3 pmol, or 54.8 pmol His+7968. Arrow indicates DNA + protein shift. Discussion In this study, we explored the transcriptional machinery associated with the jamaicamide biosynthetic gene cluster in Lyngbya majuscula. The jamaicamide cluster was chosen because it possesses a number of features commonly seen in other secondary metabolites isolated from marine cyanobacteria [3]. The https://www.selleckchem.com/products/ganetespib-sta-9090.html jamaicamides are produced by the most prolific cyanobacterial natural product producer yet GSK1120212 in vivo known (L. majuscula), are bioactive (ichthyotoxic, neurotoxic), are composed of mixed PKS/NRPS derived subunits, and contain unusual structural

features such as a vinyl chloride and alkynyl bromide rarely seen in natural products from other organisms. The first description of the jamaicamides [6] demonstrated that the cluster is composed of 17 ORFs, with 16 transcribed in the same direction. The cluster is flanked on the 5′ and the 3′ ends by transposases and hypothetical proteins. From the results of our RT-PCR

experiments, it appears that the gene cluster is preceded by an unusually long untranslated leader region (at least 844 bp), one that may be unprecedented in size for a secondary metabolite gene cluster. The function of having such a long region Akt inhibitor between the TSS and the start codon of jamA is unclear at this time, but may be important for overall regulation of the pathway. In Synechococcus PCC 7942, the psBAII and psBAIII genes encoding the photosystem II reaction center D1 protein have cis regulatory elements in addition to basal promoters. Contained in the untranslated leader region downstream

of the Glycogen branching enzyme psB TSS are light responsive elements that were found to be responsible for increased expression of the genes under high light conditions [37]. In the jamaicamide pathway, the fact that another region of DNA immediately upstream of jamA can function as a strong promoter indicates that although transcription may initiate well before the ORF start site, there could be a supplemental means of boosting transcription closer to the first protein in the cluster. The amplification of second strand cDNA from JHB RNA corresponding to all of the intergenic regions between the jamaicamide ORFs tested indicated that the pathway is transcribed in at least two pieces. The first, jamABCDEFGHIJKLMNOP, is sufficiently large (~55 kb) to assume that multiple transcripts could be needed to process this portion of the gene cluster. A similar situation was found with the microcystin gene cluster [22], in which all of the intergenic regions of the pathway aside from the bidirectional promoter were transcribed, and RACE experiments with several of these regions detected variations in intergenic TSS locations. As with microcystin, the jamaicamide pathway could contain internal promoters which, while not representing true breaks in the transcription of the pathway, can function independently if not overwritten by RNAP acting from an upstream promoter (promoter occlusion; [38]).