Researchers lyse human cells and isolate a specific messenger RNA template using gel electrophoresis. Reverse transcription polymerase chain reaction is then used to synthesize complementary DNA (cDNA) from the RNA template. Next, the cDNA is modified into an expression vector containing an optimized bacterial promoter, ribosomal binding site, and terminator sequence. After insertion of the vector into appropriate bacterial hosts, the transformed bacteria are cultured in a bioreactor and produce large quantities of a protein containing a domain that binds to a specific DNA sequence. This protein is most likely the receptor for which of the following hormones?

Question

Researchers lyse human cells and isolate a specific messenger RNA template using gel electrophoresis.  Reverse transcription polymerase chain reaction is then used to synthesize complementary DNA (cDNA) from the RNA template.  Next, the cDNA is modified into an expression vector containing an optimized bacterial promoter, ribosomal binding site, and terminator sequence.  After insertion of the vector into appropriate bacterial hosts, the transformed bacteria are cultured in a bioreactor and produce large quantities of a protein containing a domain that binds to a specific DNA sequence.  This protein is most likely the receptor for which of the following hormones?

Quizplus · Accepted Answer

11ec160b_36a7_a3b0_9249_d36b6e9530bf_MD0001_00 Prokaryotes and eukaryotes use divergent cellular machinery for protein synthesis and require different signaling sequences for efficient transcription and translation. Expression cloning is a type of DNA cloning where the signals necessary for optimal protein expression are included in the DNA vector. In this example, eukaryotic complementary DNA (containing the coding sequence for the protein of interest) is modified with prokaryotic promoter sequences (eg, the Pribnow box, −35 sequence) and a ribosomal binding site (Shine-Dalgarno sequence). The vector is then incorporated into a suitable bacterial host (eg, Escherichia coli) and subsequently transcribed and translated into protein. The protein produced in the bioreactor contains a DNA-binding domain. DNA-binding proteins are a diverse group that include transcription factors (Myc, CREB), steroid receptors (cortisol, aldosterone, progesterone), thyroid hormone receptor, fat-soluble vitamin receptors (vitamin D, retinoic acid), and DNA transcription and replication proteins. Of the choices listed, only the progesterone receptor can bind to DNA. The receptors for most steroid hormones, including progesterone, are located in the cytoplasm and translocate to the nucleus upon ligand binding. In contrast, receptors for thyroid hormone and vitamins A and D are located within the nucleus at all times. Following activation by ligand binding, both receptor types attach to DNA at hormone response elements located in the promoter region of target genes to alter gene transcription. (Choices A and E) Parathyroid hormone and glucagon act on Gs protein-coupled receptors found on the cell membrane. Binding their respective ligands subsequently activates adenylyl cyclase and increases intracellular cyclic AMP concentration. (Choice B) Growth hormone receptor is a membrane-bound receptor that works via activating the JAK-STAT pathway. (Choices C and D) The receptors for insulin-like growth factor-1 (IGF-1) and insulin are structurally similar and located at the cell membrane. The intracellular domains of these receptors have intrinsic tyrosine kinase activity, which is activated on ligand binding. Autophosphorylation of the tyrosine residues on the intracellular part of the receptors then triggers downstream signaling. Educational objective: DNA-binding proteins include transcription factors (Myc, CREB), steroid receptors (cortisol, aldosterone, progesterone), thyroid hormone receptor, fat-soluble vitamin receptors (vitamin D, retinoic acid), and DNA transcription and replication proteins. __________ References: Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors. (http://www.ncbi.nlm.nih.gov/pubmed/8223341)

Researchers Lyse Human Cells and Isolate a Specific Messenger RNA