The point mutation A34F causes dimerization of GB1

The immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a very stable, small, single-domain protein, is one of the most extensively used models in the area of protein folding and design. Variants derived from a library of randomized hydrophobic core residues previously revealed alternative folds, namely a completely intertwined tetramer (Frank et al., Nat Struct Biol 2002;9:877-885) and a domain-swapped dimer (Byeon et al., J Mol Biol 2003;333:141-152). Here, we report the NMR structure of the single amino acid mutant Ala-34-Phe which exists as side-by-side dimer. The dimer dissociation constant is 27 +/- 4 microM. The dimer interface comprises two structural elements: First, the beta-sheets of the two monomers pair in an antiparallel arrangement, thereby forming an eight-stranded beta-sheet. Second, the alpha-helix is shortened, ending in a loop that engages in intermolecular contacts. The largest difference between the monomer unit in the A34F dimer and the monomeric wild-type GB1 is the dissolution of the C-terminal half of the alpha-helix associated with a pronounced slow conformational motion of the interface loop. This involves a large movement of the Tyr-33 side chain that swings out from the monomer to engage in dimer contacts.     

Development of new laccases by directed evolution: functional and computational analyses

Laccases are blue multicopper oxidases that couple the four-electron reduction of oxygen with the oxidation of a broad range of aromatic substrates. These fungal enzymes can be used for many applications such as bleaching, organic synthesis, bioremediation, and in laundry detergents. Laccases from Pleurotus ostreatus have been successfully heterologously expressed in yeasts. The availability of established recombinant expression systems has allowed the construction of mutated, "better performing" enzymes through molecular evolution techniques. In the present work, random mutagenesis experiments on poxc and poxa1b cDNAs, using error prone PCR (EP-PCR) have been performed. By screening a library of 1100 clones the mutant 1M9B was selected, it shows a single mutation (L112F) leading to an enzyme more active but less stable with respect to the wild-type enzyme (POXA1b) in all the analyzed conditions. This mutant has been used as a template for a second round of EP-PCR. From this second generation library of 1200 clones, three mutants have been selected. Properties of the four mutants, 1M9B screened from the first library, and 1L2B, 1M10B, and 3M7C from the second library, were analyzed. The better performing mutant 3M7C presents, besides L112F, only one substitution (P494T) responsible both for the significantly increased stability and for the exhibited higher activity of this mutant. Molecular dynamics simulations have been performed on three-dimensional models of POXA1b, 1M9B, and 3M7C, and hypotheses on the structure-function relationships of these proteins have been formulated.     

Isolation and characterization of a Chlorella mutant producing high amounts of chlorophyll and carotenoids

Mutants of Chlorella that form dark green colonies under heterotrophic conditions were generated from the parental Chlorella regularisS-88 by MNNG mutagenesis. The mutant Y-21 was the highest in chlorophyll and carotenoids contents among these isolates. Y-21 grew a little slower than its parental S-88 under heterotrophic and photoautotrophic conditions. Although chloroplasts were generated in both S-88 and Y-21 during glucose starvation and under illumination, the chlorophyll and carotenoid contents of Y-21 were approximately 1.5–1.8 times those of S-88 under both growth conditions, suggesting that relative size of chloroplast is larger in Y-21 than in S-88. Consistent with these results, photosynthesis genes on chloroplast DNA were more highly up-regulated in Y-21 than in S-88 during glucose starvation and under illumination. These results lead us a model in which chloroplast generation is sugar-repressed and photoinduced in Chlorella. Y-21 appears to be a mutant in which sugar repression of chloroplast generation is weak.     

Pigments and Gas Exchange Characteristics in Leaves of Chlorophyll Mutants of Pisum sativum

Quantitative and qualitative characteristics of pigment composition and gas exchange were studied in chlorophyll mutants of pea, Pisum sativum L.: chlorotica 2004 and 2014. The mutant 2004 had light-green color, whereas the mutant 2014 has yellow-green leaves and stems; they contained about 80 and 50% of chlorophyll, respectively, compared to the initial line. cv. Torsdag. Leaves of the mutant 2004 had significantly lower carotene content and accumulated more lutein and violaxanthin. In the mutant 2014, the contents of chlorophyll and all carotenoids were reduced almost proportionally. The quantum efficiency of photosynthesis was by 29–30% lower in the mutants, and it was 1.5–2 times higher in F₁ hybrids, as compared to control plants. Our data allow us to conclude that the impairment of photosynthesis in the mutant 2014 is caused by the changed mesostructure of leaves, whereas in the mutant 2004, it may be caused by an impairment of photosystem reaction centers.     

The Endomembranes ofchlamydomonas reinhardii: A comparison of the wildtype with the wall mutants CW 2 and CW 15

Summary Ultrastructural observations on the principal endomembranes (endoplasmic reticulum, Golgi apparatus) of synchronously growing of wild type and mutant (CW 2, CW 15) strains ofChlamydomonas reinhardii have been carried out. The dictyosomes of the Golgi apparatus in all three cases are highly polar in morphology but lack intercisternal filaments. A clear spatial relationship between dictyosomes and endoplasmic reticulum is seen and a transfer of vesicles from the latter to the former is easily visualized. Coated vesicles invariably appear to be restricted to the trans-pole of the dictyosomes. The endoplasmic reticulum adjacent to the cis pole of dictyosomes is considerably hypertrophied in the case of the wild type, only partially so in the mutant CW 2 but not at all in the mutant CW 15. In the wild type this swelling is most extreme during the period of wall deposition and for several hours afterwards. The results are discussed in relation to the biosynthesis and intracellular transport of, particularly O-glycosidically linked, glycoproteins.     

Embryo-lethal mutants of Arabidopsis thaliana: analysis of mutants with a wide range of lethal phases

Summary Embryo-lethal mutants of Arabidopsis thaliana were isolated by treating mature seeds with an aqueous solution of ethyl methanesulfonate (EMS), screening the resulting M-1 plants for siliques containing 25% aborted seeds following self-pollination, and verifying the presence of induced mutations in subsequent generations. Thirty-two recessive lethals with a Mendelian pattern of inheritance were examined in detail. Developmental arrest of mutant embryos ranged from the zygotic stage of embryogenesis in mutant 53D-4A to the linear and curled cotyledon stages of development in mutants 112A-2A and 130B-A-2. These lethal phases did not change significantly when plants were grown at 18 °C rather than at 24 °C. Differences between mutant lines were found in the color of arrested embryos and aborted seeds, the percentage and distribution of aborted seeds in heterozygous siliques, the size of arrested embryos, and the extent of abnormal development. Unusual mutant phenotypes included the presence of unusually large suspensors, distorted and fused cotyledons, reduced hypocotyls, and arrested embryos without distinct cotyledons or hypocotyl tissue. The isolation of eight new mutants with a non-random distribution of aborted seeds in heterozygous siliques provides further evidence that many of the genes that control early stages of embryogenesis in plants are also expressed prior to fertilization.     

Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage λ: “cosmids”

A plasmid which contains a cos site of λ and can be packaged into lambda bacteriophage particles is termed a “cosmid”. Such plasmids can be used as gene cloning vectors in conjunction with an in vitro packaging system. The properties of a new series of cosmids based on the ColE1 replicon are described, including small temperature-sensitive plasmids which have lost mobilisation functions and carry no IS sequences. Amongst these plasmids are vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI and EcoRI. It is demonstrated that by using cosmids in particular size ranges these plasmids provide a high efficiency cloning system which yields essentially only hybrid clones without resort to a second selection or screening step, and without prior modification (e.g. phosphatase) treatment of the DNA.Attempts were made to optimise the cloning properties of the cosmid system. An Escherichia coli “gene bank” was obtained with an efficiency of 5·10⁵ clones per μg of E. coli DNA, and in which any particular unselected marker may be found in about one out of every 400 clones.It was demonstrated that deletion of mobilisation functions leads to loss of ability to form relaxation-complex without affecting copy number or segregation properties of the temperature-sensitive derivatives. The vectors are amplifiable in chloramphenicol to make up about 50% of the total cellular DNA.