Ch. 11: Gene regulations How is cloning possible?

1. Ch. 11: Gene regulationsHow is cloning possible? • Every cell has the same chromosomes • Then….. Why does a heart muscle cell look different from a skin cell? • Organisms respond to their environment by altering gene expression • Central question: what regulates gene expression?

2. Differentiation 0 • Differentiation is controlled by turning specific sets of genes on or off

3. DNA Packing 0 • eukaryotic chromosomes condense during prophase of Mitosis • helps regulate gene expression by preventing transcription • Nucleosomes • Tight helical fiber = • Supercoil = coiling of the tight helical fiber

4. 0 Metaphase chromosome Tight helical fiber (30-nm diameter) DNA double helix (2-nm diameter) Linker “Beads on a string” Nucleosome (10-nm diameter) Histones Supercoil (300-nm diameter) 700 nm Animation: DNA Packing

5. X-chromosome inactivation 0 • female mammals • one of the two X chromosomes is highly compacted and transcriptionally inactive (Barr body) • Occurs early in embryonic development, thus all cellular descendants have the same inactivated chromosome • Tortoiseshell fur coloration is due to inactivation of X chromosomes in heterozygous female cats

6. 0 Early embryo Two cell populations in adult Cell division and random X chromosome inactivation Orange fur Active X X chromosomes Inactive X Inactive X Allele for orange fur Black fur Active X Allele for black fur

7. Eukaryotic gene expression 0 • Each gene has its own promoter and terminator • Are controlled by interactions between numerous regulatory proteins and control sequences

8. 0 • Regulatory proteins • Transcription factors - help RNA polymerase bind to the promoter • Activators – • Silencers - • Control sequences • Promoter • Enhancer • Related genes located on different chromosomes can be controlled by similar enhancer sequences Animation: Initiation of Transcription

9. Enhancers Promoter 0 Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Bending of DNA Transcription

10. Alternative RNA splicing 0 • Can involve removal of an exon with the introns on either side Animation: RNA Processing

11. 0 Exons 4 1 3 2 5 DNA 4 1 3 2 RNA transcript 5 RNA splicing or 4 1 2 1 5 3 2 mRNA 5

12. Small RNAs control gene expression 0 • RNA interference (RNAi) • small, complementary RNAs bind to mRNA transcripts, blocking translation • MicroRNA (miRNA) • MicroRNA + protein complex binds to complementary mRNA transcripts, blocking translation Animation: Blocking Translation Animation: mRNA Degradation

13. 0 Protein miRNA 1 miRNA- protein complex 2 Target mRNA 4 3 Translation blocked OR mRNA degraded

14. 0 • Control of gene expression also occurs with • Breakdown of mRNA • Initiation of translation • Protein activation • Protein breakdown

15. 0 Ex. Insulin formation Folding of polypeptide and formation of S—S linkages Cleavage Active form of insulin Initial polypeptide (inactive) Folded polypeptide (inactive)

16. Epigenetic Inheritance This can be accomplished by acetylation or methylation of histones

17. Regulation of Chromatin Structure Chemical modification of histone tails can affect the configuration of chromatin and thus gene expression Histone tails DNA double helix (a) Histone tails protrude outward from a nucleosome

18. Addition of methyl groups to certain bases in DNA is associated with reduced transcription in some species Acetylated histones Unacetylated histones (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

19. NUCLEUS 0 Chromosome DNA unpacking Other changes to DNA Gene Gene Transcription Exon RNA transcript Intron Addition of cap and tail Splicing Tail mRNA in nucleus Cap Flow through nuclear envelope mRNA in cytoplasm CYTOPLASM Breakdown of mRNA Broken- down mRNA Translation Polypeptide Cleavage / modification / activation Active protein Breakdown of protein Broken- down protein

20. Why so much control over gene expression? 0 • It allows cells to respond appropriately to their environment • Signal transduction pathways convert messages received at the cell surface to responses within the cell via gene expression • Three steps: • Reception – • Amplification/transduction – • Response - transcription factor is activated, enters nucleus, transcribes specific genes

21. Signaling cell 0 Signaling molecule Plasma membrane 1 Receptor protein 2 3 Target cell Relay proteins Transcription factor (activated) 4 Nucleus DNA 5 Transcription mRNA New protein 6 Translation

22. 0 • Cloning: How? Nuclear transplantation • Replacing the nucleus of an egg cell with a nucleus from an adult somatic cell. Allow embryo to form. Embryo can be used in: • Reproductive cloning • Therapeutic cloning • Grow embryonic stem cells in culture • Induce stem cells to differentiate and grow into organs, tissues, etc.

23. 0 Donor cell Reproductive cloning Nucleus from donor cell Implant blastocyst in surrogate mother Clone of donor is born Remove nucleus from egg cell Add somatic cell from adult donor Grow in culture to produce an early embryo (blastocyst) Therapeutic cloning Remove embryonic stem cells from blastocyst and grow in culture Induce stem cells to form specialized cells

24. To clone or not to clone…. 0 • Benefits of reproductive cloning? • Disadvantages of cloning?

25. Human stem cell research 0 • Ethical concerns with reproductive cloning • Ethical concerns with therapeutic cloning? • Benefits: • Human embryos – have the greatest potential to give rise to all cell types • Adult stem cells (bone marrow) or cord blood cells • can give rise to many but not all types of cells

26. Ch 12: DNA Technology • DNA profiling • Genetically modified organisms/recombinant DNA technology • Gene therapy • Genomics

27. 0 1. DNA profiling = analysis of DNA fragments to determine whether they come from a particular individual • 3 steps: • . • Amplify (copy) markers for analysis – • Compare sizes of amplified fragments by gel electrophoresis

28. 1. Select genetic marker to analyze 0 • Short tandem repeats (STRs) are genetic markers used in DNA profiling • STRs = • STR analysis compares the lengths of STR sequences at specific regions of the genome • Current standard for DNA profiling is to analyze 13 different STR sites

29. 0 STR site 2 STR site 1 Crime scene DNA Number of short tandem repeats match Number of short tandem repeats do not match Suspect’s DNA

30. 0 2. Amplify the DNA sample • Polymerase chain reaction (PCR) = method of amplifying a specific segment of a DNA molecule • Relies upon a pair of primers = • Repeated cycle of steps for PCR: • Sample is heated to separate DNA strands • Sample is cooled and primer binds to specific target sequence • Target sequence is copied with DNA polymerase

31. 0 Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules Genomic DNA 3 3 3 5 5 5 5 DNA polymerase adds nucleotides to the 3 end of each primer Cool to allow primers to form hydrogen bonds with ends of target sequences 2 3 Heat to separate DNA strands 1 3 5 3 5 Target sequence 5 5 3 5 3 5 3 Primer New DNA

32. 3. Gel electrophoresis 0 • separates DNA molecules based on size • DNA samples placed at one end of a porous gel • Current is applied and DNA molecules move from the negative electrode toward the positive electrode • DNA fragments appear as bands, visualized through staining or radioactivity or fluorescence Video: Biotechnology Lab

33. 0 Mixture of DNA fragments of different sizes Longer (slower) molecules Power source Gel Shorter (faster) molecules Completed gel

34. 0 Crime scene Suspect 1 Suspect 2 1 DNA isolated DNA of selected markers amplified 2 Amplified DNA compared 3

35. 0 Mixture of DNA fragments Longer fragments move slower A “band” is a collection of DNA fragments of one particular length Power source Shorter fragments move faster DNA attracted to + pole due to PO4– groups

36. Applications of DNA profiling 0 • Forensics - to show guilt or innocence • Establishing paternity • Identification of human remains • Species identification • Evidence for sale of products from endangered species

37. 2. Recombinant DNA technology/ Genetically Modified organisms 0 • Recombinant DNA is formed by joining DNA sequences from two different sources: • . • . • Bacterial Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors

38. Recombinant cells and organisms can mass-produce gene products 0 • Common prokaryotic host: E. coli bacterium • Has many advantages in gene transfer, cell growth, and quantity of protein production • Common eukaryotic hosts: • Yeast: S. cerevisiae • “Pharm” animals • Will secrete gene product in milk

39. 0

40. 0 • Advantages of recombinant DNA products

41. 0 • Genetically modified (GM) • Transgenic organisms contain at least one gene from another species

42. Agrobacterium tumefaciens Plant cell DNA containing gene for desired trait 1 3 2 Ti plasmid Recombinant Ti plasmid Introduction into plant cells Insertion of gene into plasmid Regeneration of plant DNA carrying new gene Plant with new trait Restriction site

43. Pros? • GM plants • GM animals

44. Cons? 0

45. 3. Gene therapy 0 • One possible procedure: • insert functional gene into a virus • virus delivers the gene to an affected cell • Viral DNA & gene insert into the patient’s chromosome • Return the cells to the patient for growth and division

46. Cloned gene (normal allele) 0 Insert normal gene into virus 1 Viral nucleic acid Retrovirus Infect bone marrow cell with virus 2 Viral DNA inserts into chromosome 3 Bone marrow cell from patient Bone marrow Inject cells into patient 4

47. 4. Genomics 0 • Genomics = • Applications: • Evolutionary relationships: Genomic studies showed a 96% similarity in DNA sequences between chimpanzees and humans • Medical advancement: Functions of human disease-causing genes have been determined by comparisons to similar genes in yeast

48. 0

49. Human Genome Project 0 • Goals: • To determine the nucleotide sequence all DNA in the human genome • To identify the location and sequence of every human gene

50. 0 • Results of the Human Genome Project • 21,000 genes in 3.2 billion nucleotide pairs • Only 1.5% of the DNA codes for proteins • The remaining 88.5% of the DNA contains • Control regions (promoters, enhancers) • Unique noncoding DNA • Repetitive DNA