Locating genes on chromosomes

1. Locating genes on chromosomes • 2 methods to locate genes on chromosomes: • Fluorescent in situ hybridization (FISH) • Somatic cell hybridization

2. 1. Fluorescent In Situ Hybridization (FISH) • Direct method to localize genes on chromosomes • Using fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity.

3. Method • Fix chromosomes to a slide • Denature DNA by immersion in a strong base • Hybridize probe and chromosomes • Visualize probe with fluorescent scope A human telomere probe to the Pacific oyster (Crassostrea gigas)

4. 18S-28S rRNA genes (yellow) Kazuhiko Yamada, Chizuko Nishida-Umehara, Yoichi Matsuda, Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (Pelodiscussinensis, Trionychidae), Chromosome Research, Volume 13, Issue 1, Jan 2005, Pages 33 - 46

5. Human cell Mouse cell • Grow together in the presence of inactive Sendai virus • Promotes fusion of cells and nuclei 2. Somatic Cell hybridization • Somatic – body cell (not a gamete, sex cell) • Hybridization – two non-homologous cells

6. Fusion of cells • Single nucleus (unstable) • Random loss of human chromosomes • Complete set of mouse chromosome and few human chromosomes Somatic cell hybrid

7. Grow different cell lines each with different human chromosomes lost • Screen cells to determine which human chromosomes are left • Probe for gene of interest in each cell line • Survey hybrid cell lines to see which human proteins are expressed • Correlate specific proteins with specific human chromosome • E.G. Galactosidokinase

8. Galactosidokinase activity Hybrid # chromosomes 9 11 17 21 + + + + + - + - - - + - + - - - + ++- 4 2 1 1 1 2 3 4 • Galactosidokinase 1st hybrid: 9, 11,17,21 Gal + 2nd hybrid: 9, 17 Gal + 3rd hybrid: 17 Gal + 4th hybrid: 9 Gal - Which chromosome is the galactosidokinase located on? 17

9. Recombinant DNA A novel DNA molecule formed by joining together DNA sequences from different biological sources.

10. Vector Restriction enzyme Restriction enzyme: recognize and cut DNA molecules at specific nucleotide sequence (CTTAAG)

11. Restriction enzymes • Produced by bacteria as a defense mechanism against infection by viruses • More than 3500 restriction enzymes have been identified, about 150 are commonly used • Recognition sequence: a specific nucleotide sequence recognized by restriction enzymes EcoR I: CTTAAG

12. EcoRI • Recognizes 5’GAATTC 3’ • Palindrome – same sequence, antiparallel • Cuts between GA • Creates “sticky ends”

13. Vectors • They are carrier DNA molecules that transfer and help replicate inserted DNA fragments (P325-P330) • They differ in terms of the host cells they are able to enter, in the size of inserts they can carry • They should contain several restriction enzyme cleavage sitesthat allow insertion of DNA fragments to be cloned • They must be able to independently replicate themselves and any DNA fragment they carry once they are inside a host cell • Selectable marker genes: to distinguish host cells that have taken up vectors from those have not, usually an antibiotic resistance gene

14. Vectors • Plasmid – 10-20kbp of target DNA • Bacteriophage – Lambda can carry a 15 kb insert • Cosmids – a plasmid + Lambda DNA hybrid, 50 kb inserts • Bacterial artificial chromosomes (BACs) – F plasmids that can accept 150-300 kb inserts • Yeast artificial chromosomes (YACs) – can accept big inserts (~1Mb)

23. 1. Excise GFP gene and inserted into a plasmid Jellyfish GFPgene 3. Insert Keratin promoter before Coding sequence of GFP TATA Zebrafish keratin gene 2.2kb promoter GFP CDs 2. Excise keratin promoter Recombinant DNA • Expression of green fluorescent protein (GFP) in transgenic zebrafish

24. 4. Inject recombinant into embryo 5. Let embryo develop 6. GFP expressed where keratin is!! Ju B, Xu Y, He J, Liao J, Yan T, Hew CL, Lam TJ, Gong Z. 1999. Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters. Dev Genet. 1999;25(2):158-67.

25. Transgenics • Plants (or animals) carrying a foreign gene are called transgenic organisms. • Transgenic plants • Transgenic animals

26. Herbicide resistant plants http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicPlants.html Transgenic plants Golden rice, a strain genetically modified to produce β-carotene, a precursor to vitamin A

27. Transgenic Animals Transgenic Atlantic salmon (bottom) overexpressing a growth hormone (GH) gene display rapidly accelerated rates of growth compared to wild strains and nontransgenic domestic strains (top). GH salmon weigh an average of nearly 10 times more than nontransgenic strains.

28. Gene therapy • A therapeutic technique that aims to transfer normal genes into a patient’s cells • Human gene therapy began in 1990 with the treatment of a young girl named Ashanti DeSilva, who has a heritable disorder called severe combined immunodeficiency (SCID) cased by a mutation in the gene encoding the enzyme adenosine deaminase (ADA)

31. From 1990 to 1999, more than 4000 people underwent gene therapy for a variety of genetic disorders. Often failed. • Problems vectors---retrovirus are capable of causing an immune response---Jesse Gelsinger it is not yet possible to reliably target insertion of genes into specific locations in the genome

32. Ethical, Social & Legal Questions • Genetically Modified (GM) Foods --- Are they safe to eat? In Europe and Asia, labeling of food containing genetically modified ingredients is mandatory In US, such labeling is not required at present, and foods with less than 5 percent of their content from genetically modified organisms (GMOs) can be labeled as GMO-free

33. The cloning of humans??? • Technology barriers are gone “can we” “should we” • Potential abuses of the technology