MBB11003 -Molecular and Cell Biology Flashcards

1
Q

What are the universal properties of cells?

A

-contain DNA
-cell components can self-assemble
-proteins require signals
-they can respond to signals from environment
-they have feedback mechanisms

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2
Q

What are the roles of proteins?

A

-recognise specific molecules (hormones, antibodies, DNA binding proteins)
-move other molecules (eg. porin, ferritin)
-accelerate the rate of rxns (enzymes)
-structural roles (microtubules)

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3
Q

What is the primary structure of a protein?

A

the order of amino acids in a polypeptide
-involves peptide bonds (which have no rotation)

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4
Q

What is the secondary structure of a protein?

A

the folding of peptide chain into its 3D structure -alpha helix or beta sheet
-involves H-bonds
-represented by ribbon diagrams

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5
Q

What is the tertiary structure of a protein?

A

the tightly-packed thermodynamically stable 3D structure of a protein
-involved non-covalent interactions between side chains

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6
Q

What interactions occur between cysteine residues?

A

disulphide bridges
-S-H → -S-S-
-form crosslinks

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7
Q

What are protein domains?

A

areas of the protein which fold tightly and carry out a specific part of the protein’s function
-one protein will have multiple domains

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8
Q

What is the quaternary structure of a protein?

A

a protein’s complex structure made up of two or more subunits joined together
-can be a dimer (2 subunits), trimer (3 sub-us), tetramer (4 sub-us), etc

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9
Q

What happens in post-translational modification?

A

-specific parts of sequence are removed (irreversible!)
-molecules are added (methylation, glycosylation, ubiquitination, phosphorylation)

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10
Q

What is methylation?

A

addition of methyl group
eg. histones are methylated to control which parts of genome are expresses

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11
Q

What is glycosylation?

A

addition of sugars
-usually to cell surface proteins or secreted proteins

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12
Q

What is ubiquitination?

A

addition of ubiquitin (76-amino acid polypeptide -small regulatory protein)
-for degradation

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13
Q

What is phosphorylation?

A

addition of phosphate group (PO3)
-done by kinases (requires ATP, which is dephosphorylated to ADP and Pi)
-typically done to serine, threonine or tyrosine residues
-can regulate enzyme function (can change active site’s properties and alter binding of substrate)
-can be reversed by phosphatases

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14
Q

What are the Mendelian laws of inheritance?

A

-law of segregation (genes come in pairs, one of which is passed onto the offspring)
-law of independent assortment (different genes are passed onto the offspring separately)
-law of dominance (when there are two alleles of a gene, the dominant allele is expressed)

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15
Q

What is Mendel’s law of segregation?

A

genes come in pairs, one of which is passed onto the offspring

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16
Q

What is Mendel’s law of independent assortment?

A

different genes are passed onto the offspring separately

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17
Q

What is Mendel’s law of dominance?

A

when there are two alleles of a gene, the dominant allele is expressed

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18
Q

What can be concluded from Mendel’s laws of inheritance?

A

there must be a physical genetic material, which is able to be replicated, stored, expressed and varied via mutations

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19
Q

Why did Sutton use grasshoppers/Boveri use Ascaris worms when investigating genetic material?

A

-both have large chromosomes
-both only have a few chromosomes

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20
Q

What did Sutton and Boveri observe?

A

-chromosomes group in pairs and can separate
-chromosome number is reduced in gametes
-chromosomes are required for embryonic development
-chromosomes are linear structures made of genes

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21
Q

What did Sutton and Boveri investigate?

A

chromosomal inheritance

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22
Q

What did Frederick Griffith investigate?

A

whether characteristics could be swapped
investigated Streptococcus pneumoniae + whether he could make the S strain into R strain and vice versa

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23
Q

What is the difference between S strain and R strain Streptococcus pneumoniae?

A

S (smooth) strain has a polysaccharide coat (forming a protective capsule around the bacteria, protecting it from the host’s immune system) and is pathogenic
R (rough) strain does not have a polysaccharide coat (so is unprotected from host’s immune system) making it not pathogenic

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24
Q

What was the transforming principle Griffith came up with?

A

something hereditary which was causing a change in genotype
-options were DNA, proteins, polysaccharides, lipids, RNA

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25
What did Avery, MacLeod and McCarthy investigate?
what was causing the transformation Griffith had seen aka what the transforming principle actually was -they concluded it was DNA
26
What did Hershey and Chase investigate?
which component of a bacteriophage is injected into a bacteria -using bacteriophage T2 (host: E.coli) -they radiolabelled its DNA or protein, infected bacteria, separated phage ghosts and tested phage ghosts for radiolabelled DNA/protein -concluded it was DNA
27
What experimental evidence is there of DNA structure?
Chargaff's experiment into the proportions of diff bases using paper chromatography -same proportions of purines and pyrimidines -same proportions of A and T, and C and G (diff to eachother though) X-ray crystallography -X pattern (=helix) -regular pattern (=repeated, even structure) -distance between spots (=distance between turns of helix, 3.5nm)
28
What are the features of Watson and Crick's model of DNA?
-A-T and C-G hydrogen bonded base pairs (2 H-bonds for A-T, 3 for C-G) -purines and pyrimidines are base paired to eachother -DNA strands run antiparallel to eachother (one 5' to 3', the other 3' to 5') -double helical structure (each phosphate backbone forms a helix) -double helix has major and minor grooves -one complete turn of the double helix is 10.5 base pairs
29
How many hydrogen bonds are between adenine and thymine?
2
30
How many hydrogen bonds are between cytosine and guanine?
3
31
How many base pairs is a complete turn of DNA?
10.5
32
What are the structural features of eukaryotic chromosomes?
-centromere (region which links chromosomes to spindle) -telomere (repetitive sequences of DNA at end of chromosomes)
33
What is a centromere?
specialised chromosomal region where chromosomes link to spindle microtubules -directs equal segregation of chromosomes during mitosis and meiosis -not necessarily at centre of chromosome
34
What is a telomere?
repetitive DNA sequences at the end of linear chromosomes -protect ends of chromosome
35
What are the structural features of prokaryotic genomes?
-single circular chromosome w/a few million bps (more compact genome than eukaryotes) -plasmids (small circular molecule w/thousands of bps which can be passed to other prokaryotes via conjugation)
36
What are the roles of DNA binding proteins?
-regulate gene expression (eg. transcriptional regulators like lac operon) -cut DNA at specific sequences (eg. restriction endonucleases) -protect DNA (eg. histones)
37
What did Mendel and Stahl do to investigate DNA replication?
-grew bacteria in medium containing N15 (which makes DNA heavy) -transfer bacteria to a medium containing N14 (which makes new DNA lighter) -separate heavy and light molecules by ultracentrifugation -look at DNA using UV light -heavier DNA would form bands at bottom, lighter DNA would form bands at top -results showed DNA replication is semi-conservative
38
What does DNA polymerase do in DNA replication?
add DNA nucleotides one at a time onto bases from 5' to 3' using template strand
39
What does primase do in DNA replication?
generates a primer (made of RNA)
40
What does ligase do in DNA replication?
joins new sections of new DNA together -joins "gaps" in sugar-phosphate backbone to make it a continuous molecule
41
What does helicase do in DNA replication?
breaks hydrogen bonds between bases to separate the DNA strands -to unwind helix
42
What does single sided binding protein do in DNA replication?
binds to strands (separated by helicase) to stop strands reannealing
43
What does topoisomerase do in DNA replication?
relieves pressure around the replication bubble by making breaks in the DNA molecule and then resealing it
44
What is the leading strand?
the strand of DNA where 5' to 3' synthesis points towards the replication fork so it can be continuously replicated
45
What is the lagging strand?
the strand of DNA where 5' to 3' synthesis points away from the replication fork so it can not be continuously replicated -must be primed multiple times -forms Okazaki fragments which need to be joined together
46
Why is DNA replication described as semi-discontinuous?
replication is continuous on leading strand but discontinuous on lagging strand (-opposite on other side of replication bubble)
47
What is the issue with the lagging strand requiring so many primers?
each time the primer is removed (because primer is RNA so must be removed), a gap is left at the end of the chromosome which is impossible to fill (due to DNAP only being able to work from 5' to 3') so a small piece of DNA is lost each time HOWEVER only telomeres (repeating DNA sequences at end of chromosomes) are lost which can be extended by telomerase
48
What does telomerase do in DNA replication?
replenishes the telomeres (which have been lost when the primer was removed) from an RNA template
49
What is the Shelterin Complex?
specialised proteins which form a protective cap on telomeres -this hides telomeres from being detected as "cell damage" (differentiates it from DNA breakages) so that nucleases don't break it down -this recruits telomerase
50
What can base pairing be like in RNA?
canonical (Watson-Crick bps -U/A and C/G) non-canonical (wobble bps)
51
What is canonical base pairing in RNA?
Watson-Crick base paring U with T and C with G
52
What is non-canonical base pairing in RNA?
Wobble base pairing due to folding and intramolecular interactions causing unique 3D structures Non-Watson-Crick pairs eg. U with G Eg. long range tertirary structure interactions like the A-minor motif
53
What is the A-minor motif?
Common tertiary interaction in RNA where two consecutive adenine residues interact with adjacent base pairs in minor-groove (elsewhere in the RNA molecule) through ionic interactions -an example of non-canonical base pairing
54
What happens in RNA transcription?
-RNA is synthesised by DNA-dependent RNA polymerase (which has an active site containing a short RNA/DNA heteroduplex) -genetic sequence in sense strand is transcribed to RNA by nucleotide triphosphates (NTPs) being selected by base pairing with the template strand (transcription bubble) and being added to 3’ end of RNA strand -RNAP is targeted to promotor region and when it reaches the terminator region, it is released (means transcription is not across whole genome but only the section needed to make the protein)
55
What is the structure of RNA polymerase?
5 subunits: -2 alpha (which bind transcription factors) -beta and beta’ (catalytic) -1 omega (responsible for assembly and stability)
56
What is the role of the 2 alpha subunits in RNA polymerase?
to bind transcription factors
57
What is the role of the beta and beta' subunits in RNA polymerase?
catalytic roles
58
What is the role of the omega subunit in RNA polymerase?
assembly and stability
59
What is the role of a sigma factor in RNA transcription?
targets RNAP to gene promotors, which enables binding IN PROKARYOTES -causes RNAP to have an open, active conformation -DNA in active site forms transcription bubble and short RNA primer is formed -sigma factor is releases, so RNAP moves away from promotor (promotor clearance)
60
How many RNA polymerases do bacteria have?
1
61
How many nuclear RNA polymerases do eukaryotes have?
3 nuclear RNAPs (plus others in plants) (have different RNAPs in mitochondria)
62
What are the three nuclear RNA polymerases in eukaryotes?
RNA polymerase 1 -transcribes rRNA RNA polymerase 2 -transcribes mRNA and non-coding RNAs RNA polymerase 3 -transcribes tRNA and 5s rRNA
63
Which RNA polymerase transcribes rRNA?
RNAP 1
64
Which RNA polymerase transcribes mRNA and non-coding RNAs?
RNAP 2
65
Which RNA polymerase transcribes tRNA and 5s rRNA?
RNAP 3
66
Why do eukaryotes have 3 different nuclear RNAPs?
-they transcribe different RNAs -each have common and unique subunits
67
What is the role of general transcription factors (gTFs) in RNA transcription?
enables RNAP to bind IN EUKARYOTES for RNAP 2… -TATA box binding protein (TBD) in TF2D binds to TATA box in DNA, causing DNA to bend, which allows other factors to be recruited -preinitiation complex (PIC) assembles at promotor using RNAP2 and gTFs
68
What is the TATA box?
A/T rich region within promotor region of eukaryotic gene promotors -which TATA box binding protein (TBD) in TF2D binds to
69
What does transcription using RNAP 2 require?
-activators (bind to enhancers in DNA sequence to attract RNAP 2 to DNA) -mediators (allows activators, gTPs and RNAP 2 to communicate) -general transcription factors -chromatin-modifying proteins
70
What is the difference between transcription and translation being coupled or compartmentalised?
COUPLED: in prokaryotes mRNA is translated into a protein whilst it is being made by RNAP -causing rapid gene expression -quick responses to changes in environment COMPARTMENTALISED: in eukaryotes mRNA is transcribed in nucleus and then translated in cytoplasm (separate as organelles are compartmentalised) -allows regulation at diff steps -less direct but more diverse
71
What happens in eukaryotic mRNA processing?
-5' end is capped -introns are removed (pre-mRNA splicing) -3' end is processed (cleavage and polyadenylation)
72
Why is it important for the 5' end of mRNA to be capped in eukaryotes?
helps mRNA to be distinguished from other types of RNA in the cell -this helps it to be further processed and exported and aids its translation
73
What occurs in the capping of eukaryotic mRNA?
-phosphate is removed from 5' end by a phosphatase -GMP (guanosine monophosphate guanine) is added (ie. guanine added) in reverse linkage (5' to 5' insread of 5' to 3') by a guanyl transferase -methyl group is added to guanosine by a methyl transferase (usually at position 7) ∴ known as m7G cap
74
Why is the cap on eukaryotic mRNA known as the m7G cap?
extra guanine nucleotide added to 5' ∴G cap nucleotide is methylated usually at position 7 ∴m7
75
Why is eukaryotic mRNA a monocistronic transcription?
a mRNA molecule only codes for one polypeptide (in prokaryotes, one mRNA can be translated into lots of different proteins)
76
What are spliceosome complexes?
large complexes made up of RNA and a spliceosome (protein) which carry out pre-mRNA splicing by removing introns (keeping exons) -smaller RNA/protein complexes (snurps) assemble/disassemble active spliceosomes
77
What are snurps?
small nuclear ribonucleoproteins aka small RNA/protein complexes -assemble/disassemble active spliceosomes
78
How are intron-exon sequences conserved (during pre-mRNA splicing)?
splice site sequences allow recognition of intronsic and exonic sequences -intron contains a 5' splice site, a branch point and a 3' splice site -the 5' and 3' splice sites are highly conserved and allow this recognition
79
How does pre-mRNA splicing occur? (its mechanism)
2 steps (transesterification rxn) -2' hydroxy group of branchpoint adenosine attacks 3' phosphate of exon -this 5'-2' phosphodiester bond gives a looped lariat -the 3' hydroxy group (generated from the first step) attacks the 5' phosphate of the 3' exon and the lariat is released (lariat is then degraded by enzymes)
80
What appears in the electrophoresis of cellular eukaryotic RNA?
rRNA (LARGE -25s and 18s appear on agrose gel; 5.8s and 5s appear on acrylamide gel) tRNA (SMALL -only appears on acrylamide gel) N/B: mRNA is not clearly visible -cells transcribe lots of diff mRNAs which vary in length and are very unstable
81
What experiments were carried out to find out about the genetic code?
-in vitro translation (cell extract isolated and its mRNA is degraded, synthetic C14-labelled RNA/aas are added and then protein is precipitated and collected) -ribosome binding (isolated purified ribosomes are incubated with radiolabelled amino-acyl tRNAs and mRNA codon, forming a stable ribsome/aa-tRNA codon-complex which can be separated to check its radioactivity)
82
Which sequences code for the stop codon?
UAA UAG UGA
83
Which sequence codes for methionine?
AUG
84
How many codons code for a specific amino acid?
61 -there are 64 possible codons but 3 of them (UAA, UAG and UGA) code for the stop codon -degenerate: multiple codons code for same aa (synonymous codons tend to vary on 3rd position of codon) -some codons just code for one aa (eg. AUG only codes for Met)
85
What is the structure of tRNA?
-cloverleaf secondary structure (5' and 3' drawn together, aa s attached to 3' OH of 3' terminal adenine) -specific nucleotides are post-transcriptionally modified (allows wobble in base pairing) -folded into L-shape -caused by coaxial stacking of helices and base pairing between ends of loops
86
How are tRNAs charged?
aminoacyl-tRNA synthases charge tRNAs by adding on an amino acid -requires ATP (which is converted into ADP and Pi) -aa is linked to tRNA by an ester linkage between carboxy group of aa and 3' hydroxy group of terminal nucleotide of tRNA
87
Why are tRNAs charged?
for gene expression -allows aa to be delivered to ribosome for translation to produce polypeptide chain
88
What is the structure of ribosomes?
-2 RNP (ribonucleoprotein) subunits: large subunit (where peptide bond formation occurs) and small subunit (where codon/anticodon binding occurs) -3 non-overlapping tRNA binding sites at subunit intersurface: A (where acyl-RNA binds), P (where peptidyl RNA binds) and E (where non-charged tRNA bind before leaving) -peptidyl transferase centre (PTC) is RNA-rich (RNA catalyses formation of peptide bond) -have a polypeptide tunnel -have a decoding centre
89
How are ribosomes synthesised in eukaryotes?
-rRNA transcription and early rRNA processing occurs in nucleoli -processing and assembly occurs in nucleoplasm and cytoplasm -functionally active ribosomes are only generated once they've left the nucleus (translation occurs in cyctoplasm)
90
What happens in the translation elongation cycle?
-2 tRNAs bind to A and P sites (known as pre-translocation state) (each tRNA is bought to ribosome by EF1A, which hydrolyses 1 GTP) -peptidyl transferase catalyses the formation of a peptide bond between the amino acids attached to these 2 tRNAs -ester linkage between amino acid and tRNA breaks -translocation: ribosome moves along mRNA (requires EF2, so 1 GTP is hydrolysed) so these 2tRNAs enter the P and E sites (post-translocation sites) and the tRNA in the E site leaves -another tRNA binds in the A site and this repeats
91
What are the 3 tRNA binding sites at the ribosomal subunit intersurface?
A -where acyl-RNA binds P -where peptidyl RNA binds E -where non-charged tRNA bind before leaving (exit site)
92
Which GTPases are involved in translation in eukaryotes?
elongation factors EF1A (brings aminoacyl-tRNA to ribosome) and EF2 (needed for translocation) -each hydrolyse one GTP
93
Which GTPases are involved in translation in prokaryotes?
elongation factors EF-Tu and EFG
94
What are the 2 distinct methionyl-tRNAs?
initiator methionyl-tRNA -recognises AUG codon, targeted to P site elongator methionyl-tRNA -binds to AUG codon, bought to A site
95
How is translation initiated in prokaryotes?
Shine-Dalgarno (SD) sequence in mRNA is recognised by 16S rRNA by base pairing with nucleotides at the 3' end of it -SD seq is typically AGGA
96
How is translation initiated in eukaryotes?
-initiator tRNA (bound to eIF2 and small subunit) is assembled at 5' end of mRNA by interacting with the cap-binding complex (CBC) -preinitiation complex scans along mRNA using CBC's helicase activity until it locates the initiation codon (AUG) in an appropriate context -after the initiation codon is selected, the large subunit of the ribosome is recruited
97
How is translation terminated in prokaryotes?
-termination/release factors (PROTEINS) recognise stop codons -initial binding of release factor (RF1 or RF2 depending on which stop codon) triggers peptide hydrolysis (recognition) -RF3 (a GTPase) allows RF1/RF2 to be released from ribosome -additional factors dissociate ribosome complex
98
How is translation terminated in eukaryotes?
-termination/release factors (PROTEINS) recognise stop codons -initial binding of release factor eRF triggers peptide hydrolysis (recognition) -eRF3 (a GTPase) allows eRF to be released from ribosome -additional factors dissociate ribosome complex
99
What is a cis element?
the regulatory sequence in DNA (or RNA transcript) which trans-acting factors interact with
100
What is a trans-acting factor?
a protein or RNA molecule which interacts with a cis element to regulate the expression of a target gene, different to the gene it was encoded by
101
What are the two types of mutations which affect gene expression?
in cis -mutations within the same gene in trans -mutations within a different gene
102
What do cis mutations identify?
DNA/RNA sequences which affect gene regulation
103
What do trans mutations identify?
factors that regulate the expression of a target gene
104
How can gene expression be regulated at transcription level?
-trans-acting factors (activators or repressors) can activate or repress translation -substrate-product availability can regulate the expression of enzyme-coding genes (inducers or corepressors up or down regulate expression of enzyme-coding genes)
105
What are trans-acting activators?
trans-acting factors which cause the activation of gene expression by interacting with RNAP's alpha subunit to promote DNA binding -promote expression at weak promotors +ve control (transcription increased)
106
What are trans-acting repressors?
trans-acting factors which cause down regulation of transcription -ve control (transcription decreased)
107
What is the promotor region like in E.coli genes?
E.coli gene promotors have bipartite sequences (2 separate nucleotide sequences both recognised by polymerase which act together) -sequences close to eachother are stronger promotors so have high transcriptional activity -sequences divergent to eachother are weaker promotors so have lower transcriptional activity so need to be stimulated by a transcriptional activator
108
How can trans-acting factors regulate genes at transcription level?
trans-acting factors can activate or repress transcription: -trans-acting activators interact w/alpha subunit of RNAP and promote DNA binding -trans-acting repressors cause down regulation of transcription
109
How can substrate-product availability regulate enzyme-coding genes at transcription level?
-substrates can cause upregulated expression of enzymes (known as inducers) aka increase transcription -substrates can cause downregulated expression of enzymes (known as corepressors) aka decrease transcription
110
How can gene expression be regulated at RNA processing level?
pre-mRNA splicing (introns removed, exons included/excluded) -can occur in diff patterns, resulting in 2 distinct proteins being produced -alternatively, can result in a non-productive pathway, where mRNA is degraded and expression is blocked
111
What do splicing activators and repressors do?
bind to specific sequences within pre-mRNA to promote exon inclusion or exclusion -either one or the other of adjacent exons are included
112
How can gene expression be regulated at translation level?
-typically at initiation step -or gene expression can be repressed due to presence of a repressor or small metabolite or diff conditions
113
How can gene expression be autoregulated?
-gene product influences its own expression (eg. if too much is produced)
114
What is in the lac operon?
regulatory gene: Pi and lacI Plac lacO structural genes: lac Z, lac Y, lac A
115
What are the three structural genes in the lac operon?
lac Z -codes for beta galactosidase lac Y -codes for lactose permease lac A -codes for acetyltransferase
116
What is the inducer for the lac operon?
allolactose (a derivative of lactose generated by β galactosidase)
117
What is the repressor for the lac operon?
lacI
118
What is the structure of lacI?
-is a homoetetramer (has 4 identical subunits associated, but not covalently bound) -has 3 domains: tetramerisation domain (allows protein to be assembled into a tetramer), core domain (where inducer binds) and head domain (where the DNA operator sequence binds)
119
What happens when the lacI repressor binds to the operator sequence in the lac operon?
-DNA binding sites of each subunit are aligned, causing DNA to twist -makes RNAP transcriptionally inactive (it can still bind but isn't active anymore)
120
How is the lac operon regulated by catabolic repression?
-glucose is E.coli's preferred carbon source, so when glucose is present genes required for metabolism of other sugars (eg. lactose) are repressed -when lactose is present as well as glucose, lactose is only metabolised after all the glucose has been used up -causing a diauxic growth curve -lac operon requires absence of glucose and presence of lactose
121
Why are genes encoding adenylate cyclase and catabolite activator protein (CAP) needed for lac operon?
-adenylate cyclase makes cyclic adenosine monophosphate (cAMP) -cAMP binds to CAP -cAMP/CAP complex binds to CAP site on DNA -this is required for RNAP activity, allowing the structural genes (lac Z, Y and A) to be coded for mutations in the genes adenylate cyclase and CAP block the expression of the lac operon N/B: adenylate cyclase is inhibited in presence of glucose (only occurs when lactose is being metabolised)
122
How does eIF2 (eukaryotic initiation factor 2) control transcription?
-eIF2 binds to Met-tRNA (charged initiator tRNA) and brings it to a small ribosomal subunit -when initiation codon is localised, eIF2 hydrolyses GTP to GDP (eIF2 has a v. low GTPase activity so needs to be stimulates by a GTPase activating protein (GAP)) -this causes a conformational change in eIF2 -this conformational change causes eIF2 and GDP to be released from the initiator tRNA -cells can downregulate translation by depleting levels of eIF2 (known as integrated stress response)
123
What is the role of GAPs?
GAP = GTPase activating protein -stimulate activity of GTPases (eg. eIF2)
124
What is required to recycle GTPases?
a guanine exchange factor (GEF) -promotes release of GDP and GTPase is then recharged with GTP
125
What is the role of GEFs?
GEF = guanine exchange factor -promotes release of GDP -recharges GTPase with GTP
126
How do cells downregulate translation by depleting the levels of eIF2?
-protein kinases phosphorylate eIF2 -eIF2-Pi binds tightly to eIF2B (decreasing levels of eiF2B) -this acts as an inhibitor (rather than a substrate) known as an integrated stress response
127
Why is molecular cloning carried out?
-to isolate a specific region of DNA (eg. a gene, promotor, intron, etc) -downstream to be able to sequence gene, analyse mutants vs normal genes, express and purify proteins, etc
128
How is molecular cloning carried out?
-DNA is cut using restriction enzymes -DNA is placed into a vector to get recombinant DNA -recombinant DNA is transferred into a host -host w/recomb DNA is selected and replicated
129
What are restriction enzymes?
enzymes which recognise a short, specific DNA sequence -4 types (type 2 most commonly used in labs)
130
What are type 2 restriction enzymes?
restriction enzymes which recognise specific 4-8bp sequence and generate sticky or blunt ends and generate 5’ phosphate and 3’ OH groups -have a polindromic DNA sequence (reads the same 5’ to 3’ on both strands)
131
What is a palindromic DNA sequence?
a sequence which reads the same 5’ to 3’ on both strands
132
What are the different types of restriction enzymes?
type 1 and 3 -cleave DNA at random point (far) type 2 -cuts DNA at specific point (close to recognition site) type 4 -cleaves modified DNA
133
How do restriction enzymes work?
-bind non-specifically to DNA -move along DNA until it finds recognition site -specific binding triggers structural changes in enzyme and DNA -Mg2+ required -5' phosphate and 3' hydroxy ends are generated
134
Why do the overhanging ends need to be compatible when cutting DNA in molecular cloning?
to allow vector and insert to stick to eachother
135
Why is the pairing between two compatible overhanging ends not a permanent reaction? (molecular cloning)
-only hydrogen bonds -no phosphodiester bonds => can be solved by using DNA ligase (eg. phage T4) to reform phosphodiester bonds
136
How does DNA ligase catalyse the formation of phosphodiester bonds?
-AMP is transferred to a lysine residue in ligase's active site -AMP is transferred to 5' phosphate -AMP-P bond is attacked by 3' OH, forming covalent bond and releasing AMP -ATP is required to replace AMP (∴ ATP is cofactor)
137
What issues could there be with the ligation reaction in molecular cloning?
-not enough DNA -DNA mixed w/other molecules -no convenient restriction site -vector self-ligates -incorrect recombinant DNA (wrong orientation)
138
How can you solve the issue of vectors self-ligating in molecular cloning?
modifying DNA ends -removing 5' phosphate (only 1 phosphate so no phosphodiester bonds; sticky ends can base pair but can't ligate) -adding 5' phosphate -removing DNA overhang
139
What are the essential features of vectors?
origin of replication (allows it to replicate inside host) selectable marker (allows cells containing vector to survive) multiple cloning sites (where gene is cloned)
140
How can recombinant DNA be transferred into the host in molecular cloning?
transformation -electroporation (high voltage pulse to induce transient pores in membrane) -chemical transformation (heat shock causes cell membranes to change so DNA can be taken up)
141
How can you select the hosts which do have the vector as a product of molecular cloning?
by using a selectable marker on the vector eg. antibiotic resistance genes -can then treat with antibiotic and see which survive (ISSUE: can't distinguish between empty vector and vector containing recombinant DNA)
142
What is PCR used for?
to amplify DNA in vitro -specific and selective
143
Why is PCR amplification described as being exponential?
molecules of DNA double each cycle
144
What happens in PCR?
-denaturation (double stranded DNA dissociates into single stranded DNA) at 95°C -primer annealing (primers bind to comp seq) at 55-65°C (temp depends on melting temp of primer) -primer extension (DNAP synthesises new strands of DNA from 3' end of primers) at 68-72°C
145
What polymerases are often used in PCR?
Taq (used a lot, faster) Pfu (slower but more accurate and better thermostability) -which is used depends on what you need most eg. thermostability, extension rate, etc
146
What are the properties of primers used in PCR?
-specific to template -at least 17bp long (typically 20bp) -come in pairs (bind opposite strands in opposite directions) -Tm around 60-65°C (primer Tm determines what temp to use for annealing)
147
When the annealing temp (Ta) is right in PCR, what do the primers do?
primers bind to specific sequence
148
When the annealing temp (Ta) is too low in PCR, what do the primers do?
primers might bind non-specifically to other DNA sequences
149
When the annealing temp (Ta) is too high in PCR, what do the primers do?
primers might not bind at all/not bind efficiently -reduces yield
150
What issues are there with ligating the PCR product straight into the vector?
-blunt-ended cloning is inefficent -blunt-ended cloning is non-directional -PCR products have no 5' phosphate -not an issue if vector has one, but if vector does there is a risk of it self-ligating -taq polymerase adds a 3' alanine overhang to its products (can remove it/some vectors can use it!) to avoid issues: restriction sites can be incorporated into primer
151
What variants of PCR are there?
RT-PCR quantitative PCR (qPCR)
152
What are the uses of RT-PCR?
-molecular cloning of a protein coding cDNA sequence -analysing mRNA expression
153
What is done in RT-PCR?
RNA is reverse transcribed into complementary DNA (cDNA) and then PCR is used to amplify specific cDNA sequence
154
How is cDNA synthesis carried out in RT-PCR?
-reverse transcriptase synthesises first strand of cDNA -poly(dT) primers bind to poly(A) tail of mRNA -RNA is removed -second strand of CDNA is synthesised by Klenow fragment of DNAP1 -hairpin formed by reverse transcriptase acts as a primer -ssDNA loop can be digested by a nuclease
155
Why is qPCR needed?
amplifcation is exponential but after a while it plateaus because dNTPs and primers run out or DNAP looses activity -qPCR allows you to calculate the relative levels of what you had to start with
156
How can the qPCR product be measured?
using a fluorescent dye (SYBR green, which fluoresces when it binds to DNAs proportionally to amount of DNA -not sequence specific) or fluorescent probes (fluoresces when displaces from template, sequence specific, can multiplex) -fluorescence increases over time
157
What is the cycle threshold (Ct)? (in qPCR)
the point at which the fluorescence (from the dye or probes) exceeds the background level -the lower the Ct value, the less cycles are needed to reach the threshold -difference in Ct values between two samples can be used to calculate relative amounts
158
Why do we need to analyse the products of DNA cloning?
to make sure it is correct (checking they aren't empty vectors)
159
What do we carry out to get DNA out of bacteria?
a mini prep
160
How is a mini prep done?
-grow lots of bacteria -break bacteria open -genomic DNA (not plasmids -too small and compact) precipitates -get µg of purified plasmid DNA
161
What does a restriction digest do?
digest DNA with a restriction enzyme
162
What does electrophoresis do?
separate DNA fragments based on their size by using a current
163
How is electrophoresis carried out?
-DNA ladder and samples are loaded into wells -power is turned on and DNA fragments migrate through the gel from the -ve electrode to +ve electrode -fragments are then separated by size (largest fragments towards -ve, smallest towards +ve) -dye is added to visualise
164
What does restriction mapping do?
identifies which recombinant DNAs have been successful/which have the gene orientated incorrectly or are just an empty vector by using restriction enzymes -successful recombinant DNA will produce a PCR product whereas an empty vector or incorrect recombinant DNA will not
165
What is Sanger Sequencing?
specialised form of DNA synthesis where the identity of a nucleotide at a specific position can be identified (synthesis can be stopped at a known nucleotide, meaning we know what length the DNA molecule is) (can carry out with A, then repeat with other nucleotides) -use ddNTPs with fluorescent labels -aka Chain Termination Sequencing
166
What is dNTP?
deoxynucleotide phosphate
167
What is ddNTP?
dideoxynucleotide phosphate -a modified version of dNTP where there is not a 3' OH (just H)
168
How was Sanger Sequencing originally carried out compared to now?
-originally 4 separate rxns, each with a diff ddNTP, now 1 rxn with all 4 ddNTPs present, each with a diff fluorescent label -originally radioactively labelled primer -originally T7 DNAP was used, now taq DNAP is used -originally used agarose electrophoresis, now use capillary gel electrophoresis -originally more DNA templates were needed and it was more labour-intensive -now is more specific and sensitive -now is easier, quicker and cheaper
169
What methods are used to analyse gene expression?
gene expression of DNA/RNA: -PCr-based -hybridisation based gene expression of proteins: -immunological-based -fusion proteins
170
Which methods of analysing gene expression are hybridisation based?
-Northen Plot Analysis -Microarrays -Fluorescent in situ hybridisation (FISH)
171
How is Northen Blot analysis carried out?
-RNA separated by size using electrophoresis -RNA is transferred to a membrane -allows for blotting -gene-specific probe is added and hybridises to target sequence
172
What are the limitations of Northen Plot Analysis?
-slow -only gives info about 1 gene at a time
173
How is microarray carried out?
-olignonucleotides (short single-stranded DNA/RNA frags) are attached to spot on a chip -each spot has a diff oligonucleotide, specific to a gene -RNA is prepared -fluorescently labelled cDNA is made from RNA -fluorescent cDNA is applied to chip and allowed to hybridise
174
What are the limitations of microarrays?
-not easy to quantify mRNA levels -better for assessing relative levels
175
What is an oligonucleotide?
a short single-stranded DNA/RNA fragment -used in genetic analysis
176
How is FISH different to Northen Blot?
-probe is labelled with fluorescent marker and visualised using microscopy
177
What are primary antibodies?
protein-specific antibodies
178
What are secondary antibodies?
antibodies which recognise primary antibodies -have a conjugate attached -something we can detect
179
How is Western Blotting carried out?
-proteins are separated by size using electrophoresis -proteins are transferred to a membrane -proteins are detected using a primary (protein-specific) antibody and a labelled secondary antibody (often labelled w/light)
180
What can Western Blotting tell us?
-where protein is (ie. which tissue it's in) -protein levels -about post-translation modification of protein
181
How is immunofluorescence carried out to analyse protein expression and localisation?
-secondary antibody is conjugated to a fluorescent marker and is visualised using microscopy -diff fluorophores are used so that multiple molecules can be detected (NOT a live image)
182
What is a fluorophore?
fluorescent chemical compound which re-emits light upon excitation
183
How is live imaging carried out to analyse protein expression and localisation?
-fuse 2 protein-coding sequences (CDSs) -protein and fusion protein -use fluorescent fusion proteins eg. GFP
184
What is a reporter gene?
a gene which is easy to visualise or assay which is attached to a regulatory sequence of another gene
185
What methods can be used to analyse molecular interactions?
protein-protein interactions: -pull down assay -immunoprecipitation -yeast two-hybrid protein-DNA interactions -chromatin immunoprecipitation (ChIp)
186
How is a pull down assay carried out?
-make recombinant DNA -cell lysis done to obtain a cell lysate (fluid containing cell contents) -bind fusion protein (eg. GST) to affinity ligand -wash away unwanted substances to get purified recombinant protein -see what interacts with it
187
How is immunoprecipitation carried out?
-make recombinant DNA -cell lysis done to obtain a cell lysate (fluid containing cell contents) -bind antibody to affinity ligand -wash away unwanted substances to get purified recombinant protein -see what interacts with it (co-immunoprecipitation)
188
How is yeast two-hybrid carried out?
-using fusion proteins -DNA binding protein is fused to a "bait" and transcription activator is fused to "prey" -if bait and prey interact, transcription of reporter gene occurs
189
How is chromatin immunoprecipitation carried out?
-an antibody or fusion protein is used to purify protein -assay which DNA molecules are associated with that protein by DNA analysis
190
What techniques are used to measure protein levels?
Western Blot
191
What techniques are used to measure RNA levels?
Quantitative RT-PCR Northern Blot Microassay Luciferase assay
192
What techniques are used to measure protein localisation?
Immunofluorescence Live cell imaging
193
What techniques are used to measure RNA localisation?
FISH
194
What techniques are used to measure DNA localisation?
FISH
195
What techniques are used to measure protein interactions?
Immunoprecipitation Pull down assay Yeast-two hybrid ChIp (w/DNA)
196
What techniques are used to measure DNA interactions?
ChIp (w/protein)
197
What biological membranes exist in eukaryotic cells?
plasma membrane (cell boundary, controls movement in and out) organelle membrane (compartmentalises cytoplasm)
198
What are the fundamental properties of membranes?
-act as a barrier -flexible -can self repair -continuous (no edges) -selectively permeable (only certain molecules can pass through)
199
What are biological membranes made up of?
-lipids -proteins -carbohydrates (linked to lipids as glycolipids or to proteins as glycoproteins)
200
How can phospholipids move in the lateral plane of a membrane?
-rotation -flexion -flip-flop (moving from one leaflet to the other) (RARE)
201
Why are phospholipids described as ampithatic?
have hydrophilic, polar head and hydrophobic tail
202
How does the composition of a phospholipid affect its fluidity?
no. double bonds and no. carbons -more C=Cs = more fluid (unsaturated phospholipids are more mobile) -more acyl chains = more fluid
203
What are the 4 main classes of phospholipids?
-phosphatidyl-ethanolamine -phosphatidyl-serine (-ve) -phosphatidyl-choline -sphingomyelin
204
How does the saturation of phospholipids affect its fluidity?
unsaturated phospholipids have cis-double bonds so are more mobile/fluid saturated phospholipids have longer chains so are less mobile/fluid
205
What is the structure of cholesterol?
-polar head -rigid steroid ring structure -non-polar hydrocarbon chain -smaller than other lipids
206
What is the role of cholesterol in membranes?
makes membrane less permeable -packs between phospholipids, making the surface of the membrane more deformable/rigid (not the whole membrane) -at high temps, stops membrane becoming crystalleine
207
What is the role of cholesterol in membranes?
makes membrane less permeable -packs between phospholipids, making the surface of the membrane more deformable/rigid (not the whole membrane) -at high temps, stops membrane becoming crystalline
208
What can phospholipids form in aqueous solutions?
micelles -cone-shaped lipids bilayer (more energetically favourable in spheres than planar) -cyclinder-shaped lipids
209
What does Fluroscent Recovery After Photobleaching (FRAP) show about the structure of membranes?
dynamic movement of proteins in membrane -provides insight into protein's structure (proteins are tagged with GFP and some are bleached -can see the movement of the coloured proteins into the areas of bleached)
210
What are integral membrane proteins?
proteins inserted directly into membrane by hydrophobic domain -needs strong detergent to remove
211
What are peripheral membrane proteins?
proteins associated with integral membrane proteins or covalently bound to lipids inserted in the membrane
212
What are microdomains (in membranes)?
clusters of lipids -microdomains form because membrane lipids aren't all the same (aren't homologous) eg. Rafts -formed from cholesterol and spingolipids -more rigid and thicker than rest of membrane, allowing proteins with a long transmembrane protein to be selected
213
Why are red blood cells a good model for studying membranes?
in hypotonic solutions, they form ghost cells which burst, which looses all the haemoglobin so the membranes can easily be looked at by themselves
214
What is spectrin?
a cytoskeletal protein which holds cytoskeleton to plasma membrane -in red blood cells, spectrin forms lattice under cell surface, giving them flexibility
215
What are the key features of biological membranes?
-asymmetric -protein always orientated the same way -lipid composition diff in each half of bilayer
216
What is spectrin?
a cytoskeletal protein which holds cytoskeleton to plasma membrane -in red blood cells, spectrin forms lattice under cell surface, giving them flexibility
217
Why is asymmetry important in membranes in terms of blood groups?
-allows blood groups (O, A, B, AB) to be different to eachother -diff terminal sugars of oligosaccharide chains determines blood groups
218
Why is asymmetry important in membranes in terms of coagulation (clot formation)?
phosphatidylserine on platelet membranes provides a site for nucleation for coagulation cascade
219
Why is asymmetry important in membranes in terms of apoptosis?
aminophospholipids (phosphotidylserine and phosphatidylethanolamine) are transferred to outer leaflet of apoptotic cells' plasma membrane which are detected by receptors on macrophages' plasma membranes
220
How does the phospholipid bilayer act as a barrier?
-highly impermeable to ions and solutes (small, uncharged polar) (allows hydrophobic, large, polar substances through)
221
How can the permeability of membranes to different substances be investigated?
using liposomes (artificial vesicles)
222
Which membrane transport proteins are involved in passive transport?
channel proteins carrier proteins
223
Which membrane transport proteins are involved in active transport?
carrier proteins
224
What are electrochemical gradients?
combination of membrane potential (voltage across cell) and concentration gradient -established by ionic concentration differences on either side of membrane produced by ion channels, carriers and pumps -involved in lots of processes, such as driving transport, conveying electrical signals (nerves) and making ATP
225
What is ion transport like when a membrane has no membrane potential?
ions can pass through
226
What is ion transport like when a membrane has a negative membrane potential inside?
ions can pass through easily
227
What is ion transport like when a membrane has a positive membrane potential inside?
ions movement is inhibited
228
What is the difference between carrier proteins and channel proteins?
-carriers bind to solute, channels only weakly interact with solute ∴transport is faster with channels -channels passive, carriers passive or active
229
Why is transport quicker with channel proteins rather than carrier proteins?
channels only weekly interact with solute whereas carriers bind to solute
230
What are the characteristics of ion channels?
-form hydrophilic pores through membrane -specific for diff ions (∴lots of diff types) -rapid movement down conc/electrochemical gradient -rapidly close and open (intra or extracellular) -stimulated by voltage, ligand binding or mechanisms
231
What types of ion channels can there be?
voltage-gated channels ligand-gated channels mechanically-gated channels
232
How do carrier proteins transport solutes?
by undergoing a conformational change
233
What transport proteins can active transport be mediated by?
coupled carrier ATP-driven pump light-driven pump
234
What are the two types of coupled carriers?
symport -2 solutes transported in same direction antiport -2 solutes transported in diff directions
235
What are uniport carriers?
carriers which only transport one solute
236
What are symport carriers?
coupled carriers where 2 solutes are transported in the same direction
237
What are antiport carriers?
coupled carriers where 2 solutes are transport in opposite directions to eachother
238
What is transport driven by in mammalian plasma membranes?
Na+ gradients
239
What is transport driven by in bacterial, yeast and intracellular membranes?
H+ gradients
240
Why are prokaryotes considered evolutionary successful?
-largest biomass -occupy lots of diff niches -most variation
241
Why do eukaryotes have a nucleus?
separates processes of transcription and translation, enabling alternative splicing
242
Where could the nucleus hypothetically come from evolutionarily?
-invagination of membrane around DNA, forming a primitive nucleus (explains double membrane) -one prokaryote engulfing another (endosymbiosis)
243
What is the experimental evidence for the nucleus to have evolved via endosymbiosis?
using homology hit analysis to compare yeast cells with Archae -further up graph, more similar genes, more commonly related
244
How is the entire genome packaged into the nucleus?
-DNA is wrapped around nucleosomes (8 subunits of histones) -wrapped twice around each histone -tightly packed -30nm fibre
245
What is the nucleosome?
basic structure of DNA consisting of a segment of DNA being wrapped around 8 histones
246
What is heterochromatin?
dense, tightly packed chromatin
247
What is euchromatin?
loosely packed, less dense chromatin
248
What does electron microscopy staining reveal about chromatin organisation in the nucleus?
dense staining of interphase DNA = heterochromatin less dense staining of interphase DNA = euchromatin very dense staining of RNA = nucleolus (black and white staining)
249
What does Fluorescence In Situ Hybridisation (FISH) chromosomal painting about chromatin organisation in the nucleus?
evidence for heterochromatin and euchromatin -individual chromosomes can be picked out -diff chromosomes occupy specific regions (inherited so region does change) so are found in heterochromatin and euchromatin
250
Why is the nucleus described as dynamic?
location of genes within nucleus changes depending on its transcriptional status
251
What are the characteristics of the nucleus?
-compartmentalised -dynamic -occupies
252
Why is the nucleus described as being occupied?
sub-nuclear organelles are present in the inter-chromatin space -are dynamic and move in non-random ways using ATP eg. PML bodies, Cojal bodies, etc
253
How has the existence of sub-nuclear organelles been revealed?
by gene tagging and improvements in microscopy
254
What happens in the nucleolus?
ribosome synthesis
255
What happens in the speckles?
pre-mRNA processing
256
What happens in the cajal bodies?
splicing
257
What happens in the PML bodies?
storage
258
What is the structure of the nucleolus like?
-not membrane bound -collection of macromolecules incl rRNA genes, precursor rRNA, mature rRNA, RNA processing enzymes, snoRNPs, ribosomal protein subunits, partly assembled ribosomes (sometimes contain mRNA and tRNA)
259
What is the nucleolus?
a non-membrane bound area in the nucleus where ribosomes are produced using ribosomal RNA
260
What is the nuclear envelope?
a double unit membrane around the nucleus perforated by pores and supported by lamella (fibrous meshwork)
261
What is the structure of the nuclear envelope like?
-double unit membrane -has pores -supported by lamella (meshwork of lamin fibres) -lamella is found between the nuclear envelope and heterochromatin and is responsible for the nuclear envelope's asymmetric nature (as diff proteins need to be in inner membrane to associate with lamella)
262
What is the role of lamella in the nucleus?
-ensures nuclear envelope's double membrane is asymmetric (diff proteins present in outer and inner membrane as proteins in inner membrane are near lamella eg. lamella associated proteins aka LAP) -aids structural integrity -involved in gene regulation
263
What is Hutchinson-Gilford progeria syndrome caused by?
mutation in gene encoding lamin A (is an example of a laminopathy)
264
What are laminopathies?
inherited diseases due to damage to lamin in nucleus (eg. amino acid mutations) -diverse range eg. Hutchinson-Gilford progeria syndrome due to mutation in gene encoding lamin A
265
How was the existence of nuclear pores revealed?
SEM, Cryo-EM, Gold Transmission EM
266
What is the structure of nuclear pores?
-on cytoplasmic side, have fibrils -on nuclear side, have nuclear basket of fibrils
267
What is the function of nuclear pores?
to control access in and out of the nucleus in a size-dependent manner -particles > 50,000g/mol can't enter nucleus by simple diffusion but can by active signal-dependent transport (which causes nuclear pores to open up to 26nm in diameter) where a specific peptide sequence (nuclear localisation sequence) is required for entry
268
How are new phospholipids in the phospholipid bilayer synthesised?
-phospholipid synthesis adds to cytosolic side of phospholipid bilayer -scramblase catalyses these phospholipids to be flipped in the bilayer, resulting in symmetric growth of both halves of the bilayer (lipids are equilibrated) -new membrane is delivered via exocytosis -flippase catalyses specific phospholipids to be flipped to cytoplasmic monolayer, which ensures asymmetry of bilayer is maintained
269
What does scramblase do?
flips newly synthesised phospholipids in bilayer to equilibrate lipids, causing symmetric growth of bilayer
270
What does flippase do?
flips specific phospholipids to cytoplasmic monolayer, ensuring the membrane's asymmetry is maintained
271
How is the asymmetry of the phospholipids in red blood cells' plasma membrane maintained?
aminophospholipid translocase (a flippase) transfers a phosphatidylserine from extracellular leaflet of the membrane to its cytosolic leaflet
272
What is Scott's disease?
a deficiency in scramblase -results in increased bleeding
273
Why is the endoplasmic reticulum described as being dynamic?
it is continuously breaking and reforming
274
What is the structure of the endoplasmic reticulum like?
hollow tubes and flattened sacs -connected to nuclear envelope
275
What are the two types of endoplasmic reticulum?
-rough (has ribosomes sitting on pores of ER) -smooth
276
What are the functions of the endoplasmic reticulum?
-quality control -protein synthesis -lipid synthesis -storage -detoxification
277
What is the smooth endoplasmic reticulum responsible for?
-phospholipid and cholesterol synthesis -steroid hormone production -glyceride and glycogen synthesis -glyceride and glycogen storage -calcium storage
278
What are chaperone proteins?
proteins which aid the folding of newly synthesised amino acid sequences into tertiary and quaternary structures -associate with proteins in lumen of ER if they are not yet folded
279
How is the endoplasmic reticulum involved in quality control?
export translocate misfolded proteins so that they are degraded -means only correctly folded proteins enter the secretory pathway
280
How are calcium ions used in acinar cells (pancreatic cells which produce a large number of enzymes)?
-after stimulation, acinar cells release Ca2+ -this causes vesicles to fuse with cell surface membrane -digestive enzymes are released
281
What is pancreatitis?
overload of Ca2+ (can be due to failure of Ca2+ pumps) causing alcohol/biliary disease
282
How are substances transported from the endoplasmic reticulum to Golgi apparatus?
-vesicles bud off from donor compartment of endoplasmic reticulum -vesicle moves through cytoskeletan -vesicle's coat is discarded (enables it to fuse) -vesicles fuse to target compartment of Golgi apparatus, releasing the contents of its lumen into the target compartment
283
What are vesicle coats?
lattice-like cage of peripheral membrane proteins (specialised proteins) around the membrane forming the vesicle -coat aids formation of vesicle -coat must be discarded to reveal SNARE before vesicle can fuse with target compartment
284
What type of vesicle coats are there?
-clathrin -COPI -COPII
285
What do COPI-coated vesicles do?
move substances forwards and backwards through the golgi apparatus and back to the ER
286
What do COPII-coated vesicles do?
bud from ER
287
What are SNARES?
transmembrane proteins (typically with large cytoplasmic domain and small lumenal domain) which ensure the correct vesicle fuses with the correct target component
288
What are the two types of SNARES?
v-SNAREs (vesicle-SNAREs) t-SNARES (target-SNAREs)
289
How do vesicles reach the correct target?
v-SNAREs (in vesicle's membrane) recognises complementary t-SNARE (in target membrane) and they bind, forming a SNARE pair
290
Where does the Golgi apparatus transport substances to?
-endoplasmic reticulum (backwards -retrograde) -cell membrane, etc (forwards -anterograde)
291
What is retrograde transport?
transport from the Golgi apparatus back to the endoplasmic reticulum
292
What is anterograde transport?
transport from the Golgi apparatus to the plasma membrane
293
What is the structure of the Golgi apparatus?
composed of flattened sacs called cisternae, which together make a golgi stack* -cis golgi network -cis cisternae* -medial cisternae* -trans cisternae* -trans golgi network
294
What are the components of the Golgi stack in the Golgi apparatus?
-cis cisternae -medial cisternae -trans cisternae
295
What are the functions of the Golgi apparatus?
-modification and packaging of secreted proteins (diff modifications occur in diff, specific regions) -renewal and modification of plasma membrane -delivery of substances to other organelles (especially in the endocytic pathway)
296
Where do vesicles fuse to the Golgi apparatus?
cis face
297
Where do vesicles leave the Golgi apparatus?
trans face
298
What pathways can substances go to after leaving the trans face of the Golgi apparatus?
-signal-mediated diversion to lysosome or to secretory vesicle -constitutive secretory pathway to plasma membrane
299
What do cells take up?
-nutrients (macromolecules) -signals (growth factors, morphogens, etc) -antibodies -enzymes -viruses -bacteria -membranes
300
What can happen to endocytosed material?
-is delivered to early endosome (a membrane-bound organelle) -is degraded -is recycled (sent back to cell surface) -is sent to apical domain of plasma membrane (transcytosis)
301
What is transcytosis?
vesicular transport from one side of a cell to the other -substance is taken up via endocytosis (at basolateral domain of membrane), transported across using vesicles and released at the other face via exocytosis (at apical domain of membrane)
302
What different endocytic pathways are there?
large scale endocytic pathways eg. phagocytosis, macropinocytosis small scale endocytic pathways eg. clathrin-mediated, caveolar-mediated, etc
303
What is phagocytosis?
a form of endocytosis where large vesicles called phagosomes ingest large particles, such as bacteria and apoptotic cells
304
What happens in phagocytosis?
-pathogen is coated in antibodies (opsonisation) to make it detectable by phagocytes (or other cells) -bind to phagocyte surface receptors, which detect them -phagocyte produces pseudopods (ruffles) which encircle the pathogen (as they encircle it, more and more receptors bind to antibodies on surface of pathogen) until it becomes enclosed by the phagocyte's membrane
305
How is frustrated phagocytosis used experimentally to observe phagocytosis?
-cover slip is opsonised (coated w/antibodies) -phagocyte is attached -phagocyte will try to engulf antibodies but is unable to -we are then able to see what is recruited at this point and what mechanisms are in place
306
How do we know that plasma membrane is recycled rather than being being newly synthesised?
-total SA of cell membrane doesn't change -membrane taken in quicker than it can be synthesised ∴must be recycled
307
What was the first experimental indication of membrane recycling?
-macrophages taking up beads to mimic phagocytosis -SA of beads is equiv. to membrane taken in -total SA of macrophage stays the sme -membrane is taken in quicker than it can be synthesised so it must be recycled
308
What is macropinocytosis?
an actin-driven form of endocytosis where extra-cellular material is taken in non-selectively
309
What happens in clathrin-mediated endocytosis?
-more receptors are made, which are clustered in clathrin-coated pits (precursor to vesicle) -when substance (eg. LDL) binds, coated pits bud off to form clathrin-coated vesicles -vesicles discard their coats before fusing with endosome -in early endosome, substance dissociated from receptors -transport vesicles, containing receptors, bud off from endosome and recycle the receptors, bringing them back to the membrane -substance is released from endosome into lysosomes
310
What is the structure of clathrin in clathrin-coated pits and veiscles?
-clathrin triskelia (three heavy and three light chains) polymerise into a lattice -adaptor proteins attach the clathrin coat (lattice) to the receptors
311
What causes the final pinching of clathrin coated vesicles?
dynamin
312
Why is there a pH gradient from the plasma membrane to the lysosomes?
-reduction in pH aids maturing of endosomes -early endosome requires lower pH than plasma membrane for substances to dissociate from receptors -lysosomes require even lower pH for optimum enzymatic digestion
313
What do Rab proteins do?
help SNARES to mediate fusion -are specific -diff Rabs for diff organelles
314
What is cargo inside the intraluminal vesicles (vesicles inside the late endosome) destined for?
lysosomes
315
What are the functions of mitochondria?
-energy production (H+ gradient produced across inner mitochondrial membrane which drives ATPase) -involved in apoptosis
316
What is the structure of mitochondria?
-have a double membrane -inner membrane contains enzymes and transport proteins and is highly folded -folds are called cristae (increase SA) -outer membrane encloses organelle and contains enzymes for mitochondrial lipid synthesis and has porins (which allow entry of molecules <5000kDa) -inter-membrane space -matrix contains mitochondrial DNA, tRNAs, ribosomes (39S and 28S), enzymes and metabolites
317
What is the structure of the inner mitochondrial membrane?
-highly folded into cristae (increases SA) -contains transport proteins and redox-performing proteins (forming electron transport chain) -contains enzymes involved in energy production
318
What is the structure of the outer mitochondrial membrane?
-have porins (large channels) which allow molecules <5000kDa to enter -contain enzymes for mitochondrial lipid synthesis
319
Which ribosomes are present in mitochondria?
29S and 28S ribosomes
320
What is the genetics of mitochondria like?
double stranded circular chromosome about 15-17kbps long -encodes 37 genes -inherited from mother
321
How are new mitochondria produced?
-can't be newly synthesised so arise from existing mitochondria via fission
322
What occurs in the mitochondrial life cycle?
-debris is segregated -fission (mitochondria splits into two) -fusion (occurs as a stress response -two mitochondria fuse to form one) -mitophagy (mitochondria is engulfed into an autophagosome and is degraded) -biogenesis
323
What translocation proteins are embedded in the inner and outer mitochondrial membranes?
-TOM (translocator of outer membrane) -TIM (translocator of inner membrane) -SAM (sorting and assembly machinery) -OXA (cytochrome oxidase activity)
324
How do proteins enter the mitochondrial matrix?
-proteins are already synthesised (interacting proteins like chaperones are bound to synthesised proteins to stop them folding before they are docked to TOM) and then translocated into the mitochondria using signal sequencing and translocation proteins (eg. TOM, TIM, OXA, SAM) --protein precursor binds to receptor protein in TOM complex -it is then inserted into the membrane by the TOM complex and translocated into the inter-membrane space and then translocated through the TIM23 complex into the matrix (goes through TOM and TIM at same time) -polypeptide signal is cleaved off by signal peptidase
325
How are chaperones involved with transporting proteins into the mitochondria?
-chaperones bind to newly synthesised proteins, to stop them folding before they get translocated by TOM -chaperones must then be dissociated from the polypeptide chain, using ATP
326
How do proteins enter the inner mitochondrial membrane? (to stay there)
-most common: using TOM and TIM or -protein completely enters matrix -signal sequence is cleaved, which unmasks a second sequence, causing insertion into OXA (cytochrome oxidase activity) complex
327
How do proteins enter the outer mitochondrial membrane? (to stay there)
-issue: TOM can’t insert proteins into bilayers -instead, chaperones keep proteins (typically porins, which are beta-barrel proteins) unfolded as they enter the intermembrane space -SAM complex inserts the proteins into the outer mitochondrial membrane and folds them
328
What are peroxisomes?
vesicles found in all eukaryotic cells which carry out oxidation and reduction rxns
329
What is the structure of peroxisomes?
-surrounded by a single membrane -have no DNA or ribosomes
330
What is the function of peroxisomes?
carry out oxidative rxns (remove hydrogen atoms from inorganic compounds) eg. b-oxidation of fatty acids, detoxification of alcohol
331
How are new peroxisomes made?
produced by endoplasmic reticulum -peroxisomal precursor vesicle buds off from ER -peroxisomal precursor vesicle uptakes peroxisomal proteins and lipids from cytosol and grows, becoming a peroxisome or from pre-existing peroxisomes -peroxisome grows -fission occurs, producing two daughter peroxisomes
332
How do proteins get into peroxisomes?
-Pex5 recognises signal sequence (Ser-Lys-Leu) and accompanies cargo into peroxisome, is ubiquitylated and cycled back into cytosol
333
What is Zellweger syndrome?
severe brain, liver and kidney abnormalities caused by a mutation in Pex5 gene (gene involved in translocating proteins into peroxisomes)
334
What is required for cell motility?
-energy -directions -mechanical interactions (w/something outside cell) -microtubules/actin microfilaments
335
What are microtubules?
hollow tubes of alpha and beta tubulin
336
What is the structure of microtubules?
-13 protofilaments -24nm diameter -tubulin dimers of alpha and beta tubulin
337
What are some examples of things moved by microtubule-based motility?
-cells, protozoa, sperm, etc -moving fluids, reproductive tracts, etc -using cilia/flagella
338
What is the difference between cilia and flagella?
different lengths (cilia shorter than flagella)
339
What is the similarity between cilia and flagella?
same structure -both have an axomere
340
What is the structure of an axoneme?
9+2 microtubule arrangement -9 outer doublets consisting of complete fibres (13 protofilaments), incomplete fibres (10 protofilaments) and dynein arms and radical spokes -inner pair
341
How is polarity created across an axoneme?
dynein arms on one side have contact but don't have contact on other side
342
How many protofilaments are in a complete microtubule?
13
343
How many protofilaments are in the incomplete microtubules found in the outer doublets of an axoneme?
10
344
What enables axonemes to bend?
nexin crosslinks between doublets
345
How do cilia move?
via power strokes and recovery strokes -controlled by outer dynein arms
346
How do flagella move?
wave-like waveform -controlled by inner dynein arms
347
What is the function of the dynein arms in an axoneme?
control waveforms of cilia and flagella -inner arm controls waveform -outer arm controls power
348
What are basal bodies?
continuation of axoneme inside cell -shorter than axoneme
349
What is the structure of basal bodies?
9x3 microtubule array
350
How does actin treadmilling work?
-actin is disassembled at - end -actin removed is recycled (by ATP being added) by being added to the + end (causing movement in the direction of the + end)
351
What are the differences between an actin filament and a microtubule?
-microtubules are wider -actin filaments are solid whilst microtubules are hollow -actin filaments have homomeric (identical) subunits but microtubules have different subunits (alpha and beta) -actin filament subunits are 42kDa but microtubule subunits are 50kDa -actin filaments have 375 amino acids whist microtubules have 450 -actin filaments have ATP in monomer (hydrolysed to ADP during assembly so ADP in filament) but microtubules have GDP in monomer (GDP in alpha, DTP in beta)
352
How are actin filaments assembled?
-monomers added to + end -ATP hydrolysed to ADP
353
What is the function of actin binding proteins?
control movement (inhibit by binding ATP eg. profilin or activate)
354
What can actin binding proteins do?
-sequence monomers of actin filaments -nucleate monomers -polymerise monomers to produce actin filaments -cap actin filaments (end-blocking) -cross-link actin filaments -bundle actin filaments -severe actin filaments -bind actin filaments to membrane -depolymerise actin filaments (back into monomers)
355
What filament is in cilia/flagella?
microtubule
356
What filament is in cytoskeletan/muscle?
actin
357
What motor protein is in cilia/flagella?
dynein
358
What motor protein is in cytoskeletan/muscle?
myosin
359
What happens in the myosin power stroke?
-Ca2+ binds to troponin, causing it to move and reveal the active site -cross-link forms between myosin and actin -myosin head bends, releasing ADP and Pi -new ATP binds to myosin and cross-link breaks -ATP is hydrolysed to ADP and Pi, returning myosin to its original position
360
How does actin-based motility of a cell work?
-signal stimulates filopodium -lamellipodium pushes cell forwards -new adhesions form with the matrix -old adhesions at back of cell with the matrix break -cell contracts and moves forward -cycle continues
361
What is extracellular matrix?
extracellular meshwork of proteins and hydrated macromolecules (secreted)
362
What are some examples of specialised extracellular matrix? (And what makes them specialised?)
-bones (calcified) -keratin (fibrous and loose) -tendons (tensile strength) -skin (elastic) -cornea (transparent)
363
What are the functions of extracellular matrix?
-regulate migration -regulate tissue integrity and cell shape -regulate proliferation (checks cells and encourages independent growth)
364
What are the types of extracellular matrix?
-fibrous proteins (collagen, elastin) -adhesion proteins (fibronectin, laminin) -hydrated sugars
365
What is the structure of collagen?
-repetitive structure: Gly-Pro-Hyp triplet repeat -triple helix -3 alpha chains
366
What is the triplet repeat in collagen?
glycine-proline-hydroxyproline
367
What is collagen produced by?
fibroblasts and epithelial cells
368
What is Ehlers-danlos syndrome caused by?
defect in collagen
369
How is collagen synthesised?
-in a multi-step process driven by self-assembly -uses vitamin C as cofactor -involved cleavage of pro-peptides and assembly of fibrils
370
Which enzyme catalyses the conversion of tropoelastin to elastin?
lysyl oxidase
371
How does elastin gain structure as it deforms?
stretches, revealing single elastin molecules and cross links
372
What is Marfen syndrome caused by?
defect in fibrillin
373
What is the structure of GAG's glycoaminoglycans?
-repetitive disaccharide chains 70-200 units long -highly sulphated (charged) -often substituted into proteins using a link tetrasaccharide
374
What are glycoaminoglycans divided into?
proteoglycans and glycoproteins
375
What adhesion glycoproteins are there?
-laminin -fibronectin -integrins -focal adhesions
376
What is the structure of laminin?
-cruciform structure -heterotrimer (β, α and γ chains) -have globular domains and coiled-coil-α-helical domain
377
What is the structure of fibronectin?
-disulphide crosslinks -collagen binding site -cell binding site (type 3 receptors)
378
What is the structure of integrins?
-heterodimer (α and β subunits non-covalently associated) -α subunit with divalent cations bound -β subunit with cysteine rich domains -matrix binding site -COOH groups in cytosol
379
What is the function of integrins?
bind matrix through divalent cations -removing divalent cations causing cell to detach)
380
What is the structure of focal adhesions?
-integrin -cytoskeletal proteins eg. talin
381
What is the function of focal adhesions?
form mechanical links between intracellular actin and extracellular myosin -involved in signalling -transmembrane receptor
382
What can defects in integrin lead to?
-platelets binding fibroniogen to clot blood -Glonemann's thrombasthenia -bleeding gums/noses -leukocyte adhesion deficiency (LAD) syndrome -impaired expression -recurrent bacterial infections
383
What types of cell-cell junctions are there?
-cell-matrix anchoring junctions -cell-cell anchoring junctions -tight junctions -channels forming junctions
384
What are cadherins?
transmembrane proteins which mediate cell-cell adhesion -Ca2+ dependent
385
What is the structure of an adherens junction?
-bundle of actin filaments -contains cadherins -links to cytoskeletan
386
What is the difference between hemidesmosomes and desmosomes?
-hemidesmosomes attach cells to basal lamella but desmosomes attach cells to cells -hemidesmosomes contain integrins but desmosomes contain cadherins (integrins and cadherins link to intermediate filaments)
387
Where are desmosome junctions found?
between cells
388
What are hemidesmosome junctions found?
between cell and basal lamina
389
What is Pemphigus?
an autoimmune disease where skin blisters due to failure in desmoglein (α cadherin) which holds keratinocytes in epidermis
390
Where are tight junctions found?
between cells in clusters near apical face
391
What acts as experimental evidence of tight junctions?
electron dense dye
392
What is paracellular transport?
transport of substances across epithelium by passing through intercellular space (gap junctions) -passive -regulated
393
What is transcellular transport?
transport of substances across epithelium by passing through transmembrane proteins in the apical and basal membranes and the cytoplasm in between -active
394
Where are gap junctions found?
between cells -patches rather than continuous barrier -found in connective tissue, neurones, heart muscle, epithelia (places where regulation is key)
395
What proteins make up gap junctions?
connexons
396
What is the structure of connexons?
-6 subunits called connexins (can be homomeric or heteromic) -flexibility in arangements allows gap junctions to be regulated
397
What can regulate gap junctions?
-pH -membrane potential -Ca2+ -cell signalling
398
What events does the cell cycle require?
-chromosomal replication -chromosomal segregation -cell division
399
When do normal cells proliferate?
when stimulated by extrinsic factors eg. nutrient status, integrins, TGF-β receptors, G-protein coupled receptors, tyrosine kinase receptors -these can be overruled by signalling proteins (which force proliferation to stop)
400
What extrinsic factors could stimulate normal cells to proliferate?
nutrient status -integrins -TGF-β receptors -G-protein coupled receptors -tyrosine kinase receptors
401
How can stimulating extrinsic factors be overruled?
by signalling proteins
402
What is the cell cycle clock?
the way a cell decides whether to proliferate or not based on signals from environment -decides cell fate
403
What happens during the first growth (G1) phase of the cell cycle?
-cells increase in size -ribosomes and RNA are produced -preparation for DNA synthesis
404
What happens during the synthesis phase of the cell cycle?
-DNA synthesis (chromosomes duplicated)
405
What happens during the second growth (G2) phase of the cell cycle?
-fidelity of DNA is checked -preparation for nuclear division
406
What happens during the mitotic phase of the cell cycle?
-mitosis (prophase, prometaphase, metaphase, anaphase, telophase) -cytokinesis
407
What happens in the cell cycle?
-interphase (G1, synthesis and G2) -mitosis and cytokinesis
408
When in the cell cycle are there checkpoints?
-end of G1 -during synthesis -during G2 -during mitosis
409
What does the checkpoint at the end of G1 check for?
DNA damage
410
What does the checkpoint during synthesis check for?
DNA damage and stalled replication forks
411
What does the checkpoint during G2 check for?
damaged/unreplicated DNA
412
What does the checkpoint during mitosis check for?
correct attachment of chromosomes to spindle and correct separation of chromosomes to poles of cell
413
Why are checkpoints needed in the cell cycle?
-to stop cell cycle if there's a problem -to allow for increase of scheduled lengths of phases -to facilitate repair processes
414
How does the restriction point in G1 determine whether cells continue with the cell cycle or not?
-if serum and growth factors are removed before cells have completed 80-90% of G1, they revert to G0 (don't continue with cell cycle) -if serum and growth factors are removed during the final hour of G1, they proceed to synthesis, etc
415
What proteins are involved in the transition between stages of the cell cycle?
-cyclin -cyclin dependent kinases (cdk) -diff cyclins and diff cdks needed for diff phases
416
How can the way cells transition between phases of the cell cycle be investigated?
-genetic approach: looking for mutations (studying mutant cells that can't proceed into next phase of cell cycle) -biochemical approach: looking for proteins involved in transition (studying large no. cells undergoing same transition at same time)
417
Why is yeast used as a model system for genetically investigating cells transitioning between stages of the cell cycle?
-is obvious by looking at yeast cells which phase of cell cycle they are in -have fast division rate -cell cycle genes are highly conserved -yeast cells can be haploid or diploid
418
How is cell-free mitosis used as a model system for biochemically investigating cells transitioning between stages of the cell cycle?
-deplete cytoplasm of diff proteins -remove cytoplasm at diff stages to study changes over time -protein electrophoresis can be carried out
419
How do molecular switches work?
-kinases phosphorylate target proteins to turn them on (using ATP) -phosphatases dephosphorylate phosphorylated target proteins to turn them off
420
How is the activity of kinases visualised and quantified?
-extract is incubated with ATP and substrate -electrophoresis of substrate is carried out
421
What is a chromosome?
linear DNA molecule
422
What are homologous chromosomes?
chromosomes with the same genes in the same order -one from mother, one from father
423
What are chromatids?
newly copied DNA strands still joined together by centromere
424
What happens in prophase?
sister chromatids condense
425
What happens in metaphase?
mitotic spindle attaches to kinetochore by microtubules
426
What happens in anaphase?
sister chromatids separate to opposite poles of the cell
427
What happens in telophase?
nuclear envelope reassembles
428
What drives mitosis?
M-cdk complex (using cyclinB)
429
What does the M-cdk complex trigger?
-assembly of mitotic spindle -attachment of each sister chromatid to opposite poles -condensation of chromosomes -breakdown of nuclear envelope -rearrangement of cytoskeleton and golgi body
430
What happens to cdk1 for it to become an active M-cdk?
-M-cyclin binds to cdk 1, forming inactive M-cdk -cdk-activating kinase (CAK) and active phosphatase (cdc25) activate M-cdk by removing inhibitory phosphate
431
What does active M-cdk do as positive feedback?
-promote the activation of phosphatase (cdc25) -inhibit the cdk-inhibitory kinase (Wee1) -this forces the switch from G2 to M phase -increase in cyclinB expression increases the levels of M-cyclin
432
What causes the transition from G2 to M phase?
M-cdk complex
433
What causes the transition from metaphase to anaphase?
Anaphase-promoting complex (APC) -a ubiquitin ligase (promotes ubiquitination and degradation) -causes the degradation of M-cyclin in proteasome and securin
434
What are the targets of the anaphase-promoting complex (APC)?
-S and M cyclins (if they are degraded, most cdk targets will be dephosphorylated by phosphatase, which inactivates the cdks) -securin (if degraded, securin can no longer protect protein linkages holding sister chromatids together and a protease is activated that separates the sister chromatids)
435
What can go wrong in mitosis?
chromosomes end up in wrong daughter cell (chromosome non-disfunction) -causing mutations on both alleles, resulting in a phenotypic change (2 hit hypothesis)
436
What are the different parts of mitotic spindle?
-astral microtubules -interpolar microtubules -kinetochore microtubules
437
What is a centrosome?
centriole surrounded by pericentriolar matrix (highly structured matrix containing lots of proteins) to nucleate the microtubules
438
What are the astral microtubules?
microtubules released from centrosome which have contact with cell cortex to position the spindle
439
What are the kinetochore microtubules?
microtubules which attach to the chromosomes at kinetochore
440
What are the interpolar microtubules?
microtubules which overlap with the interpolar microtubules from the opposite spindle pole
441
How is the correct attachment of microtubules to kinetochores sensed?
when attached correctly, kinetochore is pulled in opposite directions but sister chromatids resist -creating tension (which is sensed)
442
What is done when the microtubules are attached incorrectly to kinetochores?
when attached incorrectly, tension is lower so inhibitory signal is released, causing microtubuial attachment to loosen
443
How does the cell know if the microtubules are attached correctly or not to the kinetochores?
correctly -tension (from kinetochores being pulled in opposite directions) incorrectly -less tension
444
How is the anaphase-promoting complex (APC) activated?
by cdc20
445
How is separase activated?
securin is degraded
446
What does activated separase do?
cleaves and dissociates the cohesins between sister chromatids, allowing them to separate as the kinetochore microtubules shorten (allows anaphase to occur)
447
What happens in anaphase A?
kinetochore microtubules shorten, pulling chromatids apart and breaking the cohesin bridges
448
What happens in anaphase B?
-astral microtubules pull poles of cells further apart by motors and depolymerisation -interpolar microtubules slide past eachother
449
What can go wrong in mitosis?
-loss of heterozygosity by non-disfunction (chromatids separated unevenly) -loss of heterozygosity by recombination (chromosome arms swap) -loss of heterozygosity by gene conversion (polymerase jumps across close strands and replicates some of other template strand)
450
How can loss of heterozygosity occur by non-disfunction?
-chromatids separated unevenly so that one daughter cell has more (3/1) -this can be eliminates by apoptosis -however, if it isn't, cell may try to divide again and extra chromosome may be lost -resulting in hemizygous cell (with only one chromosome) where no active protein can be made
451
How can loss of heterozygosity occur by mitotic recombination (in M phase)?
-arms of chromosomes swap, causing mutant alleles to be on diff arms -separation works but could result i a cell with 2 mutated alleles
452
How can loss of heterozygosity occur by gene conversion (in S phase)?
if strands are too close together, polymerase could jump across the strands and replicate some of other template strand
453
What happens in meiosis 1?
-maternal and paternal homologues pair up -genetic diversity is generated by regeneration between homologous chromosomes -1 complete chromosome is pulled apart
454
What happens in meiosis 2?
-separation into haploid daughter cells
455
What happens in meiotic prophase 1?
-homologues pair up -pairing facilitated by synaptonemal complex
456
What is the chiasma?
X-shaped site of chromosomal recombination in paired homologues during meiosis
457
What is the synaptonemal complex?
Complex formed when homologues pair up -paired homologues bought 400nm apart -complex formed by axial cores being cross-linked by transverse filaments -complex aids binding -zips replicated DNA together
458
How is the synaptonemal complex formed?
axial cores (proteins that bind chromatin using cohesion) are cross-linked by transverse filaments
459
What is crossover interference?
The observation that when one crossover is forming, other crossovers nearby are inhibited from forming -limits how much crossing over can occur
460
What are the two main ways chromosomes are anomalous (meiosis gone wrong)?
-chromosome number -structural arrangement of chromosomes
461
What is aneuploidy?
disorder of chromosome number -caused by chromosome non-disfunction -monosomy, trisomy, polyploidy, etc
462
How are aneuploidies (chromosome number disorders) visualised?
by Spectral Karyotyping (SKY)
463
Why does chromosome non-disfunction have a more serious affect when it occurs in meiosis 1, compared to meiosis 2?
-when it occurs in meiosis 1, all the gametes are affected -when it occurs in meiosis 2, only half of the gametes are affected
464
What is monosomy?
a type of aneuploidy where one chromosome is missing (only have one copy of chromosome) -embryonically lethal
465
What is trisomy?
a type of aneuploidy where there are three copies of chromosomes
466
What is polyploidy?
a type of aneuploidy where there is a whole extra set of chromosomes
467
What could the consequences of aneuploidy be?
-embryonically lethal (eg. triploid -XXX/XYY/XXY, nullsomy -missing chromosome pair, monosomy) -minor issues (eg. additional sex chromosome) -infertility (eg. missing sex chromosome -X)
468
When do structural rearrangements of chromosomes tend to occur?
during homologous recombination
469
How can cells die?
via necrosis (uncontrolled) or apoptosis (controlled)
470
When does necrosis occur?
-physical damage (extreme temps, cuts, burns) -toxins (internal or external) -acute hypoxia (low oxygen concs) -ischaemia (loss of blood supply)
471
When does apoptosis occur?
-physiological situations (tissue cell maintenance, developmental cells loss, immune response, hormone-dependent involution) -pathological situations (DNA damage, virally infected cells)
472
What are the characteristics of cells undergoing necrosis?
-compromised membrane integrity -swelling of organelles/cells -increased intracellular calcium -actolysis (enzymes start destroying their own cells) -cell lysis (bursting)
473
What are the characteristics of cells undergoing apoptosis?
-shrinkage -nuclear breakdown -apoptic bodies (vesicles containing dying parts of cells) -phagocytosis triggered -energy required (apoptosis is an active process)
474
Does apoptosis cause an inflammatory response?
no -because it is controlled
475
Does necrosis cause an inflammatory response?
yes -triggered by cell lysis (bursting)
476
In what situations may necrosis and apoptosis both occur?
-when cells in middle start dying via necrosis -detected by body -so cells surrounding them die via apoptosis to limit the spread of necrotic cell death eg. in brain ischaemia
477
What happens to cell shape in apoptosis?
shrinks/condenses
478
What happens to cell shape in necrosis?
swells
479
What happens to plasma membrane integrity in apoptosis?
maintained
480
What happens to plasma membrane integrity in necrosis?
collapsed
481
What cellular process occurs in apoptosis?
budding
482
What cellular process occurs in necrosis?
blebbing
483
What cellular control occurs in apoptosis?
packaging into apoptic bodies
484
What cellular control occurs in necrosis?
leakage to intracellular fluid
485
What happens to DNA in apoptosis?
fragments chromatin condenses
486
Is ATP required for apoptosis?
yes
487
Is ATP required for necrosis?
no
488
What mediator is essential for apoptosis?
caspase
489
What are ced genes?
genes involved in apoptosis -recognise apoptotic signal -phagocytosis of apoptotic cells
490
What ced genes are present in mammalian embryos (and C.elegans equiv)?
BH3 (EGL-1) Bcl-2(Ced 9) APAF-1 (Ced 4) Caspases (Ced 3)
491
What are caspases?
ced genes which carry out cell death (essential for apoptosis) -initiators or executioners -irreversible pathways
492
What is the structure of a caspase?
-has a cysteine residue in active site -are aspartic acids -cleavage site in target protein
493
What are initiator caspases?
caspases activated by apoptotic signals which activate executioner caspases
494
What are executioner caspases?
caspases which cleave over 1000 proteins
495
How can proteolytic cascades be amplified?
one initiator caspade can activate multiple executioner caspases
496
What do caspase targets do?
-cause breakdown of nuclear structure (eg. cleave nuclear lamins to breakdown lamella) -prevent DNA repair by cleaving poly ADP-ribose polymerase (PARP) -cause cytoskeletal changes (eg. cleave cytoskeletal proteins to breakdown actin)
497
What are the two pathways which initiate apoptosis?
-extrinsic pathway -intrinsic pathhway
498
What happens in the extrinsic pathway which initiates apoptosis?
-apoptic signal (eg. ligand on lymphocyte) binds to Fas death receptors on cell -this causes the death-induced signalling complex (DISC), which composes of caspases and FADD adaptor proteins, to assemble -caspases are activated and cleaved -executioner caspases cause the cell to undergo apoptosis
499
What happens in the intrinsic pathway which initiates apoptosis?
-apoptic signal causes mitochondria to release cytochrome c -cytochrome c activated Aparf1 (apoptic protease activating factor) -apoptosome assembles -caspase 9 is recruted -caspase 9 is activated and cleaved -executioner caspases are activated and cause the cell to undergo apoptosis
500
What triggers the intrinsic pathway which initiates apoptosis?
-stress signals (eg. DNA damage) -developmental damage
501
What triggers the extrinsic pathway which initiates apoptosis?
death ligands
502
What is cancer?
disease of aberrant cell proliferation and differentiation
503
How do we known cancer can be caused by environmental influences?
by studying people migrating -cancer trends fitted with place they moved to
504
What environmental influences cause cancer?
-infection -diet -noxious substances
505
How can chromosomal changes causing cancer be researched?
using cytogenetic analysis -condensed chromosomes from cells undergoing mitosis -identify abnormalities s -use molecular probes and FISH
506
What is the Philadelphia chromosome?
abnormality in chromosome 22 of leukaemia cells in chronic myeloid leukaemia -chromosome translocation
507
What happens when BCR binds to ABL (in chromosome translocation, causing cancer)?
-ABL (a protein kinase) can not switch itself off -causes constant proliferation
508
What genes are associated with cancer?
-oncogenes -tumour suppressor genes -DNA-repair genes
509
What are oncogenes?
genes with the potential to cause cancer by transforming cellular behaviour to cause proliferation -generally dominant
510
How do proto-oncogenes become oncogenes?
-deletion/point mutation in coding sequence -hyperactive protein produced -regulatory mutation -more of normal protein produced -gene amplification -normal protein is overproduced -chromosome rearrangement -hyperactive fusion protein produced or normal protein is overproduced
511
What is Ras?
-first human oncogene identified -small GTPase (on -RasGTP, off -RasGDP) -important in growth factor-induced growth
512
What is mutant Ras like in growth?
Ras is only in active form (GTP-bound form) -looses ability to hydrolyse GTP to GDP -causes cell to growth without any control
513
How is Ras important in growth factor-induced growth?
-tyrosine kinases dymerize in presence of growth factor -take up Grb2 and Sos -this turns Ras on -Ras turns diff pathways on eg. cell growth, gene expression, cell movement
514
What is done in cell fusion experiments?
-normal cell and cancerous cell are fused -hybrid cells are inserted into immune-compromised mouse -no tumours formed in mouse ∴normal genes must be dominant to cancerous (normal cells must expressor tumour suppressor genes, which get lost in oncogenesis)
515
What is a tumour suppressor gene?
gene which helps to prevent cancer formation -recessive genes -mutations in tumour suppressor favour cancer formation
516
How many mutations are needed for tumourigenesis?
more than one (one is not sufficient) -5-8 driver mutations
517
Why are cancer cells known as being genetically unstable?
have an increased tendency for mutations
518
What does dying chromosomes and plotting a circos plot show us?
-can visualise chromosomal translocation -see changes in copy number, interchromosomal rearrangement, intrachromosomal rearrangement
519
What does genetic instability cause defects in?
-DNA repair pathways -correction mechanisms for DNA repair errors -mechanisms for DNA segregation errors
520
What does increased cell division contribute to?
tumorigenesis
521
What does decreased apoptosis contribute to?
tumorigenesis
522
How do (normal) cells respond to cellular stresses?
though p53 signalling pathway -stable, active p53 causes cell cycle arrest, senescence and apoptosis
523
What is p53?
cell cycle checkpoint gene involving in the signalling pathways in response to cellular stresses (eg. DNA damage, telomere shortening, hyperproliferative signals)
524
What happens to p53 in most cancers?
is mutated -disrupts instrinsic apoptosis
525
What happens as a result of p53 being mutated in cancers?
-disrupts intrinsic apoptosis -contributes to genetic instability
526
What are ribozymes?
enzymes with a RNA catalytic subunit -involved in RNA splicing
527
What are the functions of the early endosome?
-receives incoming vesicles -sorts internalised cargo (either for degradation or to return to membrane) -develop into late endosomes (for degradation)