Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

G:C-->T:A and G:C-->C:G transversions are the predominant spontaneous mutations in the Escherichia coli supF gene: an improved lacZ(am) E. coli host designed for assaying pZ189 supF mutational specificity.

Summary Escherichia coli K12 strain KS40 and plasmid pKY241 were designed for easy screening of supF mutations in plasmid pZ189. KS40 is a nalidixic acid-resistant (gyrA) derivative of MBM7070 (lacZ(am)CA7020). Using in vitro mutagenesis, an amber mutation was introduced into the cloned gyrA structural gene, of E. coli, to give pKY241, a derivative of pACYC184. When KS40 containing pKY241 (designated KS40/pKY241) is transformed with pZ189, nalidixic acid-resistant GyrA protein is produced from the chromosomal gyrA gene and wild-type GyrA protein from pKY241 because of the suppression of the gyrA amber mutation by supF. It is known that the wild-type, otherwise nalidixic acid-sensitive, phenotype is dominant over the nalidixic acid-resistant phenotype. Thus, KS40/pKY241 gives rise to nalidixic acid-sensitive colonies when it carries a pZ189 plasmid with an active supF suppressor tRNA. If the supF gene on the plasmid carries an inactivating mutation then KS40/pKY241 will form nalidixic acid-resistant colonies. By using this system, the spontaneous mutational frequency of the supF gene on pZ189 was calculated to be 3.06 × 10^–7 per replication. Among 51 independent supF mutations analyzed by DNA sequencing, 63% were base substitutions, 25% IS element insertions, 9.6% deletions and 1.9% single-base frameshifts. The base substitutions included both transversions (84.8%) and transitions (15.2%), the largest single group being G:C to T:A transversions (45.4% of the base substitutions). These results demonstrate that the KS40/pKY241 system we have developed can be used to characterize the DNA sequence changes induced by mutagens that give very low mutational frequencies.     

Novel Mutations Found in Two Genes of Thai Patients with Isolated Methylmalonic Acidemia

Molecular genetic analysis of three patients diagnosed with isolated methylmalonic acidemia (MMA) revealed that one was mut ⁰ MMA, with a mutation in the MUT gene encoding the l-methylmalonyl-CoA mutase (MCM), and two were cblB MMA, with mutations in the MMAB gene required for synthesizing the deoxyadenosylcobalamin cofactor of MCM. The mut ⁰ patient was homozygous for a novel nonsense mutation in MUT, p.R31X (c.167C → T), and heterozygous for three previously described polymorphisms, p.K212K (c.712A → G), p.H532R (c.1671A → G), and p.V671I (c.2087G → A). The new MMAB mutation, p.E152X (c.454G → T), was found to be homozygous in one cblB patient and heterozygous in the other patient, who also had four intron polymorphisms in this gene.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis in Amaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light → phytochrome → G protein → guanylyl cyclase → cGMP.     

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     

Mutations induced by glyoxal and methylglyoxal in mammalian cells.

To investigate the mutation spectra of glyoxal and methylglyoxal in mammalian cells, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. The cytotoxicity and the mutation frequency increased according to the doses of glyoxal and methylglyoxal. The majority of glyoxal-induced mutations (65%) were base-pair substitutions, in which G:C-->C:G transversions were predominant. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which G:C-->C:G and G:C-->T:A transversions were predominant.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Sequence variation of chalcone synthase gene in a spontaneous white-flower mutant of Chinese cabbage-pak-choi

A spontaneous white-flower mutant of Chinese cabbage-pak-choi (Brassica campestris ssp. chinenesis, syn. B. rapa ssp. chinenesis) was found in our test fields, and all the plant characters except flower color were identical with wild type ones. We hypothesized that a mutational event had occurred in the gene coding for chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis pathway. Two genes, later designated BcCHS and BcCHS-wf, were isolated from wild type and mutant Chinese cabbage-pak-choi, respectively, using gene-specific primer pairs. Comparison of the genomic sequences revealed two mutations in BcCHS-wf, both with A to G transitions, one at position +37 bp and the other at +970 bp. Both nucleotide substitutions occurred in AGA codes for arginine into GGA for glycin at residue +13 and into AGC coding for serine at residue +229, respectively. Homologous genes of BcCHS were isolated from another four cruciferous plants, though there were some differences among the genomic and deduced amino acid sequences, the mutation locus of the mutant, as we called it, were identical to the wild type Chinese cabbage-pak-choi.     

Reduced folate carrier 80A→G polymorphism,plasma folate,and risk of placental abruption

Folate deficiency and maternal smoking are strong risk factors for placental abruption. We assessed whether the reduced folate carrier [NM_194255.1: c.80A→G (i.e., p.His27Arg)] (RFC-1) polymorphism was associated with placental abruption, and evaluated if maternal smoking modified the association between plasma folate and abruption. Data were derived from the New Jersey-Placental Abruption Study—a multicenter, case-control study of placental abruption (2002–2007). Maternal DNA was assayed for the RFC-1 c.80A→G polymorphism using a PCR-dependent diagnostic test. Maternal folate (nmol/l) was assessed from maternal plasma, collected immediately following delivery. Due to assay limitations, folate levels at ≥60 nmol/l were truncated at 60 nmol/l. Therefore, case–control differences in folate were assessed from censored log-normal regression models following adjustment for potential confounders. Distribution of the mutant allele (G) of the RFC-1 c.80A→G polymorphism was similar between cases (52.3%; n = 196) and controls (50.5%; n = 191), as was the homozygous mutant (G/G) genotype (OR 1.1, 95% CI 0.6–2.2). In a sub-sample of 136 cases and 140 controls, maternal plasma folate levels (mean ± standard error) corrected for assay detection limits were similar between placental abruption cases (63.6 ± 5.1 nmol/l) and controls (58.3 ± 4.7 nmol/l; P = 0.270), and maternal smoking did not modify this relationship (interaction P = 0.169). We did not detect any association between the RFC-1 c.80A→G polymorphism and placental abruption, nor was an association between plasma folate and abruption risk evident. These findings may be the consequence of high prevalence of prenatal multivitamin and folate supplementation in this population (over 80%). It is therefore not surprising that folate deficiency may be rare and that the RFC-1 c.80A→G polymorphism is less biologically significant for placental abruption.     

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (−11377C→G and −11282T→C), one intronic SNP (+10211T→G) and one exonic SNP (+45T→G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T→G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T→G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07–1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3–1.8; P < 10⁻⁷) and that for GG genotype, 2.10 (95% CI 1.3–3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T→G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.     

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.     

Inferring the Pattern of Spontaneous Mutation from the Pattern of Substitution in Unitary Pseudogenes of Mycobacterium leprae and a Comparison of Mutation Patterns Among Distantly Related Organisms

The pattern of spontaneous mutation can be inferred from the pattern of substitution in pseudogenes, which are known to be under very weak or no selective constraint. We modified an existing method (Gojobori T, et al., J Mol Evol 18:360, 1982) to infer the pattern of mutation in bacteria by using 569 pseudogenes from Mycobacterium leprae. In Gojobori et al.’s method, the pattern is inferred by using comparisons involving a pseudogene, a conspecific functional paralog, and an outgroup functional ortholog. Because pseudogenes in M. leprae are unitary, we replaced the missing paralogs by functional orthologs from M. tuberculosis. Functional orthologs from Streptomyces coelicolor served as outgroups. We compiled a database consisting of 69,378 inferred mutations. Transitional mutations were found to constitute more than 56% of all mutations. The transitional bias was mainly due to C→T and G→A, which were also the most frequent mutations on the leading strand and the only ones that were significantly more frequent than the random expectation. The least frequent mutations on the leading strand were A→T and T→A, each with a relative frequency of less than 3%. The mutation pattern was found to differ between the leading and the lagging strands. This asymmetry is thought to be the cause for the typical chirochoric structure of bacterial genomes. The physical distance of the pseudogene from the origin of replication (ori) was found to have almost no effect on the pattern of mutation. A surprising similarity was found between the mutation pattern in M. leprae and previously inferred patterns for such distant taxa as human and Drosophila. The mutation pattern on the leading strand of M. leprae was also found to share some common features with the pattern inferred for the heavy strand of the human mitochondrial genome. These findings indicate that taxon-specific factors may only play secondary roles in determining patterns of mutation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor:Dr. Dmitri Petrov]     

Mutational specificity of thymine deprivation-induced mutation in the lacI gene of Escherichia coli

LacI⁻ mutants obtained following 2 and 6 h of thymine deprivation were cloned and sequenced. The mutational spectra recovered were dissimilar. After 2 h of starvation the majority of mutations were base substitutions, largely G: C→C: G transversions. Frameshift mutations but not deletions were observed. In contrast, following 6 h of starvation, with the exception of the G: C→C: G transversion, all possible base substitutions were recovered. Moreover, several deletions but no frameshift events were observed. The differences in the mutational spectra recovered after two periods of thymine deprivation highlight the role of altered nucleotide pools and the potential influence of DNA replication mechanisms.     

Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.     

The mutational demography of protein C deficiency

The geographical distribution and prevalence of 256 single base-pair substitutions (105 of them being different) within the coding region of the human protein C (PROC) gene were correlated with their initial likelihoods of generation. A significant positive correlation was observed between these mutational likelihoods and the geographical dispersal of the PROC gene lesions within and between 16 different countries. This relationship could be attributed to the fact that, with few exceptions, high dispersal was only exhibited by CGTG and CGCA transitions, i.e. those substitutions that are known to arise de novo at the highest frequency. The statistical distribution of mutational likelihoods was as predicted on the basis of the PROC cDNA sequence alone, allowing however for the redundancy of the genetic code. These findings suggest (1) that genetic drift and lesion-specific selection have been of relatively minor importance in determining the mutational spectrum observed in the PROC gene and (2) that most multiple reports of particular substitutions in different geographical locations appear to reflect recurrent mutation rather than identity-by-descent.     

The paradox of viable <Emphasis Type="Italic">sup45</Emphasis> STOP mutations: a necessary equilibrium between translational readthrough,activity and stability of the protein

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q → TAA, 242R → TGA, 317L → TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.     

Molecular Cloning, Mapping, and Polymorphism of the Porcine SCG2 gene

The secretogranin II (SCG2) gene is associated with the synthesis and secretion of follicle-stimulating hormone and luteinizing hormone. In the present study, we have determined the complete cDNA sequence of pig SCG2, which was submitted to GenBank with accession no. AY870646. Its complete open reading frame of 1,851 nucleotides encodes 616 amino acids. The predicted protein shares 80–87% identity with mouse, human, and bovine SCG2 proteins, and all four species share almost complete identity in the secretoneurin and EM66 domains. Pig SCG2 is a protein of 589 amino acids and 68,132 Da, preceded by a signal peptide of 27 residues. It contains nine pairs of dibasic residues, which are used as potential cleavage sites for generation of physiologically active peptides. Analysis of the SCG2 gene across the INRA-Minnesota porcine radiation hybrid panel indicates close linkage with microsatellite marker SW2608, located on Sus scrofa chromosome 15 (SSC15) q25, which harbors several QTL for ovulation rate and meat quality. Comparative sequencing and EST analysis revealed nine SNPs in porcine SCG2 cDNA, including seven SNPs in the coding region and two SNPs in the 3′ UTR. Four nonsynonymous SNPs (G622A, G1671T, C1718T, and A1790C) resulted in amino acid substitutions of Ala→Thr, Glu→Asp, Pro→Leu, and Asn→Thr, respectively.