Replacement of C305 in Heart/Muscle-Type Isozyme of Human Carnitine Palmitoyltransferase I with Aspartic Acid and Other Amino Acids

Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.     

Sequence variation in two lignin biosynthesis genes,cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2)

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2) are genes which may influence variation in lignin content and composition within plants. Sequence variation within these genes may be responsible for changes in enzyme activity and/or specificity, which could cause variation in lignin content or composition. This study examines sequence variation within these two genes in Eucalyptus globulus, an important species used in pulp and paper-making. Twenty-one single nucleotide polymorphisms (SNPs) were identified in the exons of CCR, of which nine were neutral mutations and 12 were missense mutations. Six of the missense mutations affected highly conserved amino acids within the protein sequence of CCR. Eight SNPs were identified in the CAD2 exons, six of which were neutral mutations and two which were missense mutations. One of the missense mutations affected a highly conserved amino acid within the protein sequence. In addition, 32 SNPs were identified in the CCR introns along with four insertion/deletions and two polyA length variation regions. Polymorphism affecting highly conserved amino acids may alter enzyme function and this molecular variation may be linked to variation in lignin profiles. Selecting positive alleles which produce favourable lignin profiles would be advantageous in tree breeding programs.     

Polymorphisms in chicken extracellular fatty acid binding protein gene

In this study, we report the investigation of extracellular fatty acid binding protein gene (Ex-FABP) genetic polymorphism in a sample of 360 chicken individuals. The screening of the coding regions with their intron–exon boundaries and the proximal flanking regions was performed through a PCR-SSCP strategy. Following sequence analysis revealed 35 novel single nucleotide polymorphisms (SNPs) of chicken Ex-FABP gene. Among the 35 SNPs, twenty-five were found in the introns. And the remaining seven and three SNPs were in the coding region and the 5′UTR, respectively. Two SNPs in the coding region caused two missense mutants and the other five did not result in any amino acid changes. The nature and the distribution of Ex-FABP mutations in three chicken breeds were analyzed. Variations detected here might have an impact on Ex-FABP activity and function and underpin the development of gene markers for chicken fatty deposition and metabolism. The polymorphism, generated by C4715T mutation in exon5, was significantly associated with thickness of subcutaneous fat plus skin in cocks. Subcutaneous fat plus skin of cocks was more thick in TT genotype than in CC genotype (P < 0.05). The Ex-FABP gene could be a candidate locus or linked to a QTL that significantly affects fatty deposition and metabolism in chicken.     

Identification of a fructose-1,6-bisphosphate aldolase gene and association of the single nucleotide polymorphisms with growth traits in the clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis>

This study investigated whether there were single nucleotide polymorphisms (SNPs) in fructose-1,6-bisphosphate aldolase (FBA) gene associated with growth traits of the clam Meretrix meretrix. A FBA gene was identified in M. meretrix and its deduced amino acid residues shared high identity with type I aldolase. The FBA (MmeFBA) mRNA expression profile was examined by real-time PCR in different tissues and the significantly high expression level in foot and adduct muscle suggests that MmeFBA is a muscle type aldolase which functions in glycolytic pathway. In the MmeFBA gene, we identified four intron SNPs and three exon SNPs including a nonsynonymous SNP (mmfbae-2). These SNPs were genotyped in 205 clams from two clam populations with significantly different growth performance. Results showed that allele frequencies of three SNPs (mmfbai-1, mmfbai-3 and mmfbae-2) and the genotype frequency of mmfbai-1 were all significantly different between the two populations. The haplotype analysis further supported the three SNPs distributed differently between the two populations. This study successively characterized three growth-related SNPs in a gene involved in energy metabolism of M. meretrix. These findings could contribute the development of phenotype-selective breeding program in M. meretrix.     

2-Deoxyribose 5-phosphate aldolase: Genetic analyses of structure

A total of 33 mutant strains of Salmonella typhimurium deficient in deoxyribose 5-phosphate activity have been isolated and characterized as missense or nonsense. Three-factor transductional analyses of the mutants were used to construct a fine structure map of the deoC gene, which codes for a peptide of 28,500 molecular weight. An unusual clustering of the missense mutants was observed, where 75% of all the missense mutants mapped in an area which corresponds to 19% of the total gene length. It is suggested that this area of the protein is particularly sensitive to amino acid replacements but that other areas of the protein are reasonably tolerant of such changes. Nonsense mutations are found scattered throughout the gene. This is expected since the carboxyl-terminal tyrosine is essential for enzymatic activity.     

Pig liver carnitine palmitoyltransferase I, with low Km for carnitine and high sensitivity to malonyl-CoA inhibition, is a natural chimera of rat liver and muscle enzymes

The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.     

Substitution of glutamate-3, valine-19, leucine-23, and serine-24 with alanine in the N-terminal region of human heart muscle carnitine palmitoyltransferase I abolishes malonyl CoA inhibition and binding

The muscle isoform of carnitine palmitoyltransferase I (M-CPTI) is 30- to 100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). We have previously shown that deletion of the first 28 N-terminal amino acid residues in M-CPTI abolished malonyl CoA inhibition and high-affinity binding [Biochemistry 39 (2000) 712-717]. To determine the role of specific residues within the first 28 N-terminal amino acids of human heart M-CPTI on malonyl CoA sensitivity and binding, we constructed a series of substitution mutations and a mutant M-CPTI composed of deletion 18 combined with substitution mutations V19A, L23A, and S24A. All mutants had CPT activity similar to that of the wild type. A change of Glu3 to Ala resulted in a 60-fold decrease in malonyl CoA sensitivity and loss of high-affinity malonyl CoA binding. A change of His5 to Ala in M-CPTI resulted in only a 2-fold decrease in malonyl CoA sensitivity and a significant loss in the low- but not high-affinity malonyl CoA binding. Deletion of the first 18 N-terminal residues combined with substitution mutations V19A, L23A, and S24A resulted in a mutant M-CPTI with an over 140-fold decrease in malonyl CoA sensitivity and a significant loss in both high- and low-affinity malonyl CoA binding. This was further confirmed by a combined four-residue substitution of Glu3, Val19, Leu23, and Ser24 with alanine. Our site-directed mutagenesis studies demonstrate that Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl CoA inhibition and binding, but not for catalysis.     

Genetic Variation in FABP4 and Evaluation of Its Effects on Beef Cattle Fat Content

FABP4 is a protein primarily expressed in adipocytes and macrophages that plays a key role in fatty acid trafficking and lipid hydrolysis. FABP4 gene polymorphisms have been associated with meat quality traits in cattle, mostly in Asian breeds under feedlot conditions. The objectives of this work were to characterize FABP4 genetic variation in several worldwide cattle breeds and evaluate possible genotype effects on fat content in a pasture-fed crossbred (Angus-Hereford-Limousin) population. We re-sequenced 43 unrelated animals from nine cattle breeds (Angus, Brahman, Creole, Hereford, Holstein, Limousin, Nelore, Shorthorn, and Wagyu) and obtained 22 single nucleotide polymorphisms (SNPs) over 3,164?bp, including four novel polymorphisms. Haplotypes and linkage disequilibrium analyses showed a high variability. Five SNPs were selected to perform validation and association studies in our crossbred population. Four SNPs showed well-balanced allele frequencies (minor frequency?>?0.159), and three showed no significant deviations from Hardy-Weinberg proportions. SNPs showed significant effects on backfat thickness and fatty acid composition (P?相似文献   

Fatty acid synthase effects on bovine adipose fat and milk fat

A quantitative trait locus (QTL) was identified by linkage analysis on bovine Chromosome 19 that affects the fatty acid, myristic acid (C14:0), in subcutaneous adipose tissue of pasture-fed beef cattle (99% level: experiment-wise significance). The QTL was also shown to have significant effects on ten fatty acids in the milk fat of pasture-fed dairy cattle. A positional candidate gene for this QTL was identified as fatty acid synthase (FASN), which is a multifunctional enzyme with a central role in the metabolism of lipids. Five single nucleotide polymorphisms (SNPs) were identified in the bovine FASN gene, and animals were genotyped for FASN SNPs in three different cattle resource populations. Linkage and association mapping results using these SNPs were consistent with FASN being the gene underlying the QTL. SNP substitution effects for C14:0 percentage were found to have an effect in the opposite direction in adipose fat to that in milk fat. It is concluded that SNPs in the bovine FASN gene are associated with variation in the fatty acid composition of adipose fat and milk fat.     

Genetic and biochemical impairment of mitochondrial complex I activity in a family with Leber hereditary optic neuropathy and hereditary spastic dystonia.

A rare form of Leber hereditary optic neuropathy (LHON) that is associated with hereditary spastic dystonia has been studied in a large Dutch family. Neuropathy and ophthalmological lesions were present together in some family members, whereas only one type of abnormality was found in others. mtDNA mutations previously reported in LHON were not present. Sequence analysis of the protein-coding mitochondrial genes revealed two previously unreported mtDNA mutations. A heteroplasmic A-->G transition at nucleotide position 11696 in the ND4 gene resulted in the substitution of an isoleucine for valine at amino acid position 312. A second mutation, a homoplasmic T-->A transition at nucleotide position 14596 in the ND6 gene, resulted in the substitution of a methionine for the isoleucine at amino acid residue 26. Biochemical analysis of a muscle biopsy revealed a severe complex I deficiency, providing a link between these unique mtDNA mutations and this rare, complex phenotype including Leber optic neuropathy.     

Phylogenetic network and physicochemical properties of nonsynonymous mutations in the protein-coding genes of human mitochondrial DNA

Theories on molecular evolution predict that phylogenetically recent nonsynonymous mutations should contain more non-neutral amino acid replacements than ancient mutations. We analyzed 840 complete coding-region human mitochondrial DNA (mtDNA) sequences for nonsynonymous mutations and evaluated the mutations in terms of the physicochemical properties of the amino acids involved. We identified 465 distinct missense and 6 nonsense mutations. 48% of the amino acid replacements changed polarity, 26% size, 8% charge, 32% aliphaticity, 13% aromaticity, and 44% hydropathy. The reduced-median networks of the amino acid changes revealed relatively few differences between the major continent-specific haplogroups, but a high variation and highly starlike phylogenies within the haplogroups. Some 56% of the mutations were private, and 25% were homoplasic. Nonconservative changes were more common than expected among the private mutations but less common among the homoplasic mutations. The asymptotic maximum of the number of nonsynonymous mutations in European mtDNA was estimated to be 1,081. The results suggested that amino acid replacements in the periphery of phylogenetic networks are more deleterious than those in the central parts, indicating that purifying selection prevents the fixation of some alleles.     

Comparative understanding of UTS2 and UTS2R genes for their involvement in type 2 diabetes mellitus

Several reports have shown that urotensin 2 (UTS2) and its receptor (UTS2R) are involved in glucose metabolism and insulin resistance, which lead to development of type 2 diabetes mellitus (T2DM) in humans. In the present study, we annotated both bovine UTS2 and UTS2R genes and identified 5 single nucleotide polymorphisms (SNPs) for the former gene and 14 mutations for the latter gene. Four mutations were genotyped on a Wagyu x Limousin reference population, including 6 F₁ bulls, 113 F₁ dams and ~250 F₂ progeny. Among 12 phenotypes related to fat deposition and fatty acid composition, we observed that the UTS2 gene was significantly associated with the amount of skeletal saturated fatty acids, while its receptor (UTS2R) gene had significant effects on amounts of saturated and monounsaturated fatty acids, Δ⁹ desaturase activity for converting 16:0 into 16:1, muscle fat (marbling) score and Longissimus Dorsi muscle area. However, in this population, these markers were not associated with subcutaneous fat depth or percent kidney, pelvic and heart fat. We also found that mutations in the promoter regions altered the promoter activities in both genes and coding SNPs might affect the mRNA stability in the UTS2R gene. Overall, our present study provides the first evidence that both UTS2 and UTS2R genes regulate skeletal muscle fat accumulation and fatty acid metabolism, thus indicating their potential pathological functions related to obesity and T2DM in humans.     

Localised mutagenesis of the fts YEX operon conditionally lethal missense substitutions in the FtsE cell division protein of Escherichia coli are similar to those found in the cystic fibrosis transmembrane conductance regulator protein (CFTR) of human patients

Summary After localised mutagenesis of the 76 min region of the Escherichia coli chromosome, we isolated a number of conditionally lethal mutants. Some of these mutants had a filamentation temperature sensitive (fts) phenotype and were assigned to the cell division genes ftsE of ftsX whereas others were defective in the heat shock regulator gene rpoH. Both missense and amber mutant alleles of these genes were produced. The missense mutant ftsE alleles were cloned and sequenced to determine whether or not the respective mutations mapped to the region of the gene encoding the putative nucleotide binding site. Surprisingly, most of these mutant FtsE proteins had missense substitutions in a different domain of the protein. This region of the FtsE protein is highly conserved in a large family of proteins involved in diverse transport processes in all living cells, from bacteria to man. One of the proteins in this large family of homologues is the human cystic fibrosis transmembrane conductance regulator (CFTR), and the FtsE substitutions were found to be in very closely linked, or identical, amino acid residues to those which are frequently altered in the CFTR of human patients. These results confirm the structural importance of this highly conserved region of FtsE and CFTR and add weight to the current structural model for the human protein.     

Patterns of nucleotide substitutions inferred from the phylogenies of the class I major histocompatibility complex genes

Summary Patterns of nucleotide substitutions in human major histocompatibility complex (MHC) class I genes were estimated by using phylogenetic trees of DNA sequences. The pattern is defined as a set of 12 parameters, each of which represents the relative frequency of substitutions from a particular nucleotide to another. The pattern at the antigen recognition sites (ARS) in functional MHC genes was remarkably different from that at the remaining coding region (non-ARS). In particular, the proportion of transitions among all the nucleotide substitutions (P _s) was extremely low at the third codon positions of ARS. In the HLA-A genes, P _s at the third codon positions was only 6% in ARS, whereas it was 69% in non-ARS. In HLA-B, the corresponding values were 30% in ARS and 80% in non-ARS, respectively. On the other hand, P _s in a class I pseudogene (HLA-H) was 57%, which was in good agreement with P _s in other pseudogenes. Because pseudogenes are selectively neutral, the pattern in pseudogenes is regarded as the pattern of spontaneous substitution mutations. In general, the pattern in functional genes that are subject to selective forces deviates from the pattern in pseudogenes. At the third codon positions in coding regions, transitions scarcely cause amino acid replacements, whereas about half of transversions do cause replacements. Accordingly, P _s at the third codon positions decreases if amino acid replacements are accelerated by natural selection but increases if amino acids are conserved by functional constraint. Our observations imply that the ARS region is subject to natural selection favoring amino acid replacements, whereas the non-ARS region is subject to functional constraint. Offprint requests to: T. Gojobori     

Functional Nonequivalence of Drosophila Actin Isoforms

We show that different Drosophila actinisoforms are not interchangeable. We sequenced the sixgenes that encode conventional Drosophilaactins and found that they specify amino acidreplacements in 27 of 376 positions. To test the significance ofthese changes we used directed mutagenesis to introduce10 such conversions, independently, into the Act88Fflight muscle-specific actin gene. We challenged these variant actins to replace the nativeprotein by transforming germline chromosomes of aDrosophila strain lacking flight muscle actin.Only one of the 10 reproducibly perturbed myofibrillarfunction, demonstrating that most isoform-specific aminoacid replacements are of minor significance. In order toestablish the consequences of multiple amino acidreplacements, we substituted portions of theDrosophila Act88F actin gene with correspondingregions of genes encoding other isoforms. Only one offive constructs tested engendered normally functioningflight muscles, and the severity of myofibrillar defects correlated with the number of replacementswithin the chimeric genes. Finally, we completelyconverted the flight muscle actin-encoding gene to onespecifying a nonmuscle isoform, a change entailing atotal of 18 amino acid replacements. Transformationof flies with this construct resulted in disruption offlight muscle structure and function. We conclude thatactin isoform sequences are not equivalent and that effects of the amino acid replacements,while minor individually, collectively confer uniqueproperties.