Prevention of the flowering of a tree,silver birch

Genetic modification of trees presents great advantages but it is hampered by the possible spread of introduced genes to native populations. However, the spread would be prevented if the modified trees would be sterile. We have previously shown that the induction of sterility by the prevention of flowering is possible in tobacco and Arabidopsis by introducing a gene construct composed of the ribonuclease gene BARNASE ligated to the flower-specific promoter of the birch gene BpMADS1. In the present study, we test this gene construct in silver birch (Betula pendula Roth). When this gene construct was introduced into very early-flowering birch clones, 81 kanamycin resistant lines were obtained. In 38 lines, the vegetative development was disturbed, e.g., the leaves were small and the plants were short and bushy or the growth of plants was weak. More importantly, in 7 other lines no male inflorescences formed or they aborted early. If male inflorescences were formed, they did not contain any stamens. The initial growth of these lines was similar to the non-transgenic control lines. Later, however, the growth of the non-flowering lines differed from that of the controls in showing some dichotomic branching and a reduced number of branches. Preliminary results showed that the gene construct can prevent the development of female inflorescences as well. The results show clearly that BpMADS1::BARNASE can prevent the flowering in a tree but the prevention of flowering may cause some side effects. Studies with ordinary birch clones will show whether the side effects are a property of the early flowering clones or all birches.     

Improved production of human type II procollagen in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> in shake flasks by a wireless-controlled fed-batch system

Background  

Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.     

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell-T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell-mediated immunity.     

Manganese peroxidase of Agaricus bisporus: grain bran-promoted production and gene characterization

The main manganese peroxidase (MnP) isoenzyme of Agaricus bisporus ATCC 62459 produced in lignocellulose-containing cultures was isolated, cloned and sequenced. In liquid medium, where MnP was previously detected only in trace amounts, the production of MnP was enhanced by rye and wheat bran supplements. The pI (3.25) and N-terminal amino acid sequence (25 aa) of the enzyme from bran-containing cultures were identical to those reported from compost-isolated MnP1. MnP1 is a 328-aa long polypeptide preceded by a 26-aa leader peptide. The nucleotide sequence and putative amino acid sequence of MnP1 reveal its similarity to Pleurotus ostreatus MnP3 (62.5%), Lepista irina versatile peroxidase (VP) (61.8%) and Pleurotus eryngii VPs VPL2 and VPL1 (61.9% and 61.2%, respectively). The intron-exon structure resembles that of P. ostreatus MnP1 and P. eryngii VPL1. Despite the sequence similarity to VPs, in the A. bisporus MnP1 sequence, alanine (A163) is present instead of tryptophane (W164), distinguishing it from the veratryl alcohol oxidising P. eryngii VPLs. The MnP sequence can be used as a tool to examine the pattern of ligninolytic gene expression during the growth and fruiting of A. bisporus to optimise compost composition, fungal growth and mushroom production.     

Absence of PTPN11 mutations in 28 cases of cardiofaciocutaneous (CFC) syndrome

CFC (cardiofaciocutaneous) syndrome (MIM 115150) has been considered by several authors to be a more severe expression of Noonan syndrome. Affected patients present with congenital heart defects, cutaneous abnormalities, Noonan-like facial features and severe psychomotor developmental delay. We have recently demonstrated that Noonan syndrome can be caused by missense mutations in PTPN11(MIM 176876), a gene that encodes the non-receptor protein tyrosine phosphatase SHP-2. In this report, we have evaluated the possible involvement of mutations in PTPN11 in CFC syndrome. A cohort of 28 CFC subjects rigorously assessed as having CFC based on OMIM diagnostic criteria was examined for mutations in the PTPN11 coding sequence by using DHPLC analysis. The results showed no abnormalities in the coding region of the PTPN11 gene in any CFC patient, nor any evidence of major deletions within the gene suggesting that mutations in other gene(s) are responsible for this syndrome.     

The expression and promoter specificity of the birch homologs for PISTILLATA/GLOBOSA and APETALA3/DEFICIENS

B-function genes determine the identity of petals and stamens in the flowers of model plants such as Arabidopsis and Antirrhinum . Here, we show that a putative B-function gene BpMADS2 , a birch homolog for PISTILLATA , is expressed in stamens and carpels of birch inflorescences. We also present a novel birch gene BpMADS8 , a homolog for APETALA3 / DEFICIENS , which is expressed in stamens. Promoter-GUS analysis revealed that BpMADS2 promoter is active in the receptacle of Arabidopsis flower buds while BpMADS8 promoter is highly specific in mature stamens. BpMADS2 promoter:: BARNASE construct prevented floral organ development in Arabidopsis and tobacco. In birch, inflorescences with degenerated stamens and carpels were obtained. BpMADS8::BARNASE resulted in degeneration of stamens in Arabidopsis and birch causing male sterility. In tobacco, only sepals were developed instead of normal flowers. The results show that the BpMADS2::BARNASE construct can be used to specifically disrupt floral organ development in phylogenetically distant plant species. The stamen-specific promoter of BpMADS8 is a promising tool for biotechnological applications in inducing male sterility or targeting gene expression in the late stamen development.     

Diversity of chlorophenol-degrading bacteria isolated from contaminated boreal groundwater

Chlorophenol-degrading bacteria from a long-term polluted groundwater aquifer were characterized. All isolates degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol at concentrations detected in the contaminated groundwater (< 10 mg l^–1). Pentachlorophenol was degraded by three isolates when present alone. In two gram-positive isolates, 2,3,4,6-tetrachlorophenol was required as an inducer for the degradation of pentachlorophenol. The gram-positive isolates were sensitive to pentachlorophenol, with an IC₅₀ value of 5 mg/l. Isolates belonging to the Cytophaga/Flexibacter/Bacteroides phylum had IC₅₀ values of 25 and 63 mg/l. Isolates belonging to α-, β- and γ-Proteobacteria generally tolerated the highest pentachlorophenol concentrations (> 100 mg/l). Polychlorophenol-degrading capacity was found in strains of Nocardioides, Pseudomonas, Ralstonia, Flavobacterium, and Caulobacter previously not known to degrade polychlorophenols. In addition, six polychlorophenol-degrading sphingomonads were found. Received: 27 September 1998 / Accepted: 21 December 1998     

Advantages from diel vertical migration can explain the dominance of Gonyostomum semen (Raphidophyceae) in a small, steeply-stratified humic lake

In late summer, a large flagellated alga, Gonyostomum semen(Raphidophyceae), constituted most of the phytoplankton biomassin a small steeply-stratified humic lake. Its diel verticalmigration (DVM) was very distinct and extended at night intothe anoxic hypolimnion. After midsummer, the depletion of hypolimneticoxygen led to a gradual release of soluble reactive phosphorus(SRP) from the sediment, but one month later, irrespective ofcontinuing stratification, the concentrations again returnedto undetectable levels down to the bottom. As this coincidedwith the rapid increase in G.semen population, the latter wasprobably responsible for the depletion of SRP. The flux of SRPfrom the sediment to the epilimnion was virtually interrupted,making non-migrating phytoplankton dependent only on regeneratedand inflowing inorganic phosphorus. Besides nutrient availability,DVM also benefited G.semen in the reduction of metabolic andgrazing losses. In this lake, the remarkable multiple advantagesof DVM probably explain the dominance of the large G.semen inthe late summer phytoplankton biomass over much smaller algae.     

Initial Steps in the Pathway for Bacterial Degradation of Two Tetrameric Lignin Model Compounds

We investigated the metabolic route by which a lignin tetramer-degrading mixed bacterial culture degraded two tetrameric lignin model compounds containing β—O—4 and 5—5 biphenyl structures. The α-hydroxyl groups in the propane chain of both phenolic and nonphenolic tetramers were first oxidized symmetrically in two successive steps to give monoketones and diketones. These ketone metabolites were decomposed through C_α(=O)—C_β cleavage, forming trimeric carboxyl acids which were further metabolized through another C_α(=O)—C_β cleavage. Dehydrodiveratric acid, which resulted from the cleavage of the carbon bonds of the nonphenol tetramer, was demethylated twice. Four metabolites of the phenolic tetramer were purified and identified. All of these were stable compounds in sterile mineral medium, but were readily degraded by lignin tetramer-degrading bacteria along the same pathway as the phenol tetramer. No monoaromatic metabolites accumulated. All metabolites were identified by mass and proton magnetic resonance spectrometry. The metabolic route by which the mixed bacterial culture degraded tetrameric lignin model compounds was different from the route of the main ligninase-catalyzed C_α—C_β cleavage by Phanerochaete chrysosporium.