bioRxiv Science⌕ Search

Biology subjects

Gunbin, K. V.

Publications and source records attributed to Gunbin, K. V..

2 recordsLinked to original sources

Mitochondrial direct repeat reduction as a strategy for enhancing human longevity: the case of the common repeat

Aging, characterized by a series of functional declines correlated with advancing chronological age, has a significant mitochondrial DNA (mtDNA) component, with somatic mtDNA deletions playing a central role. In post-mitotic or slow-dividing cells like neurons and skeletal muscles, selfish mtDNA deletions clonally expand within a cell, ultimately leading to the deterioration and death of host cells and appearence of age-related phenotypes. Thus reducing the burden of somatic deletions could have far- reaching systemic benefits for the entire human body. Given the crucial role of direct nucleotide repeats in the formation of mitochondrial deletions, we hypothesize that minimizing these repeats in the human mitochondrial genome could enhance healthspan by decreasing somatic deletions. To investigate this hypothesis, we focus on the "common repeat", a 13-base pair perfect direct repeat sequence (ACCTCCCTCACCA) located at positions 8470-8482 and 13447-13459, respectively. This perfect repeat: (i) is highly prevalent, with its potential deleterious consequences affecting the majority of humans; (ii) represents one of the most fragile sites, highly prone to forming deletions; (iii) when disrupted, is associated with a decreased somatic deletion load and enhanced human healthspan; (iv) is likely to experience positive selection in the present or near future due to indirect fitness effects, such as the "grandmother effect", and direct fitness effects, such as (v) a decreased mutation rate. These observations support the argument that reducing the mtDNA somatic deletion load through targeted disruption of these repeats, or by using naturally occurring polymorphisms with disrupted repeats in mitochondrial medicine, could be an effective approach to increasing human longevity.

genetics↗

Isolation of highly purified genomic material from mitochondria of muscle tissue cells

In this work, we adapted a method for isolation of a highly purified fraction of mitochondrial DNA from muscle tissues suitable for further sample preparation of libraries without an amplification step for sequencing tasks on various NGS platforms and a method for evaluating the purity of DNA from contamination by nuclear genome regions. We optimized several techniques7,8 for enrichment of the mitochondrial fractions and purifying mtDNA. Here, we describe a protocol that allows getting from 80-100 mg of muscle tissues up to 1000 ng mtDNA, almost free from impurities of RNA and fragments of the nuclear genome.To assess the degree of purity of human mtDNA fraction from impurities of the nuclear genome, we adapted the PCR-screening technique7 for the beta-actin gene region and AluSx-repeats in the human genome. This methodology avoids false-heteroplasmy calls (PCR biases or NUMT contamination) that occur when long-range PCR amplification is used for mtDNA enrichment.

molecular biology↗