Determinants of thymotropism in Kaplan radiation leukemia virus and nucleotide sequence of its envelope region.

Radiation leukemia viruses (RadLVs) are a group of murine leukemia viruses which are induced by radiation and cause T-cell leukemia. Viral clones isolated from the BL/VL3 lymphoid cell line derived from a thymoma show variable tropism and leukemogenic potential. We have constructed chimeric viruses by in vitro recombination between two viruses, a RadLV that is thymotropic and an endogenous ecotropic virus that is nonthymotropic. We show here that, in contrast to thymotropism determinants identified previously, which lie in the long terminal repeat (LTR), it is the envelope region that is responsible for the thymotropism of BL/VL3 RadLV. The nonthymotropic virus which we have rendered thymotropic by transfer of the env region of RadLV in the present study has been shown previously to become thymotropic when the LTR of another thymotropic virus is inserted in its genome. Thus, the LTR and envelope gene may be involved in complementary action to lead to thymotropism.     

Morphogenesis of endoplasmic reticulum in Xenopus oocytes after microinjection of rat liver smooth microsomes

We have determined the kinetics of endoplasmic reticulum (ER) reconstitution following insertion of rat-liver smooth microsomes (SM) into Xenopus oocyte cytoplasm using electron microscopy as well as cytochemistry and thick-section 3-dimensional reconstruction. Oocytes were fixed 0, 10, 20, 40, 80, and 120 min after microinjection with SM and processed for thin- and thick-section electron microscopy. At 0 min postinjection, rat liver SM were observed as small vesicles and were loosely dispersed amongst oocyte organelles. At 10 min, tubules were discerned among many elongate vesicles; and these structures comprised large cytoplasmic regions delimited by mitochondria and yolk platelets. By 20 min, segregation of transplanted organelles yielded yolk-platelet-free regions composed of few vesicles but increasingly numerous, long and anastomosing tubules. By 40 min, a network with numerous tubular branches and fenestrations was observed among the few remaining vesicles. By 80 min, transformation of rat liver SM into a complex network of branching and anastomosing tubules was complete. Three-dimensional reconstruction revealed the network to be composed of interconnecting elements consisting of anastomosing tubules. The reconstituted network of anastomosing tubules in Xenopus oocytes was compared to the network of anastomosing tubules in rat liver hepatocytes and was found to be essentially identical. Network formation occurred in oocytes pretreated with either vinblastine (40 microM) or nocodazole (0.166 microM), and network organization was maintained in oocytes treated with the same drugs after microinjection and reconstitution. We conclude that SM retain sufficient molecular information for rapid self-assembly into structures resembling those in the cells from which they were derived. Both the assembly and maintenance of ER structure in oocyte cytoplasm are microtubule-independent. The formation of such structures following microinjection of SM into living cells provides a unique assay for this type of membrane subfraction.     

Specificity of Octopine Uptake by Rhizobium and Pseudomonas Strains

The octopine-utilizing strain Agrobacterium tumefaciens B6S3 and three nonagrobacteria which had the capacity to utilize this opine were compared for octopine uptake. The characteristics of uptake by Rhizobium meliloti A3 and strain B6S3 were similar. In both bacteria, uptake activity was inducible by octopine and by the related opine octopinic acid, and competition assays showed that these two opine substrates were accepted by the same uptake system with an equivalent affinity. Cells of Pseudomonas putida 203 accumulated octopine against a concentration gradient, and this activity was induced specifically by octopine. While strain 203 did not utilize octopinic acid, a spontaneous mutant with a combined capacity for octopine and octopinic acid utilization was obtained. Both opines induced octopine uptake by this mutant, but octopinic acid was not a substrate for the induced system. Thus, the Pseudomonas uptake system exhibited a different specificity for octopine than the corresponding Agrobacterium system. The nonfluorescent pseudomonad GU187j, which utilized the three related opines octopine, octopinic acid, and nopaline, was constitutive for octopine uptake. Strain GU187j possessed a system which accepted these three opines, but not arginine or ornithine, with a similar affinity.     

Defective lysis of streptomycin-resistant escherichia coli cells infected with bacteriophage f2

A lysis defect was found to account for the failure of a streptomycin-resistant strain of Escherichia coli to form plaques when infected with the male-specific bacteriophage f2. The lysis defect was associated with the mutation to streptomycin resistance. Large amounts of apparently normal bacteriophage accumulated in these cells. Cell-free extracts from both the parental and mutant strains synthesized a potential lysis protein in considerable amounts in response to formaldehyde-treated f2 RNA but not in response to untreated RNA. As predicted from the nucleotide sequence of the analogous MS2 phage, the protein synthesized in vitro had the expected molecular weight and lacked glycine. The cistron for the lysis protein overlapped portions of the coat and replicase cistrons and was translated in the +1 reading frame. Initiation at the lysis protein cistron may be favored by translation errors that expose the normally masked initiation site, and streptomycin-resistant ribosomes, known to have more faithful translation properties, may be unable to efficiently synthesize the lysis protein.     

Leakage induced in Escherichia coli cells by A protein-RNA complexes from bacteriophage f2

Complexes of f2 phage RNA and its A protein, or maturation protein, transfect Escherichia coli cells much better than does protein-free RNA. We used these complexes to introduce the bacteriophage f2 lysis gene into cells. The A protein-RNA complex was found to kill cells, probably by causing them to leak large macromolecules. Previously induced beta-galactosidase leaked from cells treated either with the A protein-RNA complex or with lethal but noninfectious complexes that had been treated with formaldehyde. This observation was consistent with an earlier finding that formaldehyde-treated f2 RNA stimulates the in vitro synthesis of a lysis protein. The complexes did not stimulate the rate of leakage of beta-galactosidase from a streptomycin-resistant mutant known to be lysis defective. On the other hand, the rate of leakage was increased in a double mutant resistant to both streptomycin and rifampin and which is lysed normally by f2 bacteriophage.     

The role of strain energy in cycloamylose substrate complexation

The cycloamyloses, a group of cyclic oligosaccharides composed of α-1,4-linked glucose units, provide an opportunity to study the driving forces responsible for enzyme-substrate binding. The cycloamylose substrate binding energy has been attributed to two sources: expulsion of high energy cavity water and release of conformational strain energy. Our studies have shown that release of strain energy plays only a minor role in overall energetics of binding.     

Crystal and molecular conformation of the fluorescent probe. 8-anilino-1-naphthalenesulfonic acid (ANS).

The crystal and molecular structure of the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS) has been determined as the ammonium monohydrate with two conformationally distinct molecules in the triclinic $P\overline{1}$ lattice. The angles between the aromatic rings and the CNC plane are ?9/22° and 112/22° respectively. There is an NH...O intramolecular hydrogen bond in each molecule indicating that hydrogen bond formation is not dependent on the anilino geometry. There are also short intramolecular H...H contacts involving the hydrogens which have anomalous proton shifts shown in a recent NMR study.     

Genetic architecture,biochemical underpinnings and ecological impact of floral UV patterning

Floral attraction traits can significantly affect pollinator visitation patterns, but adaptive evolution of these traits may be constrained by correlations with other traits. In some cases, molecular pathways contributing to floral attraction are well characterized, offering the opportunity to explore loci potentially underlying variation among individuals. Here, we quantify the range of variation in floral UV patterning (i.e. UV ‘bulls‐eye nectar guides) among crop and wild accessions of Brassica rapa. We then use experimental crosses to examine the genetic architecture, candidate loci and biochemical underpinnings of this patterning as well as phenotypic manipulations to test the ecological impact. We find qualitative variation in UV patterning between wild (commonly lacking UV patterns) and crop (commonly exhibiting UV patterns) accessions. Similar to the majority of crops, recombinant inbred lines (RILs) derived from an oilseed crop × WI fast‐plant^® cross exhibit UV patterns, the size of which varies extensively among genotypes. In RILs, we further observe strong statistical‐genetic and QTL correlations within petal morphological traits and within measurements of petal UV patterning; however, correlations between morphology and UV patterning are weak or nonsignificant, suggesting that UV patterning is regulated and may evolve independently of overall petal size. HPLC analyses reveal a high concentration of sinapoyl glucose in UV‐absorbing petal regions, which, in concert with physical locations of UV‐trait QTLs, suggest a regulatory and structural gene as candidates underlying observed quantitative variation. Finally, insects prefer flowers with UV bulls‐eye patterns over those that lack patterns, validating the importance of UV patterning in pollen‐limited populations of B. rapa.     

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (T_m) of the mutants over the native BcEST that has a T_m of 50°C was outperformed by TtEST, a naturally occurring homologue with a T_m of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.