Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp EC3

in Molecular Genetics & Genomics (2003), 270(4), 337-346

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a ... [more ▼]

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan. [less ▲]

Structure of the Telomeric Ends of Mt DNA, Transcriptional Analysis and Complex I Assembly in the Dum24 Mitochondrial Mutant of Chlamydomonas Reinhardtii

in Molecular Genetics & Genomics (2001), 266(1), 109-14

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some ... [more ▼]

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some monomers via their deleted ends. All molecules lack at least cob, nd4 and the 3' end of nd5, three adjacent genes located in the left part of the genome. We present evidence showing that in dum24, as in other deletion mutants, the deletions extend to the left telomeric end, and propose that the only replicative forms in the mutants are the dimeric DNA molecules that possess intact telomeric structures at both ends. Two abnormally large transcripts produced from chimeric genes are detected in dum24, which throws some light on the location of potential promoter sequences and processing signals in the mitochondrial genome. Using BN-PAGE analysis and immunological methods to detect complex I, we further show that dum24 mitochondria do not possess the normal multimeric complex I (850 kDa), but produce a smaller, partially assembled, complex (650 kDa), demonstrating a role for ND4 and/or ND5 subunits(s) in complex I assembly. [less ▲]