Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

The half-life of metabolically labeled pp60src of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp60src with tumor-bearing rabbit serum. These experiments showed that pp60src has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp60src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42 degrees C) or permissive (35 degrees C) temperature. The half-life of the pp60src -associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp60src. The apparent contradiction between the half-lives of the kinase activity and the [35S]methionine-labeled pp60src protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp60src, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.     

Further evidence for the existence of a viral envelope protein defect in the Bryan high-titer strain of Rous sarcoma virus.

Electron microscopy observations of purified Bryan high-titer Rous sarcoma virus (BH RSV) using the freeze-drying technique showed that progeny made in the absence of a helper virus lacked visible surface projections or spikes. Phenotypic mixing experiments employing BH RSV and a thermolabile mutant of vesicular stomatitis virus, tl 17, yielded no evidence of pseudotype formation. Since tl 17 is known to be defective for an envelope glycoprotein, the lack of successful phenotypic mixing with BH RSV is consistent with the observed absence of viral spikes.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Pollen germination in Welwitschia mirabilis Hook. f.: differences between the polyplicate pollen producing genera of the Gnetales

Pollen grains of the seed plant genera Ephedra L. and Welwitschia Hook. f. (Gnetales) are of similar size, shape, and have a polyplicate exine with alternating thicker and thinner regions. Ephedra pollen is considered inaperturate and the exine is shed during germination, leaving the male gametophyte naked. The shed exine curls up and forms a characteristic structure with transverse striations. Such upcurled exines have been found in situ in Early Cretaceous seeds with affinities to Ephedra. The purpose of this study was to document the germination of Welwitschia pollen and investigate whether they also discard their exine during this process.

The pollen grains of Welwitschia are monoaperturate with a distinct, distal sulcus. During germination, the sulcus splits open and the gametophyte expands to a spherical form that extends out of the exine. The pollen tube starts to grow one or two hours later and as in Ephedra, it is displaced towards one side. The exine is not shed but remains as a “cap” that partly covers the male gametophyte. Thus, in this respect the germination process is distinctly different from that in Ephedra and this study demonstrates that discharging the exine during pollen germination is unique to Ephedra, among the polyplicate pollen producing genera in the Gnetales.