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Housekeeping (constitutive) genes
genes expressed in all cells because they provide basic functions needed for sustenance of all cells (basic metabolic and structural functions)
part of the upstream control region of genes that are usually very accessible to DNase I (enzyme that cleaves DNA)
Cells in the brain and cells in the liver contain the same DNA. How is it that these cells are different cell types?
Each cell type has a distinct pattern of gene expression
What is a housekeeping protein and in which cell type is it found?
These are proteins associated with basic metabolism, translation, transcription, replication, and cell structure of a cell; they are generally present in all cell types
What is heterochromatin?
Heterochromatin is condensed chromatin. It tends to be transcriptionally inactive, and it is less sensitive to DNAse I digestion than euchromatin.
What is euchromatin?
Euchromatin is relaxed (i.e. non-condensed) chromatin, and is usually associated with transcriptionally active genes
What are hypersensitive sites and where are they located with respect to genes?
Hypersensitive sites are short regions of chromatin that are exceptionally sensitive (i.e. very accessible) to nucleases such as DNase I; they are often found in the upstream control regions of active genes.
locus control regions (LRCs)
they regulate gene expression indirectly by regulating chromatin organization over certain chromosomal domains
How do locus control regions (LRCs) work?
Sections of DNA containing LRCs may form loops over desired genes to enhance their expression
protein complex that can alter chromatin structure via nucleosome remodeling
-complexes are thought to act globally to increase mobility of nucleosomes throughout the genome
-some complexes may target specific genes
histone acetyltransferase (HAT)
- enzyme that catalyzes acetylation of histones (specifically at lysine residues), which leads to the unfolding of chromatin
- • increases the accessibility of transcription factors to access DNA
histone deacetylases (HDAC)
enzymes that remove acetyl groups (O=C-CH3) from an N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly
enzymes that methylate the 5th carbon in cytosine residues after DNA replication has occurred to repress gene transcription
when DNA methyltransferases create 5-methylcytosine residues in a highly specific pattern; eg. most of the methylated Cs are next to Gs
Which amino acid residue in histones is typically most susceptible to acetylation?
found in the promoters of genes that are actively transcribed in all cell types (housekeeping genes); CpG rich stretches almost always lack methylation
CpG methylation is also a key mediator of:
X- inactivation (lionization) and other epigenetic effects
The assembly of basal transcription factors requires the presence of what other class of transcription factors?
Activators bind to what regulatory regions of DNA?
What is the function of coactivators?
They allow communication between activators and basal transcription factors
Activators communicate with the basal factors through:
The complex of coactivators binds to which protein?
TATA Binding Protein (TBP)
Activator and coactivator interactions enable the basal factors to position ________________ at the start of the protein-coding region and to set the _________ in motion
RNA polymerase II
What is the function of repressors? Where do they bind on DNA?
- They block the initiation of transcription by interfering with the function of activators
- • they bind to silencers
What is the result of activator-coactivator interactions?
Activator-coactivator interactions allow basal transcription factors to position RNA polymerase II at the beginning of the protein-coding region of a gene and set the polymerase in motion
How are cells able to control the transcription of every gene individually?
Genes are generally different in respect to the combination of promoter and enhancer (or silencer) elements they carry
What happens to transcription and translation during elevated temperature?
Cells suspend transcription and translation of all genes EXCEPT for those that help with survival of cells at high temperature, like genes encoding heat shock proteins
heat-shock factor (HSF)
- a protein that after heat-shock binds to a specific DNA site up-stream of certain genes
- • binding & subsequent phosphorylation of the bound factor results in increased transcription
- • HSF is present in cells before heat-shock; it's the heat that changes it to an active form
What causes heat shock factor protein (HSF) to become active?
Heat shock (elevated temperatures) as well as other stresses (such as toxins)
What is the function of heat shock factor (HSF) proteins?
After heat shock-induced activation, HSF proteins bind to specific DNA sites upstream of certain genes and increase transcription of those genes.
Binding of heat shock factor (HSF) proteins to DNA is not sufficient to increase transcription of heat shock genes. What else must occur after binding?
the HSF must be phosphorylated for transcription of heat shock binding proteins to occur
Name three steroid hormones:
- 1) estrogen
- 2) testosterone
- 3) progesterone
Steroid hormones are all derivatives of which metabolite?
What is the mechanism by which steroid hormones affect the transcription of genes?
Steroid hormones are fat soluble so they can diffuse across membranes; they complex with their receptors and together translocate into the nucleus where they displace nucleosomes from promoter regions and cause other transcription factors to bind
What are three factoids about steroid hormones?
- 1) different hormonal signals will affect only those genes that contain the appropriate response elements
- 2) steroid receptors are not present in all cells in the same concentrations
- 3) the affected gene will remain active ONLY as long as the hormone is present in the cell
What are the DNA binding sites for steroid hormone/receptor complexes called?
Hormone response elements (or enhancers)
What are four transcription factors that mediate gene expression in SPECIFIC cell types?
- 1) Isl-1: works only in islet cells of the pancreas to regulate the Insulin gene
- 2) MyoD1: works only in the skeletal muscle to regulate the Myosin gene
- 3) Oct-2: works only in B cells to regulate Immunoglobulin heavy and light chain genes
- 4) LFB1: works only in the liver to regulate the albumin gene
• these factors are normally synthesized, or activated, only in one specific tissue, resulting in cell type-specific transcription of genes whose expression is dependent upon them
What information can be extracted from DNA microarrays?
Levels and patterns of gene expression
How are DNA fragments able to attach to the underlying slide of glass in a DNA microarray?
The DNA stick to the slide because the slides are coated with poly-L-lysine, which is positively charged (DNA is negatively charged)
DNA microarrays are useful in determining what kind of information?
The relative expression levels of genes. The sample applied to the microarray is fluorescence-labeled cDNA.
How does antisense therapy affect mRNA translation?
In antisense therapy, a fragment of nucleic acid that is complimentary to an mRNA of interest is introduced into a cell. If the nucleic acid binds to a complementary fragment of mRNA, it blocks ribosomes from translating the mRNA.
In antisense therapy, a nucleic acid fragment is introduced into a cell. How is the antisense nucleic acid fragment protected from intracellular degradation?
The oxygen atoms that make up the phosphate links between nucleotides of the DNA backbone are replaced by sulfur atoms, rendering the nucleic acid immune to immediate degradation.
What are three issues with antisense therapy?
- 1) toxicity of introduced nucleic acids
- 2) keeping nucleic acids inside the cells once they are introduced
- 3) identifying the genes that are causative of the diseases we intend to treat
RNA interference (RNAi)
a technique in which double-stranded RNAs homologous to a gene of interest are introduced inside a cell with the goal of silencing that gene
How do siRNAs silence gene expression?
siRNAs are separated into single strands which bind to the RISC complex, facilitating degradation of complementary mRNAs before they can be translated by ribosomes.
an enzyme that cleaves double-stranded RNAs into smaller double-stranded RNA fragments called siRNAs
- a protein complex that binds to single strands of siRNAs
- • the siRNAs serve as a template that recognize complementary mRNAs
- • once recognized, the RNase subunit of the RISC complex cleaves the complementary mRNAs so that they CANNOT be translated
What was the RISC complex probably originally used for in cells before it's siRNA capabilities came in handy?
it was probably present to degrade any double stranded VIRAL (harmful) RNA