Fucosidosis
William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop in Atlas of Inherited Metabolic Diseases, 2020
The gene is composed of eight exons spanning 23 kb [31]. Three patterns of mRNA were found [32] in Italian patients: two lacked mRNA; one had reduced amounts of an RNA with a cDNA by hybridization of a pattern indicating loss of a restriction site; and three had mRNA that was normal in size and content. Among mutations defined, a deletion of two exons [10] resulted in marked reduction of cross-reacting material (CRM) and absence of enzyme activity; a C-to-T transition leading to a TAA stop-codon, p.Q422X, deleting the carboxyl end of the enzyme [16, 33, 34]. This mutation causes loss of an EcoRI restriction site that is useful for molecular diagnosis [11]. A C-to-A change in exon 6 led to a stop codon p.W382X [12]. Among other mutations, most have been missense, not deletions or other major changes in gene structure [10–13, 35, 36]. Nonsense mutations were common. A homozygous nonsense mutation c1295G>A,p.W423X was found in exon 8 in a patient with unusual phenotype [23\ (v.s.). A patient with a missense mutation, p.L405R, was 46 years old despite less than 1 percent of control enzyme activity and no CRM [37]. Most of the mutations reported have led to virtual absence of activity of the enzyme, and this has been independent of the variability seen clinically.
rDNA: Evolution Over a Billion Years
S. K. Dutta in DNA Systematics, 2019
The structural map of the rDNA repeating unit was determined originally by restriction enzyme analysis and 125I labeled rRNA (5.8S, 18S, 28S) probes prepared from Balb/c mice. The size of the unit was estimated to be between 36198 and 44 kb.199 The restriction enzyme EcoRI produces three fragments which hybridize to 18S and/or 28S rRNA: (1) a fragment varying in size from 13 to 15 kb, containing most of the 18S rRNA gene, all the external transcribed spacer, and part of spacer; (2) a 6.6 kb fragment containing approximately 25% of the 18S rDNA, both internal transcribed spacers (ITS 1 and ITS 2), the 5.8S rDNA, and most of the 28S rDNA; (3) a 6.6 kb fragment containing the 3′ end of the 28S rDNA and part of the spacer downstream from the 28S rDNA. As summarized in Figure 11 these three fragments in combination with detailed restriction enzyme maps have been the starting point for the structural analysis of the mouse rDNA repeating unit.
Dealing with the invisible
Brendan Curran in A Terrible Beauty is Born, 2020
The first restriction enzyme was purified from the common bacterium Escherichia coli (E. coli among friends). The enzyme, termed called EcoR1, cuts DNA molecules wherever it finds the base sequence G-A-A-T-T-C in DNA regardless of the origin of the molecule. As this particular sequence will occur randomly in all long DNA molecules, the enzyme will cut the DNA into random fragments – about half a million of them in the case of human DNA. Because the enzyme is so specific, DNA is always cut into the same precise fragments whenever it is exposed to this particular enzyme. Furthermore, the DNA is cut in a staggered fashion to produce short overhanging segments, all ending in CTTAA. So, after human DNA has been exposed to this enzyme, it is transformed from a series of incredibly long DNA molecules of totally unknown DNA sequence into 500,000 smaller fragments. Each is still of unknown sequence but ends with the same short DNA handle on the right hand side and the precise complementary sequence on the left (Figure 5.4). Plasmids are, of course, made of DNA and are therefore sensitive to digestion by restriction enzymes. A circular plasmid cut with EcoR1 will become a linear piece of DNA and carry exactly the same left and right handle sequences as every one of the 500,000 fragments of human DNA as prepared above. The handles are very useful: they will eventually be exploited to smuggle each one of the individual fragments of human DNA into different bacterial cells.
In Vitro Generation of Anti-Osteosarcoma Cytotoxic Activity Using Dendritic Cells Loaded with Heat Shock Protein 70-Peptide Complexes
Published in Fetal and Pediatric Pathology, 2019
Yang Li, Danhui Wang, Xiutong Fang
XhoI and EcoRI restriction sites were introduced at the 5′ and 3′ ends of Hsp70 cDNA. A ligation between the DNA fragment and XhoI and EcoRI-digested pBluescript II SK (+) (Stratagene, La Jolla, CA, USA) was performed. Final construct pBS-Hsp70 was verified by double-stranded DNA sequencing (Shanghai DNA BioTech Ltd., Shanghai, China). The plasmid pBS-Hsp70 was digested with XhoI and EcoRI, and the target DNA fragment was subcloned in pET28a. Following ligation, the expression plasmid pET28a-Hsp70 was introduced into E. coli strain DH5α competent cells and cultured overnight. Plasmid DNA was prepared from the liquid cultures [Luria–Bertani (LB) liquid media containing kanamycin] of individual colonies and digested with proper restriction endonuclease. The digestion pattern was used to screen for the presence of the recombinant plasmid. The plasmid pET28a-Hsp70 from positive transformants was introduced into BL21 (DE3) competent cells (Thermo Fisher Scientific, Waltham, MA, USA). The competent cells were then plated out on LB agar plates with 50 μg/ml kanamycin and incubated overnight at 37 °C. The next day, individual colonies were suspended in 10-ml LB broth with 50 μg/ml kanamycin and incubated at 37 °C until OD600 reached 0.6–1. 0.1 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG, Sangon Biotech, Shanghai, China) was added to the culture and incubated at 37 °C for 2, 2.5, or 3 h. The protein expression of Hsp70 was analyzed with SDS-PAGE, thin layer scanning, and Western blot assay.
Tumor-targeting oncolytic virus elicits potent immunotherapeutic vaccine responses to tumor antigens
Published in OncoImmunology, 2020
Yong Luo, Chaolong Lin, Yidi Zou, Fei Ju, Wenfeng Ren, Yanhua Lin, Yale Wang, Xiaoxuan Huang, Huiling Liu, Zeng Yu, Pingguo Liu, Guowei Tan, Quan Yuan, Jun Zhang, Chenghao Huang, Ningshao Xia
HSV-1 strain KOS virus was purchased from ATCC and propagated in U-2 OS cells. The viral titer was determined in U-2 OS cells as previously described.26 For the construction of the donor plasmids, different strategies were used. To generate the 27p plasmid, the 5ʹ and 3ʹ regions flanking the genomic regions of core ICP27 promoter (from nt113422 to nt113590) were amplified with the primer pair #1 and #2, and then sequentially cloned into BamHI and PstI digested PUC57 vector with a linker containing the PmeI and SpeI sites. To generate the 27p-hTERT plasmid, the region for core ICP27 promoter in 27p was replaced with DNA fragments containing the core hTERT promoter,27 using the restriction enzymes PmeI and SpeI. To generate the fICP0 plasmid, the 5ʹ and 3ʹ regions flanking the genomic regions of ICP0 were amplified with the primer pair #3 and cloned into HindIII and EcoRI digested PUC57 vector. To generate the fICP0-GFP plasmid, the ICP0 region of picp0 was replaced with DNA fragments encoding eGFP using the restriction enzymes NcoI and SalI. To generate the PF0 plasmid, the DNA fragment covered 478 bp upstream of the ICP34.5 initiation codon to 409 bp downstream of the ICP0 stop codon was amplified with the primer pair #4 and ligated into PstI and SacI digested pMD18-T vector. The d34.5/0GFP plasmid was derived from PF0 that the ICP34.5 and ICP0 coding region was replaced by a DNA fragment encoding eGFP at the NcoI and SalI site. All primers used in this study are listed in Table S5.
Modulation of ABCG2 surface expression by Rab5 and Rab21 to overcome multidrug resistance in cancer cells
Published in Xenobiotica, 2020
Maryam Yousaf, Moazzam Ali
Mammalian expression vector pEGFP-C2 (Clontech), was used to construct N- terminally tagged ABCG2 (EGFP-ABCG2) fusion plasmid. The plasmid pSIN4-EF2-ABCG2-IRES-Neo containing full length human ABCG2 coding region was a gift from Ren-he Xu (Addgene plasmid # 25983) (Zeng et al., 2009). Fast digest EcoRI and BamHI (Thermo Fisher Scientific) were used to digest the plasmid and full-length ABCG2 fragment was ligated into EcoRI/BamHI digested pEGFP-C2 vector, followed by the transformation into E. coli (DH5-α cells). Positive transformants were sequence verified. GFP tagged expression construct of inactive Rab5A mutant, pEGFP-Rab5A-S34N (Addgene plasmid # 28045) and constitutively active Rab5A mutant pEGFP-Rab5A-Q79L (Addgene plasmid # 28046) were gift from Qing Zhong (Sun et al., 2010).
Related Knowledge Centers
- Directionality
- Endonuclease
- Escherichia Coli
- Molecular Biology
- Protein Dimer
- Restriction Enzyme
- Plasmid
- Restriction Modification System
- Sticky & Blunt Ends
- Palindromic Sequence