MARKER GENE(S) Sources: 1. Biotechnology By B.D. Singh, 2. Gene cloning and DNA analysis By TA Brown, 3. Advance and Applied Biotechnology By Marian Patrie, 4. Molecular Biotechnology By Glick et al and 5. Principle of Gene Manipulation By sandy b Primrose et al. Dr Diptendu Sarkar [email protected] Marker gene • Monitoring and detection of transformation/ transfection systems in order to know wheather the DNA has been successfully transferred into recipient cells, is done with the help of a set of genes. These are called marker genes. • Marker genes is are introduced into the plasmid along with the target gene. • Marker genes are categorized into two types: a) reporter or scorable genes [detected through highly sensitive enzyme assay] and b) selectable marker genes [detected through expression of resistance to a toxin]. • Some genes are used as selectable markers and reporter genes, whereas, some are used either as selectable markers or as reporter genes. 12/10/2019 DS/RKMV/MB 2 Reporter genes for plants • A number of screnable or scorable or reporter genes are available, that show immediate expression in the cells/tissue. A reporter gene is a test gene, whose expression results in quantifiable phenotype. A reporter system is useful to analysis of gene expression and standardization of parameters for successful gene transfer in a particular technique. • An assay for reporter gene is usually carried out at the level of protein quantification. The use of reporter gene is requires a method of gene transfer, either transient or stable. • An ideal reporter gene should have the following features: Scorable markers are very useful in 1)Detection with high sensitivity, molecular biology studies, e.g., assaying promoter strength etc., and to determine 2)Low endogenous back ground, the effectiveness of various 3)There should be a quantitative assay, transformation methods etc. they are, however, not suitable for development of 4)Assay should be nondestructive and transgenic lines. 5)Assay should requires a minimal amount of effort and expense. 12/10/2019 DS/RKMV/MB 3 About 50 selectable marker genes 12/10/2019 DS/RKMV/MB 4 12/10/2019 DS/RKMV/MB 5 Selectable marker genes for plants • Selection of transformed cells is a key factors in developing successful methods for genetic transformation. This is done by certain selectable marker genes, that are present in the vector along with the gene of interest. Selectable markers are an integral part of any transformation strategies. • Selectable marke genes enable the transformation cells to survive on media containing toxic levels of selection agent, whereas, nontransformed cells get killed. • A large number of such selectable marker genes have become available, viz., antibiotic, antimetabolite, and herbicide resistance genes, hormone biosynthesic genes, and genes conferring resistance to toxic levels of amino acid analogs. • The usefulness of a resistance marker depends on: 1) The characteristics of selection agent, 2) The resistance gene, 3) Prokaryotic or eukaryotic (plants/ animals) materials. 12/10/2019 DS/RKMV/MB 6 12/10/2019 DS/RKMV/MB 7 Reporter genes for animals Thymidine kinase gene • The nucleotides dATP, dGTP, dCTP, dTTP are produced by two independent pathways: Endogenous pathway amino acids, e.g., glycine (for dATP and dGTP), and aspartate (for dTTP), and and dihydrofolate are used to produce new nucleotides. DHFR 2. Salvage pathway. recycles the purine and pyrimidine nucleotides produced from degeneration of nuclic acid TK - • The enzyme thymidine kinase belong to the • Thymidine kinase deficient (TK ) cells are killed on HAT salvage pathway. (hypoxanthine-aminopterin-thymidine) medium, which • Thymidine kinase phosphorylates thymidine contains the drug aminopterin. to yield thymidine monophosphate (dTMP), • Aminopterin blocks the endogenous pathway of which is subsequently converted into nucleotide production by inhibiting the enzyme thymine triphosphate (dTTP). dihydrofolate reductase (DHFR), which catalyzes the first reaction of endogenous pathway in the utilization of dihydrofolate in nucleotide biosynthesis. 12/10/2019 DS/RKMV/MB 8 • Further, the TK- cells are unable to utilize the thymidine present in the medium for nucleotide production. • As a result, TK- cells die of nucleotide starvation on HAT medium. • Therefore, thymidine kinase gene can be used as a selectable marker only when TK- host cells are used for transformation. • The transformed cells are cultured on HAT medium on which only TK+ cells can survive and multiply (exogenous TK enzyme support the growth). • The requirement of TK- host cells is a serious limitation of this marker, such markers are often called dominant selectable markers. 12/10/2019 DS/RKMV/MB 9 CAD protein [carbamoyl-phosphate synthatase2, aspartate transcarbamoylase and dihydroorotase] (PALA : N- phosphonacetyl-L-aspartate resistance) • CAD protein is a multifunctional enzyme. • CAD catalyzes the first three reactions of endogenous uridine synthesis. • CAD is inhibited by PALA (N- phosphonacetyl-L-aspartate). • Mammalian cells are sensitive to PALA. • PALA resistant cell lines developed through selection overproduce CAD protein due to a high amplification of the concerned gene. • CAD gene isolated from Syrin Hamster, serves as a dominant marker, making normal mammalian cells resistant to high concentration of PALA. • This gene, becomes highly amplified in the stably transformed cell lines. 12/10/2019 DS/RKMV/MB 10 XGPRT (Mycophenolic acid resistance): • The bacterial enzyme XGPRT (Xanthine-guanine-phosphoribosyl-transferase) is analogous to the the mammalian enzyme HGPRT (hypoxanthine- guanine-phosphoribosyl-transferase). Both XGPRT (utilize xanthine) and HGPRT are enzymes of salvage pathway. it inhibits HGPRT, catalyzed conversion of IMP to XMP in salvage Normal mammalian cells are pathway sensitive to mycophenolic acid and aminopterin aminopterin which blocks endogenous purine biosynthesis. The bacterial XGPRT gene • Normal mammalian cells can not survive, since, HGPRT is serves as a dominant unable to utilize xanthine for producing GMP. selectable marker on a culture • However, mammalian cells producing bacterial XGPRT can utilize medium, media xanthine, present in the medium to produce GMP, and are able containing mycophenolic acid, to survive and proliferate. adenine and xanthine. 12/10/2019 DS/RKMV/MB 11 Neomycin phosphotransferase (G418 resistance): • Normal mammalian cells re sensitive to the aminoglycoside antibiotic G418, which inhibits protein synthesis. • Neomycin phosphotransferase encoded by bacterial transposon Tn5 and Tn 601 confers resistance to G418. • Transfected cells are efficiently selected for on a medium containing the antibiotic G418, which is powerful selection agent. 12/10/2019 DS/RKMV/MB 12 Antibiotics and antibiotic resistance gene used in gene cloning Antibiotic Mode of action Resistance gene Ampicillin Inhibits cell wall synthesis by disrupting β-lactamase (AmpR) gene product is secreted peptide glycan crosslinking and hydrolyses ampicillin. Tetracycline Inhibits binding of aminoacyl tRNA to the TetR gene product is membrane bound and 30S ribosomal subunit prevents tetracycline accumulation by an efflux mechanism Kanamycin Inhibits translation by interfering with Neomycin or aminoglycoside ribosome function phosphotransferase (NeoR) gene product inactivates kanamycin by phosphorylation Bleomycin Inhibits DNA and RNA synthesis by The BlaR gene product binds to bleomycin and binding to DNA prevents it from binding to DNA. Hygromycin B Inhibits translation in prokaryotes and Hygromycin-B-phosphotransferase (HgpR or eukaryotes by interfering with ribosome HygR) gene product inactivates hygromycin B by translation phosphorylation Chloramphenicol Binds to the 50S ribosomal subunit and Chloramphenicol acetyl transferase (CAT or inhibits translation CMR) gene product metabolizes chloramphenicol in the presence of Acetyl CoA. 12/10/2019 DS/RKMV/MB 13.