1what did Walther Flemming discover?
mitosis and chromosomes
2what 3 molecules do chromosomes contain?
1 double-stranded DNA molecule, proteins, RNA
3What is a karyotype?
the number and visual appearance of the chromosomes in the cell nuclei of an organism
4How many chromosomes are in a human cell?
23 pairs of homologous chromosomes
5What are autosomes?
the numbered chromosomes
6What are 3 advantages to studying model organisms when researching basic biological
mechanisms?
relatively short reproduction times, manipulatable in lab setting, controlled environment & mating
7What are sex chromosomes?
chromosome involved in sex determination (X and Y)
8Name 3 applications for whole genome assemblies in basic or clinical biology
personalized medicine, ancestry, GWAS
9What are ‘fluorescent tags’ and how are they used to study protein behaviors in cells?
visible proteins like GFP or RFP used to track specific proteins or DNA loci live
10What kinds of DNA sequences are in genomes?
transposons (LINEs and SINEs), introns, exons, genes, repeated sequences, unique sequences
11What is the difference between tandem and dispersed repeats?
tandem repeats are short copies of DNA sequences positioned one after another, dispersed are randomly distributed across genome
12Give an example of tandem repeats
satellite DNAs, rDNA
13Give an example of dispersed repeats
Transposable elements
14Where are most of the protein-coding genes located?
euchromatin
15Where in the chromosome is heterochromatin concentrated?
near centromeres and telomeres
16How is heterochromatin organized within the 3D nucleus?
located on the edge of a 3D nucleus, following the path of the nuclear membrane, forms a separate domain from euchromatin
17What types of DNA sequences are enriched in heterochromatin?
repeated DNA (satellites, TEs, non-protein coding RNA)
18Does heterochromatin contain protein-coding genes?
no, contains genes for maintenance of the 3D architecture, chromosome segregation, evolution, and length of the S phase.
19Why are most genome sequence assemblies incomplete?
issues from repeated DNAs, problems with cloning or sequencing, mistaken assemblies due to sequence homology
20What is the C-value paradox?
lack of correlation between biological complexity and genome size
21What type of sequence most strongly correlates with genome size?
TEs
22What features do not correlate with genome size?
biological complexity, size, number of genes, diploid number
23What are the 3 main mechanisms that drive evolution of genome size and organization?
whole genome duplication, Duplication and divergence of existing individual genes, Expansion/contraction of transposons and other repeated DNAs
24What 4 things can exchange at tandem repeats generate?
chromosome arm fusion, chromosom arm fission, sequence length contractions, sequence length expansions
25What is synteny?
organisms of relatively recent divergence show similar blocks of genes in the same relative positions in the genome
26what are the consequences of exchange between dispersed repeats?
arm and sub-arm rearrangements, which results in synteny
27How is evolution facilitated by gene duplication? Give an example
gene loss, non-overlapping functions, complementary functions, differential regulation. EX: duplicated opsin genes thatsense different wavelengths, in different combinations and in different species
28What surprising discovery led to Barbara McClintock’s Nobel Prize?
discovered mobile elements (transposons, jumping genes)
29What plant phenotype led to McClintocks's Nobel Prize discovery?
corn pigmentation
30What are the 3 types of TEs?
DNA-only, LTR retrotransposons (retroviral-like), non-LTR retrotransposons (non retroviral-like)
31In DNA transposons, what enzyme is used for movement, and how does it move?
transposase, cut-and-paste or copy-and-paste
32In retroviral transposons, what enzyme is used for movement, and how does it move?
reverse transcriptase and integrase, moves via RNA intermediate driven by promoter in LTR
33in nonretroviral transposons, what enzyme is used for movement, and how does it move?
reverse transcriptase and endonuclease, moves via RNA intermediate synthesized by neighboring promoter
34Name 3 ways that TEs can disrupt or alter gene functions
insertion into exon/enhancerepressor, rearrangements, introduction of new information
35Provide an example of TEs that have been appropriated for organismal functions during evolution
Red skin color for apples from a transposon insertion, mechanism of adaptive immunity in jawed vertebrates co-opted from a DNA transposon
36What are the 5 main stages of mitosis, and what events are central to accomplishing each stage?
prophase, metaphase, anaphase, telophase, cytokinesis
37What events in prophase
chromosomes condense, nuclear envelope breakdown, appearence of kinetochore
38What events in metaphase?
centromeres at opposite poles, chromosomes lined up and attached to mitotic spindles
39What events in anaphase?
cohesins break down, sister chromatids pulled to opposite ends, cell elongates
40What events in telophase?
chromosomes decondense at opposite poles, nuclear envelope forms around chromosomes, mitotic spindle breaks down
41what happens in cytokinesis?
cleavage furrow or cell wall separates the cells
42What drives chromosome movements in mitosis?
Kinetochores bind to microtubules and mediate chromosome movement
43What methods can you use to track chromosome dynamics in living cells?
phase contrast microscopy
44What is HiC?
a derivative of a series of chromosome conformation capture technologies used to capture all pairwise 3D associations across the genome and generates contact frequency maps
45what did HiC reveal about the structure of condensed chromosomes in mitosis
revealed the segregation of chromatin into active and inactive compartments, and the folding of DNA into self-associating domains and loops
46What are SMC proteins/complexes?
Structural Maintenance of Chromosomes, ancient DNA organizers, present in all known organisms
47SMCs have _______ which enables them to act as _________, and form __________
ATP activity, molecular motors, ring-like structures
48Give an example of an SMC complex
condensin 1 and condensin 2
49How are loops formed?
Condensin uses ATP hydrolysis
50What protein complex is responsible for chromosome condensation in mitosis?
condensin protein complex
51What is sister cohesion? (SCC)
the process by which sister chromatids are paired and held together from the time of DNA replication in S-phase until the meta to ana transition
52What is Cohesin?
protein complex that mediates sister chromatid cohesion, homologous recombination, and DNA looping
53What proteins and enzymatic activities drive cell cycle transitions?
cyclins and CDKs
54
What is a cell cycle checkpoint and in general how does it work?
points in the cell cycle where progress is delayed until errors (like DNA damage) in essential processes are fixed
55SAC-what stage in mitosis is blocked?
spindle assembly checkpoint: prevents the metaphase to anaphase transition until all chromosomes are bioriented (attached to opposite spindle poles)
56What can SAC "sense" ?
when even one kinetochore is not properly attached
57Once the SAC is ‘satisfied’ what happens at the molecular level that allows sister chromatids to separate?
APC-Complex activation leads to destruction of the remaining cohesion in centromeric region, Securin is degraded allowing Separase to be active
59What molecules and interactions move chromosomes to the poles?
APC polyubiquinates cyclin, thus inactivating CDK, and tags securin for destruction, allowing separase to cut the cohesins and allow the chromosomes to separate
60What is the definition of epigenetics?
Heritable changes in genome function without mutation
61What is Position-Effect Variegation (PEV)?
discrete markings (variegation) caused by the silencing of a gene in some cells through its abnormal juxtaposition with heterochromatin via rearrangement or transposition
62What type of chromosome change did Muller identify as causing PEV?
chromosome inversion that moved the gene closer to the heterochromatin
63What phenotypic evidence demonstrates that PEV is inherited through cell divisions?
white vs red eyes in drosophilia and yeast
64What is the composition and organization of canonical nucleosomes?
DNA wrapped around histone core (8 histones per nucleosome)
65Do nucleosomes reduce or increase access to DNA?
reduce, need to moveemove nucleosome to access DNA
66How are epigenetic modifications added to histones?
histone tails extend out of nucleosome and are chemically modified by a variety of enzymes
67Name three types of post-translational histone modifications
acetylation and phosphorylation = higher gene expression, methylation = gene silencing
68What are writers?
enzymes that add modifications
69give examples of writers
HMTs, HATs, ubiquitin ligases, kinases
70What are readers?
contains domains that bind to specific modified histones
71Give examples of readers
HP1, PRC2
72What are erasers?
enzymes that remove modifications
73Give examples of erasers
histone demethylases, HDACs, phosphatases, ubiquitin proteases
74What does HP1 bind, and what does the modification do?
H3K9me2/3, silences genes
75What does PRC2 bind, and what does the modification do?
H3K27me3, suppresses gene expression
76HP1 also interacts with this HMTase
Suv(3-9)h1
77What is the fate of nucleosomes at the replication fork?
split between old and new, will be diluted without replenishing
78Give one example of a molecular mechanism that explains how histone modifications are heritable
PROPAGATION of modifications to NEW nucleosomes based on patterns present in nearby OLD nucleosomes via binding protein or modification enzyme mechanism
79How can the molecular mechanism that explains how histone modifications are heritable explain the gene silencing that occurs when euchromatics genes are juxtaposed with heterochromatin by chromosome rearrangements?
spreading and replication are mediated by the same mechanism (HP1 binding H3K9me2/3 and recruiting Su(var)3-9)
80Su(var)
suppresor of PEV
81What domain in HP1 and PC bind to each protein’s histone modification target?
chromodomain
82How do positive and negative feedback loops contribute to loss and retention of epigenetic states?
promotes stability of epigenetic states once established but allows for stochastic or regulated reversal
83How are histone modifications and other epigenetic factors mapped to genomes?
Chromatin ImmunoPrecipitation + DNA sequencing (ChIP-seq)
84How would you demonstrate that heterochromatin can ‘spread’ along a chromosome?
ChIP analysis, Place active gene near heterochromatin and see it get inactivated as it spreads
85How do nucleosome remodelers promote genome functions (e.g. transcription)?
remodelers can move or remove nucleosomes to allow access to DNA
86What are kinetochores?
protein on centromeres where the spindle fibers attach during cell division to pull sister chromatids apart
87When do kinetochores form in the cell cycle?
prometaphase
88What evidence indicates that a specific DNA sequence is neither necessary nor sufficient for kinetochore formation and centromere propagation?
when there are 2 areas of CEN DNA, only 1 forms a centromere (not sufficient), neocentromeres form without CEN DNA (not necessary)
89Under what circumstances is plasticity useful for genome functions?
allows the centromere to move during evolution and to adapt to situations that are unfavorable for centromere identity
90What protein determines centromere identity and propagation?
CENP-A, an H3 variant
91What happens to kinetochore formation and centromere propagation when CENPA is removed?
cannot form or propagate
92What type of experiment would demonstrate that CENPA is sufficient for centromere identity and kinetochore formation? Necessary?
recruit CENP-A to non-centromeric site to see if centromere will form
93Is centromeric chromatin = heterochromatin?
no, centrochromatin
94CENP-A is present ____, but kinetochores are present ______
the entire cell cycle, only in mitosis
95How is centromere propagation similar to heterochromatin heritability?
centromere idencty and inheritance use an epigenetic mechanism
96What is a neocentromere?
new centromeres that form at a place on the chromosome that is usually not centromeric
97What is a functional dicentric chromosome?
chromosome with 2 centromeres
98What is the nuclear lamina?
dense network anchored to the inner nuclear membrane that provides mechanical support and regulates DNA replication and cell division
99What method is used to visualize chromosome territories?
Fluorescence in situ hybridization (FISH)
100Are active genes more often associated with the interior or exterior of chromosome territories?
exterior (where territories interact)
101What methods can be used to identify interactions between chromosome territories?
Hi-C and ChIP-Seq
102What are A and B compartments?
organization for chromosomes, A is active B is inactive
103What is the difference between FISH and IF?
FISH is used to detect specific DNA or RNA sequences within cells, while IF is used to detect specific proteins or other molecules
104What is a TAD, and how does it differ from A/B compartments?
topologically associated domain, it is local regulation of contact frequency, while A/B is intra and interchromosomal regulation. multiple TADs can make up an A or B compartment
105What are CTCFs and what do they do?
transcriptional repressor that binds to specific GC-rich DNA sequences, forming chromatin loops
106How are TADs generated?
boundaries form at CTCF and cohesin peaks, specifically when CTCF sites are convergent
107What complexes drive loop extrusion? Does loop extrusion require energy?
condensins 1 and 2, yes ATP
108What roles do cohesin and CTCF play in formation of TADs ?
cohesins extrude the loop until it reaches the point of convergence for 2 CTCF sites
109What happens to TADs if cohesins were degraded?
no loops, fewer contacts
110What happens to TADs if no CTCF sites?
more non-specific contacts between TADs (no "boxes" on contact map)
111How could chromosome looping positively affect gene expression? Negatively?
Cause interactions between genes and regulatory sequences (negative and positive)
112How could you quickly eliminate a protein from the cell?
Auxin-inducin degradation (AID) system will attach to the protein of interest, an SCF complex including an F-box will recognise the AID and eliminate it using a proteosome
113what effect does WAPL removal have on genome organization
alter Tads and A/B regions and eliminate long range interactions between genes
114What is WAPL?
a key regulator of the Cohesin complex
115Why is phase separation important in the context of nuclear architecture/organization?
it drives chromatin compartmentalization
116What is multivalency, and why is this property important for phase separation/condensate formation?
when a molecule has more than 2 binding domains, important because it promotes phase separation and liquidity by binding weakly at one site and letting go at another site
117How is the nucleolus formed? What nucleates nucleolus formation?
forms around transcribing rRNA, nucleates at Nucleolus Organizing Regions that contain active rDNA genes
118How would you demonstrate nucleolus formation experimentally?
take single rDNA, insert at ectopic sites in genome to see if nucleoulus forms
119Why do nucleoli contain different layers? How are they generated ?
fibrillar center, dense fibrillar component, and granular component, representing the sites of progressive stages of rRNA transcription, processing, and ribosome assembly
120What features of NPM1 help it contribute to GC formation?
pentameric (can bind to self), acidic tracts to bind to robosomal proteins, and nucleic acid binding domain to interact with rRNA
121What features of Fib contribute to DFC formation?
intrinsically disordered region to bind to self, nucleic acid binding domain to bind rRNA
122What drives FC formation?
transcription of NORs
123What are the most likely causes of nucleolar dissolution in mitosis?
nuclear envelope breakdown diluting components, CDK phosphorylation decreasing binding affinities, turning off rRNA transcription
124How are transcriptional condensates generated? Are they generated by only epigenetic mechanisms or does DNA sequence play a role?
nucleated by DNA sequence-specific binding factors whose disordered tails interact weakly with multivalent proteins
125Why are HP1 proteins good candidates for biological phase separation?
multivalent, dimerizes, can bind to 4+ molecules, including itself
126What nucleates heterochromatin condensate formation? How do they grow?
mimicks nucleoli, HP1a nucleates and recruits more multivalent partners, fusion to grow
127Describe one mechanism that can account for dissolution of heterochromatin domains during mitosis. Why dissolve nucleoli and heterochromatin condensates during mitosis?
disturbance of binding to HP1, allows for duplicated chromosomes to migrate to opposite ends of the cell
128How could liquid condensates contribute to genome organization and functions?
compartments, spreading by HP1 wetting
129What are chromocenters and how are they formed?
masses of heterochromatin that forms when satellite DNA comes together in the nucleus during interphase
130Why or how do nucleolar numbers change during the cell cycle?
mitosis has an increase in the number of nucleoli, in interphase a decrease occurs as they fuse together
131What types of structures can inhibit nucleolar fusions?
Network of actin filaments
132What features promote normal nuclear shape and material properties?
elasticity and stability, given by HP1
133What can happen to migrating cells if HP1 is removed?
nuclear softening and distortion
134What combination of histone modifications was recognized by the MCA1 antibody? Describe one experimental approach that was used to validate this result.
H3K9me3S10ph, immunofluorescence microscopy
135What kinase is required to remove HP1 from mitotic chromosomes?
Aurora B
136What changes in gene expression occurred within the neocentromere?
increased transcriptional activity and decreased heterochromatin levels
137Describe how WAPL-XP (overexpression) impacted Pcdh isoform and cohesion
dynamics
biased expression of Pcdhac2 over other pcdha genes, less cohesion
138Describe how WAPL-KO impacted Pcdh isoform and cohesion
dynamics
Cohesin enabled a longer reach of the Pcdh enhancers
139Describe how RAD21-KO impacted Pcdh isoform and cohesion
dynamics
Huge decrease in Pcdh expression levels, suggesting cohesin is critical for Pcdh isoform diversity
140How did WAPL expression change across the developmental trajectory of OSNs?
Decrease in Wapl levels was found to be correlated with the onset of stochastic Pcdh expression
141How does conventional nuclear architecture differ from the inverted nuclear architecture of mouse rod cells? Describe the position of eu- and hetero-chromatin in both conventional and inverted nuclei.
conventional is Heterochromatin at nuclear periphery/edges and euchromatin-rich center, inverted is dense heterochromatin in the center and decondensed euchromatin on the outside
142Briefly describe how the distribution of heterochromatin in inverted nuclei improves vision in low-light settings, and generally how the authors tested this hypothesis.
inverted has a convex lense, a stronger focusing, and less scattering of light. interferometric phase microscope was used to study isolated rod cells and learn about the optical properties
143What changes in OR gene expression occurred following the deletion of the Greek islands?
a reduction in trans interactions
144What changes in OR gene expression occured following the knockout of LDB1
reduced trans and long-range cis contacts between Greek islands
145Describe how LDB1 and Greek island enhancers coordinate OR gene expression
adaptor protein Ldb1 regulates the assembly and maintenance of OR compartments, Greek Island hubs, and OR transcription, providing mechanistic insight and functional support for the role of trans interactions in gene expression.