Module 5 Flashcards
The cell cycle
G1: - recovery from mitosis, growth
S phase (synthesis): is DNA duplicated
G2: pre-mitosis checkpoints
M(mitosis):chromosome segregation and cell division
G0 = temporary or permanent exit from cell cycle
- everything except m phase is interphase
Chromosome
the structural unit of genetic material consisting of double stranded
DNA and proteins.
Chromatid-
One copy of a duplicated chromosome.
Sister Chromatids
Identical copies of a chromosome joined by a centromere.
Homologous Chromosomes
Chromosome pair that includes one from each parent. Different alleles
The Goal of Mitosis
Make two genetically identical
daughter cells (i.e., keep all alleles)
Chromosome segregation in mitosis
- Duplicated chromosomes line up independently
- Each pair of sister chromatids (identical) separate
- Each daughter cell gets all of the genetic information
Mitotic Stages I
- Interphase
- period between two successive mitotic divisions. It can be further divided into three subphases: G1, S, and G2. During interphase, the cell grows, carries out its normal functions, and replicates its DNA in preparation for cell division. - Prophase
- the chromatin condenses and becomes visible as distinct chromosomes. The nuclear membrane starts to break down (nuclear envelope dissolves), and the centrioles move to opposite poles of the cell. Spindle fibres begin to form. - Prometaphase
- transitional stage between prophase and metaphase. In prometaphase, the nuclear membrane completely disintegrates, and the spindle fibres attach to the kinetochores, which are protein structures on the centromeres of the chromosomes. The chromosomes start to move toward the centre of the cell. - Metaphase
- chromosomes aligned at the metaphase plate. The spindle fibres are fully formed and connected to the centrioles at each pole. The tension exerted by the spindle fibres ensures proper alignment of the chromosomes.
Mitotic Stages II
- Anaphase
- The sister chromatids of each chromosome separate and move toward opposite poles of the cell. The spindle fibres shorten, pulling the chromatids apart. - Telophase
- The separated chromatids reach the opposite poles, and a new nuclear envelope reforms/reassembles around each set of chromosomes. The chromosomes start to decondense, and the spindle fibres disassemble. - Cytokinesis
- physical separation of the cell into two daughter cells. contractile ring forms. when it constricts, it creates a cleavage furrow.
cleavage furrow (in animal cells) or a cell plate (in plant cells).
Cohesins
Proteins that hold sister chromatids together.
Centromere
Repetitive DNA sequence that serves as a target for mitotic machinery
Kinetochore
Protein complex (one/chromatid) that links the centromere to microtubules
So why bother with sex?
- Evolution depends on novel mutations.
- In addition, new combinations of existing alleles create new
phenotypes. - Sex (reshuffling of alleles) drives evolution.
Meiosis I
- Homologous chromosomes pair.
- Recombination occurs between
homologous chromosomes. - Homologous chromosomes
separate. Sister chromatids
remain intact. - Each daughter cell gets a
different set of chromosomes. - Each daughter cell is 2n, but
contains either a maternal
or a paternal version of
the chromosome.
Meiosis II
- Sister chromatids separate.
- Four gametes form (1n)
The Goal of Meiosis:
Make four genetically distinct
daughter cells (Shuffle and reduce to 1n)
phosphorylation
Kinase: drive cell cycle
an enzyme that adds a phosphate group to its target
Phosphatase: an enzyme that removes a phosphate from its target. the target protein (substrate) can be activated or inactivated by phosphate
- 3 different amino acids that can be phosphorylated: serine, tyrosine and threonine
Heterodimeric protein kinases
Two parts:
* Cyclin-regulatory subunit
* Cyclin dependent-kinase (CDKs)-catalytic subunit
CDKs are present throughout the cell cycle.
Cyclins are cyclical (expressed at specific cell cycle stages).
CDKs are inactive unless bound by a cyclin, and the specific cyclin determines the CDK’s specificity
If you don’t have the cyclin you cant recognise what you need to phosphorylate and if you don’t have the cdk you don’t have the enzymatic activity to add that phosphate group to the residue that you’re trying to
CDKs in the eukaryotic cell cycle
G1 cdks
(G0 = inactive cdks)
G1/s cdks
S cdks
G2/M cdks
M cdks
How would you test the activity of a CDK?
Kinase assay:
- pull down cyclin/cdk complex using antibodies
- purify antibody/protein complexes
- add substrate and radioactive ATP
- quantify amount of labelled phosphate transferred to substrate on an SDS PAGE gel
ubiquitination
- Ubiquitin-Protein ligases attach Ub
to a target protein. - Process repeats multiple times. This
results in polyubiquitination. - Proteasome recognizes polyubiquitination, and destroys the protein.
Ubiquitin-Protein Ligases
facilitate the transfer of ubiquitin molecules from ubiquitin-conjugating enzymes to target proteins. They recognize specific target proteins and promote the attachment of ubiquitin to these proteins, determining their fate and influencing various cellular processes.
SCF complex (SKP1, Cullin, F-box)
- Involved in the G1-S phase transition
Anaphase Promoting Complex or cyclosome (APC/C)
- Involved in metaphase-anaphase and mitosis-G1 transitions
Phosphorylation vs. Ubiquitination
Phosphorylation is temporary and reversible (molecular switch)
Poly-ubiquitination -> degradation is permanent and irreversible
G1Cyclin-CDKs
promote S phase entry by phosphorylating and activating proteins involved in the initiation of DNA replication, e.g. TFs
These TFs drive the expression of genes for DNA replication and cell
growth.
Transcribed genes include:
* Enzymes to make dNTPs.
* DNA polymerases
* Other replication proteins
* S-phase cyclins
Their activity ensures proper progression through the cell cycle and accurate replication of the genome.
SCF Ubiquitin-protein ligase
An inhibitor of S-phase cyclin/CDKs
Push cell into S-phase by polyubiquitinating (dooming) an
inhibitor of S-phase cyclin-CDKs.
Why is S-phase cyclin/CDK inhibition needed for control of S-phase entry?
Makes the transition much more abrupt
- prevent re-replication
- coordinate with other cell cycle phases
- activate replication checkpoints
- maintain genome stability.
It ensures the accurate and orderly progression of DNA replication, contributing to the proper functioning and integrity of the cell.
- CKI (Sic1) is a poor substrate and must be phosphorylated on
multiple sites - Peak CDK activity->CKI phosphorylation and degradation (one
of the last substrates to be fully phosphorylated)
S-phase cyclin/CDKs
Phosphorylate and activate numerous proteins that go on to replicate the DNA.
The onset of DNA replication
means that we have reached S-phase.
S-phase cyclin/CDKs also help prepare the cell for mitosis (similar to the role that G1/S CDKs play in G1)
The G1/S Transition
- G1 and G1/S CDKs prepare the cell for S-phase by inducing expression
of genes required for DNA replication. - The SCF Ub-ligase degrades an inhibitor of S-phase cyclin/CDKs
so that DNA replication can begin.
Why is the G1/S transition so abrupt?
It’s a poor substrate that requires high levels of kinase to become phosphorylated.
Needs to be phosphorylated on multiple sites.
This makes it one of the last substrates to get phosphorylated in G1.
Why screen for temperature-sensitive mutants?
provides a powerful tool for studying gene function, protein activity, cellular processes, and identifying potential drug targets. By harnessing the temperature-dependent effects of mutations, researchers can gain valuable insights into the complex workings of biological systems ??
Mutagenise = grow up cells at permissive temperature, then shift them to restrictive temperature. characterise lines that fail to grow after temperature shift.
