We used tilapia species, wild populations and breeding programmes to validate our methods. The precision of the pipeline was evaluated on the basis of its ability to identify the genetic makeup of samples of known ancestry. The easy and inexpensive application of local ancestry inference in breeding programmes will facilitate the monitoring of the genetic profile of individuals of interest, the tracking of the movement of genes from parents to offspring and the detection of hybrids and their origin.


Author(s):  
Julia Katharina Wenskus ◽  
Deirdre Vincent ◽  
Maja Hempel ◽  
Konrad Reinshagen

AbstractIntroduction To date, several genes involved in the pathogenesis of HD have been recognized. Out of these, the RET gene (chromosomal locus 10q11), one of the first genes identified in combination with HD, is still considered the basis for HD development. However, even with over a hundred RET gen coding sequence mutations identified, the mutations do not fully explain the observed sex bias of HD and the elevated risk of developing HD among siblings. Thus, our aim was to evaluate the clinical relevance of an as yet undescribed genotype in a family with HD to improve genetic counseling for families with RET mutation-associated HD.Patients This case report provides an overview of a family with a history of HD with a novel, unreported autosomal dominant RET mutation.Results/Summary The family examined in this study clearly demonstrates that (1) the genotype to phenotype correlation of patients with RET mutation-associated HD is not directly related, and (2) genetic mechanisms underlying the different HD phenotypes, as well as the model of inheritance of HD, are complex and not yet fully understood. As such, a multifactorial genesis of HD appears more likely and should be the center of genetic counseling for concerned families. Having identified another RET mutation with a possible correlation of severity of HD and gender will aid in filling the gaps of the incomplete picture of the pathogenesis of HD.


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