Andreas Düsterhöft

Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c–GFP) and the nucleus (Ynl075w–GFP) as the sites of action of two proteins. Ynl080c–GFP and Ynl081c–GFP fluorescence was visible in dot‐shaped and elongated structures, whereas the Ynl022c–GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C‐terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5′–GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP–Ynl020c and GFP–Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases. Copyright

Cloning, sequence and characterization of m5C-methyltransferase-encoding gene, hgiDIIM (GTCGAC), from Herpetosiphon giganteus strain Hpa2

We have cloned the gene (hgiDIIM) encoding the methyltransferase (MTase) of the SalI isoschizomeric restriction-modification (R-M) system, HgiDII (GTCGAC), into Escherichia coli. The hgiDIIM gene has been isolated from the same plasmid library of Herpetosiphon giganteus strain Hpa2, as was the previously cloned R-M system, HgiDI [AcyI/GRCGYC; Düsterhöft et al., Nucleic Acids Res. 19 (1991) 1049-1056]. Sequencing and functional localization of hgiDIIM revealed an open reading frame (ORF) of 354 codons (39786 Da) with significant homologies to the group of m5C-, rather than the m4C-/m6A-, MTases. Subsequent cloning and analysis of adjacent chromosomal segments led to the identification of two additional ORFs upstream (ORF15, 139 codons) and downstream (ORF68, 611 codons) from hgiDIIM with the same transcriptional orientation as the hgiDIIM gene. However, the expected restriction enzyme function was not found in either of these ORFs.

Organization and gene expression within restriction-modification systems of Herpetosiphon giganteus.

We have characterized a family of related restriction-modification (R-M) systems from the soil bacterium Herpetosiphon giganteus (Hgi). A comparison of their genetic organization reveals two types of regulatory proteins, called controlling ORF C. While one of these small reading frames derived from RM.HgiCI seems to be an enhancer of its own promoter, evidence is provided for a silencer function of the other ORF C derived from the closely related AvaII-type systems RM.HgiBI/CII/EI. The respective silencer function is detected during our various attempts to clone three isoschizomers with unusually high differences in their specific activity. Sequencing and site-directed mutagenesis revealed just two amino acids as being responsible for a massive increase in specific activity of these endonucleases.

Site Directed Mutagenesis on the Restriction Endonuclease Eco RI Using Mixed Oligonucleotides

Abstract The restriction endonuclease EcoRI could be modified via site directed mutagenesis at position Arg200. Using the thiophosphate system we introduced either Lys, Glu or Gly in a one pot procedure. Although G recognition should be affected, Lys200 showed wildtype specificity.