Viral promoters and there use in genetic engineering

Vectors A vector is any vehicle used to transfer foreign genetic material into another cell. The major types of vectors are : Plasmids Bacteriophages. Other viruses. Cosmids. Artificial chromosomes.

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The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Common to all engineered vectors are: An origin of replication. A multicloning site. A selectable marker.

Viral Vectors :

Viral Vectors Viral vectors are one of the major vechicles used by scientists in gene therapy to get their sequences expressed in the proper host. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene.

Promoter :

Promoter A promoter is a regulatory region of DNA located upstream (towards the 5' region) of a gene, providing a control point for regulated gene transcription. The promoter contains specific DNA sequences that are recognized by proteins known as transcription factors. These factors bind to the promoter sequences, recruiting RNA polymerase, the enzyme that synthesizes the RNA from the coding region of the gene.

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Promoter region The non-coding nucleotide sequence at the transcription start region that is characterized by the presence of a number of conserved or consequence sequences. Internal promoter A class of enhancers which are gene-specific sequences that increase transcription.

PROMOTER ELEMENTS :

PROMOTER ELEMENTS 1. Core promoter The minimal portion of the promoter required to properly initiate transcription. 2. Proximal promoter The proximal sequence upstream of the gene that tends to contain primary regulatory elements

Enhancers :

Enhancers It enhances (modulates) the level of transcription depends on the type of tissue, developmental stage, stage of the cell cycle, induction by hormones or other molecular signals. The main features of enhancers are the following: 1) it acts distantly 2) its activity does not depend on orientation.

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Silencers DNA sequences suppressing promoter activity are called silencers. Thus enhancer/silencer sequences regulate the activity of promoters but are not themselves involved in the promoter activity perse.

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Insulators Insulators are: stretches of DNA (as few as 42 base pairs may do the trick) located between the enhancer(s) and promoter or silencer(s) and promoter of adjacent genes Their function is to prevent a gene from being influenced by the activation (or repression) of its neighbors.

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Prokaryotic promoters In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site. Eukaryotic promoters In eukaryotes, the transcriptional complex can cause the DNA to bend back on itself, which allows for placement of regulatory sequences far from the actual site of transcription.

Operons :

Operons A promoter serves to initiate the transcription of multiple structural genes that are immediately adjacent to it. This arrangement is called an operon. In operons, promoters have adjacent, juxtaposed or interspersed regulatory sites to which regulatory proteins bind. In eukaryotic promoters, the regulatory sites are spread out over a longer distance.

Promoters in transcription process :

Promoters in transcription process Transcription is the name given to the process in which RNA is constructed by cellular enzymes. Steps: RNA polymerase Promoter or enhancer region Transcription factors Coded anti-sense Centarl dogma

Role of promoters in eukaryotic transcription :

Role of promoters in eukaryotic transcription The gene is transcribed, spliced and processed to form mRNA, and The mRNA is translated into a polypeptide. Promoters comprise multiple elements: CAAT box TATA box GC box CAP site RNA polymerase II

Prokaryotic Transcription :

Prokaryotic Transcription Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins. Prokaryotic transcription and translation can occur simultaneously. Prokaryotic transcription occurs in the cytoplasm alongside translation.

Types of promoters :

Types of promoters Inducible promoters The activity of promoters,iduced by the presence or absence of biotic or a biotic factors are called inducible promoters. They are of two types: Chemically regulated promoters Physically regulated promoters

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Physically regulated promoters Heat shock promoters Heat shock responsive genes have specific sequence in their promoters which replace the common -35 and -10 region. These promoters are recognized by S32. S32 is recognized by RpOH gene. HS sigma factor is turned on when exposed to heat.

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Chemically regulated promoters IPTG is a classic example of a compound added to cells to activate a promoter. It is often used to activate the lacZ gene when cloning a new fragment of DNA and using blue/white selection. IPTG can be added to the cells to activate the downstream gene or removed to inactivate the gene.

Viral promoters :

Viral promoters Viral promoters are commonly used as regulatory elements in gene therapy vectors due to their strong activity in various cell lines in vitro. CMV promoters Human cytomegalovirus immediate-early gene (CMV) promoter. The CMV promoter induces high-level constitutive expression in a variety of mammalian cell lines.

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CaMV promoter The promoter responsible for the transcription of the whole genome of a cauliflower mosaic virus(CaMV). Closely related to human hepatitis B virus. 35S is very strong constitutive promoter. High level of gene expression in dicot plants.

Gene Expression Vectors :

Gene Expression Vectors Minimal promoters used to study gene regulatory elements such as enhancer elements.

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Constitutive promoters used to direct expression of gene products.

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Cell-specific promoters used to specify expression to target cells.

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Regulated promoters used to control the on/off expression of cloned genes.

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Expression vectors contain DNA sequences flanking the multiple cloning site (MCS) that function as sequence-specific binding sites for transcription factors. Common DNA sequence motifs found in many types of promoters are represented by SP1, CAAT and AP1 binding sites. HREs are hormone response elements.

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Comparison of bovine leukemia virus (BLV) and CMV promoter Method Comparison of CMV and BLV luciferase vectors including D17, FLK, BL3.1 and primary bovine B cells Addition of BLV Tax expression vectors /or BLV infection as well as treatment with trichostatin A (TSA).

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Results BLV promoter, while having low basal activity compared to the CMV promoter, can be induced to high-levels of activity similar to the CMV promoter in all cells tested. Tax or BLV infection specifically enhanced BLVp activity with no effect on CMVp activity. In contrast, the non-specific activator, TSA, enhanced both BLVp and CMVp activity.

Use of viral promoters in mammalian cell-based :

Use of viral promoters in mammalian cell-based Viral promoters are used because they can be express with very 'high' magnitude in a variety of cell types in the laboratory. Receptor/reporter system. viral promoters and a natural mammalian promoter.

Viral promoters in bacteria :

Viral promoters in bacteria The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell lines. The preceding cloning procedures are usually performed in Escherichia coli and foreign promoter results in an expression of the gene in bacteria.

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Results five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Thymidine kinase gene from HSV1 showed no activity. Polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. Transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences

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Conclusion promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.