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Genomics, Proteomics and Transgenics

Genomics, Proteomics and Transgenics. Genetics Spring 2014. Outline. Definition. Genomics: DNA sequence, organization, function and evolution of genomes.

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Genomics, Proteomics and Transgenics

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  1. Genomics, Proteomics and Transgenics Genetics Spring 2014

  2. Outline

  3. Definition • Genomics: DNA sequence, organization, function and evolution of genomes. • Proteomics: identification of all proteins in a cell or organism (plus post-translational modifications), as well as cellular location, functions, interactions.

  4. Genomics, Proteomics and Metabolomics The study of metabolomics is attracting a flurry of biotechs and academics, with research implications ranging from plant biology to drug discovery.

  5. Recombinant DNA: gene cloning, genetic engineering: permitted construction and replication of DNA molecules derived from different sources. • Transgenic organisms have genomes that have been modified by recombinant DNA techniques = genetically modified organisms (GMOs) or chimera. Chimera

  6. Tetragameticchimerism: Fertilization of two oocytes by two spermatozoa inducing the fusion of two zygotes into one individual with genotype 46, XY/46,XX mosaic for true hermaphrodism. • Chimera (true hermaphrodism):Individual composed of cells derived from two different populations

  7. Blood group chimera: Exchange of hematopoietic stem cells by dizygotic twins in utero. Types of twins (A) monochorionic, diamniotic MZ twins (1–5% of MZ twins) (B) monochorionic,monoamniotic MZ twins (70–75% of MZ twins) (C) dichorionic, diamniotic MZ twins with separateplacentas (20–25% of MZ twins) (D) Dizygotic twins with fused placentas (0–5% of DZ twins) (E) Dizygotic twins with unfused placentas 95–100% of DZ twins.

  8. Artificial chimerism: Transfusion and transplantation. Swedes Perform Pioneering Uterine Transplants (09/21/2012) • Transgenics: Genetic engineering of featherless chicken Patents for clinical applications genetic engineering of human genes

  9. Cloning Strategies Cloning vectors are DNA molecules that carry foreign DNA into a host cell (bacterium or yeast), replicate inside this cell and produces many copies of themselves and the foreign DNA (sequence for replication, cloning sites and selection methods).

  10. Bacterial vectors include plasmids, lambda and cosmids • Plasmid- circular DNA molecule that autonomously replicates inside the bacteria; cloning limit: 100 to 10,000 base pairs. • Phage- derivatives of bacteriophage lambda; linear DNA molecules, whose region can be replaced with foreign DNA without disrupting its life cycle; cloning limit: 8-20 kb. • Cosmids- circular DNA molecule that combines features of plasmids and phage; cloning limit - 35-50 kb. • Bacterial Artificial Chromosomes (BAC)- based on bacterial mini-F plasmids. cloning limit: 75-300 kb. • Yeast Artificial Chromosomes (YAC)- artificial chromosome that contains telomeres, origin of replication, a yeast centromere; cloning limit: 100-1000 kb

  11. Cloning libraries are large sets of clones containing many (all) genes of genome of an organism. • genomic library – contains genomic fragments. • cDNA library – contains cDNA fragments. • Colony hybridization - screens a library for desired clones (labeled or unlabeled piece of desired gene) Metagenomic library consisting of 70,000 clones were screened on skim milk plate for protease activity. The positive clone showing zone of clearance in skim milk agar plate is indicated by an arrow.

  12. Yeast vectors include various YACs that can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. Yeast plasmids use prototrophic markers (URA+) into ura- cell; select on media lacking uracil (ex. YEP24).

  13. Reverse transcriptase (RT) can create a cDNA from mRNA

  14. Useful features of a plasmid cloning vector include drug-resistance gene, origin of replication, screening (selection) method and MCS (Multiple Cloning Site).

  15. Gene inactivation of lacZuses color screening to identify inserts of recombinant clones Blue white selection Nonrecombinant plasmid containing uninterrupted lacZ region; B-galactosidase cleaves Xgal in media to give blue colonies and recombinantplasmid with donor DNA inserted into the MCS, interrupting lacZ region; colonies remain white on Xgal media.

  16. Techniques: Microarrays Microarrays assess functional genomics by evaluating transcriptional profiling (DNA microarrays or chips are used). Fluorescently tagged cDNA probes are hybridized to DNA spots in the microarray for studying differential expression of thousands of genes at a time in two mRNA samples (quantitative technique).

  17. Small part of a yeast DNA chip after hybridization where each spot is portion of gene.

  18. Techniques: Chromatin Immunoprecipitation Chromatin Immunoprecipitation (ChIP) investigate interactions between DNA and protein (enhancers and activators) as it isolates DNA-protein complexes.

  19. Techniques: Two-hybrid analysis . The nuclear protein GAL4 is a positive regulator of gene expression for the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1. GAL4 recognizes a 17 base-pair long sequence in the upstream activating sequence (uas) of these genes, GAL4 binds to the DNA as a homodimer.

  20. Identification of protein-protein interactionsin vivo. If protein 1 binds protein 2, then Gal4-DBD-1 binds Gal4-AD-2 and activates expression.

  21. Transgenics • Transgenics are constructed for research purposes, as models of disease, to produce improved crops and etc … • Transgenics start with the formation of chimera leading to either knock-out or gain-of-function homozygous transgenic organisms. • Transgenicsis often controversial (concern for safety, benefits.)

  22. Gene knockout and gene replacement can occur in stem cells

  23. Knock out experiments show that Bmp7 is involved in the formation of ………………..

  24. Nuclear Transplantation Cloning of the mammal sheep Ovis aries (Wilmut, 1997) Nuclei isolated from cultured mammary gland cells from a pregnant ewe and fused with enucleated donor oocytes. Transfer of the embryos into uteri of pregnant ewes and subsequent development gave rise to the lamb Dolly (1 out of 434 transfer). Dolly, the Finn Dorset lamb and her surrogate Scottish Blackface mother. Dolly and Bonnie, her first lamb.

  25. Therapeutic cloning(somatic nuclear transfer) has for goal to develop treatments for disease and injury from stem cells that have the same nucleus, and so DNA, as the injured or sick individual. Reproductive cloning(somatic nuclear transfer) has for goal to develop clones of the donor whom the somatic nucleus was obtained.

  26. Gene Therapy

  27. Review Problems

  28. 12.19 - You want to introduce a human gene into a bacterial vector downstream of a bacterial promoter to produce a large amount of the human protein. Should you use the genomic DNA or the cDNA? Explain your reasoning. 12.22 - A DNA microarray is competitively hybridized with human genomic DNA from females and males. The female DNA is labeled with a moiety that fluoresces red; the male DNA with one that fluoresces green. What color would you expect a spot to fluoresce if: a. it corresponds to an autosomal gene? b. it corresponds to an X-linked gene? c. it corresponds to a Y-linked gene?

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