EUKARYOTIC GENE EXPRESSION

1. EUKARYOTIC GENE EXPRESSION

2. DNA PACKING • Histones • Nucleosomes (1st) • 30nm fibers (2nd) • Looped domains (3rd) • Heterochromatin – • not transcribed, • metaphase chromatid • Euchromatin – open, • actively transcribed • Chromosome location specific

3. GENOME ORGANIZATION - DNA LEVEL • Repetitive DNA (tandem repeats) • 10-15% in mammals • short sequences (10) repeated in series, 100,000’s times • Fragile X: 100’s instead of 30 triplet repeats • Huntington’s: CAG repeats • # of repeats correlates with severity and age of onset. • Regular, mini & micro satellites • Interspersed Repetitive DNA • Scattered, 25-40%, similar but not identical • Alu elements: family of sequences 5% primate genome, about 300 bp, many transcribed; most transposons

4. Telomeres & centromeres Useful in fingerprinting

5. Chromosome puff MULTIGENE FAMILY: Collection of similar or identical genes Salamander RNA 3 kinds of rRNA after processing, S-sedimentation Rates due to differences in density

6. NON-IDENTICAL MULTIGENE FAMILY 2 related families that code for globins 4 subunits, 2α & 2β Nonfunctional, very similar to functional genes

7. Gene Amplification: the temporary increase in the number of copies of a gene rRNA in amphibians rRNA in developing ovum, large # of ribosomes  burst of protein synthesis after fertilization Extra copies cannot replicate & are broken down Selective Gene Loss: occurs in certain insects, whole chromosomes or parts of chromosomes may be lost early in development.

8. REARRANGEMENTS • Transposons: can prevent normal functioning, may ↑ or ↓production, or be activated, 10% of human genome • Retrotransposons: use an RNA intermediate • Immunoglobulin Genes: code for antibodies, genes become rearranged as immune cells differentiate

9. Retrotransposon Movement: like retrovirus reproduction, can populate the genome in huge numbers

10. DNA Rearrangementmaturation of an immunoglobulin gene The joining of V, J & C regions of DNA in random combinations  enormous variety of antibody-producing lymphocytes • Hundreds of V regions • Several Junction regions • 1-2 Constant regions

11. CONTROL OF GENE EXPRESSION • Cellular differentiation – divergence in form & function as cells become specialized during development • 3-5% expressed at any given time (liver vs skin) • DNA Methylation – genes not expressed, methyl group (CH3) attached • Barr body – heavy methylation, inactive X, heterochromatin • Genomic Imprinting – turning off alleles • Histone Acetylation – acetyl groups on histone a.a. causing shape change, grip DNA less tightly, easier access to genes for transcription

12. OPPORTUNITIES FOR CONTROL OF GENE EXPRESSION • DNA packing, methylation, acetylation • Transcription (most important) • RNA Processing • Transport to cytoplasm • Degradation of mRNA • Translation • Cleavage, Chemical modification, Transport to cellular destination • Degradation of protein

13. TRANSCRIPTIONAL CONTROLS Transcription factors • Equivalent to repressors & activators • Bind to specific sites (TATA box) • May be near promoter • Optimum binding – 50 ish factors • “read” DNA without unzipping to find appropriate gene (zinc fingers & leucine zippers) Promoter, Enhancer, Suppressor sequences

14. Transcription initiation – controlled by transcription factors (proteins) that interact w/ DNA & each other. Typical Eukaryotic Gene – promoter, terminator, distal & proximal control elements (key to high levels of transcription) Promoter regions bind to RNA polymerases I,II,III (tRNA’s)

15. Model for enhancer action Activator proteins bind to enhancer sequences DNA bends, activators closer to promoter Protein-binding domains attach to transcription factors, form active transcription initiation complex on promoter

16. DNA Binding Domain: 3-D part of a transcription factor that binds to DNA Found in many regulatory proteins α helix β sheet held by zinc atom 2 α helices w/ spaced leucines coil

17. POST-TRANSCRIPTIONAL CONTROLS • mRNA processing – mG cap? poly A tail? • Alternative splicing – same primary transcript, different introns & exons • mRNA degradation – prokaryotic mRNA’s last a few minutes; eukaryotic- hours, can be days even weeks (hemoglobin) • Translation inhibited by masked mRNA prior to fertilization (activated in embryo) • Protein processing & degradation

18. ALTERNATIVE mRNASPLICING

19. PROTEIN DEGRADATION

20. EUKARYOTIC vs PROKARYOTIC • Genes spread out • 1 promoter = 1 gene • Many introns • Processing • 2 copies of DNA (2n) • Paired, rod shaped chrom. • 3 polymerases • Nucleus, separation of transcription/translation • Operons • 1 promoter=multiple genes • Lack introns • No processing • 1 copy of DNA (n) • Single circular chrom. • 1 polymerase • No nucleus, simultaneous transcription/translation

21. CANCER Mutations in genes that regulate growth & division, chemical carcinogens, physical mutagens (X-rays, viruses) • Oncogenes – cancer causing • Proto-oncogenes – normal gene  oncogene • Tumor-suppressor genes – prevent uncontrolled division • ras gene – proto-oncogene • p53 gene – tumor suppressor gene

22. Proto-oncogenes  Oncogenes

23. ras gene • G protein • Relays growth signal • Result – stimulation of cell cycle

24. p53 – transcription factor, activates p21, product blocks CDK’s

26. MODEL: DEVELOPMENT OF COLORECTAL CANCER

27. DNA TECHNOLOGY

28. TERMINOLOGY Recombinant DNA – DNA in which genes from two different sources are combined in vitro into the same molecule. Genetic engineering – direct manipulation of genes for practical purposes. Biotechnology – manipulation of organisms or their components to make useful products. Gene cloning – method for preparing well defined, gene sized pieces of DNA in multiple identical copies.

29. BACTERIAL PLASMIDS FOR CLONING

30. RESTRICTION ENZYMES • Cut up foreign DNA. • Endonucleases • RESTRICTION SITE • Recognition sequence • Usually symmetrical • RESTRICTION FRAGMENT • Piece of DNA cut by specific enzyme • STICKY END • Single strand end of restriction fragment

31. Cloning a human gene in a bacterial plasmid

34. USING A NUCLEIC ACID PROBE TO IDENTIFY A CLONED GENE Cloned gene of interest on a plasmid, probe is short length of radioactive single stranded DNA complimentary to part of gene. 2) Result – single stranded DNA stuck to filter paper 3) Probe DNA hybridizes with complimentary DNA on filter 4) Filter laid on photographic film, radioactive areas expose film (autoradiography)

35. MAKING COMPLEMENTARY DNA (cDNA) FOR A EUKARYOTIC GENE Expression vector - Cloning vector w/ prokaryotic promoter upstream of restriction site where eukaryotic gene can be inserted. cDNA – made in vitro using mRNA template & reverse transcriptase (DNA w/o introns)

36. TERMINOLOGY Yeast artificial chromosomes (YACs) – vectors that combine the essentials of a eukaryotic chromosome ( origin for replication, centromere, 2 telomeres) w/ foreign DNA. Electroporation – application of electric pulse to solution containing cells creating temporary hole in membrane allowing DNA to enter. Genomic library – complete set of thousands of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome. cDNA library – library containing a collection of genes (represents only part of a cell’s genome – only the genes that were transcribed in the starting cells)

37. Shown - 3 of the thousands of “books” in the library. Each is a bacterial clone containing one particular variety of foreign genome fragment in its recombinant plasmid The same 3 foreign genome fragments in a phage library

38. PCR • Polymerase chain reaction • Technique to quickly amplify (copy many times) a piece of DNA w/o using cells • Start w/ double stranded DNA (“target”), add to polymerase, nucleotide supply & primers • 5 minutes per cycle

39. DNA ANALYSIS & GENOMICS Genomics – the study of whole sets of genes and their interactions Gel electrophoresis – separates macromolecules (nucleic acids or proteins) based on size, electrical charge and other physical properties. Southern blotting – hybridization technique that enables researchers to determine the presence of certain nucleotide Sequences in a sample of DNA. Restriction fragment length polymorphisms RFLPs - differences in DNA sequence on homologous chromosomes that can result in different restriction fragment patterns. Scattered abundantly throughout genomes

40. GEL ELECTROPHORESIS Negatively charged DNA migrates toward positive electrode. Longer fragments travel more slowly DNA samples arranged in bands along a “lane” according to size. Shorter fragments travel farthest 3 DNA samples placed in wells. Electrodes attached & voltage applied

41. Using restriction fragment patterns to distinguish DNA from different alleles • 2 homologous segments w/ different alleles • Electrophoresis separates the fragments; allele 1has 3 fragments, allele 2 has 2 • Addition of binding dye, fragments fluoresce pink; shown: 6 samples cut w/ a restriction enzyme

42. RESTRICTION FRAGMENT ANALYSIS BY SOUTHERN BLOTTING DNA denatured & transferred to paper Radioactivity exposes film, image forms – bands w/ DNA base-pairs w/ probe Probe complimentary to gene of interest

43. MAPPING GENOMES AT THE DNA LEVEL • Human Genome Project – effort to map the entire human nucleotide sequence for each chromosome. • Genetic (Linkage) Mapping – construction of a linkage map using various genetic markers. • Physical Mapping: Ordering DNA Fragments • chromosome walking: make fragments that overlap, then use probes of the ends to find the overlaps • Bacterial artificial chromosome (BAC): artificial version of a bacterial chromosome that can carry inserts of 100,000 – 500,000 base pairs • DNA Sequencing – determining the nucleotide sequence of a DNA segment or an entire genome. 3 sequencing methods • Alternative Approaches to Whole-Genome Sequencing

44. Chromosome walking • Prepare probe to match 3’ end • Cut starting DNA w/ 2 restriction • enzymes & clone fragments • 3) Use probe 1 to screen library II • for DNA fragments that overlap the • known gene • 4) Isolate DNA from tagged clone, • prepare probe 2 to match 3’ end of • that segment. • 5) Use probe 2 to screen library I for • an overlapping fragment farther along • 6) Repeat 4&5 with new probes & • Alternating libraries to “walk” down DNA • 7) Result – DNA map w/ series of • known markers (sequences) in a • Known order & separated by known • distances

45. SANGER METHOD • 4 portions each incubated w/ • primer • DNA polymerase • 4 deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, dTTP • Different one of the 4 nucleotides in modified dideoxy (dd) form

46. 4 portions each incubated w/ • primer • DNA polymerase • 4 deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, dTTP • Different one of the 4 nucleotides in modified dideoxy (dd) form Synthesis of new strands begins at primer & continues until a dideoxyribonucleotide is incorporated, which prevents further synthesis. SANGER METHOD SANGER METHOD Eventually, a set of labeled strands of various lengths is generated.

47. SANGER METHOD New strands separated by electrophoresis Sequence can be read from bands on autoradiograph and original template sequence deduced. Longest fragment ends with a ddG, so G must be the last base in the sequence

48. ALTNERATIVE STRATEGIES FOR SEQUENCING AN ENTIRE GENOME - The arrangement of DNA fragments in order depends on their having overlapping regions Cut DNA of chromosome into small fragments Clone fragments in plasmid or phage vectors Sequence fragments Assemble overall sequence

50. DNA microarray assay for gene expression Researcher simultaneously test all the genes expressed in particular tissue for hybridization with an array of short DNA sequences representing thousands of genes. Fluorescence intensity indicates relative amount of mRNA in tissue.