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Genetics: From Genes to Genomes

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1 Genetics: From Genes to Genomes
PowerPoint to accompany Genetics: From Genes to Genomes Fourth Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver Reference D Prepared by Malcolm Schug University of North Carolina Greensboro Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

2 Drosophila melanogaster: Genetic Portrait of the Fruit Fly
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

3 Figure D.1 Fig. D.1 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

4 Outline of Reference D Structure and organization of genome Life cycle
Techniques of genetic analysis Balancer chromosomes P-element transposons Production of mosaics Ectopic gene expression Targeted gene knockouts Drosophila genome project Genetic analysis of body plan development in Drosophila: a comprehensive example Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

5 Structure of the Drosophila Genome
Chromosomes of Drosophila Four chromosomes designated 1-4 XY sex determination (XX females, XY males) Sex determined by X:A ratio Figure D.3 Fig. D.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

6 Giant polytene chromosomes of larval salivary gland are key tools
Replicate times sister chromatids stay associated under perfect lateral register Homologous chromosome stay tightly synapsed Chromocenter – common region where centromeres coalesce Figure 14.6 Check Figure number in Chapter 14 Fig. 14.6a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

7 Drosophila Genome 170,000 kb of DNA
21% is highly repetitive satellite DNA in heterochromatin and Y chromosome 3% repeated genes for rRNA, 5s RNA and histones 9% is 50 families of transposons 2-9 kb in length Telomeres do not have simple repeats Telomeres have transposable element sequences 67% of genome-unique sequences in euchromatin comprising about 13,600 genes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

8 Life cycle Fig. D.4 Figure D.4
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.4

9 Many Adult Structures Develop from Imaginal Discs in Larvae and Pupae
Figure D.5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.5

10 Balancer Chromosomes help Preserve Linkage
Balancers carry multiple, overlapping inversions Most contain a dominant marker and recessive lethal mutation that prevents survival of homozygotes Useful in many genetic experiments Figure D.6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.6

11 Techniques of Genetic Analysis
In Drosophila, crossing over occurs only in females Absence of crossing over in males has considerable technical significance Maintain linkage relationships by inheritance through male parent In females, only a moderate amount of crossing over occurs 1-2 crossovers per meiosis No crossing over in heterochromatin or 4th chromosome Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

12 P-element Transposons are Critical Tools in Molecular Genetics
Hybrid dysgenesis Males from Drosophila strains carrying P elements crossed to females that lack P elements P element becomes highly mobile in germ line of F1 hybrids Chromosome breakage reduces fertility in hybrids Progeny of F1 flies carry many new mutations induced by P element insertions Molecular details P element primary transcript encodes transposase that catalyzes transposition Cross between P and M strain causes hybrid dysgenesis Cross between P and P strain does not Eggs produced by P female have repressor protein that prevents transposition Repressor coded for by alternatively spliced P element mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

13 Figure D.7 Fig. D.7 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

14 Transformation: the Introduction of Cloned DNA into Flies
P elements used as vectors Insert fly DNA into intact P element and then into plasmid Inject into syncytial embryos from M strain mothers Cross to P males Mimicking hybrid dysgenesis Figure D.8a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.8a

15 P Element Tagging Helps Clone Genes of Developmental Interest
P element contains visible marker such as white and plasmid with antibiotic resistance Plasmid rescue – cut DNA from mutant strain and circularize with DNA ligase Transform into E. coli Plasmid plus gene-containing fly DNA flanking P element insertions Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

16 Isolation of a DNA Sequence from a Transposon-tagged Gene
Plasmid Rescue Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

17 Isolation of a DNA Sequence from a Transposon-tagged Gene
Inverse PCR Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

18 Enhancer Trapping to Identify Genes by Expression Pattern
P element with lacZ gene downstream of promoter When mobilized, 65% of new insertions express lacZ reporter during development Promoter can only activate transcription if under control of enhancers of genes near insertion site Detects genes turned on in certain tissues Genes isolated by plasmid rescue Figure D.10 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.10

19 Genetic Mosaics Composed of cells of more than one genotype
Generated by mitotic recombination X-ray induced mutations cause crossing-over in homologues in somatic cells X-ray method has now been replaced by site-specific recombination enzyme from yeast called FLP Figure D.11 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.12 a

20 Using Mosaics to Determine if Genes Needed for Embryo Viability Also Play a Role Later in Development What if a mutant gene arrests development early when homozygous? How do you study its effects later in development? Mosaics produced by FLP, a site-specific recombination enzyme from yeast Create genes homozygous in adult that are heterozygous in early development Figure D.13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.11

21 Testing Ability of Cell and its Descendents to Contribute to Adult Structures
Minute mutations 50 minute genes throughout genome Heterozygotes cause reduced rate of protein synthesis Mitotic recombination produces Minute+/Minute+ cells in Minute+/Minute- background +/+ cells will grow big Determines fate of cells by examining tissue growth after mitotic recombination event Figure D.11b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.12 b

22 Ommatidia are facets of fly’s compound eye
Mosaics Used to Tell When and Where a Gene Must be Expressed to Generate Normal Phenotype Ommatidia are facets of fly’s compound eye During development undifferentiated cells are recruited to become photo-receptors (R1-R8) for each facet Final photoreceptor in each group is R7 Figure D.12a,b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.13 a,b

23 Mosaics generated with marked cells (white)
Sevenless (sev) and bride of sevenless (boss) mutants develop without R7 Mosaics generated with marked cells (white) Requirement for Sev+ is cell autonomous Presumptive R7 cell must make Sev+ protein Sev+ affects only cell in which it is made Figure D.12c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.13c

24 R7 cell develops properly only if R8 cell makes Boss+
Boss+ is not cell autonomous Produced in one cell (R8) but affects differentiation of different cell (R7) Figure D.12d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.13d

25 Using Mosaics to Determine Importance of Gene Product for Oogenesis
Expression outside of cells or tissue where gene is normally expressed Eyeless protein under control of heat shock promoter and introduce into genome by P element transposition Eyes appear anywhere on fly’s body when heat shocked Figure D.15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.15

26 The Drosophila Genome Project
13,600 known or predicted genes present One gene every 9 kb Half of fly proteins homologous with mammalian proteins One third homologous to nematodes 61% of human disease genes have homologues in flies 30% of genes unrelated to genes in other organisms Only 4000 genes essential for viability Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

27 To date – P element disruptions for 65% of genes
A New Direction for Drosophila Genome Project: Obtaining Mutations in All Fly Genes Isolate as many P element insertion alleles as possible of as many genes as possible To date – P element disruptions for 65% of genes New methods of gene knockout such as RNAi to circumvent difficulties with knockouts by homologous recombination Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

28 Genetic Analysis of Body Plan Development: a Comprehensive Example
How body becomes specialized along both anterior-posterior (AP) and dorsal-ventral (DV) axes Segmentation genes subdivide body into an array of identical body segments Homeotic genes assign a unique identity to each body segment How does the developing animal establish the proper number of body segments? How does each body segment know what kinds of structures it should form and what role it should play in the animal’s body? Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

29 Figure D.18 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.18

30 Replace d. with fig D.19d from 3e Fig. D.19
Figure D.19 Replace d. with fig D.19d from 3e Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.19

31 Four Classes of Genes Responsible for Formation of Segments
Maternal genes Gap genes Pair-rule genes Segmentation polarity genes Function in a hierarchy that progressively subdivides the embryo into successively smaller units Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

32 Maternal Genes Interact to Produce Morphogen Gradients
Maternal-effect mutations Recessive mutations in maternal genes that influence embryonic development Maternally supplied components account for formation of body plan between fertilization and end of 13 syncytial divisions Nusslein-Volhard and Wieschaus screened thousands of mutagen treated chromosomes by examining phenotypes of embryos from homozygous mutant mothers Focuses on stocks with homozygous mutant sterile females Identified large number of maternal genes Nobel Prize in medicine Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

33 Morphogen – substances that define different cell fates in a concentration-dependent manner
Interaction of two signaling centers located in the anterior and posterior poles of the egg pattern insect body axis Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

34 bicoid (bcd) Encodes the Anterior Morphogen
Image seems blurry – replace with Figure D.20 a and c if possible – if not delete this text box. Figure D.20 a, c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.20 a,c

35 How Bcd Protein Works Figure D. 21 Figure D. 21 top
Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

36 Figure D.21 bottom Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.21

37 Specification of Segment Number through Activation of Zygotic Genes in Successively More Sharply Defined Regions of the Embryo Zygotic genes Transcribed and translated from DNA in nuclei of embryonic cells Expression begins in syncytial blastoderm stage (roughly cycle 10) Nusslein-Volhard and Wieschaus performed zygotic mutant screen Ethyl methane sulfonate (EMS) mutagenesis Homozygous mutant embryos Recessive lethals Three classes of genes 9 gap genes 8 pair-rule genes 17 segment-polarity genes Hierarchy of gene expression Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

38 Gap genes Expressed first
Gap mutants show a gap in segmentation pattern at positions where particular gene is absent Binding sites in promoter have different affinities for maternal transcription factors Gap genes encode transcription factors that influence expression of other gap genes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

39 Zones of Expression of Four Gap Genes: Hunchback, Kruppel, Knirps, and Giant in Late Syncytial Blastoderm Embryos Figure D.22a Figure D.22a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

40 Defects in Segmentation from Mutations in Gap Genes
Figure D.22b B) C) D) A) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.22b

41 Mutations in Gap Gene Result in Loss of Segments Corresponding to Zone of Expression
Figure D.22 c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.22 c

42 Segment Polarity Genes are Lowest Level of Segmentation Hierarchy
Mutations in segment polarity genes cause deletion of part of each segment and its replacement by mirror image of different part of next segment Regulatory system complex Transcription factors encoded by pair-rule genes initiate pattern by regulating segment polarity genes Interactions between cell polarity genes maintain periodicity later in development Activation occurs after cellularization of embryo is complete Diffusion of transcription factors within syncytium ceases to play a role Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

43 Pair-rule Genes (a) zones of expression at beginning of blastoderm stage Each gene expressed in seven stripes (b) formation of Eve stripe 2 requires activation by Bcd and Hb proteins and repression by Gt and Kr proteins 700 bp upstream regulatory region of eve gene that directs the Eve second stripe contains multiple binding sites Figure D.23 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.23

44 Figure D.24 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.24

45 Figure D.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.25

46 Each Segment Establishes own Identity Through Activation of Homeotic Genes
Homeotic mutations cause different segments to develop as if located elsewhere bithorax (bx) Anterior third thoracic segment (T3) develops like second anterior thoracic segment (T2) postbithorax (pbx) posterior T3 transforms into posterior T2 Figure D.26 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.26

47 Figure D.28 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Figure D.28

48 Antennapedia Complex and the Homeobox
Homeotic selector genes Two clusters of genes on third chromosome – antennapedia complex and bithorax complex Responsible for determining segment identity Figure D.27 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. D.27

49 Antennapedia Complex and the Homeobox
Specifies the identities of segments in head and anterior thorax Five genes labial (lab) – expressed in intercalary region proboscipedia (pb) expressed in maxillary and labial segments Deformed (Dfd) – expressed in mandibular and maxillary segments Sex combs reduced (Scr) – expressed in labial and T1 segments Antennapedia (Antp) – expressed mainly in T2, but also active in lower levels in all three thoracic segments and abdomen Other genes (not homeotic) zerknullt (zen) – specifies dorsal embryonic structures fushi tarazu (ftz) – segmentation gene of pair-rule class bicoid (bcd) – encodes maternally supplied anterior determinant Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display


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