Proteomic analysis of the bacterial cell cycle

A global approach was used to analyze protein synthesis and stability during the cell cycle of the bacterium Caulobacter crescentus. Approximately one-fourth (979) of the estimated C. crescentus gene products were detected by two-dimensional gel electrophoresis, 144 of which showed differential cell cycle expression patterns. Eighty-one of these proteins were identified by mass spectrometry and were assigned to a wide variety of functional groups. Pattern analysis revealed that coexpression groups were functionally clustered. A total of 48 proteins were rapidly degraded in the course of one cell cycle. More than half of these unstable proteins were also found to be synthesized in a cell cycle-dependent manner, establishing a strong correlation between rapid protein turnover and the periodicity of the bacterial cell cycle. This is, to our knowledge, the first evidence for a global role of proteolysis in bacterial cell cycle control.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

Pseudoknots in prion protein mRNAs confirmed by comparative sequence analysis and pattern searching

The human prion gene contains five copies of a 24 nt repeat that is highly conserved among species. An analysis of folding free energies of the human prion mRNA, in particular in the repeat region, suggested biased codon selection and the presence of RNA patterns. In particular, pseudoknots, similar to the one predicted by Wills in the human prion mRNA, were identified in the repeat region of all available prion mRNAs available in GenBank, but not those of birds and the red slider turtle. An alignment of these mRNAs, which share low sequence homology, shows several co-variations that maintain the pseudoknot pattern. The presence of pseudoknots in yeast Sup35p and Rnq1 suggests acquisition in the prokaryotic era. Computer generated three-dimensional structures of the human prion pseudoknot highlight protein and RNA interaction domains, which suggest a possible effect in prion protein translation. The role of pseudoknots in prion diseases is discussed as individuals with extra copies of the 24 nt repeat develop the familial form of Creutzfeldt–Jakob disease.