Structure-Function Relationships of Elements of the Bacteriophage f1 Genome

Expression of the ten genes on the small filamentous phage f1 genome must be closely regulated during the infection cycle, since this nonlytic coliphage lives in delicate balance with its host. The sequence and context requirements of specific regions involved in f1 mRNA metabolism were investigated by: 1) relocation of an intact transcription unit on the phage genome; 2) construction of a hybrid E. coli-f1 transcription unit which could be studied in a phage or plasmid context; 3) identification and characterization of a rho-dependent transcription termination signal in f1 by cloning it between the promoter and gene of an E. coli transcription unit, and by studies of its function in its usual phage context. Expression of the f1 major coat (gene VIII) protein was studied in vivo in cells infected with variant f1 phage whose genomes had been restructured in vitro. This gene with its most proximal promoter and terminator was found to behave as an independent transcription unit. Coat protein gene expression was unaffected by transposition of the gene to the phage\u27s large intergenic region, and was independent of orientation, but was dependent upon its own intact promoter. Gene VIII was placed under control of the E. coli lac promoter and the RNA from the new transcription unit was characterized. Coat protein was expressed from a hybrid mRNA initiated at the lac promoter. This mRNA, which contained the 5\u27 36 nucleotides of the lac operon mRNA at its 5\u27end, attached to the entire gene VIII mRNA sequence, was unstable (half life -1.5 min), as is the E. coli lac operon mRNA. In contrast, authentic f1 gene VIII mRNA is extremely stable (half life -10 min). The f1 large intergenic region was shown to encode a rho-dependent transcription termination signal. The minimal sequence required for terminator function in a heterologous plasmid system encompasses about 100 nucleotides. Like most known rho-dependent terminators, the signal contains a region of dyad symmetry. It differs from previously characterized rho-dependent terminators, in that the sequence at the termination site is G-C rather than A-T rich. In a rho mutant host, f1 tranvii scripts pass through the normal termination site, and stop downstream within a region of high potential secondary structure near the f1 origin of DNA replication. A method is described for the efficient construction in vitro of recombinant DNA molecules from fragments produced by cleavage with the restriction endonuclease HgaI. The method relies upon the unique properties of HgaI and is applicable to any viral or plasmid DNA that contains several HgaI recognition sites. Using f1 DNA, it is shown that only HgaI fragments that were originally adjacent on the genome can anneal, that infectious molecules are reassembled with high efficiency from a mixture of fragments, and that recombinant genomes can be easily constructed from parental DNAs containing genetic markers which map in different HgaI fragments

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacI-galR family of regulatory genes

malR of Strepomyces coelicolor A3(2) encodes a homologue of the Lacl/Galr family of repressor proteins, and is divergently transcribed from the malEFG gene cluster, which encodes components of an ATP-dependent transport system that is required for maltose utilization. Transcription of malE was induced by maltose and repressed by glucose. Disruption or deletion of malR resulted in constitutive, glucose-insensitive malE transcription at a level markedly above that observed in the parental malR+ strain, and overproduction of MalR prevented growth on maltose as carbon source. Consequently, MalR plays a crucial role in both substrate induction and glucose repression of maltose utilization. MalR is expressed from a single promoter with transcription initiating at the first G of the predicted GTG translataion start codon

A study of the regulation of undecylprodigiosin biosynthesis in Streptomyces coelicolor A3(2)

Undecylprodigiosin is one of four secondary metabolites with antibacterial activity produced by S. coelicolor A3(2). The overall aim of this study was to further investigate the control of biosynthesis of the secondary metabolite undecylprodigiosin (Red) in Streptomyces coelicolor A3(2). Proline transport mutants (Put-) were isolated, and the over-production of Red was observed in these strains. It was hypothesised that Red biosynthesis is essential as a shunt for excess proline in the Put- mutants. Red biosynthesis was abolished by disrupting the redX structural gene in a Put- mutant. The Put- RedX- mutants were viable, demonstrating that Red is not essential in Pur mutants. Si nuclease mapping of redD and redX genes in a Put- mutant revealed that red genes are transcribed earlier in the growth phase of Put- mutants compared to the progenitor strain J802. Pwb (pigmented whilst bid) mutants had been isolated due to their ability to produce Red in a b1dA background. The regions believed to contain the mutations of Pwb-6, Pwb-9, Pwb-16 and Pwb+ were sub-cloned and sequence data obtained. An open reading frame was identified which is predicted to encode a protein showing homology to the UhpA-LuxR family of regulators. The open reading frame, contains an in-frame TTA codon. It is proposed that this gene, named redZ, mediates the b1dA dependence of Red biosynthesis. The Pwb-6 mutation was located to the putative -35 promoter region. The mutation makes the promoter more similar to the enteric bacterium major sigma factor promoter -35 consensus sequence. It is anticipated that greater transcription from the promoter causes the Pwb phenotype. Introduction of the Pwb-9 redZ gene into antibiotic biosynthesis mutants, absA and absB, did not result in Red biosynthesis

Cloning of the Complete Gene for Carcinoembryonic Antigen

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region

Regulation of expression of the human carbonic anhydrase 1 gene

The HCAI gene is expressed in a developmental stage-specific manner and also exhibits tissue-specific expression patterns. This thesis describes the cloning and characterisation of the 5'-end of the HCAI gene which contains a large intron within its 5' untranslated region. S1 nuclease and primer extension analysis were used to define the transcription start site and the site of 3'-end maturation. Bandshift assays have been used to show that there are at least six DNA sequences, based on the consensus [5 '-TT/AATCA/T-3'] and flanking the HCAI gene, which bind the erythroid-specific factor, GF-1. The presence of GF-1 binding sites is shown to increase expression from a eukaryotic promoter in erythroid cells and not in non-erythroid cells. A transient heterokaryon system was set up by fusing the erythroieukaemic cell lines MEL C88 (expressing MCAI ) and K562 SAI (a human cell line with an embryonic / foetal phenotype, not expressing CAI. RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This indicates the developmental stage-specific expression of HCAI to be regulated by trans-acti ng factors. Expression of HCAI mRNA in colon tissue was confirmed. Furthermore as in the case of mouse CAI, the HCAI colon mRNA is transcribed from a different promoter to erythroid HCAI mRNA

Development of Plasmid Vectors for Regulated Expression of Cloned Genes in Streptomyces lividans and Escherichia coli

Using pIJ486 (Ward et al., 1986) two bifunctional (E. coli/Streptomvces) expression vectors were constructed. They contained the promoters lac (pGLW49) and tac (pGLW50) just upstream of a promoter-less kanamycin phosphotransferase gene (aphll)

Regulation of Pseudomonas aeruginosa amidase expression by AmiC

Regulation of amidase synthesis in P.aeruginosa has been studied using cloned genes from strains PACl and PAC433. Using in vitro constructed rearrangements, deletions and insertions, a new gene amiC has been identified between the amidase structural gene iamiE) and the positive regulator gene (amiE). Transcomplementation studies in E.coli and P.aeruginosa and analysis of in vitro constructed amiC- mutants has shown that AmiC is a negative regulator of amidase expression. The DNA sequence of the amiC region has been determined and two potential ntrA dependent promoters identified upstream of amiC. amiC and amiR expression vectors were constructed for complementation analysis in E.coli and P.aeruginosa. Using the amiC expression vector in P.aeruginosa, AmiC was purified to homogeneity. The purified amiC was shown to have protein kinase activity in vitro. A model has been proposed whereby the transcription antitermination activity of AmiR is modified by AmiC dependent phosphorylation. DNA sequencing studies completed the sequence of the entire 5.3kb Hin-dIII-Sa/I P.aeruginosa DNA fragment containing the amidase genes and two additional open reading frames were identified, giving the gene order for the operon, amiEYCRX. A transcription terminator was identified downstream of amiE. Transcript analysis of the amidase operon has shown constitutive expression from the amiE σ70-dependent promoter. Under inducing conditions, appproximately 40% of the amiE transcripts read through the downstream terminator sequence and into amiY, C, R, X. Studies designed to investigate the role of the ntrA dependent promoters failed to identify transcripts starting from these positions. However amidase expression from the cloned genes was shown to be regulated by the enteric ntr system. These investigations showed that σ54-holoenzyme functions to down-regulate amidase expression in E.coli and P.aeruginosa. The roles of AmiY and AmiX have not been established. However AmiY contains a consensus nucleotide binding domain and amiX shows the characteristics of an integral membrane protein