## 2 Transcriptional Regulation in Integrons
### Transcription of Gene Cassettes Independent of Pc and Cassette-Borne Promoters
#### 2.4.4 Fused Gene Cassettes  
Gene cassettes generally include a single gene and an _altC_ site. However, cassette fusions may arise in several ways, such as slippage during DNA replication caused by _attC_ stem-loop structures, or as a result of illegitimate integrase-mediated intermolecular recombination events between internal regions of _attC_ and _attI_ sites, which lead to _attC_ site loss (partial or total) from the upstream cassette [5]. In this case, both complete coding regions are retained in a unique cassette, creating permanent gene arrays comparable to bacterial operons, which remain mobilizable by the _attC_ site from the second cassette [5; 27].  
Several fused cassettes have been documented, and in some cases, the fused genes could be expressed by the presence of upstream promoter regions. It has been shown that the fused gene cassette _bla\({}_{\text{OXA-10}}\)/aadA1_, created by the deletion of _bla\({}_{\text{OXA-10}}\)_attC_ site, was transcribed by their own promoter sequence present upstream _bla\({}_{\text{OXA-10}}\)_ cassette [41]. In Tn\(1331\), the promoterless _bla\({}_{\text{OXA-9}}\)_ gene, found immediately downstream of an _aadA1_ cassette with a truncated _attC_ site, was transcribed from both the Pc and from an additional promoter located at the 3'end of the _aadA1_ coding region [97]. [PERSON] et al. [90] demonstrated that the _ant(3)-li/aac(6)-lld_ fused cassette was transcribed by a promoter provided by an upstream Group II intron, generating a bifunctional protein that retained both ANT(3)-I and AAC(6)-II activities, as demonstrated by the observed extended aminoglycos resistance phenotype.  
However, in spite of the presence of specific promoters in several fused cassettes, studies on the transcription dynamics of fused gene cassettes in integrins were scarce. A previous study measured the relative expression of the _bla\({}_{\text{GES-1}}\)/aacA4_ fused cassette. This fusion was placed in the second position of a class 1 trigon, preceded by the _gcu14_ cassette of unknown function [98]. This fusion was created by partial loss of 91 bp of GES-1 _attC_ site, retaining the entire coding regions of both _bla\({}_{\text{GES-1}}\)_ and _aacA4_ (Figure 7). This study experimentally revealed that the _gcu14_ cassette, found in the first position closer to the PcW\({}_{\text{TGN-10}}\), had an increased transcription relative to _bla\({}_{\text{GES-1}}\)_ and _aacA4_. On the other hand, similar transcript amounts were observed for _bla\({}_{\text{GES-1}}\)_ and _aacA4_, both alone and in the fused form, indicating that that the fused genes were transcribed together. Northern Blot revealed the production of both monocistronic and full-length polycistronic transcripts of gene cassette array, as found elsewhere [2]. The monocistronic transcripts corresponded in size to the _gcu14_ gene cassette, but never to _bla\({}_{\text{GES-1}}\)_ or _aacA4_ alone. In fact, _bla\({}_{\text{GES-1}}\)_ and _aacA4_ were only found in full-length polycistronic transcripts corresponding to the entire gene cassette array _gcu14-bla\({}_{\text{GES-1}}\)/aacA4_, demonstrating that these genes retained in the fusion were always co-transcribed [98]. Moreover, the identification of polycistronic transcripts comprising the _gcu14_ cassette, which harbors a complete _attC_ site, gave support to the previous evidence that _attC_ does not act as a transcriptional terminator [59].  
Figure 6: Schematic representation of the acquisition dynamics of a promoter-containing ORF-less cassette by a class 1 integro. The insertion of the promoter-containing ORF-less cassette into _attI11_ site disengages the original first gene cassette A (blue arrow) from Pc promoter but provides a new promoter region.