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Enzyme Sites (enzyme + site)
Selected AbstractsA genetic polymorphism in the coding region of the gastric intrinsic factor gene (GIF) is associated with congenital intrinsic factor deficiency,HUMAN MUTATION, Issue 1 2004Marilyn M. Gordon Abstract Congenital intrinsic factor (IF) deficiency is a disorder characterized by megaloblastic anemia due to the absence of gastric IF (GIF, GenBank NM_005142) and GIF antibodies, with probable autosomal recessive inheritance. Most of the reported patients are isolated cases without genetic studies of the parents or siblings. Complete exonic sequences were determined from the PCR products generated from genomic DNA of five affected individuals. All probands had the identical variant (g.68A>G) in the second position of the fifth codon in the coding sequence of the gene that introduces a restriction enzyme site for Msp I and predicts a change in the mature protein from glutamine5 (CAG) to arginine5 (CGG). Three subjects were homozygous for this base exchange and two subjects were heterozygous, one of which was apparently a compound heterozygote at positions 1 and 2 of the fifth codon ([g.67C>G] + [g.68A>G]). The other patient, heterozygous for position 2, had one heterozygous unaffected parent. Most parents were heterozygous for this base exchange, confirming the pattern of autosomal recessive inheritance for congenital IF deficiency. cDNA encoding GIF was mutated at base pair g.68 (A>G) and expressed in COS-7 cells. The apparent size, secretion rate, and sensitivity to pepsin hydrolysis of the expressed IF were similar to native IF. The allelic frequency of g.68A>G was 0.067 and 0.038 in two control populations. This sequence aberration is not the cause of the phenotype, but is associated with the genotype of congenital IF deficiency and could serve as a marker for inheritance of this disorder. Hum Mutat 23:85,91, 2004. © 2003 Wiley-Liss, Inc. [source] Deletion hotspot in the argininosuccinate lyase gene: association with topoisomerase II and DNA polymerase , sites ,HUMAN MUTATION, Issue 11 2006John Christodoulou Abstract Molecular analysis of argininosuccinate lyase (ASAL) deficiency has led to the identification of a deletion hotspot in the ASL gene. Six individuals with ASAL deficiency had alleles that led to a complete absence of exon 13 from the ASL mRNA; each had a partial deletion of exon 13 in the genomic DNA. In all six patients, the deletions begin 18 bp upstream of the 3, end of exon 13. In four cases, the deletions were 13 bp in length, and ended within exon 13, whereas in two other patients the deletions were 25 bp and extended into intron 13. The sequence at which these deletions begin overlaps both a putative topoisomerase II recognition site and a DNA polymerase , mutation/frameshift site. Moreover, the topoisomerase II cut site is situated precisely at the beginning of the deletions, which are flanked by small (2- and 3-bp) direct repeats. We note that a similar concurrence of these two putative enzyme sites can be found in a number of other deletion sites in the human genome, most notably the ,F508 deletion in the CFTR gene. These findings suggest that the joint presence of these two enzyme sites represents a DNA sequence context that may favor the occurrence of small deletions. Hum Mutat 27(11), 1065,1071, 2006. © 2006 Wiley-Liss, Inc. [source] Biochemical and mutational analyses of the cathepsin c gene (CTSC) in three North American families with Papillon Lefèvre syndromeHUMAN MUTATION, Issue 1 2002Y. Zhang Abstract Papillon Lefèvre syndrome (PLS) is an autosomal recessive disorder characterized by palmoplantar hyperkeratosis and severe periodontitis. The disease is caused by mutations in the cathepsin C gene (CTSC) that maps to chromosome 11q14. CTSC gene mutations associated with PLS have been correlated with significantly decreased enzyme activity. Mutational analysis of the CTSC gene in three North American families segregating PLS identified four mutations, including a novel mutation p.G139R. All mutations were associated with dramatically reduced CTSC protease enzyme activity. A homozygous c.96T>G transversion resulting in a p.Y32X change was present in a Mexican PLS proband, while one Caucasian PLS proband was a compound heterozygote for the p.Y32X and p.R272P (c.815G>C) mutations. The other Caucasian PLS proband was a compound heterozygote for c.415G>A transition and c.1141delC mutations that resulted in a p.G139R and a frameshift and premature termination (p.L381fsX393), respectively. The c.415G>A was not present in more than 300 controls, suggesting it is not a CTSC polymorphism. Biochemical analysis demonstrated almost no detectable CTSC activity in leukocytes of all three probands. These mutations altered restriction enzyme sites in the highly conserved CTSC gene. Sequence analysis of CTSC exon 3 confirmed the previously reported p.T153I polymorphism in 4 of the 5 ethnically diverse populations studied. © 2002 Wiley-Liss, Inc. [source] An in vitro recombination method to convert restriction- and ligation-independent expression vectorsBIOTECHNOLOGY JOURNAL, Issue 3 2008Feng Guo Abstract In recent years, restriction-less recombination cloning systems based on site-specific recombinase with high efficiency have been proven to be very successful. Thus, it is desirable to convert existing conventional vectors to recombination vectors. In this report, we describe the conversion of a set of widely used conventional vectors to Gateway® recombination expression vectors. An attB cassette flanked by several restriction enzyme sites was inserted in a cloning vector, and then subcloned into existing vectors to be converted to construct intermediate vectors containing the attB cassette, which were then converted to recombination expression vectors by in vitro recombination. The intermediate vectors generated in this study can be used for releasing the attB cassette to convert other vectors using the same protocol described here. With the increasing number of recombination vectors constructed with this protocol, the likeliness of releasing the attB cassette from an existing vector, rather than synthesizing it with PCR, will increase. The final expression vectors can also be used for releasing the attR cassette for constructing new vectors. [source] | ||||||||||