Organization of the Chorion Genes of BOMBYX MORI, a Multigene Family. II. Partial Localization of Three Gene Clusters

Chorion genes of the inbred stock Ascoli have been localized to three linked clusters by analysis of testcross progeny. Electrophoretic variants screened by isoelectric focusing served as markers. The clusters are designated Ch 1, Ch 2, and Ch 3. The gene order is Ch 1–Ch 2–Ch 3–Y, with relative map distances of approximately 0.4 m.u. for Ch 1–2, 3.3 ± 0.9 m.u. for Ch 2–3, and 21 m.u. for Ch 3-Y. In a separate testcross using different markers, two chorion regions were localized 2.3 m.u. and 3.1 m.u. from p. These markers could not be assigned to Ch 1, 2, or 3 because there is at present no test for allelism in this system.     

Organization and developmental timing of the Bombyx mori chorion gene clusters in strain C108

We have standardized the map of chorion structural gene clusters in Bombyx mori strain C108 by analyzing quantitative and qualitative chorion electrophoretic markers in recombinant progeny from four independent crosses. In all we assigned 22 markers to three gene clusters, representing about one-third of the total number of chorion genes: 2 to Ch 1, 9 to Ch 2, and 8 to Ch 3. Three additional markers belong either to Ch 7 or Ch 2. By referring to published chorion protein synthesis patterns, we show that the clusters are restricted in their developmental specificities: Ch 3 appears to be an early locus, carrying all of the mapped early markers (4) and half of the early middles (3/6), while Ch 1 and Ch 2 carry predominantly middle (4/5) and all late, Hc (6) markers, along with some early middle markers (3). We cite evidence to show that Ch 1 and Ch 2 compose the left and right halves of a single gene cluster, which we formally designate as Ch 1–2.     

Development of T. aestivum L.-H. californicum Alien Chromosome Lines and Assignment of Homoeologous Groups of Hordeum californicum Chromosomes

Hordeum californicum （2n = 2x = 14, HH） is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring （CS）-H. californicum amphidiploid （2n = 6x = 56, AABBDDHH） was established. By genomic in situ hybridization （GISH） and multicolor fluorescent in situ hybridization （FISH） using repetitive DNA clones （pTa71, pTa794 and pSc119.2） as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheat--alien chromosome lines, including four disomic addition lines （DAH1, DAH3, DAH5 and DAH6）, five telosomic addition lines （MtH7L, MtHIS, MtH1L, DtH6S and DtH6L）, one multiple addition line involving H. californicum chromosome H2, one disomic substitution line （DSH4） and one translocation line （TH7S/1BL）, were identified from the progenies derived from the crosses of CS-H. californicum amphidiploid with common wheat varieties. A total of 482 EST （expressed sequence tag） or SSR （simple sequence repeat） markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2, H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5Hc, 2He, 6Hc, 3Hc and 1Hc, respectively. The chromosomes H1 and H6 were designated as 7Hc and 4Hc, respectively, by referring to SSR markers located on rye chromosomes.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca²⁺. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) of 105 ± 15 kcal/mol, in the absence of Ca²⁺. In the presence of Ca²⁺ concentrations sufficient to saturate its sites on Factor IX, the T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca²⁺, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca²⁺, Factor IXα displayed thermograms characterized by a T_M of 51.0 ± 0.5 °C and a ΔH_c of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca²⁺ (the values in the presence of saturating Ca²⁺ are given in parentheses), undergoes thermal denaturation with a T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca²⁺ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.     

Organization of the Chorion Genes of BOMBYX MORI, a Multigene Family. I. Evidence for Linkage to Chromosome 2

The chorion genes of silkmoths comprise a multigene family that codes for 50 or more highly specialized structural proteins found in the eggshell. A detailed study of the chromosomal organization of these genes was initiated, using inbred stocks of Bombyx mori as a source of electrophoretic variants for genetic markers. Chorion protein patterns were screened on thin-slab polyacrylamide isoelectric focusing gels. A wide range of polymorphism was observed between stocks. However, isoelectric focusing patterns obtained within a stock were nearly homogeneous, indicating that inbreeding has produced a high degree of homozygosis. Testcrosses were carried out to examine the linkage relationships between electrophoretic markers in four inbred stocks. One race (C108) was selected as a standard against which to compare the inheritance of the variants found in the other three stocks. Chorion markers behaved like codominant Mendelian traits in F₁ crosses. A total of 15 out of 16 C108 markers cosegregated in subsequent testcrosses, indicating that they are linked. These genes were mapped to the second chromosome, using markers Gr and Y.     

A microsatellite-based genetic linkage map of the cichlid fish, Astatotilapia burtoni (Teleostei): a comparison of genomic architectures among rapidly speciating cichlids

Cichlid fishes compose an astonishingly large number of species and formed species flocks in record-breaking time. To facilitate efficient genome scans and comparisons of cichlid genomes, we constructed a medium-density genetic linkage map of microsatellite markers of Astatotilapia burtoni. The mapping cross was derived from two inbred laboratory lines to obtain F₂ progeny by intercrossing. The map revealed 25 linkage groups spanning 1249.3 cM of the genome (size ~950 Mb) with an average marker spacing of 6.12 cM. The seven Hox clusters, ParaHox C1, and two paralogs of Pdgfrβ were mapped to different linkage groups, thus supporting the hypothesis of a teleost-specific genome duplication. The A. burtoni linkage map was compared to the other two available maps for cichlids using shared markers that showed conservation and synteny among East African cichlid genomes. Interesting candidate genes for cichlid speciation were mapped using SNP markers.     

Hyperdiversity of Genes Encoding Integral Light-Harvesting Proteins in the Dinoflagellate Symbiodinium sp

The superfamily of light-harvesting complex (LHC) proteins is comprised of proteins with diverse functions in light-harvesting and photoprotection. LHC proteins bind chlorophyll (Chl) and carotenoids and include a family of LHCs that bind Chl a and c. Dinophytes (dinoflagellates) are predominantly Chl c binding algal taxa, bind peridinin or fucoxanthin as the primary carotenoid, and can possess a number of LHC subfamilies. Here we report 11 LHC sequences for the chlorophyll a-chlorophyll c ₂-peridinin protein complex (acpPC) subfamily isolated from Symbiodinium sp. C3, an ecologically important peridinin binding dinoflagellate taxa. Phylogenetic analysis of these proteins suggests the acpPC subfamily forms at least three clades within the Chl a/c binding LHC family; Clade 1 clusters with rhodophyte, cryptophyte and peridinin binding dinoflagellate sequences, Clade 2 with peridinin binding dinoflagellate sequences only and Clades 3 with heterokontophytes, fucoxanthin and peridinin binding dinoflagellate sequences.     

Generation of allantoin from the oxidation of urate by cytochrome c and its possible role in Reye's syndrome

Allantoin in the presence of calcium ions has been implicated as a potential toxic agent in Reye's syndrome. An investigation of possible alternative sources of allantoin in humans, which lack the enzyme uricase, has been initiated. Urate is a strong reducing agent which can reduce cytochrome c nonenzymatically, with the concomitant production of CO₂ and H⁺. The stoichiometries measured for the various reactants and products were 1 urate:2 cytochrome c:1 H⁺:1 CO₂. The initial reaction rate depended on the concentrations of both urate and cytochrome c, with reaction kinetics that were first order with respect to urate and second order with respect to cytochrome c. The participation of molecular oxygen in this reaction could not be detected. The pH and ionic strength optima for this reaction were determined to be 9.5–10.5 and 10⁻⁵m, respectively. Based on the results reported here, the following balanced equation can be written: urate⁻² + 2 cytochrome c⁺³ + 2 H₂O → allantoin + 2 cytochrome c⁺² + H⁺ + HCO₃⁻. We propose that allantoin can be generated from the oxidation of urate by cytochrome c⁺³, and that this is a potential source of allantoin in human tissues.     

An aluminoborate cluster [AlB12O14(OH)12]: Synthesis and structure of [C5H6N][AlB12O14(OH)12]

A new aluminoborate, [C₅H₆N][AlB₁₂O₁₄(OH)₁₂], has been hydrothermally synthesized at 200 °C. The single-crystal diffraction study reveals that it crystallizes in space group C2/c. It consists of aluminoborate clusters [AlB₁₂O₁₄(OH)₁₂]⁻ and counterions [C₅H₆N]⁺. The aluminoborate cluster contains an Al(OH)₆ octahedron as a core that is capped by two raft-like polyborate units [B₆O₇(OH)₆]. These clusters are further interlinked by extensive hydrogen bonding to form a three-dimensional (3D) network, containing large channels along the b-axis, in which the [C₅H₆N]⁺ cations are located.     

Obstruction of Transmembrane Helical Movements in Subunit a Blocks Proton Pumping by F1Fo ATP Synthase

Subunit a plays a key role in promoting H⁺ transport-coupled rotary motion of the subunit c ring in F₁F_o ATP synthase. H⁺ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F_o subunit c. H⁺ are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2–5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H⁺ pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H⁺ pumping by 85–90%. H⁺ pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H⁺ access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H⁺ release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function.     

Whole-Genome Optical Mapping and Finished Genome Sequence of Sphingobacterium deserti sp. nov., a New Species Isolated from the Western Desert of China

A novel Gram-negative bacterium, designated ZW^T, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5–42°C with an optimum of 30°C, and at pH 7.0–11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c or C_16:1 ω6c/C_16:1 ω7c) (39.22%), iso-C15:0 (27.91%), iso-C_17:0 3OH (15.21%), C_16:0 (4.98%), iso-C_15:0 3OH (3.03%), C16:0 3OH (5.39%) and C_14:0 (1.74%). The major polar lipid of strain ZW^T is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZW^T represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZW^T (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZW^T, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZW^T features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZW^T with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZW^T to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant.

Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552,+0.19 V, is markedly less than that of horse heart cytochrome c. (2) ?H_ox³ of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) ?H_ox³ and ?S_red³ of cytoochrome c-552 are more negative than those of horse heart cytochrome c. (4) k_red of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

Algoriphagus boseongensis sp. nov., a member of the family Cyclobacteriaceae isolated from a tidal flat

A Gram-stain negative, aerobic, non-motile, non-spore-forming and rod-shaped bacterial strain, BS-R1^T, was isolated from a tidal flat at Boseong, South Korea. Strain BS-R1^T was found to grow optimally at 30 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BS-R1^T belongs to the genus Algoriphagus, clustering consistently with the type strain of Algoriphagus mannitolivorans, with which it exhibited 98.4 % sequence similarity. Sequence similarities between strain BS-R1^T and the type strains of the other Algoriphagus species were between 92.7 and 97.0 %. Strain BS-R1^T was found to contain MK-7 as the predominant menaquinone and iso-C_15:0, iso-C_17:0 3-OH and summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) as the major fatty acids. The major polar lipids of strain BS-R1^T were identified as phosphatidylcholine, phosphatidylethanolamine and one unidentified lipid. The DNA G+C content was determined to be 42.3 mol% and its mean DNA–DNA relatedness values with A. mannitolivorans KACC 11349^T was 17 ± 5 %. The phylogenetic and genetic distinctiveness and differential phenotypic properties demonstrated that strain BS-R1^T is distinguishable from the other Algoriphagus species as well as A. mannitolivorans. On the basis of the data presented, strain BS-R1^T is considered to represent a novel species of the genus Algoriphagus, for which the name Algoriphagus boseongensis sp. nov. is proposed. The type strain is BS-R1^T (=KCTC 32580^T = CECT 8446^T).     

<Emphasis Type="Italic">Streptomyces thermoalkaliphilus</Emphasis> sp. nov., an alkaline cellulase producing thermophilic actinomycete isolated from tropical rainforest soil

During an investigation exploring potential sources of novel thermophilic species and natural products, a novel thermophilic and alkaliphilic actinomycete with alkaline cellulase producing ability, designated strain 4-2-13^T, was isolated from soil of a tropical rainforest in Xishuangbanna, Yunnan province, China. The morphological and chemotaxonomic characteristics of strain 4-2-13^T are consistent with those of the members of the genus Streptomyces. The strain forms extensively branched aerial mycelia and substrate mycelia. Spiral spore chains were observed on aerial mycelia; spores were oval to cylindrical, with smooth surfaces. The organism was found to contain ll-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan. The whole cell hydrolysates were found to contain glucose and ribose. The cellular fatty acid profile mainly consists of anteiso-C_17:0 and iso-C_16:0. The menaquinones were identified as MK-9(H₈), MK-10(H₆) and MK-9(H₆). The polar lipids profile were found to consist of diphosphatidylglycerol, phosphatidylmethylethanolamine, a ninhydrin-positive glycophospholipid, phosphatidylinositol, phosphatidylglycerol and unidentified glycolipids. The 16S rRNA gene sequence analysis showed that the organism belongs to the genus Streptomyces and in the 16S rRNA gene tree it formed a distinct phyletic line together with the closely related type strain Streptomyces burgazadensis Z1R7^T (95.2% sequence similarity). However, the phenotypic characteristics of strain 4-2-13^T are significantly different from those of S. burgazadensis Z1R7^T. Based on the phenotypic, chemotaxonomic and phylogenetic characteristics, strain 4-2-13^T represents a novel species in the genus Streptomyces, for which the name Streptomyces thermoalkaliphilus sp. nov. is proposed. The type strain is 4-2-13^T (= DSM 42159^T = CGMCC 4. 7205^T).     

Geodermatophilus siccatus sp. nov., isolated from arid sand of the Saharan desert in Chad

A novel Gram-positive, aerobic, actinobacterial strain, CF6/1^T, was isolated in 2007 during environmental screening of arid desert soil in the Sahara near to Ourba, Chad. The isolate was found to grow best in a temperature range of 20–37 °C and at pH 6.0–8.5 and showed no NaCl tolerance, forming black-coloured and nearly circular colonies on GYM agar. Chemotaxonomic and molecular characteristics determined for the isolate match those previously described for members of the genus Geodermatophilus. The DNA G + C content of the novel strain was determined to be 74.9 mol %. The peptidoglycan was found to contain meso-diaminopimelic acid as the diagnostic diamino acid. The main phospholipids were determined to be phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, diphosphatidylglycerol and traces of phosphatidylglycerol; MK-9(H₄) was identified as the dominant menaquinone and galactose as the diagnostic sugar. The major cellular fatty acids were found to be the branched-chain saturated acids iso-C_16:0 and iso-C_15:0, as well as C_17:1ω8c. The 16S rRNA gene sequence shows 97.5–97.9 % sequence identity with the four validly named or at least effectively published members of the genus: Geodermatophilus obscurus (97.5 %), Geodermatophilus arenarius (97.7 %), Geodermatophilus ruber (97.9 %) and Geodermatophilus nigrescens (97.9 %). Based on the results from this polyphasic taxonomic analysis and DNA–DNA hybridizations with all type strains of the genus, we propose that strain CF6/1^T represents a novel species, Geodermatophilus siccatus, with the type strain CF6/1^T = DSM 45419^T = CCUG 62765^T = MTCC 11414^T.     

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H₂O₂, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H₂O₂-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H₂O₂-induced liposome leakage was abolished by cyanide presumably competing with H₂O₂ for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H₂O₂ permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H₂O₂, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.     

Hymenobacter qilianensis sp. nov., isolated from a subsurface sandstone sediment in the permafrost region of Qilian Mountains,China and emended description of the genus Hymenobacter

A red–pink, Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain DK6-37 was isolated from the permafrost region of Qilian Mountains in northwest of China. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that this isolate represents a novel member of the genus Hymenobacter, with low sequence similarities (<97 %) to recognized Hymenobacter species. Optimum growth was observed at 28 °C, pH 7.0 and 0 % NaCl. The strain was found to contain MK-7 as the predominant menaquinone. The polar lipids were identified as phosphatidylethanolanmine, two unknown aminophospholipids, one unknown aminolipid and three unknown polar lipids. The major fatty acids were identified as summed feature 3 (C_16:1 ω7c/C_16:1 ω6c as defined by MIDI), summed feature 4 (anteiso-C_17:1 B/iso-C_17:1 I), C_16:1 ω5c, iso-C_17:0 3-OH, iso-C_15:0 and C_18:0. The DNA G + C content was determined to be 67.4 mol %. On the basis of the polyphasic evidence presented, it is proposed that strain DK6-37 represents a novel species of the genus Hymenobacter, for which the name Hymenobacter qilianensis sp. nov. is proposed. The type strain is DK6-37^T (= CGMCC 1.12720^T = JCM 19763^T).