Presentation is loading. Please wait.

Presentation is loading. Please wait.

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display D-1 PowerPoint to accompany Genetics: From Genes to Genomes Fourth.

Similar presentations


Presentation on theme: "Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display D-1 PowerPoint to accompany Genetics: From Genes to Genomes Fourth."— Presentation transcript:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 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? 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 Mosaics produced by FLP, a site-specific recombination enzyme from yeast Create genes homozygous in adult that are heterozygous in early development Create genes homozygous in adult that are heterozygous in early development Fig. D.11

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

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

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

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

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

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

27 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 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 Isolate as many P element insertion alleles as possible of as many genes as possible To date – P element disruptions for 65% of genes 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 New methods of gene knockout such as RNAi to circumvent difficulties with knockouts by homologous recombination

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

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

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

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

32 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Maternal Genes Interact to Produce Morphogen Gradients Maternal-effect mutations Maternal-effect mutations Recessive mutations in maternal genes that influence embryonic development 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 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 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 Focuses on stocks with homozygous mutant sterile females Identified large number of maternal genes Identified large number of maternal genes Nobel Prize in medicine Nobel Prize in medicine

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

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

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

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

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

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

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

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

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

42 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 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 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 Regulatory system complex Transcription factors encoded by pair-rule genes initiate pattern by regulating segment polarity genes Transcription factors encoded by pair-rule genes initiate pattern by regulating segment polarity genes Interactions between cell polarity genes maintain periodicity later in development Interactions between cell polarity genes maintain periodicity later in development Activation occurs after cellularization of embryo is complete Activation occurs after cellularization of embryo is complete Diffusion of transcription factors within syncytium ceases to play a role Diffusion of transcription factors within syncytium ceases to play a role

43 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Pair-rule Genes (a) zones of expression at beginning of blastoderm stage (a) zones of expression at beginning of blastoderm stage Each gene expressed in seven stripes 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 (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 700 bp upstream regulatory region of eve gene that directs the Eve second stripe contains multiple binding sites Fig. D.23

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

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

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

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

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

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


Download ppt "Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display D-1 PowerPoint to accompany Genetics: From Genes to Genomes Fourth."

Similar presentations


Ads by Google