Crossing over is the process of exchange of genetic material or segments between non-sister chromatids of two homologous chromosomes. Crossing over occurs due to the interchange of sections of homologous chromosomes.
Meaning of Crossing Over:
If linkage is complete, there should be all parental combinations only and no recombination. But actually there is no absolute linkage, thus allowing for some recombination. How does recombination take place? At the beginning of the 20th century, Janssens had made cytological observations on meiotic chromosomes in salamanders.
He found that chromosomes showed cross-shaped configurations and suggested that they represented a break and exchange of chromosome segments. A few years later, Morgan supplemented his genetical studies on Drosophila with cytological observations and explained linkage on the basis of breakage and exchange in synapsed chromosomes.
He could thus account for the greater frequency of parental combinations and also why linkage was not absolute so that recombinant types occurred in F2 progeny. Morgan termed the cross-shaped configuration observed by Janssens as chiasma. The term crossing over referred to the actual exchange of segments between homologous chromosomes and could take place due to breakage and reunion in the paired homologues.
Recombination is a genetic outcome of breakage and exchange of segments. It cannot be observed cytologically, but can by inferred genetically from experiments. Crossing over is the process of exchange of genetic segments which cannot be observed cytologically but can be estimated genetically from the frequency of recombinants in the F2 progeny.
Mechanism of crossing-over:
Mechanism of crossing-over can be explained under the following heads:
The homologous chromosomes pair lengthwise due to a force of mutual attraction in zygote of prophase-I in meiosis. The pairing starts at one or more points and proceeds along the whole length in a zipper fashion. The process of pairing is called synapsis. The paired homologous chromosomes are called bivalents. During synapsis, a molecular scaffold called synaptonemal complex aligns the DNA molecule of two homologous chromosomes side by side.
(ii) Duplication of chromosomes:
Synapsis is followed by the duplication of chromosomes which changes the bivalent nature of chromosome to four- stranded stage or tetravalent. Four stranded stage of chromatids occurs due to splitting of homologous chromosomes into sister chromatids attached with un-splitted centromeres.
Fig. Crossing over at 4-stranded stage results in 50% recombinant and 50% parental types of gametes.
Every pair of homologous chromosomes usually forms at least one chiasma somewhere along its length. There is a characteristic average number of chiasmata for each type of chromosome. In general, the longer the chromosome the greater the number of chiasmata. Moreover, the further apart two genes are located on a chromosome, the greater the chance for a chiasma to occur between them.
The percentage of crossover (recombinant) gametes formed by a given genotype indicates the frequency of chiasma formation between the genes in question. When chiasma forms in one cell between two gene loci, only half of meiotic products will be of crossover type. Therefore, chiasma frequency is twice the frequency of crossover products.
Chiasma % = 2 x crossover %
Crossover % = 1/2 Chiasma %
In other words, if chiasma forms between the loci of genes A and B in 30 % of the tetrads (paired homologous chromosomes) of an individual of genotype AB/ab, then 15% of the gametes will be recombinant (Ab or aB), and 85% will be parental (AB or ab). Further, the map distance between A and B would be 15 units apart.
It is noteworthy that the Drosophila male shows complete linkage due to absence of crossing over. In the cross between a normal red-eyed long-winged fly and purple vestigial, the F1 hybrids are all red-eyed and long-winged.
If heterozygotes from the F1 progeny are used as male parents and backcrossed with purple vestigial females, only two phenotypes appear in the progeny: the homozygous purple vestigial and the heterozygous red-eyed long-winged.
The re-combinations are absent demonstrating absence of cross over and presence of complete linkage in Drosophila male. If a reciprocal testcross is performed using F1 heterozygotes as females and purple vestigial as male, recombinants appear in the progeny.
Frequency of Crossing Over:
In experiments on linkage, the proportions of parental phenotypes and the new combinations can be counted. From the percentage of recombinants the amount of crossing over can be calculated.
In the cross between purple long and red round sweet peas described earlier, the sum of the new combinations (106 + 117) = 223 when divided by total progeny (1528 + 106 + 117 + 381) = 2132 and multiplied by 100 indicates 10.4% recombination or frequency of crossing over in F1 gametes. In this cross the parental types equal 89.6%.
The fact that recombinants occurred in F2 indicates that the distance between genes for flower colour and pollen shape allowed crossing over to take place between one parental chromosome and its homologue from the other parent.
Now if distance between two linked genes is more, there are greater chances of chiasma formation between them resulting in higher percentage of recombinants in the progeny. Contrarily, if distance between linked genes is less, chiasma formation between them will be less, and corresponding reduction in the number of recombinants in the progeny.
Thus it is possible to locate genes on chromosomes on the basis of their crossover frequency. The distance between genes is measured in map units. According to Sturtevant one map unit is equal to 1% crossing over. In other words, if one gamete out of 100 gametes carries a crossover chromosome for two linked genes, we say that the two genes are one map unit apart.
The correlation between crossing over and distances between genes may not be true for all genes on a chromosome. This is because chiasma formation does not occur at random throughout the length of a chromosome. It occurs with different frequency in different parts of the chromosome.
In the two large chromosomes of Drosophila there is less crossing over near the centromere and towards the ends, but more in the middle of the two arms. This would suggest that genes near the centromere are closer when actually they are further apart than the crossover percentage indicates. After determining positions of several linked genes in Drosophila, Morgan hypothesized that genes occurred in linear order along the length of the chromosome. The position of each gene was called the locus.
The Three-Point Cross:
When two genes are mapped by performing a cross, it is called a two-point cross. The three- point cross Involves three pairs of linked genes, is a valuable method for determining positions of genes in relation to each other and for mapping distances between genes.
In 1926, Bridges and Olbrycht used this method for mapping three recessive sex-linked genes in Drosophila: scute sc (without bristles), echinus ec (rough eyes), and crossveinless wings cv (absence of transverse veins). The cross involved mating of a scute crossveinless fly with an echinus fly, and then testcrossing female F1 heterozygotes with the recessive hemizygous male showing all three recessive phenotypes.
In the data of Bridges and Olbrycht clearly the first group of progeny represents the parental combinations, and the second two groups and are re-combinations.
Since chiasma formation takes place between linked genes, in order to determine crossover percentage in a three point cross, the genes must be analysed two at a time, ignoring the third gene each time. Thus we can examine three combinations: cv – ec, cv – sc, and ec – sc in each group of progeny. Considering genes cv – ec, these are found to be present in progeny of the first group in the same way as in the parent.
That is cv is present in its normal wild form (+) on one homologue along with ec; in the other homologue cv is present along with the wild form of ec. Thus in the first group of progeny cv and ec are present as cv + and ec +. Since this is the way they are present in the parent, we can infer that there is no recombination between cv and ec.
The second group of progeny again shows cv + on one homologue and ec + on the other as in the parents. Therefore, the second group of progeny also does not represent recombination between genes cv and ec. In the third group of progeny both cv and ec are present on one homologous chromosome whereas the other homologue has wild forms (+ +) of both cv and ec. This arrangement of cv and ec i.e., + and + is different from that in the parental combination and has arisen due to crossing over between the loci for cv and ec.
The data in Fig. 8.4 shows that there were 192 flies in the third group of progeny. We can calculate the percentage recombination between cv and ec by dividing 192 by the total progeny (1638 + 150 + 192 = 1980) and multiplying by 100. The amount of crossing over between cv and ec is thus found to be 9.7%.
Let us now consider genes sc and cv. In the first group of progeny they are present as in the parental combinations, that is ++ on one chromosome and as sc cv on the other. In the second group of progeny their arrangements are different, sc + being together in one chromosome and cv + together on the other homologue.
Their changed positions indicate recombination between sc and cv. In the third group of progeny also sc and cv are present differently than in parental combinations. Thus all the 150 members of second group of progeny and 192 of the third group represent recombination between sc and cv. The recombination percentage between sc and cv is calculated as described, i.e.,
(150 + 192 x 100)/1980 = 17.3%
Analysing the genes ec-sc in the same way we find that in the first group of progeny they are present as in the parental combinations. In the second group they are present as ++ on one chromosome, and as sc ec on the other i.e. they represent recombination. In the third group of progeny they are present as in the parental combinations.
Therefore, percentage recombination between sc-ec is (150 X 100)/1980 = 7.6%. We have thus found that percentage recombination between scute and crossveinless is 17.3, between scute and echinus 7.6 and between crossveinless and echinus 9.7. The recombination percentage also represents crossover percentage and map distance between the genes.
With the data available, it is now possible to map the genes. The chromosome is drawn as a line and the two genes showing lowest recombination frequency are marked first (Fig. 8.5a). In this case they are scute and echinus 7.6 map units apart. Next mark cv and ec which are 9.7 units apart by indicating cv either on the left or right of ec.
If we mark cv 9.7 map units on the left of ec, then as seen in Fig. 8.5b, the distance between cv and sc would equal 9.7 minus 7.6 equal to 2.1 units. This does not agree with the data on recombination percentage found experimentally. But if we mark cv on the right of ec 9.7 units apart, then it indicates a map distance between sc and ev equal to 9.7 plus 7.6 i.e., 17.3 which is identical to the experimental data.
Double Crossing Over:
In a three-point cross involving three genes A, B and C there are eight possible combinations of genes, namely ABC, AbC, ABc, abC, aBc, Abe, abc. Sometimes one or more of the expected combinations do not appear in the progeny. This is due to two crossovers occurring simultaneously in two regions (Fig. 8.7).
When a single crossover occurs, two genes are exchanged resulting in the formation of a crossover gamete. But if at the same time a second crossover also takes place between the next two genes, the original combination of genes is restored on each chromatid resulting in a parental combination. It happens then that in such a cross a double crossover is represented as a non-crossover, giving a recombination frequency lower than the actual.
This also reveals that Sturtevant’s statement that 1% crossing over equals one map unit is not always justified. Clearly, crossover percentage is not always equal to recombination percentage. When there are double crossovers between same two chromatids, the number of recombinants in the progeny is less than the number of crossover gametes.
The crossover percentages are important for mapping genes accurately. Some geneticists prefer to use Morgan for map units, one Morgan being equal to one per cent recombination frequency and one centimorgan equal to 0.01 Morgan.
Double crossovers cannot occur between genes that are located close to each other. In Drosophila it has been found that double crossovers cannot occur between genes closer than 10 or 15 map units apart. Moreover, the class of progeny that occurs least frequently represents the double crossovers. It also indicates greater map distance between two genes.
Maximum frequency of double crossovers can occur between gene loci at each end of the chromosome. In any case, more than 50% recombination cannot be expected between two genes because only two of the four chromatids in a paired meiotic bivalent are involved in a crossover.
Sturtevant pointed out that certain parts of chromosomes were more liable to exchange segments than others. Thus if we consider three hypothetical genes A, B and C the probability of a crossover occurring between A and B may be 10%, and between B and C may be 15%.
But what is the probability that two crossovers between A and B and between B and C should occur simultaneously? We know that the probability of two chance events occurring simultaneously is equal to the product of the individual probabilities.
In the hypothetical cross stated above, the probability that two crossovers occur between A and B, and B and C would be 10% x 15% or 0.1 x 0.15 = 0.015 = 1.5%. It has also been found experimentally that the actual percentage of double crossovers is a little less than that expected theoretically. This is due to interference, a term coined by Muller. Accordingly, the occurrence of one crossover reduces the chance of a second crossover in its neighbourhood.
Although some explanations have been put forward for interference, both at the cytological and molecular levels, none is considered satisfactory. Muller further proposed the terms coefficient of coincidence to describe the strength or degree of interference. The coefficient of coincidence is equal to the ratio of the observed percentage of double crossovers to the expected percentage of double crossovers.
The extent of interference is different between different pairs of genes. The value of coincidence falls and the value of interference rises when the distance between genes decreases. Based on the coefficient of coincidence interference can be described to range from absolute (no double crossovers) to partial (doubles less frequent than expected), none (doubles equal to expected frequency) or negative (doubles more frequent than expected).
Cytological Basis for Crossing Over:
At the zygotene stage of meiosis homologous chromosomes come together and start pairing. By pachytene pairing is stabilized, and each ribbon-like chromosome actually consists of two homologues paired (synapsed) close to each other called bivalents. Each homologue in a bivalent consists of two identical sister chromatids.
Chromatids belonging to two different homologues in a bivalent are called non-sister chromatids. Due to presence of four chromatids, the pachytene bivalent is sometimes called a tetrad.
Crossing over takes place between non-sister chromatids and involves breakage and reunion of only two of the four chromatids at a given point on the chromosomes. Figure 8.8 illustrates how one homologous pair of chromosomes goes through meiosis to form four gametes.
Two of the gametes receive a chromosome with genes linked in the same way as in the parental chromosomes (ABC and abc). These gametes represent non-crossovers or parental types and are produced from chromatids that were not involved in crossing over. The other two gametes, (ABc, abC) represent the recombinant or crossover types and were produced after crossing over and recombination between the originally linked genes.
Cytological Proof of Crossing Over:
In 1917 Goldschmidt proposed that recombination takes place due to exchange of alleles without exchange of chromosome segments. He assumed that at metaphase genes get detached from the chromosome. Later during meiosis the genes get reabsorbed on the chromosome either in the same or in a different place.
In 1930 Winkler put forth his “gene conversion” hypothesis. Accordingly, if gene replication occurs in closely synapsed homologous chromosomes, the wrong allele may get replicated. When this occurs at only one locus it appears like a crossover. Both the above theories presented difficulties in understanding.
In 1931 John Belling proposed a theory of crossing over based on exchange of chromosome segments in lily plants. Belling studied the morphology of bead-like chromomeres which are arranged linearly on the chromonema. Since the structure and arrangement of chromomeres is identical in a pair of homologous chromosomes, Belling thought they might represent genes.
He explained recombination by assuming that the chromomeres were synthesised first, and the chromonemata which were synthesised later, became connected to the chromomeres.
Wherever the strands of chromonemata did not get connected with chromomeres in the original linear order, they crossed over and passed through another chromomere resulting in recombination and genetic exchange. The name copy choice was later given to Belling’s mechanism for recombination. However, the idea was disproved due to lack of evidence from genetic tests.
Significance of Crossing Over:
Crossing over occurs in living organisms ranging from viruses to man. It constitutes evidence for sexual reproduction in an organism. Its widespread occurrence in organisms ensures exchange of genes and production of new types which increase genetic diversity.
This increases phenotypic diversity, which at the species level is responsible for genetic polymorphism. The occurrence of polymorphism is of advantage to a species because it leads to groups of individuals becoming adapted to a wider range of habitats. This increases the potential for evolutionary success.
FACTORS AFFECTING CROSSING OVER
- High temperature and exposure to radiation rays such as x-rays increase the frequency of crossing over.
As the age advances the frequency of crossing over decreases.Some mutation decreases the frequency.
- Crossing over is less frequent near centromeres and the tips of the chromosomes.
- Inversion of chromosome segments suppresses the crossing over.
- A rearrangement of linear array of genes on chromosome in such a way that their order in the chromosome is reversed is called Inversion.
- Crossing over at point reduces the chances of another crossing over in adjacent regions. In other words chiasma formation at one point prevents the chiasma formation in the vicinity. This phenomenon is called Interference .
- Certain chemicals and radiomimetic substances have been found to increase somatic crossing over.
- Some chemicals have been found to decrease crossing over.
E.g. Colchicine, Selenium in its excess amount.