Theme 3: Responding to the Environment - Module 4: Turning Off the Signal Flashcards
How can the relative amount of gene products produced by an organism vary?
varies depending on changing conditions and different signals
How do organisms ensure that the right amount of each gene is present at the right time?
the expression of genes is regulated
when can gene expression be regulated?
at many different points during the synthesis of a functional protein
what are some different points during synthesis in which the expression of a gene can be regulated?
- transcription initiation
- RNA processing
- overall stability of the RNA molecule
- protein synthesis
- protein modifications and transport
- protein degradation
what does multi-level regulation enable the cell to do?
enables a cell to rapidly alter the levels of active proteins in response to internal and external signals
To understand how cells work, what is it important to know?
when and where specific genes/groups of genes are expressed within an organism
what is a straight forward war to examine the patterns of gene expression of specific genes?
identify the mRNA products that are produced
How can the mRNA be detected?
with a complementary probe that has a fluorescent tag attached to it
what can in situ hybridization be used for?
utilized to study the expression of one or a few genes of interest
How can we use in situ hybridization to understand any temporal differences that may exist within the genes of organisms?
can do the in situ hybridization at different stages of development and compare them
How many genes were we able to assess using in situ hybridization?
a few
what technique was developed that allowed us to examine the expression of thousands of genes at once?
DNA microarray technique
what is the DNA microarray technique largely based on?
base-pair interactions and binding of complementary strands of nucleic acids
what time of adaptation is this to the in situ hybridization?
in vitro adaptation
Since the entire genome of different organisms is not known it is possible to set up glass slides that contain what?
tiny spots with known DNA sequences or genes
What do DNA microarray chips contain?
DNA molecules that act as probes
What do probes in the DNA microarray do?
detect gene expression (also referred to as the transcriptome or the set of mRNA transcripts that are expressed by various genes)
what can genomic DNA sequences as probes be used to investigate?
whether specific genes of interest are transcribed and looking at groups of genes to determine whether they’re expressed in any specific coordinated manner
what does the DNA microarray technique make possible?
possible to gain insights into possible interacting gene networks within a genome
it is possible to manufacture DNA microarrays containing up to how many oligonucleotides?
100, 000 (each representing a different gene)
what are oligonucleotides?
short fragments of nucleic acids
are all of our genes active at once since we contain identical genetic material throughout our body?
no
what are DNA microarrays useful for?
visualizing variation in gene expression during different developmental stages, across different cell types and in response to different signals
investigations about which genes are active or not in different cells can provide information about what?
how the cells function normally and what changes when gene expression is altered
DNA microarrays can also be utilized to identify differences in gene expression levels between what kinds of cells?
normal and cancerous
what did different types of cancer cells use to be identified by?
the organs in which the tumours developed
what does DNA microarray analysis make possible to differentiate with regards to normal vs. cancerous cells?
patterns of gene activity
to answer questions about what genes are involved in transforming normal cells to tumour cells what needs to be done?
- growing both cell types in culture
- isolating gene products or mRNA that is transcribed by the two cell types
- performing DNA microarray analysis
when the mRNA has been isolate from the cells what can these molecules serve as?
templates for making complementary cDNA molecules to mRNA
what enzyme is used to make complementary cDNA molecules to the mRNA
reverse transcriptase enzyme
what is used during the reverse transcription process?
- fluorescent nucleotides - they become part of the newly synthesized cDNA molecule
what is it important to label the cDNA from both cell types with? why?
- different coloured fluorescent dyes
- so they can be easily identified during microarray analysis
what does a DNA microarray chip consist of in large numbers?
single stranded DNA fragments
what do the single stranded fragments represent?
different genes
how are these DNA fragments organized in the glass slide for the microarray analysis?
tightly spaced array
what does the DNA fixed to the chip represent?
all genes known in the cell (can be differences in gene expression at any time)
what can be done with the normal and cancerous dyed cells?
they can be combined in equal amounts and tested for hybridization with the single-stranded DNA molecules that are present in the microarray chip
what is measured after this combination takes place?
use scanner at each spot on the microarray slide to measure relative differences in colour and intensity
if a particular gene is active what will it produce?
many molecules of mRNA
what happens as a result of producing many mRNA molecules?
more labelled cDNA molecules available after reverse transcription which will be able to hybridize to the DNA on the microarray chip
when many molecules of mRNA are produced, more cDNA molecules are available and able to hybridize to the DNA microarray chip, what observation does this lead to?
many bright spots
what happens when an equal mixture of labelled cDNA of both normal epithelial cells and breast carcinoma cells are applied to a microarray chip?
they will compete for the synthetic complementary of DNA fragments that re distributed across all spots of the microarray chip
what does a green fluorescent spot mean?
gene is more expressed in normal epithelial cells (decreased expression/downregulated in breast carcinoma cells)
what does a red fluorescent spot mean?
specific gene is unregulated - more expressed in breast carcinoma cells compared to the normal epithelial cells
what does a yellow fluorescent spot mean?
gene is equally expressed in both cell types
how can relative gene activity be measured?
measuring intensity of fluorescence
how are brighter fluorescent spots produced?
by genes that are more active (produce more mRNA)
what does it mean if there is no fluorescences at all in a particular spot?
gene may be inactive
is it vital that gene expression be regulated at many different points during the synthesis of a functional protein?
yes
is it sufficient to remove proteins from the cell if mRNAs are continually transcribed into new proteins?
no
how can gene expression be stopped?
mRNAs should be degraded
what is one way to regulate mRNA stability?
through the length of the polyA tail
are there more ways to regulate mRNA stability?
yes
is there evidence that some genes can be transcribed into mRNA but the mRNA never ends up being translated?
yes
how can genes be transcribed into mRNA but the mRNA is never translated?
occurs due to the activation of RNA interfering machinery
what is RNA interfering machinery activated by?
short, noncoding regulatory double stander RNA molecules
what is an example of a small regularity RNA?
microRNA molecules
what are microRNA molecules transcribed from?
from protein-encoding genes using the same RNA polymerase that transcribes other RNA molecules
what do the transcribed microRNA often form?
form hairpin loops
why do the microRNA form hairpin loops?
due to complementary base-pairing within these microRNA precursor transcripts
what are the hairpin loops processed into? what can they do?
- processed into smaller, single stranded mRNA fragments
- can activate the RNA interference machinery
what do the processed microRNA become a part of?
incorporated as part of a RNA-induced silencing complex (RISC complex)
the single stranded microRNA molecules contain sequences that are complementary to what?
specific target mRNA sequences that require regulation
the microRNA in the RISC complex with bind in what manner to the target mRNA sequence?
non-exact
what happens once the microRNA bind to the target mRNA sequence?
the proteins within the RISC complex can inhibit translation
what type of RNAs are transcribed and process in a similar manner as the microRNAS
small interfering RNAs/siRNAs
do siRNAs become associated with the RISK complex?
yes
what is the difference in which the small regulatory microRNAs and siRNAs are able to regulate gene expression?
due to manner in which they interact with the target mRNA sequence and how the target mRNA is regulated
are siRNAs exact complements of their mRNA target?
yes
onto of binding to the complementary sequence by an association with the RISC complex, what else do they do?
also induce cleavage or cutting of the target mRNA
what does the cleavage due to the target mRNA?
destabilizes the target mRNA and further contributes to pot-transcriptional regulation of gene expression
what are RNA interference mechanisms?
endogenous processes that regulate gene expression
why have researchers widely adopted this tool?
- used as a technique to turn off desire genes and examine and obtain information about the role of the gene in cells
- can figure out how misrelated/altered gene expression can lead to various diseases
when is one of the final opportunities to control gene expression?
after translation
what are some of the post-translational modifications that can allow for the production of mature, functional proteins?
- cleavage
- disulphide bond formation
- acetylation
- phosphorylation
- methylation
what does post-translational modifications allow?
cell to activate or inactivate specific proteins
what does selective degradation do?
limits the length of time in which a protein functions in a cell
what are proteasomes? where are they found? what are they able to do? what happens as a result?
- very large protein complexes
- found in cells
- able to break peptide bonds
- as a result they degrade unneeded or damaged proteins
by degraded unneeded or damaged proteins, what does this enable a cell to do?
to regulare the concentrations of specific proteins
how are the cells able to regulate the concentrations of specific proteins?
by breaking long polypeptides into small fragments of a few amino acids in length
what happens after the polypeptides are broken into small fragments of a few amino acids?
the small fragments of a few amino acids in length can be further degraded into amino acids and used in subsequent rounds of translation
what does the cell need to do with regards to protein degradation?
accurately identify and regulate which proteins will be degraded
identification of proteins for degradation is largely accomplished through what?
a tagging mechanism
what happens during protein degradation?
target proteins are tagged though an enzymatic cascade
what are proteins tagged with?
small ubiquitin proteins
what is the tagging process largely dependent on?
ATP
what is the tagging processes facilitated by?
ubiquitin activation, conjugation to the target substrate protein, and ligation
what does ubiquitin activation, conjugation to the target substrate protein, and ligation result in?
polyubiquitin chain
what is the polyubiquitin chain attached to? what does it allow?
- substrate target protein
- allows for the proteasome to degrade the tagged protein
what else is largely ATP dependent?
the passage of the target protein through the proteasome
what does the the passage of the target protein through the proteasome allow?
allows for the unfolding of the protein and subsequent cleavage into smaller fragments
what is another mechanism that can turn off gene expression signals? what does it result in?
the ubiquitin-proteaseome pathway
- results in degradation of thousands of proteins in the cell
what can those degraded proteins include?
- transcription factors
- cell-cycle realtors
- mis-folded or aggregated proteins
