Differential Gene Expression in Development Flashcards
(60 cards)
It is the process by which cells become different from one another based upon the unique combination of genes that are active or “expressed.”
Differential gene expression
The three postulates of differential gene expression
- Every somatic cell nucleus of an organism contains the complete genome established in the fertilized egg. In molecular terms, the DNAs of all differentiated cells are identical.
- The unused genes in differentiated cells are neither destroyed nor mutated; they retain the potential for being expressed.
- Only a small percentage of the genome is expressed in each cell, and a portion of the RNA synthesized in each cell is specific for that cell type.
It regulates which of the nuclear genes are transcribed into nuclear RNA.
Differential gene transcription
It regulates which of the transcribed RNAs (or which parts of such a nuclear RNA) are able to enter into the
cytoplasm and become messenger RNAs.
Selective nuclear RNA processing
It regulates which of the mRNAs in the cytoplasm are translated into proteins.
Selective messenger RNA translation
It regulates which proteins are allowed to remain and/ or function in the cell.
Differential protein modification
To know the specific time and place of gene expression, one needs to use procedures that locate a particular type of messenger RNA or protein within a cell. These techniques include:
northern blots, RTPCR, in situ hybridization, microarray technology for transcripts, and western blots and immunocytochemistry for proteins
To ascertain the function of genes once they are located, scientists are using new techniques, such as
CRISPR/Cas9-mediated knockouts, antisense, RNA interference, morpholinos(knockdowns), Crelox analysis, and ChIPSeq techniques.
These techniques enable researchers to compare thousands of mRNAs and computer-aided synthetic techniques can predict interactions between proteins and mRNAs.
RNA analysis by microarrays, macroarrays, and RNAseq
The main principle is to take advantage of the single-stranded nature of mRNA and introduce a complementary sequence to the target mRNA by in situ probes that enables visualization or detection. What technique is this?
In situ hybridization
This technique uses dyes to allow researchers to look at entire embryos (or their organs) without sectioning them, thereby observing large regions of gene expression next to regions devoid of expression.
Hybridization occurs between the probe anti-sense RNA and the targeted mRNA. To visualize the cells in which hybridization has occurred, researchers apply an antibody that specifically recognizes the target mRNA.
In situ hybridization
It is based on two highly specific interactions. One is the binding of a transcription factor or a modified nucleosome to very particular sequences of DNA (such as enhancer elements), and the other is the binding of antibody molecules specifically to the transcription factor or modified histone being studied.
Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq)
It enables a researcher to use known transcription factors as bait to isolate the DNA sequences they specifically recognize. Researchers then use these identified enhancer regions to generate transgenic reporter constructs and organisms that enable visualization of gene expression in live cells and organisms.
Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq)
It takes advantage of the high throughput capabilities of next-generation sequencing technology to sequence and quantify the RNA present in a cell. Next-generation sequencing can analyze these transcripts for both nucleotide sequence and quantity.
RNA-Seq
It has been particularly powerful for comparing transcriptomes between identical samples differing only in select experimental parameters.
RNA-Seq
It had an enormous effect on genetic research, making
gene editing faster and less expensive than ever and making it relatively simple in organisms from E. coli to
primates. This technique uses a system that occurs naturally in prokaryotes for defending against invading viruses.
CRISPR/CAS 9 Genome Editing
Cells will naturally try to repair double-strand breaks through a process called
nonhomologous end-joining (NHEJ)
It is a stretch of DNA containing short regions that when transcribed into RNA serve as guides (short guide RNAs or sgRNAs) for recognizing segments of viral DNA.
CRISPR (clustered regularly interspaced short palindromic repeats)
It uses homologous recombination to place two Cre recombinase recognition sites (loxP sequences) within
the gene of interest, usually flanking important exons.
CRE-LOX System
It allows for control over the spatial and temporal pattern of a gene knockout and gene misexpression.
Researchers have inserted stop codons flanked with
loxP sites to prevent transcription of a given gene until the stop codon is removed by Cre-recombinase.
CRE-LOX System
It is found in “developmental control genes,” where they regulate the synthesis of the transcription factors and other developmental regulatory proteins used in
the construction of the organism. The default state of these promoters is “on,” and they have to be
actively repressed by histone methylation.
High CpG-content promoters (HCPs)
It is found in those genes whose products characterize
mature cells. The default state of these promoters is
“off,” but they can be activated by transcription factors. The nucleosomes on these promoters have relatively few modified histones in the repressed state.
Low CpG-content promoters (LCPs)
It is the addition of methyl groups to DNA by enzymes called DNA methyltransferases.
DNA methylation
DNA methylation appears to act in two ways to repress gene expression
- It can block the binding of transcription factors to enhancers
- A methylated cytosine can recruit the binding of proteins that facilitate the methylation or deacetylation of histones, thereby stabilizing the nucleosomes.
