The F1F0-ATPase of Escherichia coli. The substitution of alanine by tyrosine at position 25 in the c-subunit affects function but not assembly

A site-directed mutation in the gene which codes for the c-subunit of the F1F0-ATPase, resulting in the substitution of Ala-25 by Tyr, has been constructed and characterized. A plasmid carrying the mutation was used to transform strain AN943 (uncE429). The resulting strain is unable to grow on succinate as sole carbon source and possesses an uncoupled growth yield. Membranes prepared from the mutant possess low levels of ATPase activity and are proton-impermeable. The F1-ATPase activity was found to be inhibited by 80% when bound to the membrane. When carried on a plasmid, the mutation is dominant in complementation tests with all mutant unc alleles tested and when transformed into wild-type strain AN346, the mutation results in an uncoupled phenotype. A mutant which overcomes this dominance was isolated and found to possess an 11-amino-acid deletion extending from Ile-55 to Met-65 within the c-subunit. These results are discussed in relation to the previously isolated Ala-25 to Thr mutant (Fimmel, A.L., Jans, D.A., Hatch, L., James, L.B., Gibson, F. and Cox, G.B. (1985) Biochim. Biophys. Acta 808, 252-258) and in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).     

Distinct phospholipase C-gamma-dependent signaling pathways in the Drosophila eye and wing are revealed by a new small wing allele

The Drosophila genome contains a single phospholipase C-gamma (PLC-gamma) homolog, encoded by small wing (sl), that acts as an inhibitor of receptor tyrosine kinase (RTK) signaling during photoreceptor R7 development. Although the existing sl alleles behave genetically as nulls, they may still produce truncated Sl products that could in theory still provide limited PLC-gamma function. Both to identify a true null allele and to probe structure-function relationships in Sl, we carried out an F(1) screen for new sl mutations and identified seven new alleles. Flies homozygous for any of these alleles are viable, with the same short-wing phenotype described previously; however, two of the alleles differ from any of those previously isolated in the severity of the eye phenotype: sl(9) homozygotes have a slightly more extreme extra-R7 phenotype, whereas sl(7) homozygotes have an almost wild-type eye. We determined the mutant defect in all seven alleles, revealing that sl(9) is a molecular null due to a very early stop codon, while sl(7) has a missense mutation in the highly conserved Y catalytic domain. Together with in vitro mutagenesis of the residue affected by the sl(7) mutation, these results confirm the role of Sl in RTK signaling and provide evidence for two genetically separable PLC-gamma-dependent pathways affecting the development of the eye and the wing.     

Spectrum of mutations in the major human skeletal muscle chloride channel gene (CLCN1) leading to myotonia.

Autosomal dominant myotonia congenita and autosomal recessive generalized myotonia (GM) are genetic disorders characterized by the symptom of myotonia, which is based on an electrical instability of the muscle fiber membrane. Recently, these two phenotypes have been associated with mutations in the major muscle chloride channel gene CLCN1 on human chromosome 7q35. We have systematically screened the open reading frame of the CLCN1 gene for mutations by SSC analysis (SSCA) in a panel of 24 families and 17 single unrelated patients with human myotonia. By direct sequencing of aberrant SSCA conformers were revealed 15 different mutations in a total of 18 unrelated families and 13 single patients. Of these, 10 were novel (7 missense mutations, 2 mutations leading to frameshift, and 1 mutation predicted to affect normal splicing). In our overall sample of 94 GM chromosomes we were able to detect 48 (51%) mutant GM alleles. Three mutations (F413C), R894X, and a 14-bp deletion in exon 13) account for 32% of the GM chromosomes in the German population. Our finding that A437T is probably a polymorphism is in contrast to a recent report that the recessive phenotype GM is associated with this amino acid change. We also demonstrate that the R894X mutation may act as a recessive or a dominant mutation in the CLCN1 gene, probably depending on the genetic background. Functional expression of the R894X mutant in Xenopus oocytes revealed a large reduction, but not complete abolition, of chloride currents. Further, it had a weak dominant negative effect on wild-type currents in coexpression studies. Reduction of currents predicted for heterozygous carriers are close to the borderline value, which is sufficient to elicit myotonia.     

Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients.

Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in >1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients.     

Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K, P409A, g10208delAA [two families]). Haplotype analyses indicated that the families with the R112C or g10208delAA mutations were not related. The proband with the D266E lesion had a severe classic phenotype, having developed renal failure at 15 years. In contrast, the patient with the S247C mutation had a variant phenotype, lacking the classic manifestations and having mild renal involvement at 64 years. CONCLUSIONS: These results further define the heterogeneity of alpha-Gal A mutations causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis in these families, and provide additional genotype/phenotype correlations in this lysosomal storage disease.     

High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes

We have analyzed 640 Spanish cystic fibrosis (CF) families for mutations in the CFTR gene by direct mutation analysis, microsatellite haplotypes, denaturing gradient gel electrophoresis, single-strand conformation analysis and direct sequencing. Seventy-five mutations account for 90.2% of CF chromosomes. Among these we have detected seven novel CFTR mutations, including four missense (G85V, T582R, R851L and F1074L), two nonsense (E692X and Q1281X) and one splice site mutation (711+3A→T). Three variants, two in intronic regions (406-112A/T and 3850-129T/C) and one in the coding region (741C/T) were also identified. Mutations G85V, T582R, R851L, E692X and Q1281X are severe, with lung and pancreatic involvement; 711+3A→T could be responsible for a pancreatic sufficiency/insufficiency variable phenotype; and F1074L was associated with a mild phenotype. These data demonstrate the highest molecular heterogeneity reported so far in CF, indicating that a wide mutation screening is necessary to characterize 90% of the Spanish CF alleles. Received: 3 July 1997 / Accepted: 20 August 1997     

Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes

Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.     

Effects of a temperature sensitivity mutation in the J1R protein component of a complex required for vaccinia virus assembly

Vaccinia virus J1R protein is required for virion morphogenesis (W. L. Chiu and W. Chang, J. Virol. 76:9575-9587, 2002). In this work, we further characterized the J1R protein of wild-type vaccinia virus and compared it with the protein encoded by the temperature-sensitive mutant virus Cts45. The mutant Cts45 was found to contain a Pro-to-Ser substitution at residue 132 of the J1R open reading frame, which is responsible for a loss-of-function phenotype. The half-life of the J1R-P132S mutant protein was comparable at both 31 and 39 degrees C, indicating that the P132S mutation did not affect the stability of the J1R protein. We also showed that the J1R protein interacts with itself in the virus-infected cells. The N-terminal region of the J1R protein, amino acids (aa) 1 to 77, interacted with the C-terminal region, aa 84 to 153, and the P132 mutation did not abolish this interaction, as determined by two-hybrid analysis. Furthermore, we demonstrated that J1R protein is part of a viral complex containing the A30L, G7L, and F10L proteins in virus-infected cells. In immunofluorescence analyses, wild-type J1R protein colocalized with the A30L, G7L, and F10L proteins in virus-infected cells but the loss-of-function P132 mutant did not. Furthermore, without a functional J1R protein, rapid degradation of A30L and the 15-kDa forms of the G7L and F10L proteins was observed in cells infected with Cts45 at 39 degrees C. This study thus demonstrated the importance of the J1R protein in the formation of a viral assembly complex required for morphogenesis.     

A homozygous nonsense mutation in SOX9 in the dominant disorder campomelic dysplasia: a case of mitotic gene conversion

Campomelic dysplasia (CD; MIM 114290), an autosomal dominant skeletal malformation syndrome with XY sex reversal, is caused by heterozygous de novo mutations in and around the SOX9 gene on 17q. We report a patient with typical signs of CD, including sex reversal, who was, surprisingly, homozygous for the nonsense mutation Y440X. Since neither parent carried the Y440X mutation, possible mechanisms explaining the homozygous situation were a de novo mutation followed by uniparental isodisomy, somatic crossing over, or gene conversion. As the patient was heterozygous for six microsatellite markers flanking SOX9, uniparental isodisomy and somatic crossing over were excluded. Analysis of intragenic single-nucleotide polymorphisms suggested that the homozygous mutation arose by a mitotic gene conversion event involving exchange of at least 440 nucleotides and at most 2,208 nucleotides between a de novo mutant maternal allele and a wild-type paternal allele. Analysis of cloned alleles showed that homozygous mutant cells constituted about 80% of the leukocyte cell population of the patient, whereas about 20% were heterozygous mutant cells. Heterozygous Y440X mutations, previously described in three CD cases, have been identified in seven additional cases, thus constituting the most frequent recurrent mutations in SOX9. These patients frequently have a milder phenotype with longer survival, possibly because of the retention of some transactivation activity of the mutant protein on SOX9 target genes, as shown by cell transfection experiments. The fact that the patient survived for 3 months may thus be explained by homozygosity for a hypomorphic rather than a complete loss-of-function allele, in combination with somatic mosaicism. This is, to our knowledge, the first report of mitotic gene conversion of a wild-type allele by a de novo mutant allele in humans.     

Interallelic complementation at the Arabidopsis CRE1 locus uncovers independent pathways for the proliferation of vascular initials and canonical cytokinin signalling

The differentiation of vascular tissue plays a central role in root architecture and its functionality. Regardless of its importance, the molecular mechanisms involved in the inception of vascular morphogenesis and their interaction with hormones are only now beginning to be understood. The characterisation of the WOODEN LEG (wol/cre1 mutant), impaired in procambial cell proliferation and the identification of WOL/CRE1 as a cytokinin receptor, provided the first genetic evidence pointing to a role of cytokinins in the formation of vascular initials. However, the striking wol phenotype in vascular differentiation is unique among all the available cre1 alleles collection. In this work, we identified a mutant with identical deficiencies in vascular differentiation as wol. Complementation analysis revealed that this mutant rescued the wol short-root phenotype. However, genetic characterisation of the mutant showed that the mutation was located at the CRE1 locus, indicating that both alleles displayed interallelic complementation. Trans-heterozygotes characterisation showed that these plants fully restored the deficiency in vascular differentiation but not the canonical cytokinin signalling. Furthermore, we show that, as measured in root growth inhibition, calli regeneration assays and northern analysis, the original wol allele is in fact more sensitive to cytokinins than the trans-heterozygous plants, or some cre1 alleles showing wild-type vascular morphogenesis. Thus, there is no strict correlation between the phenotype in vascular differentiation displayed by the cre1/wol alleles and canonical cytokinin signalling. These results indicate that at least partially independent regulatory circuits may operate in procambial cell proliferation and in cytokinin responsiveness exerted through the CRE1 receptor.     

Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates

Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.     

Mutational analysis of 85 mucopolysaccharidosis type I families: frequency of known mutations, identification of 17 novel mutations and in vitro expression of missense mutations

The lysosomal storage disorder, mucopolysaccharidosis type I (MPS I), is caused by a deficiency of the enzyme alpha-L-iduronidase, which is involved in the breakdown of dermatan and heparan sulphates. There are three clinical phenotypes, ranging from the Hurler form characterised by skeletal abnormalities, hepatosplenomegaly and severe mental retardation, to the milder Scheie phenotype where there is aortic valve disease, corneal clouding, limited skeletal problems, but no mental retardation. In this study, 85 MPS I families (73 Hurler, 5 Hurler/Scheie, 7 Scheie) were screened for 9 known mutations (Q70X, A75T, 474-2a>g, L218P, A327P, W402X, P533R, R89Q, 678-7g>a). W402X was the most frequent mutation in our population (45.3%) and Q70X was the second most frequent (15.9%). In 30 families, either one or both of the mutations were not identified, which accounted for 25.9% of the total alleles. Therefore, all 14 exons of the alpha-L-iduronidase gene were screened in these patients and 23 different sequence changes were found, 17 of which were previously unknown. The novel sequence changes include 4 deletions (153delC, 628del5, 740delC, 747delG), 5 nonsense mutations (Q60X, Y167X, Q400X, R619X, R628X), 6 missense mutations (C205Y, G208V, H240R, A319V, P496R, S633L), a splice site mutation (IVS12+5g>a), and a rare polymorphism (A591T). The polymorphism and novel missense mutations were transiently expressed in COS-7 cells and all of them except the polymorphism showed complete loss of enzyme activity. In total, 165 of the 170 mutant alleles were identified in this study and despite the high frequency of W402X and Q70X, the identification of many novel mutations unique to individual families further highlights the genetic heterogeneity of MPS I.     

The traT protein is able to normalize the phenotype of a plasmid-carried permeability mutation of Salmonella typhimurium

The isolation of different classes of antibiotic-supersensitive outer membrane permeability mutants of Salmonella typhimurium has been described previously (Sukupolvi et al., 1984, Journal of Bacteriology 159, 704-712). One of these, the SS-A mutation, sensitizes the bacteria to gentian violet and to hydrophobic antibiotics. The phenotype of the SS-A mutant was restored to normal when a cloned fragment of the F plasmid, or the R plasmid R6-5, carrying the genes traS, T and D was introduced on a multicopy plasmid. The introduction of a plasmid carrying only the traT gene showed that this gene was sufficient to restore the phenotype. Only clones with functioning traT (irrespective of copy number) restored the normal antibiotic-resistant phenotype in the SS-A mutant. An incompatibility test using a donor strain which carried transposon Tn10 in the 60 MDa plasmid of S. typhimurium and a recipient in which Tn5 was placed close to the SS-A mutation indicated that the SS-A mutation was located in the 60 MDa virulence plasmid (previously called the cryptic plasmid) of S. typhimurium. The introduction of the large virulence plasmid carrying the SS-A mutant allele into wild-type S. typhimurium or Escherichia coli resulted in strains with a phenotype identical to that of the original SS-A mutant.     

Towards a model to explain the intragenic complementation in the heteromultimeric protein propionyl-CoA carboxylase

Mutations in the PCCA or PCCB genes coding for alpha and beta subunits of propionyl CoA carboxylase can cause propionic acidemia. To understand the molecular basis of the intragenic complementation previously reported at the PCCB locus, we now examine the complementation behaviour of four carboxy-terminal and 11 amino-terminal naturally occurring mutant alleles both using cell fusion and reconstructing the complementation event by transfecting the mutant cDNAs to generate multimeric hybrid proteins. Alleles carrying mutations p.R410W and p.W531X are able to complement with 10 out of 11 amino-terminal mutations assayed. Only the unstable p.R512C, p.L519P and p.G112D mutants fail to complement. The results analyzed in the framework of the crystal structure of the homologous 12S transcarboxylase from Propionibacterium shermanii show that all mutant alleles studied are located at beta subunits interfaces, complementing alleles at the inter-trimer interface, where the catalysis probably happens, and non-complementing alleles at the intra-trimer interface, probably disrupting the trimer formation. Our results also show a remarkable stabilization effect when p.R410W is cotransfected with p.G246V. We propose a model for intragenic complementation requiring the production of two different beta subunits carrying carboxy and amino-terminal mutations that allow regenerating functional active sites and in which a stabilization effect between subunits could be relevant to ameliorate the biochemical phenotype of each mutation separately.     

The demographics and distribution of type B Niemann-Pick disease: novel mutations lead to new genotype/phenotype correlations

We have collected demographic and/or mutation information on a worldwide sample of 394 patients with type B Niemann-Pick disease (NPD). The disorder is panethnic, with the highest incidence occurring in individuals of Turkish, Arabic, and North African descent. Only five of the 394 patients were Ashkenazi Jewish, revealing that, unlike the type A form of NPD, type B NPD does not occur frequently within this population. Mutation analysis of the acid sphingomyelinase (ASM) gene (designated "SMPD1") was performed on 228 patients (324 unique alleles), and several novel, "common" mutations were found. Among these were the L137P, fsP189, and L549P mutations, which accounted for approximately 75% of the alleles in Turkish patients, the H421Y and K576N mutations, which accounted for approximately 85% of the alleles in Saudi Arabian patients, the S379P, R441X, R474W, and F480L mutations, which accounted for approximately 55% of the alleles in Portuguese/Brazilian patients, and the A196P mutation, which accounted for approximately 42% of the alleles in Scottish/English patients. The previously reported DeltaR608 mutation occurred on approximately 12% of the alleles studied. Overall, a total of 45 novel mutations were found, and several new genotype/phenotype correlations were identified. In particular, the L137P, A196P, and R474W mutations were consistent with a less severe form of type B NPD, whereas the H421Y and K576N mutations led to an early-onset, more severe form that was specific to Saudi Arabia. These data provide the first extensive demographic assessment of this disorder and describe several new mutations that can be used to predict phenotypic outcome and to gain new insights into the structure and function of ASM.     

Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen

The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype ayw mutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtype adr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, the adr variant I97L replicates as well as its parental adr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by the trans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.     

Structure of the SLC7A7 gene and mutational analysis of patients affected by lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of cationic amino acid transport caused by mutations in the SLC7A7 gene. We report the genomic structure of the gene and the results of the mutational analysis in Italian, Tunisian, and Japanese patients. The SLC7A7 gene consists of 10 exons; sequences of all of the exon-intron boundaries are reported here. All of the mutant alleles were characterized and eight novel mutations were detected, including two missense mutations, 242A-->C (M1L) and 1399C-->A (S386R); a nonsense mutation 967G-->A (W242X); two splice mutations IVS3 +1G-->A and IVS6 +1G-->T; a single-base insertion, 786insT; and two 4-bp deletions, 455delCTCT and 1425delTTCT. In addition, a previously reported mutation, 1625insATCA, was found in one patient. It is noteworthy that 242A-->C causes the change of Met1 to Leu, a rare mutational event previously found in a few inherited conditions. We failed to establish a genotype/phenotype correlation. In fact, both intrafamilial and interfamilial phenotypic variability were observed in homozygotes for the same mutation. The DNA-based tests are now easily accessible for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.     

Genetic identification of residues involved in association of alpha and beta G-protein subunits.

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpa1 protein. A mutation at position E307 of the Gpa1 protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant alpha subunit to physically associate with a specific mutant G beta subunit. The mutation in the Gpa1 protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the alpha subunit with the wild-type beta subunit. Yeast cells constructed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified G alpha subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.     

水稻苗期耐盐突变体的遗传分析及基因定位

在籼稻品种R401辐射诱变的M2群体中筛选到一个苗期耐盐突变体, 在150 mmol/L的NaCl溶液处理下对照植株枯萎死亡, 而突变体植株依然存活。以粳稻品种Nipponbare(不耐盐)和耐盐突变体作亲本, 构建了一个F2群体, 调查该群体在150 mmol/L的NaCl溶液胁迫下的表现, 发现Nipponbare和耐盐突变体苗期耐盐性的差异受单个主基因控制, 耐盐为隐性, 将该基因暂时命名为SST(t)。利用该F2群体, 采用集团分离分析(Bulked segregant analysis, BSA)法将SST(t)定位在第6染色体上, 进一步对F2群体中137个典型的耐盐单株的分子标记进行分析, 将该基因定位在InDel标记ID26847和ID27253之间, 约2.3 cM (或406 kb)的区间内, 与两标记分别相距1.2 cM和1.1 cM。     

Importance of opgHXcv of Xanthomonas campestris pv. vesicatoria in host-parasite interactions

Tn5 insertion mutants of Xanthomonas campestris pv. vesicatoria were inoculated into tomato and screened for reduced virulence. One mutant exhibited reduced aggressiveness and attenuated growth in planta. Southern blot analyses indicated that the mutant carried a single Tn5 insertion not associated with previously cloned pathogenicity-related genes of X. campestris pv. vesicatoria. The wild-type phenotype of this mutant was restored by one recombinant plasmid (pOPG361) selected from a genomic library of X. campestris pv. vesicatoria 91-118. Tn3-gus insertion mutagenesis and sequence analyses of a subclone of pOPG361 identified a 1,929-bp open reading frame (ORF) essential for complementation of the mutants. The predicted protein encoded by this ORF was highly homologous to the previously reported pathogenicity-related HrpM protein of Pseudomonas syringae pv. syringae and OpgH of Erwinia chrysanthemi. Based on homology, the new locus was designated opgHXcv. Manipulation of the osmotic potential in the intercellular spaces of tomato leaves by addition of mannitol at low concentrations (25 to 50 mM) compensates for the opgHXcv mutation.