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.SapovadiyaAssistant Research

ScientistDepartment of Genetics

& Plant Breeding College of Agriculture

J.A.U., Junagadh

Submitted By:Vekariya Trang

AshokbhaiRoll No.: 31

M.Sc.(Agri.) 1st semesterDepartment of Genetics

& Plant BreedingCollege of Agriculture,

J.A.U., Junagadh

FINE STRUCTURE OF GENE

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.

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.

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.

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.

Seymour Benzer’s Concept (1955, 1957, 1959, 1961)

Modern Definition of GeneGene 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:

CISTRONThe 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.

MUTON 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.

RECON 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

PROKARYOTIC Gene structure

Genes based on their activity:1.House keeping genes2.Specific genes.

STRUCTURAL FEATURES:Simple gene structure.Small genomes(0.5 to 10 million bp). Prokaryotic genes are collinear with their proteins.

a. CODING REGIONb. PROMOTER ELEMENTSc. TERMINAL REGION OR TERMINATOR.

PROKARYOTIC Gene structure

a. Coding region- Starts with an initiator codon and ends with termination codonNo introns (uninterrupted).Collinear to its mRNA.

Eukaryotic gene structureExons IntronsPromoter sequencesTerminator sequencesUpstream sequencesDownstream sequencesEnhancers and silencers(upstream or downstream)Signals(Upstream sequence signal for addition of cap.Downstream sequences signal for addition of poly A tail.)

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.

Eukaryotic gene.

INTRAGENIC CROSSING-OVER & COMPLIMENTATION

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

Complementation

TYPES OF COMPLEMENTATIONIntra genic Complementation: Complementation take place between Two

different genes

Intergenic Complementation: Complementation take place between

Two different allele of the same gene

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

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

Some mutations in the phage’s genetic material can alter the ability of the phage to produce plaquesThus, 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 plaquesThis 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

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

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

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

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.

Benzer carefully considered the pattern of complementation & noncomplementationHe 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 testSo, if two mutations occur in the same cistron,

they cannot complement each other

A cistron is equivalent to a gene

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

DESCRIBES THE GENERAL STRATEGY FOR INTRAGENIC MAPPING OF RII PHAGE MUTATIONS

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

SPLIT GENE

SPLIT GENEDefenition: 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

Split Genes and RNA Splicing

intronexon exon

•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

SPLIT GENES HAVE TWO TYPES OF SEQUENCES normal sequences

interrupted sequences

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

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.

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

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).

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

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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

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.

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.

Thank you !!