Biomedical subjects

P M Smooker

Publications and source records attributed to P M Smooker.

6 recordsLinked to original sources

PubMed1992

Atypical (mild) forms of dihydropteridine reductase deficiency: neurochemical evaluation and mutation detection.

We investigated two patients with an atypical (mild) form of dihydropteridine reductase (DHPR) deficiency. Both responded to the loading test with tetrahydrobiopterin; their plasma phenylalanine levels were lowered from 278 mumol/L to 85 and 48 mumol/L and from 460 mumol/L to 97 and 36 mumol/L after 4 and 8 h, respectively. In one of the patients, a combined loading test with phenylalanine followed by tetrahydrobiopterin was also carried out and showed a profile typical for DHPR deficiency. The phenylalanine hydroxylation rate was calculated to be 43 and 87%, 4 and 8 h after cofactor administration, respectively. Diagnosis was confirmed by the absence of DHPR activity in the patient's erythrocytes. In cultured fibroblasts, residual activity of 4 and 10%, respectively, was found. Excretion of urinary pterins was essentially normal, and the biopterin to neopterin ratio in cerebrospinal fluid was increased. Although in both patients cerebrospinal fluid homovanillic acid was found to be normal, and 5-hydroxyindoleacetic acid was substantially reduced, there was no sign of neurologic alterations until the age of 2 y. However, one of the patients recently developed deceleration of head growth, whereas psychomotor development continued to be normal for age. Using the chemical cleavage method on the amplified cDNA, mismatches of T to G at nucleotide 659 and of G to A at nucleotide 475, respectively, were identified. These results also demonstrate that screening for tetrahydrobiopterin deficiency by urinary pterin analysis alone can miss some newborns with mild DHPR deficiency and that all children with tetrahydrobiopterin defects need full neurochemical evaluation together with analysis of the enzyme activity.

Amino Acid Metabolism, Inborn Errors↗

PubMed1991

The nuclear:organelle distribution of chloroplast ribosomal proteins genes. Features of a cDNA clone encoding the cytoplasmic precursor of L11.

The majority of chloroplast ribosomal proteins are encoded in the nuclear genome. In order to characterize these proteins through their mRNA, we have previously constructed a spinach cDNA expression library and raised antisera to several spinach chloroplast ribosomal proteins. Here we describe the immuno isolation of cDNA clones encoding protein L11 and its chloroplast-targeting presequence. The cytoplasmic precursor form of L11 is 224 amino acid residues long (Mr 23,662); the mature L11 and the transit sequence are predicted to be of approximately 159 and approximately 65 residues, respectively. The predicted chloroplast L11 is significantly longer than the E coli L11, but similar (in size) to archaebacterial and yeast cytoplasmic L11. In sequence it is closer to E coli L11 (54% identity) than to the archaebacterial (32%) or yeast (23%) proteins. These results and the conservation of the contexts of the 3 methyl modified residues found in E coli L11 are discussed in the light of the endosymbiont theory and nuclear relocation of the rp/KAJL gene cluster.

Amino Acid Sequence↗

PubMed1990

Ribosomal protein L35: identification in spinach chloroplasts and isolation of a cDNA clone encoding its cytoplasmic precursor.

We describe the isolation of spinach chloroplast ribosomal protein L35 and characterization of a cDNA clone encoding its cytoplasmic precursor. This protein was only recently identified in ribosomes, but the sequences of four L35 genes have now been reported and confirm its presence in eubacteria, chloroplasts, and cyanelles. Using N-terminal sequence data, oligonucleotides were designed and a cDNA library was screened. The nucleotide sequence of the cDNA clones shows that the spinach L35 protein is encoded as a precursor of 159 residues, comprising a mature protein of 73 residues and a transit peptide of 86 residues. The cleavage site for forming the mature protein is deduced to be Thr-Val-Phe-Ala decreases Ala-Lys-Gly-Tyr. The L35 protein in the photosynthetic organelle of the protozoan Cyanophora paradoxa is encoded in the organelle DNA [Bryant & Stirewalt (1990) FEBS Lett. 259, 273-280]. The corresponding gene has not been found in the chloroplast DNA of a lower plant (liverwort) and two higher plants. Our results demonstrate that the L35 protein in a higher plant (spinach) is encoded in the nucleus. This finding, in light of the endosymbiont hypothesis, suggests an organelle to nucleus transfer of the L35 gene at the evolutionary beginnings of land plants.

Amino Acid Sequence↗

PubMed1990

A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms. Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence.

The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21. This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants. We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor. The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues). The extra length is in peptide extensions at both amino and carboxyl termini. The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far. The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts. The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence of a cDNA encoding the lipoate acetyl transferase (E2) of human heart pyruvate dehydrogenase complex differs from that of human placenta.

Tissue specific isoforms of an enzyme autoantigen were sought in an attempt to explain a possible disease-associated translocation of the enzyme. A human heart cDNA clone (0.66 kb) coding for part of the lipoate acetyl transferase component of pyruvate dehydrogenase complex, recently identified as one of the major autoantigens of primary biliary cirrhosis was isolated. The cloned cDNA corresponded to nucleotides 1545-2201 of a previously published placental sequence, but showed some differences which give rise to differences in the inferred amino acid sequences of proteins. This may indicate the existence of tissue-specific isoforms of the lipoate acetyl transferase component of pyruvate dehydrogenase complex coded for by a multi-gene family.

Acetyltransferases↗

PubMed1988

A mitochondrial intergenic mutation affecting processing of specific yeast mitochondrial transcripts.

The mutation in the temperature-conditional mit- mutant h56, mapped previously to the var1 gene region of Saccharomyces cerevisiae mitochondrial DNA, results in a specific inhibition of var1 protein synthesis in cells incubated at the non-permissive temperature, 36 degrees C (1). We have now characterized the mutation present in mutant h56 by DNA sequencing and found it to be an A to T transversion located 109 nucleotides upstream of the var1 reading frame. Two spontaneous revertants of mutant h56 restore the parental strain sequence at residue -109, confirming that this single base change within the 5'-untranslated region of the var1 mRNA is responsible for defective synthesis of the var1 protein. A comparison of var1 transcripts in the parental and mutant strains has shown that the mutation specifically blocks formation of var1 mRNA at 36 degrees C and leads to accumulation of precursor transcripts. Expression of the oli1 gene, co-transcribed with the var1 gene in primary transcripts, is not affected. It is concluded that the mutation in mutant h56 alters the secondary structure of the precursor RNA, inhibiting an endonucleolytic cleavage required to generate the 5' end of var1 mRNA.

Base Sequence↗