Course 4 Flashcards
What is the function of the cell cycle?
- cell reproduction - parent cell -> 2 daughter cells
- Assumptions of division into 2 identical daughter cells:
O completely duplicating genetic material ( DNA replication)
O duplication of other functional capacities of the cell ( organelle duplication) - cell cycle duration varies from cell to cell, in mammalian somatic cells it is about
20 hours
What are the different phases of the cell cycle?
- nuclear and cell division (mitosis + cytokinesis) and interphase
- phases of the cell cycle
O G 1 phase (gap 1) - dormancy period
O S phase (synthesis) - DNA replication
O G 2 phase - dormancy period
O M phase (mitosis) - mitosis & cytokinesis - phase-specific changes in cell morphology and physiology - the cell is not round (“blebs” & “microvilli”), it is usually rounded only when entering mitosis (disconnection from the extracellular matrix, RNA and protein synthesis)
What are the functional stages of the cell cycle?
- basic functional stages of the cell cycle are DNA replication (S phase), nuclear division (mitosis, M phase), cell division (cytokinesis, M phase) and doubling of cell functional capacities (G1 -> G2)
- The cell cycle is actually several coordinated functional cycles , which in certain circumstances function independently of each other => for example, the
growth cycle, the cycle of DNA synthesis, nuclear division and cell division
O separate operation of cycles are for instance grooving eggs ( growth cycle is excluded), meiosis ( after one cycle of DNA synthesis), followed by two cycles of nuclear fission) and endoreduplication ( DNA synthesis only)
S phase of the cell cycle: DNA replication
- DNA replication has a semiconservative character ( the resulting double helices have one original fiber and one newly produced one)
- DNA replication organization - replication unit (replica), origin of replication , replication fork
- mechanism of DNA replication
O DNA polymerase - ensures its own DNA replication
O primase - synthesizes the primer on which it anneals (DNA polymerase)
O helicase - untangles/unwinds double-stranded DNA
§ leading strand - new fiber in the 5 ‘-> 3’ direction (DNA polymerase δ), the
template is a 3 ‘-> 5’ fiber
§ lagging strand -
new strand in the 3 ‘-> 5’ direction (DNA
polymerase α)
* Okazaki fragments - synthesize in the
5 ‘-> 3’ direction, DNA ligase joins the
individual fragments
O coupled histone synthesis - histone synthesis
it usually takes place simultaneously with the S phase
§ the leading strand keeps histones, the lagging strand gets new histones
Nuclear division (mitosis) - M phase of the cell cycle
- Mitosis and cytokinesis are mostly interrelated processes
- the chromosomes condense 10,000x
- centrosome cycle - involves duplication of the centrosome (centriole)
What are the phases of mitosis?
- prophase - chromosome condensation (two sister chromatids)
O the centrosomes move slowly into place - prometaphase - the disintegration of the nuclear envelope (nuclear lamina)
O attachment of mitotic spindle microtubules to chromosomes (via kinetochore microtubules connected to the centromere) - metaphase - chromosomes in the equatorial plane
O metastable state - the cell is waiting for an instruction to the next phase - anaphase - division of sister chromatids
O the released chromatids travel to the poles of the mitotic spindle - telophase - reintegration of nuclear packaging
What is the structure and function of the mitotic spindle?
O 3 types of microtubules
O kinetochore - interconnect kinetochores
§ anchored in the centrosome by their minus end
O polar - push the mitotic spindle apart
the cells connect with the polar MTs of the second centrosome
§ they allow centrosomes to push away from each other
O astral - they attach to the cell cortex, they go in all directions from centrosomes (forms a star, therefore astral)
How are chromosomes segregated by the mitotic spindle?
O anaphase A - detachment of chromatids from each other by shortening kinetochore microtubules
O anaphase B - moving centrosomes and chromatids apart
work of polar and astral microtubules
What is the disintegration and reintegration of the nuclear envelope based on?
disintegration and reintegration of the nuclear envelope is based on phosphorylation and dephosphorylation of nuclear lamina proteins
Cell division (cytokinesis)
- Cytokinesis usually begins during mitotic anaphase, but doesn’t take a part in the M phase
- it ensures approximately even distribution of the components of the parent cell into 2 daughter cells (organelles, cytosol)
- a new cell boundary arises in the equatorial plane of the mitotic spindle
- mechanisms of cytokinesis
O animal cells - grooving process
§ contractile ring - ticks the cell, made up of actin and myosin
§ the need for a new plasma membrane
O cells of higher plants - a new cell boundary arises in the middle like a cell plate (fragmoplast)
§ it grows from the center to the regions where it connects to the cell membrane of the parent cell
O algae and mushrooms - invagination (edge-to-center sticking) plasma membranes
Doubling of functional capacities of a cell
- exact duplication of nuclear DNA vs. duplication of other cell components
O protein and RNA synthesis - during the whole interphase
O organelle duplication
§ simple membrane organelles (ER, GA) - before entering cytokinesis there are twice as many, they cannot create them de novo, they
can only add or enlarge them
§ complex “cell-like” organelles (MIT, chloroplasts) - autonomous reproduction by fission
What are resting cells?
- cells permanently in G 0 phase (G 0 not part of the cell cycle)
- does not participate in the cell cycle
- transition G 1 -> G 0 and G 0 -> G 1 it is associated with changes in cell physiology and requires some time
- to return from G 0 to G 1 Myc protein synthesis is required
- G 0 have a lower level of Myc protein and is more resistant to stress
O Myc protein = transcription factor - for example, stem cells and cells in stable terminal differentiation stages
- the cell cycle of the Xenopa embryo does not include G 1 phase
What are the mechanisms of cell cycle regulation?
- two basic levels of cell cycle regulation
O activation- move a cell from G 0to G 1
§ primary (early) response gene expression -> secondary gene expression
O progress - cell passage through the cell cycle - G cell passage through the cell cycle - G 1 -> S -> G 2 -> M -> G 1 -> …
§ two blocks in the cell cycle - G1 block and G 2 block (we can stop the passage of the cell)
How are the mechanisms for regulating the cell cycle activated?
- activation takes place in two functional steps
O primary (early) response gene expression - genes encoding most secondary gene transcription factors
O expression of secondary genes - the products of the primary genes are controlled
What is the function of the primary response genes?
- primary response gene expression - direct response to a stimulus signal
O expression of no mediating protein is required - their products regulate the expression of secondary genes
- important genes of the primary response
O c-myc - key gene activation
O c-fos & c-jun - heterodimer of Fos and Jun proteins (transcription factor AP-1)
What is the function of secondary genes?
- the expression of secondary genes is mediated by transcription factors encoded by the primary response genes
- secondary genes encode effector proteins of cell cycle realization
- examples of secondary genes - CDK genes (cyclin depending kinase; serine-threonine kinases), cyclin genes, c-myb
What is the function of the Rb protein?
- Rb family proteins - pRb, p107, p130
- they are key regulators of the cell cycle
Rb proteins regulate (inhibit) the cell cycle at the level of activation (G 0 -> G 1) and progression (passage G1 and and transition G1—WITH)
O Rb proteins bind to a transcription factor E2F thereby inhibiting it; The Rb protein is inhibited by phosphorylation, which alters its
conformation -> release occurs, E2F becomes active -> uncontrolled proliferation - positive feedback in the cell cycle applies - active proteins that ensure progression through the cell cycle deactivate Rb
What is the mechanism of progression of the cell cycle?
- progression through the individual phases of the cell cycle takes place on the basis of the gradual activation of certain CDKs
- cyclin dependent kinase (CDKs) - are serine-threonine kinases that play a central role in regulating progression
- for CDK activation it is necessary to bind cyclins (hence the name - cyclin dependent kinase)
O CDK is a stable catalytic subunit
O cyclin is a regulatory unit degraded during the cell cycle - further modifications (eg phosphorylation / dephosphorylation) are often required for complete activation of the cyclin / CDK complex
- active CDK phosphorylates the relevant proteins, thereby inducing a certain process of the cell cycle (entry into the S phase, entry into mitosis)
How is the progression of the cell cycle-regulated?
- changes in cyclin / CDK complex activity - CDK level remains the same, cyclin level changes cyclically
O in general - linear increase in cyclin level -> binding to CDK -> activation of CDK -> induction of the relevant process (eg
entry into mitosis) -> cyclin degradation -> CDK inactivation - entry into anaphase - cleavage of sister chromatids is ensured by the enzyme separase, which is blocked by the protein securine
O active APC (anaphase promoting complex) comes and cuts securin - separase is free and divides chromatids
Cyclin / CDK complexes
- cell cycle progression is controlled by a system of several CDKs and relevant cyclins
O cyclin D / CDK4, CDK6 - passage of G1 phases
O cyclin E / CDK2 - transition from G1 to S phase (starts a new cell cycle)
O cyclin A / CDK2 - passage with phase
O cyclin A / CDK1 - passage of G2 phases
O cyclin B / CDK1 - regulates cell entry into M phase (mitosis)
§ CDK1 is also called cdc2
What are some CDK inhibitors?
- by binding to the cyclin / CDK complex, they inhibit its activity
- types of CDK inhibitors
O family p21 - p21
§ p21 acts at the cyclin level E / CDK2
O INK4 family - p15, p16 - p53 allows the blocking of potentially very dangerous replication of damaged DNA
O DNA damage -> activation of p53 -> induction of p21 expression -> p21 -> inhibition of cyclin E / CDK2 -> blocking of S entry phase
What are the steps in synthesis of pyrimidine nucleotides?
1) synthesis of carbamoyl phosphate in the cytoplasm of the cell, carbamoyl synthetase 2 (CAD enzyme activity)
O glutamine + HCO3 + ATP -> carbamoyl phosphate
2) carbamoyl phosphate + aspartate -> join together , the phosphate is cleaved
3) dehydration - splitting the water, the circle joins and forms dihydroorotate ( dihydroorotic acid)
O reactions 1, 2 and 3 catalyze one large enzyme - CAD
4) dehydrogenation - dihydroorotic acid -> orotic acid
O enzyme dihydroorotate dehydrogenase , it sits in the inner mitochondrial membrane and looks outward
O electron acceptor is here Coenzyme Q (respiratory chain)
5) orotic acid + PRPP -> orotidine monophosphate (OMP)
O PRPP - important compound , it is called activated ribose, properly phosphoribosil pyrophosphate
PhosphoRibosil PyroPhosphate is a universal ribose donor; occurs when ribose-5-phosphate receives pyrophosphate from ATP
O PRPP and PRDP (PhosphoRibosil DiPhosphate) are exactly the same
O upon formation of the bond between orotate and PRPP, the pyrophosphate is released and, due to the enzyme pyrophosphatase , disintegrates, thereby
releases enough energy to form an N-glycosidic bond
6) decarboxylation - orotidine monophosphate -> uridine monophosphate (UMP) + CO 2
O reactions 5 and 6 catalyze the same enzyme - orotidine monophosphate synthase - OMP synthase
- Synthesis of cytidine monophosphate - UMP + glutamine -> CMP + glutamate
- Synthesis of thymidine monophosphate - deoxyuridine monophosphate (dUMP) + methylene tetrahydrofolate -> TMP + dihydrofolate
O thymidylate synthase enzyme; important site for tetrahydrofolate metabolism
O Thymidine has deoxyribose, unlike uridine and cytidine, which have ribose
How are purine nucleotides synthesized?
- in the synthesis of pyrimidines a base was built and ribose was attached to it, in the case of purines it is the other way around
- the whole synthesis takes place in the cytosol
- PRPP acts as the basis on which substrates are bound and bases are formed (phosphoribosil synthetase = important regulatory enzyme (feedback), ensures the formation of PRPP
1) The amide group from Gln is transferred to C1 PRPP , the pyrophosphate is released again and disintegrates
O PRPP + Gln -> 5-Phosphoribosil-1-amine + Glu
O this step is regulated by the enzyme amidophosphoribosil transferase which is positively regulated PRPP = the more nucleotides are formed
2) results in inosine monophosphate (IMP)
O substrates for these reactions: glutamine ( donates amide group), glycine (whole), aspartate ( amine group),
WHAT 2 ( whole), formyl tetrahydrofolate ( 2 carbon residues)
O the IMP base is called Hypoxanthine (Hyx)
- amination of IMP (aspartate is used) produces AMP
- dehydrogenation of IMP produces Xanthidine monophosphate, in the next step amide is added (glutamine is used) and GMP is formed
- Both AMP and GMP inhibit their synthesis from IMP in reverse, regulating the balance
between AMP and GMP
Ribonucleotides -> Deoxyribonucleotides
- enzyme ribonucleotide reductase ( cofactor is iron)
- thioredoxin protein is an electron donor (due to its SH groups) and NADPH supplies thioredoxin electrons
- general equation: ribonucleotide diphosphate -> deoxyribonucleotide diphosphate
Tetrahydrofolate (derivative B9)
- under normal circumstances, tetrahydrofolate is the donor of mono-carbon residues - methyl / formyl / methylene-tetrahydrofolate transfers a single-carbon residue, tetrahydrofolate is formed and it is “charged” again, eg by the reaction serine -> glycine
O the exception is thymidine synthesis , wherein upon transfer of the carbonaceous residue, the tetrahydrofolate is oxidized to
dihydrofolate, which is completely useless and must first be reduced back to tetrahydrofolate in order to be recovered and useful
O if the enzyme dihydrofolate reductase that reduces dihydrofolate is blocked, all reactions that use tetrahydrofolate as a cofactor will be blocked as well
- the tetrahydrofolate forms are freely convertible with one another, except for methyl tetrahydrofolate
O methyl tetrahydrofolate cannot be converted into anything else and the only way to get that monocarbon residue is to get rid of (and thus convert to tetrahydrofolate) is the synthesis of methionine from homocysteine
O synthesis of methionine from homocysteine has one cofactor - vitamin B12
O it follows that in the absence of B12, methyl tetrahydrofolate will accumulate, so the body will not have enough tetrahydrofolate for nucleic acid synthesis
§ = we need vitamin B12 to recycle vitamin B9
§ = in case of B12 deficiency, the body will also show signs of B9 deficiency = important !!
Nucleotide recycling
- binding of PRPP , more common in purines
- adenine binds to PRPP to form AMP; APRT enzyme (adenine phosphoribosil transferase)
- guanine binds to PRPP, GMP is formed; HGPRT enzyme (hypoxanthine-guanine phosphoribosil transferase)
- the HGPRT defect is called Lesch-Nyhan syndrome
O symptoms - hyperuricemia, behavioral disorders (extreme psychotic self-harm, mental retardation),high concentration of PRPP, which stimulates further synthesis of products (thanks to positive feedback) = overproduction of waste products
Degradation of pyrimidines
- complete decomposition except for water and ammonia
- 2 intermediates: β-alanine (in uracil and cytosine) and β-amino isobutyrate (for thymine)
Degradation of purines
- all purines decompose to the final product which is uric acid (trihydroxypurine)
- uric acid is poorly soluble in water => even a small increase leads to crystallization and gout disease
- the enzyme that forms uric acid is Xanthine oxidase ( hypoxanthine -> xanthine -> uric acid)
O Xanthine oxidase contains an element Molybdenum
DNA replication
- one identical helix is created from one double helix
- semiconservative character - each of the daughter helices has one original
(template) and one new (complementary) helix - the synthesis proceeds from the 5 ‘to the 3’ end ( 5 ‘is the 5th carbon on deoxyribose bearing a phosphorus moiety and 3’ is the 3rd carbon on deoxyribose bearing an OH group; The 5 ‘end is thus the end of the helix where the phosphorus group is free, the 3’ end is where the OH is free)
O the newly formed fiber is therefore oriented 5 ‘-> 3’
O and because the helices join each other in opposite directions, the template
must be 3 ‘-> 5’
Replicon
Replicon (a segment of DNA): replication unit with its own replication origin.
Procaryotic chromosome: one replicon
Eucaryotic chromosome: many (hundreds and thousands) replicons
Replication origin: a specific sequence of DNA (rich in A-T pairs) where
the replications starts.
Replication fork: the replication continues from the replication origin in
both opposite directions → two replication forks moving apart (the shape of letter Y).
Procaryotic chromosome: replication fork moves at 1000bp/s.
Eucaryotic chromosome: replication fork moves at 100bp/s.
DNA polymerase
it catalyzes the formation of phosphodiester bond between two nucleotides (3´end and 5´end of deoxyriboses) via relevant
phosphate.
The newly added nucleotide of growing DNA strand: first, complementary pairing with the base of relevant nucleotide of the
template, afterwards, the formation of phosphodiester bond with the previous
nucleotide of growing strand
Nucleotide enters the reaction as nucleoside triphosphate.
Energy released by freeing pyrophosphate (PPi) is used for polymerization reaction.
Two important and limiting properties of DNA polymerase:
* It can synthesize new DNA strand only in 5´→3´ direction (according to the template in 3´→5´ direction)!
* It is unable to start the synthesis of new DNA strand, it can only extend the existing strand of nucleic acid.
DNA polymerases of eukaryotic cell:
* DNA polymerase α
* DNA polymerase δ
and other types (DNA polymerase β)
What are some other proteins of replication machinery?
- Helicase: after the binding to replication origin, it unwinds the double helix of DNA (energy from ATP is used).
- Single-strand binding protein: molecules of the protein stabilize single-stranded DNA by binding to it.
- Primase: it starts the replication by the formation of a short RNA strand (primer).
Primer provides DNA polymerase with 3´ end, DNA polymerase continues the synthesis of new DNA strand according to the
template. primer in procaryotic cell: 5 bp
primer in eucaryotic cell: 10 bp - Protein sliding clamp: it keeps DNA polymerase attached to the template strand and it allows DNA polymerase to slide along the strand.
What are the mechanisms of DNA replication?
leading strand
Synthesis of new strand in 5´→3´on template 3´→5´
synthesis runs continuously (DNA polymerase δ)
Lagging strand
Synthesis of new strand in 3´ →5´ direction on 5´→3´ template:
Synthesis runs discontinously (DNA polymerase α)
Lagging strand:
DNA polymerase „skips“ here forward along the template and then it synthesizes backwards in proper direction 5´→3´.
The synthesis of new strand is performed piece after piece and these pieces are referred to as Okazaki fragments (each fragment starts with its own primer).
Afterwards, RNA primers are removed, missing DNA is synthesized by
relevant DNA polymerase and finally individual fragments are joined by
DNA ligase.
Okazaki fragments of procaryotic cell: about 1000 nucleotides
Okazaki fragments of eucaryotic cell: about 200 nucleotides
REPLICATION OF ENDS OF EUCARYOTIC
CHROMOSOMES
A problem of synthesizing the lagging strand at the end of chromosomes (telomere): it is solved by telomerase
Telomerase: adds short repeats of a DNA sequence to the 3´end
it uses a RNA template that is part of the enzyme
Repetitive DNA sequence then acts as a template to complete replication of the end of lagging strand.
PROOFREADING
It is a correcting activity of DNA polymerase on new DNA strand in 3´→5´ direction while it synthesizes new strand in 5´→3´
direction.
Functioning of DNA polymerase before binding a new nucleotide:
* It verifies whether previously bound nucleotide has the base complementary to the template
* If yes, it continues by binding a new nucleotide
* If no, it removes the previous wrong nucleotide and, instead of this
nucleotide, the corresponding nucleotide is bound
Proofreading activity of DNA polymerase explains why DNA polymerase has only 5´→3´ polymerase activity and proofreading in
3´→5´ direction.
Proofreading in 5´→3´ direction (hypothetical polymerization in 3´→5´
direction) is not possible from the chemical point of view.
MISMATCH REPAIR
Mismatch repair: it corrects wrongly paired bases of newly synthesized DNA strand (it corrects mistakes of replication machinery).
Proteins, involved in mismatch repair, recognize pairing which is not
complementary (mismatch) due to the deformation of DNA double helix.
Afterwards, they remove wrong segment of new DNA strand and synthesize this segment again.
Replication machinery: 1 error/107 nucleotides
Mismatch repair: correction of 99% errors of replication machinery
→Overall accuracy of DNA replication: 1 error/109 nucleotides
MECHANISMS OF ACCIDENTAL DNA DAMAGE
- Depurination: release of guanine or adenine from DNA (spontaneous)
- Deamination: conversion of cytosine to uracil (spontaneous)
- Formation of pyrimidine (thymine) dimers: caused by UV irradiation
MECHANISMS OF THE REPAIR OF
ACCIDENTALLY DAMAGED DNA
General steps of the repair of damaged DNA:
* recognition of the damage of DNA strand → Excision of the damaged DNA by specific nucleases
* synthesis of removed DNA according to complementary strand by repair DNA polymerases
* rejoining newly synthesized DNA segment with repaired DNA strand by DNA ligase (ligation)
* Base excision repair
* Nucleotide excision repair
The stability of DNA and thus also the stability of genetic information depends on mechanisms of DNA repair.
What is the essence of transcription?
Transcription: synthesis of new complementary RNA strand according
to DNA template (copying genetic information from DNA).
Synthesis of new RNA strand is catalyzed by RNA polymerase.
During transcription, one strand of DNA double helix serves as a template (template strand) while the second strand is referred to as coding strand.
RNA POLYMERASE
RNA polymerase: it catalyzes the formation of phosphodiester bond
between subsequent ribonucleotides.
Properties of RNA polymerase:
* It has only 5´→3´ polymerase activity (similarly like DNA polymerase)
according to template in 3´→5´ direction
- It is able to start the synthesis of new RNA strand (on the contrary to DNA polymerase)
3 types of RNA polymerase in eucaryotic cell:
* RNA polymerase I: genes for most of rRNAs (with the exception of 5S rRNA)
* RNA polymerase II: all genes encoding proteins, genes of some small RNAs
* RNA polymerase III: genes encoding tRNAs, gene for 5S rRNA, genes of some small RNAs
What is the mechanism of transcription?
Initiation of transcription:
RNA polymerase binds to the promoter of gene which will be translated.
Promotor: a sequence of DNA that indicates the point where the transcription starts. Promoter is located before the first transcribed nucleotide. It contains TATA box (formed mainly by T and A nucleotides).
RNA polymerase binding to promoter:
General transcription factors are involved in the binding.
Promotor is asymmetrical and it always binds RNA polymerase in one direction while RNA polymerase can transcribe DNA templates only in a 3´→5´ direction.
→Proper strand is only transcribed, i.e. template strand.
What happens after RNA polymerase binds to the promoter?
RNA polymerase unwinds in front of it a short segment of DNA double
helix (accessibility of template DNA strand for transcription)
→
RNA polymerase adds to the first transcribed DNA nucleotide complementary RNA nucleotide and thus the transcription is started.
Elongation phase of transcription:
RNA polymerase continues along the template DNA strand, it unwinds
ahead a short segment of DNA double helix and at the same time it synthesizes new RNA strand on the basis of complementary pairing
with the bases of template strand.
During transcription, a short hybrid double-stranded segment of DNA
RNA is formed transiently. However, newly synthesized RNA strand is released from binding to DNA soon.
How is transcription terminated?
In bacteria, signal for the termination is a specific DNA sequence (terminator). In eucaryotes, the mechanism of termination differs for individual RNA polymerases (so far it is not fully clarified)
→
The release of RNA polymerase from binding to DNA and at the same time the release of newly transcribed RNA strand.
Transcription machinery: 1 error/104 nucleotides
POSTTRANSCRIPTIONAL RNA PROCESSING
Posttranscriptional RNA processing:
modifications of RNA after its transcription in the nucleus of the eucaryotic cell and before its transport into cytoplasm and its translation.
Modifications of both ends of transcribed RNA strand:
* RNA capping: 7-methylguanosine is bound to the 5´end by the unusual 5´→5´ linkage via a bridge made of 3 phosphates (cap).
- RNA polyadenylation: repeated adenine nucleotides (100-200) are bound to the 3´end (poly-A end).
These two modifications increase the stability of mRNA
RNA splicing
noncoding sequenses (introns) are removed from primary transcript and coding sequenses (exons) are joined in given order.
Completed mRNA, which is ready for translation, is formed by the
posttranscriptional processing.
What is RNA splicing?
Introns are often longer than exons.
Exons (in an average gene): ~ 1000 nucleotides
Introns (in an average gene): 5000-20 000 nucleotides
Spliceosomes: large complexes of ribonucleoproteins and proteins
which carry out splicing.
Present ribonucleoproteins contain small nuclear RNAs (snRNAs).
Small nuclear RNAs recognize exon-intron boundaries and they form small nuclear ribonucleoprotein particles (snRNPs) referred to as „snurps“.
„Snurps“ form the core of spliceosome.
What is the mechanism of RNA splicing?
at the place of relevant intron, a loop is formed due to the interaction of
spliceosome with RNA → ends of neighboring exons get closer → RNA
strand between intron and exon is interrupted and corresponding ends
of neighboring exons are joined → the loop of excised intron is released and at the same time components of spliceosome are
released
Evolutionary significance of intron existence:
* Possibility of alternative splicing (more proteins from one gene).
* Increased probability of genetic recombination between exons of
different genes.
What is the essence of translation?
Translation: the synthesis of new polypeptide chain (protein) according to genetic information saved in corresponding mRNA.
The sequence of amino acids is determined by the sequence of bases in corresponding mRNA. It happens on the basis of genetic code.
It is a turn (translation) from the language of nucleotides into the language of amino acids.
Translation apparatus reads mRNA in 5´→3´ direction.
New polypeptide chain is synthesized from the N end towards the C end
Where is translation carried out?
Translation is carried out on ribosomes.
Together with mRNA (it is carrying genetic information), tRNA plays a key role during translation.
Individual types of tRNA function as adaptors recognizing corresponding codon for amino acid which they carry
What is the structure and function of ribosome?
Ribosome: a large complex that comprises several types of rRNA and a big number of various proteins.
Eucaryotic ribosome (80S ribosome):
* Small subunit: 1 type of rRNA (18S) & 33 proteins.
* Large subunit: 3 types of rRNA (5S, 5.8S, 28S) & 49 proteins.
There are 4 specific binding sites on ribosome:
* mRNA binding site
and three tRNA binding sites, i.e.
* A site (aminoacyl-tRNA): it binds tRNA carrying relevant amino acid
* P site (peptidyl-tRNA): it binds peptidyl-tRNA (peptide bound to tRNA)
* E site (exit): tRNA is released from ribosome here
What is the structure and function of tRNA?
tRNA: about 80 nucleotides, it contains some minor bases and nucleotides derived from them (pseudouridine - ψ, dihydrouridine - D).
Structure of tRNA: 4 short double-stranded segments (complementary pairing)
3 double-stranded segments are terminated by single-stranded loop
it has a character of clover leaf
Relevant amino acid is bound at the 3´end (terminal sequence of CCA) by energy-rich bond.
Energy of this bond is used for the formation of peptide bond during translation.
What is an anticodon?
sequence of 3 nucleotides of tRNA which is complementary to the triplet of bases on mRNA encoding amino acid carried by this
tRNA.
Some of tRNAs require, for binding to mRNA during translation, precise complementary pairing only on the first two positions of codon.
→
For amino acid encoded by more triplets with the same first two bases, only one tRNA is sufficient for translation.
Thus only 31 types of tRNA are sufficient for 20 amino acids encoded by
61 codons.
Aminoacyl-tRNA synthetase
performs binding of amino acid
to relevant tRNA with corresponding anticodon. Each amino acid has own specific
aminoacyl-tRNA synthetase.
These synthetases carry out decoding of the genetic code
What is the mechanism of translation?
The translation of mRNA always starts with the codon AUG which encodes methionine. →
Every new polypeptide chain starts with methionine.
A specific initiator tRNA carrying methionine is required for the initiation of
translation.
Initiation of translation:
Binding of the initiator tRNA together with other initiator factors to the small ribosomal subunit
→ complex binds to the 5´end of mRNA (it recognizes the cap)
→ it slides along mRNA in 5´→3´ direction until it recognizes the first AUG codon
→ the large subunit binds (ribosome is completed) while the initiator
tRNA is bound at the P site
→ further aminoacyl-tRNA, with the anticodon corresponding to the
second codon of mRNA after AUG, binds at the A site
Elongation of translation
It starts by binding of further aminoacyl-tRNA at the A site
→ polypeptide chain is released from tRNA at the P site and it is bound by peptide bond to the amino acid carried by the tRNA at the A site
→ at the same time tRNA carrying prolonged polypeptide chain is shifted from the A site to the P site and now free tRNA is shifted from
the P site to E site
→ thus, the A site is free for binding of further aminoacyl-tRNA
→ in the last step, tRNA is released from the E site and whole the cycle
of elongation can be repeated
Peptidyl transferase: it catalyzes the formation of peptide bond, it is a
part of the large ribosomal subunit.
How is translation terminated?
It takes place when one of the three stop codons (UAA, UAG, UGA) appears at the A site
→ stop codons are not recognized by any tRNA and protein release
factors are bound instead of tRNA
→ a consequence is that peptidyl transferase catalyzes the binding of water molecule instead of amino acid
→ polypeptide chain is terminated and it is released from the ribosome
Polyribosomes: the initiation of translation happens repeatedly on individual mRNA molecules → several ribosomes perform translation on one mRNA molecule at the same time
DNA study methods
- DNA diagnostics deals with gene sequences and repetitive sequences of non-coding regions
- RNA diagnostics and protein diagnostics deal with gene expression
- nucleic acid sources
O DNA - all nuclear cells (ideally blood)
O RNA - only cells where the gene is expressed (because there is a different RNA in each tissue due to cell differentiation)
DNA diagnostics
-are able to detect:
O monogenic and polygenic hereditary diseases
O some types of tumors (protooncogenes and tumor suppressor genes)
O infection present (pathogens being sought)
O disease progression during treatment
O identification of people in forensic medicine (individual people differ from each other mainly by repetitive sequences)
O HLA-typing (HLA is a complex of genes responsible for the detection of foreign particles) - during transplantation
Preventive diagnostics
- preimplantation - embryo testing before artificial insemination
- prenatal - prenatal screening in high-risk pregnancies (not without risk for the child, parental consent required)
- presymptomatic - neonatal screening
DNA variability
- DNA sequence variability between different individuals of the same species
- DNA polymorphism - physiological; the occurrence of the allele in at least 1% of the population determines the predisposition to polygenic diseases
- mutation - pathological; the occurrence of the allele is not even in 1% of the population, it causes monogenic diseases
Characteristics of DNA diagnostics
- a polymorphism of a given predisposing gene is detected
- targeted analysis - we know the sequence and localization of the gene, we know the nature of the mutation (deletion / substitution / …)
O We are going to be sure to look at a specific point in DNA
O DNA is isolated -> PCR (multiplication of isolated DNA) + further analysis -> visualization (electrophoresis)
O examination of the family is not necessary - complete analysis - we know the sequence and localization of the gene, but we do not know exactly which mutation it is
O necessary examination of other family members (because we are trying to find out exactly what nucleotides are located in
this sequence of genes of a healthy and a sick family member, compare them and find a mutated nucleotide)
DNA isolation
- basic steps
O lysis of the cells and release of the DNA into solution
O removal of proteins by cleavage, adsorption or extraction
§ we do not want proteins in solution because they would inhibit the following steps
O precipitation of DNA - DNA precipitates and can be washed -> removal of impurities
O dissolving DNA in water or buffer
Purity and concentration of DNA
- protein concentration is measured
- spectrophotometry - adsorption maximum for DNA is 260nm, for proteins 280nm
O with the ratio 260/280 we find the purity of DNA (the higher the ratio the cleaner the DNA) - gel electrophoresis with fluorescent dyes
O The DNA with the bound colors is visible in the gel
O the gel contains a DNA sample of known concentration - then only the light intensities are compared (highly indicative)
Gel electrophoresis
- size distribution of DNA fragments (RNA, proteins) on the principle of moving charged particles in an electric field
- DNA contains negatively charged phosphate groups -> movement from cathode (-) to anode (+)
- the speed of movement is inversely proportional to the length of the fragment
- gel - network structure of polymer molecules with pores
O polyacrylamide - able to distinguish DNA fragments differing by one nucleotide
O agarose - divides nucleotides differing by at least 10 nucleotides, preferably used on DNA - ethidium bromide - the color added to the gel binds to DNA and glows under UV
Polymerase chain reaction (PCR)
- principle: multiplication (amplification) of a selected section of DNA
- the only PCR control constant is the temperature
- basic components of PCR
O DNA sample
O two types of primers
O free nucleotides (dATP, dTTp, dCTP, dGTP) O DNA polymerase with buffer
Primers
- short DNA oligonucleotides (20-30 nucleotides) binding to a specific site on the DNA
- each primer binds to one strand of a double helix, one primer is called forward and the other reverse
- serves as a replication start
- delimit the target region of the DNA we want to amplify (template strand for replication must be 3 ‘-> 5’ and the primer indicates the start of replication, so the primers bind to the 3 ‘edge of the target DNA)
- annealing of the trimmers is affected by temperature - it is necessary to create hydrogen bonds, the length of the primers and the content of nucleotides raise the required temperature (CG needs a higher temperature than AT)
The course of PCR
- PCR takes place in cycles (30-40) and each cycle has 3 steps
1) denaturation - reversible separation of individual DNA strands by breaking the hydrogen bonds between complementary nucleotides at a temperature of at least 94 ° C
2) annealing - attaching primers to separate strands of DNA
O the minimum temperature depends on the primer used
3) extension (elongation) - elongation, synthesis of a new DNA strand using DNA polymerase
O we need 72 ° C, but human DNA polymerase would not withstand it, so living bacteria DNA polymerase is used in hot springs, so-called TAQ polymerase
Types of PCR
- PCR with allele-specific primers (ASO-PCR)
O when we know exactly what mutation there is -> the primer will only fit on it
O targeted analysis - 2 separate tubes are used
§ 2 primers are used - one of the pair binds directly to the site of the possible mutation and one somewhere to the surroundings § in the 1st tube the allele-specific primer is complementary to the healthy allele, it only mounts if the mutation did not happen
§ in the 2nd tube the allele-specific primer is complementary to the mutation, it only mounts if the mutation has occurred
§ if a reaction occurs in both tubes -> heterozygous - PCR with general primers
O amplification always occurs, then targeted, complete analysis
O we use it if we don’t know what it is - we need more general primers
O we will use general primers in targeted and complete analysis
PCR subtypes
- nested PCR - 2x PCR with general primers, another PCR reaction is performed on the result of one PCR
O used for old or bacterial-contaminated samples
O PCR primers may be (purely coincidentally) complementary to non-human DNA, so we do not know if the result is a copy of human DNA, so it is mixed with other primers and 2. PCR is either performed (human DNA) or not (foreign DNA) O it is determined whether the DNA sample belonged to a human or not - multiplex PCR - two or more PCRs take place in one reaction mixture at a time
PCR analysis of the PCR product with general primers
- unknown mutation - complete analysis
O sequencing - searching for the complete (exact) order of nucleotides in an amplified section of DNA - known mutation - targeted analysis
O hybridization - We analyze the PCR product using a labeled probe
§ nucleotide linkage based on complementarity = hybridization
§ the DNA fragment is placed in a tube together with the labeled probe, which is complementary to a healthy part of the gene, if there is a mutation then the fragment does not associate with the probe
O restriction fragment length polymorphism (RFLP)
§ The PCR product is specifically digested with restriction enzymes ( restriction endonuclease - restrictase) § the endonuclease recognizes and cleaves them in a specific sequence (eg … CCAAGG … -> … CCA and AGG …)
§ Inspired by bacterial enzymes, enzymes can now be made to order in laboratories as required § the cleavage result is suitable for gel electrophoresis
Gene expression
- the process by which genetic information stored in individual genes is realized as functional molecules of encoded proteins (ie the transfer of genetic information from DNA -> RNA -> protein)
- the expression of individual genes is regulated in cells => cells can quickly adapt to changes in the environment
- cell differentiation - expression of different genes leads to different cell types
Levels of gene expression regulation
1) genome (DNA)
2) transcription (DNA -> RNA / primary transcript)
3) posttranscriptional modifications (RNA / primary transcript -> mRNA)
4) mRNA
5) translation (mRNA -> polypeptide chain)
6) post-translational modifications (polypeptide chain -> functional protein) 7) protein degradation (functional protein -> degraded protein)
Genome
- gene amplification - the number of copies of one gene will increase -> transcription will overwrite it more -> expression will increase
- genome rearrangement - the genome is not 100% stable, eg transposons (semiparasitic segments of DNA capable of changing their position and replicating in the genome) or
genes encoding immunoglobulins - chromosome condensation / decondensation - transcription factors and RNA polymerase need developed DNA to work -> heterochromatin or barr body are not expressed
O histone deacetylase - cleaves the acetyl group from the histone to condense the DNA
- DNA methylation - higher degree of methylation -> lower degree of gene expression
O in general, genes with methylated DNA are not expressed (Barr body, imprinting)
O methylase - catalyzes the methylation of cytosine at 5’C in DNA (5-methylcytosine)
O methylation is also a mechanism of gene imprinting
Transcription
- is regulated by proteins that bind to regulatory DNA sequences
- DNA regulatory sequences - 10-10,000bp long sequences that are involved in turning specific genes on and off
O important regulatory sequences - promoters and enhancers (special DNA sequences capable of triggering transcription-specific gene, even at a distance of up to 1,000,000bp) - proteins regulating gene transcription
O general transcription factors - ubiquitous, attached to everything
O specific transcription factors - genes also have their own transcription factors, they are the main regulators
O they contain several DNA binding motifs
§ homeodomain - has 3 α-helix parts
-zinc finger- a zinc atom is attached here - leucine zipper- contains a lot of leucine
How is transcription regulated in eukaryotic cells?
- general transcription factors - they associate with RNA polymerase at the promoter via binding to the TATA box
- TATA box - a DNA sequence on a promoter that usually contains nucleotides A and T
- activator and enhancer - the activator binds to the enhancer, together through mediator proteins, and general transcription factors help to bind RNA polymerase to the promoter and trigger transcription
O the activator and enhancer need not be at all near the transcribed gene - combinatorial control - Several protein regulators work together to regulate the expression of a particular gene - many of these regulators are needed for activation
How is transcription regulated in prokaryotic cells?
O operon - a set of genes transcribed from a single promoter
O operator - a site on the promoter where the protein can bind and function as transcriptional repressor / activator
O repressor operon - trp operon - if there is too much tryptophan, it mounts the operator and blocks further synthesis
O activator operon - lac operon - if there is too much lactose, it mounts the operator and activates the synthesis of the enzyme,
which can metabolize lactose
Posttranscriptional modifications
- RNA capping and RNA polyadenylation (increase mRNA stability)
- post-transcriptional modifications affecting gene expression
O alternative montage - allows the coding of several genes with the same gene, thanks to the use of other combinations of exons of that one RNA itself (exons may be omitted, but their order must be maintained)
§ different cell types make different proteins from the same gene
O RNA editing - after transcription, nucleotides in the transcribed RNA can be inserted, deleted or substituted
§ modification of transcribed genetic information - discovery of new initiation and stop codons , associated with a change in reading frame
mRNA
- mRNA degradation - mRNA viability affects expression ( the more stable the higher the expression level)
- microRNA - short regulatory RNAs that regulate gene expression by regulating the degradation of the respective mRNA
O it looks for complementary sections of RNA as soon as it finds such a section it sits down and begins to degrade the whole fiber - mRNA viability can also be regulated by nucleotide sequences in the 3 ́ untranslated region of the mRNA e.g. IRE (iron responsive element) , to which it is bound IRP (iron regulatory protein)
Translation
- it can be regulated by the binding of a specific protein to untranslated sections of mRNA
- e.g. IRE in the untranslated 5 ‘region of the mRNA , to which it is bound IRP and translation is blocked
Posttranslational adjustments
- Post-translational modifications of a polypeptide chain involve a number of mechanisms, including chain cleavage and molecular binding
- a) removing methionine from the N-terminus
- b) removing the signal sequence ( which determines the target of the protein)
- c) proteolytic cleavage - forming a functional protein by cleaving the precursor polypeptide chain
O proinsulin -> insulin + C peptide - d) forming disulfide bridges - they form between adjacent cysteines, they stabilize the structure of the protein
- e) chemical modification of amino acids - phosphorylation (phosphate-binding) and hydroxylation (OH binding)
O only tyrosine, serine, histidine, and threonine can be phosphorylated - f) glycosylation - binding of the oligosaccharide chain, glycoproteins are formed in this way (this is not possible in bacteria)
- g) bonding of a prosthetic group - non-amino acid / non-protein molecule
required for protein function (heme)
Degradation of proteins
- a method of regulating the concentration of a particular protein in a cell
- proteasome - a large complex of proteolytic enzymes arranged in a cylindrical shape
with a catalytic site inside - ubiquitin - a small protein, covalently attached to proteins, indicates them for
degradation
What are gametes?
Gametes are germ cells
- gametogenesis - differentiation of highly specialized germ cells capable of creating a new individual after fertilization
- egg (ovum) - various sizes, carries a lot of nutrients
- sperm - small, motile
Origin of primordial cells
- germ cells can be recognized very early in the embryo
O they can be seen for the first time in the yolk sac of the embryo around the 24th day of development
- -6. week they migrate through the mesenchyme to the places where the gonads should form
- after migration, germ cells (which have multiplied along the way) induce genital development
- oogony - from several thousand, mitosis multiplies to + - 7 million (roughly during the 2nd - 5th month of development)
O around the 7th month, oogonia ceases to multiply and enters the prophase of the first meiotic division - spermatogony - they enter meiosis only during puberty and never lose the ability of mitosis
Spermatogenesis
- the process that leads from spermatogonia to sperm
- 3 phases - multiplication (mitotic division), maturation (meiosis) and differentiation
- It takes about 9 weeks + a few weeks after the sperm are in the epididymis before they are able to function
- the onset of sperm development takes place in the seminiferous tubules of the testis
O seed-forming channels contain germinal epithelium - they pass through the entire height of the epithelium Sertolli cells, among which sperm occur at various stages of development
§ Sertolli cells help with differentiation and supply them with the necessary nutrients § stem cells sit on the basement membrane and divide mitotically
§ the cells gradually differentiate towards the lumen
- among the seminiferous tubules are Leydig cells, which produce testosterone
Spermatogenesis - development of sperm
- stem cells (spermatogonia), which attach to the basement membrane, divide mitotically, giving rise to spermatogonia A and spermatogonia B
O spermatogonia - remain at the basement membrane, acts as stem cell
O spermatogonia B - already further they do not divide mitotically
O penetrates the barrier that holds spermatogonia A in place and becomes the primary spermatocyte
- primary spermatocyte - meiotic division begins at puberty, but gets stuck in prophase I for a long time (3 weeks)
O one primary spermatocyte divides into two secondary spermatocytes - secondary spermatocytes - it is further divided into 4 spermatids
- spermatids (spermatozoa) - get rid of unnecessary organelles and begin spermiogenesis
Spermiogenesis
- the process of maturation of spermatids in sperm
- some organelles are transformed, others are depleted by the cell
O chromatin it concentrates in the nucleus (heterochromatin) and moves to one side of the cell - it forms a header
O Golgi apparatus turns into acrosome - the organelle that surrounds the front of the nucleus forms such a cap
§ It is full of enzymes that help sperm penetrate the egg
O microtubules and dynein they begin to form an axoneme - a microtubule structure inside the flagellum
O centrioles - one serves as an anchor for the flagellum, the other gives the material to form the axonema
O mitochondria they begin to move to MT and surround them - the so-called mitochondrial helix ( they supply whip energy for movement)
O everything else (including the cytoplasm) is thrown out of the cell in the form of residual bodies that phagocytose the surrounding
Sertoli cells
Sperm maturation
- newly formed sperm ripen in the epididymis
O sperm are formed, which, however, are still not able to fertilize - the so-called capacitance
- capacitance - the final stage of maturation - preparation for the release of enzymes from the acrosome + changes in the sperm membrane
O takes place only in the female reproductive system - sperm hyperactivation - increase in sperm movement
- acrosomal reactions - fusion of the acrosome with the plasma membrane, formation of an acrosomal process
Sertoli cells
- supporting cells - provide a microenvironment for spermatogenesis
- are anchored in the basal lamina
- they have an oval core, a distinct nucleus
- Neighboring Sertoli cells are connected by a tight junciton, forming hemotesticular barrier
O it divides the epithelium into basal compartment ( spermatogonia A) an adluminal compartment ( residue)
O it also has immunological significance - germ cells in the spermatocyte stage are genetically different from wear cells and
they could be recognized as foreign and discarded by autoimmune processes
O function of the selective transport mechanism
Leydig cells
- are placed in groups between the testicular canals near the capillaries
- produce androgens - testosterone
- contain fat droplets, rich smooth endoplasmic reticulum and tubular type mitochondria
Oogenesis
- at birth - 400,000 oocytes are surrounded by a layer of follicular cells
O only about 400 of them mature, the rest degenerate - closely related to the ovarian cycle (controlled by the hypothalamic-pituitary system)
- in addition to the development of the egg, the follicle (shell) around the egg also develops
Egg
- forms the stores of yolk as a reservoir of energy, phospholipids and cholesterol to build the membranes of the embryo
- The adult egg is the largest cell in the body
- contains a lot of proteins - other energy stores, transcription factors, signaling molecules
- ribosomes and tRNA - proteosynthesis after fertilization
- mRNA - early development - morphogenetic factors (transcription and growth
factors) - It moves through lashes and muscle contractions
- saved in cumulus oophorus
Layers surrounding the egg
- zona pellucida - a lot of glycoproteins, GAGs, hyaluronic acids etc.
O produced by the oocyte as a shield through which nothing passes
O it only has a sperm receptor
O after fertilization, the chemical composition changes - corona radiata - packaging made of follicular cells
O connection to the oocyte using nexus
Stages of follicles
- primordial follicle - single-layer flat epithelium just below the surface of the ovary, diameter up to 40 μm
O resting stage of the egg - primary follicle - monolayer cubic epithelium, diameter up to 100 μm
O zona pellucida forms between the oocyte and the follicle - secondary follicle - multilayered epithelium, diameter 200 μm
O oocyte enlargement, stromal cells line up circularly around the follicle (theca folliculi) - tertiary follicle = Graafian follicle diameter up to 2.5 cm
O oocyte enveloped by a thick zona pellucida
O theca folliculi divided into theca interna (epithelial cells) and theca externa (myofibroblasts) - yellow body - corpus luteum - what is left when Graaf’s follicle bursts and the oocyte gets out
- atresia - death of follicles
Ovulation
- rupture of the mature Graafian follicle
- a sharp rise in LH concentration in the blood
- the oocyte completes the 1st maturation division and enters the 2nd maturation division (stops in metaphase - the division is completed only after fertilization of the oocyte) → mature
Graafian follicle bursts and empties (theca externa myofibroblasts) → the oocyte is captured by the fallopian tube, where it remains until becoming fertilized (approximately 3 to 4 days; if the oocyte is not fertilized, the 2nd maturation division is not completed and the oocyte is expelled from the body)
O the movement of the egg in the fallopian tube is ensured by the movement of the cilia and the contraction of the muscle of the fallopian tubes
Corpus luteum
- the rest of the Graafian follicle that remains in the ovary gives rise yellow body - follicular cells are transformed into granulosal lutein cells and theca foliculi cells are transformed into theca-lutein cells
O granulosa-lutein (large lutein) cells - they secrete gestagen, which induces reproductive tissue differentiation system and maintains pregnancy
O theca-lutein (small lutein) cells - they secrete androgens - the corpus luteum is formed at the LH signal (= first signal; promotes progesterone production) - the corpuscle is then itself functional for about 12-14 days -> when the
egg is not fertilized, luteolysis occurs - a scar remains = corpus albicans - fertilization → embryo produces hCG ( one of the markers of pregnancy tests) = the second signal for the corpus luteum to produce progesterone (massively
mainly in the first two months) and grow strongly (corpus luteum graviditatis) - it produces only progesterone, not estradiol because it has no aromatase
Menstrual cycle
- desquamation phase ( menstrual phase) - the pars functionalis breaks down, the blood vessels open
- proliferation phase = follicular - growth of new epithelium from the remnants of glands, the mucosa grows rapidly, under the influence of FSH
- secretory phase - The endometrium is the thickest, the endometrial glands are filled with secretions and twists glycogen, proteins, strom are stored in stromal cells.
- eggs are capable of transcription even during meiotic division
Fertilization
- it most often takes place in the ampular section of the fallopian tube
- approx. 24 hours: penetration of sperm into the oocyte -> completion of the 2nd maturation division -> connection of maternal and paternal chromosomes in ->
formation of diploid zygote -> first mitotic division
Sperm penetration into the egg
- sperm must get through the corona radiata and the zona pellucida → in the zona pellucida, the sperm binds to its receptor (ZP3) and triggers acrosome response ( release of enzymes from the acrosome) → this creates channels through which sperm can penetrate
- the cytoplasmic membrane at the sperm head merges with the oocyte membrane
- the fusion of the membranes initiates a cortical reaction that prevents other sperm from entering the egg
- during fertilization, the entire content of sperm enters the egg (the cytoplasmic membrane remains outside - it merges with the membrane of the egg) → part of the content is
used by the egg for its own use (centriole + chromosomes), the rest is destroyed - imprinting - disabling some genes depending on whether they are from the mother or the father; important for the differentiation of the embryoblast into the trophoblast (in the
father, the genes for the embryoblast are turned off so that only those on the trophoblast are affected and vice versa)
What happens in the egg after sperm penetration
- completes the 2nd maturation division (a mature oocyte and a secondary pole body are formed)
- chromosomes are enveloped by a nuclear envelope - maternal primordial nucleus
- the sperm nucleus is decondensed and its nuclear envelope is renewed - paternal primordial nucleus
- followed by the approach of both primordial nuclei → decay of nuclear envelopes → chromosomes are captured on a common dividing spindle → diploid zygote
- the rest of the sperm is destroyed by zygote enzymes
Polyspermia prevention
- rapid blockage of polyspermia - change in electric potential (Ca 2+)
- slow blockage of polyspermia - enzymes from cortical granules prevent the binding of any other sperm by destroying other binding sites in the zona
pellucida (ZP2 and ZP3) - fertilization envelope - space between pellucida and egg - GAG, peroxidase and hyaline
- zonal reaction - leaching of hydrolytic enzymes from the oocyte, takes place in the zona pellucida
The result of fertilization
- the restoration of diploid chromosome number (new combination of genetic information)
- gender is determined
- initiation of scoring (development of a new individual) - sperm contributed to this by a mitotic spindle
Grooving
- it is a mitotic division without cell growth and proteosynthesis - the number of cells increases without increasing the volume / size of the whole embryo
O the cells divide mitotically without synthesizing new organelles after mitosis - mitotic division is equal and total
O 40 hours - 4 cells
O 3ED - 6-12 cells
O 4ED - 16-32 cells - this stage of development is called morula
PROCESS OF DIFFERENTIATION
Differentiation: process of generating diverse types of cells (cell specialization)
zygote → more than 200 distinct cell types in vertebrates
DETERMINATION
Determination: process in which the cell becomes commited to certain fate
Cell usually becomes determinated before it starts to differentiate.
BIOLOGICAL SIGNIFICANCE OF
DIFFERENTIATION
- Development (ontogenesis) of a multicellular organism
- Renewal of tissues and organs
Development (ontogenesis) of multicellular organism:
zygote → mature organism
Ontogenesis is realized in coordination with cell proliferation.
Renewal of tissues & organs:
* Physiological cell renewal
* Repair: repair regeneration
wound healing
BASIC PRINCIPLES OF DIFFERENTIATION
The genome remains intact during differentiation.
Different cells of organisms do not contain different genes but they express different genes.
Cell memory: cells differentiate and remain differentiated after the signal for differentiation disappears. Cells keep in their memory the effects of past signals and pass them on to their descendants.
Mechanisms of cell memory: positive feedback (self-activation of
gene expression)
* Positional information: cell is capable of retaining information reflecting its location in an organism.
Degree of cell capability to differentiate into various cell types
- Totipotence: cells of early stages of cleavage
- Pluripotence: stem cells (blood cells)
- Unipotence: stem cells (epidermis)
SIGNALING MOLECULES OF
DIFFERENTIATION
Signaling molecules are involved in the regulation of cell differentiation
during development.
Morphogen (signaling molecule): concentration gradient of a morphogen
forms positional signal for individual cells → relevant differentiation
MECHANISMS CONTROLLING
DIFFERENTIATION
- Asymmetry: chemical and other asymmetries of the egg →cleavage results in cell with different quantities of determinative
molecules - Embryonic induction: interaction of cells → certain fate of one or both participants
- Positional signal: formation of concentration gradient of signaling molecule (morphogen) → positional signal for cells
- Response timing: cells change their internal status during the time (intracellular clock) → they respond differently to the same signal
during different time periods
HOM COMPLEX /HOX COMPLEXES
Homeotic genes: they determinate anteroposterior character of Drosophila body segments
Homeotic genes are organized within Hom complex in one chromosome.
Hox genes: in vertebrates homologous with homeotic genes of Drosophila
Hox genes are organized within Hox complexes.
Hox complexes: 4 Hox complexes in mammals (Hox A, Hox B, Hox C,
Hox D)
individual Hox complexes are localized on different chromosomes
Hox complexes are involved in generation of positional information (positional signal): proteins coded by individual Hox genes function as molecular markers of a body region.
The sequence of genes within chromosome corresponds with the spacial sequence of expression of genes within body
PROCESS OF CELL SENESCENCE
Cell senescence: cell after a certain number of realized cell divisions is losing its ability to proliferate and finally it inevitably dies (state of terminal differentiation)
Biological significance of cell senescence: protection against uncontrolled
proliferation
Genetic control of cell senescence
HAYFLICK LIMIT
The life span of the cell is not determined by certain time but by certain number of realized cell cycles.
Hayflick limit: programmed life span of somatic cell of certain organism
expressed as a number of cell cycles
Hayflick limit of human embryonic cells: 50 cell cycles
Immortality of cells: cancer cells
cells of germinal lines
CAUSES OF CELL SENESCENCE
- Accumulation of mutations and injuries: wear-and-tear theory
- Oxidative damage
- Damage of mitochondrial genome
- Role of insulin signaling: inhibition of the expression of proteins
protecting against injuries - Shorterning of telomeres
OXIDATIVE DAMAGE
Production of ROS (reactive oxygen spiecies): result of metabolism
ROS, on the basis of oxidation, they damage membranes, proteins and
nucleic acids.
DAMAGE OF MITOCHONDRIAL GENOME
Mutation rate in mitochondrial DNA is significantly higher (10-20x) than
mutation rate in nuclear DNA.
Damage of mitochondrial genome: defects in energy production
ROS production
apoptosis induction
SHORTENING OF TELOMERES
Mechanism of telomere shortering: insufficient function of telomerase
Consequences of telomere shortering: DNA damage → p53 activation
CELL DEATH
Programmed cell death: cell has genetically encoded program for selfdestruction, after receiving relevant signal the cell is active
participant of selfdestruction
Apoptosis: type of programmed cell death in animals (characterized by caspase activation)
it is accomplished under physiological conditions
Necrosis: nonprogrammed cell death
necrosis is accomplished under nonphysiological conditions
it results from irreversible cell damage
What are some other types of programmed cell death?
- Necroptosis (necrosome, permeabilization of plasma membrane)
- Lysosomal cell death (permeabilization of lysosomal membrane)
- Pyroptosis (inflammasome: activation of caspase-1)
- Ferroptosis (iron accumulation, lipid peroxidation, permeabilization of
plasma membrane) - Autophagic cell death (autophagy/autophagosome)
Comparison of apoptosis and necrosis
Apoptosis
* active process (it requires
energy supply)
* early DNA degradation
* chromatin condensation
* plasma membrane stays intact
* cell shrinking
* formation of apoptotic bodies
* without an inflammatory response
Necrosis
* passive process
* DNA stays intact
* chromatin stays intact
* loss of function and integrity of
plasma membrane
* cell swelling
* disintegration of plasma
membrane → cell lysis
* accompanied by inflammatory
response
BIOLOGICAL SIGNIFICANCE OF APOPTOSIS
- Regulation of cell number
- Formation of organisms during development
- Function of the immune system
- Pathological states
ROLE OF APOPTOSIS IN CELL NUMBER
REGULATION
Cell elimination: cells with damaged DNA, virally infected cells
- Maintenance of tissue homeostasis: maintenance of steady state
cell number
Tissue homeostasis: balance between the production of new cells by proliferation and loss of cells by apoptosis
