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Foundational genetic > Regulation genetic > Flashcards

Regulation genetic Flashcards

1
Q

How many are expressed at a given time

A

-some gene are turn on all the time while other are only turned on when needed
Constitutive expression – essential, ‘housekeeping’ genes
Regulated expression – specialised function
-around 50-75 percent at a given time

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2
Q

Why regulate gene expression?

A
  • to conserve energy and resources. does not make gene product that would not be used
  • Development. some gene are only turned non to facliltate develoment and is turn off after development is complete
  • Cell and tissue. different tissue would need different gene to be turn on or off for specialise fuction
  • Interaction with the enviroment
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3
Q

How is gene expression regulated

A
  • transcription
  • translation
  • MRNA stability
  • protein stability
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4
Q

Principles of transcriptional gene regulation for cis acting sequence

A

cis-acting sequences
RNA polymerase recognises the promoter sequence
Repressor binds to operator sequence prevent transcription
Activator binds to activator binding site (initiator sequence) allow for transcription
Activators promote transcription for positive control
Repressors inhibit transcription for negative control

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5
Q

Effectors

A

Effectors modify the properties of regulatory proteins

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6
Q

Origin of E. coli lac operon

A

–cell require carbon/glucose to fuction properly, ecoli is the same
-Glucose is preferred but ecoli can ultilise other carbon source When lactose is available in the environment, express genes for lactose uptake and catabolism
BUT, only when glucose is not available.

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7
Q

Overview of the lac operon

A

IT CONSISST OF THESE ITEM IN THIS ORDER
Inducer gap Promoter Operator Structural genes
lacI lacP lacO lacZ lacY lacA

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8
Q

What is used for Genetic analysis of the lac operon

A

IPTG is used
Inducer, but not a substrate
No inducer Very low lacZ,Y,A (few copies per cell)
With Inducer High lacZ,Y,A (1000s copies per cell)

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9
Q

Genetic analysis of the lac operon lacl

A

Most common class of mutants were constitutive mutants in lacI
Bacterial conjugationà (partial) diploid of lac operon
lacI– is recessive to lacI ( it is written as F) a negative strand is added
lacI+ is trans-acting (it is a diffusible product) by adding a positive strand onto a negative gene

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10
Q

Genetic analysis of the lac operon lacOc

A

-wild type is inductable
-lacOc is constitutive
lacOC is cis-acting (the mutation affects adjacent genes) (when different stand is added, not all gene are turned on)

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11
Q

Genetic analysis of the lac operon lacI

S

A

Non-inducible (super-repressor) mutants à lacI
S (rare), dominant
lacIS is dominant and trans-acting

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12
Q

Carbon catabolite repression of lac operon

A

-when glucose is high, low cAMP
-when glucose is low, high CAMp
crp gene encodes CAP
CAP and cAMP-binding to promoter activates transcription by RNAP

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13
Q

Genetic analysis of crp

A

Z Y
crp+ CBS+ Z+ Y+/ – + – + Wild type is inducible
2 crp– Z+ Y+ /– – – – crp– are non-inducible
3 crp– Z+ Y+/F’ crp+ Z– Y–/ – + – + crp– is recessive in trans
4 cbs– Z+ Y+ /– – – – cbs– is non-inducible
5 cbs– Z– Y+/F’ cbs+ Z+ Y–/ – + – – cbs– is dominant in cis

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14
Q

Summary of positive versus negative control

mutation effect

A

Positive controlled by activator. loss of fuction mutation is common, lead to recessive and non- inductable. rare alterfuction lead to constitutive dominant
Negative controlled by recessor, loss of fuction lead to constitutive, common, recessive. rare altered fuction lead to dominant non-inductable

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15
Q

What is different about eukaryotes gene regulation

A

Eukaryotic genes are not arranged in operons
Co-regulated genes can be dispersed in the genome
The default transcriptional state in eukaryotes is OFF (chromatin has an important role here)

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16
Q

General principles of eukaryotic gene expression

A

RNA polymerase II binds to promoter, BUT insufficient to activate transcription
General Transcription Factors associate with RNA pol II and promoter proximal elements, present in many genes
- promoter proximal elements are upstream CAT box (-100) and GC rich box (-200)
Regulatory proteins contain one or more domains
Transcriptional regulators often operate as protein complexes

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17
Q

How do we know these promoter proximal elements are important

A

point mutations in the β-globin gene

-mutation in these area lead to lower level of transcription

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18
Q

regulator protein of eukaryotic gene expression

A

RNA polymerase II binds to promoter, BUT insufficient to activate transcription
General Transcription Factors bind to promoter proximal elements
Transcription Factors bind to enhancers and silencers
(can be proximal or distal to promoters)

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19
Q

Galactose utilisationin yeast

A

Structural genes required for galactose utilisation:
Galactose uptake protein and several metabolic enzymes
Regulatory genes GAL4, GAL80 and GAL3
Gal 2 to transport yeat inside and Gal 1,7,10 breake it down into glucose

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20
Q

Gal4 transcription factor

A

gal4 loss-of-function mutants are recessive, non-inducible > Gal4 is an activator for positive control
DNA-binding domain recognises upstream activator sequences > UAS (Upstream activator sequence) are enhancers
Gal4 functions as a dimer and has a DNA-binding domain (BD) and an activation domain (AD)
Both domains can function independently

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21
Q

Gal4 recruits transcriptional machinery

A

Gal4 AD binds to TATA-binding protein (TBP) > promotes transcription by RNA polymerase II
Gal4 interacts with mediator complex >Recruits RNA polymerase to promoter

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22
Q

Mediator complex

A

Mediator is a protein complex, which interacts with TFs and RNAP
A co-activator facilitates activation by a transcription factor but does not bind directly to DNA

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23
Q

Gal80 regulatory protein

A

gal80 loss-of-function mutants are recessive, constitutive>Gal80 is a repressor for negative control
ànot all transcriptional regulators are DNA-binding proteins
Gal80 interacts with Gal4 AD to inhibit activity
>AD activity is the switch, not DNA-binding

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24
Q

Gal3 regulatory protein

A

gal3 loss-of-function mutants are recessive, non-inducible >Gal3 is an co-activator and a galactose sensor
-bind to Gal 80 and dissassociate it from Gal4

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25
Q

Variegated eyes in Drosophila

A

Herman Muller(1946 Nobel prize)
Wild type red eye w+/w+
white eye w-/w-
X-ray mutagenesis lead to mosaic white eye w+/w+
The position of the white locus, but not the genotype, has changed

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26
Q

Chromatin structure

A

Eukaryotic DNA is packaged into chromatin
150 bp DNA per nucleosome around 8 subunit 4 type
Different regions of the chromosome have different chromatin structure
Least condensed euchromatins to heterochrome most condense

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27
Q

Histone modifications

A

-Histonine have many tails which can be modified
Post-translational modification of lysines in histone tails by:
• Histone deactylases (HDACs) condense
• Histone acetyltransferases (HATs) relax
• And others…

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28
Q

Histone modifications in the GALsystem

A

Mig1 is a DNA-binding protein (repressor of GAL1) bind to mig1 binding site
Tup1 is a co-repressor, which recruits an HDAC > Histone deactylation condenses chromatin around GAL1

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29
Q

Chromatin remodelling

A

Nucleosomes are not fixed in position so DNA can slide back and forth
-when the promoter is hidden by the nucleosome, it is inactive
Shifting of nucleosomes can expose regulatory elements

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30
Q

Maintenance of chromatin state during DNA replication

A

-The post-translational modifications of histones defines the histone code
-the state of chromatin are preserved after replication
àChromatin state can be inherited (epigenetic inheritance)

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31
Q

Mating-type switching in yeast

A

-Mating requires the fusion of two haploid yeast cells
Two mating types: ‘a’ and ‘⍺’ which can be express to determine the gender of the spore
-Haploid cells are able to switch mating type This is controlled by the MAT locus (can switch around)

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32
Q

MAT gene in yeast

A

MATa and MAT⍺encode transcriptional regulators of mating-type genes eg, pheromones production, pheromone receptors etc.
MAT locus is flanked by transcriptionally silenced copies of MATa(HMRa) and MAT⍺ (HML⍺)
During mating-type switching, the silent copies are transferred into the MAT locus by recombination
silent information regulator (sir) mutants (affect the chromosomal structure)
express both a and ⍺genes, mutants are sterile
Sir2 is a HDAC histone deacetylation condenses chromatin

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33
Q

SWI-SNF complex

A

Two genetic screens in yeast identified mutants in the same locus
switch (swi) – unable to switch mating type
sucrose non-fermenting (snf) – cannot grow well on sucrose-media
swi2/snf2 mutants
• could not grow on sucrose, could not switch mating type
• Swi2/Snf2 protein is part of the SWI-SNF complex > Required for gene activation by chromatin remodelling

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34
Q

Role fo SWI-SNF

A

TATA sequences is wrapped around nucleosome > RNA polymerase cannot access promoter
SWI-SNF complex associates with promoter, Shifts nucleosomes, using ATP
TATA sequence now accessible,RNA polymerase can initiate transcription

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35
Q

Variegated eyes in Drosophila expalination

A

Inversion positions white locus near heterochromatin (centromere)
àHeterochromatin spreads over to silence the white+ geneThis is called position-effect variegation (PEV)

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36
Q

Barrier insulators

A

Barrier insulators prevent spreading of heterochromatin. it is at the edge of heterochromatin and recruit HAT
maintain euchromatin by localised histone acetylation

37
Q

Why regulate translation

A

-another way of controlling gene expression

~ 80% cellular energy is dedicated to protein synthesis

38
Q

transcription factor fuction in eukaryote translation

A
  1. 5’-cap recognition
    -Cap-dependent initiation of translation in eukaryotes, eIF4E bind to the G7m 5’ and recruit other IF, turn into eIF4F
    -eIF4F complex recruits small ribosomal subunit
  2. Met-tRNAi
    eIF2 is required to place the Met-tRNAiin the P Site
39
Q

Rapamycin

A
  • extract from Streptomyces hygroscopicus in easter island
    -have anti fungal property
    useful in
    • Immunosuppressant
    • Cancer treatment
    • Ageing
40
Q

A genetic screen in yeast for rapamycin-resistance

A

WT is unable to grow on Rapamycine, tor1 mutant able to

tor1 is a mutant of mTOR, a kinase protein (involve in modification of other protein)

41
Q

Regulation of translation in eukaryotes 5’-cap recognition

A

-4E-Binding Protein prevents formation of eIF4F complex,
-Blocks initiation of translation
on nutrient rich situation:
-mTOR is activated, 4EBP is phosphorylase
Phosphorylation of 4E-BP by mTOR prevents binding to eIF4E ->cap-dependent initiation of translation proceeds
In lo nutrition condition, mTOR is inactive, 4eBP is not phosphorilised and bind to eIF4E, prevent translation
mTOR is a global regulator of translation

42
Q

Regulation of translation in eukaryotes: Cap-dependent initiation of translation in eukaryotes

A

-DURING STRAVATION OF AMINO ACID, GCN4( increase in amino acid generation) and GCN2 (inhibition of translation ) is recruited
Phosphorylation of eIF2 by GCN2 inhibits the formation of Met-tRNAi
Reduced availability of Met-tRNAiinhibits initiation of translation

43
Q

Protein localisation

A
  • transcription regulator must be transport in or out of the nucleus to assume their function
    -nuclear localisation sequence (NLS) is regconise by importins which trnasport it pass the nuclear pore complex, into the nucleus (found in the internal of the protein)
    nuclear export sequence (NES) is regconised by exportin which transport it out
44
Q

Nuclear localisation of the glucocortocoid receptor

A

GR is a enhancer which need to bind to the active binding site for gene expression
In the absence of steroid, glucocorticoid receptor (GR) is bound to HSP90 in the cytoplasmIn the presence of steroid, HSP90 is displaced to reveal NLS, recognised by importin
In the nucleus, GR binds to glucocorticoid response elements (GRE) to activate transcription

45
Q

Ubiquitination

A

Ubiquitination is a eukaryote-specific post-translational modification

  • when a protein need to be removed, ubiqitine bind to it forming a ubiqutine chain. Ubiquitin is added to proteins by a ubiquitin ligase
  • the ubiquinated protein is then degrade by 26s proteasome
46
Q

Ubiquitination in cell cycle control

A

Levels of cyclin proteins are regulated through cell cycle progression
Anaphase-promoting complex (APC/C) is a ubiquitin ligase which add Uq to cyclin

47
Q

Development in animals

A

Some animal have direct development from their juvenile form into adult form ( growth)
other have drastic change from their juvenile form to adult form metamorphosis

48
Q

Embryogenesis in a newt

A
  • egg is fertilized by the sperm into a blastocyte
  • Series of rapid cell divisions into blastula (Cleavage)
  • Cells move inwards to generate a multilayered embryo know as Gastrula (Gastrulation()
  • Formation of the tissues, organs and structures of the body (organogesesis)
49
Q

distinct process in embryogenesis

A

Four distinct but overlapping processes occurring during embryogenesis
Determination, Morphogenesis, Differentiation and Growth

50
Q

Determination

A

Determination – establishment of the fate of a cell before specialised characteristics have developed

51
Q

Morphogenesis

A

Cellular activities during embryogenesis:
cells divide, move around, change shape and come together to form functional units (organs)
Collectively these cellular activities are called morphogenesis
Process that generates the structures of an organism (tissues and organs) – controls the spatial distribution of cells in the developing organism

52
Q

Differentiation

A

Differentiation – process by which cells acquire their distinct characteristics and functions
Associated with differences in gene expression
House-keeping Expressed in all cells
Cell specific Expressed in some cells

53
Q

Homeotic gene mutants in Drosophila

A

Homeotic mutants display homeosis – replacementof one body part by another （ultrabithorax antennapedia
-occur in determination

54
Q

Organisers

A

Oraganisers is a cell that establish the idendity of nearby cell
transplatn of organiser can have drastic effect on the embryo
-transplant of organiser from the endoderm to ectoderm result in conjoined zebra fish
-transplat of ZPA from anterior to posterior lead to extra digit in chicken

55
Q

Why is certain organism used as role model in genetic

A
  • Small gene size
  • Short generation time and large amount of offspring
  • Developmental mutants easy to observe
56
Q

Arabidopsis mutants in homeotic genes affect organ identity

A
  • flower first from different layer of swril and each swril develop into different organ/structure
  • mutant develop all swril into one structure
57
Q

role of zebre fish

A

Zebrafish embryogenesis is fast and visualised in vitro

  • use to infer for vertabrate
  • see through embryo and newvborn
58
Q

Establishment of cell fate in C. elegans

A

It is possible to follow the fate of every cells in nematode embryogenesis >generate a lineage map
Cells become progressively more restricted in cell fate as development proceeds
Position 1 = Zygote gives rise to 7 different cell types
Position 2 = EMS cell gives rise to 5 different cell types
Position 3 = C cell gives rise to 3 different cell types
Establishment of cell fate involves changes in gene expression that allow a cell to proceed down one of a series of alternative pathways

59
Q

Conserved genetic control of animal development

A

Early embryogenesis is highly similar in vertebrates (and invertebrates)
This ‘modularity’ facilitates evolution of body parts/organs

60
Q

Development is gene regulation in space and time (how is homeotic gene expressed)

A

Homeotic genes are expressed in specific spatial domains and determine identity of body parts

61
Q

Genes involved in development

A

Regulatory genes
genes that control expression of other genes (e.g. transcription factors, signalling proteins)
These genes play a major role in determination
Effector genes
genes that determine properties of the cell (e.g. cytoskeletal proteins, trafficking proteins, adhesion molecules etc.)
These genes play a major role in morphogenesis and differentiation

62
Q

Cell communication coordinates gene expression

A

Spatial domains express distinct sets of genes in an organised manner
Groups of cells (called organisers) influence gene expression in other groups of cells
Without cell communication, organisation is lost

63
Q

Cell communication via a signaling pathway

A

Cells separated by plasma membranes must communicate through extracellular molecules –ligands
Ligands bind to and activate receptors on another cell . Active the receptor leading to phosporilasation of TF leading to active or inactive. cis regulation

64
Q

Establishment of the body plan nomalculture

A

from head to tail is anterior - posterior
from front to back is dorsal ventral
z axis is mediolateral

65
Q

Pattern formation along the Dorsoventral axis

A

Cells along the dorsoventral axis express genes depending on their relative spatial positions within the embryo
from dorsal to ventral: ZEN,DPP, THISBE, TWIST

66
Q

twist ROLE

A

Twist is expressed in ventral cells and promotes furrow formation

67
Q

Morphogens provide positional information

A
  1. Establish a morphogen concentration gradient
  2. Cells perceive morphogen conveys positional information via concentration
  3. Cell fate established according to positionpattern formation in response to morphogen concentration
68
Q

French Flag model

A

Different genes expressed in response to specific concentrations of the morphogen
Threshold concentrations mark the transition from one pattern of gene expression to another

69
Q

The morphogen Dorsal

A

Positional information along the dorsoventral axis is generated by a gradient of nuclear-localised Dorsal morphogen – determined by an external signal

70
Q

Role of Dorsal in pattern formation

A

Transcriptional activity of genes respond to different concentrations of Dorsal
twist has an upstream cis-acting regulatory element (enhancer)with two low-affinity Dorsal binding sites
>twist expression is activated in the ventral region of the embryo where there is a high conc. of Dorsal
-thisbe has upstream cis-acting regulatory elements (enhancer)with three high-affinity Dorsal binding sites
àthisbe expression is activated in the lateral region of the embryo where there is an intermediate conc. of Dorsal

71
Q

The Dorsal genetic network

A

Twist is a transcription factor àRegulates 100s genes
ONE OF THEM IS FOG promotes apical constriction
àVentral furrow formation

72
Q

Fog causes myosin contraction

A

Fog is secreted from the apical side of cells àmyosin contraction and apical constriction

73
Q

Anteroposterior patterning in Drosophila

A

Maternal effect genes > Establish anteroposterior axis
Gap genes > Formation of block of segments
Pair-rule genes > Segment periodicity (marking of segment)
Segmentation-polarity genes > Patterning within segments
Homeotic genes > Fate determination

74
Q

Examples of mutants in segmentation genes

A

mutation of gap gene lead to kruppel, lack large segment of embryo (posterior region)
Pair-rule genes have either evenskip or odd skip mutation ( lack segment at odd/even)
mutation in segment polarity lead to goosberry mutant, iregualr polarity
all are lethal

75
Q

bicoid is a maternally expressed gene

A

bicoid mRNA is produced in the nurse cells of the egg chamber and is deposited into the oocyte
bicoid mRNA is anchored at the anterior end of the egg. Bicoid marks the anterior end of the developing egg

76
Q

Bicoid protein gradient in the zygote

A

Early stages of embryogenesis – all nuclei contained within one cytoplasm – a syncitium
When bicoid mRNA is translated, Bicoid protein diffuses to form a concentration gradient
zygote post fetalization is immunolocalization (many nuclei in one cell)
Maternal-acting gene bicoid encodes a transcription factor

77
Q

Establishment of Gap gene expression

A

Gap genes are expressed in specific regions along the anteroposterior axis
Gap genes are regulated by the maternal-effect genes present in the anterior and posterior regions of the embryo (e.g. Bicoid)
Gap genes also regulate each other (e.g. Hunchback)

78
Q

Bicoid activates hunchback

A

The Gap gene hunchback is activated by Bicoid
The hunchback promoter has three Bicoid binding sites
Hunchback active in anterior region where there are high levels of Bicoid
Sharp posterior boundary might arise from a threshold
-when a reporter gene is added to measure the effect of mutant, the less bicoid binding site, the smaller affected area

79
Q

Regulators of Krüppel

A

Hunchback is an activator of Krüppel
But high levels of Hunchback are associated with a lack of Krüppel expression
Gap genes can also restrict each others expression domains through repression
Giant and Knirps repress Krüppel
High Hunchback concentration activates Giant
Giant represses Krüppel.

80
Q

Pair-rule genes are expressed in stripes

A

there are 14 segement , the dorsal are fushi tarazu(ftz) while the anterior is even-skipped (eve)
Expressed in a periodic pattern:
7 alternating transverse stripes - every second segment

81
Q

How are the stripes of pair-rule genes generated

A

-it is regulated by both maternal gene and gap gene

82
Q

Control of even-skipped expression

A

cis-acting regulatory elements of eve, different stripe are coded separately
Stripes turned on one by one – independently regulated
on the stripe 2 enhancer sequence, there is 5 acitivator sequnece for bicoid and 1 for hunch back. ontop of that there is 3 krippple and giant binding site (repressor)

83
Q

Homeotic genes controlling segment identity

A 
Homeotic genes in Drosophila
Conserved homeobox (Hox) transcription factors, 8 in drosophilla with distinct pattern of expression along to anterior - posterior but not segment related
84
Q

Ubx in formation of limb

A

Ubx regulate Distal-less (Dll) is a Hox target gene > promotes the formation of appendages (eg. limbs). each segment is mark by enrail (segement polarity)

  • mutation in Ubx lead to Dll express in A1
  • mutation in both Ubx and abd-A lead to mutation in most of the abdomen => both gene work to control dll
  • mutation in engrail sequence prevent the binding of regulatory gene can also lead to mutation
85
Q

Hox genes contribute to evolution of development

A 
In dipteran (fly) wg and DSRF gene is bind by Ubx > one pair of wing
in lepidopteran (buterfly) Ubx cis-elements in target genes are absent
86
Q

Hox genes are conserved in animals

A

Hox genes are present throughout the animal kingdom
Many have almost identical AA in the homeodomain
These animals last shared a common ancestor over 500 mya
Chicken Hox genes can perform the function of fly Hox genes

87
Q

Hox in rat

A
  • expasion in number of copies (4) and gene loss lead to 39 hox gene
  • retain the arrangement of gene in the chromosome
  • genome order of hox gene still similar eventhough does not develop segmently
  • hox 1,3,4 in anteior while hox 8-13 in posterior
88
Q

Hox genes specify vertebrate identify

A

hox10 mutant Lumbar >thoracic (thorax should end at t13)
hox11 mutant Sacral >lumbar
Hox10 and Hox11 act in posterior segments to repress (more) anterior identity

89
Q

Translational regulation of cell identity in C. elegans

A

-during the 4 cell stage :anterior ABa, ABp, P2, EMS posterior
-glp-1 mRNA is present in all cells at four-cell stage
BUT GLP-1 protein is only present in ABa/ABp cells
Spatial Control Region (SCR) in glp-1 3’UTR is required for repression of GLP-1 in EMS and P2 (posterior) cells
Translational repression of GLP-1 in EMS and P2 also requires Gld-1
GLD-1 expression is high in posterior cells, and associates with glp-1 3’UTR

Foundational genetic (15 decks)

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