Determination of the size of the Azotobacter vinelandii chromosome

The chromosome of Azotobacter vinelandii UW was digested separately with the rape cutter restriction endonucleases Swal (5-ATTTAAAT), PmeI (5GTTTAAAC) and Pacl (5-TTAATTAA) and the products were separated by pulsed-field gel electrophoresis. The size of the chromosome was determined to be approximately 4.5 megabase pairs (Mb) based on the sum of the sizes of the restriction fragments. This is almost the same as the size of the chromosome of Escherichia coli. The inability of the undigested DNA to enter the gel has led us to infer that the chromosome is circular.     

Structure of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex: new prosthetic groups,Q-space,and the ‘hors d’oeuvres hypothesis’ for assembly of the complex

3-Å crystal structures of the cytochrome b₆f complex have provided a structural framework for the photosynthetic electron transport chain. The structures of the 220,000 molecular weight dimeric cytochrome b₆f complex from the thermophilic cyanobacterium, Mastigocladis laminosus (Kurisu et al. 2003, Science 302: 1009–1014), and the green alga, Chlamydomonas reinhardtii (Stroebel et al. 2003, Nature 426: 413–418), are very similar. The latter is the first structure of a integral membrane photosynthetic electron transport complex from a eukaryotic source. The M. laminosus and C. reinhardtii structures have provided structural information and experimental insights to the properties and functions of three native and novel prosthetic groups, a chlorophyll a, a -carotene, and a unique heme x, one copy of which is found in each monomer of the cytochrome b₆f complex, but not the cytochrome bc₁ complex from the mitochondrial respiratory chain of animals and yeast. Several functional insights have emerged from the structures including the function of the dimer; the properties of heme x; the function of the inter-monomer quinone-exchange cavity; a quinone diffusion pathway through relatively narrow crevices or portals; a modified reaction scheme for n-side quinone redox reactions; a necessarily novel mechanism for quenching of the bound chlorophyll triplet state; a possible role for the bound chlorophyll a in activation of the LHC kinase; and a structural and assembly role for the four small PetG, L, M, and N subunits. An hors doeuvres hypothesis for assembly of the complex is proposed for the small hydrophobic stick or picket fence polypeptides at the periphery of the complex, based on the cis-positive orientation of the small hydrophobic subunits and the toothpick binding mode of the -carotene.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

Association of polymorphic markers of the lipid metabolism genes with diabetic polyneuropathy in type 1 diabetes mellitus

A possible association of the polymorphic markers 2/3/4 of the apolipoprotein E gene (APOE) and I /D of the apolipoprotein B gene (APOB) with diabetic polyneuropathy (DPN) was analyzed in patients with type 1 diabetes mellitus (T1DM) with (N=86) or without (N=94) clinical signs of DPN. The two groups did not differ significantly in allele and genotype frequencies of the 2/3/4 polymorphic marker of the APOE gene. Analysis of the allele and genotype frequency distributions of the I/D polymorphic marker of the APOB gene showed that risk of DPN is higher in carriers of allele I or genotype I/I (OR=1.66 and 2.01, respectively) and lower in carriers of allele D (OR=0.60). The results implicate the APOB gene, which codes for one of the major components of the lipid metabolism system, in DPN development in patients with T1DM.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 230–234.Original Russian Text Copyright © 2005 by Voronko, Yakunina, Strokov, Lavrova, Nosikov.     

SCM2, a tryptophan permease in Saccharomyces cerevisiae, is important for cell growth

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm21-4) cause slow growth, whereas one disrupted allele (scm25) is lethal. Cells with both the scm21 and trp1-101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm21 or trp1-101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.     

Localization of the Laevigatum powdery mildew resistance gene to barley chromosome 2 by the use of RFLP markers

Summary The powdery mildew disease resistance gene Ml(La) was found to belong to a locus on barely chromosome 2. We suggest that this locus be designated MlLa. Linkage analysis was carried out on 72 chromosome-doubled, spring-type progeny lines from a cross between the winter var Vogelsanger Gold and the spring var Alf. A map of chromosome 2 spanning 119cM and flanked by two peroxidase gene loci was constructed. In addition to the Laevigatum resistance locus the map includes nine RFLP markers, the two peroxidase gene loci and the six-row locus in barley.     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Satellited chromosomes,systematics and phylogeny inTaraxacum (Asteraceae)

A survey is made of the occurrence, nature and frequency of satellited chromosomes in the agamospermous genusTaraxacum. Species belonging to the 10 sections thought to be most primitive in the genus lack satellited chromosomes. In most other sections, a characteristic satellited chromosome is seen with a large euchromatic region distal to the presumed nucleolar oraniser region (NOR). In sections of a precursor type, there is always one chromosome of this Taraxacum type per haploid genome. In sections thought to be of an advanced type the number of such satellited chromosomes is very unstable, sometimes even within the same tissue. In sectionHamata, two such satellited chromosomes are invariably found in triploids. This finding strongly supports the integrity of this section, suggests that the species of the section are monophyletic, and have evolved from a single ancestor subsequent to the occurrence of obligate agamospermy. In three sections of the genus, satellited chromosomes of the conventional type with a very small distal euchromatic region distal to the NOR are reported for the first time in the genus.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Modification of lignin by Geotrichum klebahnii

¹³C-NMR spectroscopic analysis indicates that the yeast-like species Geotrichum klebahnii is an efficient microorganism for lignin biodegradation. This strain modified beechwood lignin even if it was the only carbon source by C-C side chain cleavage, C-oxidations, aromatic ring cleavage and reductive reactions. The obtained results outline prospective application of G. klebahnii for biotechnological pre-treatment of lignocellulosic materials.     

Excision and transposition of two Ds transposons from the bronze mutable 4 derivative 6856 allele of Zea mays L

Summary The regulatory mutation bronze mutable 4 Derivative 6856 (bz-m4 D6856) contains a complex 6.7 kb Dissociation (Ds) element tagged with a duplication of low copy bz 3 flanking sequences (Klein et al. 1988). This creates a unique opportunity to study the transposition of a single member of the repetitive family of Ds elements. Eighteen full purple revertants (Bz alleles) of bz-m4 were characterized enzymatically and by genomic mapping. For 17 of the Bz alleles, reversion to a wild-type phenotype was caused by excision of the 6.7 kb Ds transposon. Nine of these Bz alleles retained the transposon somewhere in their genome. In this study we show that like Ac (Schwartz 1989; Dooner and Belachew 1989), the 6.7 kb Ds element can transpose within a short physical distance, both proximal and distal to its original position. Additional bz sequences have been mapped immediately distal to the mutant locus in bz-m4 D6856; genetic evidence suggests these are flanked by two additional Ds elements. The remaining Bz revertant, Bz :107, arose from excision of a more complex 13 kb Ds element.     

Projecting population-level response of purple sea urchins to lead contamination for an estuarine ecological risk assessment

As part of an ecological risk assessment casestudy at the Portsmouth Naval Shipyard (PNS), Kittery,Maine, USA, the population level effects of leadexposure to purple sea urchin, Arbaciapunctulata, were investigated using a stage-classifiedmatrix population model. The model divided the lifehistory of A. punctulata into five classes,incorporating both, the developmental stages of thisspecies and the endpoints from a laboratory bioassay. Finite population growth rate () was themetric relating population level impact to leadexposure. An inverse relationship was observed betweenlead tissue residues in A. punctulata and. Bioassay treatments which resulted insignificant impacts on fertilization success and zygoteviability did not translate into significant effects on, unless those treatments also negativelyimpacted adult survival. These results paralleled theelasticity (relative sensitivity) analysis of themodel, which indicated that was mostsensitive to adult and subadult survival and wasrelatively insensitive to fecundity, fertilizationsuccess, or zygote survival. Model results indicatedthat the environmental lead levels observed at PNSshould not pose significant ecological risk to seaurchin populations. Additionally, the model resultsindicated that impacts to the early life stagesroutinely used in toxicity testing do not necessarilytranslate directly into impacts at the populationlevel.     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation

Summary An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique BglII site. A kanamycin resistance cartridge was cloned into the BglII site. This construct was mobilized into A. vinelandii CA12 ( nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.     

Sex identification by male-specific growth hormone pseudogene (GH-Ψ) in Oncorhynchus masou complex and a related hybrid

It is often difficult to identify sexes of many fish species by conventional cytological method because of the lack of heteromorphic sex chromosomes. Isolation of sex-specific molecular markers is thus important for sexing and for understanding sex chromosome evolution in these species. We have identified genetic sexes by PCR-based male-specificity of a growth hormone pseudogene (GH-) in masu and Biwa salmon, two subspecies of the Oncorhynchus masou complex, and their hybrid Honmasu. PCRs with primers designed from sequences of chinook salmon GH genes amplified GH-I and GH-II fragments in both sexes, but a third GH- fragment was detected in predominant proportion of males and very few phenotypic females. The consistency of phenotypic sex with genetic sex identified by GH- for masu salmon, Biwa salmon and Honmasu was 93.1, 96.7 and 94%, respectively. The remaining individuals showed inconsistency or deviation from sex-specificity: a few phenotypic males lacked the GH-, whereas a few phenotypic females possessed the GH-. Sequence of the putative GH- fragment from such females was identical to that from genetic males, and shared about 95% homology with the corresponding GH- fragment from chinook salmon. This result confirmed that these females were really GH--bearing individuals. PCR analyses with primers designed from masu salmon GH- gave identical results, indicating that the absence of GH- in a few males was not resulted from primer mismatching. These GH--bearing females and GH--absent males were more likely to originate from spontaneous sex reversion than from crossing-over between GH- and the sex determination gene/region.