Gene concept

Recombinant DNA technology

1. Gene Concept: Classical concept, Fine structure of the gene, Molecular concept of the gene , Pseudogenes , Overlapping genes, Oncogenes Promila Sheoran Ph.D. Biotechnology GJU S&T Hisar

2. Gene Concept  Although the role of hereditary units (factors) in transfer of genetic characters over several generations in organisms was advocated by Gregor John Mendel, yet the mystry of the 'hereditary units was unravelled during early 1900s. In 1909, W. Johanson coined the term 'gene' that acts as hereditary units. However, early work done by several workers proposes various hypotheses to explain the exact nature of genes . In 1906, W. Bateson and R.C. Punnet reported the first case of linkage in sweet pea and proposed the  presence or absence theory.  According to them the dominant character has a determiner, and the recessive character tacks determiner . In 1926, T.H. Morgan discarded all the previous existing theories and put forth the  paniculate gene  theory. He thought that genes are arranged in a linear order on the chromosome and look like beads on a string. 

3. This theory of gene was well accepted by the cytologists. In 1933, Morgan was awarded Nobel prize for advocating the theory of genes . After the discovery of DNA as carrier of genetic information, the Morgan's theory was discarded. Therefore, it is necessary to understand both, the classical and modern concepts of gene . According to the classical concepts a gene is a unit of  ( i )  physiological functions,  (ii)  transmission or segregation of characters, and  (iii ) mutation.

4. Genes are discrete particles inherited in Mendelian fashion that occupies a definite locus in the chromosome and responsible for expression of specific phenotypic character. Numbers of genes in each organism is more than no. of chromosome, hence several genes are located on each chromosome. The genes are arranged in a single linear order like beads on string. Each gene occupies specific location called locus. If the position of gene changes then character changes. Gene can be transmitted from parents to offsprings . Genes may exist in many alternate forms called alleles. Genes may undergo sudden changes in position and composition called mutation. Genes are capable of self duplication producing their own copies.

5. In 1969, Shapiro and co-workers published the first picture of isolated genes. They purified the  lac   operon of DNA and took photographs through electron microscope. In 1941, G.W. Beadle and E.L. Tatum working at St Standford university clearly demonstrated one-gene-one enzyme hypothesis, based on experiments on  Neurospora crassa . They made it clear that genes are the functional units and transmitted to progenies over generations; also they undergo mutations. They treated  N. crassa   with X-rays and selected for X-ray induced mutations that would have been lethal. Their selection would have been possible when  N. crassa   was allowed to grow on nutrient medium containing vitamin B6. This explains that X-rays mutated vitamin B6 synthesing genes. They concluded that a gene codes for the synthesis of one enzyme. In 1958, Beadle and Tatum with Lederberg received a Nobel prize for their contribution to physiological genetics.

6. Fine Structure of Gene A gene expresses itself through a series of steps involved in a sequential synthesis of a product and, therefore, may have one or more functional units . There can be several sites in a gene, each capable of being independently involved in mutational and recombinational events. A gene thus is neither a functional, nor a mutational or a recombinational unit, but is a complex locus, whose fine structure should be studied. Such fine structure has been studied in a number of cases using higher resolving power of recombination technique. 

7. Fine structure of  rII   locus in T4 phage The most refined analysis of a single gene ever conducted is the one undertaken by  Seymour Benzer   for a locus in T4 bacteriophage infecting  E. coli.  This locus is known as  rII   locus and a mutant at this locus is responsible for the formation of rough plaques or colonies  Fig. A mottled plaque showing  rII   mutant among a large number of normal plaques.

8. This locus had largest number of rapid lysing (r) mutants, and is called  rII locus. It can be distinguished from other  r  loci, by the inability of  rll   mutants to produce plaques on lysogenic 'K' strain of  E. coli. The   rII   mutants, may though infect 'K' strain, but can not cause lysis   and  are, therefore, unable to produce any plaques. In contrast, these  rII   mutants make large sharp plaques on  E. coli,  strain B. The wild type phage T4 ( rII + ) will make  small  and  fuzzy plaques,  both on B and K strains.

9. Further, when 'K' was infected simultaneously by  rII +   and  rII ,  large plaques were formed, since  rII +   helps in lysis so that  rII   may express. These distinguishing features enabled Benzer to identify mutants and wild type phages with high efficiency . Complementation test. In   order to find out complementation relations between different  rII   mutant alleles, Benzer used two different  rII   mutants, arbitrarily designated as  rII x   and  rII y .  He allowed mixed infection of K strain by these two mutants. Although in most cases, this does not result into lysis and plaque formation, in some cases it does lead to plaque formation. If two mutants did not form plaques on mixed infection, they were placed in the same group, but if plaques are produced, the two mutants involved in mixed infection were placed in two different groups.

10. In this manner, two groups A and B could be established in  rII   region. All mutants with the help of complementation test could be classified in these two groups, in such a manner that two mutants from group A or two mutants from group B could not cause plaque formation but mixed infection by one mutant of group A and another of group B, could cause plaque formation . Since groups A and B are distinguished on the basis of  cis -trans  test, these were termed as  cistron   A and  cistron   B.

11. Mutants belonging to the same cistron i.e. A or B would exibit   cis -trans   phenomenon, meaning that they would give wild type only in   cis   configuration and not in  trans  configuration. Two mutants from different cistrons (A and B) would give wild type (plaque formation) even in  trans  configuration, which in other words is called complementation.   Fig . Results of mixed infection by (a) a double mutant (having two  rII   mutations A1 and A2 belonging to same  cistron   A) and the wild type strain, phage; (b) two of T4 phage mutant strains, having different  rll   mutations ( A1 and A2) belonging to same  cistron   A.

12. From the complementation test, it is obvious that in  rII   region, two cistrons A and B are independent functionally and must be responsible for sequential synthesis of two separate products, which presumably are polypeptide chains. Therefore , all mutants belonging to one cistron share a common deficiency, which is different from the deficiency due to mutants belonging to the second cistron . When two mutants belong to same cistron , both are deficient for same product and therefore, they can not complement, but when two mutants belong to two different  cistrons ,  they, being deficient for different products, can complement, and may express wild phenotype  i.e.  lysis and plaque formation.

13. Molecular concept of the gene In molecular terms, a gene commonly is defined as  the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide.   According to this definition, a gene includes more than the nucleotides encoding the amino acid sequence of a protein, referred to as the  coding region.  A gene also includes all the DNA sequences required for synthesis of a particular RNA transcript. In some prokaryotic genes, DNA sequences controlling the initiation of transcription by RNA polymerase can lie thousands of base pairs from the coding region.

14. In eukaryotic genes, transcription-control regions known as enhancers can lie 50 kb or more from the coding region. Other critical noncoding regions in eukaryotic genes are the sequences that specify 3′ cleavage and polyadenylation   [poly(A) sites]  and splicing of primary RNA transcripts. Mutations in these RNA processing signals prevent expression of a functional mRNA and thus of the encoded polypeptide.  Most bacterial genes have no introns , whereas most genes of multicellular organisms do. The introns in human genes encoding average-size proteins are often much longer than the exons .

15. Many bacterial proteins with related functions are encoded by contiguous genes regulated by a single transcription-control region. This type of gene cluster, called an  operon , is transcribed into a single, polycistronic mRNA, which is translated to yield several different proteins. Most eukaryotic transcription units are transcribed into monocistronic mRNAs, each of which is translated into a single protein. The primary transcript produced from a simple eukaryotic transcription unit is processed into a single type of mRNA. The primary transcript produced from a complex eukaryotic transcription unit can be processed into two or more different mRNAs depending on the choice of splice sites and/or polyadenylation sites. In the case of many complex units, one mRNA is produced in one cell type, while an alternative mRNA is produced in a different cell type.

16. Pseudogenes   In muiticellular organisms, a wide variety of DNA sequences are found, which are of no apparent use. Some of these sequences are defective copies of functional genes and are, therefore, called  pseudogenes . These pseudogenes have been reported in human beings, mouse and  Drosophila.  The most popular examples of these pseudogenes include the following, ( i ) Human α - globin   and β - globin pseudogenes   , found in each of the two globin gene clusters. Complete nucleotide sequence of pseudo alpha globin gene is now known and it has been shown that both these genes are non-translatable, since they may have mutations in initiation codon and also frame-shift mutations along their length, (ii) In mouse also there are two alpha globin pseudogenes (ψ) ,  one of them (ψα3) is different from other pseudogenes since it has no introns which are present in functional α - globin genes  as well as in other pseudogenes .

17. Overlapping gene An  overlapping gene  is a gene whose expressible nucleotide sequence partially overlaps with the expressible nucleotide sequence of another gene. In this way, a nucleotide sequence may make a contribution to the function of one or more gene products.   Bacteriophage ΦX174 contains a single stranded DNA approximately 5,400 nucleotides in length. The genome of ΦX 174 consists of nine cistrons . From the information about proteins coded, an estimate could be made of the number of nucleotides required. This estimate of number of nucleotides exceeds 6,000 which is much higher than the actual number of nucleotides present i.e., 5,400. Therefore, it was difficult to explain how these proteins could by synthesized from a DNA segment which is not long enough to code for the required number of amino acids.

18. On detailed study of the system, it was discovered that sequences in the same segment could be utilized by two different cistrons coding for different proteins. Such overlapping of cistrons will be theoretically possible if the two cistrons have to function at different times and their nucleotide sequences are translated in two different reading frames. In 1976  Barrell   and his co-workers discovered that in ΦXl74, having nine cistrons (A, B, C, D, E, J, F, G, H), cistron E is present between D and J and that the cistron E overlaps cistron D. It could be shown that amber mutations in cistron E lie within the cistron D and these amber mutations do not influence the translation of cistron D into its protein. Similarly some other nonsense mutations for cistron E also lie in cistron D suggesting that the cistrons D and E overlap in the DNA sequences and that the cistron D and E are translated in two different reading frames so that amber codon in mRNA of one cistron will not be read as termination codon during the translation of mRNA of the other cistron .

19. Oncogene An  oncogene  is a gene that has the potential to cause cancer.  In tumor cells, they are often mutated or expressed at high levels. Most normal cells will undergo a programmed form of rapid cell death (apoptosis) when critical functions are altered. Activated oncogenes can cause those cells designated for apoptosis to survive and proliferate instead. Most oncogenes require an additional step, such as mutations in another gene, or environmental factors, such as viral infection, to cause cancer. Since the 1970s, dozens of oncogenes have been identified in human cancer

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