The multicopper oxidase CutO confers copper tolerance to Rhodobacter capsulatus

The cutO gene of the photosynthetic purple bacterium Rhodobacter capsulatus codes for a multicopper oxidase as demonstrated by the ability of the recombinant Strep-tagged protein to oxidize several mono- and diphenolic compounds known as substrates of Escherichia coli CueO and multicopper oxidases from other organisms. The R. capsulatus cutO gene was shown to form part of a tri-cistronic operon, orf635-cutO-cutR. Expression of the cutO operon was repressed under low copper conditions by the product of the cutR gene. CutO conferred copper tolerance not only under aerobic conditions, as described for the well-characterized E. coli multicopper oxidase CueO, but also under anaerobic conditions.     

A new class of LINEs (ATLN-L) from Arabidopsis thaliana with extraordinary structural features.

The Arabidopsis thaliana genome has about 250 copies of LINEs (here called ATLNs). Of these, some, called ATLN-Ls, have an extra sequence of about 2 kb in the region downstream of two consecutive open reading frames, orf1 and orf2. Interestingly, the extra sequences in these ATLN-L members have another open reading frame, designated as orf3. Each member is flanked by direct repeats of a target site sequence, showing that ATLN-L members with the three open reading frames have retrotransposed as a unit. The ATLN-L members are also distinct from other ATLN members: orf1 terminates with TAA (or TAG) and is located in the same frame as orf2, and the ATG initiation codon of orf2 is not present in the proximal region. A sequence that may form a pseudoknot structure in ATLN-L mRNA was present in the proximal region of orf2, therefore the TAA (or TAG) termination codon of orf1 is assumed to be suppressed to produce an Orf1-Orf2 transframe protein during the translation of the ATLN-L mRNA. The region between orf2 and orf3 is several hundred bp long, suggesting that orf3 expression is independent of orfl-orf2. The amino acid sequences of the proteins Orf1 and Orf3 are highly homologous in their N-terminal half regions that have a retroviral zinc-finger motif for RNA binding. Orf3, however, has a leucine-zipper motif in addition to the zinc-finger motif. The C-terminal regions of the Orf1 and Orf3 proteins have poor homology, but seem to have nuclear localization signals, suggesting that these proteins are involved in the transfer of ATLN-L mRNA to nuclei. A phylogenetic tree shows that Orf3 proteins form a branch distinct from the branches of the Orf1 proteins encoded by ATLN-L members. This indicates that an ancestor element of ATLN-Ls has incorporated the orf1 frame carried by another ATLN member into its distal region to orf1-orf2 during evolution.     

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of orf1 was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.