Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency.

We have devised an allele-specific amplification method with a TaqMan fluorogenic probe (TaqMan-ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele-specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles without post-PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end-point PCR products, therefore allowing greater flexibility in designing allele-specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan-ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases.

Binding of THZif-1, a MAZ-like zinc finger protein to the nuclease-hypersensitive element in the promoter region of the c-MYC protooncogene.

A detailed analysis is reported of the binding of the zinc finger protein THZif-1 to the nuclease-hypersensitive element (NHE) in the promoter region of the c-MYC gene using the electrophoretic mobility shift assay and a series of mutants of a fusion protein composed of glutathione S-transferase and THZif-1. The THZif-1 protein bound specifically to the single-stranded (ss) pyrimidine-rich DNA of the NHE (ss c-myc NHE-C) with an apparent dissociation constant (Kd (app)) of 0.077 microM. By contrast, no binding to the single-stranded purine-rich DNA of the NHE (ss c-myc NHE-G) was detected. Moreover, the binding affinity of THZif-1 protein was 2-fold higher for the single-stranded 5-methyl-2'-deoxycytidine derivative of NHE (ss c-myc NHE-me5C) than for the unmethylated NHE. In the case of the binding of THZif-1 to methylated double-stranded (ds) NHE (ds c-myc NHE-me5CG), no significant binding to the DNA was observed. The decrease in binding to DNA of THZif-1 was significant in the case of mutated ds c-myc NHE, in which more than two sites of deoxycytidine residues were methylated. However, the binding affinity of THZif-1 protein for methylated and for unmethylated triple-helical DNA of the NHE was almost identical. Moreover, the domain of the THZif-1 protein that made the major contribution to binding to ss c-myc NHE-C or ss c-myc NHE-me5C corresponded to the amino-terminal second zinc finger motif. Taken together, the results indicate that the THZif-1 protein exhibits preferential DNA-binding activity with ss c-myc NHE-C, ds c-myc NHE-CG, and ts c-myc NHE but not with ss c-myc NHE-G and ds c-myc NHE-me5CG in vitro.

Members of the MAZ family: a novel cDNA clone for MAZ from human pancreatic islet cells.

A human recombinant cDNA clone that encoded a zinc-finger protein (Myc-associated zinc-finger protein of human islet; MAZi) was cloned by screening a cDNA library prepared from human pancreatic islet cells. The encoded protein showed a high degree of homology to the Myc-associated zinc-finger protein MAZ (ZF87 or Pur-1). However, differences between the cDNAs for MAZi and MAZ were found in the length of the encoded polyalanine stretch and in the sequence of the 5'-end leader. MAZi transcripts were significantly more abundant in rat pancreatic islet carcinoma tissue than in normal rat islet cells. Moreover, MAZi protein bound specifically to the pyrimidine-rich strand of the CT-element of the c-myc gene in vitro and strongly induced the expression of chloramphenicol acetyltransferase (CAT) from a c-myc promoter/ CAT reporter construct in human pancreatic cells. Our results suggest that a distinct member of the MAZ family is expressed in human islet cells and enhances the transcriptional activity of the c-myc gene.

Cooperatively between an upstream TATA-like sequence and a CAA repeated element mediates E1A-dependent negative repression of the H-2Kb class I gene.

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2Kb basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2Kb class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2Kb gene is one of the elements that is required for Ad12-E1A-mediated negative repression.

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA.

We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3' termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I from Escherichia coli, which has 3'-->5' exonuclease and replacement activities as well as its main 5'-->3' polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.

Inhibitory effect of oligoribonucleotide phosphorodithioates against the 3'-exonuclease activity.

Oligoribonucleotides consisted of modified nucleotides at 3'-turmini were chemically synthesized on the solid phase method. Moreover, these analogues were investigated the stability in a prokaryotic cell-free translation system and the resistance against a few kind of nucleases containing 3'-exonuclease activity.

Solid phase synthesis of mRNA by the phosphoramidite approach using 2'-O-1-(2-chloroethoxy)ethyl protection and its stability in E. coli system.

The decaoligoribonucleotides containing initiation codon AUG of the phage O beta-A protein mRNA were synthesized on a solid phase by the phosphoramidite approach using 2'-O-1-(2-chloroethoxy)ethyl (Cee) protection. The Cee group is completely stable under the acidic conditions required to remove the 5'-terminal protecting groups in oligoribonucleotide synthesis on a solid support, and yet is easily removable at pH 2.0 for the final unblocking step. Stabilization of the synthetic mRNA to in the cell-free translation system from E. coli A19 was measured. It was found that the oligomers with selected 2'-O-methylation were degraded completely in the translation system.

Use of new phosphonylating and coupling agents in the synthesis of oligodeoxyribonucleotides via the H-phosphonate approach.

New phosphonylating and coupling agents for the synthesis of oligodeoxyribonucleotides via H-phosphonate approach have been developed. Tris(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite, prepared by the reaction of lithium salt of 1,1,1,3,3,3-hexafluoro-2-propoxide with PCl3, reacts with deoxyribonucleosides in the presence of a catalytic amount of triethylamine to produce in the high yield the corresponding deoxyribonucleoside 3'-H-phosphonate units. The use of a new coupling reagent, 1,3-dimethyl-2-chloro-imidazolinium chloride (DMCI) for the internucleotidic H-phosphonate bond formation via the H-phosphonate approach is also discussed in detail.

Solid phase synthesis of oligoribonucleotides using the 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp) group for the protection of the 2'-hydroxy functions and the H-phosphonate approach.

The solid phase synthesis of oligoribonucleotides using the H-phosphonate approach and the 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp) and dimethoxytrityl (DMTr) groups, respectively, for the protection of the 2'- and 5'-hydroxy functions is described. The use of a new reagent, tris-(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite for the preparation of nucleoside H-phosphonate units is also discussed in detail.

Synthesis and properties of the box 9R and 9R' sequences of Tetrahymena rRNA.

The chloroethoxyethyl (CEE) group is completely stable under the acidic conditions required to remove the 5'-protecting groups in the oligoribonucleotide synthesis, but can be cleaved under the similar condition to that of the tetrahydropyranyl (THP) group in region of pH 2-3. The oligoribonucleotides were synthesized by the phosphoramidite method on solid supports.