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T Fukao

Publications and source records attributed to T Fukao.

At least 109 records · Page 6Linked to original sources
PubMed1994

Molecular cloning and nucleotide sequence of complementary DNA for human hepatic cytosolic acetoacetyl-coenzyme A thiolase.

Complementary DNA for human cytosolic acetoacetyl-CoA thiolase (CT) was cloned with the use of anti-[human CT] antibody. The human CT cDNA clone (HCT10) has a 1479-bp insert and a 1191-base open reading frame encoding 397 amino acid residues. Partial polypeptide sequences from purified human CT were present in the deduced sequence. In vivo expression analysis showed that HCT10 encoded potassium-ion non-activated acetoacetyl-CoA thiolase with no 3-ketooctanoyl-CoA thiolase activity, which is characteristic for CT. The deduced amino acid sequence has a 34-57% homology with 4 other human thiolases and 4 acetoacetyl-CoA thiolases of microorganisms.

Acetyl-CoA C-Acetyltransferase↗
PubMed1994

Identification of a nonsense mutation in ALD protein cDNA from a patient with adrenoleukodystrophy.

The molecular basis of X-linked adrenoleukodystrophy (ALD) was investigated. Six (A to 50) fragments of cDNA for ALD protein (Mosser et al. Nature 361: 726-730, 1993) from an adult patient with adrenomyeloneuropathy were amplified by PCR and mutations were screened by Mutation Detection Enhancement gel electrophoresis. A single base substitution (2154 C-->T, which resulted in the formation of a termination codon for glutamine (Q590STOP) and deletes Pst I site (CTGCAG-->CTGTAG), was detected. Eight other ALD patients did not have this mutation. A family study revealed the presence of both the mutant and normal alleles in the mother, a sister and a niece, indicating that these individuals were carriers. A nephew with childhood ALD who died 10 years earlier had the same mutant allele as detected by Pst I restriction assay. This report is the first description of a mutant allele for ALD, at the cDNA level, and presents confirmatory evidence of ALD protein as the primary etiology of ALD.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1994

A physical map across chromosome 11q22-q23 containing the major locus for ataxia telangiectasia.

We have constructed a long-range physical map for 12 markers, including genes for GRIA4, IL1BC, and ACAT, across 9 Mb of chromosome 11q22-q23 in the region of the major locus for ataxia-telangiectasia (A-T). The markers fall into proximal and distal groups with respect to the centromere. We have linked the proximal and distal groups by hybridization to a 2.7-Mb NotI fragment and a 4.6-Mb MluI fragment. The following locus order was obtained: centromere-CJ52.75-J12.1C2-Y11B11R-IL1BC-+ ++hbcDNA-GRIA4-CJ52.3-Y11B29L-ACAT- CJ52.193-J12.8-Y11B06R-telomere. We show that hbcDNA/GRIA4 and CJ52.3 are very closely linked to each end, respectively, of the 2.7-Mb NotI fragment, thereby fixing the position of the complete contig. Our results indicate that the gene for A-T is flanked by the markers GRIA4 and J12.8, which are no more than 3 Mb apart, on a 4.6-Mb MluI fragment. The physical map allows rapid positioning of markers, and this will facilitate the construction of a YAC contig across the region.

Ataxia Telangiectasia↗
PubMed1994

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation.

Acetyl-CoA C-Acetyltransferase↗
PubMed1993

Molecular studies of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in the two original families.

We describe mutations identified in stored skin fibroblast cell lines from two original probands (JB and JM), first reported with 2-methylacetoacetic aciduria, and shown later to have a deficiency of the K(+)-activated enzyme, mitochondrial acetoacetyl-coenzyme A thiolase (T2). JB is homozygous for a 4-base insertion (GCAG) which is derived mutation. The primary mutation is an AG/gt to AG/gc transition at the 5'-splice-junction site in intron 11. An alternative splice site 4 bp downstream (Ggcag/gt) is used which causes a frame shift and replaces 39 C-terminal residues by 70 nonfunctional residues. JM is homozygous for a mutation in the translation-initiation codon (ATG to AAG). By expression analyses the JB mutation (IVS11nt2) causes an unstable T2 polypeptide and the JM mutation (M1K) severely impairs T2 mRNA translation. The JB allele associates with Dutch ancestry (no consanguinity) and the JM allele with Chilean ancestry (distant consanguinity).

Acetyl-CoA C-Acetyltransferase↗
PubMed1993

Biochemical and immunochemical study of seven families with 3-ketothiolase deficiency: diagnosis of heterozygotes using immunochemical determination of the ratio of mitochondrial acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase proteins.

The possibility of identifying heterozygotes of 3-ketothiolase deficiency, an inborn error of metabolism caused by a defect of mitochondrial acetoacetyl-CoA thiolase (T2), was tested in seven unrelated families by using enzymatic assay of thiolase activity and immunoblot analysis. The ratio of acetoacetyl-CoA thiolase activities, in the presence and absence of K+ ion (+K/-K ratio), in fibroblasts from 15 normal controls was around 2.0 (1.8 to 2.4), whereas the +K/-K ratio in eight patients was always 1.0. The ratio for the 13 obligate carriers ranged from 1.4 to 1.9, causing a minor overlap with control. Identification of heterozygote cells by immunoblot analysis, using anti-T2 antibody alone as a probe, was difficult, as previously reported. We therefore carried out immunoblot analysis, using as probes a mixture of anti-T2 antibody and the antibody against mitochondrial 3-ketoacyl-CoA thiolase (T1), another mitochondrial thiolase, and determined the ratio of the intensities of the T2 and T1 bands (T2/T1 ratio) using a densitometer. When the T2/T1 ratio was calculated, there was no overlap between the heterozygotes and normal controls. Hence, the heterozygotes can be unambiguously identified using this method. The thiolase activities and T2/T1 proteins in immunoblotting were detectable in peripheral lymphocytes, rectal mucosa, amniocytes, and liver. Thus, the postnatal diagnosis of 3-ketothiolase deficiency can be readily made using lymphocytes or rectal mucosa. The applicability of these methods in amniocytes indicates that prenatal diagnosis of this disease should be possible.

Acetyl-CoA C-Acetyltransferase↗
PubMed1992

Molecular basis of 3-ketothiolase deficiency: identification of an AG to AC substitution at the splice acceptor site of intron 10 causing exon 11 skipping.

3-Ketothiolase deficiency (3KTD) is the result of a deficiency in mitochondrial acetoacetyl-CoA thiolase (T2). The molecular basis of 3KTD was analyzed in a patient (GK10) and his family at the protein, cDNA and gene levels. Protein analyses showed that GK10's T2 protein was undetectable in fibroblasts even with the pulse-protein labeling method and that his parents were carriers of 3KTD. Complementary DNA analyses with PCR showed that T2 cDNA in the patient lacked the normal exon 11 sequence and that his parents were obligatory carriers of the DNA sequence which canceled exon 11. When the PCR-amplified genomic fragments around exon 11 were sequenced, an AG to AC mutation at the 3' splice site of intron 10 was detected. This mutation is presumed to be responsible for exon 11 skipping.

Acetyl-CoA C-Acyltransferase↗
PubMed1992

Identification of TaqI polymorphism in the mitochondrial acetoacetyl-CoA thiolase gene and familial analysis of 3-ketothiolase deficiency.

We analyzed the mitochondrial acetoacetyl-CoA thiolase gene (T2) by Southern blotting. Fifteen unrelated healthy individuals and members of five families with 3-ketothiolase deficiency (3KTD) were analyzed. We found a TaqI polymorphism, the heterozygosity of which was calculated to be 0.5 among healthy Japanese individuals. This restriction fragment length polymorphism (RFLP) proved to be useful for detecting 3KTD patients and its obligatory carriers, at the DNA level and in two out of five 3KTD families. This polymorphism was found to be generated by the presence/absence of a TaqI site in intron 9 of the T2 gene. With in vitro amplification of the genomic region around the TaqI site, this RFLP can be detected within 2 days.

Acetyl-CoA C-Acetyltransferase↗
PubMed1992

Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency.

3-Ketothiolase deficiency (3KTD) stems from a deficiency of mitochondrial acetoacetyl-coenzyme A thiolase (T2). We analyzed the molecular basis of 3KTD in two generations of a family. A boy (patient 2, GK04), his father (patient 1, GK05), his mother, and his brother were studied; three mutant alleles of T2 gene were identified. Patient 1 is a compound heterozygote: one allele has a point mutation of G to A at position 547 on his T2 cDNA, causing Gly150 to Arg substitution of the mature T2 subunit, and the other allele has GT to TT transition at the 5' splice site of intron 8, causing exon 8's skipping of the T2 cDNA. Patient 2 is also a compound heterozygote: one allele inherited from his mother has AG to CG transition at the 3' splice site of intron 10, causing exon 11's skipping of the T2 cDNA, and the other allele derived from patient 1 has the G to A mutation (Gly to Arg). The brother of patient 2 is an obligatory carrier with the mutant allele causing the exon 8 skipping. This report seems to be the first complete molecular definition of 3KTD at the gene level.

Acetyl-CoA C-Acetyltransferase↗
PubMed1992

Further analysis of mutant thiolase protein in fibroblasts from a Japanese boy with 3-ketothiolase deficiency.

We examined the mutant protein of mitochondrial acetoacetyl-CoA thiolase (mutant T2) in fibroblasts from a Japanese boy with 3-ketothiolase deficiency. The molecular size of the mutant T2 protein, determined by pulse labeling and SDS/PAGE, was intermediate between the mature subunit and the precursor of T2. To characterize the mutant T2 protein, pulse-labeling and rhodamine 6G inhibition of mitochondrial transport in fibroblasts, cell-free translation experiments, and family studies by thiolase assay, immunoblotting, and pulse-labeling were carried out. The mutant T2 was detectable as early as a 10-min pulse. The probable precursor of the mutant T2 was not detectable in either the rhodamine 6G inhibition or cell-free translation experiments. In the parents, the K+ ion dependency of acetoacetyl-CoA thiolase activity was low and the T2 bands in immunoblots were faint. It would thus appear that the parents are heterozygotes of this disease. In pulse-labeling, only a band for the mutant T2 was detected in the patient and a single band for the normal mature subunit of T2 in the father; both bands were detected in the mother. These findings suggested that the mutant T2 in the patient was inherited from the mother, and that the expression of another mutant allele of the father may be either abolished or scanty.

Acetyl-CoA C-Acetyltransferase↗
PubMed1992

Biochemical investigation of a Brazilian patient with a defect in mitochondrial acetoacetylcoenzyme-A thiolase.

A case report of 3-ketothiolase deficiency due to a defect of mitochondrial acetoacetyl-CoA thiolase protein in a Brazilian boy and its biochemical investigation is presented. The child had moderate generalized hypotonia, EEG alterations and crises of metabolic acidosis following infections. Hypotonia and EEG abnormalities disappeared with a low protein diet, and physical and mental development are normal. Urinary organic acid excretion was typical of 3-ketothiolase deficiency, showing consistently high levels of 2-methyl-3-hydroxybutyric acid and tiglylglycine. Activation of acetoacetyl-CoA thiolase activity by potassium (K) ion in cultured fibroblasts was not observed, demonstrating the lack of activity of mitochondrial acetoacetyl-CoA thiolase. In addition, the signal for the mitochondrial acetoacetyl-CoA thiolase protein was undetectable in the immunoblot analysis. In the pulse-chase experiments, the signal for mitochondrial acetoacetyl-CoA thiolase was detected after a 1-h pulse but not after a 24-h chase. These results indicate that the deficiency was caused by an unstable mitochondrial acetoacetyl-CoA thiolase protein.

Acetyl-CoA C-Acetyltransferase↗
PubMed1991

Structure and expression of the human mitochondrial acetoacetyl-CoA thiolase-encoding gene.

To examine 3-ketothiolase deficiency at the gene level, we analyzed the structure of the human mitochondrial acetoacetyl-CoA thiolase (MAT; EC 2.3.1.9)-encoding gene (MAT). From the genomic library of a normal subject in lambda EMBL3, we isolated seven overlapping clones covering the entire length of MAT and the structural organization was determined. The gene spans approx. 27 kb and contains twelve exons interrupted by eleven introns. The 5'-flanking region of the gene lacks a conventional TATA box, but is G + C-rich and contains two CAAT boxes. Included are a putative binding site for the transcription factor, Sp1, and sequences resembling the binding sites of several other transcription factors, all features characteristic of housekeeping genes. A CAT assay revealed that a 101-bp DNA fragment immediately upstream from the cap site has promoter activity, and suggested that a DNA fragment from bp -888 to -102 probably contains a negative regulatory element(s).

Acetyl-CoA C-Acetyltransferase↗
PubMed1991

Morquio disease: isolation, characterization and expression of full-length cDNA for human N-acetylgalactosamine-6-sulfate sulfatase.

We cloned and sequenced a full-length cDNA of human placental N-acetylgalactosamine-6-sulfate sulfatase, the enzyme deficient in Morquio disease. The 2339-nucleotide sequence contained 1566 nucleotides which encoded a polypeptide of 522 amino acid residues. The deduced amino acid sequence was composed of a 26-amino acid N-terminal signal peptide and a mature polypeptide of 496 amino acid residues including two potential asparagine-linked glycosylation sites. Expression of the cDNA in transfected deficient fibroblasts resulted in higher production of this sulfatase activity than in untransfected deficient fibroblasts. The cDNA clone was hybridized to only a 2.3-kilobase species of RNA in human fibroblasts. The amino acid sequence of N-acetylgalactosamine-6-sulfate sulfatase showed a high degree of homology with those of other sulfatases such as human arylsulfatases A, B or C, glucosamine-6-sulfatase, iduronate-2-sulfatase and sea urchin arylsulfatase.

Amino Acid Sequence↗