Fragmentation (cell biology)

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Fragmentation describes the process of splitting into several pieces or fragments. In cell biology, fragmentation is useful for a cell during both DNA cloning and apoptosis. DNA cloning is important in asexual reproduction or creation of identical DNA molecules, and can be performed spontaneously by the cell or intentionally by laboratory researchers. Apoptosis is the programmed destruction of cells, and the DNA molecules within them, and is a highly regulated process. These two ways in which fragmentation is used in cellular processes describe normal cellular functions and common laboratory procedures performed with cells. However, problems within a cell can sometimes cause fragmentation that results in irregularities such as red blood cell fragmentation and sperm cell DNA fragmentation.

Contents

DNA Cloning

DNA cloning can be performed spontaneously by the cell for reproductive purposes. This is a form of asexual reproduction where an organism splits into fragments and then each of these fragments develop into mature, fully grown individuals that are clones of the original organism (See reproductive fragmentation). DNA cloning can also be performed intentionally by laboratory researchers. Here, DNA fragmentation is a molecular genetic technique that permits researchers to use recombinant DNA technology to prepare large numbers of identical DNA molecules. In order for DNA cloning to be completed, it is necessary to obtain discrete, small regions of an organism's DNA that constitute specific genes. Only relatively small DNA molecules can be cloned in any available vector. Therefore, the long DNA molecules that compose an organism's genome must be cleaved into fragments that can be inserted into the vector DNA. [1] Two enzymes facilitate the production of such recombinant DNA molecules:

1. Restriction Enzymes
Restriction enzymes are endonucleases produced by bacteria that typically recognize small base pair sequences (called restriction sites) and then cleave both strands of DNA at this site. [2] A restriction site is typically a palindromic sequence, which means that the restriction-site sequence is the same on each strand of DNA when read in the 5' to 3' direction.
For each restriction enzyme, bacteria also produce a modification enzyme so that a host bacterium's own DNA is protected from cleavage. This is done by modifying the host DNA at or near each potential cleavage site. The modification enzyme adds a methyl group to one or two bases, and the presence of this methyl group prevents the restriction endonuclease from cutting the DNA. [3]
Cut that creates a sticky end EcoRI restriction enzyme recognition site.svg
Cut that creates a sticky end
Cut that creates a blunt end SmaI restriction enzyme recognition site.svg
Cut that creates a blunt end
Many restriction enzymes make staggered cuts in the two DNA strands at their recognition site, which generates fragments with a single stranded "tail" that overhangs at both ends, called a sticky end. Restriction enzymes can also make straight cuts in the two DNA strands at their recognition site, which generates blunt ends. [4]
2. DNA ligase
During normal DNA replication, DNA ligase catalyzes end-to-end joining (ligation) of short fragments of DNA, called Okazaki fragments. For the purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends. They are covalently ligated together through the standard 3' to 5' phosphodiester bonds of DNA. [5]
DNA ligase can ligate complementary sticky and blunt ends, but blunt-end ligation is inefficient and requires a higher concentration of both DNA and DNA ligase than the ligation of sticky ends does. [6] For this reason, most restriction enzymes used in DNA cloning make staggered cuts in the DNA strands to create sticky ends.

The key to cloning a DNA fragment is to link it to a vector DNA molecule that can replicate within a host cell. After a single recombinant DNA molecule (composed of a vector plus an inserted DNA fragment) is introduced into a host cell, the inserted DNA can be replicated along with the vector, generating a large number of identical DNA molecules. [7] The basic scheme for this can be summarized as follows:

Vector + DNA Fragment
↓
Recombinant DNA
↓
Replication of recombinant DNA within host cell
↓
Isolation, sequencing, and manipulation of purified DNA fragment

There are numerous experimental variations to this scheme, but these steps are essential to DNA cloning in a laboratory. [8]

Apoptosis

Fragmentation is the third and final step of cell disassembly during apoptosis (right side of scheme). Apoptotic cell disassembly.png
Fragmentation is the third and final step of cell disassembly during apoptosis (right side of scheme).

Apoptosis refers to the demise of cells by a specific form of programmed cell death, characterized by a well-defined sequence of morphological changes. [10] Cellular and nuclear shrinkage, chromatin condensation and fragmentation, formation of apoptotic bodies and phagocytosis by neighboring cells characterize the main morphological changes in the apoptosis process. [11] Extensive morphological and biochemical changes during apoptosis ensure that dying cells leave minimal impact on neighboring cells and/or tissues.

Genes involved in controlling cell death encode proteins with three distinct functions: [12]

The cleavage of chromosomal DNA into smaller fragments is an integral part, and biochemical hallmark, of apoptosis. Apoptosis involves the activation of endonucleases with subsequent cleavage of chromatin DNA into fragments of 180 base pairs or multiples of 180 base pairs (e.g. 360, 540). This pattern of fragmentation can be used to detect apoptosis in tests such as a DNA laddering assay with gel electrophoresis, a TUNEL assay, or a Nicoletti assay. [13] Apoptotic DNA fragmentation relies on an enzyme called Caspase-Activated DNase (CAD). [14] CAD is usually inhibited by another protein in the cell, called Inhibitor of caspase-activated DNase (ICAD). [15] In order for apoptosis to begin, an enzyme called caspase 3 cleaves ICAD so that CAD becomes activated. CAD then cleaves the DNA between nucleosomes, which occur in chromatin at 180 base pair intervals. The sites between nucleosomes are the only parts of the DNA that are exposed and accessible to CAD. [16]

Irregularities

DNA fragmentation can occur under certain conditions in a few different cell types. This can lead to problems for a cell, or it may lead to a cell receiving a signal to undergo apoptosis. Below are a couple of examples of irregular fragmentation that can occur in cells.

1. Red blood cell fragmentation
A blood smear from a patient with hemolytic anemia, showing schistocytes Schizocyte smear 2009-12-22.JPG
A blood smear from a patient with hemolytic anemia, showing schistocytes
A fragmented red blood cell is known as a schistocyte and is generally the result of an intracellular mechanical injury to the red blood cell. [17] A wide variety of schistocytes may be observed. Schistocytes are usually seen in relatively low numbers and are associated with conditions in which the normally smooth endothelial lining, or endothelium, is roughened or irregular, and/or the vascular lumen is crossed by strands of fibrin. [18] Schistocytes are commonly seen in patients that have hemolytic anemia. They are also a feature of advanced iron deficiency anemia, but in this case the observed fragmentation is most likely a result of the fragility of the cells produced under these conditions.
2. Sperm cell DNA fragmentation
In an average male, less than 4% of his sperm cells will contain fragmented DNA. However, partaking in behaviors such as smoking can significantly increase DNA fragmentation in sperm cells. There is a negative correlation between the percentage of DNA fragmentation and the motility, morphology, and concentration of sperm. There is also a negative association between the percentage of sperm that contain fragmented DNA and the fertilization rate and embryo cleavage rate. [19]

Related Research Articles

<span class="mw-page-title-main">DNA ligase</span> Class of enzymes

DNA ligase is a type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms may specifically repair double-strand breaks. Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA.

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

In the field of genetics, a suicide gene is a gene that will cause a cell to kill itself through the process of apoptosis. Activation of a suicide gene can cause death through a variety of pathways, but one important cellular "switch" to induce apoptosis is the p53 protein. Stimulation or introduction of suicide genes is a potential way of treating cancer or other proliferative diseases.

<span class="mw-page-title-main">Library (biology)</span>

In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation, though this term is used for other procedures as well. In a restriction digest, DNA molecules are cleaved at specific restriction sites of 4-12 nucleotides in length by use of restriction enzymes which recognize these sequences.

DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be done intentionally by laboratory personnel or by cells, or can occur spontaneously. Spontaneous or accidental DNA fragmentation is fragmentation that gradually accumulates in a cell. It can be measured by e.g. the Comet assay or by the TUNEL assay.

<span class="mw-page-title-main">Subcloning</span>

In molecular biology, subcloning is a technique used to move a particular DNA sequence from a parent vector to a destination vector.

<span class="mw-page-title-main">DNA shuffling</span>

DNA shuffling, also known as molecular breeding, is an in vitro random recombination method to generate mutant genes for directed evolution and to enable a rapid increase in DNA library size. Three procedures for accomplishing DNA shuffling are molecular breeding which relies on homologous recombination or the similarity of the DNA sequences, restriction enzymes which rely on common restriction sites, and nonhomologous random recombination which requires the use of hairpins. In all of these techniques, the parent genes are fragmented and then recombined.

A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

Cellular waste products are formed as a by-product of cellular respiration, a series of processes and reactions that generate energy for the cell, in the form of ATP. One example of cellular respiration creating cellular waste products are aerobic respiration and anaerobic respiration.

<span class="mw-page-title-main">Apoptotic DNA fragmentation</span> Cleavage of DNA into tiny pieces during apoptosis

Apoptotic DNA fragmentation is a key feature of apoptosis, a type of programmed cell death. Apoptosis is characterized by the activation of endogenous endonucleases, particularly the caspase-3 activated DNase (CAD), with subsequent cleavage of nuclear DNA into internucleosomal fragments of roughly 180 base pairs (bp) and multiples thereof (360, 540 etc.). The apoptotic DNA fragmentation is being used as a marker of apoptosis and for identification of apoptotic cells either via the DNA laddering assay, the TUNEL assay, or the by detection of cells with fractional DNA content ("sub G1 cells") on DNA content frequency histograms e.g. as in the Nicoletti assay.

Topoisomerase-based cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3'-end of the PCR products. This characteristic is exploited in "sticky end" TOPO TA cloning. For "blunt end" TOPO cloning, the recipient vector does not have overhangs and blunt-ended DNA fragments can be cloned.

pUC19

pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix and the abbreviation for the University of California, where early work on the plasmid series had been conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the most widely used vector molecules as the recombinants, or the cells into which foreign DNA has been introduced, can be easily distinguished from the non-recombinants based on color differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is reversed.

<span class="mw-page-title-main">Caspase-activated DNase</span> Protein-coding gene in the species Homo sapiens

Caspase-activated DNase (CAD) or DNA fragmentation factor subunit beta is a protein that in humans is encoded by the DFFB gene. It breaks up the DNA during apoptosis and promotes cell differentiation. It is usually an inactive monomer inhibited by ICAD. This is cleaved before dimerization.

Gibson assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who is the chief technology officer and co-founder of the synthetic biology company, Telesis Bio.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology


Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">In vitro recombination</span> Process of isolation and amplification of DNA segments

Recombinant DNA (rDNA), or molecular cloning, is the process by which a single gene, or segment of DNA, is isolated and amplified. Recombinant DNA is also known as in vitro recombination. A cloning vector is a DNA molecule that carries foreign DNA into a host cell, where it replicates, producing many copies of itself along with the foreign DNA. There are many types of cloning vectors such as plasmids and phages. In order to carry out recombination between vector and the foreign DNA, it is necessary the vector and DNA to be cloned by digestion, ligase the foreign DNA into the vector with the enzyme DNA ligase. And DNA is inserted by introducing the DNA into bacteria cells by transformation.

<span class="mw-page-title-main">Ligation (molecular biology)</span>

Ligation is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA, whereby DNA fragments are joined to create recombinant DNA molecules (such as when a foreign DNA fragment is inserted into a plasmid). The ends of DNA fragments are joined by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'-phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Eukaryotic cells ligases belong to ATP type, and NAD+ - dependent are found in bacteria (e.g. E. coli).

Rate-zonal centrifugation is a centrifugation technique employed to effectively separate particles of different sizes. The tube is first filled with different concentrations of sucrose or another solute establishing layers with different densities and viscosities, forming a density gradient, within which the particles to be separated are added. The larger particles will be able to travel to the bottom layer because they are more massive. The greater mass allows the particles to travel through layers with a greater viscosity, while the smaller particles will remain at the top, as they lack the mass to travel through the more viscous layers. Once the centrifugation is over, fractions are collected.

References

  1. ↑ Lodish, Harvey, Arnold Berk, Chris A. Kaiser, Monty Kriger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, and Matthew P. Scott. Molecular Cell Biology. 7th ed. New York: W.H. Freeman and, 2013. Print.
  2. ↑ Rao, Desirazu N., Swati Saha, and Vinita Krishnamurthy. "ATP-Dependent Restriction Enzymes." Progress in Nucleic Acid Research and Molecular Biology 64 (2000): 1-63. Print.
  3. ↑ Rao, Desirazu N., Swati Saha, and Vinita Krishnamurthy. "ATP-Dependent Restriction Enzymes." Progress in Nucleic Acid Research and Molecular Biology 64 (2000): 1-63. Print.
  4. ↑ Lodish, Harvey, Arnold Berk, Chris A. Kaiser, Monty Kriger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, and Matthew P. Scott. Molecular Cell Biology. 7th ed. New York: W.H. Freeman and, 2013. Print.
  5. ↑ Tomkinson, Alan E., and Zachary B. Mackey. "Structure and Function of Mammalian DNA Ligases." Mutation Research/DNA Repair 407.1 (1998): 1-9. Print.
  6. ↑ Hung, Mien-Chie, and Pieter C. Wensink. "Different Restriction Enzyme-generated Sticky DNA Ends Can Be Joined in Vitro." Nucleic Acids Research 12.4 (1984): 1863-874. Print.
  7. ↑ "Ch 20." Avonapbio /. N.p., n.d. Web. 20 Nov. 2012. <http://avonapbio.pbworks.com/w/page/9429274/Ch%2020>.
  8. ↑ Lodish, Harvey, Arnold Berk, Chris A. Kaiser, Monty Kriger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, and Matthew P. Scott. Molecular Cell Biology. 7th ed. New York: W.H. Freeman and, 2013. Print.
  9. ↑ Smith, Aaron; Parkes, Michael AF; Atkin-Smith, Georgia K; Tixeira, Rochelle; Poon, Ivan KH (2017). "Cell disassembly during apoptosis". WikiJournal of Medicine. 4 (1). doi: 10.15347/wjm/2017.008 .
  10. ↑ Lodish, Harvey, Arnold Berk, Chris A. Kaiser, Monty Kriger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, and Matthew P. Scott. Molecular Cell Biology. 7th ed. New York: W.H. Freeman and, 2013. Print.
  11. ↑ Hua, Xhang J., and Ming Xu. "DNA Fragmentation in Apoptosis." Cell Research 10 (2000): 205-11. Nature. 17 July 2000. Web. 19 Nov. 2012.
  12. ↑ Lodish, Harvey, Arnold Berk, Chris A. Kaiser, Monty Kriger, Anthony Bretscher, Hidde Ploegh, Angelika Amon, and Matthew P. Scott. Molecular Cell Biology. 7th ed. New York: W.H. Freeman and, 2013. Print.
  13. ↑ Bortner, Carl D., Nicklas B.E. Oldenburg, and John A. Cidlowski. "The Role of DNA Fragmentation in Apoptosis." Trends in Cell Biology 5.1 (1995): 21-26. Print.
  14. ↑ Jog, Neelakshi R., Lorenza Frisoni, Qin Shi, Marc Monestier, Sairy Hernandez, Joe Craft, Eline T. Luning Prak, and Roberto Caricchio. "Caspase-activated DNase Is Required for Maintenance of Tolerance to Lupus Nuclear Autoantigens." Arthritis and Rheumatism 64.4 (2012): 1247-256. Print.
  15. ↑ Kutscher, Daniel, Alfred Pingoud, Albert Jeltsch, and Gregor Meiss. "Identification of ICAD-derived Peptides Capable of Inhibiting Caspase-activated DNase." FEBS Journal 279.16 (2012): 2917-928. Print.
  16. ↑ Bortner, Carl D., Nicklas B.E. Oldenburg, and John A. Cidlowski. "The Role of DNA Fragmentation in Apoptosis." Trends in Cell Biology 5.1 (1995): 21-26. Print.
  17. ↑ Bessman, JD. "Red Blood Cell Fragmentation. Improved Detection and Identification of Causes." American Journal of Clinical Pathology 90.3 (1988): 268-73. Print.
  18. ↑ "Schistocytes." Schistocytes. N.p., n.d. Web. 20 Nov. 2012. <http://ahdc.vet.cornell.edu/clinpath/modules/rbcmorph/schisto.htm>.
  19. ↑ Sun, J. G., A. Jurisicova, and R. F. Casper. "Detection of Deoxyribonucleic Acid Fragmentation in Human Sperm: Correlation with Fertilization in Vitro." Biology of Reproduction 56.3 (1997): 602-07. Print.
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