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From Flashcardlet > Advance Genetics Test I > Flashcards

Advance Genetics Test I Flashcards

1
Q

Chromatin

Two proteins:

A

DNA and and protein interactions

  • Histone
    2. Non histone chromosomal proteins
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2
Q

Histones

A
  • Small protein with a strong positive charge

- Bind to a neutralise the negatively charged DNA with a chromatin

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3
Q

Nucleosome

A
  • Assembly of a nucleosome is formed when 8 separate histone protein subunits attach to the DNA molecules
  • The combined tight loop of DNA and protein is the nucleosome
  • Multiple nucleosomes are coiled together and these are stacked on each other. These are called chromatin
  • It is looped and further packaged using other proteins. This folding makes the DNA fit inside the nucleus of each cell (chromosome).
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4
Q

Non Histone Proteins

A
  • Some play purely structural roles.
  • Helps package DNA into structures distinct from the histone containing nucleosomes
  • In chromatin, those proteins that remain after histones have been removed.
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5
Q

Nucleosome Def

A
  • fundamental unit of chromosomal packaging

- DNA coiled around a core of histones

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6
Q

Function of chromatin

A
  • To package DNA into a smaller volume to fit the cell.
  • Controls gene expression, DNA replication/repair
  • Changes in chromatin structure are regulated by chemical modifications of histone proteins
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7
Q

Nucleosomes and Gene Expression

A

How DNA is wound around nucleosome determines if and how certain proteins interact with specific DNA sequences

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8
Q

Histone Modification

A

Euchromatin (extended)

  • Acetylation results in the loosening of chromatin
  • Replication and transcription

Heterochromatin (condensed)

  • Deacetylation holds DNA together strongly
  • Restricts access to various enzymes
  • Prevents transcription, RNA pol to big to enter

Histone modification controls the transition between these two states

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9
Q

Histone modifications impact:

A
  • Regulation of DNA replication
  • DNA repair
  • Gene transcription
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10
Q

Effect of Histone Modification

A

Acetylation results in the loosening of chromatin= Euchchromatin
->Replication and transcription

Deacetylation holds DNA together strongly= Heterochromatin
->Restricts access to various enzymes

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11
Q

Activators of genes transcription modify histones:

A
  • Acetylation of histones

- Transcription factors and RNA pol binds to the promoters

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12
Q

Repressors of transcription modify histones:

A
  • Deacetylation of histones
  • Transcription factors and RNA pol unable to bind to promoter
  • Nucleosome need to open up along DNA so can access promoters
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13
Q

Remodelling Complexes

A

Allow access to condensed DNA for machinery.

  • On gene activation, DNA binding activators bind the UAS
  • Recruitment of SAGA and SWI/SNF complexes
  • -these are non histone proteins, help with DNA unwinding and help with repositioning
  • -Multi subunit complex that disrupts chromatin (SWI-SNF) and gives transcription machinery access to the promoter
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14
Q

DNA Methylation

A
  • Results in repression of transcription
  • Heavy methylated regions found near transcription start sites
  • Histones are deacetylated, compact chromatin
  • Proteins binding methylated DNA form complexes with proteins involved in histone deacetylation
  • Histone acetylated, promoting transcription

e.g. genomic imprinting, X chromosome inactivation

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15
Q

Cell cycle

A

Repeated cell growth followed by division

4 Phases

  • Gap 1 (G1) phase
  • S Phase (synthesis)
  • Gap 2 (G2) phase
  • M phase
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16
Q

G1 Phase

A
  • Cells grow larger
  • Cell synthesises mRNA and proteins in preparation for subsequent steps leading to mitosis
  • G1 phase ends when the cells moves into the S phase of interphase
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17
Q

S Phase

A
  • Starts with DNA synthesis commences
  • All chromosome become replicated
  • DNA in cell doubles
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18
Q

G2 Phase

A
  • DNA is checked by enzymes for mistakes and repaired
  • Significant biosynthesis occurs, production of microtubules required for mitosis
  • Inhibition of protein synthesis during G2 phase prevents the cell from undergoing mitosis
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19
Q

M Phase

A
  • When ready, cell division begins=mitosis

- Nuclear membrane dissolves, chromosome, cytoplasm and cell membrane double forming two cells

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20
Q

Control of the cell cycle.

A
  • Signalling molecules (cytokines, growth factors) bind receptors activating pathways controlling genes
  • Have checkpoints in these cell cycles to make sure the cell has got to a certain point=cyclins
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21
Q

Gap 0 (G0) Phase

A
  • Non proliferative cells enter G0 state from G1

- Cells temporarily or reversibly stop dividing

22
Q

Interphase

A
  • Before division, a cell needs to take in nutrients
  • These preparations are done during the interphase
  • Interphase proceeds in three stages: G1, S phase, G2 Phase
  • Interphase is preceded by the previous cycle of mitosis and cytokines
23
Q

Cyclins

A
  • Proteins involved in controlling the cell cycle
  • Controls cell progression
  • Cyclins activate cyclin dependent protein kinases (cdk)
  • Cyclins/cdk add phosphate groups to proteins=inhibition/activation
24
Q

Cyclin D (G1)

A
  • cdk4 and cdk6 interact with cyclin D as it gets produced. Making it an active complex
  • Phosphorylates proteins within the cell
  • Leads to the transcription of cyclin E.
25
Q

Cyclin E (G1/S)

A
  • cdk2 interacts with cyclin E. Making it an active complex
  • Phosphorylates proteins within the cell
  • Promotes the expression of cyclin A
26
Q

Cyclin A (S/G2)

A
  • cdk1 interacts cyclin A making it an active complex
  • Phosphorylates proteins within the cell
  • Promotes the expression of cyclin B
27
Q

Cyclin B (G2/M)

A
  • cdk1 interacts with cyclin B making an active complex
  • Necessary for the progression of cells into M phase
  • Low levels lead to apoptosis
28
Q

Initiation of replication

A

-Eukaryotic chromosome is linear, have multiple ori on each chromosome which replicative forks work in both directions

29
Q

Ori

A

Origin of replication

  • Sequence in genome where replication is initiated
  • Replication proceeds bidirectionally or unidirectionally
30
Q

Pre-replication complex

A
  • Protein complex that forms at the ori during the initiation step of DNA replication
  • Needed for DNA replication to occur
31
Q

Negative Control of Replication

A
  • Proteins exist to prevent DNA replication
  • Ensures only one round of DNA replication occurs during each cell cycle

Example: Geminin (GMNN)
-Prevents assembly of the pre replicative complex (pre-RC)

32
Q

Separation of DNA Strands

A
  • Separated by helicases
  • Problem occurs due to DNA not being free to rotate on its own axis
  • DNA separation results in overwinding of DNA
  • Generates positive supercoils ahead of the replicative fork
33
Q

Three DNA Topology

A
  1. Supercoiling: coiling of the DNA double strand
  2. Knotting: entanglement of a single DNA molecule
  3. Catenation: linking of two or more DNA molecules
34
Q

Topoisomerases

A
  • Facilitate DNA transcription or replication

- Able to change DNA typology

35
Q

Type I Topoisomerase

A
  • Enzyme breaks one strand of a supercoiled double helix
  • Introduces a swivel or point of free rotation
  • Relaxes that supercoiling by cutting that one strand
36
Q

Type II Topoisomerase

A
  • Removes catenanes at the end of replication
  • Enzyme breaks two strands of the DNA helix
  • Involves ATP
37
Q

4-5-4 Pyrosequencing

A

-Method amplifies DNA inside water droplets in an oil solution
-Each droplet contains a single DNA template attached to a single primer-coated bead that then forms a clonal colony
(Bead has multiple copies of the reaction)
-The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzyme

-Uses luciferase to generate light for detection of the individual nucleotides added one base at a time to the DNA
(Since light is given off, easy to tell if nucleotides have been added)

38
Q

Illumina (Solexa) Sequencing

A
  • Technology based on reversible dye terminators
  • DNA molecules have adaptor attached. Adaptors attach DNA to slides
  • Amplification of DNA so local clonal colonies are formed
  • 4 types of reversible terminator fluorescently labelled nucleotides are added and non incorporated nucleotides are washed away.
  • Camera takes images of the fluorescently labelled nucleotides
  • Dye are terminal 3’ blocker is chemically removed from the DNA allowing the next cycle

Amplify the signal so it can be read. Cam add nucleotides at the same time as different colours are given off

39
Q

Ion Torrent Sequencing

A
  • Based on the detection of hydrogen ions that are released during the polymerisation of DNA
  • Microwells containing a single template DNA strand is flooded with a single type of nucleotide
  • If the nucleotide is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand.

Relies on the natural process of how nucleotides are added.

40
Q

MinION

A
  • USB powered DNA sequencer, laptop power
  • Uses ‘disruptive nanopore based technology’
  • Sequence DNA, RNA and proteins
  • Nanopore is an organic molecule penetrated by a very small hole
  • Nanopore is mounted on the membrane
  • Voltage difference placed between the two holes of the fluid. Nanopore hole forms a path
  • Fluid contains mobile ions. Ion current passes through the nanopore centre taking nearby molecules, proteins, RNA, DNA with it.
  • Molecules disrupt the flow of ions in a characteristic manner which can be detected and interpreted.
41
Q

Advances in sequencing techniques benefitting human genetics.

A
  • Lower costs
  • Easier to use
  • Can use it in the field
  • High throughput sequencing allows production of 1000s or millions of sequences at once
  • Take longer reads`
42
Q

Two common fluorescent methods

A
  1. Dye fluorescent: non specific fluorescent dyes that intercalate with the dsDNA
  2. Fluorescent reported probes:
    - sequence specific DNA probes labelled with a fluorescent report
    - detection after hybridisation of the probe with its complementary DNA target
43
Q

Dye fluorescence

A

e.g. SYBR Green

  • DNA binding dye binds to all dsDNA in PCR, causes fluorescence of dye
  • Increase in DNA product during the PCR, increases fluorescence intensity
  • PCR reaction is prepared as usual with the addition of fluorescent dsDNA.
  • After each cycle, the level of fluorescence is measured with a detector
  • Dye only fluoresces when bound to the dDNA (PCR product)
  • Allows DNA concentration to be quantified.
44
Q

Dye fluorescence problems

A
  • dsDNA dyes will bond to all dsDNA PCR products e.g. primer dimers (primers binding to themselves)
  • Potentially interfere with or prevent accurate quantification of the intended target sequence
45
Q

Taqman Probes

A
  • Have two primers and a probe specific to a gene. Probes sits between the primers and also bonds to the gene we are interested in
  • Probe contains a fluorescent protein and a quencher
  • Together on the probe, no fluorescence is given off as quencher quenches the fluorescence
  • During a round of PCR, the primers and probe will hybridise to the specific region, and will get extension
  • DNA becomes double stranded but doesn’t rely on it for the fluorescence to be given off
  • As polymerase reads along, starts to degrade the probe
  • The fluorescent proteins get separated from the quencher and fluorescence is given off

-Need to have lots of probe in reaction as each time its getting broken down.

46
Q

miRNA

A
  • miRNA mediate RNA interference (RNAi)
  • miRNA are processed from larger primary transcripts
  • Play a role in the regulation of at lease 70% of each organisms protein coding genes
  • Responsible for the post transcriptional regulation of many mRNAs via translational repression
  • Generated from non coding RNA
47
Q

miRNA processing

A

-miRNAs undergo transcription, generates short dsRNA. RNA at some point has to form a hairpin look to form double strand, then does on to make miRNA
(artificially making dsRNA)
-Pri-miRNA is processed in the cell nucleus by Drosha and Pasha enzymes. Pasha holds RNA while Drosha cuts it up. it becomes pre-miRNA
-Pre-miRNA leaves through a nuclear pore into the cytoplasm
-Dicer processes dsRNA substrates. Goes to the RISC complex and Dicer cuts it up into smaller pieces.

48
Q

miRNA and RNAi

A
  • Perfect complementarity to target RNA= Causes mRNA cleavage
  • With less complementarity= causes some type of inhibition of translation
49
Q

Small interfering RNA (siRNA)

A
  • siRNA pathway is responsible for detecting exogenous dsRNA. Destroys transcripts derived from invading RNA viral detection pathways
  • Pathway protects cells from invading viral dsRNA’s by destroying those RNAs.
50
Q

siRNA Mechanism

A
  • dsRNA usually arise in the cell from an exogenous sources (virus)
  • Both miRNA and siRNA are processed by the enzyme dicer and incorporated into the complex called RISC.
  • Resulting single stranded RNA interferes with expression of a gene containing the complementary sequence.

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