In this article we will discuss about:- 1. Introduction to Crossing Over of Genes 2. Factors Affecting Crossing Over of Genes 3. Cytological Basis of Crossing Over 4. Molecular Mechanism Crossing Over 5. Hybrid DNA Models of Crossing Over.
Contents:
- Introduction to Crossing Over of Genes
- Factors Affecting Crossing Over of Genes
- Cytological Basis of Crossing Over of Genes
- Molecular Mechanism Crossing Over of Genes
- Hybrid DNA Models of Crossing Over of Genes
1. Introduction to Crossing Over of Genes:
Recombination of genes on the same chromosome is accomplished by crossing over, a process by which parts of homologous chromosomes are interchanged. Crossing over is important in evolution because it generates variation in a species. Crossing over and independent assortment are mechanisms that produce new combination of genes.
The important features of the concept of crossing over are:
i. The loci of the genes on a chromosome are arranged in a linear sequence.
ii. The two alleles of a gene in a hetero- zygote occupy corresponding positions in the homologous chromosomes, that is, allele A occupies the same position in homologue 1 that allele a occupies in homologue 2. iii. Crossing over involves the breakage of each of two homologous chromosomes (actually chromatids) and the exchange of parts.
iv. Crossing over occurs at pachytene after the synapsis of the homologous chromosomes in prophase I of meiosis. Since chromosome replication occurs during interphase, meiotic crossing over occurs in the post replication tetrad stage with four chromatids for each pair of homologous chromosomes (Fig. 8.6).
v. Chromosomes with recombinant combinations of linked genes are formed by occurrence of crossing over in the region between the two loci.
vi. The probability of crossing over occurring between two loci increases with increasing distance between the two loci on the chromosome.
2. Factors Affecting Crossing Over of Genes:
The frequency of crossing over between linked genes is affected by several factors:
1. Distance between genes:
ihe frequency of crossing over between two genes is positively associated with the distance between their locations in the chromosome.
ii. Distance from centromere:
Genes located in the vicinity of centromeres show a relatively lower recombination rate than those located away from them.
iii. Chromosomal aberrations:
Translocations, inversions (crossover suppressor) reduce recombination between the genes located within the altered segment.
iv. Sex:
The frequency of recombination is influenced by the sex, e.g., male Drosophila does not show recombination.
v. Genotype:
Some genes affect occurrence of crossing over, e.g., c3G gene of Drosophila prevents recombination.
vi. Age of female:
A progressive decline of recombination frequency with the advancing age of female Drosophila is noticed.
vii. Association with heterochromatin:
Hetero- chromatic segments influence crossing over.
viii. Environmental factors:
Temperature, radiation (X-ray, γ-ray), chemicals (antibiotics EMS) also affect the frequency of crossing over.
3. Cytological Basis of Crossing Over of Genes:
Morgan first proposed crossing over to explain the formation of recombinant combinations of genes that were shown to be linked by genetic data. He hypothesized that the linkage was the result of the location of these genes on the same chromosome. If crossing over occurs, one might expect to be able to observe it (or its consequences) cytologically.
The cross shaped structures, in which two of the four chromatids of homologous chromosome pairs appear to exchange pairing partners, are readily detected in cytological studies of meiosis in many organisms. These cross shaped structures were first detected in amphibians and called chiasmata by F. Janssens.
Studies in which chiasma frequency was correlated with recombination frequency have shown a direct relationship between crossing over and chiasmata.
The cytological evidence that homologous chromosomes exchange parts during crossing over was first noted in 1931 by Stern in Drosophila and Creighton and McClintock in maize. Normally the two chromosomes of any homologous pair are morphologically indistinguishable.
Stern, Creighton and McClintock identified homologues that were morphologically distinguishable, that is, they were not entirely homologous. The chromosome pairs were homologous along most of their length. They paired arid segregated normally during meiosis. The ends of the homologues were different and could be recognized by microscopy (heteromorphic pair).
Stern’s Experiment in Drosophila:
In Stern’s experiment, stocks carrying structural chromosomal changes were crossed to produce a female which had section of Y chromosome attached to one of its X chromosomes. The other X chromosome was composed of two approximately equal fragments, each with its own centromere. Each of the two X chromosomes was, therefore, uniquely recognizable under the microscope.
In addition, one of the X chromosome fragments carried the mutant alleles for carnation (car, recessive light eye colour) and Bar (B, semi-dominant narrow eye shape), while the long X – Y chromosome carried the normal alleles for these genes. In the absence of crossing over, such females would be expected to produce only car B or ++ gametes.
When fertilized by, a carnation-eyed male (car +), the female non-crossover offspring of the mating should therefore appear as carnation, bar-eyed (car +/car +) and wild type (++/ car+).
Genetic crossing over, however, will produce more different female phenotypes – carnation (car + / car +) and bar (+B / car +), and more interestingly, will also produce uniquely recognizable chromosomes if chromosomal exchange accompanies genetic crossing over.
The two crossover products produced by the female are one long X chromosome and a short X fragment with attached Y section. When combined with the normal long X chromosome of the male, the crossover female offspring’s are thus cytologically distinct from the non-crossovers.
Stern found the predicted genotype – X chromosome correlation in almost every crossover female examined. Such cytological observations suggested that genetic crossing over was accompanied by an actual exchange of chromosome segments (Fig. 8.9).
Creighton and McClintock’s Experiment in Corn:
Creighton and McClintock also demonstrated the correlation between genetic crossing over and the exchange of parts of homologous chromosomes. They analysed crosses involving two loci on chromosome 9 of maize.
One gene controlling the kernel colour (C – coloured; c – colourless) and the other controlling the type of carbohydrates in the kernel (Wx – starchy; wx – waxy). They made use of a chromosome with a densely stained “knob” at one end and an extra (trans-located) piece of chromosome at the other end (Fig. 8.10).
The plant was heterozygous for coloured aleurone and starchy (non-waxy) endosperm characters and carried these genes in repulsion phase, i.e., Cwx / cWx. Cwx was carried on the knobbed chromosome and cWx on the knobless chromosome. Such a plant was test crossed with plant homozygous recessive for both characters, i.e., colourless and waxy (cwx / cwx).
If the chromosome region between the knob and the c gene is represented as I region and that between c and Wx as II region, then one would expect two types of non-crossover gametes (Cwx and cWx) and six types of crossover gametes including single and double crossovers. The progeny can be classified into eight types based on phenotypes and cytological observations.
The following observations in phenotype and cytology of progeny (Fig. 8.11) suggested that actual exchange of chromosome segments was involved in genetic crossing over:
i. Association of knob in chromosome with the phenotype, colourless seed (c), and non-waxy endosperm (Wx), showed crossing over in region I, as the phenotypes are associated with knobless chromosome in the parents.
ii. A ring of four chromosomes is formed in meiosis due to presence of translocation in heterozygote condition. In this case, in meiotic metaphase I, the presence of a ring of four chromosomes without a knob suggested cytological exchange of chromosome segments, because the knob was associated with the 9th chromosome carrying translocation in the parent.
iii. Presence of knob in the bivalents instead of being with the quadrivalents, can be treated as an evidence for cytological crossing over, since the knob was originally associated with the translocation and the translocation usually results in the formation of quadrivalents.
4. Molecular Mechanism Crossing Over of Genes:
The models that have been proposed to account for crossing over are of two general types:
(i) Copy choice and
(ii) Breakage – reunion (Fig. 8.12).
Copy Choice Models:
These are based on the assumption that molecules of DNA in the process of being synthesized could switch over from using DNA of one homologue as template in one region to using the DNA of the other homologue as template in another region. Most such copy choice models of crossing over were based on the assumption of conservative DNA synthesis.
Copy choice models rapidly lost support once DNA replication was proven to be semi-conservative. Pure copy choice (with no breakage and reunion) is not mechanistically compatible with semi-conservative DNA replication. But a small amount of copy choice DNA repair synthesis is associated with crossing over by breakage and reunion.
This copy choice repair synthesis may be responsible for gene conversion or non-reciprocal recombination. Crossing over, therefore, occurs by a complex mechanism that includes some aspects of both breakage and reunion and copy choice models.
Breakage and Reunion Hypothesis:
It involves the breakage of two homologous chromatids and the reunion of the parts in recombinant arrangements. Extensive data now document the occurrence of breakage and reunion during crossing over process.
Direct evidence for breakage and reunion has been obtained from Taylor’s experiments in eukaryotes by labelling chromosomes with radioactive thymidine [3H] and following the distribution of radioactive chromatids by autoradiography. Labelled and unlabeled segments of chromatids were often observed to be interchanged.
They did not exclude, however, the possibility that the unlabeled segment is the result of new synthesis rather than the reunion of an unlabeled segment of a parental chromosome.
5. Hybrid DNA Models of Crossing Over of Genes:
The currently popular models to explain the molecular mechanism of crossing over, are based on hybrid DNA (heteroduplex DNA) formation. In these models, released broken strands of the DNA duplex pair crosswise with unbroken strand of non-sister chromatid resulting in the formation of hybrid DNA segments.
These models took into account all the various types of genetic data that must be consistent with the mechanisms of crossing over in terms of breakage and reunion with associated repair synthesis.
Single Strand Break:
Holliday model and Whitehouse model, named after the scientists who proposed them in 1964, involve single strand break of two DNA duplexes belonging to non-sister chromatids. In the model (Fig. 8.13) proposed by H.L.K. Whitehouse, the two strands participating will have opposite polarity (5’→ 3′) and (3′ → 5′), but in the model proposed by Robin Holliday, their polarity will be similar (Fig. 8.14).
The Holliday model has been widely accepted and the possible pathways for occurrence of crossing over based on Holliday model has been discussed below. This pathway (Fig. 8.15A) begins with an endonuclease that cleaves single strands of each of the two parental DNA molecules (breakage).
Segments of the single strands on one side of each cut are displaced from their complementary strands, probably with the aid of DNA binding proteins, helix destabilizing protein, and DNA unwinding proteins (also called helicases).
The displaced strands then exchange pairing partners, base-pairing with the intact complementary strands of homologous chromosomes.
This process is also aided by certain proteins (rec A protein). As the single strand of one double helix “invades” (displaces the identical or homologous strand and base pairs with the complementary strand) a second homologous double helix, the displaced single strand of the second double helix, in turn, similarly invades the first double helix.
Fig. 8.15B: Double-strand-break model of recombination
The protein (rec A protein) mediates such a reaction by binding to the unpaired strand of DNA, promoting the displacement of a segment of one strand of the double helix by the unpaired strand, once a homologous double helix is found. The cleaved strands are subsequently covalently joined in recombinant arrangements (reunion) by DNA ligase.
If the original breaks in the two strands do not occur at exactly the same site in the two homologues, some “tailoring” will be required before DNA ligase can catalyze the reunion step; this tailoring involves excision of a limited number of bases by an exonuclease and repair synthesis by a DNA polymerase. This sequence of events produce an X-shaped recombination intermediate (called a “chi” form).
A similar sequence of enzyme catalyzed breakage and reunion events, involving the other two single strands, occurs to complete the process of crossing over.
Double Strand Break:
Homologous recombination possibly occurs by more than one mechanism. Evidence from studies of the yeast, Saccharomyces cerevisiae, shows that ends of molecules produced by double strand cuts or breaks are recombinogenic. Thus, a double strand break model of crossing over was proposed by Szostak in 1983 (Fig. 8.15B).
The major difference between this model and Holliday model is that recombination is mediated by a double strand break in one of the parental double helices’ and not by just single strand breaks.