Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria.

Abstract

We have used the TEM beta-lactamase transposon Tn901, located on Escherichia coli plasmid pRI46, to introduce in vivo a genetic marker into plasmid pUH24, present in the cyanobacterial strain Anacystis nidulans R-2. Restriction enzyme analysis and heteroduplex studies of the 8.3 x 10(6)-dalton plasmids pCH1-pCH5, present in the ampicillin-resistant A. nidulans R-2 colonies obtained after transformation with pRI46, demonstrated that these plasmids consist of the complete sequence of Tn901 inserted at different places into plasmid pUH24. The pUH24::Tn901 recombinant plasmids transform A. nidulans R-2 with a frequency of 10(-4)--10(-5) per microgram of plasmid DNA and contain a single cleavage site for the restriction enzyme Xho I. From pCH1 a plasmid of 5.5 x 10(6) daltons,pUC1, was constructed with only a part of the Tn901 sequence and an additional single cleavage site for the restriction enzyme BamHI. This plasmid, as well as plasmids pCH1-pCH5, are potentially useful as vectors for cloning genes in cyanobacteria and for studying cyanobacterial plasmid biology.

A new approach for molecular cloning in cyanobacteria: cloning of an anacystis nidulans met gene using a Tn 907-induced mutant

“…One of these recombinant plasmids, pBHTl87 (15.1 kb), was analyzed further. Restriction analysis of the purified EcoRI met::Tn902 DNA fra~ent revealed the presence of two BumHI sites, one located in the transposon Tn901 ( Van den Hondel et al,, 1980), the second in the cloned A. niduluns R-2 DNA fragment. Fig.…”

“…As an alternative to classical genetic analysis, we have begun to develop a molecular cloning system in A. niduluns R-2 ( Van den Hondel et al, 1980).…”

“…The small plasmid pUH24 has been genetically marked by in vivo insertion of the TEM P-lactamase transposon Tn90Z ( Van den Hondel et al, 1980). This recombinant plasmid pCH 1 (pUH24: :Tn 901) efficiently transforms A. nidulans R-2, is stably maintained, and confers on the cells resistance to l--2 pgg/ml of ampicillin.…”

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

“…The freshwater unicellular cyanobacterium Synechococcus elongatus PCC 7942, formerly Anacystis nidulans R2, carries two endogenous plasmids, pANS (also called pUH24) and pANL (also called pUH25) (van den Hondel et al, 1980;Van der Plas et al, 1992). These two plasmids have been identified not only in the closely related strain S. elongatus PCC 6301, but also in Cyanobium (Synechococcus) sp.…”

Modelling and multiobjective optimization for simulation of cyanobacterial metabolism

“…Its genome was sequenced, annotated and published in 1980 (van den Hondel et al, 1980;Van der Plas et al, 1992;Chen et al, 2008). Shastri and Morgan (2005), which leads to short-cuts in flux distributions when optimised.…”