Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus

Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in whole blood have been recovered. The mutant line Ldh1 ^a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1 ^a7Neu, Ldh1 ^a11Neu, and Ldh1 ^a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1 ^a2Neu carried an A/T → G/C transition in codon 112 (in exon 3), resulting in an Asn → Asp substitution; Asn112 is part of the helix αD, which is involved in the coenzyme-binding domain. Ldh1 ^a7Neu contained an A/T → C/G transversion within the codon for residue 194 in exon 4, causing an Asp → Ala substitution, which may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1 ^a11Neu in exon 7: the transition C/G → T/A, a silent mutation, and two transversions C/G → A/T and C/G → G/C, both missense mutations, which led to the amino acid replacements Ala319 → Glu and Thr321 → Ser, respectively, located in the αH helix structure of the COOH tail of LDHA. We suggest that the mutation is the result of a gene conversion event between Ldh1 ^a wild-type gene and the pseudogene Ldh1-ps. The alteration Ile → Thr of codon 241 in exon 6 caused by the base pair change T/A → C/G was identified in the mutation Ldh1 ^a12Neu; IIe241 is included in the helix α2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical and physiological alterations. Received: 3 July 1997 / Accepted: 30 September 1997     

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases

We used multiplex PCR followed by sequencing to screen for mutations in the 14 exons of theRPE65 gene in early-hildhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber’s congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the visual cycle and mutations identified in the gene could have implications for vitamin A-based therapeutic intervention. We were able to identify a homozygous mutation (AAT → AAG) in exon 9 in an arRP patient and a heterozygous missense transversion (AAT → AAG) also in exon 9 of an LCA patient. We also identified a polymorphism in exon 10 (GAG → GAA) in an arRP as well as an LCA patient. Mutation screening would be greatly facilitated by multiplex PCR which could cut down costs, labour and time involved. The nucleotide changes observed in this study could bede novo. Though a larger study has been undertaken, from the preliminary results it appears that in India theRPE65 gene seems to be less involved in causation of LCA.     

Lipopolysaccharide ofRhodospirillum salinarum 40: structural studies on the core and lipid A region

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) ofRhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4 bisphosphorylated β(1→6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlCN→GlcN, 2,3-diamino-2,3-dideoxy-d-Glc-(DAG)→DAG, and DAG→GlcN. Lipid A ofR. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked andcisΔ¹¹-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cisΔ¹¹-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine phosphorylethanolamine, nor by 4-amino-4-deoxy-l-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1→4)HexA(1→5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS ofR. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10⁻¹–10⁻² of that reported forSalmonella abortus equi LPS, and it was also capable of inducing TNFα and IL6 in macrophages of C57BL/10ScSN mice.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997     

Two new mutations in the sterol 27-hydroxylase gene in two families lead to cerebrotendinous xanthomatosis

This report concerns two new mutations in the sterol 27-hydroxylase gene in two patients with cerebrotendinous xanthomatosis (CTX). In a Surinam-Creole patient (patient A), a G deletion on position cDNA 546/547 in exon 3 led to a frameshift and the introduction of a premature termination codon. In a Dutch patient (patient B), a C→T transition at position 496 in exon 3 also led to a premature termination codon. Patient A was homozygous for the mutation, whereas patient B was compound heterozygous, a C→T transition also being found in exon 6 at position 1204. The two new mutations were confirmed by restriction analysis with the restriction enzymes FokI and MaeI, respectively. Received: 24 July 1996 / Revised: 9 August 1996     

A Gln-281 to Arg substitution in α-l-fucosidase is responsible for a common polymorphism detected by isoelectric focusing

A common polymorphism of human -l-fucosidase consists of three phenotypes (Fu 1, Fu 2, and Fu 2-1) assigned by isoelectric focusing. The phenotypes are determined by two codominant alleles (Fu¹ and Fu²). Isozymes with the Fu 2 phenotype have more basic pI than Fu 1, while Fu 2-1 is a mixture of Fu 2 and Fu 1. Recently, a missense mutation (A⁸⁶⁰G) in the -l-fucosidase gene was described that did not affect -l-fucosidase activity. The mutation causes the substitution of Arg (pKa^Guan=12.5) for Gln-281, which has no ionizable side chain. Isoelectric focusing profiles of extracts of COS-1 cells transfected with wild-type or mutant -l-fucosidase cDNAs had phenotypes of Fu 1 and Fu 2, respectively. Next, 20 human lymphoid cell lines were examined for the occurrence of the A⁸⁶⁰G mutation and expression of the Fu 1, Fu 2, and Fu 2-1 phenotypes. Eight lines with Fu 2 were homozygous for the A⁸⁶⁰G mutation; six lines with Fu 1 were homozygous for the normal nucleotide (A⁸⁶⁰); and six lines with Fu 2-1 were heterozygous. Thus, the A⁸⁶⁰G mutation is the molecular basis for the protein phenotypes and the Fu¹ and Fu² alleles. The normal nucleotide (A⁸⁶⁰) is responsible for Fu¹ and the A⁸⁶⁰G mutation for Fu².     

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.     

Comparative analysis of four <Emphasis Type="Italic">β</Emphasis>-galactosidases from <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> NCIMB41171: purification and biochemical characterisation

Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351 and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na⁺ and/or K⁺ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg⁺⁺ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence or absence of cations, with Mg⁺⁺, Mn⁺⁺ and Ca⁺⁺ ions exerting the most positive effect. Determination of the specificity constants (k _cat/K _m) clearly indicated that BbgI (6.11 × 10⁴ s⁻¹ M⁻¹), BbgIII (2.36 × 10⁴ s⁻¹ M⁻¹) and especially BbgIV (4.01 × 10⁵ s⁻¹ M⁻¹) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 10⁴ s⁻¹ M⁻¹) than lactose (1.48 × 10³ s⁻¹ M⁻¹). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different way to the bacterial physiology.     

Eight novel CARD15 variants detected by DNA sequence analysis of the CARD15 gene in 111 patients with inflammatory bowel disease

We performed a limited DNA sequence analysis of the CARD15 gene in 89 patients with Crohn’s disease (CD), 19 patients with ulcerative colitis (UC), and three patients with indeterminate colitis (IC), who were heterozygous carriers of one of the common CARD15 mutations [c.2104C>T (p.R702W), c.2722G>C (p.G908R), or c.3019_3020insC (p.Leu1007fsX1008)], the c.2462+10A>C variant, or of a new amino acid substitution in the 3′-end of exon 4. CARD15 exons 4, 5, 6, 8, and 11 were amplified by PCR and completely sequenced, thereby theoretically covering 73.9% of the described CARD15 variants and 96.6% of the mutated alleles. Using this approach, eight novel amino acid substitutions [c.1171C>T (p.R391C), c.1387C>G (p.P463A), c.2138G>A (p.R713H), c.2278C>T (p.R760C), c.2368C>T (p.R790W), c.2371C>T (p.R791W), c.2475C>G (p.N825K), and c.2546C>T (p.A849V)] were detected in six CD and two IC patients, and one UC patient. A severe disease phenotype was observed especially in patients who are compound-heterozygous for a common and a novel CARD15 mutation.Schnitzler and Brand contributed equally     

Relationship between indoor and outdoor airborne fungal spores, pollen, and (1→3)-β-D-glucan in homes without visible mold growth

In this exploratory study, indoor and outdoor airborne fungal spores, pollen, and (1→3)-β-D-glucan levels were determined through long-term sampling (24-h) using a Button Personal Inhalable Aerosol Sampler. The air samples were collected in five Cincinnati area homes that had no visible mold growth. The total count of fungal spores and pollen in the collected samples was conducted under the microscope and Limulus Amebocyte Lysate (LAL) chromogenic assay method was utilized for the determination of the (1→3)-β-D-glucan concentration. For the combined number concentration of fungal spores and pollen, the indoor and outdoor geometric mean values were 573 and 6,435 m⁻³, respectively, with a geometric mean of the Indoor/Outdoor (I/O) ratio of .09. The geometric means of indoor and outdoor (1→3)-β-D-glucan concentrations were .92 and 6.44 ng m⁻³, respectively, with a geometric mean of the I/O ratio equal to .14. The I/O ratio of (1→3)-β-D-glucan concentration was found to be marginally greater than that calculated based on the combined number concentration of fungal spores and pollen. This suggests that (1→3)-β-D-glucan data are affected not only by intact spores and pollen grains but also by the airborne fragments of fungi, pollen, and plant material, which are ignored by traditional enumeration methodologies. Since the (1→3)-β-D-glucan level may elucidate the total exposure to fungal spores, pollen, and fungal fragments, its I/O ratio may be used as a risk marker for mold and pollen exposure in indoor environments.     

Hb Costa Rica or · 2 ‚ 277(EF1)His→Arg: the first example of a somatic cell mutation in a globin gene

We have identified a minor hemoglobin component (∼5%) in the blood of a healthy Costa Rican female, but not in her mother and two brothers (father not studied), that has an His→Arg replacement at position β77 (Hb Costa Rica). No other amino acid replacements were observed and no β- or γ-chain-like peptides were present. Hb Costa Rica has a normal stability. Sequence analyses of numerous polymerase chain reaction (PCR)-amplified segments of DNA that contain exon 2 of the β gene failed to identify a CAC→CGC (His→Arg) mutation. The same was the case when cDNA was sequenced, indicating that a β-Costa Rica-mRNA could not be detected with this procedure. Gene mapping of genomic DNA with BglII, BamHI, and HindIII gave normal fragments only and with the same intensity as observed for the fragments of a normal control. The quantities of the β chain variants Hb J-Iran and Hb Fukuyama with related mutations at β77 vary between 30% and 45% in heterozygotes, whereas that of Hb F-Kennestone with the same His→Arg mutation but in the ^Gγ-globin gene, is a high 40%–45% (as percentage of total ^Gγ) in a heterozygous newborn. These different observations exclude a heterozygosity of the A→G mutation at codon β77, as well as a deletion comparable to that of Hbs Lepore or Kenya, or a β-globin gene duplication, and point to a nontraditional inheritance of Hb Costa Rica. Allele-specific amplification of cDNA with appropriate primers identified the presence of a low level of mutated mRNA in the reticulocytes of the patient, which was confirmed by dotblot analysis of the same material with ³²P-labeled probes. Comparable amplification products were not observed in genomic DNA. The A→G mutation apparently occurred in a somatic cell at a relatively early stage in the development of the hematopoietic cell system, and Hb Costa Rica accumulated through rapid cell divisions in patchy areas in the bone marrow (somatic mosaicism). An unequal distribution of Hb Costa Rica over the red cells supports this possibility. Received: 25 August 1995 / Revised: 13 December 1995     

Hypertrophic cardiomyopathy in young Maine Coon cats caused by the p.A31P cMyBP-C mutation - the clinical significance of having the mutation

Background  

In Maine Coon (MC) cats the c.91G > C mutation in the gene MYBPC3, coding for cardiac myosin binding protein C (cMyBP-C), is associated with feline hypertrophic cardiomyopathy (fHCM). The mutation causes a substitution of an alanine for a proline at residue 31 (p.A31P) of cMyBP-C. The pattern of inheritance has been considered autosomal dominant based on a single pedigree. However, larger studies are needed to establish the significance of cats being heterozygous or homozygous for the mutation with respect to echocardiographic indices and the probability of developing fHCM. The objective of the present study was to establish the clinical significance of being homozygous or heterozygous for the p.A31P cMyBP-C mutation in young to middle-aged cats.     

Purification,characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from <Emphasis Type="Italic">Curvularia lunata</Emphasis>

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein⁻¹ under the conditions, using diosgenin-3-O-α-L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin. This project was supported by the National Natural Science Foundation of China (NSFC; 30572333).     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

Genotype-phenotype correlation in cystic fibrosis patients compound heterozygous for the A455E mutation

Cystic fibrosis (CF) has a high incidence in the French-Canadian population of Saguenay Lac-Saint-Jean (Quebec). The A455E mutation accounts for 8.3% of the CF chromosomes. This mutation was shown to be associated with a milder lung disease in the Dutch population. Twenty two CF patients distributed in 17 families and compound heterozygotes for the A455E mutation have been followed at the Clinique de Fibrose Kystique de Chicoutimi. Fourteen patients also carried the ΔF508 mutation while the remaining eight patients had the 621+ 1G→T mutation. Each patient was matched by sex and age to a patient homozygous for the ΔF508 mutation. The pairs were analyzed for several clinical and laboratory variables. The A455E compound heterozygotes were diagnosed at a later age (P = 0.003) and had chloride concentrations at the sweat test lower than those homozygous for the ΔF508 mutation (P = 0.007). More patients were pancreatic sufficient (P = 0.004). They had a higher Shwachman score (P = 0.001) and better pulmonary function tests (P < 0.02). CF patients compound heterozygous for the A455E mutation have a milder pancreatic and lung disease than the ΔF508 homozygotes. Therefore, the A455E should be associated with a better prognosis. Received: 24 February 1997 / Accepted: 16 June 1997     

Mice hypomorphic for Atr have increased DNA damage and abnormal checkpoint response

The ATR checkpoint pathway responds to DNA damage during the S/G2 phases of the cell cycle and is activated early in tumorigenesis. Investigation of ATR’s role in development and tumorigenesis is complicated by the lethality of homozygous knockout mice and the limited effects of heterozygous deficiency. To overcome this limitation, we sought to create mice with a hypomorphic Atr mutation based on the ATR mutation in the human disease Seckel syndrome-1 (SCKL1). Homozygous SCKL1 mice were generated by targeted knock-in of the A → G SCKL1 mutation. Western blot and RT-PCR analysis established that homozygotes have no reduction in Atr protein or increase in missplicing as is seen in humans. Thus, the A → G substitution alone is not sufficient to reproduce in mice the effects that are seen in humans. However, homozygous SCKL1 mice that retain the neo cassette used for targeting have an estimated 66-82% reduction in total Atr protein levels due to missplicing into the neo cassette. Under conditions of APH-induced replication stress, primary fibroblasts from homozygous mice displayed an increase in overall chromosome damage and an increase in gaps and breaks at specific common fragile sites. In addition, mutant cells display a significant delay in checkpoint induction and an increase in DNA damage as assayed by Chk1 phosphorylation and γ-H2ax levels, respectively. These mice provide a novel model system for studies of Atr deficiency and replication stress. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical characterization of the minichromosome maintenance protein from the archaeon Thermoplasma acidophilum

Minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. Studies have shown that the MCM complex from the thermoacidophilic euryarchaeon Thermoplasma acidophilum (TaMCM) has some properties not reported in other archaeal MCM helicases. Here, the biochemical properties of the TaMCM are studied. The protein binds single-stranded DNA, has DNA-dependent ATPase activity and ATP-dependent 3′ → 5′ helicase activity. The optimal helicase conditions with regard to temperature, pH and salinity are similar to the intracellular conditions in T. acidophilum. It is also found that about 1,000 molecules of TaMCM are present per actively growing cell. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Specificity of recA441-mediated (tif-1) mutational events.

Summary To investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI ^– mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G : C C : G, G : C T : A and A : T T : A transversion events were specifically enhanced after SOS induction. A preferential 5-Y-Purine-3 neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5-C/GTGG-3 sequences is also observed. Fifty events were recovered at the lacI frameshift hotspot site and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA⁺ background and is a consequence of the fourfold induction of the (–)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI ^– mutations.     

Quorum sensing inhibitors from the red alga,<Emphasis Type="Italic">Ahnfeltiopsis flabelliformis</Emphasis>

Three new acylhomoserine lactone (AHL) inhibitors have been isolated from the Korean red alga.Ahnfeltiopsis flabelliformis, via bioactivity-guided fractionation using the recombinantAgrobacterium tumefaciens liquid culture bioassay. Unlike the majority of AHL inhibitors reported to date, these compounds were α-d-galactopyranosyl-(1→2)-glycerol (floridoside) (1), betonicine (2), and isethionic acid (3), all of which are structurally unrelated to AHLs.     

Propionic and methylmalonic acidemia: antisense therapeutics for intronic variations causing aberrantly spliced messenger RNA

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5′ or 3′ cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients’ fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of ¹⁴C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.