Finn Kirpekar

The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA

tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic‐producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification is dependent on the presence of the methyl group donor, S‐adenosyl methionine. Analysis of the 74‐mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches, indicating that TlrB is the first member to be described in a new subclass of rRNA methyltransferases that are implicated in macrolide drug resistance.

Oligodeoxynucleotide analogues containing 3′-deoxy-3′-C-threo-hydroxymethylthymidine: Synthesis, hybridization properties and enzymatic stability

Abstract Novel Oligodeoxynucleotide analogues containing 3′- C - threo -methylene phosphodiester internucleoside linkages were synthesized on automated DNA-synthesizers using the phosphoramidite approach. The sugar modified phosphoramidite building block 5 was obtained by phosphitylation of 1-(2,3-dideoxy-5- O -(4,4′-dimethoxytrityl)-3- C -hydroxymethyl-β-D- threo -pentofuranosyl)thymine ( 4 ) which was synthesized in only three steps from 5′- O -(4,4′-dimethoxytrityl)thymidine ( 1 ). The hybridization properties and enzymatic stability of the oligonucleotide analogues were studied by UV experiments. 17-Mers having one or three modifications in the middle or two modifications in each end hybridized to DNA with moderate lowered affinity compared to unmodified 17-mers (ΔT m 1–3°C per modification). Furthermore, the end-modified and all-modified oligonucleotides were stable towards snake venom phosphodiesterase.

Branched oligodeoxynucleotides: Automated synthesis and triple helical hybridization studie

Abstract Automated synthesis of novel mono-branched (“Y-shaped”) and double-branched (“H-shaped”) oligodeoxynucleotide analogues has been accomplished by use of phosphoramidite chemistry. The synthetic strategy involves stereoselective 3′- O -phosphitylation of nucleoside 2 to give building block 3 and fully automated stereoselective elongation and subsequent branching of amidite 3 on a DNA-synthesizer. Thus, this strategy allows construction of branched nucleic acid molecules with sequences of arbitrary branch length and base composition. Compared to the corresponding linear analogues, the novel branched oligodeoxynucleotides exhibited increased thermal stability in intramolecular and intermolecular hybridization experiments.

Mutational analysis of a variant of ARS1 from Saccharomyces cerevisiae

SummaryA naturally occurring single base-pair G to A transition, creating a 10/11 near-match close to the essential 11 base-pair core consensus of ARS1, was used to investigate the importance of near-match sequences. The 10/11 near-match can not substitute for the core consensus since an ARS- phenotype is observed when the core consensus is deleted. However, deletion mutations revealed that this near-match together with a short palindromic sequence, also situated in the B-flanking region, comprise a single element crucial for optimal ARS function. The palindrome has the potential of forming a stemloop structure. Rather precise observations concerning the borders of the B-region were achieved. The four base pairs separating the near-match from the core consensus perform a spacing function where the identity of the bases are unimportant. However, this spacing is highly important since deletion of these four base pairs leads to an ARS- phenotype.

The yeast Rad6 protein: A mediator of homologous recombination across the scaffold attached region at the replication origin ARS1

Here we show that the ubiquitin‐conjugating enzyme Rad6p plays a crucial role in locus‐specific replacement recombination in the TRP1‐ARS1 region. In rad6‐1 strains, where this ubiquitination activity is modified, homologous recombination across a 150 bp continuous region is completely abolished. Our results unambiguously identified the ARS1 scaffold attached region (SAR) as being the region where this impediment for replacement recombination is located, since a merging of the location of the recombination impediment and binding properties in a scaffold exchange assay with deletion mutations was observed. Our observations strongly support the notion of torsionally separated chromosomal domains being organized by SARs and scaffold proteins, and being dynamically realigned as a consequence of ubiquitination and proteolysis.