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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Genes : Nature, Concept and Synthesis

 
     
 

Split Genes (or Introns)

During 1970, in some mammalian viruses (e.g. adenoviruses) it was found that the DNA sequences coding for a polypeptide were not present continuously but were split into several pieces. Therefore, these genes were variously named as split genes or introns (Gilbert, 1978), interrupted genes or intervening sequences (Lewin, 1980), inserts (Weismann, 1978), Junk DNA. For the discovery of split genes in adenoviruses and higher organisms, Richards J.Roberts and Phillip Sharp were awarded Nobel prize in 1993.

  The split genes have exons separated by introns. Removal of introns through RNA splicing.  
 

Fig. 2.7. The split genes have exons separated by introns. Removal of introns through RNA splicing.

 


As shown in Fig 2.7 a DNA sequence codes for rnRNA but the complete corresponding sequence of DNA is not found in mRNA. Certain sequences of DNA are missing in mRNA. The sequences present in DNA but missing in mRNA are called intervening sequences or introns, and the sequences of DNA found in RNA are known as exons. The exons code for mRNA.

After transcription a limited RNA transcript has the intron sequences present in the interrupted genes. Genes coding for rRNA and tRNA may also be intervened. The cistrons are found only in prokaryotes except cyanobacteria and archaeobacteria (archaea).

 

Content

Chemical nature of DNA

 

Chemical composition

 

Nucleotides, nucleosides

 

Polynucleotides

 

Chargaff's rule of equivalence

Physical nature of DNA

 

Watson and Cricks model of DNA

 

Circular and superhelical DNA

 

Organization of DNA in eukaryotes

Structure of RNA

Gene concept

Units of a gene

 

Cistron

 

Recon

 

Mutan

Split genes (introns)

 

RNA splicing

 

Ribozyme

 

Evolution of split genes

Overlapping gene

Gene organization

Gene expression

Gene regulation

 

Transcription

 

 

The lac operon (structural gene, operator gene, promoter gene and repressor gene)

Artificial synthesis of genes

 

Synthesis of a gene for yeast alanine tRNA

 

Synthesis of a gene for bacterial tyrosine tRNA

 

Synthesis of a human leukocyte interferon gene

Gene synthesis by using mRNA

Gene machine

The PCR

 

Amplification of DNA (melting of target DNA, annealing of primers, primer extension)

 

Application of PCR technology

   

For some time it was not certain how mRNA is synthesized from a DNA containing introns? Some possible explanations for the mechanism of mRNA synthesis were given: (i) DNA rearrangement occurs during transcription with the removal of introns, (ii) during transcription RNA polymerase skips the introns and transcribes only exons, (iii) individual exon transcribes separately and rejoins to form the complete mRNA, and (iv) RNA polymerase may synthesize both introns and exons, and processing of transcripts occurs later on. The transcripts corresponding to introns are removed. Later on it was shown that the fourth mechanism operates in transcription of mRNA.


RNA Splicing
In the initial stage, RNA transcript introns are synthesized which are removed later on by a process called RNA splicing (Fig. 2.7). The junctions of intron-exon have a GU sequences at the intron's 5'-end, and an AG sequence at its 3'OH end. These two sequences are recognized by the special RNA molecules known as small nuclear RNA (snRNA) or snurps (Steitz, 1988).

These together with proteins form small nuclear ribonucleoprotein particles called snRNPs. Some of the snRNPs recognize the splice junctions and splice introns accurately. For example, the UI-snRNP recognizes the 5'-splicing junction, and the U5 snRNP recognizes the 3' splicing junction. Consequently pre-mRNA is spliced in a large complex called a spliceosome (Guthrie, 1991). The spliceosome consists of pre-mRNA, five types of snRNPs and non-snRNP splicing factors (Rosbash and Seraphin, 1991).

Robert and Sharp, the Nobel prize winners in 1993, independently hybridized the mRNA of adenovirus with their progeny or DNA segments of virus. The mRNAs hybridized the ssDNA of virus where the complementary sequences were present. The mRNA-DNA complexes were observed under electron microscope to confirm which part of viral genome had produced the mRNA strand. It was found that mRNA did not hybridize DNA linearly but showed a discontinuous complexes pattern. Huge loops of unpaired DNA between the hybridized complexes clearly revealed the large chunk of DNA strand that carried no genetic information and did not take part in protein synthesis. The adenovirus mRNA contained four different regions of the DNA.

The 5-globin genes of mice and rabbits, and tRNA genes of yeast tyrosine-tRNA consists of eight genes three of which have been studied in detail. Each gene contains 14 bases (ATTT-AYCAC-TACGA) as intron in the middle. In the same way the pre-tRNA genes contain introns of 18-19 bases. In all the genes introns are present near anticodon. Similarly, a few rRNA genes arc also known to contain introns and some of pre-rRNA are self splicing.


Ribozyme
For the first time, Thomas Cech (1986) discovered that pre-rRNA isolated from a ciliated protozoa, Tetrahymena thermophila is self splicing. Thereafter, S. Altman showed that ribonuclease cleaves a fragment of pre-tRNA from one end, and also contains a piece of RNA. This RNA fragment catalyses the splicing reaction i.e. acts as enzyme. Therefore, this RNA segment catalyzing the splicing reaction is called ribozyme. For this discovery Cech and Altman were awarded the Nobel prize in 1989 in chemistry. The best studied ribozyme activity is the self-splicing of RNA. This process is widespread and occurs in T. thermophila pre-tRNA, mitochondrial mRNA of yeast and other fungi, chloroplast tRNA, rRNA and mRNA, and mRNA of bacteriophage.

The rRNA intron of T. thermophila is 413 nucleotide long. The self-splicing reaction needs guanosine and is accomplished in three steps: (i) the 3'-G attacks the 5' group of introns and cleaves the phosphodiester bond, (ii) the new 3'-OH group on the left exon attacks the 5'-pfoosphate on right exon. Consequently two exons join and remove the intron; and (iii) the 3'-OH Ofintron attacks the phosphate bond of nucleotide 15 residues from its end releasing the terminal fragment and cyclizing the intron (Cech, 1986).


Evolution of Split Genes
Before the discovery of split genes in 1977, all genes analyzed in detail were the bacterial genes. Bacteria were considered to resemble the simpler cell from which eukaryotes must have been evolved. Now, it is supposed that split genes are the laciest condition and bacteria lost their introns only after evolution of most of their proteins,  for the ancient origin of introns has been obtained by the examination of the gene that  the ubiquitous enzyme, triosephosphate isomerase (TPI). The TPI is coded by a gene that  six introns (in vertebrates), five of these are present at the same position as in maize. This shows that five introns were present in the gene before evolution of eukaryotes about 109 years ago (Nyberg and Cronhjort, 1992).

The TPI plays a key role in cell metabolism that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, a central step in glycolysis and glucogenesis. By comparing this enzyme in various organisms it appears that the TPI evolved before the divergence of prokaryotes and eukaryotes from a common ancestor cell, progenote (Nyberg and Cronhjort, 1992).

The unicellular organisms under a strong selection pressure minimized the superfluous genome in their cell, whereas there was no such pressure on multicellular organisms. That is why Aspergillus has five introns and Saccharomyces has none. Precise loss of introns would have occurred by deletion in prokaryotes. The TPI is thought to be evolved to its final three dimensional structure before eubacteria, archaeobacteria and eukaryotic lineage split off from progenote (Nyberg and Cronhjort, 1992).

 
     
 
 
     



     
 
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