Gene: Introduction, Concepts and Structure | Cell Biology

In this article we will discuss about Gene:- 1. Introduction to Gene 2. The Changing Concept of Gene 3. Fine Structure.

Introduction to Gene:

Mendel’s, (1865) experiments with Garden pea plant showed that certain hereditary “factors” were concerned in determining the appear­ance of certain morphological traits.

Such Mendelian “factors” were described as “gene” by Johanssen (1909) and these genes were shown to be present on chromosome as beads on string and this was the basis of the chromosome theory of heredity proposed (1902-1903) by Shutton and Boveri.

Thus on the basis of these classical observations, a gene was considered in the early days as a single, small and indivisible hereditary unit that occurred at a definite point on the chromosome and was responsible for a specific phenotypic character. As the knowledge of gene increased day by day in the subsequent studies, the classical concept about gene was changed and modified accordingly.

The Changing Concept of Gene:

The discovery of many phenomena like crossing over, gene-recombination and gene mutation have provided another set of information about gene. But recombination was not believed to occur only between the beads or genes.

Hence the gene was not considered sub-divisible. Thus a gene is considered to control the inheritance of one character, to be indivisible by recombination and to be the smallest unit capable of mutation.

It was soon realised that a gene, in true sense, is not responsible for the expression of one trait by itself, although it may exercise the major control on its development.

That genes express themselves through synthe­sis of enzyme was demonstrated for the first time in 1941 by G. W. Beadle and E. L. Tatum due to their discovery of biochemical mutations in Neurospora. Based on their work, Beadle and Tatum proposed a concept called “one gene-one enzyme” hypothesis.

Thus it became evident that a gene controlled a biochemical reaction by directing the production of a single enzyme. But it was also realised that one gene produces a single polypeptide and not one enzyme as the latter may consist of more than one polypeptide. Thus, the gene may now be defined as a segment of DNA which contains the information for a single polypeptide (the functional unit).

Each functional unit consists of a series of nucleotides that specifies the sequence of amino acid residue of polypeptide chain such as those of the A and B chains of the tryptophan synthetase enzyme or the α and β chains of haemoglobin. But it is shown that a change in as little as one nucleotide of the polypeptide specifying gene may mutate and produce a variant of the wild type chain that differs in one amino acid residue.

So the functional gene or unit is not the same as the mutational gene, but appears to consist of many mutable sites. The gene must also be considered from the standpoint of the nature of the sites of which recombination may occur.

The functional gene, therefore, appears to be composed of many mutational as well as re-combinational sub-units. The first evidence that the gene was sub-divisible by mutation and recombination came from studies of the X- linked lozenge locus of Drosophila melanogaster by C.P. Oliver in 1940.

Oliver demonstrated that crossing over oc­curred between two mutants such as alleles lzs and Izg of the sex linked lozenge locus of D. melanogaster at a low frequency of 0.2%. This was the first evidence for intragenic recom­bination.

According to classical concept a gene is not sub-divisible in that crossing over does not occur within a gene; it always occurs between two separate genes. But Oliver’s studies first indicated that the gene was, in fact, more complex than a bead on a string.

They were first steps towards the present concept of the gene as a long sequence of nucleotide pairs that is capable of mutating and recombining at many different sites along its length.

(i) Cis-Trans Test:

This is an indirect experimental evidence to prove that a gene is sub-divisible. The standard phenotype, i.e., parental form, without any mutation, is called wild type. The genes present in the wild type organism is generally designated by ‘+’ sign for comparison with mutant gene.

Before going to discuss the cis-trans test, it is reasonable to understand the meaning of cis and trans arrangement of gene. Cis arrangement means the condition in which a double heterozygote has received two linked mutations from one parent and their wild type alleles from the other parent, e.g., ab/ab x ++/.++-produces heterozygotes ab/++ (Fig. 15.1).

Trans arrangement means the condition in which a double heterozygote has received a mu­tant and a wild type allele from each parent— for example a+/a+x+b/-l-b produces a+/+b (Fig. 15.2).

In a cis-trans test the phenotypes produced in cis and trans heterozygotes for two mutant alleles are compared with each other. In a cis heterozygote, both mutant alleles are located in the same chromosome and their wild type alle­les are present in the homologous chromosome, i.e., mutant alleles are linked in the coupling phase.

Thus it is expected to produce the wild type phenotype (unless the mutant alleles are dominant or co-dominant) irrespective of whether the two mutant, alleles are located in the same gene or in two different genes.

On the other hand, in case of trans heterozygotes one, mutant alleles are located in the homologous chromosome—they are linked in the repulsion phase. Hence, in trans heterozygotes, it is expected to produce the mutant phenotype if the two alleles are located in the same gene. But if they are located in two different genes, the wild type phenotype would be produced.

Hence simply by comparing the phenotypes for any two mutant alleles it is possible to determine if they are located in the same gene or in two different genes.

They are located in the same gene if their cis heterozygotes produce the wild type phenotype, while their trans heterozygotes have the mutant phenotype. But if both their cis and trans-heterozygotes have the wild type phenotype they are considered to be located in two different genes.

(ii) Complementation Test:

The production of wild type phenotype in a trans-heterozygote for two mutant alleles is termed as complementation and such a study is known as complementation test. The results obtained from complementation tests are highly precise and reliable and they permit an oper­ational demarcation of gene.

Mutant alleles present in the same gene do not show com­plementation, while those located in different genes show complementation. Actually, this concept is generally true in prokaryotes but in eukaryotes several noteworthy exceptions are known.

The basis of complementation test (Fig. 15.3) may be simply described as follows. A gene produces its effect primarily by directing the production of an active enzyme or polypeptide. On the other hand, a mutant allele of this gene directs the production of an inactive form of the enzyme as a result of which it produces the mutant phenotype.

In the cis heterozygote, one of the two homologous chromosomes has the wild type allele(s) of the gene(s). This wild type allele will direct the synthesis of active enzyme—thereby producing the wild type phe­notype.

In trans heterozygotes, if the mutant alleles are present in the same gene, the enzyme molecules produced by them will be inactive and capable of producing only the mutant phe­notype. But if two mutant alleles are located in two different genes, one chromosome of trans heterozygote will have the wild type allele of the other gene.

Therefore, the trans heterozygote will have functional product of both the genes and the wild type phenotype will be produced by complementation. The complementation test has proven to be useful in delimiting genes.

But, in many cases, this test does not provide evidences to delimit gene.

These are:

a. Dominant or co-dominant mutation.

b. Genes in which mutations occur that show intragenic complementation.

c. Polar mutation, i.e., mutation that affects the expression of adjacent genes.

d. The gene in question does not produce a diffusible gene product, e.g., proteins.

Some other genes—such as operator and promotor genes which generally occur in the operon—do not code for a polypeptide or an enzyme. Hence they can act only in the cis position and they cannot show complementa­tion. Therefore, such genes are called ‘cis- acting gene.’

Fine Structure of Gene:

We have already discussed that there can be several sites in a gene, each capable of be­ing independently involved in mutational and re-combinational events. Therefore, a gene is neither a functional nor a re-combinational unit but is a complex locus whose fine structure should be studied.

The most extensive study on the fine structure of gene was undertaken by Seymour Benzer for a locus in T₄ bacteriophage infecting E. coli. This locus is known as rπ locus.

T4 bacteriophage contains a linear molecule of DNA of about 200,000 base pair long which is packed within its head (Fig. 15.4). When T₄ bacteriophage infects E. coli the bacterial cell lyses in about 20-25 minutes releasing 200-300 progeny phage particles.

When the inoculum of E. coli cells are plated in a petridish containing semi-solid nutrient medium, it will produce an uniform confluent growth or lawn on the surface of nutrient medium after certain period of incubation at the appropriate temperature [Fig. 15.5(a)], If the isolated T₄ bacteriophage particles are placed at different sites on the surface of bacterial lawns, T4 bacteriophage infect the bacterial cell and all the E. coli cells in the immediate surrounding vicinity of phage will be destroyed.

This leads to the development of a clear area in the bacterial lawn. The clear areas are called plaques which indicate the areas of infection and lysis of bacterial cell due to infection by phage and is characteristic of phage.

The plaques are surrounded by a fuzzy or turbid margin called halos which are produced due to a phenomenon called lysis inhibition [Fig. 15.5(b)], It is a delay in lysis of T₄ infected E. coli cells as a consequence of a subsequent infection by another T₄ particle. The ability of T₄ phage to cause lysis of bacterial cell is controlled by gene(s) present in a specific locus called ‘r’ locus (r = rapid lysis).

S. Benger isolated over thousands of inde­pendent mutant strains carrying mutation in the r locus (Fig. 15.6). Most of the r mutants map and classify into three distinct loci called r^Ir^II and r^III. The mutants can be recognised to some extent on the basis of the morphology of plaques, the ability of mutants to cause lysis of bacterial cell.

Mutants in the r^II locus are easily recognised due to their inability to multiply in E. coli strain K₁₂(λ) which has the chromosome of phage A integrated in its chromosome. How­ever, r^II mutants grow rapidly in other strains of E. coli such as strain B and strain K_i2 lacking the λ chromosome.

The wild phage T₄ r^II+ makes small and fuzzy plaques both on B and K strains, whereas the r^II mutants make large sharp plaques on E. coli strain B and K strains (Fig. 15.7). These distinguishable properties enabled Benzer to distinguish mutants and wild type phage with high efficiency. The r^II mutants axe conditional lethals unable to grow in K₁₂(λ); this property was exploited by Benzer for a fine genetic analysis of the r^II locus.

Benzer isolated over 3,000 independent mu­tants of the r^II locus and subjected them to complementation test. Phage carrying r^II mu­tation can be easily identified by sterile tooth­pick transfers of phage from individual plaques growing on E. coli strain B (r^II-permissive)

“Lawns” to lawn of E. coli strain K₁₂ (λ) (r^II restrictive) and lawns of E. coli strain B (Fig. 15.8). Each plaque to be tested (left side of Fig. 15.8) is stabled with a sterile toothpick which is subsequently touched to maxked axea in a petridish with a K₁₂(λ) lawn (in the center of Fig. 15.8) and then to an identically marked area in a dish with an E. coli B lawn (right side of Fig. 15.8).

Mutants that fail to grow (are lethal) on K₁₂(λ) (left side of the centre plate) can be recovered from the plaques on the E. coli B plates (right side of the Fig. 15.8).

If plaques develop on the E. coli B lawn, it indicates complementation between the two r^II mutants used for co-infection, while an absence of plaques signifies a lack of complementation. Mutants at the r^I and r^III loci as well as r⁺ phage (right side of the central plate) will grow on both K₁₂(λ) and B. Benzer placed all r^II mutants in two arbitrary groups named be A and B.

All the r” mutations were found to located in one of the two genes of cistron. Benzer designated these two genes r^IIA and r^IIB (Fig. 15.9).

The r^II A region appears to consist of about 2,000 deoxyribonucleotide pairs. The A region transcribes a messenger RNA that translates an A polypeptide; the B region is similarly responsible for a B polypeptide. B polypeptides are needed for lysis of K type E. coli cells. The wild type (r⁺) phages produces both A and B polypeptides. A mutant produces normal B polypeptide but not A, and vice versa.

Hence infection only by identical r^II A mutants or by identical r^II B mutant alone can cause lysis of the host cells, because none of the phages can produce both A and B polypeptide (Fig. 15.10).

On the other hand, infection by two different mutants (one an r^II A mutant and the other an r ^II B mutant) on the same host cell does result in lysis (Fig. 15.11). It indicates that regions A and B are functionally different and show complementation.

Benzer observed that with infection by two phages—one the wild type (r⁺) and the other mutant in either A or B region, i.e., with mutation in the cis position—lysis occurred. But the lysis did not occur when the mutation A or B were in the trans configuration.

Thus, it was clear that mutation in one functional region (A or B) Eire complementary only to mutations in the other region and complementation is detectable by cis-trans test.

Each functional region is responsible for the production of a given polypeptide chain. Benzer defined the functional unit as cistron and conformed op­erationally more closely to what we commonly think as gene. This cistron, therefore, may be thought of as the gene at the functional level. There can be over a hundred points within a functional unit wherein a mutation can take place and cause a detectable phenotypic effect.

This means that a cistron is over hundred nucleotide pairs in length and there is some evidence that some cistrons may be as long as 30,000 nucleotide pairs. Actually each cistron represents a part of a gene which is responsible for coding of only one polypeptide chain of an enzyme that has two or more different polypep­tide chains in its complete enzymatic unit.

A cistron also includes initiating, terminating and any un-transcribed nucleotides.

(a) The Muton:

It is the smallest unit of DNA which, when altered, can give rise to a mutation. Study of the genetic code makes it clear that an alteration of a single nucleotide pair in DNA may result in a missense codon in transcribed mRNA (e.g., AGC—>AGA) or nonsense (e.g., UGC—> UGA). So a cistron may be expected to consist of many mutable units or mutons. The term muton was given by Benzer.

(b) The Recon:

It is the smallest part of DNA which is inter­changeable through crossing over and recombi­nation. Extremely delicate studies of recombi­nation in E. coli indicate that a recon consists of not more than two pairs of nucleotides, may be only one.

A recon may occur within a cistron. Thus a gene of classical concept is made up of a number of functional units—the cistrons— which consist of a number of recons and mutons (Fig. 15.12).

(i) Recombination Frequency:

The complementation test shows that all the r^II mutants were located within A and B cistrons. In order to estimate the frequency of recom­bination between r^II mutants, E. coli strains B cells are infected with a mixture of the two r^II mutants.

If the crossing occurs between two chromosomes of mutant strain it yields one wild type and one double mutant type for each crossing over event (Fig. 15.13). Therefore, some of the progeny phage present in the lysate of the B strain (infected by a mixture of two r^II mutant) would be of wild type.

The frequency of the wild type phage in the lysate is determined by plating lysate on the lawn of K₁₂(λ) strain. Each wild type phage would produce a plaque on this lawn. This is a highly efficient selection system for wild type phage and as many as 10® progeny phage may be examined in a single petridish.

The number of plaques produce on Kj₂(λ) represents the number of wild type phage particles in the lysate. A equal number of phage would have the double mutant produced due to recombination. Therefore, the number of recombination phage in the lysate would be twice the number of plaques produced on Ki₂(λ).

Thus, the frequency of recombination may be measured as follows:

However, to map 3,000 mutations by only standard recombination test is a highly labo­rious task and is practically impossible because the number of all possible two-point crosses only (infection of E. coli cells by mixture of two mutants at a time) will be about 4 ½ illion, i.e., 3,000 x 2,999/2.

Hence Benzer was able to avoid such a laborious undertaking by developing a shortcut method of mapping that used overlapping deletion mutation. This technique is known as deletion mapping. It permits the deletion of recombination value of 0.0001 or even 0.00001%.

(ii) Deletion Mapping:

Benzer first mapped a number of r^II mutants using the data of recombination test. He noted that some of these mutants did not show recom­bination with some other r^II mutants. These mutants also failed to undergo reverse muta­tion, i.e., mutation to wild type r⁺.

Benzer classified these non-reverting, non-recombining r^II mutations as deletion mutation. Benzer also proposed that these deletion mutations (multisite mutations) resulted from the deletion or loss of segments of DNA. These deletions were arranged in sets of overlapping deletions representing segments of different sizes in r^II regions as shown in Fig. 15.14.

The principle involved in this method was that if a particular mutation presents in the region of a deletion represented by a r^II mutant, then, on mixed infection with this deletion mutant, the point mutation will not be able to give rise to wild type, but if it falls outside the deletion regions it will be able to give rise to wild type and recombinant type.

The extents of the deleted segments can be analysed by crossing the deletion mutants to a set of reference point mutations which are previously mapped. Once a set of overlap­ping deletion has been mapped, their end- point will divide the region resolved by the longest deletion in a set of intervals A, B, C, D (Fig. 15.15).

When an unknown new mutant carrying a point mutation is isolated, the mutant can immediately be mapped to a defined interval by crossing the mutant with each of the overlapping deletion mutants. A mutant in interval D will not produce any wild type recombinant progeny in any of the four crosses. A mutation in interval C will recombine with deletion IV (Fig. 15.15) but not with the other three deletions, and so on.

In this manner, Benzer characterised with deleted segments of a large number of r^II deletion mutants. This permitted him to divide the entire r^II locus into 47 small segments (Fig. 15.16). A set of seven of these deletion mutants permitted him to divide the r ^II locus into 7 regions like A₁ – A₆ and B.

Each new r ^II mutant to be mapped was crossed pairwise with each of these seven deletion mutants and the presence of wild type (recombinants) phage particles counted in the progeny.

On the basis of this data a new r^II mutant was localised in one of the- seven segments. (Table 15.1 and Fig. 15.17.) Once an unknown r^II mutant is pointed in a segment, it is crossed to another set of deletion mutant’s which allows its locali­sation in a smaller sub-division of that segment.

The final mapping of r^II mutants is done on the basis of recombination data from two and point crosses among mutants located within the concerned subsection of the r^II locus. Benzer et al identified more than 300 sites of mutation that were separable by recombination. The progeny of mutation at different sites is highly variable.

Electron Microscope Heteroduplex Mapping:

The presence of genetically well-defined dele­tion mutations at the r^II locus can also be de­termined using a technique called heteroduplex mapping. A DNA heteroduplex is a DNA molecule in which the two strands are not complementary.

One strand of a DNA double helix may contain one allele of the gene and the other strand may not be totally complementary and may carry of different alleles of the gene. The non complementary portions of DNA then form a heteroduplex which may vary in size from one mismatched base pair to large segments of the molecule.

Heteroduplex mapping involves in vitro preparation of DNA hetero-duplexes and their analysis by electron microscope. The heteroduplex may be prepared by mixing the denatured single-stranded DNA segments of wild type and mutant type followed by DNA renaturation.

The prepared hetero-duplexes between DNA from T₄r⁺ phage and DNA from each of several genetically well characterised r^II deletion mutants. Thereafter they are analysed by electron microscope.

The results obtained estimates of 1,800±70 nucleotide pairs and 845 ± 50 nucleotide-pairs for the sizes of the r^II A and r^II B genes, respectively. These results combined with the extensive genetic data of Benzer et al provide a fairly clear picture of the fine structure of the r^II locus.

(c) Overlapping Genes:

The presence of overlapping gene provides an interesting information for the study of fine structure of a gene. It is generally accepted that the boundaries of neighbouring genes do not overlap.

The study of nucleotide sequences of φ x 174 bacteriophage has clearly resolved that out of total 10 genes of φ x 174, two are located entirely within the coding sequences of two different genes. A third overlaps the sequences of three different genes. This surprising result has important genetic implication for the study of fine structure of gene.

(d) Fine Structure of Genes in Eukaryotes:

Complementation and recombination study have been used to prepare fine structure maps of several eukaryotic genes. By this technique, genetic fine structure maps have now been constructed for many genes of Drosophila; maps have also been worked out for several other higher animals and higher plants.

One of the best examples of such a gene is the rosy (ry) eye locus of Drosophila which codes for the enzyme xanthine dehydrogenase. The different alleles of ry locus map at 10 different sites on the basis of recombination frequency (Fig. 15.18).

Many of the ry mutants do not show complementation (shown in upper line of the Fig. 18.18) while several others show complementary (shown in the lower line of the figure). The complementary allele may be located at the same site or at a site very close to one where non-complementary alleles are located.

The complementation of ry alleles is a case of intragenic complementation. The recovery of wild type recombination is very easy because rosy mutants are conditional lethals. As a result, wild type recombination produced from the heterozygotes for ry² ry³ alleles can be easily isolated and counted by growing their progeny on a purine supplemented medium, on this medium only wild type progeny would survive.

Besides rosy locus, fine structure maps of gene of many other eukaryotes have been prepared like white (w) eye, notch (N) wing, lozenge (lz) eye, zetse (another eye colour locus near the white locus) etc. loci of Drosophila, waxy (wx) and other loci of maize, some loci of yeast.

Analysis and mapping of eukaryotic gene have also got some limitations due to:

i. Examining enough progeny of a cross to detect rare intragenic recombination in eukaryotes is a laborious job.

ii. In many cases, determining how many genes are present at a locus has proven difficult in eukaryotes. This problem arises due to the presence of complex loci.

iii. Complementation tests have often yielded ambiguous results—due to the occurrence of intragenic complementation. Delimiting genes of eukaryotes by complementation test should be done, whenever possible, using amorphic or null mutation (mutation resulting in no gene product) to minimize the possibility of confounding effects of intragenic complementation.

In eukary­otes, however, some genes have interesting structural features which are not found in most prokaryotes.

Therefore, our view of fine structure of any gene as discussed earlier may be partly ambiguous due to use of a specific recombination system. Further, the distances between genes on a genetic map may not correspond to the distances between them in the DNA molecule of which they are a part at the molecular level. There may also be present gaps or a genetic map due to non-availability of mutants in that region.

At the molecular level the fine structure of a gene can be resolved by modern genetic mapping through determination of nucleotide sequence of the concerned DNA segment. Alternatively we can prepare a genetic map by breaking the DNA at specific sites with the help of restriction endonucleases which are specific in recognising very short DNA sequences and cutting the DNA at these specific sites.

These sites of breakage can be identified and mapped in eukaryotes. This modern technique for in prokaryotes to give rise to a restriction genetic mapping at the molecular level has been map.