Metal-binding, nucleic acid-binding finger sequences in the CDC16 gene of Saccharomyces cerevisiae.

The CDC16 gene is involved in the process of chromosome segregation in mitosis and a cdc16ts mutant accumulates the predominant microtubule-associated protein at the nonpermissive temperature. We find that the CDC16 gene open reading frame (ORF) is capable of encoding a protein whose calculated molecular weight and pI are 94,967 and 6.60, respectively. This hypothetical protein contains 16 cysteine residues; five are clustered at the N-terminal, 4 are placed about 3 residues apart in the middle of the peptide, and 3 are located close to the C-terminal. Each of these could form a metal-binding, nucleic acid-binding domain, suggesting this protein acts either as a repressor of the microtubule-associated protein gene or as a component necessary for spindle elongation, possibly interacting with the DNA. The start of the CDC16 ORF is only 95 bp downstream from the end of the MAK11 ORF. In this region there are two TATA boxes in tandem, but there is no room for a UAS or other regulatory sequences. An ATG is present 5 bp upstream of the start of the large ORF. Its frame terminates after only two amino acids

The essential roles of cytidine diphosphate-diacylglycerol synthase in bloodstream form Trypanosoma brucei

Funded by Wellcome Trust: Senior Research Fellowship, Grant Number: 067441 and Wellcome Trust, Grant Numbers: 082596, 093228.Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host in several fundamental ways. This has lead to the validation of a plethora of novel drug targets, giving hope of novel chemical intervention against this neglected disease. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate for several pathways in both prokaryotes and eukaryotes, being produced by CDP-DAG synthase (CDS). However, nothing is known about the single T. brucei CDS gene (Tb927.7.220/ EC 2.7.7.41) or its activity. In this study we show TbCDS is functional by complementation of a non-viable yeast CDS null strain and that it is essential in the bloodstream form of the parasite via a conditional knockout. The TbCDS conditional knockout showed morphological changes including a cell-cycle arrest due in part to kinetoplast segregation defects.Biochemical phenotyping of TbCDS conditional knockout showed drastically altered lipid metabolism where reducing levels of phosphatidylinositol detrimentally impacted on glycoylphosphatidylinositol biosynthesis. These studies also suggest that phosphatidylglycerol synthesised via the phosphatidylglycerol-phosphate synthase is not synthesised from CDP-DAG, as was previously thought. TbCDS was shown to localised the ER and Golgi, probably to provide CDP-DAG for the phosphatidylinositol synthases.Publisher PDFPeer reviewe