The gene scaS is amplified by PCR and cloned into the pFE4 vector using EcoRI and HindIII restriction sites.
Extract
[...] Of these different restriction sites, we can rule out the BamHI and XbaI restriction sites, since they are also present in the coding sequence of scas (according to figure 1). We can rule out the SalI restriction site since it is present in two copies in the vector, and using the latter would lead to a loss of the replication origin (noted "Ori"). We then have the EcoRI and HindIII restriction sites for the cloning of scas in the cloning vector. [...]
[...] The m1 mutation, which corresponds to a small deletion of 10 nucleotides, is located in intron situated between exon 3 and exon 4 of the gene Dm. The m2 mutation is a point mutation of the substitution type, replacing one nucleotide with another, and is located in intron 4 of the gene Dm, in the coding sequence of the version 2 protein. These mutations can have different consequences: -The m1 mutation, which is located in intron 3 of the gene Dm, is not located in the coding sequence of the protein, and will therefore a priori no incidence on the peptide sequence of the protein produced by the gene Dm. [...]
[...] A transcriptional fusion corresponds to the fusion of the promoter of the gene of interest with the sequence of a reporter gene, with the coding sequence of the gene of interest and translation signals. It is different from a translational fusion since the latter also contains the signals necessary for translation as well as the first nucleotides of the coding region. While the transcriptional fusion makes it possible to specifically study the transcription of the gene of interest, the translational fusion makes it possible to study both the transcription and translation of a gene of interest. 7. What can you conclude about the expression of scaY? of scaS? [...]
[...] We know that scaS code for a protein involved in the survival of P. mirabilis in the presence of a significant amount of copper, and the gene scaY a function unknown gene is found in all strains containing the gene scaS. For more information, a study of the transcriptional fusions of scaS-lacZ and scaY-lacZ notably in wild-type strains. We can see that in a wild-type strain, the ?-galactosidase activity of the transcriptional fusion of scaY-lacZ is 3000 units in the absence as well as in the presence of copper, while the ?-galactosidase activity of the transcriptional fusion of scaS-lacZ is 90 units in the absence of copper and 1900 in the presence of copper. [...]
[...] Balance scheme of the functions of ScaS and ScaY in P. mirabilis. Subject II: Eukaryotic molecular genetics: 1. Represent the different primary and mature RNAs of the gene Dm. You will indicate on your diagram the position of the start and stop codons of translation as well as the UTR sequences on mature RNAs. The different primary and mature RNAs of the gene Dm are represented below, note that the primary RNA, numbered 1 by our care, corresponds after splicing to the mature RNA bearing the same number. [...]
