Search PubMed⌕ Search

Biomedical subjects

F D Ledley

Publications and source records attributed to F D Ledley.

At least 109 records · Page 6Linked to original sources
PubMed1988

Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes.

Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. We report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human alpha 1-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating form the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the alpha 1-antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

Animals↗
PubMed1988

Biochemical defect of the hph-1 mouse mutant is a deficiency in GTP-cyclohydrolase activity.

A hyperphenylalaninemic mouse mutant, hph-1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid-dihydropteridine reductase activity is also normal. GTP-cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP-cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4-dihydroxyphenylalanine (DOPA) and serotonin.

Aminohydrolases↗
PubMed1988

Localization of mouse phenylalanine hydroxylase locus on chromosome 10.

Mouse phenylalanine hydroxylase has been localized on chromosome 10C2----D1 by in situ hybridization using a mouse phenylalanine hydroxylase cDNA clone. This locus is distinct from the hyperphenylalaninemia locus on chromosome 14 and the locus for tyrosine hydroxylase on chromosome 7.

Animals↗
PubMed1988

Mapping of human methylmalonyl CoA mutase (MUT) locus on chromosome 6.

Methylmalonyl CoA mutase (MCM) catalyzes an essential step in the degradation of several branch-chain amino acids and odd-chain fatty acids. Deficiency of this apoenzyme causes the mut form of methylmalonic acidemia, an often fatal disorder of organic acid metabolism. An MCM cDNA has recently been obtained from human liver cDNA libraries. This clone has been used as a probe to determine the chromosomal location of the MCM gene and MUT locus. Southern blot analysis of DNA from human-hamster somatic-cell hybrid cell lines assigned the locus to region q12-p23 of chromosome 6. In situ hybridization further localized the locus to the region 6p12-21.2. A highly informative RFLP was identified at the MCM gene locus which will be useful for genetic diagnostic and linkage studies.

Animals↗
PubMed1987

Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes.

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.

Animals↗
PubMed1987

GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria.

Classical Phenylketonuria (PKU) is an autosomal recessive human genetic disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). We isolated several mutant PAH cDNA clones from a PKU carrier individual and showed that they contained an internal 116 base pair deletion, corresponding precisely to exon 12 of the human chromosomal PAH gene. The deletion causes the synthesis of a truncated protein lacking the C-terminal 52 amino acids. Gene transfer and expression studies using the mutant PAH cDNA indicated that the deletion abolishes PAH activity in the cell as a result of protein instability. To determine the molecular basis of the deletion, the mutant chromosomal PAH gene was isolated from this individual and shown to contain a GT-- greater than AT substitution at the 5' splice donor site of intron 12. Thus, the consequence of the splice donor site mutation in the human liver is the skipping of the preceding exon during RNA splicing.

Amino Acid Sequence↗
PubMed1987

Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli.

A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine. Recombinant human phenylalanine hydroxylase produced in E. coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver. The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase. These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell. The availability of recombinant human phenylalanine hydroxylase produced in E. coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.

Amino Acids↗
PubMed1987

P-chlorophenylalanine does not inhibit production of recombinant human phenylalanine hydroxylase in NIH3T3 cells or E. coli.

P-chlorophenylalanine is an irreversible inhibitor of rat phenylalanine hydroxylase in vivo and in rat hepatoma cells and is frequently administered to rodents to create an animal model for phenylketonuria. We investigated the effect of p-chlorophenylalanine on production of human phenylalanine hydroxylase in human hepatoma cells and cells transformed with the recombinant human phenylalanine hydroxylase gene. P-chlorophenylalanine inhibited production of the human enzyme in human hepatoma cells and transformed mouse hepatoma cells but had no effect on the production of the enzyme in transformed NIH3T3 cells or in E. coli. Thus, phenylalanine hydroxylase inhibition does not result from a simple interaction between the drug and enzyme.

Animals↗
PubMed1987

Physical and genetic localization of quinonoid dihydropteridine reductase gene (QDPR) on short arm of chromosome 4.

A portion of a cDNA clone corresponding to the 3' end of the human quinonoid dihydropteridine reductase (QDPR) mRNA was used as a probe to physically map the QDPR gene by analysis of somatic cell hybrid lines. The provisional assignment of QDPR to chromosome 4, based on expression of the human enzyme in hybrids, was confirmed. The gene was further regionally localized on the short arm to 4p16.1----4p15.1. This physical localization places QDPR in the same area of the genome that contains the defect causing Huntington's disease (HD). The QDPR probe revealed a restriction fragment length polymorphism with the enzyme BanII, permitting determination of its genetic proximity to D4S10, an anonymous DNA marker tightly linked to HD. QDPR is only loosely linked to D4S10, excluding any primary role for the gene in HD.

Animals↗
PubMed1987

Assignment of human tryptophan hydroxylase locus to chromosome 11: gene duplication and translocation in evolution of aromatic amino acid hydroxylases.

A cDNA clone for rabbit tryptophan hydroxylase was used as a probe to identify human tryptophan hydroxylase gene fragments in a panel of hamster-human somatic cell hybrids and determine its chromosomal location in man. A single locus was identified for tryptophan hydroxylase on chromosome 11. Tryptophan hydroxylase is a member of the superfamily of pterin-dependent aromatic amino acid hydroxylases which includes tyrosine hydroxylase, located at 11p15.5-p15, and phenylalanine hydroxylase, located at 12q22-q24.1 in human. The locations of these genes and the evolutionary distance between their sequences suggest that at least three distinct genetic events have occurred during the evolution of the aromatic amino acid hydroxylase superfamily: two sequential gene duplications giving rise to the three distinct hydroxylase loci, and a translocation which separated the tryptophan and tyrosine hydroxylase loci on chromosome 11 from the phenylalanine hydroxylase locus on chromosome 12.

Animals↗
PubMed1987

Selection for phenylalanine hydroxylase activity in cells transformed with recombinant retroviruses.

Cells deficient in phenylalanine hydroxylase (PAH) are tyrosine auxotrophs and will not survive in tyrosine-free media. PAH activity can be constituted in cultured cells by infection with recombinant retroviruses carrying a human PAH cDNA. Mouse hepatoma cells transformed with recombinant PAH will grow in tyrosine-free media since these cells constitutively synthesize the cofactor tetrahydrobiopterin which is essential for PAH activity. NIH3T3 cells transformed with the PAH cDNA express the PAH apoenzyme, but this enzyme is inactive in vivo since these cells do not synthesize biopterin. We describe a method of selection for PAH in the fibroblast-like NIH3T3 cells involving tyrosine-free media supplemented with biopterin, reducing agents, and antioxidants. Cells transformed with the recombinant PAH gene exhibit PAH activity in culture and will grow in the biopterin-supplemented tyrosine-free media. Metabolic selection for PAH activity provides a new selectable marker for gene transfer experiments. This method is shown to be useful in the production of high titers of recombinant retroviruses carrying PAH and provides a model for experiments in somatic gene therapy of phenylketonuria.

Animals↗
PubMed1987

Retroviral mediated transfer and expression of human alpha 1-antitrypsin in cultured cells.

Genetic deficiency of alpha 1-antitrypsin in man is a predisposing factor to emphysema and a disorder potentially correctable by somatic gene therapy. A full-length human alpha 1-antitrypsin cDNA was cloned into a retroviral vector and introduced into cells which package the recombinant gene in a retroviral capsule. Cells infected with the recombinant retrovirus express human alpha 1-antitrypsin mRNA and protein. The recombinant protein is glycosylated, secreted and exhibits anti-protease activity against human neutrophil elastase.

Cloning, Molecular↗
PubMed1987

Correlation between polymorphic DNA haplotypes at phenylalanine hydroxylase locus and clinical phenotypes of phenylketonuria.

Eight polymorphic sites for seven restriction endonucleases have been reported at the human phenylalanine hydroxylase locus. The composite profile of the presence or absence for each of the eight polymorphic sites within an allele defines the haplotype of the corresponding allele. Twelve such haplotypes associated with normal and mutant phenylalanine hydroxylase alleles have been identified in 33 Danish families with children with phenylketonuria. Of the 66 mutant alleles analyzed, 59 (89%) were associated with only four haplotypes. The identification of individual phenylalanine hydroxylase alleles by haplotype analysis enables correlation of the hyperphenylalaninemic phenotypes of the patients with their genotypes. Patients who were either homozygous or heterozygous for the mutant alleles of haplotypes 2 and 3 had a severe clinical course. Patients who had a mutant allele of either haplotype 1 or 4 usually had a less severe clinical phenotype. The recent demonstration that the mutation responsible for classic phenylketonuria associated with haplotype 3 is not present in mutant alleles of other haplotypes provides unambiguous evidence that there are multiple mutations in the phenylalanine hydroxylase gene and supports the hypothesis that different combinations of mutant alleles may be responsible for the clinical diversity of phenylketonuria.

Child↗
PubMed1987

Structure and expression of human dihydropteridine reductase.

Dihydropteridine reductase (DHPR; EC 1.6.99.7) catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin and is an essential component of the pterin-dependent aromatic amino acid hydroxylating systems. A cDNA for human DHPR was isolated from a human liver cDNA library in the vector lambda gt11 using a monospecific antibody against sheep DHPR. The nucleic acid sequence and amino acid sequence of human DHPR were determined from a full-length clone. A 112 amino acid sequence of sheep DHPR was obtained by sequencing purified sheep DHPR. This sequence is highly homologous to the predicted amino acid sequence of the human protein. Gene transfer of the recombinant human DHPR into COS cells leads to expression of DHPR enzymatic activity. These results indicate that the cDNA clone identified by antibody screening is an authentic and full-length cDNA for human DHPR.

Amino Acid Sequence↗
PubMed1987

Retroviral gene transfer into primary hepatocytes: implications for genetic therapy of liver-specific functions.

The liver is an important target for potential gene therapy because of the critical role it plays in intermediary metabolism and synthesis of serum proteins. We report the use of retroviral vectors for transfer of recombinant genes into primary mouse hepatocytes. Hepatocytes were grown in a defined serum-free medium and expressed liver-specific functions for up to 14 days. Hepatocytes were transformed to Genticin (G418) resistance by infection with recombinant retroviruses carrying the Tn5 neomycin-resistance gene. The G418-resistant cells exhibited characteristic hepatocyte morphology and continued to express liver-specific gene function. A retrovirus that expresses neomycin resistance driven by a herpes simplex thymidine kinase promoter produced the most efficient transformation compared with viruses using the retroviral long terminal repeat promoter or the simian virus 40 early-region promoter. These experiments indicate that primary hepatocytes can be successfully cultured and transformed with recombinant genes using retroviral vectors. These results provide a model for future somatic gene replacement therapy in which functional genes can be introduced into hepatocytes by viral-mediated gene transfer.

Animals↗