Changes in the connexin 26 gene (<Emphasis Type="Italic">GJB2</Emphasis>) in Russian patients with hearing loss: Results of long-term molecular diagnostics of hereditary nonsyndromic hearing loss

Molecular testing for mutations in the connexin 26 gene (GJB2) is a routine diagnostic analysis for subjects with hereditary hearing loss worldwide. However, till now there is no assessment of the diagnostic significance of this analysis for Russian patients, and there are difficulties in interpretation of the results of DNA diagnostics. In the present study, a sample of 705 patients with nonsyndromic autosomal recessive hearing loss from different regions of Russian Federation was investigated. A portion of DFNB1 hearing loss caused by mutations in the GJB2 gene among the sample was 46%. The frequency of DFNB1 hearing loss was 1:1000, that is, the frequency of isolated autosomal recessive hearing loss 1:500 in the population. It was found that each sixteenth individual in Russia is a heterozygous carrier of the mutation in the GJB2 gene. Totally, 20 pathological GJB2 alleles were detected; among them, a c.35delG mutation with the allelic frequency 81% prevails. Six most frequent mutations (c.35delG, c.313_326del14, c.23+1G>A (IVS1+1G>A), c.235delC, c.167delT, and p.Glu120del), which account for 95% of pathological GJB2 alleles, were detected. Mutations previously not described in the GJB2 gene (c.129delG, p.Gly200Arg, and c[Arg127His, Gly160Ser]) were found. An optimal algorithm of molecular testing of Russian patients which detects up to 100% of mutations in the GJB2 gene was suggested. Data concerning a clinical significance of p.Met34Thr and p.Val37Ile mutations are confirmed in the study. Eight polymorphic substitutions in the GJB2 gene which do not have clinical significance (p.Val27Ile, c.*3C>A, p.Val153Ile, p.Gly160Ser, c.Arg127His, p.Glu114Gly (c.341A>G), c.-45C>A, and p.Ala149Thr) were also detected.     

Genetic Testing of Non-familial Deaf Patients for <Emphasis Type="Italic">CIB2</Emphasis> and <Emphasis Type="Italic">GJB2</Emphasis> Mutations: Phenotype and Genetic Counselling

CIB2 and GJB2 genes variants contribute significantly in familial cases of prelingual recessive hearing loss (HL). This study was aimed to determine the CIB2 and GJB2 variants and associated phenotype in 150 non-familial individuals with HL. After getting informed consent, 150 non-familial deaf patients were enrolled and blood samples were obtained for DNA extraction. Pure tone air conduction audiometry was performed. Coding exons of CIB2 and GJB2 genes were Sanger sequenced. A tetra primer ARMS assay was developed for recurrent CIB2 variant. Four bi-allelic GJB2 variants, c.71G>A p.(Trp24*), c.231G>A p.(Trp77*), c.235delC p.(Leu79Cysfs3*) and c.35delG p.(Gly11Leufs24*), were found in nine hearing impaired individuals. We also found four homozygotes and five carriers of c.380G>A p. (Arg127His) variant of controversial clinical significance. CIB2 sequencing revealed single recurrent variant c.272T>C p. (Phe91Ser) segregating with HL in ten individuals. Among our patients, c.71G>A (p.Trp24*) was the most common variant, accounted for 45% of GJB2 variants. Two known GJB2 variants, c.235delC p. (Leu79Cysfs3*) and c.310del14 p. (Lys105Argfs2*), are reported here for the first time in Pakistani population. Our data further support the benign nature of c.380G>A p. (Arg127His) variant. For CIB2, c.272T>C p. (Phe91Ser) is the second common cause of HL among our sporadic cases. Phenotypically, in our patients, individuals homozygous for GJB2 variants had profound HL, whereas CIB2 homozygotes had severe to profound prelingual HL. Our results suggest that GJB2 and CIB2 are common cause of HL in different Pakistani ethnicities.     

Spectrum and frequency of mutations in the connexin 32 gene (<Emphasis Type="Italic">GJB1</Emphasis>) in hereditary and sensory neuropathy type 1X patients from Bashkortostan

Hereditary motor and sensory neuropathy type 1X (HMSN 1X) is the second most frequent form of demyelinating polyneuropathies and is caused by mutations in the gene for connexin 32 protein (Cx32, GJB1). The contribution of HMSN 1X to the structure of HMSN in the Republic of Bashkortostan was determined. The GJB1 mutations were detected in 18 out of 131 unrelated patients, which constituted 13.7%. The four missense mutations identified were represented by: p.Pro87Ala (c.259C > G) with the frequency of 10%; p.Arg22Gln (c.65G > A) (2.98%); p.Arg15Gln (c.44G > A); and p.Thr86Ile (c.257C > T) (0.8%). The latter mutation was never described before. The frequent mutation p.Pro87Ala was tested for linkage disequilibrium with the alleles of five polymorphic microsatellite DNA loci associated with the GJB1. It was demonstrated that 10 out of 13 chromosomes carrying the mutation mentioned had common DXS8111-DXS983-DXS8107-DXS8052 haplotype. This finding suggested the distribution of this mutation on the territory of the Republic of Bashkortostan as a result of the founder effect. The mutational spectrum of GJB1 and mutation frequencies observed in the HMSN 1X patients examined were characterized by ethnic heterogeneity. This finding will provide development of most optimal algorithm of the HMSN DNA diagnostics in the region.     

Frequence and the mutation spectrum of <Emphasis Type="Italic">GJB2</Emphasis>-related hearing loss in children of Dagestan as compared with the central European part of Russia

The sequencing of the entire coding region and the donor site of the splicing of the GJB2 gene has shown that prelingual neurosensory nonsyndromic autosomal recessive deafness/poor hearing in Dagestan Republic is genetically more heterogeneous than in the central European part of Russia. Thus, the number of the revealed mutations in the GJB2 gene in Dagestan was only 28% of the total number of alleles in patients tested. The main mutations in the GJB2 gene in Dagestan were represented by three forms typical for West Asia: 35delG mutation (22% of all mutant alleles), ΔE120 mutation (22%), both in the coding region, and IVS 1+1 G>A (44%) in the donor site of splicing. In addition, a novel mutation, ΔE187, was revealed in the coding region. All mutations were found in a compound heterozygous or a heterozygous state. This result allows one to explain the deafness/poor hearing by pathogenic mutations in the GJB2 gene only in 19% of Dagestan families with neurosensory nonsyndromic autosomal recessive disorders of hearing. We suggest that the origin and maintenance of the allele diversity of inherited deafness/poor hearing in Dagestan can be explained by the national and cultural peculiarities of small populations of nations of North Caucasus.     

<Emphasis Type="Italic">Ban</Emphasis>I/D13S141/D13S175 Represents a Novel Informative Haplotype at the <Emphasis Type="Italic">GJB2</Emphasis> Gene Region in the Iranian Population

Non-syndromic sensorineural hearing loss (NSHL) represents the most common cause of hearing loss in the Iranian patients. In view of the large numbers of mutations identified in GJB2, mutations analysis of the gene has been time-consuming and cost-ineffective. Alternatively, molecular markers that are highly linked to the GJB2 gene have proven to be useful in carrier detection and prenatal diagnosis of NSHL families. These markers usually show a population-dependent-based haplotype frequency. However, to date, no information on the genotyping and frequency of the markers is present for the Iranian population. In this study, genotyping and analysis of the haplotype frequency of three markers, including BanI, D13S141, and D13S175, at the GJB2 region were investigated. The haplotype frequency was estimated using PHASE program. The input data contained two alleles (+ and –) for BanI, four alleles for D13S141, and seven alleles for D13S175. Among the 42 possible haplotypes examined, four haplotypes showed relatively high frequencies (≥5%). Therefore, a combination of BanI/D13S141/D13S175 could be suggested as an informative haplotype for possible carrier detection and prenatal diagnosis of NSHL in the Iranian population.     

Low incidence of GJB2, GJB6 and mitochondrial DNA mutations in North Indian patients with non-syndromic hearing impairment

Mutations at the DFNB1 locus which encode connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively, are main cause for sporadic and familial non-syndromic hearing impairment (NSHI) in many populations. 342-kb deletion [del (GJB6-D13S1830)] of Cx30 gene is second most common connexin mutation. Specific mitochondrial DNA (mtDNA) mutations have been found to be associated with NSHI. In this study, we screened 210 NSHI patients for GJB2 mutations, ΔGJB6-D13S1830 deletion and three point mutations in mtDNA (A1555G, A3243G, A7445G) using PCR, DHPLC and sequencing in North Indian cohort. 35delG was found to be the most common mutation (10.9%), followed by W24X (3.8%) and W77X (1.9%) mutations. We did not observe GJB6-D13S1830 deletion and three mitochondrial point mutations in our cohort. Most of patients (50/58) carried monoallelic variations. Our results reveal different spectrum of GJB2 mutations specific to North Indian cohort, with 35delG being most prevalent. These results suggest that different types of GJB2 mutations affect autosomal recessive NSHI according to ethnic background.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

<Emphasis Type="Italic">KRAS</Emphasis>, <Emphasis Type="Italic">BRAF</Emphasis>, <Emphasis Type="Italic">EGFR</Emphasis> and <Emphasis Type="Italic">HER2</Emphasis> gene status in a Spanish population of colorectal cancer

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical–pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.     

Interaction of gene <Emphasis Type="Italic">HSM3</Emphasis> with genes of the epistatic <Emphasis Type="Italic">RAD6</Emphasis> group in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In eukaryotes, damage tolerance of matrix DNA is mainly determined by the repair pathway under the control of the RAD6 epistatic group of genes. This pathway is also a main source of mutations generated by mutagenic factors. The results of our recent studies show that gene HSM3 participating in the control of adaptive mutagenesis increases the frequency of mutations induced by different mutagens. Mutations rad18, rev3, and mms2 controlling various stages of the RAD6 pathway are epistatic with mutation hsm3 that decreases UV-induced mutagenesis to the level typical for single radiation-sensitive mutants. The level of mutagenesis in the double mutant srs2 hsm3 was lower than in both single mutants. Note that a decrease in the level of mutagenesis relative to the single mutant srs2 depends on the mismatch repair, since this level in the triple mutant srs2 hsm3 pms1 corresponds to that in the single mutant srs2. These data show that the mutator phenotype hsm3 is probably determined by processes occurring in a D loop. In a number of current works, the protein Hsm3 was shown to participate in the assembly of the proteasome complex S26. The assembly of proteasomes is governed by the N-terminal domain. Our results demonstrated that the Hsm3 protein contains at least two domains; the N-terminal part of the domain is responsible for the proteasome assembly, whereas the C-terminal portion of the protein is responsible for mutagenesis.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

Zinc Uptake Regulator (<Emphasis Type="Italic">zur</Emphasis>) Gene Involved in Zinc Homeostasis and Virulence of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in Rice

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn²⁺ or Fe³⁺ at a concentration of 500 μM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae. Wanfeng Yang and Yan Liu contributed equally to this work     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

Effect of lipopolysaccharide mutation on oxygenation of linoleic acid by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing CYP102A2 of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The effects of cell wall mutation on the oxygenation of linoleic acid (M.W. 280) by recombinant Escherichia coli expressing the CYP102A2 gene encoding self-sufficient P450 monooxygenase of Bacillus subtilis was investigated. After the CYP102A2 gene was heterologously expressed in E. coli W3110 and its isogenic lipopolysaccharide (LPS) structural mutant strains, their whole-cell biotransformation activities were compared. The mutants used in this study had previously been designated as MLK53, MLK1067, and MLK986. These strains carry one or two defined mutations in the secondary acyl fatty acids of the LPS lipid A constituent. The CYP102A2 gene was overexpressed in both wild type E. coli W3110 and its mutant strains, with the specific activity ranging from 1.7 to 2.1 U/mg protein. Interestingly, the whole-cell biotransformation activity of those recombinant biocatalysts differed significantly. Indeed, MLK986 possessing the tetraacylated LPS showed a higher oxygenation activity of linoleic acid than those in wild type or other mutant strains having hexa- or penta-acylated LPSs. These results suggest that the biotransformation efficiency of E. coli-based biocatalysts, especially for medium- to large-sized lipophilic organic substrates, can be enhanced via engineering their LPS, which is known to function as a formidable barrier for hydrophobic molecules.     

The mouse mutants <Emphasis Type="Italic">recoil wobbler</Emphasis> and <Emphasis Type="Italic">nmf373</Emphasis> represent a series of <Emphasis Type="Italic">Grm1</Emphasis> mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP₃). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.     

Identification and Characterization of <Emphasis Type="Italic">sanH</Emphasis> and <Emphasis Type="Italic">sanI</Emphasis> Involved in the Hydroxylation of Pyridyl Residue During Nikkomycin Biosynthesis in <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis>

Nikkomycins are highly potent inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned from Streptomyces asochromogenes. Two cytochrome P450 monooxygenase genes (sanQ, sanH) and one ferredoxin gene (sanI) were found in the cluster. It was reported that SanQ is involved in the hydroxylation of l-His, a key step in 4-formyl-4-imidazolin-2-one base biosynthesis. Here, we have studied the function of sanH and sanI. Disruption of sanH abolished the production of nikkomycin X and Z, but it accumulated one dominant component nikkomycin Lx, which is the nikkomycin X analog lacking the hydroxy group at the pyridyl residue. The sanI disruption mutant accumulated predominantly nikkomycin Lx in addition to nikkomycin X and Z. The nikkomycin production profile of the sanH and sanI double disruption mutant was the same as that of the sanH disruption mutant. These results confirmed that SanH is essential for the hydroxylation of pyridyl residue in nikkomycin biosynthesis of S. ansochromogenes and first demonstrated that SanI is an effective electron donor for SanH, but not for SanQ in vivo.     

Mutations in the <Emphasis Type="Italic">S</Emphasis> gene region of hepatitis B virus genotype <Emphasis Type="Italic">D</Emphasis> in Turkish patients

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.     

Marital Structure,Genetic Fitness,and the <Emphasis Type="Italic">GJB2</Emphasis> Gene Mutations among Deaf People in Yakutia (Eastern Siberia,Russia)

Autosomal recessive deafness type 1A (DFNB1A) caused by mutations in the GJB2 gene (Cx26) is the main cause of nonsyndromic hearing impairment in many populations worldwide. It is considered that widespread prevalence of DFNB1A can be due to the long tradition of intermarriages between deaf people (assortative marriages) combined with their increased social adaptation and genetic fitness after widespread introduction of sign language. For the first time, the data on mating structure and reproduction of deaf people living in Yakutia (Eastern Siberia, Russia) are presented in comparison with contribution of the GJB2 gene mutations to the etiology of hearing impairment. The relative fertility of deaf people compared to their hearing siblings is 0.78 (mean number of children 1.76 ± 0.10 and 2.24 ± 0.09 to deaf and their hearing siblings, respectively, p = 0.0018). The rate of assortative marriages among deaf people is 77.1% (81 of 105 marriages). Biallelic mutations in the GJB2 gene were found in 42.2% (43 of 102) of examined deaf people, which corresponded to diagnosis DFNB1A for these patients. A comparison of deaf marital partners by GJB2 status revealed a proportion of noncomplementary marriages (24%) in which hearing loss in both partners was caused by the presence of biallelic GJB2 gene mutations resulting in the birth of only deaf children in such couples. Thus, the set of obtained data including a relatively high genetic fitness (expressed as relative fertility) of deaf people in Yakutia in combination with a high rate of assortative marriages among them and high incidence of DFNB1A indicates a possible weakening of selection against such trait as “deafness” and a possible increase in the frequency of GJB2 mutant alleles in subsequent generations.     

Fine genetic mapping and physical delimitation of the lesion mimic gene <Emphasis Type="Italic">spotted leaf 5</Emphasis> (<Emphasis Type="Italic">spl5</Emphasis>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Spotted leaf 5 (spl5), a lesion mimic mutant, was first identified in rice (Oryza sativa L.) japonica cv. Norin8 in 1978. This mutant exhibits spontaneous disease-like lesions in the absence of any pathogens and resistance to rice blast and bacterial blight; however, the target gene has not yet been isolated. In the present study, we employed a map-based cloning strategy to finely map the spl5 gene. In an initial mapping with 100 F₂ individuals (spl5/spl5) derived from a cross between the spl5 mutant and indica cv. 93-11, the spl5 gene was located in a 3.3-cM region on chromosome 7 using six simple sequence repeat (SSR) markers. In a high-resolution genetic mapping, two F₂ populations with 3,149 individuals (spl5/spl5) were derived from two crosses between spl5 mutant and two indica cvs. 93-11 and Zhefu802 and six sequence-tagged site (STS) markers were newly developed. Finally, the spl5 gene was mapped to a region of 0.048 cM between two markers SSR7 and RM7121. One BAC/PAC contig map covering these markers’ loci and the spl5 gene was constructed through Pairwise BLAST analysis. Our bioinformatics analysis shows that the spl5 gene is located in the 80-kb region between two markers SSR7 and RM7121 with a high average ratio of physical to genetic distance (1.67 Mb/cM) and eighteen candidate genes. The analysis of these candidate genes indicates that the spl5 gene represents a novel class of regulators controlling cell death and resistance response in plants.