1. Genetics: From Genes to Genomes
PowerPoint to accompany
Genetics: From Genes to Genomes
Fourth Edition
Leland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. Silver
Prepared by Mary A. Bedell
University of Georgia
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2. 12 The Eukaryotic Chromosome CHAPTER OUTLINE PART IV
How Genes Travel on Chromosomes
CHAPTER
The Eukaryotic Chromosome
CHAPTER OUTLINE
12.1 Chromosomal DNA and Proteins
12.2 Chromosome Structure and Compaction
12.3 Chromosomal Packaging and Function
12.4 Replication and Segregation of Chromosomes
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3. Chromosomal DNA and proteins
Chromosomes have a versatile, modular structure for packaging DNA that supports flexibility of form and function Chromatin is the generic term for any complex of DNA and protein found in a nucleus of a cell Chromosomes are the separate pieces of chromatin that behave as a unit during cell division Chromatin is ~ 1/3 DNA, 1/3 histones, 1/3 nonhistone proteins DNA interaction with histones and nonhistone proteins produces sufficient level of compaction to fit into a cell nucleus
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4. Histone proteins
Histones – small, positively-charged, and highly conserved
Bind to and neutralize negatively charged DNA
Make up half of all chromatin protein by weight
Five types - H1, H2A, H2B, H3, and H4
Core histones (H2A, H2B, H3, and H4) make up the nucleosome
Posttranslational modifications of histones H3 and H4
Methylation and acetylation of histone tails
Affect chromatin structure and gene expression in specific chromosomal regions
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5. Nonhistone proteins
Hundreds of other proteins that make up chromatin and are not histones
200 – 200,000 molecules of each kind of nonhistone protein
Large variety of functions
Structural role – chromosome scaffold (see Figure )
Chromosome replication – e.g. DNA polymerases
Chromosome segregation – e.g. kinetochore proteins (see Figure 12.3)
Active in transcription – largest group
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6. Different levels of chromosome compaction
Table 12.1
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7. The nucleosome is the fundamental unit of chromosomal packaging
DNA wraps twice around nucleosome core octamer (Figure ) and forms 100 Å fiber
Results in 7-fold compaction of DNA
Spacing and structure of nucleosomes affect genetic function
Determines whether DNA between nucleosomes is accessible for proteins that initiate transcription, replication and further compaction
Arrangement along chromatin is highly defined and transmitted from parent to daughter cells during DNA replication
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8. Nucleosomes look like "beads on a string" in the electron microscope
Diameter of DNA helix (string) is 20 Å Diameter of nucleosome core (bead) is 100 Å
Fig. 12.4
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9. The nucleosome core is an octamer of two each of histones H2A, H2B, H3, and H4
160 bp of DNA wraps twice around a nucleosome core 40 bp of linker DNA connects adjacent nucleosomes Histone H1 associates with linker DNA as it enters and leaves the nucleosome core
Fig. 12.5
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10. X-ray crystallography of a nucleosome
DNA bends sharply at several places as it wraps around the core histone octamer Base sequence dictates preferred nucleosome positions along the DNA
Fig. 12.6
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11. Models of higher-order packaging
100 Å fiber is compacted into 300 Å fiber by supercoiling
Results in an additional 6-fold compaction of DNA
Fig. 12.7a
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12. The radial loop-scaffold model for higher levels of compaction
Several nonhistone proteins (NHPs) bind to chromatin every kb and tether the 300 Å fiber into structural loops Other NHPs gather several loops together into daisylike rosettes
Fig. 12.7b
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13. The radial loop-scaffold model for higher levels of compaction (cont)
Condensins may further condense chromosomes into a compact bundle for mitosis
Fig. 12.7c
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14. The karyotype of a human female examined by high-resolution G-banding
Metaphase chromosomes stained with Giemsa have alternating bands of light and dark staining Each band is contains many DNA loops and ranges from 1 to 10 Mb in length Banding patterns on each chromosome are highly reproducible
Fig. 12.9
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15. Locations of genes in relation to chromosomal bands
Short arm = p arm Long arm = q arm Within each arm, light and dark bands are numbered consecutively
Fig
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16. Comparison of chromosomes from humans and great apes
Banding patterns in some chromosomes (i.e. Chr 1) are nearly identical The metacentric Chr 2 in humans was formed by fusion of two acrocentric chromosomes in great apes
Chromosome 1
Acrocentric chromosomes of great apes
Fig
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17. Chromosomal packaging and function
Heterochromatin – highly condensed, usually inactive transcriptionally
Darkly stained regions of chromosomes
Constitutive – condensed in all cells [e.g. most of the Y chromosome and all pericentromeric regions (see Fig )]
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 transcriptionally
Lightly stained regions of chromosomes
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18. Position-effect variegation (PEV) in Drosophila
White+ (w+) gene is normally located in euchromatin Chromosomal inversion can result in w+ gene being located adjacent to heterochromatin
w+ gene is expressed in all cells (red pigment)
w+ gene is silenced in some cells (no pigment) but is expressed in other cells (red pigment)
Fig a
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19. PEV in Drosophila (cont)
Gene silencing can be caused by spreading of heterochromatin into nearby genes Spreading can occur over > 1000 kb of chromatin Heterochromatin spreads further in some cells than in others
Fig b
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20. Identification of molecules involved in heterochromatin formation
Genetic screens in Drosophila were used to identify mutations that enhance or suppress the extent of PEV
Mutation in enhancer of PEV results in more cells having gene inactivation
Encodes protein that localizes to heterochromatin
Mutation in suppressor of PEV results in fewer cells having gene inactivation
Encodes protein that adds methyl group to lysines on histone H3, signal for chromatin condensation
Barriers – specific sites on DNA that block the spread of heterochromatin
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21. X-chromosome inactivation in female mammals occurs through heterochromatin formation
Example of facultative heterochromatin
Dosage compensation in mammals so that X-linked genes in XX and XY individuals are expressed at same level
Random inactivation of all except one X chromosome in XX
Barr bodies – darkly stained heterochromatin masses observed in somatic cells at interphase
XX person has one Barr body
XXX person has two Barr bodies
XXY person has one Barr body
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22. X chromosome mosaicism
In very early embryo, both X chromosomes are active
In humans, random X-inactivation occurs ~ 2 weeks after fertilization
Some cells have maternal X inactivated, other cells have paternal X inactivated
All cell descendants have the same inactive X
Adult females are mosaic at X-linked genes
In females heterozygous for X-linked mutation:
Some cells have wild-type allele inactivated
Some cells have mutant allele inactivated
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23. X-inactivation is initiated by expression of the Xist gene
Xist, X inactivation specific transcript
One of the few genes expressed on the inactive X but is not expressed on the active X
Xist RNA is a large, non-coding, cis-acting regulatory RNA
Binds to the X-chromosome that it was expressed from
Initiates histone modifications (methylation, deacetylation) that result in heterochromatin formation
Deletion of the Xist gene abolishes X inactivation
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24. Transcription is controlled by chromatin structure and nucleosome position
Spacing and structure of nucleosomes affect transcription
Three major mechanisms can regulate chromatin patterns
Histone modifications – addition of methyl or acetyl groups
Remodeling complexes can alter nucleosome patterns
Change accessibility of promoter sequences
Assays for DNase hypersensitive sites (Figure 12.14)
Remove or reposition promoter-blocking nucleosomes
Histone variants can cause different nucleosomal structures, e.g. CENP-A at centromeres
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25. Assay for chromosomal regions that lack nucleosomes
DNase hypersensitive sites - promoters of transcribed genes are more susceptible to nuclease digestion than promoters of non-transcribed genes
Fig
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26. Origins of replication in eukaryotes
Rate of DNA synthesis in human cells ~ 50 nt/sec
Most mammalian cells have ~ 10,000 origins
It 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 )
Accessible regions of DNA that are devoid of nucleosomes
Replication unit (replicon) – DNA running both ways from one origin to the endpoints
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27. Origins of replication in yeast are "autonomously replicating sequences" (ARSs)
ARSs permit replication of plasmids in yeast cells AT-rich consensus sequence found in all ARS elements, flanked by sequences that promote replication initiation ARS1 (below) is the first ARS to be characterized
Fig
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28. Telomeres are "caps" that protect the ends of eukaryotic chromosomes
Telomeres consist of specific repetitive sequences and don't contain genes
Species-specific sequences
e.g. TTAGGG in humans, TTGGGG in Tetrahymena
repeats with variable number between different cell types
Prevent chromosome fusions and maintain integrity of chromosomal ends
Fig
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29. Replication at the ends of chromosomes
After removal of the last RNA primer, DNA polymerase cannot replicate some of the sequences at the 5' end
DNA synthesis occurs only in 5'-to-3' direction
Without a special mechanism, DNA would be lost from every new DNA strand at each cell cycle
Fig
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30. Telomerase is a ribonucleoprotein that extends telomeres
Telomerase RNA is complementary to telomere repeat sequences Serves as template for addition of new DNA repeat sequences to telomere Additional rounds of telomere elongation occur after telomeres translocate to newly-synthesized end
Fig
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31. Telomerase activity and cell proliferation
In yeast that has deletion of telomerase, telomeres shorten by 3 bp per generation
Eventually the chromosomes break and the cells die
In humans, the levels of telomerase and cellular life-span varies between different types of cells
Most somatic cells have low expression of telomerase
Telomeres shorten slightly at each cell division
Senescence after < 50 generations in culture
Germ cells, stem cells, and tumor cells have high expression of telomerase
At each generation, telomere length is maintained
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32. Chromosome duplication includes reproduction of chromatin structure
Chromatin fiber unwinds before DNA synthesis occurs
Synthesis of histones and their transport into nucleus must be tightly coordinated with DNA synthesis
Newly-synthesized DNA must associate with either preexisting histones or with newly-synthesized histones
After DNA replication, nucleosomal DNA must produce the same level of compaction as before replication
In differentiating cells, a slightly different chromatin condensation pattern can appear after replication
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33. Segregation of condensed chromosomes depends on centromeres
During anaphase of mitosis and meiosis II, sister chromatids must segregate to different daughter cells
During anaphase of meiosis I, sister chromatids do not separate and homologous chromosomes segregate to different daughter cells
Centromeres have two functions:
Hold sister chromatids together (through action of cohesin)
Attachment sites for chromosome segregation machinery (through formation of kinetochore)
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34. Characteristics of centromeres
Appear as constrictions in chromosomes
Can be in middle of chromosome (metacentric) or near one end of the chromosome (acrocentric)
Consist of satellite DNAs, which are repetitive, noncoding sequences
Tandem repeats of 5 – 300 bp long, can extend over megabases of DNA
Have different chromatin structure and higher-order packaging than other chromosomal regions
Predominant human satellite DNA is a-satellite
171 bp repeat, present in > 1 Mb block of tandem repeats
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35. Action of cohesin during mitosis
Cohesin is a protein complex that holds sister chromatids during metaphase At anaphase, cohesin is enzymatically cleaved and sister chromatids are released from each other
Fig a
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36. Action of cohesin during meiosis I
At anaphase I, cohesin along chromosome arms is enzymatically cleaved but cohesin at centromeres is not cleaved Shugoshin protects centromeric cohesin from degradation
Fig b
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37. Action of cohesin during meiosis II
After entry into metaphase II, shugoshin is removed and centromeric cohesin is degraded
Fig c
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38. Structure of centromeres in higher organisms
Centromeres hold sister chromatids together and contain information for construction of a kinetochore Cohesin binds sister chromatids together at the centromere
Fig
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39. Structure and DNA sequence organization of yeast centromeres
Fig
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40. Histone variants at centromeres
Specialized 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 transcribed
Surrounded by regions of heterochromatin interspersed with euchromatin
Histone variant CENP-A is present in central core of all eukaryotes examined
Differs from histone H3 in N-terminal region
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