This document discusses several key concepts related to gene structure: 1. Genes in eukaryotes contain both coding (exon) and non-coding (intron) sequences. Introns are removed during RNA processing to form mRNA. 2. Benzer performed fine structure analysis of phage T4 genes which demonstrated that genes can undergo intragenic recombination, or crossing over within the gene. This established that genes have an internal structure smaller than previously believed. 3. Split genes were discovered, which have interrupted sequences (introns) between coding sequences (exons). RNA splicing removes introns to form mature mRNA from split genes.

1. FINE STRUCTURE OF GENE, ALLELIC COMPLEMENTATION
AND SPLIT GENES
COURSE TITLE: PRINCIPLES OF GENETICS
(2+1)
COURSE NO.: GP 501
Submitted To:
Dr. M.H.Sapovadiya
Assistant Research Scientist
Department of Genetics & Plant
Breeding
College of Agriculture
J.A.U., Junagadh
Submitted By:
Vekariya Trang Ashokbhai
Roll No.: 31
M.Sc.(Agri.) 1st semester
Department of Genetics &
Plant Breeding
College of Agriculture,
J.A.U., Junagadh

2. FINE STRUCTURE
OF GENE

3. INTRODUCTION
A gene is a specific sequence of DNA containing
genetic information required to make a specific
protein
Prokaryotic gene is uninterrupted.
In Eukaryotic gene the coding sequences (exon)are
seprated by non-coding sequences called introns.
In complex eukaryotes, introns account for more
than 10 times as much DNA as exons.

4. What is a gene?
•The gene is the
Functional unit of
Heredity.
•Each gene is a segment of DNA that give rise to a
protein product or RNA.
•A gene may exist in alternative forms called alleles.
•Chromosome in fact carry genes.
•Each chromosome consists of a linear array of genes.

5. BEAD THEORY
 Structure: gene is indivisible by crossing over.
Crossing over always occurs between the genes
but never within them.
 Function: gene is the fundamental unit of function.
Parts of gene cannot function.
 Change: gene is also treated as a fundamental unit
of change or mutation. It changes from one allelic
form to another. There are no smaller components
within it that can be changed.

6. BEAD THEORY
 Seymour Benzer in 1950s showed that bead theory
was not correct.
 Benzer was able to use genetic system in which
extremely small level of recombination could be
detected.
 The smallest units of mutation and recombination
are now known to be correlated with single
nucleotide pairs.

7. Modern Definition of Gene
Gene as a Fundamental and Indivisible unit of genetic
information and linked together
After the discovery of DNA, its parallel behaviour with that of
chromosomes and proper understanding of most of the
molecular phenomena which may interplay in the
determination of a phenotypic trait, the gene has been defined
as follows:

8. The portion of DNA specifying a single polypeptide
chain is termed as cistron, which is a synonym for
the termed, the gene of physiological function.
Haemoglobin, therefore, would require two cistrons
for its globin protein fraction, one each for the α and
β chains.
A cistron for α -chain has at least 141 X 3=423
nucleotides and the citron for the β-chain 146 X
3=438 nucleotides.

9.  There are many positions or sites within a cistron where
mutations can occur. Therefore, the gene as a unit of mutation
is smaller. i.e., it consists of fewer nucleotides than a
cistron. Benzer coined the word muton to that smallest length of
DNA capable of mutational change.
 Thus, a muton can be defined as the smallest unit of genetic
material which when changed or mutated produces a phenotypic
effect.
 A muton may thus be delimited to a single nucleotide or some
part of nucleotide.
 Different forms of a mutationality defined genes are called
homoalleles.
 For example, in bacteria muton may be nucleotide pair and in
cistron for haemoglobin the muton may be single nucleotid.

10.  Sometimes crossing over or recombination occurs in a
cistron and this provides still, other sub-divisional
concept of the cistron, namely the recon.
A recon is the smallest unit of DNA capable of
recombination or of being integrated by transformation
in bacteria.
Recombinationally separable forms of a cistron are
called heteroalleles

11. PROKARYOTIC Gene structure
Genes based on their activity:
1.House keeping genes
2.Specific genes.
STRUCTURAL FEATURES:
Simple gene structure.
Small genomes(0.5 to 10 million bp).
 Prokaryotic genes are collinear with their proteins.
a. CODING REGION
b. PROMOTER ELEMENTS
c. TERMINAL REGION OR TERMINATOR.

12. PROKARYOTIC Gene structure

13. a. Coding region-
Starts with an initiator codon and ends with termination codon
No introns (uninterrupted).
Collinear to its mRNA.

14. Eukaryotic gene structure
Exons
Introns
Promoter sequences
Terminator sequences
Upstream sequences
Downstream sequences
Enhancers and silencers(upstream or downstream)
Signals
(Upstream sequence signal for addition of cap.
Downstream sequences signal for addition of poly A
tail.)

15. EXONS –coding sequence, transcribed and
translated. Coding for amino acids in the polypeptide
chain.
Vary in number ,sequence and length. A gene starts and
ends with exons.(5’ to 3’).
Some exon includes untranslated(UTR)region.
INTRONS- coding sequences are separated by non-
coding sequences called introns.
Any nucleotide sequence that are removed when the
primary transcript is processed to give the mature RNA
are called introns.
All introns share the base sequence GT in the 5’end
and AG in the 3’end.
Introns were 1st discovered in 1977 independently by
Phillip Sharp and Richard Roberts.

16. Eukaryotic gene.

18. INTRAGENIC CROSSING-OVER &
COMPLIMENTATION

19. Complementation is the
production of a wild type
phenotype when two haploid
genomes bearing different
recessive mutations are united in
the same cell.

20. TYPES OF COMPLEMENTATION
Intra genic Complementation:
Complementation take place between Two different
genes
 Intergenic Complementation:
Complementation take place between
Two different allele of the same gene

21. WHAT IS INTRAGENIC CROSSING OVER?
 This simply means recombination within a gene.
 In early 1950s Seymour Benzer undertook a
detailed examination of a single locus, rII,in
phage T4
 He successfully designed experiments to recover
the extremely rare genetic recombinations
arising as a result of intragenic exchange.
 He demonstrated such recombination occurs
between DNA of individual bacteriophages
during simultaneous infection of the host
bacterium E.coli
 His work is described as fine structure analysis
due to extremely detailed information provided
from his analysis

23. WHAT ARE PLAQUES?
 A plaque is a clear area
on an otherwise opaque
bacterial lawn on the agar
surface of a petri dish
 It is caused by the lysis
of bacterial cells as a
result of the growth &
reproduction of phages

24.  Some mutations in the phage’s genetic
material can alter the ability of the phage
to produce plaques
 Thus, plaques can be viewed as traits of
bacteriophages
 Plaques are visible with the naked eye
 So mutations affecting them lend themselves to
easier genetic analysis
 An example is a rapid-lysis mutant of
bacteriophage T4, which forms unusually
large plaques
 This mutant lyses bacterial cells more rapidly
than do the wild-type phages
 Rapid-lysis mutant forms large, clearly defined plaques
 Wild-type phages produce smaller, fuzzy-edged plaques

25. Benzer’s fine-structure mapping of phage T4 used similar
experiments involving the rII gene.
a. Different rII mutations of T4 were used, each with the
characteristic large clear plaques and limited host range.
b. T4 with the wild-type r+ gene infects E. coil strains B
and K12(λ). But For rII T4(mutant), strain B is
permissive but K12(λ) is nonpermissive.
 In E. coli B
 rII phages produced unusually large plaques that had poor
yields of bacteriophages
 The bacterium lyses so quickly that it does not have time to
produce many new phages
 In E. coli K12S
 rII phages produced normal plaques that gave good yields of
phages
 In E. coli K12(λ)has phage lambda DNA integrated into
its chromosome)
 rII phages were not able to produce plaques at all

26. BENZER’S GENERAL PROCEDURE FOR DETERMINING THE NUMBER OF R+
RECOMBINANTS FROM A CROSS INVOLVING TWO RII MUTANTS OF T4

27. COMPLIMENTATION
 Benzer collected many rII mutant strains
that can form large plaques in E. coli B
& none in E. coli K12(λ)
 But, are the mutations in the same
gene or in different genes?
 To answer this question, he conducted
complementation experiments

28. For the production of phenotype the presence of both wild type genes is
required. So if the mutation is present on two different genes of the parents
the progeny will still have one wild type gene from each parent, i.e. the
genes will compliment each other while in the second case mutation is
present on one gene in both parents , i.e. progeny will have only one wild
type gene which will be insufficient to give phenotype.

29.  Benzer carefully considered the pattern of
complementation & noncomplementation
 He determined that the rII mutations occurred in
two different genes, which were termed rIIA & rIIB
 Benzer coined the term cistron to refer to the
smallest genetic unit that gives a negative
complementation test
 So, if two mutations occur in the same cistron,
they cannot complement each other
 A cistron is equivalent to a gene

30. At an extremely low rate, two noncomplementing strains of viruses can
produce an occasional viral plaque, if intragenic recombination has
occurred

31. DESCRIBES THE GENERAL STRATEGY FOR INTRAGENIC MAPPING
OF RII PHAGE MUTATIONS

33. THE DATA FROM FIGURE CAN BE USED TO ESTIMATE THE DISTANCE
BETWEEN THE TWO MUTATIONS IN THE SAME GENE
 The phage preparation used to infect E. coli B was diluted by
108 (1:100,000,000)
 1 ml of this dilution was used & 66 plaques were produced
 Therefore, the total number of phages in the original
preparation is 66 X 108 = 6.6 X 109 or 6.6 billion phages
per milliliter
 The phage preparation used to infect E. coli k12(λ) was
diluted by 106 (1:1,000,000)
 1 ml of this dilution was used & 11 plaques were produced
 Therefore, the total number of wild-type phages is
 11 X 106
 In this experiment, the intragenic recombination produces an
equal number of recombinants
 Wild-type phages & double mutant phages
 However, only the wild-type phages are detected in the
infection of E. coli k12(λ)
 Therefore, the total number of recombinants is the number
of wild-type phages multiplied by two
or 11 million phages per milliliter

35. SPLIT GENE

36. SPLIT GENE
Defenition:
 genes with interrupted sequence of nucleotides are
referred to as split genes
 Usually a gene has a continuous sequence of
nucleotides.
 In other words, there is no interruption in the
nucleotide sequence of a gene. Such nucleotide
sequence codes for a particular single polypeptide
chain.
 However, it was observed that the sequence of
nucleotides was not continuous in case of some
genes; the sequences of nucleotides were
interrupted by intervening sequences

37. Split Genes and RNA Splicing

38. •Split genes were independently discovered by Richard
J. Roberts and Phillip A. Sharp in 1977, for which they
shared the 1993 Nobel Prize in Physiology or Medicine.
P.A. Sharp (Biology, MIT) Richard J. Roberts
The first observations of interrupted (split) genes, i.e.,
genes in which there are noncoding intron sequences
between the coding exon sequences, were made in animal
viruses in 1977

39. SPLIT GENES HAVE TWO TYPES OF
SEQUENCES
 normal sequences
 interrupted sequences

40. SPLIT GENE
 i. Normal Sequence (exons):
 This represents the sequence of nucleotides which
are included in the mRNA which is translated from
DNA of split gene (Fig. 13.2). These sequences
code for a particular polypeptide chain and are
known as exons

41. SPLIT GENE
 ii. Interrupted Sequence (introns):
 The intervening or interrupted sequences of split
gene are known as introns. These sequences do
not code for any peptide chain. Moreover,
interrupted sequences are not included into mRNA
which is transcribed from DNA of split genes.
 In prokaryotes such kind of introns are very less,
while in case of prokaryotes there are large
numbers of introns.

42. IMPORTANT FEATURES OF INTERRUPTED GENES:
 Each interrupted gene begins’ with an exon and
ends with an exon.
 The exons occur in the same precise order in the
mRNA in which they occur in the gene.
 The same interrupted gene organisation is
consistently present in all the tissues of organisms.
 Most introns are blocked in all reading frames i.e.,
termination codons occur frequently in their three
reading frames. Therefore, most introns do not
seem to have coding functions

43. SIGNIFICANCE OF SPLIT GENES:
 The significance of split organisation of eukaryotic genes
is not clear.
 In some cases, different exons of a gene code for
different active regions of the protein molecule, e.g., in
the case of antibodies. Thus, it has been suggested that
introns are relics of evolutionary processes that brought
together different ancestral genes to form new larger
genes. It is also possible that some introns have been
introduced within certain exons during evolution.
 Introns may also provide for increased recombination
rates between exons of a gene and thus may be of some
significance in genetic variation
 Introns are known to code for enzymes involved in the
processing of hn RNA (heterogenous RNA).

44. EVIDENCE FOR SPLIT GENES
 Most higher eukaryotic genes coding for mRNA, tRNA
and a few coding for rRNA are interrupted by unrelated
regions called introns
 Other parts of the gene, surrounding the introns, are
called exons
 Exons contain the sequences that finally appear in the
mature RNA product
 Genes for mRNAs have been found with anywhere from 0 to
362 introns
 tRNA genes have either 0 or 1 intron
14-
44

45. 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

46. 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.

47. 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.

48. Thank you !!