Locating genes on chromosomes

1. Locating genes on chromosomes • Fluorescent in situ hybridization (FISH) • Somatic cell hybridization

2. 1. Fluorescent In Situ Hybridization (FISH) • 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 microscope A human telomere probe to the Pacific oyster (Crassostrea gigas)

4. 18S-28S rRNAgenes (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 2 2 1 2 3 4 • Galactosidokinase 1st hybrid: 9, 11, 17, 21Gal + 2nd hybrid: 9, 17 Gal + 3rd hybrid: 11, 17Gal + 4th hybrid: 9, 11Gal - Which chromosome is the galactosidokinase located on? 17

9. Cloning • Standard Cloning – isolate and identify gene of interest to make lots of it to study for basic research and sequencing. • ExpressionCloning– isolate/identify a gene of interest to make its protein for medicine, bioremediation, antibodies etc. • Whole-animalCloning – to generate transgenic animals, to assist infertile couples, and for tissue regeneration and transplant.

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

11. Restriction enzymes • Produced by bacteria as a defense mechanism against infection by viruses • Recognition sequence: a specific nucleotide sequence recognized by restriction enzymes

12. Restriction enzymes

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

14. 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

15. 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)

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

17. Plasmid vector

18. Xgal : bromo-chloro-indolyl-galactopyranoside

22. Cosmid Vector

25. 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 Example • Expression of green fluorescent protein (GFP) in transgenic zebrafish

26. 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.

27. DNA libraries • One clone represents one part of a genome • cDNA library: a collection of cDNA clones • Genomic library: collection of recombinant DNA-bearing bacteria or phages

28. Collection of bacteria with inserts is a library Reverse transcriptase

29. Probes • Probes: used to identify specific sequences • Probes can be radioactive, fluorescent, or chromatic dyes

30. Radioactive probe searching a phage library

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

33. 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

34. 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.

35. 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)

37. Ethical, Social & Legal Questions • The cloning of humans??? Technology barriers are gone “can we” “should we” • Potential abuses of the technology • More readings (Chapter 24)

38. Video • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#