Answers:
Protein splicingis an intramolecular reaction of a particular protein in which an internal protein segment (called an intein) is removed from a precursor protein with a ligation of C-terminal and N-terminal external proteins (calledexteins) on both sides. The protein splicing reactions which are known now do not require exogenous cofactors or energy sources such as ATP, GTP.
Autoproteolysis is a self degradation process in which protein (intein and extein) lysis occur without further ligation while in protein splicing intiens are removed and extiens are ligated back.
2) Some vectors contain a gene which complements a function missing in their host cells eg. Gene lacZ alpha in pUC vector, whch complement such lacZ- E. coli strain in which lacZ alpha is deleted. in such case the DNA insert is so placed that it disrupt the function of lacZ alpha in the recombinant DNA.therefor E.coli cells containing the r-DNA are deficient in B-galactosidase and produse white colony on X-gal containing media, while other cell producing blue colonies.this is we can differentiate them.
3) It is a simple method for generating cDNA libraries from sub microgram quantities of mRNA. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.
Following are the stages:-
Stage1: Synthesis of First-strand cDNA Catalyzed by Reverse Transcriptase
Stage2: Second-strand Synthesis
Stage 3: Methylation ofcDNA
Stage 4: Attachment of Linkers or Adaptors
Stage5: Fractionation of cDNA by Gel Filtration through SepharoseCL-4B
Stage 6: Ligation of cDNA to Bacteriophage Arms
This describes the conversion of poly(A)+ mRNA into first-strand cDNA in a reaction catalyzed by a murine RNaseH- reverse transcriptase and primed by oligo(dT), random hexamers,or prime-adapters.
Answers :
otein splicing is defined as the excision of an intervening protein sequence (the INTEIN) from a protein precursor and the concomitant ligation of the flanking protein fragments (the EXTEINS) to form a mature extein host protein and the free intein . Protein splicing results in a native peptide bond between the ligated exteins.
Extein ligation differentiates protein splicing from other forms of autoproteolysis
Q2. Functional Complementation is helpful in screening of transformed cells from untransformed cells can be explained with an example of blue/white colony screening in Ecoli. Strains of Ecoli like JM109, DH5α have a mutation in LacZ that deletes part of the β-galactosidase (lacZ) gene. By using a plasmid that contains the deleted portion, or α-fragment, the function of the β-galactosidase gene can be restored once the plasmid has been incorporated into the bacterium.
For blue/white colony screening, the plasmids have a multiple cloning region within the coding sequence of the α-fragment. When a sequence is inserted into this cloning region, the reading frame is disrupted, and a non-functional α-fragment is produced. This fragment is incapable of α-complementation. Growing the transformed bacteria on a plate containing 5-bromo-4-chloro-3-indoyl-β -D-galactopyranosidase (X-gal) will allow you to distinguish between bacterial colonies formed from cells that contain plasmid with insert from those containing plasmid without insert. Any colony containing the plasmid (and therefore the functioning β-galactosidase gene) will turn blue, a result of the β-galactosidase activity. This is called α-complementation (Functional Complementation)
For blue/white colony screening, the plasmids have a multiple cloning region within the coding sequence of the α-fragment. When a sequence is inserted into this cloning region, the reading frame is disrupted, and a non-functional α-fragment is produced. This fragment is incapable of α-complementation. Growing the transformed bacteria on a plate containing 5-bromo-4-chloro-3-indoyl-β -D-galactopyranosidase (X-gal) will allow you to distinguish between bacterial colonies formed from cells that contain plasmid with insert from those containing plasmid without insert. Any colony containing the plasmid (and therefore the functioning β-galactosidase gene) will turn blue, a result of the β-galactosidase activity. This is called α-complementation (Functional Complementation)
Q3. The Gubler-Hoffman method, is a simplified version of Okayama and Berg's method used for non directional cDNA cloning.
- The cDNA first sysnthesis is primed by oligo(dt) and catalaysed by reverse transcriptase.
- The second strand synthesis is achieved by nick translation repair of the cDNA:mRNA hybrid, mediated by E.coli DNA polymerase I, RNase H, and E.coli DNA ligase.
- Methylate cDNA
- Attach linkers or adaptors for cloning
- Fractionate cDNA by size (select 2-8 kb)
- Ligate cDNA into bacteriophage arms
