Principles of Cell Biology (BIOL2060)

Department of Biology
Memorial University of Newfoundland

Sexual Reproduction, Meiosis, and Genetic Recombination

Mitosis leads to cell proliferation and is essential for asexual reproduction including
    1) mitotic division of unicellular organisms,
    2) budding of offspring from the parent's body and
    3) regeneration from pieces of a parent organism.
Asexual reproduction produces new individuals that are genetically identical to the parent.

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.

Meiosis

In the first part of meiosis (meiosis I) an unusual type of cell division produces two haploid cells that have chromosomes made up of two sister chromatids.
The chromosomes pair up (a process called synapsis) to form a structure known as either a bivalent (two chromosomes) or a tetrad (four chromatids).
Prophase I is the stage of meiosis where the homologous chromosomes pair and exchange DNA (genetic recombination).
Prophase I comprised of five stages; 1) leptotene, 2) zygotene, 3) pachytene, 4) diplotene and 5) diakinesis.
    1) Leptotene (Greek for "thin threads"):  chromosomes begin to condense.
    2) Zygotene (Greek for "paired threads"):  chromosomes become closely paired.
    3) Pachytene (Greek for "thick threads"): crossing over occurs.
    4) Diplotene (Greek for "two threads"): homologous chromosomes begin to separate but remain attached by the chiasmata.
    5) Diakinesis (Greek for "moving through"): chromosomes condense and separate until terminal chiasmata only connect the two chromosomes.
The chiasmata can be formed anywhere along the chromosome but during diakinesis, the chiasmatic connections are translated to the chromosomes' ends.
The homologous chromosomes are held together during synapsis by the synaptonemal complex.
Metaphase I:  Bivalent chromosomes attach to the spindle and align at the metaphase plate.
The bivalents are randomly oriented with respect to the poles such that chromosomes (maternal or paternal or both) are evenly sorted.
Only the chiasmata hold the paired homologues together.
Anaphase I:  Homologous chromosomes resolve the chiasmata and move (as a bivalent) to opposite ends of the cell.
Telophase I: Chromosomes arrive at the poles and nuclear envelopes form and cytokinesis occurs.
Meiosis II:  The second part of meiosis (meiosis II) is very similar to mitotic division except that DNA synthesis does not occur between the two stages.

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.

Genetic Recombination

Random assortment of the different alleles of genes on different chromosomes depends upon the segregation and independent assortment of the chromosomes during meiosis I.
Crossing-over of the chromosomes during meiosis I leads to genetic recombination of different alleles of genes on the SAME chromosome.
When genes are located near each other on a chromosome, they act as if they are linked and parental allele combinations are more often than not inherited together by the grandchildren.
Genetic variability is produced by genetic recombination through the process of crossing over when the chromosomes pair during meiotic prophase.
Parental homologous chromosomes exchange segments during crossing over to produce recombinant chromosomes.
Genetic mapping based upon the measurement of recombination frequencies is used to map gene locations.
Co-infection of bacterial cells with bacteriophage can lead to genetic recombination.
Transformation and transduction involve recombination of the bacterial genome with naked DNA or bacteriophage DNA.
Bacterial conjugation is a modified sexual activity that facilitates genetic recombination.

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 Technology (review)

In modern biology, genetic recombination is an applied process.
In recombinant DNA technology (slang= genetic engineering), a number of techniques are used to replicate and manipulate specific pieces of DNA in "large" amounts.

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, 8th edition
Hardin, Bertoni & Kleinsmith, 2012
Figures copyright of Pearson Education Inc.
email me at bestave@mun.ca