A simple immunological detection method for the direct screening of genes from clone banks

A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.     

Approaches used to examine the mechanism and regulation of hexose transport in rat myoblasts

This review discusses some of the approaches and general criteria that we have used to examine the properties of the hexose transport system in undifferentiated L6 rat myoblasts. These approaches include studying the kinetics of hexose transport in whole cells and plasma membrane vesicles, the effects of various inhibitors on hexose transport, the isolation and characterization of hexose transport mutants, and the use of cytochalasin B (CB) to identify the transport component(s). Transport kinetics indicated that two transport systems are present in these cells. 2-Deoxy-D-glucose is transported primarily by the high affinity system, whereas 3-O-methyl-D-glucose is transported by the low affinity system. Furthermore, these two transport systems are inactivated to different extents by CB. CB has a higher binding affinity for the low affinity hexose transport system. The inhibitory effect of various hexose analogues also revealed the presence of two hexose transport systems. The effects of various ionophores and energy uncouplers on hexose transport suggest that the high affinity system is an active transport process, whereas the low affinity system is of the facilitated diffusion type. The high affinity system is also sensitive to sulfhydryl reagents, whereas the low affinity system is not. Further evidence for the presence of two transport systems comes from the characterization of hexose transport mutants. Two of the mutants isolated are shown to be defective in the high affinity transport system, but not in the low affinity transport system. These mutants are also defective in the CB low affinity binding site. Based on our results a tentative working model for hexose transport in L6 rat myoblasts is presented.     

The Formation of Otoliths in the Frog Rana esculenta. Scanning Electron Microscopic and X-Ray Diffraction Studies

Two crystal forms of calcium carbonate were observed: calcite (utricle) and aragonite (saccule, lagena, endolymphatic sac). The first step in otolith formation is the appearance of organic structures in the macula. The subsequent step is characterized by fast growing primitive crystals with a prismatic habitus that successively transform into adult or mature crystals. With the metamorphosis, the aragonite crystals of the endolymphatic organ show clear signs of erosion that can be related to a process of CaCO₃ mobilization from such deposits.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Description of Thecavermiculatus andinus n.sp. (Meloidoderidae), a Round Cystoid Nematode from the Andes Mountains of Peru

Thecavermiculatus andinus n.sp. is described and illustrated from Oxalis tuberosa originally collected in the vicinity of Lake Titicaca high in the Andes mountains of southern Peru. This new species differs markedly front the other two species in the genus, especially in having a much greater female vulval-anal distance and annules with lined punctation on most of the female body with a lacelike pattern restricted to the posterior portion, particularly at the vulva and anus which do not protrude. Females are essentially spherical with protruding neck, white to yellowish in color, and can easily be mistaken for potato cyst nematodes. Among the dozen or more known weed and crop host plants are potato and eggplant. In order to accommodate this new species, the genus Thecavermieulatus is emended. A key to the species of this genus is presented.