In this article we will discuss about: 1. Introduction to Modern Concept of Gene 2. Concept of Gene – Classical Vs. Molecular 3. Subdivision 4. Fine Structure 5. Multi-Gene Families 6. Overlapping 7. Mobile Genetic Elements.

Contents:

  1. Introduction to Modern Concept of Gene
  2. Concept of Gene – Classical Vs. Molecular
  3. Subdivision of Gene
  4. Fine Structure of Gene
  5. Multi-Gene Families
  6. Overlapping Genes
  7. Mobile Genetic Elements


1. Introduction to Modern Concept of Gene:

The concept of gene first came from the work of Mendel. Although the term ‘gene’ was not used at that time, Mendel from his experiments on pea plants, suggested that the characters of an organism are controlled by factors located with­in its body.

Flemming recorded certain deeply stained bodies within the cell, which he termed ‘chromosomes’. Meischer on the other hand, extracted ‘nuclein’ a nucleoprotein constituent from the sperm. Afterwards, due to researches by subsequent workers (Kossel and others), an idea gradually developed on the chemical structure of chromosome.

After the chemical analysis of chromosome carried out by Meischer and his successors, chromosomes were found to consist chiefly of protein and nucleic acid. It was regar­ded as continuous frame-work of basic protein or histone, having active regions located on DNA the genes, the hereditary material.


2. Concept of Gene – Classical Vs. Molecular:

The term gene was coined by Johannsen (1909) for the hereditary factors of Mendel. The concept of gene is more important because its physical and chemical nature is the foundation of all genetic principles. The classical concept of gene is that it is a unit of function occupying a definite position on the chromosome (not sub­-divisible by recombination) and is responsible for a particular phenotypic character.

According to Watson and Crick (1953), Wilkins (1962), gene was defined as a macromolecule attached to undifferentiated nucleoprotein thread (chromonema) which can pass from cell to cell and from one generation to another generation. T. H. Morgan discussed about the nature of gene, its characters, function which leads to classical concept of gene.

Further research on gene at molecular level have revolutionized the gene concept. Our cur­rent molecular concept is, a gene is the unit of inheritance which may undergo intragenic recombination. Gene is the unit of function cod­ing for one polypeptide; operationally defined by the complementation test; may be divisible by recombination test.

The changing concept of gene from classical to molecular level may be summarized as follows:

i. Gene governs the inheritance which is bi-parental, i.e., both male and female parents contribute equally in the inheritance of charac­ters to next generation (exception cytoplasmic inheritance or maternal inheritance).

ii. Characters of an individual are deter­mined by paired genes, situated in definite num­ber of chromosome pairs or linkage groups.

iii. Many genes are present on each chro­mosome and are inherited together, called linked genes.

iv. Each gene-has a particular position on a particular chromosome being called as locus. Chromosomal aberration of translocation or inversion type may change the position of a locus.

v. During gamete formation each pair of genes gets segregated and gametes possess only one gene of that kind.

vi. Pairs of genes which are in different chromosomes or in different linkage groups are assorted independently.

vii. Genes lie in a linear order on a chro­mosome and the order remains same unless translocation or inversion takes place.

viii. A single gene may occur in several forms called alleles. Normally it has two forms — dominant and recessive; when more than two forms occur, called multiple alleles.

ix. The number of a particular gene may be increased in a ‘cell due to polyploidy. Aneuploidy also causes increase (hyperpoidy) or decrease (hypoplotdy) in the number of a parti­cular gene in a cell.

x. Mutation causes a gene to be changed from its wild form.

xi. Genes in a particular chromosome may be shifted to its homologous pair due to crossing over causing recombination.

xii. Sometimes two or more genes interact together to produce a trait (interaction of genes).

xiii. Replication of each chromosome during cell division causes the self-duplication of genes of that chromosome.

xiv. Inbreeding causes homozygosity within the gene pair, whereas outbreeding leads to heterozygosity of the gene pairs.

xv. Genes are chemically DNA, a double helical structure composed of nucleotides con­taining adenine, guanine, thymine and cytosine together with deoxyribose sugar and phosphate. The nucleotides are grouped into triplets presen­ting 64 combinations called as genetic code.

xvi. Genes play a vital role in cells:

(i) Fun­damental unit of heredity,

(ii) Direct or indirect control on cellular metabolic activity,

(iii) Repro­duction of the cell.

All these activities are medi­ated by different RNAs (mRNA, rRNA and tRNAs) involved in protein synthesis.

17. Gene directs the synthesis of protein, through transcription and translation, that func­tions as enzyme which leads to ‘one gene – one enzyme’ hypothesis.

xviii. Proteins are made up of polypeptides and the concept has got further changed to ‘one gene – one polypeptide’.

xix. Fine structure analysis of gene has resulted in the concept of ‘cistron’, a functional unit of gene; which is further sub-divisible into unit of recombination, ‘recon’ and the ‘muton’, smallest length of DNA undergoing mutations.

xx. DNA replication occurs at many points along the eukaryotic chromosome and each replicating region is called ‘replicon’.

xxi. Gene may be splitted in nature, which is made up of ‘introns’ (non-translatable portions of DNA) and ‘exons’ (translatable portions of DNA).

xxii. Overlapping genes refer to the DNA segment which is being used to produce more than one different polypeptides. Genes may even be included, i.e., within a particular DNA seg­ment of a gene another gene may be included.

xxiii. The concept of fixed location of gene has got changed with the invent of jumping gene, the transposable unit of genes are called ‘transposons’ or IS (insertion) elements.

xxiv. Oncogenes are present in the cells as proto-oncogenes which on stimulation converted to oncogenes and induce immortalization of cells.

xxv. Defective copies of functional gene may exist called pseudo-genes. Despite being detri­mental to the organisms, some genes are propa­gated in cells called ‘selfish genes’.

xxvi. A locus may bear several genes which interact -in complicated pattern called complex loci (homeotic genes). A gene may be present as multiple copies on the same or different chromo­somes (multi-gene family).


3. Subdivision of Gene:

A gene is believed to be a hereditary unit, controlling a function leading to appearance of a phenotypic trait. It is also regarded as a smallest unit that can undergo mutation and can take part in recombination. But several studies have led to change in this concept on the basis of study of structure of gene at the intragenic level.

Position Effect (Bar Eye in Drosophila):

When a chromosome rearrangement involves no change In the amount of genetic material but only in the position of genes, the term position effect is used to describe any asso­ciated phenotypic alteration. Such type of position effect can be exempli­fied by the experiment done by Sturtevant and his coworkers on the Bar eye characters of Drosophila in 1925.

In Drosophila, the wild type eye (B+/B+) has 779 facets, heterozygote condition of Bar eye (B+/B) with 358 facets and homozygote condition of Bar eye (B/B) with 68 facets.

The frequency of mutation from homozy­gous Bar to wild type and also to the Ultrabar (Bu/Bu) type (with 24 facets) occurred at a very low frequency. But Sturtevant observed that fre­quency of these events was much higher than what is expected.

The alternative explanation that was given by him was unequal crossing over. For supporting this phenomenon he took two marker genes on either sides of this Bar locus, one is forked bristles (f) and another is fused veins (fu) to evidence recombination.

He test crossed the fB+/+Bfu with fBfu/fBfu and got the following four types of combinations (Fig. 14.4).

Appearance of Ultrabar and Wild

(i) Ultrabar eye, forked bristles and fused veins (fBufu/fBfu);

(ii) Wild type eye, wild type bristles and wild type veins (+B++/fBfu);

(iii) Wild type eye, forked bristles and fused veins (f B+fu/f Bfu);

(iv) Ultrabar eye, wild type bristles and wild type veins (+Bu+/f Bfu).

Phenotypes between Homozygous Bar and Heterozygous Ultrabar

Sturtevant demonstrated that phenotype of flies with two Bar genes on one chromosome and none on the other, i.e., double Bar heterozygous, was different from those with one Bar gene on each chromosome. This indicates that position of gene with respect to adjacent

Later Bridges, through the study of salivary gland chromosomes of Drosophila, demonstra­ted cytologically that duplication of 16A segment in X-chromosome is associated with Bar charac­ter and two such repeats in the same chromo­some leads to Ultrabar formation. The conclusion made from above is that the gene is not a point, but has Its dimensions, and alleles of a gene or locus may recombine with each other.

Pseudo alleles (Lozenge Locus in Drosophila):

The different states of the same gene are termed as alleles. The alleles control respective phenotypes in a homozygous state. In a heterozygote, the dominant allele is expressed and in later generations, the alleles normally segregate with­out any recombination between them.

Sometimes genes located at the same site on the linkage map show recombination, indicating thereby that although they are functional as alletes, structu­rally they are non-allelic. Such alternative states of the same gene are called pseudo alleles.

The classical test of allelism, to find out whether two mutant alleles in question are alle­lic to each other or not, is the recombination test. The test involves whether the F1 individual gives rise to the wild types in the F2 generation or not (Fig. 14.5). Recombination is possible only when the mutant alleles are non-allelic.

Test of Allelism

Oliver conducted experiments using diffe­rent mutants of Drosophila at the sex-linked mutant locus lozenge (Iz) responsible for smaller, darker and more elliptical eyes. Wild type recombinants could be obtained at a very low frequency in F2 generation of a cross between two mutants (Iz1 and Iz2) at lozenge locus.

Green (student of Oliver) marked the lozenge locus on either sides by marker genes to confirm the appearance of wild type due to recombination in a cross of heterozygous (Iz1 Iz2) female with either Iz1 or Iz2 male (Fig. 14.6).

If wild type was the result of recombination, he expected that marker genes would also recom­bine. In his experiments, Green observed that not only wild type flies appeared for lozenge, but the marker genes a and b also recombined sugges­ting that Iz1 and Iz2 could recombine. This suggested that two mutants may be separated by a distance within the gene.

Later from a cross of apricot eyed and white eyed flies, Lewis obtained F1 having intermediate eye colour. In F2, he had expected segregation only for apricot and white, but recovered wild type also at a very low frequency.

In both the experiments, it was indicated that mutant alleles apparently recombined arid therefore proved to be non-allelic according to classical concept of allelomorphism. Since these alleles behaved as non-allelic, Lewis preferred to call them pseudo alleles and the phenomenon as pseudo-allelism.

Green's Experiment on Lozenge Locus of Drosophila

Pseudo alleles are closely linked genes, having similar phenotypic effect, which behave ordinarily as alleles but have been shown by extensive experiments to be separable by crossing over.

Cis-trans Effect (Position Pseudoallelism):

Though the appearance of wild type in F2 gene­ration could be explained on the basis of pseu­doallelism, but it was difficult to explain why F1 was not wild type. This could be explained on the basis of lack of complementation due to different arrangements (cis-trans) of mutant alle­les. In F1 heterozygotes, two arrangements are possible (Fig. 14.7).

cis-trans Effect

(i) Both mutant alleles on the same chromo­some and their wild counterparts on the other homologue (cis),

(ii) Two mutant alleles on two different homologues (trans).

Heterozygote for Iz1, and Iz2 (two mutants) may show both cis and trans arrangements. In cis configuration wild phenotype expresses, but trans configuration in F1 individuals does not allow the wild type to be expressed.

This may presumably be due to position effect, where a mutant allele does not allow adjacent region to express wild phenotype, so it may be called as position pseudo-­allelism. This has been termed as cis-trans effect by Lewis. Since intragenic inter-allelic crossing over is possible, as a result + + and Iz1lz2 gametes are produced from F1 individuals (trans). Thus in F2 generation, the wild type will express.

Test for Functional Allelism (Cis-Trans Complementation Test):

When two mutations have the same pheno­typic effect and map close together, they may comprise alleles. However, they could also rep­resent mutations in two different genes whose proteins are involved in the same function. The complementation test is used to determine the functional allelism of any two recessive genes, i.e., whether two mutations lie in the same or different genes. The test consists of making a F1 heterozygote for the two mutations (by mating parents homozygous for each mutation) and to find out whether the F1 individual exhibits wild phenotype or not.

When the two mutations lie in the same gene, the expression of wild phenotype could be explained on the basis of their cis-trans configu­rations. It is mutant when the mutations lie in trans, and must be wild type when they lie in cis. Thus mutants can complement in cis form but not in a trans form.

This comparison provides the basis for the cis-trans complementation test. In contrast, when the mutations lie in different genes, the configuration is irrelevant. In either case there is one copy of each mutant gene and one copy of each wild type gene.

Thus complementation is tested by deter­mining whether the trans-heterozygote shows wild phenotype (mutations lie in different genes) or mutant phenotype (mutations lie in the same gene). When the two mutations fail to complement in trans, the inference is that both the mutations affecting the same function, they are included in the same complementation group (Fig. 14.8).

Active Polypeptide Production

Fourteen alleles can be located on at least four mutational sites of lozenge locus of Drosophila. Alleles of different mutational sites when subjected to cis-trans test, complementa­tion relationships showed lack of complementa­tion among all the 14 alleles. Lack of comple­mentation among the different mutants of lozenge locus indicates that they belong to only one functional unit.


4. Fine Structure of Gene:

The concept of gene controlling a single character must involve the expression of several reactions and in its constituents involves a num­ber of proteins. The work of Beadle and Tatum demonstrated that the relationship between gene and enzyme is 1:1.

Evidently, if a gene is respon­sible for the synthesis of a single polypeptide then the gene as visualized by Mendel is cer­tainly not the ultimate unit of inheritance. The concept of ultimate indivisible unit of gene underwent complete change following the work of different scientists.

Concept of Cistron, Recon, Muton (Rll Locus in T4 Phage):

Benzer carried out most refined analysis of rll locus in T4 bacteriophage. A mutant at this locus is responsible for the formation of rough plaques or colonies on B strain of E. coli, but unable to produce any plaques on K strain.

Complementation Test:

This test was car­ried out by Benzer in order to find out comple­mentation relations between different rll mutant alleles. He allowed mixed infection of K strain of E. coli by two rll mutants. In most cases it did not result in plaque formation, but in some cases plaque formation occurred.

If two mutants did not form plaques on mixed infection, they were placed in the same complementation group, but if plaques were produced, the two mutants involved in mixed infection were placed in two different groups. In this manner, two groups A and B could be established in rll 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. 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. 14.9).

Complementation Test between Mutants

From the complementation test, it is obvious that in rll region, two cistrons A and B (Fig. 14.10) of 2500 and 1500 nucleotide pairs respectively 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 mutations belonging to the second cistron.

When two mutants belong to same cistron, both are deficient for same product and, therefore, they cannot 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.

Recombination Test:

After rll mutants were classified in cistron A and cistron B, Benzer was interested in analysing mutants, belonging to same cistron. It was, therefore, necessary to subject them to recombination test to find out whether they are located on same site or different sites separable by recombination.

Deletion mutation of rll locus were arranged in a sets of overlapping deletion representing segments of different sizes.

The principle involved in this technique was that if a particular point mutant lies in the region of a deletion represented by a rIl mutant, then on mixed infection with this deletion mutant, the point mutant will not be able to give rise to a wild type, but if it falls outside the deletion region, it will be able to give wild type recombinant.

rll Region of T4 Phage

Using deletion mutants having successive overlapping deletions of smaller lengths, one could locate a mutant to a fairly small region. All point mutants located in this particular small seg­ment of rll region, could then be subjected to recombination test.

For this purpose, two mutants at a time were used for mixed infection on E. coli B strain and the lysate produced on plaques formed on B strain was used for infection on K strain to find out the frequency of wild type phage particles produced.

Benzer eventually estimated 400-500 mutational sites in rll region and called each of them a unit of mutation or a muton. Thus Benzer was not only able to divide rll region into cistrons A and B, but was able to classify mutants belonging to same cistron into hundreds of muta­tional sites separable due to recombination.

Cistron, Recon, Muton:

Thus a gene is not a unit of either function or recombination or mutation. Benzer, in view of his work, coined the terms cistron (unit of function), recon (unit of recombination) and muton (unit of mutation). Cistron was defined as a unit, the elements (alle­les) of which exhibit cis-trans phenomenon.

The smallest unit capable of undergoing recombina­tion is called recon. A recon is further sub-divisi­ble into units of mutations called mutons, and several mutons in a recon will not be separable due to recombination. Therefore, cistron, recon and muton are the units, in the descending order of size and structurally the gene of classical authors is comparable to cistron of Benzer.

Deletion Mapping:

At the intragenic level, with the help of recombination studies, micro-maps have been prepared for different genes. Deletion mutations have a critical use in genetic mapping. The dele­tion can recombine with point mutations on either side. By obtaining a series of partially over­lapping deletions, any point mutation can be mapped by testing the ability to recombine among them.

When two deletions both fail to recombine with a point mutation, the site of mutation must lie in the region common to the deletions. When one deletion recombines and other does not, the site of mutation must lie in the region in which the deletions do not overlap.

When a new mutant carrying a point muta­tion is isolated, the mutation can quickly be mapped to a defined interval by crossing the mutant strain with each of the overlapping dele­tion mutants.

Suppose a new point mutation r1 has been obtained and recombination experi­ment shows no recombination with deletion I segment but can recombine with other deletion II, III ad IV, so the site of point mutation must be within the segment A (Fig. 14.11).

Use of Overlapping Deletions in Genetic Fine Structure Mapping

Results of Crosses of Five Point Mutants

Benzer characterized a large number of deletion mutants that divided the rll locus into 47 small segments. The extents of the deleted segments has been determined by crossing the deletion mutants to a set of reference point mutants previously mapped by two- and three- factor crosses (Fig. 14.12).

Deletion

Deletion Mapping

Complex Loci in Eukaryotes and Intragenic Complementation:

Genetic fine structure maps have been con­structed for many genes of higher animals (Drosophila) and plants. In case of extensive ana­lysis of gene, sometimes several mutable sites separable by recombination has been detected. A conventional gene is identified at the level of the genetic map by a tightly linked cluster of non-complementing mutations.

Whereas intra­genic complementation indicates two or more mutable sites in a gene which are separable by recombination.

Such as, most genes in D. melanogaster are relatively short, uninterrupted and have a few, short introns. But some loci are over a large map distance and often displaying a complex or ambiguous patterns of complementation. These mutations may fall into different overlapping complementation groups.

The individual mutations may have different and complex morpho­logical effect on phenotype. Such loci with unknown number of genes are called as ‘com­plex loci’ which are large, generally more than 100 kb. Mutation can occur anywhere within a large genomic region.

Genetic organization of these loci may take several forms:

i. More than one gene may reside in a com­mon expression unit, e.g., BX-C locus.

ii. Multiple genes expressed separately, but similar mutant phenotype at any region, e.g., Achaete-Scute locus.

iii. Single gene but displays, unusual comple­mentation patterns, e.g.. Notch locus.

iv. Complex locus but not large, produces multifunctional protein products, e.g. Rudimentary locus.

BX-C (bithorax complex), an example of complex locus, affects development of thorax and morphological changes in abdomen. The genetic map of BX-C is given in Fig. 14.13, the locus falls into three domains (3 coding units).

Bithorax Locus has 3 Coding Units

A crucial feature of this locus is that mutations affecting particular segments lie in the same order on the genetic map as the corresponding segments in the body of the fly. The complex or ambiguous patterns of complementation of complex loci is sometimes correlated with intragenic complementation.

It is a phenomenon totally distinct from inter-genic complementation, on which cis-trans is based. The functional forms of certain enzymes are dimers or higher multi-mers consisting of two or more polypeptides.

These polypeptides may be either homologous, the products of a single gene, or non-homologous, the products of two or more distinct genes.

When the active form of the enzyme contains two or more homologous polypeptides (it may or may not also contain non-homologous polypeptides), intragenic com­plementation may occur, in organisms that are homozygous for the wild type allele of a given gene, all the protein dimers or higher multi-mers will contain identical wild type polypeptides.

Similarly, organisms that are homozygous for any mutation in that gene will contain dimers or higher multi-mers, all of which contain identical copies of the mutant polypeptide (Fig. 14.14). An organism that is heterozygous for two different mutations in the gene will usually produce some dimers or higher multi-mers that contain one or more of each of the two different mutant polypeptides.

These hetero-multi-mers (protein multi-mers composed of the polypeptide products of two different alleles of a gene) may have partial or complete (wild type) activity. As a result, trans-heterozygotes may have a wild-type phenotype or a phenotype intermediate between the mutant and wild type.

Mechanism of Intragenic Complementation


5. Multi-Gene Families:

Some genes exist as a number of copies that can be grouped into families. Advances in molecular genetics have revealed that many eukaryotic genes belong to multi-gene families. A multi-gene family is a group of similar, but not totally identical sequences, each sequence rep­resenting a gene, so that the gene is present in multiple copies.

All of the genes in the family may occur in the same locus, e.g., five members of growth hormone gene family are clustered on chromosome 17 of human.

They may occur at different loci, e.g., five members of aldolase gene family are on different human chromosomes. The genes of a family may exist as a series of clusters on different chromosomes, e.g., the homeo-box genes which occur as four clusters on separate chromosomes, each containing- about 10 individual genes.

These multi-gene families can be classified into at least 3 groups:

(i) The different sequences of a multi-gene family may function in different tissues of the same organism, or may function in the same tissue at different times. For example, globin gene of human has six non-identical sequences which get expressed at different times, situated in a region of 50 kb on chromosome II.

The ε gene is expressed in embryo, the two γ genes are expressed in foetus, and δ and β genes are expressed after birth and in the adult (Fig. 14.15).

Multigene Family

(ii) All members (sequences) of a gene fami­ly may function in a specific tissue or at a specific time or they may function toge­ther when the product is required more. For example, heat shock genes expressed more during heat shock, storage protein genes expressed more during seed forma­tion.

(iii) In other cases, there may be multi-gene families, the members of which will ordi­narily function in all living cells at all times, as in case of rRNA genes or SnRNA genes.


6. Overlapping Genes:

Overlapping genes are those genes which can be read or translated in two different ways to produce two different proteins. From the information about proteins coded by the genome of φx174, an estimate could be made of the number of nucleotides required (number of bp should be 3 times the number of amino acids in protein).

This estimate of number of nucleotides exceeds 6000 which is much higher than the actual number of nucleotides present in single stranded DNA of φx174 (the actual number of nucleotide is 5400). Therefore, it was difficult to explain how these proteins could be coded from a DNA segment which is not long enough to code the required number of amino acids.

Genetic Map

On detailed study of the system, it was dis­covered that a single sequence can be utilized by two different cistrons coding 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 Barnell and his co-workers dis­covered that the genome of φ x 174 consists of 9 cistrons (A, B, C, D, E, F, G, H, J). Cistron E is pre­sent between C and J, and that cistron E overlaps cistron D. Again stop codon of gene D is over­lapping with the start codon of gene J (Fig. 14.18). Gene B also overlaps with gene A.

In the base sequence TAATG, the first triplet code TAA, is the stop codon of gene D and last three (ATG) is the start codon of gene J (Fig. 14.19). Here the mid­dle base (third from left) is the base where both codons meet.

Secondly, it has also been shown that gene E and B are completely included in gene D and A respectively. Both these genes produce their own protein in considerable amount. Such genes are named as included genes. Probable significance attributed to these genes is to meet the economy of space or area without affecting the production of needed proteins.

Overlapping and included Genes


7. Mobile Genetic Elements:

The discovery of mobile sequences in chromosome causing genetic instability is an important event in genetics. The mobile genetic elements are specific DNA sequences having the capacity of movement from one location to another in the chromosome. Such sequences, often referred to as mobile sequences or transposable sequences or jumping genes, were first identified in maize by Barbara McClintock.

Transposable sequences, identified both in prokaryotic and eukaryotic organisms, have been classified into two distinct categories, namely insertion sequences which are short, of about 1000 bases and the longer transposons which may even be several thousand bases long sequences or IS elements, so named because they can insert at different regions of bacterial chromosomes and plasmids through illegitimate recombination.

These are typically short and contain only genes involved in regulation of transposition. IS elements were identified as spontaneous insertion in some bacterial operons which inactivate the gene and do not allow trans­cription and translation. IS elements have been detected in certain lac mutations of E. coli.

These mutations have the unusual property of reverting to wild type a high frequency. Molecular ana­lysis eventually revealed that these unstable mutations possessed extra copies of DNA in or near the lac genes. In revertants, these extra DNA sequences are lost.

Later, similar insertion sequences were found in many other bacteria.

All of them are characterized by:

(i) The presence of inverted terminal repeats required for transposition;

(ii) The ability to create duplication of flanking DNA at the site of insertion, target site dupli­cations; and

(iii) Presence of open reading frames coding for enzyme transposase, which catalyzes transposition (Fig. 14.20A).

Structure of IS Elements

Presence of INverted Repeats

Prokaryotic Transposons:

The longer transposable sequences in bacteria, carrying genes for transposition and antibiotic resistance, are called transposons, denoted by the symbol Tn. All transposons studied so far contain inverted repeats at both ends. Characteristic stem and loop formation in each strand on strand separa­tion due to denaturation is an evidence for inverted repeats (Fig. 14.20B).

Composite trans­posons are created when two IS elements insert near each other (Fig. 14.21 A) and the region between them can be transposed by the joint action of the flanking elements/sequences. In effect, two IS elements capture a DNA sequence that is otherwise immobile.

Insertion of a Transposon

In different In elements, there are diffe­rences in orientation and/or composition of flanking sequences and composition of middle transposed sequences (Fig. 14.21 B), which usually contain genes for transposase and anti­biotic resistance.

Genetic Organization of Composite Transposons

For example, in Tn9, the flank­ing IS elements are in direct orientation with respect to each other and antibiotic resistance gene it contains is chloramphenicol. In Tn5 and Tn10, the orientation of IS elements is inverted; Tn5 contains kanamycin, bleomycin and strepto­mycin resistance genes and Tn10 contains tetracycline resistance gene.

Sometimes, the flanking IS elements in a composite transposon are not quite identical. For instance, in Tn5, the element in the right, called IS50R, is capable of producing a transposase, but the element on the left, called IS50L, produces a defective enzyme. This difference is due to single nucleotide.

Tn3 elements have other notable features. This group is the largest family of prokaryotic transposon.

They do not have IS elements at their ends. Instead, the Tn3 elements produce target site duplication when they insert into host DNA. There are three genes, tnpA, tnpR and bla, encoding respectively, a transposase (catalyzes transposition), a resolvase/repressor (inhibits transposition) and an enzyme β-lactamase (con­fers the resistance to ampicillin).

Medical Significance:

As many bacterial transposons carry genes for antibiotic resistance, consequently, it is relatively a simple matter for these genes to move from one DNA molecule to another, for instance, from chromosome to plasmid or vice versa or from one plasmid to another plasmid (Fig. 14.22).

This genetic flux has a profound medical significance because many of the DNA molecules that acquire anti­biotic resistance genes can be passed on to daughter cells – thus spreading the resistance both horizontally as well as vertically.

Transposition of a Transposon

This process has been observed in several species, pathogenic to humans, and today, many diseases are becoming difficult to control only due to this transposon activity. The spread of multiple drug resistance in bacterial populations has been accelerated by the evolution of conjugative R plasmids that carry the resistance genes.

Eukaryotic Transposons:

Geneticists have found many different types of transposons in ‘eukaryotes. They vary in size, structure, compo­sition and behaviour. Some are abundant in genome while others are rare. McClintock first detected them in maize and are being called as ‘controlling elements’ or ‘mobile genetic ele­ments’.

All these transposons have inverted repeats at their termini and create site-specific duplication when they insert into host DNA molecules.

Ac-Ds System (Ac-‘Activator’ for Ds, Ds- ‘Dissociation’ factor for breakage):

McClintock discovered the Ac and Ds elements by studying chromosome breakage in maize. She used gene­tic markers like maize kernel-pigmentation (anthocyanin gene activity loci) to detect the breakage events. When a particular marker was- lost, McClintock inferred that the chromosome segment on which it was located had been lost, an indication that a breakage event had occurred.

The loss of the marker was detected by a change in the colour of the aleurone of maize kernels.

McClintock found that the breakage respon­sible for the mosaic kernels, occurred at a parti­cular site of maize chromosome 9. She named the factor that produced these breaks Ds, for ‘Dissociation’. However, by itself, this factor was unable to induce chromosome breakage.

In fact, she noticed that Ds had to be stimulated by another factor Ac, for ‘Activator’. This Ds factor is present in some maize lines but absent in others. When different stocks were crossed, Ac could be combined with Ds to create condition that led to chromosome breakage.

This two-factor Ac/Ds system provided an explanation for genetic instability that McClintock observed on chromosome 9. Experiments by McClintock could prove that both Ac and Ds can move, and when these elements insert in or near the genes, the functions of genes are altered – sometimes completely abolished.

McClintock called the elements as controlling elements due to their influence on gene expression. DNA sequencing has shown that an Ac element con­sists of 4563 nucleotide pairs bounded by inver­ted repeats that are 11 nucleotide pairs long.

Each element is flanked by an 8-nucleotide pairs direct repeats which are created at the time that the element inserts into the chromosome, they are target site duplications, not integral parts of the element. Genetic analysis has provided some infor­mation about the mechanism whereby Ac (and presumably Ds) elements transpose.

After an Ac element has been replicated as a part of the DNA in a chromosome, it can excise from its position and move to a new one.

Once the replication fork has passed over the Ac element, a copy of that element can transpose to a site ahead of the replication fork. When the repli­cation process is finished, there will be two sister chromatids — one with single copy of Ac (in the new location only) and another with two copies (one in the new and the other in the old location).

The Ac element does not replicate itself during transposition, rather it is copied by the normal replication machinery before and after movement. The actual transposition of an Ac element is considered to be non-replicative (Fig. 14.23).

Mechanism of Transposition of AC Element

All the AC elements in the maize genome appear to be structurally similar, if not identical. This is not the case with the Ds elements, in which considerable structural heterogeneity has been observed (Fig. 14.24).

Structural Heterogeneity of Ds Elements

(i) One class of Ds elements is derived from Ac elements by deletions of internal sequences.

(ii) The terminal inverted repeats are same as in Ac, also sub-terminal sequences are same, but the remainder of the DNA is different. These unusual forms are aberrant Ds element.

(iii) Third class is characterized by a pecu­liar piggybacking arrangement. One Ds element is inserted into another, but in an inverted posi­tion. These double Ds elements are responsible for chromosome breakage.

The activating function of Ac elements is associated with a protein that they synthesize. Since this protein is involved in transposition, it is sometimes called the transposase of Ac-Ds family. In presence of Ac, Ds may undergo trans­position or may cause chromosome breaks (Fig. 14.25).

Consequences of Incluence of the Activator

If Ac+ and Ds+ denote the absence of those two elements then the result of the cross invol­ving Ac-Ds system would show the behaviour represented in Table 14.1.

Result of a Cross Involving Ac-Ds System

A number of different mutable waxy alleles could also be produced due to insertion of Ds element at different positions within the locus Wx. In addition to Ds, Ac may also get inserted into the target gene, making it unstable. The inserted Ac may become Ds due to deletions or inactivation of the gene encoding transposase.

After the discovery of Ac/Ds system from maize, Spm element was isolated from maize, a few other transposons, such as En/I, stowaway, were characterized from snapdragon. Petunia, and rice plant. Their characters are similar to those of Ac/Ds, but unlike Ds often they are complete in organization and function, i.e., they can move (transpose) without participation of additional elements.

Their existence was noted in human, mammals, and other organisms also (Alu in mammals, Copia in Drosophila, Ty element in yeast). At present, the mobile DNA sequences or transposons are known to be a common feature of living organisms, and they are the tools of evolution.

Retro-Transposons:

In addition to transposon, eukaryotic genomes contain transposable elements whose movement depends on the reverse transcription of RNA into DNA. This reversal in the flow of genetic information has led geneticists to call these elements retro-trans­posons, from a Latin prefix meaning “back­ward”.

There are two main classes of retro-trans­posons: the retrovirus- like elements and retroposons. The members of the first class resemble the chromosomes of a group of viruses that depend on reverse transcription for their propa­gation, and the members of the second-class have a structure reminiscent of poly-adenylated RNA.

Evolutionary and Genetical Significance:

Transposable elements have the following signi­ficance:

1. Evolutionary significance as nature’s loop of genetic engineering.

2. Cause mutation and chromosome breakage.

3. May he used as genetic markers.

4. Can be used as mutagens for inducing muta­tions.

5. For tagging desirable genes.

6. May be used as transformation vectors.

The evolutionary importance of transposon and insertion sequences is more and more being realised in recent times. The best example of evolutionary importance of transposable sequences has been brought up by the scientists Norwich group in pea. It has been shown that recessive wrinkled character of seeds of pea as used in Mendel’s experiment is due to the absence of branched starch in the wrinkled peas.

The dominant round seeded character in pea is due to the formation of branched starch which is synthesized by the enzyme SBE (starch bran­ching enzyme), that is absent in the recessive wrinkled type.

The molecular studies have shown that the recessive character is due to presence of 0.8 kilo base insert in the molecular frame of the normal branched starch producing enzyme which is about 3.2 kilo base long. As such the recessive wrinkled seed character is not due to absence of enzyme coding sequence but due to the distortion of the sequence.

The transposon is now extensively used in genetic manipulation in tagging desirable genes for mapping gene loci, as well as for inducing gene mutation. They have also been used as vectors to transform the organisms genetically.


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