Huckle, James William (1993) "Prokaryotic Metallothionein gene isolation, Nucleotide sequence and expression". Doctoral thesis, Durham University.
Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins, which are proposed to have roles in essential trace metal homoeostasis and in the detoxification of metal ions. The genes encoding MTs have been isolated from a wide range of eukaryotes, although MT genes have not previously been isolated from prokaryotes. The polymerase chain reaction (PGR) was initially used to isolate a prokaryotic MT gene fragment from Synechococcus PCC 6301. PGR fragments were amplified using inosine-containing primers designed from the amino acid sequence of a prokaryotic MT. Subsequent cloning and nucleotide sequence analysis revealed that the deduced amino acid sequence of the PGR product corresponded to the amino acid sequence of the prokaryotic MT. The amplified product was thus part of the gene encoding the MT, and was designated smtA. The same primers used in the initial amplification were subsequently utilised for anchored PGR, to amplify the remainder of the coding region and the 3' and 5' flanking regions of the smtA gene. A genomic library was produced from Synechococcus PGG 7942 DNA and screened using the PGR products described above as probes. A genomic clone was isolated, nucleotide sequence analysis revealed the structure of the smt locus, two open reading frames, smtA and smtB, arranged in a divergent orientation about the smt operator/promoter region. The operator/promoter region contains the transcriptional and translational signals for the two genes and three regions that are candidate sites for interaction of regulatory proteins. The transcript start sites of the two genes were mapped within the operator/promoter region by primer extension analysis. An increase in the relative abundance of transcripts of both smt genes was studied in response to various metal ions in a series of northern blots. Inhibitor studies confirmed that the smtA gene is regulated at the transcriptional level. The 5' flanking region of the smtA gene conferred metal specific induction of the reporter gene lacZ. SmtB has sequence similarity to several prokaryotic regulatory proteins and contains a putative helix-turn-helix structural domain. Deletion analysis suggests that SmtB is a repressor of smtA expression. Subsequent work has confirmed that SmtB is a trans-acting repressor of expression from the smt operator/promoter.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||16 Nov 2012 10:53|