Crossing-Over: Meaning, Mechanism and Significance

In this article we will discuss about:- 1. Meaning of Crossing-Over 2. Example of Crossing Over in Drosophila 3. Theories 4. Mechanism 5. Kinds 6. Percentage 7. Cytological Evidence 8. Factors 9. Cytological Evidence 10. Significance 11. Interference and Coincidence.

Meaning of Crossing-Over:

The phenomenon of complete linkage occurs rarely since the linked genes tend to separate during meiotic divisions. This mechanism of the genes as a result of interchange of chromosomal segments was termed crossing over by Morgan.

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Crossing over may be defined as an interchange of chromosomal parts between non-sister chromatids of a homologous pair of chromosomes resulting in the recombination of genes at Meiosis Prophase I, diplotene stage.

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The exchange of segments between the inner situated chromatids of homologous chromosomes is called crossing over.

The exchange of homologous segments with perfect correctness between non-sister chromatids of homologous chromosomes, responsible for recombination between linked genes is said as crossing over. It takes place during diplotene after the homologous chromosomes have undergone four-strand (4-chromatids) stage.

This produces a cross (X) like figure at the point of exchange of the chromatid segments; this figure is called chiasma, (Janssen (1909). Crossing over may take place at several points in one tetrad, resulting in the formation of several chiasmata whose number varies with the length of the chromosomes. It should be realized that chiasma is the result and not the cause of crossing over.

Although chiasma formation has been studied by several workers, detail of this process is till not definitely known. The cause leading to the breakage of the chromatids and their union, are not clear. According to recent findings chromosomal breakage and reunion may be the result of enzymatic action. The enzyme endonuclease brings about breakage and ligase is responsible for reunion.

Example of Crossing Over in Drosophila:

In Drosophila the factors for pink eyes (r) and curled wings (c) are recessive, while red eyes (R) and straight wings (C) are dominant. If a pure breeding fly with red eyes and straight wings is mated with another fly having pink eyes and curled wings, all the F₁ hybrids are red eyed and straight winged.

When a female from these hybrids is back crossed with a male having pink eyes and curled wings, the F₂ generation consists of 49% with red eyes and straight wings. 49% with pink eyes and curled wings 1% with red eyes and curled wings and 1% with pink eyes and straight wings. The middle two classes represent the cross-over or recombined types.

Both the factors for each allelomorphic pair, in this case, are situated in the same chromosome. If they were in different chromosomes, four classes of individuals would have appeared in equal numbers after the back cross. On the other hand, if they were completely linked, only the two grand parental types would have appeared in the F₂.

Theories of Chromosomal Crossing Over:

1. The ‘Contact First’ Theory:

This theory was proposed by Serebrovsky. It states that two chromatids (non-sister chromatids of inner side) first contact and then cross each other. The breakage comes at the point of contact and broken segments reunite to form new combination.

2. The ‘Breakage First’ Theory:

This theory was proposed by Muller. According to him, two chromosomes (inner non-sister chromatids) first break in to two segments without crossing over and then broken segments reunite with each other forming new arrangement and resulting in a new combination. This theory is widely accepted one.

3. Strain Theory (Precosity theory):

According to Darlington, chromosomal breakage occurs as a result of strain or tension during pairing. When two chromosomes pair and become spirally twisted around each other termed as relational coiling there develops a sort of tension on chromatids as a result of which they become broken at the point of contact and recombination occurs. Broken ends further rejoin but not compulsorily with the same segment from which they were detached.

When broken ends of different chromosomes get joined, an exchange in genetic material occurs. Darlington claims that it is the only chiasmata which hold the broken chromatids together as tested with Meiosis I anaphase. Thus, this theory correlates close pairing between homologous chromosomes and crossing over.

If there is no pairing, both homologous will not form or align in a line themselves and will go the same pole causing non disjunction i.e., pairing is necessary for disjunction or separation.

Recently Grell (1964) includes two more types i.e., exchange pairing and distributive pairing in Darlington theory. In ‘exchange pairing’ homologous chromosomes pair and later crossing over occurs but, distributive pairing is unaccompanied by homologous pairing and crossing over.

4. Belling’s Hypothesis:

According to this hypothesis, crossing over is not caused by breakage and reunion of chromosomes but by newly duplicated genes. Belling (1933) proposed that crossing over is the result of an exchange between new chromatids during the period of their formation.

According to this cytologist new chromomeres are formed first along side their respective sister chromosomes and the interconnecting fibers are formed later. If a relational coil exists between the homologues, the interconnecting fibres may some times be formed between non- sister chromomeres, thus producing a cross-over.

Mechanism of Crossing Over:

1. Classical Theory:

This theory was proposed and advanced by Morgan and Sharp (1934) respectively, also called two-plane theory because it is assumed that adjacent loops would be present in different planes at right angles to each other. According to this theory, chiasma formation occurs when a chromatid of a chromosome comes to associate with the non-sister chromatid of a chromosome.

Generally the sister chromatids of the two chromosome of a bivalent remain attached with each other during synapsis. But in many divisions the sister chromatids separate from each other and become attached with non-sister chromatids of the homologous chromosomes thus producing chiasma.

During diplotene when the homologous chromosomes begin to separate from each other, the chromatids involved in chiasma formation are subjected to physical tension or strain (due to equational separation and reductional separation) at the point of chiasma. This may cause breakage of the two chromatid at this point, a reunion between the chromatids segments thus produced would lead to crossing over or recombination between linked genes.

According to classical theory:

(i) A chiasma is formed after non-sister chromatids of homologous chromosomes become attached during synapsis.

(ii) Chiasma formation is not the result but cause of the crossing over.

(iii) Each chiasma does not lead the phenomenon of recombination or crossing over.

The above available experimental findings do not support this hypothesis and it is of only a historical significance. This theory, however, now stands rejected.

2. Chiasma Type Theory (Breakage & reunion theory):

It was firstly proposed by Janssens (1909) and later on expanded by him in 1924. Further, fully developed by Belling and Darlington. This is also said as one plane theory because in this theory, one would expect reductional separation of chromatids on either sides of a chiasma.

According to this theory, breakage in non-sister chromatids of homologous chromosomes, followed by the reunion of the chromatid segments resulting in crossing over. When the homologous chromosomes begin to move away from each other during diplotene, chiasmata are formed at those points where crossing over has taken place.

Thus (i) chiasma is the result of crossing over.

(ii) Only sister chromatids are attached with each other through out the whole bivalent, where as non-sister chromatids become associated to produce chiasmata

(iii) Each chiasma is the consequence of a crossing over event, therefore

(iv) 1:1 ratio is expected between the numbering of frequencies of chiasma and crossing over.

Almost all the available evidence supports the chiasma type theory. Beadle (1932), Brown and Zohary (19-55) have strongly supported this theory since there is one to one correspondence between chiasmata and genetic crossing over explaining structure of gene maps and frequencies etc.

However, Kaufmann (1934) and Cooper (1949) objects this theory since chiasmata are also formed in some tissues of male Drosophila. Because genetic crossing over is lacking in male Drosophila, these chiasmata can not be explained by chiasma type theory. Thus, chiasma type theory seems to be a universally accepted one.

(3) Copy-Choice Theory:

This theory was proposed by J. Ledeberg (1955), according to this hypothesis, the duplication process and recombination occur simultaneously. In other words the paired chromatids duplicate by synthesizing new genes. Then it is followed by the development of new connection between these genes. Thus the recombination’s are produced by newly synthesised genes.

There are mainly two objections—one is that only two of the four chromatids are involved in crossing over. Thus two original strands remain intact while two newly formed strands would be altered by recombination. Secondly, this theory states that duplication should occur during late meiotic prophase but now evidence indicates that DNA replication occurs even before synapsis.

Kinds of Crossing Over:

Crossing over may be single, multiple depending upon the number of chiasmata present in the chromosomes as follows:

1. Single Crossing Over:

When there occurs only one chiasma or crossing over at one point in chromosome pair then it is referred to as single crossing over. The gametes which are produced by this crossing over are said as single cross-over gametes.

2. Double Crossing Over:

Occasionally or some times crossing over occurs at two points in the same chromosome pair. This is said as double crossing over. The gametes produced by this crossing over are called double cross-overs. It occurs seldom than single crossing over.

3. Multiple Crossing Over:

Crossing over may also occur at three, four or more points in the same chromosomes pair and correspondingly said as triple, quadruple, or multiple crossing over.

The Percentage of Crossing Over:

The crossing over may vary depending upon the genes and their locations. The chance of crossing over are greater over long distances than over short distances.

In the figure it is quite clear that there are more chances of crossing over between A and C than between A & B i.e., percentage of crossing over of two genes is related with the distance between those 2 genes. If these genes will be near, crossing over will be minimum and likewise if they will be located at distant places, the crossing over will be greater.

Therefore, the percentage of crossing over is directly proportional to the distances of genes between which crossing over occurs. This is of particular importance for drawing the chromosome maps.

The percentage of crossing over may be taken as indication of the comparative linear distance between any two required genes. The unit of crossing over was termed morgan by Haldane and adapted by Crew and others. Suppose there is 15% crossing over between any two genes then these genes are 15 ‘morgans’ along the length of chromosomes.

Somatic Crossing Over:

Pairing in the somatic cells followed by crossing over was discovered by Stern (1931) in Drosophila, Jones in zea mays and G. Pontecovor et al in Aspergillus. In most organisms the meiotic division is restricted to the organs concerned with the production of gametes in which pairing of chromosomes occur in germ cells. Stern found that this pairing of chromosomes may also come in other vegetative tissues having somatic cells.

In Drosophila this crossing over exceptionally produced a patch or spot of cross-over tissues in certain parts, while other body parts constitute non-cross-over tissues. Thus, fly will be mosaic for cross- over and non-cross over tissues. Such types of crossing over in somatic cells is referred to as somatic crossing over.

Cytological Evidence of Crossing Over:

Morgan and his collaborators established the genetic basis of crossing over and linkage describing the exchange of parts between the homologous chromosomes and linear arrangement of linked genes along chromosomes. This genetic inclination or bias could not be demonstrated cytologically since we can not observe the homologous chromosomes (being all identical) under the microscope because of the following reasons.

(i) Crossing over occurs between homologous chromosomes. Such chromosomes are alike in appearance and it is not possible to distinguish between them in microscope.

(ii) During crossing over, the four chromatids are intimately coiled around one another.

(iii) In living cells, crossing over can not be seen. In fixed and stained cells one can not say that chromatids have exchanged parts or not.

For nearly twenty years crossing over remained only a working hypothesis with geneticists. Finally the cytological evidence which established beyond doubt the occurrence of crossing over, was given by S. Stern on Drosophila and H.B. Creigton and B.Mc Clintock on maize.

(1) Stern’s Experiments or Drosophila:

Stern discovered a variety of Drosophila in which a part of Y chromosome had broken off and became attached to the end of one of the X-chromosome. Likewise, he described another variety in which one of the X-chromosomes was broken.

Usually in Drosophila normal fly has red round (++) eyes. Two mutant genes, one carnation (car) causing darkish red eyes and being recessive to red (+)eye colour and other bar (B) causing narrow eyes and dominant to round (+) eyes are both in X chromosome. The female fly is XX and male has XY chromosome.

In female (XX), one X chromosome was broken in to two by X-ray or other means and contained mutant genes car and B. The other X contains normal allele (+ for red and + for round eye i.e., normal) and its end a segment of Y chromosome was attached. This breaking of X chromosome and attaching Y segment is a plan or scheme to distinguish between cross-overs and non-cross-overs progeny.

The carnation barred female (car B+ +) produces four types of gametes, Out of which two are cross-overs and two non-cross overs formed without interchange of homologous segments of chromosomes. When these were mated with males (XY) having carnation round eyes (car +), the non-cross-overs are carnation bar and red round while cross-over contain red bar and carnation round.

Thus, cytological basis of crossing over can be established by distinguishing chromosomes under microscope. (Stern’s experiment was an unique demonstration of the hypothesis that interchange of chromosomal material takes place between homologous chromosomes).

2. Creighton and Mc Clintock’s Experiments on Maize:

Similar demonstration of cytological crossing over was demonstrated in maize by these workers. They observed a corn plant which had a pair of chromosomes whose two members could be held apart cytologically. Among them, one was normal and another had a trans-located piece of another chromosome at one end. The other end was like a knob (hard round protuberance).

The normal chromosome carried ‘c’ for colourless endosperm and W for starchy endosperm. Other knobbed chromosome had alleles ‘C’ for coloured and ‘w’ for waxy endosperm. Creighton and Mc Clintock crossed this plant with a plant having homologous chromosome with recessive genes i.e., colourless waxy ‘ccww’.

As a gametogenesis, two non cross-overs and two cross-overs gametes will be formed which after union will form four kinds of offsprings.

The non-crossover plants i.e., colourless starchy and colourless waxy obtained from parent either knobbed chromosome or normal but cross-over plants had one chromosome of this particular pair i.e., colourless waxy (ccww) contained a chromosome with trans-located piece but no knob, where as coloured starchy (CcWw) showed knobbed chromosome but no trans-located piece.

Thus cross-overs showed cytological evidence of crossing over i.e., exchange of homologous chromosome parts during maturation of germ cells.

Factors affecting Crossing Over:

There are various genetical, physiological and environmental factors which affect the frequency of crossing over i.e., promotes or suppresses the percentage of crossing over between 2 loci.

1. Sex:

It has been observed in male Drosophila that the crossing over is completely suppressed or masked and there is also tendency of reduction of crossing over in male mammals. In silk-moth (Bombyx) crossing over does not occur in females. Thus, the sex of an individual may also affect the frequency.

It was demonstrated that X-rays may bring about definite amount of crossing over as in male Drosophila. In general, it has been found that the heterogametic sex shows comparatively lower crossing over frequencies than the homogametic sex of the same species.

2. Mutation:

Gene mutation may affect the frequency of crossing over. Gowen has found a mutation in Drosophila which reduces the percentage of crossing over in all chromosomes.

3. Age:

The age of the individual may also affect the frequency of crossing over. Bridges already found in Drosophila that as the female becomes older, the crossing over tends to decrease.

4. Inversion:

These are intra-segmental changes within the chromosomes. In a given segment of chromosomes crossing over is reduced due to inversion. The part of the chromosomes which is not involved in inversion, the frequency of crossing over increases. The cause for this is still not clear.

5. X—radiation:

Hanson showed that irradiations by radium also increase the frequency of crossing over. Muller also found increase in the frequency of crossing over after the application of X-rays.

6. Temperature:

According to Plough, low and high temperature increase the frequency of crossing over. Example is female Drosophila.

7. Centromere vicinity:

It has been observed that near centromeres and at the tips of chromosomes, crossing over is frequent. Some factors not clearly understood influence crossing over in some somatic cells also that is said as somatic or mitotic crossing over. It is, however, known that certain mutant genes called minute or small bristles increase the frequency of somatic crossing over since a single cell gives rise to a mass of somatic cells.

It follows that a somatic cell in which crossing over occurs, gives rise only to a patch of cross-over tissue. Further, such somatic cells do not produce gametes. This type of crossing over has no genetic importance. Somatic crossing over was first of all seen & studied by Stern in Drosophila. It should be borne in the mind that factors which affect crossing over, effect linkage inversely.

Significance of Crossing Over:

(1) It has a great significance in genetics. Crossing over, a wide spread phenomenon provides a direct evidence of the linier arrangement of genes. Construction of chromosome maps and tracing linkage groups has been greatly facilitated by data obtained from the study of crossing over.

(2) It increases the frequency of variation which are vital for evolution. It causes formation of many combinations which can be acted by natural selection. The established linkage groups and linier order of genes gives much light on the nature and mechanism of genes.

Chromosome Maps:

The chromosome maps represent the condensed graphic representation of the relative distance of the genes in a linkage group, expressed in the percentage recombination located and single group of chromosome. From the examples we have studied linkage and crossing over, it can be said that linkage can be taken as an exception to the second law of Mendel, and the characters of an organism are due to genes located in the chromosomes.

Moreover, genes are believed to occur in a number of linkage groups. The linkage groups correspond to the number of chromosomes. The linkage group in Drosophila melanogaster are four in number and there are four pairs of chromosomes in that fly.

In Morgan’s hypothesis of crossing over it has been assumed that the genes had a linier arrangement in the chromosome. It was also thought that the distances between the two genes on the chromosome is correlated with the amount of crossing over shown by two corresponding alleles. The percentage of crossing over is directly proportional to the distance of alleles showing cross in the chromosomes.

These two facts made to represent in the form of a map which represents that:

(i) The genes are arranged in a linier row along the chromosome.

(ii) The percentage of crossing over between two genes is proportional to their distance apart.

Thus, chromosome map may be defined as a line, number of lines being equal to linkage groups on which genes are represented by points showing particular traits or characters proportional to the amount of crossing over.

These chromosome maps are also referred to as cross-over maps since they are sketched by the amount of crossing over.

The percentage of crossing over is calculated by test crosses. In mapping genes a unit of distance is used. This unit used is one percent of crossing over called as Map unit or morgan. The crossing over between linked genes may be as little as 1/10 of 1% or up to 50% depending upon their kinds.

The first two chromosome maps were made in 1911 by Sturtevant and Bridges. Later in 1920, Morgan and his associates worked extensively on Drosophila and constructed chromosome maps. Then these maps were made in maize, chicks, tomato etc.

Location of Genes:

In fixing the exact position of a gene on the chromosome map, the cross-over frequency of a gene in relation to another is the criterian. The procedure adopted for constructing a chromosome map can be explained with reference to a three point test cross. A three point test cross is one in which the F₁ resulting from a cross involving three linked genes is back crossed to a triple recessive.

In Drosophila the three genes scute (sc), echinus (ec) and crossveinless (cv) are sex linked genes. Scute is condition in which many body bristles are missing, echinus means rough eyes and crossveinless indicates absence of cross veins on the wings, since these are recessive mutation, the F₁ females resulting from a cross between these recessive types and wild type resembles the wild type phenotypically. This is because the females drive one sex chromosome from their mother.

When the F₁ females are back crossed to triple recessive males, eight phenotypes are obtained as given below:

In constructing the chromosome map the distance between the two linked genes is indicated by their cross-over frequency i.e., percentage. Since the cross-over frequency of scute in relation to echinus is 7.6%, these two genes are marked 7.6% unit apart. Again the cross-over frequency between echinus and crossveinless is 10.1%; these 2 genes are 10.1 unit apart.

In order to determine the sequence of the three genes it is necessary to find out the cross-over frequency between scute and crossveinless. The genes scute and crossveinless and the wild type alleles were introduced in to the cross by the same parent, one of these is present in the progeny without the other in 352 (151+201) cases.

To these figures the two double cross-overs may be added. The total number of cross-overs between scute and crossveinless is thus 354 or 17.7%. This is the sum and not the difference between 7.6% and 10.1. Therefore, the gene crossveinless lies beyond echinus; it means sequence is sc, ec and cv.

Interference and Coincidence:

Besides single crossing over, having only one chiasma, there may be double or multiple crossing over. It has been discovered by H.J.Muller (1911) that when there are two double cross-overs (suppose a and b) then one cross-over (a) tries to prevent the formation of other cross over (b) This tendency of one cross-over to interfere with the other cross over is termed as interference. Suppose frequency of ‘a’ crossover is 10 and frequency of ‘b’ cross-over is 12, then their total frequency will not be 10+12 = 22 as required but will be less than 22 due to interference.

When the two things happen the same time and at the same place, they then coincide or intermix and this occurrence may be considered coincidence. This coincidence refers to the occurrence of two or more distinct cross-over (double or multiple) at about the same time in the same chromosomal region. Double cross-overs are the result of coming together (coincidence) of two single cross-overs.

When doubles occur in regular expected ratio, coincidence is said to be 100%, where as interference will be nil. But when there are no doubles (coincidence) the interference is nil i.e., coincidence is inversely proportional to the interference.

According to Muller (1916) the coefficient of coincidence is the ratio between the observed and expected frequencies of double cross overs.Coefficient of coincidence = Actual number of double cross-overs/Expected number of double cross-overs.