Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

The 85-kilodalton phosphoprotein (pp85) of human herpesvirus 7 is encoded by open reading frame U14 and localizes to a tegument substructure in virion particles.

A family of antigenically related proteins present in cells infected with human herpesvirus 7 (HHV-7), designated phosphoprotein 85 (pp85), comprises a complex of proteins, of which the 85-kDa species is phosphorylated. pp85 is a major determinant of human response to HHV-7 infection (L. Foà-Tomasi, E. Avitabile, L. Ke, and G. Campadelli-Fiume, J. Gen. Virol. 75:2719-2727, 1994; L. Foà-Tomasi, M. P. Fiorilli, E. Avitabile, and G. Campadelli-Fiume, J. Gen. Virol. 77:511-518, 1996; J. B. Black et al., Clin. Diagn. Lab. Immunol. 3:79-83, 1996). By immunoscreening of a cDNA library from HHV-7-infected cells with monoclonal antibody (MAb) 5E1, directed to the proteins of the pp85 complex, we mapped the gene encoding pp85 to the U14 open reading frame of the HHV-7 genome. A prokaryotically expressed fusion protein containing the U14 open reading frame reacted with MAb 5E1 in an immunoblot assay. A functional role for pp85 was defined by immunoelectron microscopy studies. Immunogold labeling of cryosections of HHV-7-infected cord blood mononuclear cells at high resolution localized the reactivity of MAb 5E1 to the outer surface of the virion tegument. This finding demonstrates that pp85, the product of the U14 gene, is a component of the HHV-7 tegument and suggests that the HHV-7 tegument is not a homogeneous structure but rather is composed of substructures, including an outermost layer containing pp85. The present findings, together with previously reported properties of MAb 5E1, including its ability to react with formalin-fixed paraffin-embedded samples, make this antibody a specific tool useful for etiopathogenetic studies of HHV-7 infection in humans and provide the basis for further development of pp85 into a specific recombinant diagnostic reagent.     

Demonstration of retinal afferents in the RCS rat,with reference to the retinohypothalamic projection and suprachiasmatic nucleus

In the Royal College of Surgeons (RCS) rat, characterized by inherited retinal dystrophy, retinal projections to the brain were studied using anterograde neuronal transport of cholera toxin B subunit upon injection into one eye. The respective immunoreactivity was found predominantly contralateral to the injection site in the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, medial terminal nucleus of the accessory optic tract, and bilateral hypothalamic suprachiasmatic nuclei. Although terminal density was somewhat reduced in dystrophic rats, the projection patterns in these animals appeared similar to those seen in their congenic controls and were comparable to the visual pathways described for the rat previously. In dystrophic rats, the number of cell bodies exhibiting immunoreactivity to vasoactive intestinal polypeptide, viz. a population of suprachiasmatic neurons receiving major retinohypothalamic input, was reduced by one-third, and some differences were observed in the termination pattern of the geniculohypothalamic tract, as revealed by immunoreactivity to neuropeptide Y in the suprachiasmatic nucleus.This study was supported by grants from the DFG (Re 644/2-1) and the NMFZ, Mainz (to S.R.).     

Five new microsatellite polymorphisms at the q21 region of human chromosome 21

Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel. These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62. The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel. The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map. They have also been localised in the CEPH/ Généthon YAC panel, providing a refined localisation of these polymorphic sequences. These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21.     

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

Interaction of tobacco mosaic virus protein with synthetic polynucleotides containing a fluorescent label: optical properties of poly(A,epsilonA) and poly(C,epsilonC) copolymers and energy migration from the tryptophan to 1,N6-ethenoadenine or 3,N4-ethenocytosine residues in RNP.

A spectrophotometric method for determination of the modification degrees and molar extinction coefficients for poly(A, epsilonA) and poly(C, epsilonC) copolymers has been developed. Dependence of some absorption and fluorescence parameters of the copolymers on the modification degree has been studied. Distribution of modified residues in copolymers differs from random and depends on modification conditions. Interaction between the TMV protein and copolymers has been investigated. The protein interacts with poly(A, epsilonA) of low or medium modification degree and displays no activity with respect to poly(epsilonA). On the contrary, introduction of epsilonC to the polynucleotide promotes complex formation between poly(C, epsilonC) and TMV protein. Analysis of the fluorescence emission and excitation spectra has revealed energy transfer from tryptophan to epsilonA or epsilon C in the RNP to occur and permits one to estimate the average distance between Trp (presumably Trp 52)o and the RNA base binding region in the virus to be 17 to 20 A.     

Nitrogenase from Rhodospirillum rubrum Relation between ‘switch-off’ effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios

Nitrogenase activity of ‘membrane-free’ extracts, produced from nitrogenstarved Rhodospirillum rubrum to which 4 mM NH⁺₄ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this ‘switch-off/switch-on’ effect and the function of the membrane component is discussed.Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein ratios. The $\text{ATP}\text{2 e}{}^{\mathrm{?}}$ values are 4–5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.