Somatic cell genetics

Genetics

1. SOMATIC CELL GENETICS Term paper presentation GP-501 SUBMITTED TO: Dr. D. Shivani PROFESSOR DEPT. OF GENETICS AND PLANT BREEDING SUBMITTED BY: ANIL KUMAR RAM/2020-68

2. CONTENTS Introduction Somatic cell hybridization Spontaneous and Induced fusion Gene mapping by Somatic cell hybridization Selection of Hybrids Gene mapping using chromosomal rearrangements Deletion mapping Duplication mapping Translocation mapping Case study References

3. INTRODUCTION The study of mechanism of inheritance in animals and plants by using cells in culture. In such cells, chromosomes and genes can be reshuffled by parasexual methods, rather than having to depend upon the chromosome segregation and genetic recombination that occur during the meiotic cell division preceding gamete formation and sexual reproduction. Genetic analysis is concerned with the role of genes and chromosomes in the development and function of individuals and evolution of species.

4. SOMATIC CELL HYBRIDIZATION: SOMATIC CELL HYBRIDIZATION 1 st demonstrated by G. Barski et.al in 1960. The technique of hybrid production through the fusion of somatic cells under invitro condition, is called somatic hybridization. It may be heterokaryon ( fusion of cells of two different species) or homokaryon ( two parental cell come from two different species)

5. Spontaneous and Induced fusion The frequency of spontaneous cell fusion is very low. It can be increased either by addition of UV-inactivated Sendai virus or the chemical polyethylene glycol.

6. Gene mapping by Somatic cell hybridization CHROMOSOMAL MAPPING

7. CELL FUSION Mouse-human somatic cell hybrids have been particularly useful for mapping human genes. The fusion is usually mediated chemically with polyethylene glycol, which affect cell membrane or with inactivated virus. Cell fusion 1 st produce binucleate hybrid cell. Cell fusion followed by nuclear fusion to produce uninucleate hybrid cell.

8. Selection of Hybrids Somatic cell hybridization experiments are done using selection procedure that prevent the growth of parental cells. The most commonly used selection medium is HAT medium (hypoxanthine-aminopterin-thymidine). In HAT medium, if one parental cell type is deficient for the enzyme thymidine kinase (TK - ) and other parental cell type is deficient for enzyme hypoxanthine phosphoribosyl transferase (HPRT - ), the parental cell type will not grow. Thus HAT medium select only hybrid cells. The hybrid cell survive as long as they retain the human copy of TK + allele.

9. Chromosomal loss & mapping The mouse-human somatic cell hybrids have four important features that make them especially useful for genetic analysis:- The mouse and human chromosomes are easily distinguishable. All mouse chromosomes are usually retained in hybrid cells but only few human chromosomes are retained in hybrid colons. Human chromosomes are eliminated at random during subsequent mitotic division of hybrid cells. Both sets of chromosomes, human and mouse are expressed in hybrid cells. Genes are shown to be located on specific human chromosome by correlating the human gene product in hybrid cell colons.

10. Gene mapping using chromosomal rearrangements With the somatic cell technique, we can map a gene to specific region of a chromosome if informative chromosome rearrangements are available. Three types of rearrangement that is valuable in regionalizing a gene locus are translocation, deletion & duplication. Deletions and duplications have been extraordinarily useful in locating genes on the cytological maps of Drosophila chromosome.

11. DELETION MAPPING The basic principle in deletion mapping is that a deletion that uncovers a recessive mutation must lack a wild type copy of the mutant gene This fact localizes that gene is within the boundaries of deletion. This approach has been most thoroughly developed in Drosophila genetics, where the large, banded polytene chromosomes make it possible to define deletion and duplication accurately. As an example of cytogenetic mapping, let’s consider ways of localizing the X-linked white gene of Drosophila, a wild-type copy of which is required for pigmentation in the eyes.

12. CONT… If the white gene has been deleted from the Df chromosome, then the w/Df heterozygotes will not be able to make eye pigment. The eyes of the w/Df heterozygotes will therefore be white (the mutant phenotype). If, however, the white gene has not been deleted from the Df chromosome, then the w/Df heterozygotes will have a functional white gene somewhere on that chromosome, and their eyes will be red (the wild phenotype). By looking at the eyes of the w/Df heterozygotes, we can therefore determine whether or not a specific deficiency has deleted the white gene. If it has, white must be located within the boundaries of that deficiency.

13. DUPLICATION MAPPING We can also use duplications to determine the cytological locations of genes. The procedure is similar to the one using deletions, except that we look for a duplication that masks the phenotype of a recessive mutation. The basic principle in duplication mapping is that a duplication that covers a recessive mutation must contain a wild type copy of the mutant gene. This fact localizes that gene within the boundaries of the duplication

14. Only one of these duplications, Dp2 , masks the white mutation; thus a wild-type copy of white must be present within it.

15. TRANSLOCATION MAPPING Consider an example in which human hybrid cell line were developed from a patient with a reciprocal translocation between X chromosome and chromosome 14. Long arm of X chromosome was translocated on the tip of chromosome 14 and small piece of chromosome 14 was translocated into X chromosome. Only those hybrids survived on HAT medium which has chromosome 14 with long arm of X attached. Since they survived on HAT medium, they must be HPRT + ,and since the HPRT gene is known to be x linked, the HPRT gene must map to the long arm of x chromosome. Two other x linked genes were also expressed in these hybrids: PGK and G6PD. These loci must also map to the long arm of x chromosome.

16. CASE STUDY Name of Journal: BMC biotechnology NAAS Score: 8.30 Received: 21 March 2017 Accepted: 1 June 2017 Published: 6 June 2017

17. BACKGROUND Somatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar ‘Iwa’ using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent.

18. RESULTS Following 5 days’ exposure of potato cell suspension cultures to 20 μg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type ‘Iwa’ potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants

19. The arrow indicates a rare potato cell colony rescued from cell suspension culture growing on media containing 20 μ g/l chlorsulfuron (each Petri dish was seeded with approximately 5 × 104 cells) An in vitro potato plant regenerated from a cell colony with resistance to chlorsulfuron A B Somatic cell selection and regeneration of chlorsulfuron resistance in potato

20. Conclusions Somatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS ( acetohydroxyacid synthase) genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato.

21. REFERENCES Barrell, P., Latimer, J., Baldwin, S., Thompson, M., Jacobs, J., & Conner, A. (2017). Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene.  BMC Biotechnology ,  17 (1). doi : 10.1186/s12896-017-0371-4 Gardner, E., Simmons, M., & Snustad , D. (2010).  Principles of genetics  (pp. 183-186). New Delhi: Wiley India. sarkar . (2021). Cell cell hybridization or somatic cell hybridization. Retrieved 6 March 2021, from https://www.slideshare.net/Subhradeepsarkar/cell-cell-hybridization-or-somatic-cell-hybridization Snustad , D., Simmons, M., Jenkins, J., Crow, J., & Price, H. (1997).  Principles of genetics  (pp. 166-172). Chichester: John Wiley. Somatic Cell Genetics. (2021). Retrieved 6 March 2021, from https://www.ncbi.nlm.nih.gov/books/NBK216404/

22. THANK YOU!