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rDNA: Evolution Over a Billion Years
Published in S. K. Dutta, DNA Systematics, 2019
Digestion of DNA samples with restriction endonucleases and separating the DNA fragments on a size basis using electrophoresis in an agarose or Polyacrylamide gel is the first step in characterizing the rDNA of an organism. Transfer of the DNA fragments to nitrocellulose218 and hybridization with radioactive rRNA (or a DNA sequence from a gene region from one of the organisms detailed in the preceding section) allows the preliminary mapping of the rDNA unit. A basic parameter which is essential to know in an uncharacterized system is the repeat size of the rDNA unit (see Table 1); with restriction enzyme digests this is best determined by a partial digest with an enzyme which cuts only once. Hexanucleotide recognition restriction endonucleases such as Bam HI, Hind III, EcoRI, and XbaI are often useful. The periodicity of the “ladder” which results from a partial digest of a tandem array of sequences gives the repeat length of the unit; the large size of some eukaryotic rDNA repeating units, it should be noted, precludes the use of such ladders.
Pathogenesis: Molecular mechanisms of osteoporosis
Published in Peter V. Giannoudis, Thomas A. Einhorn, Surgical and Medical Treatment of Osteoporosis, 2020
Anastasia E. Markatseli, Theodora E. Markatseli, Alexandros A. Drosos
With regard to the gene encoding ERα, several polymorphisms have been examined in terms of their relationship with BMD. The two most important polymorphisms are those that are recognized by the restriction enzymes XbaI and PvuII. These polymorphisms are located within intron 1 and regulate the transcription of the ERa gene (134). A meta-analysis that included more than 5000 women from 22 published studies (11 in Caucasians and 11 in Asians) revealed an association between polymorphism XbaI (rs9340799), BMD, and fractures (135). Another meta-analysis of data from 18,917 people at eight European centers confirmed the presence of a correlation of this polymorphism with an increased risk of fracture, but not with BMD (136). Regarding polymorphism PvuII (rs2234693), a meta-analysis recorded a weak association with BMD at the femoral neck (137). Other ERa gene polymorphisms have also been associated with either hip fractures (138) or with BMD (139,140). Polymorphisms of the gene encoding estrogen receptor ERb have been studied to a lesser extent compared to those of the ERa gene for their relation with BMD and fracture risk. Loss of function mutations of the CYP19A1 gene, which encodes aromatase, have been identified in patients with osteoporosis (141,142). A recent study conducted in postmenopausal women recorded a significant correlation between polymorphisms of the CYP19A1 gene and BMD. However, it is worth highlighting the important role of age in this study, because statistical significance was noted only in a subset of individuals at or above 67 years of age and not in younger individuals (143). In addition, two previous studies have documented an association between polymorphisms of the CYP19A1 gene and BMD in early postmenopausal women (144,145). The CYP17A1 gene plays an important role in the synthesis of estrogen and androgen. Loss of function mutations of the CYP17A1 gene lead to osteoporosis and reduce skeleton growth (146).
Gut-derived bacterial flagellin induces beta-cell inflammation and dysfunction
Published in Gut Microbes, 2022
Torsten P.M. Scheithauer, Hilde Herrema, Hongbing Yu, Guido J. Bakker, Maaike Winkelmeijer, Galina Soukhatcheva, Derek Dai, Caixia Ma, Stefan R. Havik, Manon Balvers, Mark Davids, Abraham S. Meijnikman, Ömrüm Aydin, Bert-Jan H. van den Born, Marc G. Besselink, Olivier R. Busch, Maurits de Brauw, Arnold van de Laar, Clara Belzer, Martin Stahl, Willem M. de Vos, Bruce A. Vallance, Max Nieuwdorp, C. Bruce Verchere, Daniël H. van Raalte
The pRE118-pheS-ΔfliC construct was generated the same as above. Primer pairs used to amplify the PCR fragments are FljB-P1 (5’-GCACGTCTAGAGTGACCTTTATCGTCATCTCACCGT-3’) plus FljB-P2 (5’-GTACCCAGCTGAGTCTGGGATTTGTTCAGGTTGTT-3’), and FljB-P3 (5’- AGACTCAGCTGGGTACTGCTGCGTTAATCTGCGTTA-3’) plus FljB-P4 (5’-GACAGTGAGCTCGTACAGCTATTCGCTGCATAACGA-3’), respectively. This results in a 955-bp fragment containing the upstream of fljB and a 950-bp fragment containing the downstream of the fljB, respectively. These two PCR fragments were then mixed and used as the template for a secondary PCR (with primer pairs FljB-P1 containing a XbaI restriction enzyme site and FljB-P4 containing a SacI restriction enzyme site). The 16-bp overlapping sequence (underlined) in primers FljB-P2 and FljB-P3 allows the amplification of a 1,905-bp PCR product. This PCR product was digested with XbaI and SacI, and directly cloned into the E. cloacae suicide vector pRE118-pheS.
Genetic and epigenetic disease modifiers in an Italian C9orf72 family expressing ALS, FTD or PD clinical phenotypes
Published in Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, 2022
Antonia Ratti, Silvia Peverelli, Elisabetta D'Adda, Claudia Colombrita, Michele Gennuso, Alessandro Prelle, Vincenzo Silani
Genomic DNA (10 µg) was XbaI-digested and run on 0.7% agarose gel. After gel washes in 0.25N HCl for 20 min, in 0.4N NaOH and 0.6M NaCl for 30 min and in 0.5M Tris pH 7.5 and 1.5M NaCl for 30 min, DNA was transferred to a Hybond N + membrane (Amersham) in 20X Saline Sodium Citrate (SSC) buffer. A unique-sequence probe (466bp) within C9orf72 first intron was PCR amplified (forward 5′-CTTTCTCCAGATCCAGCAGCCTCC-3′ and reverse 5′-CTGAGTTCCAGAGCTTGCTACAG-3′) and α-32P-dCTP radiolabelled by random primer labeling kit (Thermo Fisher). Probe hybridization was carried out in 50% formamide, 5X SSC, 5X Denhardt, 0.5% SDS and 25 µg/µl salmon sperm at 42 °C overnight. The membrane was washed in 2X SSC/0.1% SDS buffer at room temperature and in 0.5X SSC/0.1% SDS buffer at 60 °C for 20 min each and visualized by autoradiography.
The contribution of genetic variants of SLC2A1 gene in T2DM and T2DM-nephropathy: association study and meta-analysis
Published in Renal Failure, 2018
I. Stefanidis, M. Tziastoudi, E. E. Tsironi, E. Dardiotis, S. V. Tachmitzi, A. Fotiadou, G. Pissas, K. Kytoudis, M. Sounidaki, G. Ampatzis, P. R. Mertens, V. Liakopoulos, T. Eleftheriadis, G. M Hadjigeorgiou, M. Santos, E. Zintzaras
Figure 1 presents a flowchart of retrieved and excluded articles. The characteristics of each study are shown in Table 7. Across all available studies examining SLC2A1 variants, 8 genetic variants were studied (XbaI SNP, HaeIII SNP, Enhancer-1 SNP, Enhancer-2 SNP 1, Enhancer-2 SNP 2, Enhancer-3 SNP, HpyCH4V and rs3820589). Out of the aforementioned variants, only five variants examined in two studies or more and so meta-analyzed (XbaI SNP, HaeIII SNP, Enhancer-2 SNP 1, Enhancer-2 SNP 2 and HpyCH4V). Only XbaI SNP produced significant results in analysis using diseased controls versus cases and healthy controls versus cases giving a summary ORG of 1.428 (1.086, 1.877) and 1.581 (1.007, 2.482), respectively (Table 8). The studies comprised 1812 cases, 1763 diseased controls and 949 healthy controls and they were published between 1998 and 2015 [10,35–44]. Figures 2–4 are forest plot representations of variant rs841853.