Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system

The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Film reviews

Frozen tombs of Siberia

The Mystery of the Frozen Tombs of Siberia. Directed by Françoise Lévie; camera, Sacha Wiernik; sound, Sarah Moon Howe; editing, Michèle Maquet; produced by Sofidoc Production. 1994,26 minutes, color and black & white, English narration.

Imbalu, a ritual of manhood

Imbalu: Ritual of Manhood of the Bagisu of Uganda. A film by Richard Hawkins and Suzette Heald; director: Richard Hawkins; camera: David and Judith MacDougall; editor: Suzette Heald and Richard Hawkins; sound: Jack Reed. 1988, 95 minutes, colour, PAL video, Lugisu and English. Rental [UK only]: £9.00 (VHS‐PAL); sale: £40/ US$72 (VHS‐PAL), £80/US$110 (VHS‐NTSC) [plus VAT and postage]. Distributor: Royal Anthropological Institute, 50 Fitzroy Street, London, W1P 5HS, U.K.

The earth is our mother

The Earth is Our Mother and The Journey Back (The Earth is Our Mother, Parts I and II). Directed by Peter Elsass; produced by Casablanca Film Group and the Danish National Film Board; camera: Morten Bruus; editing: Niels Pagh Anderson; sound: Morten Bottzau; research: Guillermo Padilla. Distributed by TV2/Danmark Sales, Sortedam Dossering 55A, DK‐2100 København Ø, Denmark (Tel: +45 35 37 22 00; fax: + 45 35 37 22 27). 1987,55 minutes, color. Narration and subtitles in English.     

Role of TSP-5/COMP in pseudoachondroplasia

Pseudoachondroplasia (PSACH) is a well-characterized dwarfing condition associated with disproportionate short stature, abnormal joints and osteoarthritis requiring joint replacement. PSACH is caused by mutations in cartilage oligomeric matrix protein (COMP). COMP, the fifth member of the thrombospondin (TSP) gene family, is a pentameric protein found primarily in the extracellular matrix of musculoskeletal tissues. Functional studies have shown that COMP binds types II and IX collagens but the role of COMP in the extracellular matrix remains to be defined. Mutations in COMP interfere with calcium-binding and protein conformation. PSACH growth plate and growth plate chondrocytes studies indicate that COMP mutations have a dominant negative effect with both COMP and type IX collagen being retained in large rER cisternae. This massive retention causes impaired chondrocyte function with little COMP secreted into the matrix and premature loss of chondrocytes. Deficiency of linear growth results from loss of chondrocytes from the growth plate. Secondarily, the matrix contains minimal COMP, which may be normal and/or mutant, and little type IX collagen. This deficiency results in abnormal joints that are easily eroded and cause painful osteoarthritis. Unlike other misfolded proteins that are targeted for degradation, much of the retained COMP escapes degradation, compromises cell function, and causes cell death. Gene therapy will need to target the reduction of COMP in order to restore normal chondrocyte function and longevity.     

Using transplanted oyster (Crassostrea virginica) beds to improve water quality in small tidal creeks: a pilot study

The Eastern oyster, Crassostrea virginica, may improve water quality by filtering large quantities of particulate matter (both organic and inorganic) and nutrients from the overlying water column. Additionally, oyster reefs alter hydrodynamic conditions, further increasing the removal of particulate matter from the water column. This study examined the effects of small-scale oyster additions on sediment loading, chlorophyll a, nutrient concentrations, and flow in small tidal creeks. Two reefs were established in Hewletts Creek, New Hanover County, North Carolina. Total suspended solids (TSS), chlorophyll a, and ammonium were measured upstream and downstream of each created reef and in an adjacent control channel that lacked a reef. Data were collected monthly during ebb tides over a 10-month period between September 2000 and June 2001. In the first month after initial reef placement, mean TSS concentrations downstream of reef placement were slightly lower than those upstream of the reef. Although not statistically significant, TSS concentrations downstream of the reefs were less than upstream concentrations for five out of nine and five out of seven post-reef sampling months for the upland and the lower creek sites, respectively. Chlorophyll a concentrations were not significantly affected by initial reef placement (2×3 m), but were reduced substantially after reef enlargement (3×4 m) in one of the experimental creeks. Reef placement resulted in significant increases in ammonium concentrations downstream of the transplanted-reefs. In addition, deposition of feces and pseudofeces by the oysters resulted in accumulation of finer-grained materials in the treated channel relative to the control channels. Oyster filtration was most effective three hours following high tide, when the ratio of flow discharge to reef surface area was the highest. This work demonstrates that small oyster reefs established and maintained in some small tributary channels can reduce TSS and chlorophyll a concentrations and that the magnitude of the effect may vary over the course of the tidal cycle.     

Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition

The Rag1 and Rag2 proteins initiate V(D)J recombination by introducing site-specific DNA double-strand breaks. Cleavage occurs by nicking one DNA strand, followed by a one-step transesterification reaction that forms a DNA hairpin structure. A similar reaction allows Rag transposition, in which the 3′-OH groups produced by Rag cleavage are joined to target DNA. The Rag1 active site DDE triad clearly plays a catalytic role in both cleavage and transposition, but no other residues in Rag1 responsible for transesterification have been identified. Furthermore, although Rag2 is essential for both cleavage and transposition, the nature of its involvement is unknown. Here, we identify basic amino acids in the catalytic core of Rag1 specifically important for transesterification. We also show that some Rag1 mutants with severe defects in hairpin formation nonetheless catalyze substantial levels of transposition. Lastly, we show that a catalytically defective Rag2 mutant is impaired in target capture and displays a novel form of coding flank sensitivity. These findings provide the first identification of components of Rag1 that are specifically required for transesterification and suggest an unexpected role for Rag2 in DNA cleavage and transposition.     

Normal myoblast fusion requires myoferlin

Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.