Theme 4 Flashcards
stem cells
found in early embryos
- unspecialized cells that can reproduce indefinitely and can differentiate into specialized cells
binary fission
the way by which prokaryotic cells divide
write out the process
FtsZ
a gene in (prokaryotic) bacteria that regulates cell division
- it encodes for a specific protein that assembles and forms a ring at the site of constriction
mitotic cell division
how eukaryotic cells divide
- first requires breakdown of nuclear envelope and DNA needs to be separated, later rebuilding of nuclear envelope is necessary
2 stages of eukaryotic cell division
M phase
interphase
M phase
parent cell divides into 2 daughter cells, last about an hour
- 2 parts: mitosis and cytokinesis
mitosis
the separation of chromosomes into 2 nuclei
cytokinesis
divison of a single cell into 2 separate cells
- can begin before mitosis is even complete
interphase
the time between M phases where the cell preps for division
3 phases: G1 phase, S-phase, and G2 phase
G1 phase
b/w end of M and start of S phases
- regulatory proteins are activated (many are kinases) and promote activity of enzymes that synthesize DNA
- basically prep for S-phase
S-phase
replication of DNA in the nucleus
S for DNA Synthesis
G2 phase
between end of S phase and start of M phase
- size and protein content of cell increase in preparation for division
- preparation for mitosis and cytokinesis (M-phase)
G0 phase
before when cell would enter into G1 phase
- when cells pause in the cell cycle
ex. nerve cells, cells in the lens of the eye, cells permanently in G0 are non-dividing cells
time for cell cycle completion in eukaryotes
about 12-24 hours for actively dividing cells
chromosome
each contains a single molecule of DNA that codes for a specific set of genes
karyotype
the portrait formed by the number and shape of chromosomes representative of a species
- most human cells (not incl gametes) have 46 chromosomes), of these 22 are homologous (identical pairs) numbered 1-22 from longest to shortest. also has one pair of sex chromosomes
haploid vs diploid cell
haploid cell: has one complete set of chromosomes
diploid cell: has 2 complete sets of chromosomes
sister chromatids
2 identical copies of a chromosome are attached by a centromere
- each daughter ell needs to receive the same number of chromosomes as present in the parent cell
prophase
chromosomes condense and become visible
- in cytosol, cell begins to assemble mitotic spindle made up of microtubules which pulls chromosomes into 2 daughter cells
- centrosomes migrate to opposite poles and tubulin dimers assemble around them, forming microtubules that radiate from each centromere which serve as a guide for later chromosome movement
centrosome
a compact structure that is the microtubule organizing centre from where mitotic spindles radiate
- it duplicates during S phase and 1 migrates to each pole of cell at start of prophase
prometaphase
chromosomes attach to mitotic spindle
- nuclear envelope breaks down
- microtubules attach to kinetochore regions of centromeres and pull sister chromatids apart
kinetochores
2 protein complexes associated with the centromere
- one is located on either side of the constriction and one associated with either of the sister chromatids on either side of the centromere
metaphase
chromosome align in the centre of the cell at the metaphase plate (a region, not a structure)
- microtubules lengthen or shorten to position chromosomes at the centre
anaphase
sister chromatids fully separate as kinetochore microtubules shorten and centromere splits, chromatids are then pulled towards opposite poles
- after separation, each chromatids is considered a full chromosome!
telophase
nuclear envelope reforms around each set of chromosomes and creates 2 new nuclei
- cell prepares division into 2 cells
- microtubules of mitotic spindles break down
- as 2 new nuclei become more distinct, the chromosomes contained decondense and become less visible under a microscope
cytokinesis (animal cells)
a ringe of actin fibers (called the contractile ring) forms at the equator of a cell which contracts, pinching the cytoplasm of the cell and dividing it into 2
cytokinesis (plant cells)
division is achieved by constructing a new cell wall
- a phragmoplast (overlapping microtubules containing cell wall components) moves to the middle of the cell
- the fragments fuse to form a cell wall called the cell plate which fuses to the original cell where it’s about to split and then creates 2 new daughter cells
regulation of the cell cycle
done by cyclin dependent kinases (CDKs)
cyclin
a protein that activates kinases
- levels rise and fall with each cell cycle
kinase
an enzyme that activates or inactivates other proteins by phosphorylating key AAs on the target proteins
cyclin-dependent kinases (CDKs)
phosphorylate target proteins involved in promoting cell division
- always present but active only when bound to cyclin
cyclin-CDK complex
- triggers cell division events to occur by phosphorylating target proteins that promote cell division
types of cyclin-CDK complexes that regulate the cell cycle
G1/S cyclin-CDK complex
S cyclin-CDK complex
M cyclin-CDK complex
G1/S cyclin-CDK complex
active near the end of G1 phase, necessary for the cell to enter the S phase
- activates a protein that promotes expression of histone proteins needed for packaging newly replicated DNA
S cyclin-CDK complex
necessary for the cell to initiate DNA synthesis
- activates enzymes and proteins needed for DNA replication
- once replication has begun, the complex prevents the proteins from reassembling in the same place again and re-replicating the same DNA sequence
M cyclin-CDK complex
initiates multiple events associated with mitosis by activating structural proteins in the nucleus that break down the nuclear envelope
- also activates proteins that assemble tubulin into microtubules and promote mitotic spindle formation
cell cycle checkpoints
mechanisms to block cyclin-CDK activity required for the next steps of the cell cycle
- pauses cell division until preparation is complete or damage is repaired
DNA damage checkpoint
before entering S phase
- when DNA is damaged by radiation, a protein kinase is activated that phosphorylates a protein called p53
p53
when phosphorylated, it binds to DNA and turns on expression of a gene that codes for a protein that binds to and blocks activity of G1/S cyclin-CDK complex and arrests the transition to allow for time to repair the damaged DNA before synthesis is initiated
DNA replication checkpoint
at the end of G2, it checks to see that all the DNA has been replicated
spindle assembly checkpoint
before anaphase, it checks to see if all chromosomes are attached to the mitotic spindles
- regulatory proteins assoc. w spindle assembly monitor degree of attachment to sister chromatids
- these proteins area activated by a lack of tension in the centromere area and create a wait signal. metaphase only continues once spindles attach to kinetochore regions and spindle checkpoint proteins can then be removed
separase
the enzyme that breaks sister chromatid attachments
semiconservative replication
after replication, each new DNA duplex consists of one strand from the parent and one newly synthesized strand
conservative replication
original DNA (parent strands) remain intact after DNA replication and the daughter duplex is entirely new
dispersive replication
all 4 strands are a combination of old and new strands of DNA
Meselson and Stahl experiment
supports semiconservative replication
- write this out: method, results, and everything
leading strand
3’ end being synthesized is pointed towards the replication fork so that as the double helix unwinds, nucleotides can be added to the 3’ end continuously as one polymer
lagging strand
synthesized discontinuously
- 5’ end pointed toward replication fork but DNA isn’t synthesized in that direction, instead, it grows away from the replication fork
- as the parental duplex unwinds further, a new daughter strand is initiated with its 5’ end near the rep fork and is elongated in the 3’ direction as usual until it reaches the piece in front of it
Okazaki fragments
the short pieces of DNA that are synthesized as parts of the lagging strand
RNA primase
synthesizes a short stretch of RNA complementary to the DNA template and doesn’t require a primer
RNA primer
since DNA polymerase can only elongate the end of an existing DNA or RNA, each new DNA strand must begin w a short strand of RNA that serves as a primer/ starter for DNA synthesis
- for the lagging strand, then the growing fragment comes in contact with the primer, a different DNA polymerase takes over and removes the RNA primer, and extends the growing fragment w DNA nucleotides where the primer was
DNA ligase
joins (ligates) adjacent fragments of DNA in the lagging strand
- also repairs breaks in the DNA backbone
components of the replication complex
topoisomerase II
helicase
single stranded binding proteins
topoisomerase II
works upstream from replication fork to minimize stress on double helix that results from unwinding
helicase
separates the strands of DNA duplex at the replication fork by breaking H bonds between base pairs
single-stranded binding proteins
bring to split strands of DNA (stabilize them) so that they don’t re bind before elongation begins
coordinated synthesis of leading and lagging strands
ensures that both strands of the double helix are replicated at the same rate
- DNA polymerase complexes stay in contact with one another so that the leading and lagging strands pass through the same direction, req’s the lagging strand to be looped around
- 3’ ends of both stands are elongated at similar rates
DNA proofreading
DNA polymerase detects mispairing of nucleotides and corrects it by cutting out the incorrect one and inserting the correct nucleotide in tis place
origin of replication
each point where DNA synthesis is initiated
- organisms with circular chromosomes (prokaryotes) only have one origin of replication, but replication still occurs in both directions
replication bubble
formed by the opening of the double helix at each origin of replication
- has a replication fork on each side with a leading and lagging strand
- also has each of the components of the replication complex (enzymes)
- DNA synthesis occurs at each replication fork, and as they move in opposite directions, the bubbles get larger and when 2 bubbles meet, the fuse to form one larger bubble
shortening of linear chromosomes during DNA replication
during synthesis of the lagging strand, where RNA primer is found on the 5’ end, there is no DNA polymerase coming to replace these nucleotides and they are lost more and more after each replication (100bp/time)
telomere
repeating, non-coding regions of DNA found on the end of eukaryotic chromosomes
often 3’-GGGATT-5’
telomerase
an enzyme that restores telomeres once lost during synthesis of the lagging strand in DNA replication
- contains an RNA molecule complementary to the telomere sequence so that a new segment of the lagging strand can then be formed with another RNA primer and DNA polymerase so the template strand is completely restored w more telomerase repeats
telomerase activity
high in germ cells, and stem cells (cells w high turnover rates)
- nearly inactive in adult cells and shortening of telomeres limits the number mitotic cells can undergo thus contributing to aging
- in cancer cells, telomerase is reactivated and helps support uncontrolled growth of abnormal cells
polymerase chain reaction (PCR)
what is it, how is it set up?
DNA replication in vitro developed by Kary Mullis (1985)
- allows scientists to amplify DNA millions of time from a sample
setup incl: a buffered sol’n, DNA sample 2 primers that are complementary to template DNA, a DNA polymerase (ex Taq polymerase), and 4 dNTPS (nucleotide base pairs)
- put in thermocycler
steps of PCR
- denaturation: separation of DNA double helix into 2 target strands by heating the sample
- annealing: 2 primers anneal to their complementary sequence on DNA template
- extension: DNA polymerase synthesizes new DNA strands by extending primers in 5’ to 3’ direction using dNTPs
2^n copies are produced for n cycles of PCR
gel electrophoresis
a technique that allows us to separate DNA fragments based on the rate of movement through an agrose gel in an electric field
how does gel electrophoresis work?
- dyed DNA samples are placed in wells and when an electrical current is passed through the gel, the molecules will travel towards the positive end (anode) since DNA is negatively charged (bc of the phosphate group)
- smaller fragments move further than larger ones
- a standardized ladder of known length (bp) is added to the gel at the same time as the samples
- DNA appears orange under UV light and we can estimate the length of DNA fragments based on distance travelled relative to the ladder
steps for whole genome sequencing
- sequence DNA
- assemble sequences
- annotate sequences
write out each process
somatic mutations
occur in non-germline cells and cannot be inherited
- lead to a patch of mutated cells only occurring at specific cells in specific tissues
- the earlier the dev’t of the mutation, the larger the spread of the mutated cells throughout the body
- negligible if mutation occurs in a cell in G0 phase
germline mutation
occur in germline cells (cells that rise to gametes)
- can be inherited, all cells inherit mutation in offspring because it affects the developing embryo
Lederburg experiment
determined that mutations are random, not directed
write out the process
mutagens that cause mutations
X rays
oxygen radicals
UV light
replication errors
DNA repair mechanisms after mutations
- base excision repair
- nucleotide excision repair
- mismatch repair
consequences of mutations
cell death
cancer
aging
disease
mismatch repair
the mismatched nucleotide base (only one) puts a kink in the DNA backbone that is recognized by DNA polymerase proofreading mechanism
- DNA nuclease enzyme breaks the DNA backbone a distance from the mismatched region and removes a succession of nucleotides from the DNA strand
- DNA polymerase and DNA ligase close the gap by DNA synthesis of complementary strand
base excision repair
- uracil in DNA signals repair process detected by DNA uracil glycoslase which cleaves uracil from the deoxyribose sugar
- AP endonuclease detects the lack of a nitrogenous base and cleaves the backbone and removes the sugar, leaving an open gap
- gap is closed by enzymes, DNA polymerase and DNA ligase
nucleotide excision repair
repair process is signalled by one or more damaged bases
- enzyme cleaves DNA backbone at sites flanking the region, the region with damaged bases gets removed and the gap is filled by DNA synthesis
small scale mutations
point mutations including synonymous mutation nonsynonymous mutation nonsense mutation missense mutation insertions deletions frameshift mutations
large scale mutations
chromosomal mutations including duplications deletions inversions reciprocal translocations
point mutation
most common type is a single nucleotide pair substitution where one base pair is incorrectly matched to another aka single nucleotide polymorphism (SNP)
synonymous mutation
an nucleotide substitution that does NOT change the AA product - due to redundant nature of the genetic code
non synonymous mutation (missense)
a nucleotide substitution that changes the AA product
ex. in sickle cell anemia it results in the inability for Hgb to bind O2
nonsense mutation
a nucleotide mutation creates a stop codon that causes translation to stop prematurely
insertions vs deletions
degree of impact depends on size of insertion/deletion
insertion: 1 or more extra nucleotides are inserted into replicating DNA
deletion: skipping/removing 1 or more nucleotides during DNA replication
frameshift mutations
occur when the number of nucleotides inserter or deleted is NOT a multiple of 3
- results in an improper grouping of nucleotides downstream, most likely resulting in a missense (changed AA product) or nonsense (premature stop codon) due to incorrect grouping of codons
chromosomal duplication
tends to cause little harm to diploid chromosome
- duplication and divergence: extra gene copies could prove to be advantageous and possibly lead to new genes being formed with a similar function to the original gene
chromosomal deletion
- losing a fragment of a chromosome can result in the loss of entire genes
- if centromere is lost, often entire chromosome is lost after a few divisions bc of improper separating of DNA for daughter cells
- if occurs in embryo, will often lead to embryo death or fatal abnormalities in babies that are born
- most likely to have detrimental effects
chromosomal inversion
occurs when normal order of a gene sequence is reversed
- a chromosomal fragment breaks off and reattaches to the chromosome but backwards
- don’t usually have serious consequences, but problems could occur during gamete production
- can also explain chromosomal evolution in large populations
reciprocal translocations
when a portion of 1 chromosome is able to attach to a portion of another nonhomologous chromosome
ex. exchanging terminal fragments
- usually occur in noncoding regions of DNA in large genomes so don’t usually disrupt function
- could have problems in offspring if occurring in gametes
- can also have nonreciprocal translocations
gene evolution
a result of duplications and divergence can lead to similar but different copies of a gene in a gene family
ex. there are 5 different copies of the globin gene, all are expressed in various points in life and all have similar function; evolution dates back to 200 MYA from a single B-globin gene
