Cloning of a Gene

The cloning of a gene is the central operation of recombinant DNA technology. Gene is the basic unit of hereditary information located on a chromosome of the cell. Chromosomes are threadlike structures found in the nucleus, which are comprised primarily of DNA. The 

4 A hydrogen bond between the two chains is a weak attractive force between a covalently bounded hydrogen atom (H—N) and a negatively charged keto oxygen atoms (C=0). 

5 The twenty amino acids found in proteins are Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamic acid (Glu), Glutamine (Gin), Glycine (Gly), Histidine (His), Isoleucine (He), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Trvptophan(Trp), Tyrosine (Tyr), and Valine (Val). 

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Furukawa K, T Miyazaki (1986) Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes. J Bacterial 166 392-398. 

Gantz, I., Schaffer, M., DelValle, J. et al. (1991). Molecular cloning of a gene encoding the histamine H2 receptor. Proc. Natl. Acad. Sci. USA 88, 429-33. 

Sherrington, R., Rogaev, E. I., Liang, Y. et al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer s disease. Nature 375 754-760,1995. 

Gantz I, Schaffer M, DelValle J Molecular cloning of a gene encoding the histamine Hj receptor. Proc Natl Acad Sci USA 1991 88 5937. 

Baudry B, Fasano A, Ketley J, Kaper JB (1992) Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae. Infect Immun 60 428-434... 

Phenol is commonly used to separate cellular DNA from proteins, one step in the process that leads to the cloning of a gene. An aqueous phenol solution dissolves DNA, but it complexes with the proteins and causes them to precipitate, Centrifugation removes the precipitated proteins. Finally, addition of ethanol causes the purified DNA to precipitate so that it can be collected. 

Nambiar KP, Stackhouse J, Stauffer DM, Kennedy WP, Eldredge JK, Benner SA. Total synthesis and cloning of a gene coding for the ribonuclease S protein. Science 1984 223(4642) 1299-1301. 

AD, Fraser PE, Rommens JM, George-Hyslop PH (1995) Cloning of a gene bearing misserise mutadoris in eariy-oriset familial Alzheimer s disease. Natui e 375 754—760. 

Figure 2.2 A general method for the cloning of a gene. (Reproduced from Mishra, 2002, with the permission of John Wiley Sons.)...

In 1990, Seeburg et al. reported the first cloning of a gene expresing a functional ionotropic glutamate receptor... 

Levi, A., Eldridge. J. D., and Paterson, B. M. (1985). Molecular cloning of a gene. sequence regulated by nerve growth factor. Science 229, 393-395. 

CHANG, P.-K., SKORY, C.D., LINZ, J.E., Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus, Curr. Genet., 1992,21,231-233. 

The 3- and/or 4"-0-acyl derivatives of several tylosin-related macrolides have also been prepared by analogous bioconversion methods the 3-0-acetyl-4"-0-isovaleryl derivative of macrocin (3" -0-demethyltylosin) had properties similar to those of the corresponding ester of tylosin [90]. This research has now been further extended with the reported cloning of a gene encoding the 4"-0-isovaleryl acylase enzyme and its expression by formation of a new hybrid macrolide, 4"-0-isovalerylspiramycin [91]. These results indicate that future contributions can be anticipated from the area of molecular genetics on biochemical processes involved in macrolide biosynthesis, with the promise of production of novel hybrid macrolide structures. 

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