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J J Babon

Publications and source records attributed to J J Babon.

10 recordsLinked to original sources
PubMed2004

Novel TP53 gene mutations in tumors of Russian patients with breast cancer detected using a new solid phase chemical cleavage of mismatch method and identified by sequencing.

Mutations in the tumor-suppressor p53 gene TP53 are frequent in most human cancers including breast cancer. A new solid phase chemical cleavage of mismatch method (CCM) allowed rapid and efficient screening and analysis of the TP53 gene in DNA samples extracted from tumors of 89 breast cancer patients. The novel CCM technique utilized silica beads and the potassium permanganate/tetraethylammonium chloride (KMnO(4)/TEAC) and hydroxylamine (NH(2)OH) reactions were performed sequentially in a single tube. Mutation analysis involved amplification of five different fragments of the TP53 gene using DNA from the 89 tumor samples, then pairing of the 391 labeled PCR products and forming heteroduplexes. A total of 41 unique signals were revealed in the analysis of TP53 exons 5-9 and eight were identified by direct sequencing. The three novel mutations detected are c.600T>G (p.Asn200Lys), c.601T>G (p.Leu201Val), and c.766-768delACA (p.Thr256del). The detected mutations c.638G>T (p.Arg213Leu), c.730G>T (p.Gly244Cys), and c.758C>T (p.Thr253Ile) have not been reported in breast cancer but have been recorded in tumors of other organs. A previously reported mutation c.535C>T (p.His179Tyr) and a heterozygous polymorphism c.639A>G were also detected. Of the 41 unique signals, 36 were not identified as a sequence change. As direct sequencing requires the mutant allele concentration to be greater than 30% when the mutant allele is present in a mixture with the wild-type allele, the CCM method represents a more sensitive technique requiring a lower mutant allele concentration in the wild-type mixture compared with direct sequencing. This reveals the advantage of CCM for unknown point mutation detection in DNA samples of cancer patients.

Adult↗
PubMed2003

The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection.

Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and, more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.

Algorithms↗
PubMed2001

Interaction of linear homologous DNA duplexes via Holliday junction formation.

Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA-DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA-protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.

Animals↗
PubMed2001

COL4A4 mutation in thin basement membrane disease previously described in Alport syndrome.

BACKGROUND: Carriers of autosomal-recessive and X-linked Alport syndrome often have a thinned glomerular basement membrane (GBM) and have mutations in the COL4A3/COL4A4 and COL4A5 genes respectively. Recently, we have shown that many individuals with thin basement membrane disease (TBMD) are also from families where hematuria segregates with the COL4A3/COL4A4 locus. This study describes the first COL4A4 mutation in an individual with biopsy-proven TBMD who did not have a family member with autosomal-recessive or X-linked Alport syndrome, inherited renal failure, or deafness. METHODS: The index case and all available family members were examined for dysmorphic hematuria> 50,000/mL using phase contrast microscopy and for segregation of hematuria with the COL4A3/COL4A4 and COL4A5 loci using DNA satellite markers. COL4A4 exons from the index case were then studied using the enzyme mismatch cleavage method, and exons that demonstrated abnormal cleavage products were sequenced. RESULTS: Hematuria in this family segregated with a haplotype at the COL4A3/COL4A4 locus (P = 0.031) but not with haplotypes at the COL4A5 locus. A mutation in COL4A4 that changed C to T resulting in an arginine residue being replaced by a stop codon (R1377X) was demonstrated in exon 44, which encodes part of the alpha 4(IV) collagen sequence close to the junction with the noncollagenous domain. This mutation was present in all five family members with hematuria, but not in the four unaffected family members, 33 unrelated individuals with TBMD, or 22 nonhematuric normals. CONCLUSIONS: R1377X has been described previously in a compound heterozygous form of autosomal-recessive Alport syndrome. Our observation is evidence that TBMD can represent a carrier state for autosomal-recessive Alport syndrome in at least some individuals.

Basement Membrane↗
PubMed1999

Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol.

Many mutation detection techniques rely upon recognition of mismatched base pairs in DNA hetero-duplexes. Potassium permanganate in combination with tetraethylammonium chloride (TEAC) is capable of chemically modifying mismatched thymidine residues. The DNA strand can then be cleaved at that point by treatment with piperidine. The reactivity of potassium permanganate (KMnO4) in TEAC toward mismatches was investigated in 29 different mutations, representing 58 mismatched base pairs and 116 mismatched bases. All mismatched thymidine residues were modified by KMnO4/TEAC with the majority of these showing strong reactivity. KMnO4/TEAC was also able to modify many mismatched guanosine and cytidine residues, as well as matched guanosine, cytidine and thymidine residues adjacent to, or nearby, mismatched base pairs. Previous techniques using osmium tetroxide (OsO4) to modify mismatched thymidine residues have been limited by the apparent lack of reactivity of a third of all T/G mismatches. KMnO4/TEAC showed no such phenomenon. In this series, all 29 mutations were detected by KMnO4/TEAC treatment. The latest development of the Single Tube Chemical Cleavage of Mismatch Method detects both thymidine and cytidine mismatches by KMnO4/TEAC and hydroxylamine (NH2OH) in a single tube without a clean-up step in between the two reactions. This technique saves time and material without disrupting the sensitivity and efficiency of either reaction.

Adenine↗
PubMed1999

Mutation detection using fluorescent enzyme mismatch cleavage with T4 endonuclease VII.

Mutation detection techniques are often limited by sensitivity, ease of use and short fragment lengths. Enzyme mismatch cleavage (EMC) is a technique capable of rapidly scanning 1 kbp fragments of DNA for mutations. It relies on the ability of a bacteriophage resolvase enzyme, T4 endonuclease VII, to cleave DNA at single base pair mismatches and small heteroduplex loops. Originally the process was performed using radioactively labeled DNA and the results analysed after denaturing polyacrylamide gel electrophoresis and autoradiography. However, access to systems capable of detecting fluorescent species migrating through a gel and the widespread availability of fluorescently tagged primers have greatly improved upon the original technique. A number of mutations were detected using fluorescent EMC and the results compared to performing the technique using radiolabeled DNA. Fluorescent EMC detected the presence, position and number of mutations in DNA fragments as large as 1 kbp. The fluorescent method was found to have advantages over the original method in its ease of use, increase in signal-to-noise ratio and the ability to multiplex samples by labeling DNA fragments with different fluorophores. This improvement on an already established method provides a sensitive, robust technique for mutation detection.

Animals↗
PubMed1995

Evolution of transthyretin in marsupials.

The evolution of the expression and the structure of the gene for transthyretin, a thyroxine-binding plasma protein formerly called prealbumin, was studied in three marsupial species: the South American polyprotodont Monodelphis domestica, the Australian polyprotodont Sminthopsis macroura and the Australian diprotodont Petaurus breviceps. The transthyretin gene was found to be expressed in the choroid plexus of all three species. In liver it was expressed in P. breviceps and in M. domestica, but not in S. macroura. This, together with previous studies [Richardson, S. J., Bradley, A. J., Duan, W., Wettenhall, R. E. H., Harms, P. J., Babon, J. J., Southwell, B. R., Nicol, S., Donnellan, S. C. & Schreiber, G. (1994) Am. J. Physiol. 266, R1359-R1370], suggests the independent evolution of transthyretin synthesis in the liver of the American Polyprotodonta and the Australian Diprotodonta. The results obtained from cloning and sequencing of the cDNA for transthyretin from the three species suggested that, in the evolution of the structure of transthyretin in vertebrates, marsupial transthyretin structures are intermediate between bird/reptile and eutherian transthyretin structures. In marsupials, as in birds and reptiles, a hydrophobic tripeptide beginning with valine and ending with histidine was found in transthyretin at a position which has been identified in eutherians as the border between exon 1 and intron 1. In humans, rats and mice, the nine nucleotides, coding for this tripeptide in marsupials/reptiles/birds, are found at the 5' end of intron 1. They are no longer present in mature transthyretin mRNA. This results in a change in character of the N-termini of the subunits of transthyretin from hydrophobic to hydrophilic. This change might affect the accessibility of the thyroxine-binding site in the central channel of transthyretin, since, at least in humans, the N-termini of the subunits of transthyretin are located in the vicinity of the channel entrance [Hamilton, J. A., Steinrauf, L. K., Braden, B. C., Liepnieks, J., Benson, M. D., Holmgren, G., Sandgren, O. & Steen, L. (1993) J. Biol. Chem. 268, 2416-2424].

Amino Acid Sequence↗
PubMed1994

Evolution of marsupial and other vertebrate thyroxine-binding plasma proteins.

Binding of radioactive thyroxine to proteins in the plasma of vertebrates was studied by electrophoresis followed by autoradiography. Albumin was found to be a thyroxine carrier in the blood of all studied fish, amphibians, reptiles, monotremes, marsupials, eutherians (placental mammals), and birds. Thyroxine binding to transthyretin was detected in the blood of eutherians, diprotodont marsupials, and birds, but not in blood from fish, toads, reptiles, monotremes, and Australian polyprotodont marsupials. Globulins binding thyroxine were only observed in the plasma of some mammals. Apparently, albumin is the phylogenetically oldest thyroxine carrier in vertebrate blood. Transthyretin gene expression in the liver developed in parallel, and independently, in the evolutionary lineages leading to eutherians, to diprotodont marsupials, and to birds. In contrast, high transthyretin mRNA levels, strong synthesis, and secretion of transthyretin in choroid plexus from reptiles and birds indicate that transthyretin gene expression in the choroid plexus evolved much earlier than in the liver, probably at the stage of the stem reptiles. NH2-terminal sequence analysis suggests a change of transthyretin pre-mRNA splicing during evolution.

Amino Acid Sequence↗