Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. [1] Hybrids have been produced either between different varieties of the same species (e.g. between non-flowering potato plants and flowering potato plants) or between two different species (e.g. between wheat Triticum and rye Secale to produce Triticale).
Uses of somatic fusion include making potato plants resistant to potato leaf roll disease. [2] Through somatic fusion, the crop potato plant Solanum tuberosum– the yield of which is severely reduced by a viral disease transmitted on by the aphid vector – is fused with the wild, non-tuber-bearing potato Solanum brevidens, which is resistant to the disease. The resulting hybrid has the chromosomes of both plants and is thus similar to polyploid plants. Somatic hybridization was first introduced by Carlson et al. in Nicotiana glauca. [3]
The somatic fusion process occurs in four steps: [4]
The procedure for seed plants describe above, fusion of moss protoplasts can be initiated without electric shock but by the use of polyethylene glycol (PEG). Further, moss protoplasts do not need phytohormones for regeneration, and they do not form a callus. [5] Instead, regenerating moss protoplasts behave like germinating moss spores. [6] Of further note sodium nitrate and calcium ion at high pH can be used, although results are variable depending on the organism. [7]
Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are useful in a variety of ways, e.g.,
Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse somatic cells. Generally, human fibrocytes or leucocytes are fused with mouse continuous cell lines.
When human and mouse cells (or cells of any two mammalian species or of the same species) are mixed, spontaneous cell fusion occurs at a very low rate (10-6). Cell fusion is enhanced 100 to 1000 times by the addition of ultraviolet inactivated Sendai (parainfluenza) virus or polyethylene glycol (PEG).
These agents adhere to the plasma membranes of cells and alter their properties in such a way that facilitates their fusion. Fusion of two cells produces a heterokaryon, i.e., a single hybrid cell with two nuclei, one from each of the cells entering fusion. Subsequently, the two nuclei also fuse to yield a hybrid cell with a single nucleus.
A generalized scheme for somatic cell hybridization may be described as follows. Appropriate human and mouse cells are selected and mixed together in the presence of inactivated Sendai virus or PEG to promote cell fusion. After a period of time, the cells (a mixture of man, mouse and 'hybrid' cells) are plated on a selective medium, e.g., HAT medium, which allows the multiplication of hybrid cells only.
Several clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic and appropriate biochemical analyses for the detection of enzyme/ protein/trait under investigation. An attempt is now made to correlate the presence and absence of the trait with the presence and absence of a human chromosome in the hybrid clones.
If there is a perfect correlation between the presence and absence of a human chromosome and that of a trait in the hybrid clones, the gene governing the trait is taken to be located in the concerned chromosome.
The HAT medium is one of the several selective media used for the selection of hybrid cells. This medium is supplemented with hypoxanthine, aminopterin and thymidine, hence the name HAT medium. Antimetabolite aminopterin blocks the cellular biosynthesis of purines and pyrimidines from simple sugars and amino acids. However, normal human and mouse cells can still multiply as they can utilize hypoxanthine and thymidine present in the medium through a salvage pathway, which ordinarily recycles the purines and pyrimidines produced from degradation of nucleic acids. Hypoxanthine is converted into guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), while thymidine is phosphorylated by thymidine kinase (TK); both HGPRT and TK are enzymes of the salvage pathway. On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+) enzymes can proliferate, while those deficient in these enzymes (HGPRr- and/or TK-) can not divide (since they cannot produce purines and pyrimidines due to the aminopterin present in the HAT medium). For using HAT medium as a selective agent, human cells used for fusion must be deficient for either the enzyme HGPRT or TK, while mouse cells must be deficient for the other enzyme of this pair. Thus, one may fuse HGPRT deficient human cells (designated as TK+ HGPRr-) with TK deficient mouse cells (denoted as TK- HGPRT+). Their fusion products (hybrid cells) will be TK+ (due to the human gene) and HGPRT+ (due to the mouse gene) and will multiply on the HAT medium, while the man and mouse cells will fail to do so. Experiments with other selective media can be planned in a similar fashion.
Note: The table only lists a few examples, there are many more crosses. The possibilities of this technology are great; however, not all species are easily put into protoplast culture.
Cross | Crossed with |
---|---|
Oat | Maize |
Brassica sinensis | B. oleracea |
Torrentia fourneri | T. bailloni |
Brassica oleracea | B. campestris |
Datura innoxia | Atropa belladonna |
Nicotiana tabacum | N. glutinosa |
Datura innoxia | D. candida |
Arabidopsis thaliana | Brassica campestris |
Petunia hybrida | Vicia faba |
Table: Reference #5
Ploidy is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and pseudoautosomal genes. Sets of chromosomes refer to the number of maternal and paternal chromosome copies, respectively, in each homologous chromosome pair, which chromosomes naturally exist as. Somatic cells, tissues, and individual organisms can be described according to the number of sets of chromosomes present : monoploid, diploid, triploid, tetraploid, pentaploid, hexaploid, heptaploid or septaploid, etc. The generic term polyploid is often used to describe cells with three or more chromosome sets.
A monoclonal antibody is an antibody made by cloning a unique white blood cell. All subsequent antibodies derived this way trace back to a unique parent cell.
Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, often used in biological and medical research and commercially in the production of recombinant therapeutic proteins. They have found wide use in studies of genetics, toxicity screening, nutrition and gene expression, particularly to express recombinant proteins. CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics.
A salvage pathway is a pathway in which a biological product is produced from intermediates in the degradative pathway of its own or a similar substance. The term often refers to nucleotide salvage in particular, in which nucleotides are synthesized from intermediates in their degradative pathway.
Protoplast, is a biological term coined by Hanstein in 1880 to refer to the entire cell, excluding the cell wall. Protoplasts can be generated by stripping the cell wall from plant, bacterial, or fungal cells by mechanical, chemical or enzymatic means.
Lesch–Nyhan syndrome (LNS) is a rare inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This deficiency occurs due to mutations in the HPRT1 gene located on the X chromosome. LNS affects about 1 in 380,000 live births. The disorder was first recognized and clinically characterized by American medical student Michael Lesch and his mentor, pediatrician William Nyhan, at Johns Hopkins.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is an enzyme encoded in humans by the HPRT1 gene.
Hybridoma technology is a method for producing large numbers of identical antibodies. This process starts by injecting a mouse with an antigen that provokes an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibody producing B-cells are then harvested from the mouse and, in turn, fused with immortal B cell cancer cells, a myeloma, to produce a hybrid cell line called a hybridoma, which has both the antibody-producing ability of the B-cell and the longevity and reproductivity of the myeloma. The hybridomas can be grown in culture, each culture starting with one viable hybridoma cell, producing cultures each of which consists of genetically identical hybridomas which produce one antibody per culture (monoclonal) rather than mixtures of different antibodies (polyclonal). The myeloma cell line that is used in this process is selected for its ability to grow in tissue culture and for an absence of antibody synthesis. In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical.
Thymidine kinase is an enzyme, a phosphotransferase : 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, EC 2.7.1.21. It can be found in most living cells. It is present in two forms in mammalian cells, TK1 and TK2. Certain viruses also have genetic information for expression of viral thymidine kinases. Thymidine kinase catalyzes the reaction:
Genetics, a discipline of biology, is the science of heredity and variation in living organisms.
HAT Medium is a selection medium for mammalian cell culture, which relies on the combination of aminopterin, a drug that acts as a powerful folate metabolism inhibitor by inhibiting dihydrofolate reductase, with hypoxanthine and thymidine which are intermediates in DNA synthesis. The trick is that aminopterin blocks DNA de novo synthesis, which is absolutely required for cell division to proceed, but hypoxanthine and thymidine provide cells with the raw material to evade the blockage, provided that they have the right enzymes, which means having functioning copies of the genes that encode them.
Purine nucleoside phosphorylase, PNP, PNPase or inosine phosphorylase is an enzyme that in humans is encoded by the NP gene. It catalyzes the chemical reaction
Nucleic acid metabolism is the process by which nucleic acids are synthesized and degraded. Nucleic acids are the polymers of nucleotides. Nucleotide synthesis is an anabolic mechanism generally involving the chemical reaction of phosphate, pentose sugar, and a nitrogenous base. Destruction of nucleic acid is a catabolic reaction. Additionally, parts of the nucleotides or nucleobases can be salvaged to recreate new nucleotides. Both synthesis and degradation reactions require enzymes to facilitate the event. Defects or deficiencies in these enzymes can lead to a variety of diseases.
Purine metabolism refers to the metabolic pathways to synthesize and break down purines that are present in many organisms.
In enzymology, a dihydropyrimidine dehydrogenase (NADP+) (EC 1.3.1.2) is an enzyme that catalyzes the chemical reaction
Thymidine kinase 1, soluble, is a human thymidine kinase.
Thymidine phosphorylase is an enzyme that is encoded by the TYMP gene and catalyzes the reaction:
Cathepsin E is an enzyme that in humans is encoded by the CTSE gene. The enzyme is also known as slow-moving proteinase, erythrocyte membrane aspartic proteinase, SMP, EMAP, non-pepsin proteinase, cathepsin D-like acid proteinase, cathepsin E-like acid proteinase, cathepsin D-type proteinase) is an enzyme.
The purinosome is a putative multi-enzyme complex that carries out de novo purine biosynthesis within the cell. It is postulated to include all six of the human enzymes identified as direct participants in this ten-step biosynthetic pathway converting phosphoribosyl pyrophosphate to inosine monophosphate:
Physical map is a technique used in molecular biology to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter.
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