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#selectable marker

10 public questions tagged with this topic.

Transgenic Plant -2

Selectable marker used in Flavr Savr tomato transformation was:

Selectable marker deployed for Flavr Savr regeneration was neomycin phosphotransferase II gene nptII from Tn5 transposon of Escherichia coli, conferring resistance to aminoglycoside antibiotics kanamycin, neomycin, G418 and paromomycin. Enzyme mechanism involves transfer of gamma phosphate from ATP to 3' hydroxyl of aminoglycoside, inactivating drug ability to bind 16S rRNA of 30S ribosomal subunit, preventing misreading and bactericidal action. Tomato cotyledon explants co-cultivated with Agrobacterium harboring antisense PG plus nptII under nos promoter placed on Murashige and Skoog organogenesis medium supplemented with kanamycin 100 mg/L, carbenicillin to kill Agrobacterium, and phytohormones zeatin and IAA for shoot induction. Only transformed cells expressing NPTII detoxify antibiotic and proliferate, untransformed bleached die. Selection efficiency typically 5-15% transformation. nptII favored because plants lack endogenous resistance, assayable by ELISA and NPTII dot blot, and safety profile extensive showing rapid proteolysis in simulated gastric fluid with half-life

Ref: Beck et al. Gene 1982 Tn5 nptII; Coruzzi et al. EMBO J 1984 Plant selectable markers; NIH.

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Restriction Enzyme and Vector

Which selectable marker suppresses yeast mutations?

In yeast genetics, suppressor tRNA markers are used to monitor nonsense mutation suppression. SUP4 encodes a tyrosine tRNA with anticodon mutated to recognize ochre stop codon UAA, suppressing ade2-101 and other ochre alleles, leading to phenotypic color change in adenine biosynthesis pathway. In YAC vectors, SUP4 is placed within cloning site; disruption by foreign DNA abolishes suppression. Auxotrophic markers URA3 and TRP1 provide selection for transformation, while LacZ and GFP are bacterial reporter genes. SUP4 thus acts as insertional marker that visibly reports insertion by altering suppression phenotype in yeast.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which is a selectable marker in yeast artificial chromosome (YAC)?

Yeast artificial chromosomes require selectable markers functional in auxotrophic yeast strains to ensure maintenance. URA3 encodes orotidine-5'-phosphate decarboxylase involved in uracil biosynthesis, complementing ura3 mutant phenotype. Yeast lacking functional URA3 cannot grow without uracil, so only transformants harboring YAC with URA3 survive on minimal medium. Similar markers include TRP1 for tryptophan and LEU2 for leucine selection. URA3 also allows counter-selection with 5-fluoroorotic acid for manipulating constructs. Bacterial markers like ampR are not selectable in yeast, making eukaryotic auxotrophic markers indispensable.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Cloning Process

Which selectable marker is associated with kanamycin resistance?

Kanamycin resistance in cloning vectors is generally mediated by kanamycin resistance determinant encoding aminoglycoside 3'-phosphotransferase type II, product of nptII or kanR gene derived from bacterial transposon Tn5. This enzyme phosphorylates kanamycin, inactivating its ability to bind 30S ribosomal subunit and cause mistranslation and bacterial cell death. Cells expressing the resistance grow on media containing kanamycin at 25-50 µg per mL for selection. Neomycin resistance similarly uses nptII, while AmpR encodes β-lactamase and TetR encodes tetracycline efflux pump with distinct antibiotic specificities unrelated to kanamycin.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Cloning Process

Which of the following is a selectable marker gene?

Selectable markers confer a growth advantage under selective conditions, permitting only transformed cells to propagate. Ampicillin resistance gene bla encoding β-lactamase hydrolyzes the β-lactam ring of ampicillin, allowing Escherichia coli harboring plasmid to grow on ampicillin-containing medium. Non-transformed cells are eliminated. LacZ, GFP, and luciferase are scorable or reporter markers that produce color, fluorescence, or luminescence for visual screening but do not provide survival selection. Thus antibiotic resistance genes remain the primary selectable markers for plasmid maintenance and clone isolation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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