Nucleotide cloning

The amount of sample required is quite small as little as 10 mole is typical So many peptides and proteins have been sequenced now that it is impossible to give an accurate count What was Nobel Prize winning work m 1958 is routine today Nor has the story ended Sequencing of nucleic acids has advanced so dramatically that it is possible to clone the gene that codes for a particular protein sequence its DNA and deduce the structure of the protein from the nucleotide sequence of the DNA We 11 have more to say about DNA sequencing m the next chapter... 

DNA from a gene contains hundreds to thousands of nucleotide units for which the sequence is needed in order to interpret its code. Sequencing methods require only small amounts (5 (tg) of purified DNA, which can be produced by cloning. Automated sequencers are available that can daily sequence DNA containing hundreds of nucleotide units. 

In the human cell there are 23 pairs of chromosomes containing approximately 3000 million base pairs of DNA. Short sequences of DNA, perhaps with as few as 20 nucleotide units and sometimes radiolabeled, can be obtained either by chemical synthesis (gene machine) or from cloning. These short sequences can be used to probe for a complementary sequence by looking for the position to which they bind to any DNA sample under investigation, from blood for example. Such probes can detect as little as 100 fg of DNA and are the basis of forensic genetic fingerprinting tests. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

After a desired clone is obtained and mapped with restriction enzymes, further analysis usually depends on the deterrnination of its nucleotide sequence. The nucleotide sequence of a new gene often provides clues to its function and the stmcture of the gene product. Additionally, the DNA sequence of a gene provides a guidepost for further manipulation of the sequence, for example, lea ding to the production of a recombinant protein in bacteria. 

Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-depen-dent, and they do not modify DNA by methylation or other means. Most importantly, they cut DNA within or near particular nucleotide sequences that they specifically recognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coU has a restriction enzyme, coRI, that recognizes the hexanucleotide sequence GAATTC ... 

Devine, J. H., et al. (1993). Luciferase from the east European firefly Luciola mingrelica cloning and nucleotide sequence of the cDNA overexpression in Escherichia coli and purification of enzyme. Biochim. Bio-phys. Acta 1173 121-132. 

Knowledge of DNA sequences permits deduction of the primary structures of polypeptides. DNA sequencing requires only minute amounts of DNA and can readily yield the sequence of hundreds of nucleotides. To clone and sequence the DNA that encodes a partic-... 

A cloned complementary DNA to a neurotoxin precursor RNA extracted from the venom glands of Laticauda semifasciata was isolated and its nucleotide sequence was identified 11). The cloning of neurotoxin should aid the understanding of structure—function relationship eventually. 

Yanisch-Perron, C., Vieira, J. and Messing, J. 1985. Improved M13 phage cloning vectors and host strains nucleotide sequences of the M13mpl8 and pUCl9 vectors. Gene 33 103-1 19. 

The amino acid sequence of the human erythrocyte glucose transporter was deduced from the nucleotide sequence of a cDNA clone in 1985 [106]. Polyclonal antibodies raised against the protein were used to screen a Xgtl I cDNA library prepared from the human hepatocellular carcinoma cell line HepG2. (Like many other transformed... 

Molecular cloning and nucleotide. sequencing of amino acid permease genes... 

Five structural genes for amino acid uptake systems have been cloned in Saccharomyces cerevisiae by functional complementation, and their putative amino acid sequences deduced from the respective nucleotide sequences (Fig. 2). 

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