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 rIIx and rIIy.
•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

20. Thank you