Characterization of a pollen-specific cDNA from sunflower encoding a zinc finger protein.

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.     

Smoothelin-like 2 Inhibits Coronin-1B to Stabilize the Apical Actin Cortex during Epithelial Morphogenesis

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Expression of functional human acid beta-glucosidase in COS-1 and Spodoptera frugiperda cells

A cDNA encoding human acid beta-glucosidase (N-acylsphingosyl-1-O-beta-D-glucoside: glucohydrolase, EC 3.2.1.45) expressed catalytically active enzyme in transfected COS-1 or infected Spodoptera frugiperda (Sf9) cells. The expression plasmid p91023(B) (p91023B/Glc) and a Baculovirus (AcMNPV/Glc) containing the cDNA were constructed and used with the respective cells. By immunoblotting a glycosylated, 63-kilodalton human acid-beta-glucosidase was detected in the transfected or infected cells. A 56-kilodalton human polypeptide was obtained after complete deglycosylation with N-Glycanase. The expressed human enzymes also had partial endoglycosidase H sensitivity. The human enzyme expressed at high levels in Sf9 cells and had normal immunologic properties. With the partially purified enzyme from Sf9 cells, intact function of active site was indicated by normal kcat and Kmapp or Kiapp values for alternative substrates or potent inhibitors, respectively. The expressed enzyme was also activated normally by the negatively charged lipid, taurocholate. The results of these studies indicate that the Baculovirus expression system could provide a convenient source of normal human enzyme for structure/function investigations. In addition, this expression system should prove useful for the identification and evaluation of putative etiologic point mutations in Gaucher disease variants with kinetically altered residual enzymes.