Gene activation, functional proteins

The prior description of mRNA regulation (see Figure 1.3) has many steps. Thus, it is not surprising to find that other processes can also influence the rate and extent to which the information in a gene can be manifested as an active functional protein. Such translational level controls can entail competition for ribosomes by the numerous mRNAs from different genes. Alternately, the base interactions that occur in a duplex DNA molecule that lead to the a-hehx can also result in structural organization in mRNA. For example, the bases within an mRNA strand can self-complement thereby leading to the formation of hairpin loops. Such secondary structures that result from a primary structure (the base sequence) can influence how fast and successfully the ribosomal-mediated translation process occurs. 

The family of apelin peptides is derived from a single gene, activate a single G-protein-coupled receptor and are substrates for the angiotensin converting enzyme-2 (ACE2). Apelins regulate cardiovascular function and fluid homeostasis. The apelin receptor also functions as a co-receptor for infection of CD4-positive cells by human immunodeficiency vims ( HIV). 

According to the NC-IUPHAR Subcommittee on Opioid Receptois it was proposed to term ORL-1 recqrtor as NOP receptor [1]. The human NOP receptor gene encodes a protein of370 amino acids. Splice valiants have been found in the human and mouse NOP recqrtor with no known functional significance. NOP receptors are widely distributed throughout the brain and in the spinal cord. They are also present in immune cells. A functional role for N/OFQ has been proposed in nociception, locomotoric activity, reward, stress, and immunomodulation. 

Regulatory regions are transcriptional control sequences, which consist of promoters, response elements, enhancers and possibly silencers, located upstream of the start site of transcription. The overall effect on gene transcription is a sum of the contributions of these elements and the activities of proteins recruited to these sites. Promoters are located immediately upstream of the start site and initiate transcription. They often contain tissue- or cell-specific elements if the gene is not ubiquitously expressed. Enhancers are positive regulatory elements which function independently of orientation and distance from the genes they regulate. 

Eight exons of the AR gene encode a protein of around 917 aa depending on two polymorphic regions of polyglutamines (CAG) and polyglycines (GGN) in the N-terminal activation domain. Two isoforms are detected in tissues the predominant (80%) 110 kD (B isoform) and 87 kD (A isoform). It is not clear whether the two isoforms also serve different functions. 

By far, the most widely used application of the yeast two-hybrid system as intimated in Introduction is the identification of protein partners for a test protein of either known or unknown function. Here, the DNA encoding the test protein or the domain of a test protein is cloned in frame into the bait vector. The fish vector contains cDNA and it is constructed so that there is one cDNA molecule per vector. Fish and bait vectors are cotransformed into the appropriate strain of competent yeast, and the resultant transformed yeast cells are screened for growth on SD media and for reporter gene activities. Putative positive clones are then isolated and characterized further. In the next section, each of these stages is discussed in detail. 

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