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Molecular Approaches Towards the Isolation of Pediatric Cancer Predisposition Genes
Published in John T. Kemshead, Pediatric Tumors: Immunological and Molecular Markers, 2020
Analysis of the localization of the infrequent cutting sites in the genome shows that they tend to be clustered within small regions (0.5 to 2 kb) of the genome.127 In addition to the rare cutting enzymes, the 4-base pair recognition sequence from the Hpa II restriction enzyme (CCGG) is also frequently located within these islands. Digestion of the human genome with Hpa II produces tiny fragments,129 the Hpa II-tiny-fragments (HTF). A clustering of rare cutting sites occurs throughout the genome within the “HTF-islands”128 which are frequently, but not always, located 5’ to genes.129 The sequences flanking the HTF islands are usually highly methylated and it has been suggested that these islands, in some way, are responsible for the regulation of gene activity.129 There are an estimated 30,000 HTF islands in the human genome which is strikingly close to the total estimated number of human genes.129 The exciting possibility in this area is that, in combination with PFGE and chromosome jumping strategies, “island hopping” and, by implication “gene hopping”, may be possible.
HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
The restriction enzyme TaqI is favored by many investigators because it permits distinction between the majority of DRB-DQB-DQA haplotypes.5-9 An underlying reason for this is related to the recognition sequence within DNA of TaqI (5′-TCGA-3′) which contains the nucleotide dimer CpG. Restriction sites containing CpG dimers show a higher frequency of polymorphism in human DNA than other restriction sites, a probable result of CpG to TpG transition mutation within the dimer sequence. Hence, high level mutation at these sites is detectable by TaqI RFLP typing.3
rDNA: Evolution Over a Billion Years
Published in S. K. Dutta, DNA Systematics, 2019
The methylation of wheat rDNA has been analyzed by Flavell et al.106 using the restriction enzymes Hpa II (sensitive to methylation of C residues in the recognition sequence and Msp I (insensitive to methylation of the C residue which affects Hpa II107). These authors found varying levels of methylation among rDNA units with a major proportion having the sequence methylated. The number of rDNA units methylated increases as the number of rDNA units per cell increases in a way which suggests that excess rDNA is actually more efficiently methylated. This observation, taken together with the fact that wheat containing a 1U (from A. umbellulata, discussed above) substitution has the wheat rDNA preferentially methylated, suggests that methylation correlates with gene inactivity; as discussed by Flavell et al.106 this agrees with observations in several other systems. It is interesting that the Hpa II site assayed by Flavell et al.106 is in the vicinity of the start of transcription; the equivalent site in flax rDNA tends to be preferentially under-methylated and it has been speculated that methylation of this region may be particularly critical in determining gene activity.108 While methylation is very likely one of the prerequisites for gene inactivity in vivo, it alone is probably not sufficient since in Xenopus laevis it has been demonstrated that highly methylated rDNA can be efficiently transcribed in oocytes.109,110
Survivin as a biological biomarker for diagnosis and therapy
Published in Expert Opinion on Biological Therapy, 2021
Yuming Li, Wenshu Lu, Jiarun Yang, Mark Edwards, Shisong Jiang
One possible way to avoid this is to use a nonfunctional version of SVN, such as a recombinant overlapping peptide (ROP) as the immunogen instead of the wild-type protein. In an ROP construct, a synthetic gene is created that encodes a complete set of overlapping peptides derived from the native wild-type protein sequence. Each component peptide overlaps its adjacent sequences by 10–20 amino acids, linked by a short recognition sequence for a lysosomal protease. The resultant ROP therefore encodes all T cell epitope information from the wild-type protein, along with some B cell epitopes. However, the wild-type conformation will be comprehensively disrupted, and the ROP will lack the functions of the native protein. Once an ROP enters antigen presenting cells, either through phagocytosis (subunit vaccines) or expressed exogenously after delivery by a vector (vector-based vaccines), the ROP will be cleaved into fragments and presented to stimulate T and B cells. Thus, ROPs represent a powerful immunogenic vaccine platform that lacks the safety concerns associated with expression of native antigens – such as SVN – with intrinsic physiological functions. Future perspective
An update on the therapeutic potential of calpain inhibitors: a patent review
Published in Expert Opinion on Therapeutic Patents, 2020
Proteolytic enzymes such as calpain generally bind to the extended-strand conformation of substrates, which usually requires 4–5 residues for sequence recognition [26]. Therefore, a successful approach to protease inhibitor design is to lock the recognition sequence into a specific conformation within a cyclic peptide. This concept has been explored to design calpain inhibitors [27]. The advantages of cyclization include reduced entropic penalty for binding to the target protein [28,29] and enhanced stability to proteolytic degradation in vivo [30]. Low et al. [31] applied the concept to designed calpain inhibitors based on calpastatin, which is a highly specific natural inhibitor of calpain [32]. The group proposed that peptidomimetics that incorporate the conserved β-turn residues (i.e., KLGE) of calpastatin in the region where it binds to the active site of calpain should display calpastatin-like specificity and potency.
History of radiation genetics: light and darkness
Published in International Journal of Radiation Biology, 2019
Because so many mice are required for the SLT assays, it is impossible to conduct similar genetic studies in an ordinary-sized laboratory. However, more recent developments permitted many loci to be examined for each individual. At RERF, Asakawa improved the 2DE method and made it possible to examine about 1,000 genes for the purpose of screening for deletion mutations (loss of one allele from the ordinary two alleles) (Asakawa et al. 2004). Strictly speaking, the study’s focus is not on genes, but on radio-labeled DNA fragments. Because the NotI restriction enzyme (recognition sequence: GCGGCCGC) is used to cut and label the DNA, it is likely that the resulting fragments can contain coding genes or nearby sequences. Assuming that the mutation induction rate detected by the SLT method applies to all genomic regions, 20 to 40 mutations were expected to be obtained following the screening of 500 offspring born to 4 Gy irradiated sires; specifically, 500 offspring × 103 fragments/animal×(1 or 2)×10−5 mutations/Gy × 4 Gy. In contrast to expectations, however, only four deletion mutations were found among 500 offspring.