A novel aberrant splicing mutation of the PEX16 gene in two patients with Zellweger syndrome

Human Pex16p, a peroxisomal membrane protein composed of 336 amino acids, plays a central role in peroxisomal membrane biogenesis. A nonsense mutation (R176ter) in the PEX16 gene has been reported in the case of only one patient (D-01) belonging to complementation group D of the peroxisome biogenesis disorders. We have now identified two patients belonging to group D (D-02 and D-03) whose fibroblasts were found to contain no peroxisomal membrane structure ghosts. Molecular analysis of the PEX16 gene revealed aberrant cDNA species lacking 65 bp, corresponding to exon 10 skipping caused by a splice site mutation (IVS10 + 2T -->C). Both patients, although unrelated, were homozygous for this mutation. This mutation changes the amino acid sequence starting from codon 298 and introduces a termination codon at codon 336. As a consequence, the cell's ability to membrane synthesis and protein import is disrupted, which implies that the changed C terminus of the Pex16p in these patients likely affects its function.     

Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate kinase-associated neurodegeneration

The PANK2 gene encodes the human pantothenate kinase 2 protein isoforms, and PANK2 mutations are linked to pantothenate kinase-associated neurodegeneration. Two PanK2 protein forms are proteolytically processed to form a mitochondrially localized, mature PanK2. Another isoform arose from a proposed initiation at a leucine codon and was not processed further. The fifth isoform was postulated to arise from an alternative splicing event and was found to encode an inactive protein. Fourteen mutant PanK2 proteins with single amino acid substitutions, associated with either early or late onset disease, were evaluated for activity. The PanK2(G521R), the most frequent mutation in pantothenate kinase-associated neurodegeneration, was devoid of activity and did not fold properly. However, nine of the mutant proteins associated with disease possessed catalytic activities that were indistinguishable from wild type, including the frequently encountered PanK2(T528M) missense mutation. PanK2 was extremely sensitive to feedback inhibition by CoA thioesters (IC50 values between 250 and 500 nM), and the regulation of the active PanK2 mutants was comparable with that of the wild-type protein. Coexpression of the PanK2(G521R) and wild-type PanK2 did not interfere with wild-type enzyme activity, arguing against a dominant negative effect of the PanK2(G521R) mutation in heterozygous patients. These data described the unique biochemical features of the PanK2 isoforms and suggested that catalytic defects may not be the sole cause for the neurodegenerative phenotype.     

A missense mutation in the oxi-3 gene of the [mi-3] extranuclear mutant of Neurospora crassa

We have determined the DNA sequence of the oxi-3 gene and its 5' flanking region in the extranuclear [mi-3] mutant of Neurospora crassa. The oxi-3 gene encodes subunit 1 of cytochrome c oxidase, a protein known to be altered in the [mi-3] mutant (Bertrand, H., and Werner, S. (1979) Eur. J. Biochem. 98, 9-18). When the sequence from [mi-3] was compared to previously published sequences of the same region of mtDNA from wild-type N. crassa, a total of five differences was found. Four of these differences can be accounted for as either genetic polymorphisms or previous errors in DNA sequence determination. The remaining difference is a G/C to T/A transversion that changes a codon specifying an aspartic acid residue (GAC) to one that would specify tyrosine (TAC) at amino acid 448 of the 555 amino acid mature subunit 1 protein. This alteration was also found in the mtDNA of two separate heterokaryotic strains that had acquired the [mi-3] phenotype after repeated subculturing of heterokaryons forced between an [mi-3] strain and a strain containing a wild-type cytoplasm. The particular aspartic acid residue that would be affected by the mutation observed in [mi-3] is conserved in a diversity of species as either aspartic acid or glutamic acid, suggesting that an acidic residue at this position is important for the correct function of the subunit 1 protein. For these reasons, we consider it likely that the observed missense mutation is responsible for the [mi-3] phenotype.     

FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.     

Biochemical and genetical characterization of a fiber-defective temperature-sensitive mutant of type 2 adenovirus.

The adenovirus type 2 fiber mutant H2 ts 125 synthesized an unstable, temperature-sensitive fiber polypeptide with an apparent mol. wt. smaller by 2500 than the wild-type (62 K). The polypeptide of 59.5 K was found to be stable at the permissive temperature (33 degrees C). H2 ts 125 fiber synthesized in reticulocyte lysates had the same apparent mol. wt. of 59.5 K as the mutant fiber produced in vivo. Neither structural nor functional differences between wild-type and mutant fibers were detected in the N-terminal and C-terminal sequences, excluding the occurrence of a new initiation or termination codon. Restriction analysis of H2 ts 125 DNA also ruled out the hypothesis of a deletion mutant. The 59.5 K mutant fiber unit was normally glycosyated, N-acetylated, assembled into 6S oligomeric fiber and incorporated into virions. DNA sequencing of the H2 ts 125 fiber gene revealed two point mutations at nucleotides 3970 (C*TT leads to T*TT) and 4958 (GC*T leads to GT*T), corresponding to two amino acid changes at positions 105 and 434, respectively. The 105 mutation consisted of a conservative change Leu leads to Phe; the 434 interchange was Ala leads to Val, usually considered as nonconservative. The possibility of a donor site for splicing created by the mutation at codon GTT was eliminated on the basis of S1 nuclease analysis data. All these results suggested that either one or both mutations concerned highly organized domain(s) of the fiber polypeptide chain, resulting in aberrant mobility in SDS-polyacrylamide gels and temperature-sensitivity.     

A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division.     

A frameshift mutation that elongates the penicillinase protein of Bacillus licheniformis

A penicillinase mutant penP102, isolated after ICR (acridine mustard) mutagenesis of Bacillus licheniformis strain 749/C, retains about 50% of the wild-type penicillinase specific activity. The penicillinase produced by this mutant differs from the wild-type protein in its sensitivity to pH and its electrophoretic behaviour. The penP102 mutation appears to have several other phenotypic effects, including an increase in the efficiency of release of the extracellular form of the enzyme.The penP102 penicillinase has been purified and its amino acid sequence compared to that of the wild-type enzyme. The mutation has resulted in the replacement of the last three amino acids of the wild-type enzyme and the addition of 17 residues at the carboxy-terminus. Comparison of the wild-type and mutant amino acid sequences shows that the mutational event is a single nucleotide deletion from the codon for asparagine²⁶⁵. Consideration of the possible nucleotide sequence for the region beyond the carboxy-terminus of the wild-type protein shows that there are no possible termination codons until four and six triplets beyond the codon for the carboxy-terminal lysine, indicating that the carboxy-terminus of the wild-type extracellular penicillinase is generated by proteolytic cleavage of a larger precursor protein.     

A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G-->C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium.     

Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.     

DNA sequence analysis of the dnaK gene of Escherichia coli B and of two dnaK genes carrying the temperature-sensitive mutations dnaK7(Ts) and dnaK756(Ts).

The DNA sequence of the dnaK gene of Escherichia coli was analyzed. The nucleotide sequence of the wild-type dnaK gene of E. coli B differed from that of E. coli K-12 in 15 bp, none of which altered the amino acid sequence. Two temperature-sensitive dnaK mutations were examined by cloning and sequence analyses. Results showed that one dnaK mutation, dnaK7(Ts), was a one-base substitution of T for C at nucleotide position 448 in the open reading frame yielding an amber nonsense codon. The other mutation, dnaK756(Ts), consisted of base substitutions (A for G) at three nucleotide positions, 95, 1364, and 1403, in the open reading frame resulting in an aspartic acid codon in place of a glycine codon.     

A mutation in the gene encoding the vaccinia virus 37,000-M(r) protein confers resistance to an inhibitor of virus envelopment and release.

Plaque formation in vaccinia virus is inhibited by the compound N1-isonicotinoyl-N2-3-methyl-4-chlorobenzoylhydrazine (IMCBH). We have isolated a mutant virus that forms wild-type plaques in the presence of the drug. Comparison of wild-type and mutant virus showed that both viruses produced similar amounts of infectious intracellular naked virus in the presence of the drug. In contrast to the mutant, no extracellular enveloped virus was obtained from IMCBH-treated cells infected with wild-type virus. Marker rescue experiments were used to map the mutation conferring IMCBH resistance to the mutant virus. The map position coincided with that of the gene encoding the viral envelope antigen of M(r) 37,000. Sequence analysis of both wild-type and mutant genes showed a single nucleotide change (G to T) in the mutant gene. In the deduced amino acid sequence, the mutation changes the codon for an acidic Asp residue in the wild-type gene to one for a polar noncharged Tyr residue in the mutant.     

ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipoproteinemia. Haplotype analysis of the apoB alleles from this patient and his family members revealed him to be a genetic compound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by sequence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones contained a total of approximately 24 kb of apoB gene sequence, including regions 5' and 3' to the coding region, 29 exons, and the intron/exon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB allele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (TGA). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of macrophage colony stimulating factor (Csf1) causes osteopetrosis in the tl rat

Osteopetrosis results from a heterogeneous group of congenital bone diseases that display inadequate osteoclastic bone resorption. We recently mapped tl (toothless), a mutation that causes osteopetrosis in rats, to a genetic region predicted to include the rat Csf1 gene. In this study, we sequenced the coding sequence of the rat Csf1 gene to determine if a mutation in Csf1 could be responsible for the tl phenotype. Sequencing revealed a 10-base insertion in the coding sequence of mutant animals that produces a frameshift and generates a stop codon early in the mutant Csf1 coding sequence. The 41 amino acid polypeptide predicted to be produced from the Csf1 promoter would have only the first nine amino acids of the wild-type rat protein. These data suggest that osteopetrosis develops in tl/tl rats because they cannot produce functional mCsf, a growth factor required for osteoclast differentiation and activation.     

A missense mutation in the RING finger motif of PEX2 protein disturbs the import of peroxisome targeting signal 1 (PTS1)-containing protein but not the PTS2-containing protein

SK24 and PT54 mutant cells, which are peroxisome-deficient Chinese hamster ovary (CHO) cells isolated using peroxisomal forms of green fluorescent protein (GFP), were found to be defective in the PEX2 gene. The nucleotide sequences of PEX2 cDNA from the mutant cells were determined to identify mutation sites in the mutant cells. The mutation in SK24 cells changed cysteine to tyrosine at amino acid position 258, which is a component of the RING finger (C(3)HC(4)) motif in the carboxyl terminus of the protein. PT54 cells contained a nonsense mutation in the codon for glutamine at position 101, resulting in premature termination. The immunocytochemical analyses revealed distinct phenotypes between mutant cells defective in the PEX2 gene. Both mutant cells exhibited cytosolic mislocalizations on catalase and urate oxidase containing PTS1. On the other hand, on 3-ketoacyl-CoA thiolase containing PTS2, PT54 cells exhibited cytosolic mislocalization, but SK24 cells exhibited peroxisomal localization. When wild-type or mutant-type PEX2 cDNA was transfected into both mutant cells, the stable transformants restored the phenotype in accordance with the transfected cDNA. These observations indicate that an amino acid substitution, cysteine-258 to tyrosine, in the RING finger motif of PEX2 protein, whose function is required for peroxisomal localizations of both PTS1- and PTS2-containing proteins, results in a complete defect in the PTS1 pathway but not in the PTS2 pathway.     

Pleiotropic effects induced by modification deficiency next to the anticodon of tRNA from Salmonella typhimurium LT2.

A strain of Salmonella typhimurium LT2 was isolated, which harbors a mutation acting as an antisuppressor toward an amber suppressor derivative, supF30, of tRNATyr1. The mutant is deficient in cis-2-methylthioribosylzeatin[N6-(4-hydroxyisopentenyl)-2-me thylthioadenosine, ms2io6A], which is a modification normally present next to the anticodon (position 37) in tRNA reading codons starting with uridine. The gene miaA, defective in the mutant, is located close to and counterclockwise of the purA gene at 96 min on the chromosomal map of S. typhimurium with the gene order mutL miaA purA. Growth rate of the mutant was reduced 20 to 50%, and the effect was more pronounced in media supporting fast growth. Translational chain elongation rate at 37 degrees C was reduced from 16 amino acids per s in the wild-type cell to 11 amino acids per s in the miaA1 mutant in the four different growth media tested. The cellular yield in limiting glucose, glycerol, or succinate medium was reduced for the miaAI mutant compared with wild-type cells, with 49, 41, and 57% reductions, respectively. The miaAI mutation renders the cell more sensitive or resistant toward several amino acid analogs, suggesting that the deficiency in ms2io6A influences the regulation of several amino acid biosynthetic operons. We suggest that tRNAPhe, lacking ms2io6A, translates a UUU codon in the early histidine leader sequence with lowered efficiency, leading to repression of the his operon.     

Molecular cloning and characterisation of the ribC gene from Bacillus subtilis : a point mutation in ribC results in riboflavin overproduction

A mutation leading to roseoflavin resistance and deregulated riboflavin biosynthesis was mapped in the genome of the riboflavin-overproducing Bacillus subtilis strains RB52 and RB50 at map position 147°. The chromosomal location indicates that the deregulating mutation in RB52 and RB50 is an allele of the previously identified ribC mutation. We cloned the ribC gene and found that it encodes a putative 36-kDa protein. Surprisingly, RibC has significant sequence similarity to flavin kinases and FAD synthases from various other bacterial species. By comparing the deduced amino acid sequence of RibC from the wild-type parent strain of RB50 with the RibC sequence from the riboflavin-overexpressing RB50 mutant we identified a point mutation that resulted in a Gly to Ser exchange in the C-terminal region of the product Received: 3 June 1996 / Accepted: 19 October 1996     

Amino acid substitution of the largest subunit of yeast RNA polymerase II: effect of a temperature-sensitive mutation related to G1 cell cycle arrest

A mammalian temperature-sensitive mutant tsAF8 shows cell cycle arrest at nonpermissive temperatures in mid-G1 phase. DNA sequence comparison of the largest subunit of RNA polymerase II (Rpb1) from the wild-type and the mutant shows that the mutant phenotype results from a (hemizygous) C-to-A variation at nucleotide 944 in one rpb1 allele, giving rise to an Ala-to-Asp substitution at residue 315 in the protein. This amino acid substitution was introduced into the Schizosaccharomyces pombe rpb1 gene. Whereas tsAF8 cells showed growth defects and altered Rpb1 distribution at nonpermissive temperatures, yeast cells harboring this amino acid substitution did not show apparent temperature sensitivity. The effect of another temperature-sensitive Rpb1 mutation was also small. These results suggest that mutation of the rpb1 gene, which is critical in mammalian cells, may not be deleterious in yeast cells.     

Premature chromosome condensation is induced by a point mutation in the hamster RCC1 gene.

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.