Genetics and exercise: an introduction
Adam P. Sharples, James P. Morton, Henning Wackerhage in Molecular Exercise Physiology, 2022
Two important events of meiosis contribute to human genetic diversity: Independent assortment of chromosomes. Of each chromosome pair in somatic cells, gametes receive only one chromosome. Is it the mothers or fathers chromosome? This is a random process and so each spermatocyte or oocyte will have a random combination of chromosomes from maternal and paternal origins.Homologous recombination. During meiosis, before chromosomes migrate to daughter cells, chromosomes cross over and exchange equivalent segments. For instance, a crossing over may occur between maternal and paternal chromosome 3, resulting in an exchange of DNA between the two chromosomes. About 50–60 recombinations take place between all pairs of homologous chromosomes (i.e. pairs of chromosomes of maternal and paternal descent) during meiosis.
Adenoviral Vectors for Gene Therapy of Inherited and Acquired Disorders of the Lung
Kenneth L. Brigham in Gene Therapy for Diseases of the Lung, 2020
The details of plasmid design and common methods of use have been described in recent reviews (18,19) and therefore will not be reiterated in detail here (Fig. 1). In brief, the most widely used method involves three major steps. First, the new coding sequence with appropriate transcriptional start and stop regulatory sequences is added to a multiple cloning site within the deleted El region of a plasmid containing a portion of the left-hand (5') end of the adenovirus genome. Second, this plasmid vector containing the new coding sequence is cotransfected into 293 cells with a second plasmid that contains the entire adenovirus genome with an El deletion modified to contain a “stuffer fragment” of plasmid DNA. The stuffer fragment not only contains the plasmid origin of replication and antibiotic resistance gene for bacterial propagation, but it is sufficiently large to prevent that adenoviral DNA from being packaged into a stable viral particle. Homologous recombination occurs be tween the two plasmids so that the El region containing the coding sequence of interest replaces the plasmid stuffer within the otherwise intact genome, and the E1 proteins made by the 293 cells activate the recombinant genome replication with the result that recombinant virus is made. The third step is a series of plaque purifications with screening assays at each step to eliminate undesired wild type virus that is generated by homologous recombination between the viral sequences within 293 cells and the adenoviral plasmid with the stuffer fragment.
Role of Engineered Proteins as Therapeutic Formulations
Peter Grunwald in Pharmaceutical Biocatalysis, 2019
Homologous recombination techniques are based on high sequence homology of the parental gene sequences. The resultant chimeric gene shows an amalgamation of characteristic features of the combining parental sequences. The homologous recombination methods have been classified into in vitro and in vivo homologous recombination methods. DNA shuffling is the most prominent in vitro homologous recombination technique developed by Stemmer and coworkers (Stemmer, 1994b). Later advancements in the DNA shuffling method resulted in improvised schemes such as family shuffling (Crameri et al., 1998), and DOGs (degenerate oligonucleotide gene shuffling) (Gibbs et al., 2001). Other in vitro homologous recombination methods include: random priming in vitro recombination (RPR) (Shao et al., 1998), truncated metagenomic gene-specific PCR (TMGS-PCR) (Wang et al., 2010), staggered extension process (StEP) (Zhao et al., 1998), random chimeragenesis on transient templates (RACHITT) (Coco, 2003), synthetic shuffling (Ness et al., 2002). The in vivo homologous recombination methods include: cloning performed in yeast (CLERY) (Abecassis et al., 2003), Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) (Gonzalez-Perez et al., 2014), and phage assisted continuous evolution (PACE) (Esvelt et al., 2011).
Combining inhibition of immune checkpoints and PARP: rationale and perspectives in cancer treatment
Published in Expert Opinion on Therapeutic Targets, 2022
Martina Catalano, Luigi Francesco Iannone, Federica Cosso, Daniele Generali, Enrico Mini, Giandomenico Roviello
The exchange of DNA strands between a pair of DNA segments of the double helix that have a very similar or identical sequence is defined as homologous recombination. This exchange allows one stretch of DNA double helix to act as a template to restore lost or damaged information to the other stretch. It is particularly challenging to repair DNA damage affecting both strands of the double helix that occur in somatic cells during meiosis [16]. Germline mutations in several other HR genes have been linked to tumor predisposition. The most relevant include tumor suppressors BRCA1 and BRCA2 mutations that have a key role in different stages of HR, mainly associated with familial breast and ovarian cancers [17]. However, around 20–25% of epithelial ovarian cancer patients have pathogenic variants in several genes that mostly encode for proteins involved in DDR pathways [18]. Indeed, next generation sequencing revealed that beyond BRCA1/2, mutations in HR genes, such as PALB2, RAD51, ATM, BRIP1, BARD1, and CHEK2 occurs in up to a fifth of the patients with high-grade serous ovarian cancer [18]. Failure in the HR repair system can compromise the elimination of genome mutations and increasing the risk of oncogenesis after the accumulation of DNA damage events [19].
The biology and management of dyskeratosis congenita and related disorders of telomeres
Published in Expert Review of Hematology, 2022
Hemanth Tummala, Amanda Walne, Inderjeet Dokal
Biallelic RTEL1 variants were first identified in 2013 in a subset of patients with features of HH [44,45]. An autosomal recessive founder mutation was also been reported in HH patients from two unrelated families of Ashkenazi Jewish ancestry [46]. RTEL1 functions as a helicase (Figure 3). It is critical for telomere maintenance. It also has a role in homologous recombination. Patients with biallelic RTEL1 variants usually have very short telomeres as there is impaired resolution of T-loops. However, patient cells do not have significant defects in homologous recombination, since peripheral blood lymphocytes usually have normal chromosomal breakage score following treatment with diepoxybutane or mitomycin-C [45]. These patients have very homogeneous clinical features which include global BM failure, immune deficiency, and cerebellar hypoplasia. Subsequently, it has been observed heterozygous RTEL1 variants are responsible for some cases of pulmonary fibrosis [47]. Furthermore, a subgroup of patients with a combination of MDS and liver disease are associated with heterozygous loss of function variants in RTEL1 [48].
Targeting the DNA damage response in pediatric malignancies
Published in Expert Review of Anticancer Therapy, 2022
Jenna M Gedminas, Theodore W Laetsch
Double stranded DNA breaks can be repaired using nonhomologous end joining repair (NHEJ) or homologous recombination (HR). The repair mechanism used is based on the stability of the end of the DNA breaks [11]. NHEJ directly ligates broken DNA without the need for a homologous template [12]. Because it does not rely on a template, it is able to repair double stranded breaks in any phase of the cell cycle, however, it is also more prone to error than homologous recombination. Homologous recombination is responsible for the reactivation of stalled replication forks and repair of double stranded DNA breaks and inter-strand crosslinks [13]. The repair process occurs in three steps. The broken end of the chromosome if first paired with the homologous region on the sister chromatid. That strand is then invaded to form a Holliday junction, or DNA crossover, which generates a DNA duplex from the two different chromatids [14]. The Holliday junction is then translocated along the DNA and eventually cleaved by endonucleases to again form separate DNA molecules [14]. These two processes are activated by several kinase pathways, ataxia telangiectasia mutated (ATM), ataxia telangiectasia related (ATR), and DNA-PK, which when mutated, result in defective double-strand break repair [15].