Genetics: From genes to genomes - Chapter 12: How genes travel on chromosomes

PowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of Georgia*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display*PART IVCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12The Eukaryotic Chromosome12.1 Chromosomal DNA and Proteins12.2 Chromosome Structure and Compaction12.3 Chromosomal Packaging and Function12.4 Replication and Segregation of ChromosomesHow Genes Travel on ChromosomesCHAPTER OUTLINECHAPTERChromosomal DNA and proteinsChromosomes have a versatile, modular structure for packaging DNA that supports flexibility of form and functionChromatin is the generic term for any complex of DNA and protein found in a nucleus of a cellChromosomes are the separate pieces of chromatin that behave as a unit during cell divisionChromatin is ~ 1/3 DNA, 1/3 histones, 1/3 nonhistone proteinsDNA interaction with histones and nonhistone proteins produces sufficient level of compaction to fit into a cell nucleus Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Histone proteinsHistones – small, positively-charged, and highly conservedBind to and neutralize negatively charged DNAMake up half of all chromatin protein by weightFive types - H1, H2A, H2B, H3, and H4Core histones (H2A, H2B, H3, and H4) make up the nucleosomePosttranslational modifications of histones H3 and H4Methylation and acetylation of histone tailsAffect chromatin structure and gene expression in specific chromosomal regionsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Nonhistone proteinsHundreds of other proteins that make up chromatin and are not histones200 – 200,000 molecules of each kind of nonhistone proteinLarge variety of functionsStructural role – chromosome scaffold (see Figure 12.2)Chromosome replication – e.g. DNA polymerasesChromosome segregation – e.g. kinetochore proteins (see Figure 12.3)Active in transcription – largest groupCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Different levels of chromosome compactionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Table 12.1The nucleosome is the fundamental unit of chromosomal packagingDNA wraps twice around nucleosome core octamer (Figure 12.5) and forms 100 Å fiberResults in 7-fold compaction of DNASpacing and structure of nucleosomes affect genetic functionDetermines whether DNA between nucleosomes is accessible for proteins that initiate transcription, replication and further compactionArrangement along chromatin is highly defined and transmitted from parent to daughter cells during DNA replicationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Nucleosomes look like "beads on a string" in the electron microscopeDiameter of DNA helix (string) is 20 ÅDiameter of nucleosome core (bead) is 100 ÅCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.4The nucleosome core is an octamer of two each of histones H2A, H2B, H3, and H4Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.5160 bp of DNA wraps twice around a nucleosome core40 bp of linker DNA connects adjacent nucleosomesHistone H1 associates with linker DNA as it enters and leaves the nucleosome coreX-ray crystallography of a nucleosomeDNA bends sharply at several places as it wraps around the core histone octamerBase sequence dictates preferred nucleosome positions along the DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.6Models of higher-order packaging100 Å fiber is compacted into 300 Å fiber by supercoilingResults in an additional 6-fold compaction of DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.7aThe radial loop-scaffold model for higher levels of compactionSeveral nonhistone proteins (NHPs) bind to chromatin every 60-100 kb and tether the 300 Å fiber into structural loopsOther NHPs gather several loops together into daisylike rosettes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.7bThe radial loop-scaffold model for higher levels of compaction (cont)Condensins may further condense chromosomes into a compact bundle for mitosisCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.7cThe karyotype of a human female examined by high-resolution G-bandingCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.9Metaphase chromosomes stained with Giemsa have alternating bands of light and dark stainingEach band is contains many DNA loops and ranges from 1 to 10 Mb in length Banding patterns on each chromosome are highly reproducible Locations of genes in relation to chromosomal bandsShort arm = p armLong arm = q armWithin each arm, light and dark bands are numbered consecutivelyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.10Comparison of chromosomes from humans and great apesBanding patterns in some chromosomes (i.e. Chr 1) are nearly identicalThe metacentric Chr 2 in humans was formed by fusion of two acrocentric chromosomes in great apes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.11Chromosome 1Acrocentric chromosomes of great apesChromosomal packaging and functionHeterochromatin – highly condensed, usually inactive transcriptionallyDarkly stained regions of chromosomesConstitutive – condensed in all cells [e.g. most of the Y chromosome and all pericentromeric regions (see Fig 12.12)] Facultative – condensed in only some cells and relaxed in other cells (e.g. position effect variegation, X chromosome in female mammals) Euchromatin – relaxed, usually active transcriptionallyLightly stained regions of chromosomesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Position-effect variegation (PEV) in DrosophilaWhite+ (w+) gene is normally located in euchromatinChromosomal inversion can result in w+ gene being located adjacent to heterochromatinCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.13aw+ gene is silenced in some cells (no pigment) but is expressed in other cells (red pigment)w+ gene is expressed in all cells (red pigment)PEV in Drosophila (cont)Gene silencing can be caused by spreading of heterochromatin into nearby genesSpreading can occur over > 1000 kb of chromatinHeterochromatin spreads further in some cells than in othersCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.13bIdentification of molecules involved in heterochromatin formationGenetic screens in Drosophila were used to identify mutations that enhance or suppress the extent of PEVMutation in enhancer of PEV results in more cells having gene inactivationEncodes protein that localizes to heterochromatinMutation in suppressor of PEV results in fewer cells having gene inactivationEncodes protein that adds methyl group to lysines on histone H3, signal for chromatin condensationBarriers – specific sites on DNA that block the spread of heterochromatin Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*X-chromosome inactivation in female mammals occurs through heterochromatin formationExample of facultative heterochromatinDosage compensation in mammals so that X-linked genes in XX and XY individuals are expressed at same levelRandom inactivation of all except one X chromosome in XXBarr bodies – darkly stained heterochromatin masses observed in somatic cells at interphaseXX person has one Barr bodyXXX person has two Barr bodiesXXY person has one Barr bodyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*X chromosome mosaicismIn very early embryo, both X chromosomes are activeIn humans, random X-inactivation occurs ~ 2 weeks after fertilizationSome cells have maternal X inactivated, other cells have paternal X inactivatedAll cell descendants have the same inactive XAdult females are mosaic at X-linked genesIn females heterozygous for X-linked mutation:Some cells have wild-type allele inactivatedSome cells have mutant allele inactivatedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*X-inactivation is initiated by expression of the Xist geneXist, X inactivation specific transcriptOne of the few genes expressed on the inactive X but is not expressed on the active XXist RNA is a large, non-coding, cis-acting regulatory RNABinds to the X-chromosome that it was expressed fromInitiates histone modifications (methylation, deacetylation) that result in heterochromatin formationDeletion of the Xist gene abolishes X inactivationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Transcription is controlled by chromatin structure and nucleosome positionSpacing and structure of nucleosomes affect transcriptionThree major mechanisms can regulate chromatin patternsHistone modifications – addition of methyl or acetyl groupsRemodeling complexes can alter nucleosome patternsChange accessibility of promoter sequencesAssays for DNase hypersensitive sites (Figure 12.14)Remove or reposition promoter-blocking nucleosomesHistone variants can cause different nucleosomal structures, e.g. CENP-A at centromeresCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Assay for chromosomal regions that lack nucleosomesDNase hypersensitive sites - promoters of transcribed genes are more susceptible to nuclease digestion than promoters of non-transcribed genesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.14Origins of replication in eukaryotesRate of DNA synthesis in human cells ~ 50 nt/secMost mammalian cells have ~ 10,000 originsIt would take 800 hours to replicate the human genome if there was only one origin of replication!Many origins are active at the same time (see Fig. 12.15)Accessible regions of DNA that are devoid of nucleosomesReplication unit (replicon) – DNA running both ways from one origin to the endpointsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Origins of replication in yeast are "autonomously replicating sequences" (ARSs)ARSs permit replication of plasmids in yeast cellsAT-rich consensus sequence found in all ARS elements, flanked by sequences that promote replication initiationARS1 (below) is the first ARS to be characterizedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.16Telomeres are "caps" that protect the ends of eukaryotic chromosomesTelomeres consist of specific repetitive sequences and don't contain genesSpecies-specific sequencese.g. TTAGGG in humans, TTGGGG in Tetrahymena250-1500 repeats with variable number between different cell typesPrevent chromosome fusions and maintain integrity of chromosomal endsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.17Replication at the ends of chromosomesAfter removal of the last RNA primer, DNA polymerase cannot replicate some of the sequences at the 5' endDNA synthesis occurs only in 5'-to-3' directionWithout a special mechanism, DNA would be lost from every new DNA strand at each cell cycle Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.18Telomerase is a ribonucleoprotein that extends telomeresTelomerase RNA is complementary to telomere repeat sequencesServes as template for addition of new DNA repeat sequences to telomereAdditional rounds of telomere elongation occur after telomeres translocate to newly-synthesized endCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.19Telomerase activity and cell proliferationIn yeast that has deletion of telomerase, telomeres shorten by 3 bp per generationEventually the chromosomes break and the cells die In humans, the levels of telomerase and cellular life-span varies between different types of cellsMost somatic cells have low expression of telomerase Telomeres shorten slightly at each cell divisionSenescence after 1 Mb block of tandem repeatsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Action of cohesin during mitosisCohesin is a protein complex that holds sister chromatids during metaphaseAt anaphase, cohesin is enzymatically cleaved and sister chromatids are released from each otherCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.20aAction of cohesin during meiosis IAt anaphase I, cohesin along chromosome arms is enzymatically cleaved but cohesin at centromeres is not cleavedShugoshin protects centromeric cohesin from degradationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.20bAction of cohesin during meiosis IIAfter entry into metaphase II, shugoshin is removed and centromeric cohesin is degradedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.20cStructure of centromeres in higher organismsCentromeres hold sister chromatids together and contain information for construction of a kinetochoreCohesin binds sister chromatids together at the centromereCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.21Structure and DNA sequenceorganization of yeast centromeresCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*Fig. 12.22Histone variants at centromeresSpecialized chromatin in central core of centromere marks this region for attachment of kinetochore proteins Central core of each centromere:Composed of unique chromatin that does not recombine and is not transcribedSurrounded by regions of heterochromatin interspersed with euchromatinHistone variant CENP-A is present in central core of all eukaryotes examinedDiffers from histone H3 in N-terminal regionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 12*