The Scientific Method Flashcards
In proteomics, what enzyme is used to digest proteins.
A proteolytic enzyme, trypsin
Potential mechanisms by which change at the DNA level could be involved in the activation of a specific gene
- DNA loss
- DNA amplification
- DNA rearrangement
- DNA is unchanged but is differentially expressed
The closest example of gene loss in cells
- Red blood cells lose their nucleus therefore their DNA .
- Stable mRNA remain allowing for transcription of globin and other proteins.
- Eventually these are also lost to allow the cell to fill up with hemoglobin and oxygen.
Why is the red blood cel not a great example of gene loss.
The loss of the nucleus is primarily intended to allow the cell to fill up with hemoglobin and to assume a shape allowing for oxygen uptake, rather than a means of gene control.
DNA amplification
These occur in situations where a very high level of mRNA/ protein production is required in a short time such that a single copy of a gene could not produce enough mRNA/protein.
DNA rearrangement
The deletion of of base pairs to bring differet coding sequences together to create a variety of different proteins. Also brings regulatory sequences closer to structural sequences allowing for activation and thus transcription to mRNA.
Example of Gene Loss
The loss of the nucleus and therefore DNA in red blood cells.
Examples of gene amplification
- Globin gene aplification in red blood cells before the deletion of the nucleus to make space for oxygen.
- The amplification of eggshell genes in Drosophila melanogaster.
Examples of gene rearrangement
Immunoglobulin genes are rearranged to create a variety of antibodies to protect the body.
Pulse Labeling
The synthesis of any particular RNA can be measured by adding a radioactive nucleotide to the cell and measuring how much radioactivity is incorporated into RNA specific for the gene of interest.
Problem with pulse labeling
The amount of radioactive ribonucleotide is far less than the normal ribonucleotides in the cell. This decreases the amound of radioactive ribonucleotides incorporated into the RNA. This makes it less affective.
Nuclear run on-assay
- Removable of the cytoplasm where most of the normal ribonucleotides are this will result in there not being enough ribonucleotides for transcription.
- Radioactive ribonucleotides are then inserted directly into the nucleus allowing for transcription to start again using the radioactively labeled ribonucleotides.
Polytene chromosome
Large chromosomes containing approximately 1000 DNA molecules.
In polytene chromosome the puff in the chomosome mean.
It represents the site of intense transcriptional activity.
What are the two levels the transcriptional control.
Chromatin structure: Altering the chromatin structure so that constitulively active regulatory molecules could gain access and switch on transcription.
Primary RNA transcript: activation of transcriptional regulatory proteins inducing transcription to produce the primary RNA transcription.
Length of miRNA
20-25 bases
Length of siRNA
21-24 bases
Length of piRNA
21-31 bases
Is the miRNA a single or double stranded RNA
Single
Is the siRNA a single or double stranded RNA
Double
Is the piRNA a single or double stranded RNA
Single
Distribution of miRNA
All cell types
Distribution of siRNA
All cell types
Distribution of piRNA
Primarily germ cells, also in some other cells types.
Do miRNA Require dicer protein for maturation.
Yes
Do siRNA Require dicer protein for maturation.
Yes
Do piRNA Require dicer protein for maturation.
No
Are miRNA found in plants and in animals
Yes
Are siRNA found in plants and in animals
Yes
Are piRNA found in plants and in animals
Only Animals
Function of miRNA
Control of cellular gene expression
Function of siRNA
Control of viral and cellular gene expression
Function of piRNA
Inhibition of transposons and control of cellular gene expression
Steps of processing miRNA precursors.
- The pri-miRNA precursor is transcribed by RNA polymerase 2 and forms a haipin loop structure which binds the drosha protein.
- Cleavage by drosha generates a pre-miRNA containing the hairpin loop which is transported to the cytoplasm.
- Pre-miRNA binds to the dicer protein
- The dicer cleaves off the single-stranded loop. This releases double-stranded RNA.
- One strand of which is degraded, with the remaining strand constituting the mature. MiRNA.
Steps for processing siRNA for viruse control.
- Transgene is transcribed in the sense and antisense direction.
- Double-stranded RNA formed which is then cleaved by the dicer eventually forming siRNA.
- The siRNA can degrade the transgene mRNA and the endogenous mRNA.
Steps of processing of siRNA in gene controls.
- Functional gene and homologous pseudogene are transcribed into RNA that become double stranded RNA.
- Dicer cleave the double stranded RNA eventually turning it into siRNA.
- SiRNA represses the mRNA transcribed from the functional gene.
The processing of Piwi-interacting RNA
- The genes encoding piRNAs are transcribed to produce a primary RNA transcript.
- This is then shortened at its 5’ and 3’ ends.
- The piwi protein associates with the mRNA.
- The RNA then undergoes further shortening at its 3’ end to produce the mature piRNA.
HOW does the piRNA stop the transposable elements from moving around in the genome.
- PiRNA bind to Transposon RNA degrading transposon RNA, preventing it from moving around in the DNA.
- The binding of piRNA can also iduce the copying of the transposon RNA to produce small 22G-RNA , theses 22G-RNA can enhance the inhibition of transposon gene transcription.
Which two small RNAs work pre and post transcriptionally.
SiRNA and piRNA
How do miRNAs move from cell to cell .
- Microvesicles
2. RNA-binding protein
How are long regulatory RNA transcribed.
- From the regulatory region of a protein-coding gene.
- From transcribed region of a protein-coding gene where the regulatory RNA is usually transcribed from the opposite strand of the DNA.
- DNA strand of a completely different region of the genome
Antisense RNA Transcribed from the protein-coding gene can regulate DNA expression How?
- Regulatory RNA can recruit a chromatin-modifying complex to altere chromatin structure. (POST -TRANSCRIPTIONAL)
- Can also form a double-stranded RNA molecule by binding to the protein—coding RNA, this can turn into siRNA.
WHAT happens if the pseudogene is transcribed in the antisense direction.
The pseudogene RNA will be complementary to the protein coding RNA forming a siRNA.
What happens if the pseudogene is transcribed in the sense direction.
The pseudogene RNA will be the same as the Protein coding RNA thus miRNA that would usually bind to the protein coding RNA now bind to the pseudogene RNA. Allowing for enhanced transcription of the protein-coding RNA.
What happens when long noncoding RNAs are transcribed in the antisense direction.
- The noncoding regulatory RNA can recruit a protein complex which organizes the target gene into a tightly packed chromatin structure.
- The noncoding regulatory RNA can compete for proteins which normally bind to the gene regulatory region and stimiulates transcription.
Example of miRNA regulation
- Gene lin-4 encodes a miRNA that will inhibit the translation of the mRNA coded for by gene lin-14.
- This happens in round worms at the embryonic stage.
