Comparative analysis of <Emphasis Type="Italic">Agropyron intermedium</Emphasis> (Host) Beauv 6Ag<Superscript>i</Superscript> and 6Ag<Superscript>i</Superscript>2 chromosomes in bread wheat cultivars and lines with wheat–wheatgrass substitutions

A comparative study of wheat–wheatgrass substituted cultivars and lines resistant to leaf rust developed by the Agricultural Research Institute for Southeast Regions (Multi 6R, Belyanka, Favorit, Voevoda, Lebedushka) and Samara Agricultural Research Institute (Tulaikovskaya 5, Tulaikovskaya 10, Tulaikovskaya 100, Tulaikovskaya Zolotistaya) breeding was conducted. A complex analysis using molecular cytogenetic (C-differential banding, fluorescent (FISH) and genomic (GISH) in situ hybridization), molecular (PLUG markers), and biochemical (electrophoretic analysis of gliadins) markers demonstrated that they have a substitition of wheat chromosome 6D by the chromosomes 6Agⁱ and 6Agⁱ2 belonging to the J(=E) Agropyron intermedium (Host) Beauv (=Thinopyrum intermedium (Host) Barkworth & D.R. Dewey) subgenome. In spite of the fact that the chromosomes 6Agⁱ and 6Agⁱ2 differ in the C-banding pattern and demonstrated minor differences in the blocks of gliadin components, they had the identical pattern of pSc119.2 and pAs1 probe distribution and conjugated between themselves with insignificant disturbance. Thus, it was demonstrated that 6Agi and 6Agⁱ2 are homologous chromosomes; however, the question about allelism of their leaf rust resistance genes between themselves requires special studies. Nevertheless, using STS and SCAR markers and taking into account the type of reaction to Puccinia triticina, their non-allelism to the Lr9, Lr19, Lr24, Lr29, Lr38, and Lr47 genes was established. It was revealed that the 6Agⁱ and 6Agⁱ2 chromosomes have a different level of transmission in hybrid F₂ populations depending on the hybrid combination gene background.     

Localization of the rice stripe disease resistance gene, Stv-bi, by graphical genotyping and linkage analyses with molecular markers

 We used graphical genotyping and linkage analyses with molecular markers to determine the chromosomal location of the rice stripe disease resistance gene, Stv-b ⁱ . The stripe resistance gene from the indica rice (Oryza sativa) cv ‘Modan’ was introgressed into several Japanese rice varieties. We found 4 RFLP markers in ‘Modan’, five susceptible parental rice varieties (‘Norin No. 8’, ‘Sachihikari’, ‘Kanto No. 98’, ‘Hokuriku No.103’ and ‘Koganebare’) and four resistant progeny varieties (‘St. No. 1’, ‘Aichi No. 6’, ‘Aoisora’ and ‘Asanohikari’). Graphical genotyping of the resistant progeny revealed a chromosomal segment ascribable to ‘Modan’ and associated with stripe resistance. The chromosomal segment from ‘Modan’ was located at 35.85 cM on chromosome 11. Linkage analysis using 120 F₂ individuals from a cross between ‘Koshihikari’ (susceptible) and ‘Asanohikari’ (resistant) revealed another 8 RFLP markers in the same chromosome. We performed a bioassay for rice stripe resistance in F₃ lines of the F₂ individuals using infective small brown planthoppers and identified an 1.8-cM segment harboring the rice stripe disease resistance gene, Stv-b ⁱ , between XNpb220 and XNpb257/ XNpb254. Furthermore, Stv-b ⁱ was linked by 0.0 cM to a RFLP marker, ST10, which was developed on the basis of the results of RAPD analysis. These DNA markers near the Stv-b ⁱ locus may be useful in marker-assisted selection and map-based cloning of the Stv-b ⁱ gene. Received: 26 September 1997 / Accepted: 4 November 1997     

Periplasmic domain of CusA in an <Emphasis Type="Italic">Escherichia coli</Emphasis> Cu<Superscript>+</Superscript>/Ag<Superscript>+</Superscript> transporter has metal binding sites

The resistance nodulation division (RND)-type efflux systems are utilized in Gram-negative bacteria to export a variety of substrates. The CusCFBA system is the Cu⁺ and Ag⁺ efflux system in Escherichia coli, conferring resistance to lethal concentrations of Cu⁺ and Ag⁺. The periplasmic component, CusB, which is essential for the assembly of the protein complex, has Cu⁺ or Ag⁺ binding sites. The twelve-span membrane protein CusA is a homotrimeric transporter, and has a relatively large periplasmic domain. Here, we constructed the periplasmic domain of CusA by joining two DNA segments and then successfully expressed and purified the protein. Isothermal titration calorimetry experiments revealed Ag⁺ binding sites with Kds of 10⁻⁶–10⁻⁵ M. Our findings suggest that the metal binding in the periplasmic domain of CusA might play an important role in the function of the efflux pump.     

Genetic control for resistance to leaf rust in wheat-Agropyron lines: Agro 139 and Agro 58

Leaf rust resistance lines of Triticum aestivum carry highly effective Lr genes from Agropyron intermedium (Host) Beauv. This Agro 58 and Agro 139 resistance segregated independently of Agropyron leaf-rust resistance genes Lr-19, Lr-24 and Lr-9 from Ae. umbellulata. Monosomic analysis showed that the Lr gene in Agro 139 was incorporated into wheat chromosome 6D. C-banding analysis could not determine the C-banding pattern of A. intermedium in wheat -Agropyron lines Agro 58 and Agro 139. It is assumed that the transfers occurred from the euchromatin regions of the Agropyron chromosomes to the euchromatin regions of the wheat chromosomes. It is suggested that the Lr gene from Agro 139 be designated LrAg ⁱ-1 and the Lr gene from Agro 58 designated LrAg ⁱ-2.     

Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme on which the pentose phosphate pathway was checked. In this study, purification of a G6PD enzyme was carried out by using rat erythrocytes with a specific activity of 13.7 EU/mg and a yield of 67.7 and 155.6‐fold by using 2′,5′‐ADP Sepharose‐4B affinity column chromatography. For the purpose of identifying the purity of enzyme and molecular mass of the subunit, a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out. The molecular mass of subunit was calculated 56.5 kDa approximately. Then, an investigation was carried out regarding the inhibitory effects caused by various metal ions (Fe²⁺, Pb²⁺, Cd²⁺, Ag⁺, and Zn²⁺) on G6PD enzyme activities, as per Beutler method at 340 nm under in vitro conditions. Lineweaver–Burk diagrams were used for estimation of the IC₅₀ and K_i values for the metals. K_i values for Pb⁺², Cd⁺², Ag⁺, and Zn⁺² were 113.3, 215.2, 19.4, and 474.7 μM, respectively.     

Chromosomal location and molecular mapping of a tan spot resistance gene in the winter wheat cultivar Red Chief

The winter wheat cultivar Red Chief has been identified as the wheat cultivar most resistant toPyrenophora tritici-repentis (Ptr). This study was undertaken to determine the inheritance, chromosomal location and molecular mapping of a tan spot resistance gene in Red Chief. χ² analysis of the F₂ segregation data of the hybrids between 21 monosomic lines of the susceptible wheat cultivar Chinese Spring and the resistant cultivar Red Chief revealed that tan spot resistance in cv. Red Chief is controlled by a single recessive gene located on chromosome 3A. Linkage analysis using SSR markers in the Red Chief/Chinese Spring F2 population showed that thetsr4 gene is clustered in the region aroundXgwm2a, on the short arm of chromosome 3A. This marker has also been identified as the closest marker to thetsr3 locus on chromosome 3D in synthetic wheat lines. Validation analysis of this marker for thetsr3 andtsr4 genes using 28 resistant and 6 susceptible genotypes indicated that the 120 bp allele (thetsr3 gene) specific fragment was observed in 11 resistant genotypes, including the three synthetic lines XX41, XX45 and XX110, while the 130 bp allele was amplified only in cv. Red Chief and Dashen.Xgwm2a can be used to trace the presence of the target gene in successive backcross generations and pyramiding of thetsr3 &tsr4 genes into a commonly grown and adaptable cultivar.     

Fine mapping of <Emphasis Type="Italic">qSTV11</Emphasis><Superscript><Emphasis Type="Italic">TQ</Emphasis></Superscript>, a major gene conferring resistance to rice stripe disease

The indica rice cultivar, Teqing, shows a high level of resistance to rice stripe virus (RSV). It is believed that this resistance is controlled by the gene, qSTV11 ^TQ . For positional cloning of the resistance gene, a set of chromosome single segment substitution lines (CSSSLs) was constructed, all of which had the genetic background of the susceptible japonica cultivar, Lemont, with different single substituted segments of Teqing on chromosome 11. By identifying the resistance of the CSSSLs-2006 in a field within a heavily diseased area, the resistance gene qSTV11 ^TQ was mapped between the markers Indel7 and RM229. Furthermore, in that region, six new markers were developed and 52 subregion CSSSLs (CSSSLs-2007) were constructed. The natural infection experiment was conducted again at different sites, with two replicates used in each site in order to identify the resistance phenotypes of the CSSSLs-2007 and resistant/susceptible controls in 2007. Through the results of 2007, qSTV11 ^TQ was localized in a region defined by the markers, CAPs1 and Indel4. In order to further confirm the position of qSTV11 ^TQ , another set of subregion CSSSLs (CSSSLs-2009) was constructed. Finally, qSTV11 ^TQ was localized to a 55.7 kb region containing nine annotated genes according to the genome sequence of japonica Nipponbare. The relationship between qSTV11 ^TQ and Stvb-i (Hayano-Saito et al. in Theor Appl Genet 101:59–63, 2000) and the reliability of the markers used on both sides of qSTV11 ^TQ for marker-assisted breeding of resistance to rice stripe disease are discussed.     

A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli

A barley cDNA clone encoding a cysteine proteinase inhibitor was characterized. The deduced amino acid sequence of this barley cystatin (Hv-CPI) contains the motif QXVXG conserved among members of the cystatin superfamily. The gene (Icy), located on chromosome 2, was expressed in embryos, developing endosperms, leaves and roots as assessed by northern blot analysis. Western blot analysis detected a slightly retarded band in leaves that was not present in roots or seeds. In these two organs a more precise location of Hv-CPI was done by immuno-histochemical analysis, with polyclonal antibodies raised against the recombinant CPI protein expressed in Escherichia coli. This protein efficiently inhibited papain (K _i 2.0×10^–8 M) and ficin (K _i 2.2×10^–8 M) and, to a lesser extent, chymopapain (K _i 1.6×10^–7 M) and was inactive against bromelain. The Icy mRNA expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 °C).these authors contributed equally to this work     

A Single Genetic Determinant that Prevents Sex Reversal in C57BL-YPOS Congenic Mice

Sex determination in the mammalian embryo begins with the activation of a gene on the Y chromosome which triggers a cascade of events that lead to male development. The mechanism by which this gene, designated SRY in humans and Sry in mice (sex determining region of the Y chromosome), is activated remains unknown. Likewise, the downstream target genes for Sry remain unidentified at present. C57BL mice carrying a Y chromosome from Mus musculus musculus or molossinus develop normally as males. In contrast, C57BL/6 mice with the Y chromosome from M. m. domesticus often show sex reversal, i.e., develop as XY females. It has been documented that C57BL mice with the Y chromosome from Poschiavinus (Y^POS), a domesticus subtype, always develop as females or hermaphrodites. This suggests that a C57BL gene either up- or downstream of Sry is ineffective in interacting with Sry, which then compromises the processes that lead to normal male sex development. Nonetheless, by selective breeding, we have been able to generate a sex reversal-resistant C57BL/6-congenic strain of mice in which the XY^POS individuals consistently develop as normal males with bilateral testes. Because the resistance to sex reversal was transferred from strain 129S1/Sv (nonalbino) by simple selection over 13 backcross generations, it is inferred that a single autosomal gene or chromosomal region confers resistance to the sex reversal that would otherwise result. XY^POS normal males generated in these crosses were compared to XY^POS abnormal individuals and to C57BL/6 controls for sexual phenotype, gonadal weight, serum testosterone, and major urinary protein (MUP) level. A clear correlation was found among phenotypic sex, MUP level, and testis weight in the males and in the incompletely masculinized XY^POS mice. The fully masculinized males of the congenic strain resemble C57BL/6 males in the tested parameters. DNA analysis confirmed that these males, in fact, carry the Y^POS Sry gene.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.This Paper is dedicated to the memory of Professor James B. Boyd     

A suicide vector for allelic recombination involving the gene for glutamate 1-semialdehyde aminotransferase in the cyanobacterium Synechococcus PCC 7942

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C₅ pathway for the synthesis of δ-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 μM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 μM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemL ^GR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemL ^WT had retained its sensitivity. Southern hybridisation using gene probes for hemL, amp ^r and cm ^r confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 μM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine. Received: 7 October 1996 / Accepted: 15 January 1997     

SSR markers closely associated with genes for resistance to root-knot nematode on chromosomes 11 and 14 of Upland cotton

Molecular markers closely linked to genes that confer a high level of resistance to root-knot nematode (RKN) [Meloidogyne incognita (Kofoid & White) Chitwood] in cotton (Gossypium hirsutum L.) germplasm derived from Auburn 623 RNR would greatly facilitate cotton breeding programs. Our objectives were to identify simple sequence repeat (SSR) markers linked to RKN resistance quantitative trait loci (QTL) and map these markers to specific chromosomes. We developed three recombinant inbred line (RIL) populations by single seed descent from the crosses of RKN-resistant parents M-240 RNR (M240), developed from the Auburn 623 RNR source, moderately resistant Clevewilt 6 (CLW6), one of the parents of Auburn 623 RNR, and susceptible parent Stoneville 213 (ST213). These crosses were CLW6 × ST213, M240 × CLW6, and M240 × ST213. RILs from these populations were grown under greenhouse conditions, inoculated with RKN eggs, scored for root gall index, eggs plant⁻¹, and eggs g⁻¹ root. Plants were also genotyped with SSR markers. Results indicated that a minimum of two major genes were involved in the RKN resistance of M240. One gene was localized to chromosome 11 and linked to the marker CIR 316-201. This CIR 316-201 allele was also present in CLW6 but not in Mexico Wild (MW) (PI593649), both of which are parents of Auburn 623 RNR. A second RKN resistance gene was localized to the short arm of chromosome 14 and was linked to the SSR markers BNL3545-118 and BNL3661-185. These two marker alleles were not present in CLW6 but were present in MW. Our data also suggest that the chromosome 11 resistance QTL primarily affects root galling while the QTL on chromosome 14 mediates reduced RKN egg production. The SSRs identified in this study should be useful to select plants with high levels of RKN resistance in segregating populations derived from Auburn 623 RNR.     

Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat

Genomic in situhybridization (GISH) to root-tip cells at mitotic metaphase, using genomic DNA probes from Thinopyrum intermedium and Pseudoroegneria strigosa, was used to examine the genomic constitution of Th. intermedium, the 56-chromosome partial amphiploid to wheat called Zhong 5 and disease-resistant derivatives of Zhong 5, in a wheat background. Evidence from GISH indicated that Th. intermedium contained seven pairs of St, seven J^S and 21 J chromosomes; three pairs of Th. intermedium chromosomes with satellites in their short arms belonging to the St, J, J genomes and homoeologous groups 1, 1, and 5 respectively. GISH results using different materials and different probes showed that seven pairs of added Th. intermedium chromosomes in Zhong 5 included three pairs of St chromosomes, two pairs of J^S chromosomes and two pairs of St-J^S reciprocal tanslocation chromosomes. A pair of chromosomes, which substituted a pair of wheat chromosomes in Yi 4212 and in HG 295 and was added to 21 pairs of wheat chromosomes in the disomic additions Z1, Z2 and Z6, conferred BYDV-resistance and was identical to a pair of St-J^S tanslocation chromosomes (StJ^S) in Zhong 5. The StJ^S chromosome had a special GISH signal pattern and could be easily distinguished from other added chromosomes in Zhong 5; it has not yet been possible to locate the BYDV-resistant gene(s) of this translocated chromosome either in the St chromosome portion belonging to homoeologous group 2 or in the J^S chromosome portion whose homoeologous group relationship is still uncertain. Among 22 chromosome pairs in disomic addition line Z3, the added chromosome pair had satellites and belonged to the St genome and homoeologous group 1. Disomic addition line Z4 carried a pair of added chromosomes which was composed of a group-7 J^S chromosome translocated with a wheat chromosome; this chromosome was different to 7 Ai-1, but was identical to 7 Ai-2. The leaf rust and stem rust resistance genes were located in the distal region of the long arm, whereas the stripe rust resistance gene(s) was located in the short arm or in the proximal region of the long arm of 7 Ai-2. A pair of J^S-wheat translocation chromosomes, which originated from the WJ^S chromosomes in Z4, was added to the disomic addition line Z5; the added chromosomes of Z5 carried leaf and stem rust resistance but not stripe rust resistance; Z5 is a potentially useful source for rust resistance genes in wheat breeding and for cloning these novel rust-resistant genes. GISH analysis using the St genome as a probe has proved advantageous in identifying alien Th. intermedium in wheat. Received: 17 May 1999 / Accepted: 22 June 1999     

The detection of mitotic and meiotic aneuploidy in yeast using a gene dosage selection system

Summary A system is described in which spontaneous and chemically-induced mitotic and meiotic hyperploidy can be assayed in the same diploid culture of Saccharomyces cerevisiae. Monitoring gene dosage changes at two loci on chromosome VIII, the test utilizes a leaky temperature-sensitive allele arg4-8 and low level copper resistance conferred by the single copy allele cup1 ^s. An extra chromosome VIII provides simultaneous increased dosage for both genes, resulting in colonies that are both prototrophic for arginine at 30° C and copper resistant. During mitotic cell divisions in diploids, spontaneous chromosome VIII hyperploids (trisomes and tetrasomes) occur at a frequency of 6.4×10^-6 per viable cell. Among ascospores, the spontaneous chromosome VIII disome frequency is 5.5×10^-6 per viable spore. The tubulin-binding reagent methyl benzimidazol-2-yl carbamate (MBC) elicits enhanced levels of mitotic and meiotic aneuploidy relative to control levels. The system represents a novel model for examining chromosome behavior during mitosis and meiosis and provides a sensitive and quantifiable procedure for examining chemically induced aneuploidy.     

Analysis of Inheritance of Morphological and Biochemical Characters Introgressed into Common Wheat from <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch.

Genetic control of some morphological traits and the gliadin composition were examined in plants of two lines of common wheat carrying genes introgressed from the wild diploid cereal Aegilops speltoides. Leaf hairiness was shown to be controlled by a single introgressed dominant gene that was not allelic to the known common wheat gene Hl1. Waxlessness of the whole plant is controlled by the introgressed from Ae. speltoides inhibitor gene allelic to gene W1 ^I located on chromosome 2B. This gene was epistatic to the introgressed gene controlling spike waxlessness. The introgressed gene of spike color was shown to be allelic to Rg1 located on chromosome 1B of common wheat. However, the former gene proved to be linked to an allele of the Gli-B1 locus other than in wheat.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 793–799.Original Russian Text Copyright © 2005 by Pshenichnikova, Lapochkina, Shchukina, Berezovskaya, Trufanov.     

The genetic basis of negative selection of Tcrb-Vll+ T cells

Non-H-2 genes responsible for negative selection of Tcrb-V 11⁺ T cells were examined using backcross mice of various strains with C58, which does not delete Tcrb-V 11⁺ T cells. Two independently segregating genes were found: one leading to partial deletion was closely linked toLy-2/Ly-3 on chromosome 6, and the second giving virtually complete deletion has not yet been mapped. The A strain had only the former, whereas BALB/c, BALBK, B10.BR, CBA-T6, C3H/He, and DBA/2 expressed both of these genes. Although a gene(s) of the NIH strain led only to partial deletion, the chromosomal localization of the gene(s) has not yet been determined: no informative polymorphic molecules are expressed from genes on chromosome 6 of this strain.     

Genetic and physical mapping of the S<Subscript>H</Subscript>3 region that confers resistance to leaf rust in coffee tree (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.)

Resistance to coffee leaf rust is conferred by S_H3, a major dominant gene that has been introgressed from a wild coffee species Coffea liberica (genome L) into the allotetraploid cultivated species, Coffea arabica (genome C^aE^a). As the first step toward the map-based cloning of the S_H3 gene, using a bacterial artificial chromosome (BAC) library, we describe the construction of a physical map in C. arabica spanning the resistance locus. This physical map consists in two homeologous BAC-contigs of 1,170 and 1,208 kb corresponding to the subgenomes C^a and E^a, respectively. Genetic analysis was performed using a single nucleotide polymorphism detection assay based on Sanger sequencing of amplicons. The C. liberica-derived chromosome segment that carries the S_H3 resistance gene appeared to be introgressed on the sub-genome C^a. The position of the S_H3 locus was delimited within an interval of 550 kb on the physical map. In addition, our results indicated a sixfold reduction in recombination frequency in the introgressed S_H3 region compared to the orthologous region in Coffea canephora.     

Genetic position and amino acid replacements of several mutations in ribosomal protein S5 from Escherichia coli.

Summary The relative genetic position of the following four mutations of ribosomal protein S5 has been determined: spc-13, a mutation to spectinomycin resistance; str ⁱ _N421 and str ⁱ _d1023, mutations suppressing dependence on streptomycin and sup _0–1, a mutation suppressing partially the temperature-sensitive phenotype of an alanyl-tRNA synthetase mutation. The transduction experiments performed indicate that the spc-13 site is located in the S5 cistron proximal to the strA locus, that sup _0–1 maps proximal to the aroE gene and that the str ⁱ _N421 and str ⁱ _d1023 loci are located between these two mutational sites.Proteinchemical analysis of the amino acid replacement in protein S5 of strain N421 (carrying the str ⁱ _N421 allele) has shown that an arginine residue is replaced by leucine which results in the appearance of a trypsin intensitive bond between the tryptic peptides T2 and T16. The same alteration has been previously found by Itoh and Wittmann (1973) in the S5 protein of strain d1023.Determination of the alteration of ribosomal protein S5 of strain 0–1 (sup _0–1 allele) revealed that the C-terminal tryptic peptide is altered. It differs from that of the wild-type protein by the lack of five amino acids and the appearance of a C-terminal glycine residue instead of a lysine residue. This change can be explained by the deletion of eleven nucleotides in the S5 cistron of strain 0–1.The recent determination of the primary structure of ribosomal protein S5 (Wittmann-Liebold and Greuer, 1975) allows the ordering of the S5 alterations employed: The order is spc-13-str ⁱ _d1023 (str ⁱ _N421)-sup _0–1 with the spc-13 amino acid replacement being located at the NH₂-terminal portion of the S5 sequence and the alteration of strain 0–1 at the COOH-terminal end. The proteinchemical results are therefore in full agreement with the genetic data and unambiguously allow the conclusion that the S5 cistron is transcribed counterclock-wise on the Escherichia coli chromosome.     

SilE is an intrinsically disordered periplasmic “molecular sponge” involved in bacterial silver resistance

Ag⁺ resistance was initially found on the Salmonella enetrica serovar Typhimurium multi‐resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag⁺ resistance, encoded by the sil operon from pMG101, involves export of Ag⁺ via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag⁺ (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo‐form but folds to a compact structure upon optimal binding to six Ag⁺ ions in its holo‐form. Sequence analyses and site‐directed mutagenesis established the importance of histidine and methionine containing motifs for Ag⁺‐binding, and identified a nucleation core that initiates Ag⁺‐mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions.