Sexual reproduction allows the genetic information
of two parents to recombine to form a new individual.
One great advantage, from the population biology point-of-view, is
that sexual reproduction produces a great deal of genetic variation through
the shuffling of both beneficial and deleterious mutations.
Sexual reproduction requires diploidy (the state of having two
sets of chromosomes) with a set of chromosomes from each parent which allows
greater genetic flexibility than haploidy does.
Diploid cells may be either homozygous or heterozygous for any given
gene.
However, the gametes (sperm and ova) are specialized haploid cells
produced by meiosis.
The life cycles of sexual organisms have both diploid and haploid phases.
Some fungi spend much of their lives as haploid (1n) and only become
diploid (2n) to produce gametes.
The haploid gamete must undergo a specialized form of cell division
known as meiosis, a process that divides a diploid cell into four haploid
cells.
Sperm and ova are produced by two main processes 1) meiosis and 2) specialized
cell differentiation.
Gametogenesis differs greatly between spermatogenesis
and oogenesis.
Spermatogenesis converts the spermatocyte into four spermatids.
During oogenesis, asymmetric cell division produces one large cell
and three small ones that degenerate into three polar bodies.
Meiosis produces genetic diversity by recombining the diploid cell's
genetic complement to generate a haploid gamete.
This diversity depends upon the segregation and assortment of combination
of alleles.
Importantly, diploid organisms can bear recessive alleles of genes
that are can be completely masked by the other (usually wild type) allele.
In the mid-1800's, Gregor Mendel formulated his "Laws of Inheritance"
from his famous pea experiments.
Mendel's "Law of Segregation" ensures
that alleles of each gene separate from each other during gamete formation.
Mendel's (more controversial) "Law of Independent
Assortment" suggests that alleles of each gene separate independently
of the other genes.
Chromosomal behavior provides strong support for the laws of segregation
and independent assortment.
After all, the known DNA sequences of homologous chromosomes are essentially
the same.
The Chromosomal Theory of Inheritance (Sutton, early 1900's) was based
on five points:
1) Nuclei contain two sets of homologous chromosomes
(1 maternal and 1 paternal).
2) Chromosome retain identity and are genetically
continuous through the life cycle.
3) The two sets of homologous chromosomes are functionally
equivalent.
4) Maternal and paternal homologous chromosomes
synapse during meiosis then move to opposite poles.
5) Maternal and paternal homologous chromosomes
segregate independently.
Five examples of genetic exchange between homologous DNA molecules involves
homologous recombination…
1) prophase I of meiosis (gametogenesis)
2) coinfection of bacteria with related bacteriophages
3) transformation of Bacteria (DNA)
4) transduction of bacteria (transducing phages)
5) bacterial conjugation
Homologous recombination depends upon controlled breakage-and-exchange
of DNA has been demonstrated by experiments.
1) Co-infection of bacteria with labeled bacteriophage
showed exchange of DNA (lable).
2) Labeling eukaryotic chromosomes revealed
that post-meiotic chromosomes are composed of mixtures of the parental
chromosomes and correlates well with the genetic recombination rates of
known genes on the chromosome.
The Holliday Model of Homologous Recombination
The current model of the mechanism of exchange
of DNA between two homologous chromosomes explains gene conversion and
genetic recombination.
1) A double-stranded DNA molecule undergoes a single-stranded
break.
2) The single-stranded DNA invades the complementary
region of the double-stranded homologue.
3) DNA repair (DNA synthesis) of the dsDNA using
the invading ssDNA as template begins.
4) Reciprocal invasion results in the formation
of the "double crossover" or Holliday Junction.
5) Branch migration (movement of the crossover structure)
is the result of DNA unwinding and rewinding.
6) Resolution of the Holliday Junction will result
in either a cross over event or gene conversion (without a cross over)
event.
The synaptonemal complex develops only when single-stranded DNA successfully carries out the process of "homology searching" to facilitate the exchange process.
Recombinant DNA molecules are produced by ...
1) cleaving DNA from two different sources with
restriction endonucleases (restriction enzymes),
2) mixing the fragments together to allow the ends
of the fragments to interact and
3) linking the fragments with DNA ligase.
The cloning of specific DNA fragments usually
involve:
1) Insertion of DNA into a vector (a recombinant
vector)
2) Introduction of recombinant vector into cells
(usually E. coli)
3) Amplification of recombinant vector in the cells
4) Selection of cells that carry the recombinant
vector.
5) Identification of correct recombinant clone.
Often a "shotgun" approach is used to produce clones.
This means that instead of starting with a known specific fragment
of DNA, "all" the DNA from a source (as relatively random pieces
is cloned into a vector) to result in a library of clones.
If the source of the DNA is the genome of an organism, then the library
is refered to as a genomic library.
To examine the expressed genes of an organism, the mRNA can be "converted"
into a complementary DNA (cDNA) library through
the use of the enzyme reverse transcriptase.
cDNA is made by annealing poly-T primers to the poly-A tails of isolated
mRNA and synthesizing ssDNA from the mRNA template with reverse transciptase.
The RNA is hydrolysed and a DNA polyermerase generates the second strand
to make dsDNA.
The cDNA is then inserted into a vector and propagated as above.
As the techniques improve, larger segments of DNA can be cloned as
a continuous piece in specialized vectors such as cosmids and Yeast Artificial
Chromosomes (YACs).
Advantages:
1) Recombinant technology allows us to produce large amounts of medically
important proteins including insulin (diabetes), blood clotting factors
(hemophilia), growth hormone (dwarfism), tissue plasminogen activator (treating
blood clots), plus much more.
2) Genetic engineering of plant crops depends
upon the Ti plasmid to integrate a DNA fragment of interest into the plant
cell's chromosomal DNA.
With propagation the recombinant T DNA becomes stably incorporated
into the genome of every cell of the plant.
3) To model human diseases, mice that have specific genes inactivated
(knock-out mice) are produced through recombination
in embryonic stem cells followed by generation of chimeric transgenic mice.
4) Gene therapy, when a patient whose disease is caused by defective
copies of a gene is treated with a functional copy of that gene.
One mechanism employed to carry out gene therapy is to first remove
certain cells from a patient, introduce the gene in vitro then return
the cells to the patient.
The application of genetic recombination science may provide the basis
for many significant advances in science.
Notes prepared from Becker's World of the Cell, 9th edition
Hardin & Bertoni, 2015
Figures copyright of Pearson Education Inc.
email me at bestave@mun.ca
