Notes in BIOC 4332

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Status	Last Update	Fields
Published	04/10/2024	Human genome is {{c1::3.2 billion}} base pairs long
Published	04/10/2024	{{c1::Shotgun cloning}}: take random pieces of the genome and clone them, sequence them, and then assemble them to make the complete genome.
Published	04/10/2024	Challenges in the post genomic era: Elucidate the genes, genetic networks and their contribution to cellular and organismal phenotypes (use {{c1:…
Published	04/10/2024	{{c1::Classical genetics}} involves finding mutant phenotypes and then isolating the gene(s) responsible.
Published	04/10/2024	{{c1::Reverse genetics}}: start with a gene and find what phenotypes arise when that gene is altered. This was not possible without sequenced genomes.…
Published	04/10/2024	Proteins that cross cell membranes could be important {{c1::receptors}} or {{c1::transport proteins}}, which are often good drug targets.
Published	04/10/2024	{{c1::Membrane spanning proteins}} need to have regions of {{c2::hydrophobic}} amino acids.
Published	04/10/2024	Hydrophobic regions have very few {{c1::charged}} amino acids.
Published	04/10/2024	{{c1::Hydropathy plots}} can identify membrane spanning domains in proteins. It calculates the hydrophic index.
Published	04/10/2024	On a hydropathy plot, a protein has a lot of hydrophobic amino acids in a row, there will be a {{c1::positive::positive or negative}} peak.
Published	04/10/2024	Every {{c1::DNA manipulation}} starts with cutting DNA: the more {{c2::specifically}} you can cut, the better.
Published	04/10/2024	{{c1::Restriction enzymes}} cut too frequently so they are not very useful. The majority of gene targeting technologies utilize the formation of {{c2:…
Published	04/10/2024	Editing a gene utilizes {{c1::double stranded break repair pathways}}.
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Published	04/10/2024	EcoRI finds {{c1::GAATTC}}
Published	04/10/2024	{{c1::Restriction enzymes}} recognize short specific sequences. EcoRI would find over 68k recognition sites in chromosome 1 alone.
Published	04/10/2024	{{c1::Meganucleases}} is the modern approach to restriction enzymes.
Published	04/10/2024	Meganucleases are also known as {{c1::homing endonucleases}}, they contain both the {{c2::DNA recognition and cleavage functionalities}}; have a large…
Published	04/10/2024	Proteins are formed by distinct {{c1::domains}}. These are often the {{c2::functional units}} of the protein, allowed for easier evolution of new prot…
Published	04/10/2024	Domains can be "swapped" to engineer {{c1::chimeric proteins}}.
Published	04/10/2024	{{c1::Helix-turn-helix}} is one of the most common DNA binding domains. It is {{c2::7-9}} amino acids and can be found in the {{c3::major groove}}. A …
Published	04/10/2024	The {{c1::zinc finger}} and {{c1::leucine zipper}} are also common DNA bindign domains.
Published	04/10/2024	{{c1::Zinc finger}} utilized early on to develop gene editing processes because it can recognize about {{c2::3}} bp specifically. Putting multiple tog…
Published	04/10/2024	Human genome contains about {{c1::700}} zinc finger proteins, almost all of these are {{c2::transcription}} factors.
Published	04/10/2024	{{c1::Alpha helicies}} can recognize specific sequences, these are exploited to engineer proteins.
Published	04/10/2024	To make a zinc finger nuclease, fuse the {{c1::3-zinc-finger recognition domain}} to a {{c2::nuclease domain}}. The endonuclease {{c3::Fok1}} is found…
Published	04/10/2024	ZFNs have {{c1::3}} fingers ({{c1::9}} bp recognition site). All ZFNs come in pairs, thus the recogntion site is actually {{c1::18}} bp.
Published	04/10/2024	{{c1::Affinity}} of each finger varies, and the necessity to stitch together each individual finger with exact spacing between {{c2::fingers}} and the…
Published	04/10/2024	Zinc fingers were supplanted by {{c1::TALENs}}: TAL Effectors
Published	04/10/2024	TALE is a {{c1::transcription activator-like effector}}
Published	04/10/2024	TAL effectors are transcription factors that re-engineer the plant genome to help establish a {{c1::productive bacterial infection}}. As such, they ha…
Published	04/10/2024	DNA binding domain contains a repeated highly conserved {{c2::33-34}} amino acid sequence with divergent {{c2::12th and 13th}} amino acids. These two …
Published	04/10/2024	TALEs DNA binding "code": each {{c1::RVD}} binds to a {{c1::specific DNA base}}.
Published	04/10/2024	To make a site-speicific endonuclease: {{c1::fuse TALEs with the Fok1 endonuclease}}.
Published	04/10/2024	TALEN limitation is that it is {{c1::difficult to construct and express}}
Published	04/10/2024	CRISPR: {{c1::clustered regularly interspaced palindromic repeats}}. The nearby genes were then called "Cas" - {{c2::CRISPR associated}}.
Published	04/10/2024	Little bits of DNA in E.coli were identified as {{c2::bacteriophage/foreign bacterial DNA}}, which suggests a possible bacterial {{c1::immune system}}…
Published	04/10/2024	{{c1::Immunization}}: during the initial infection little pieces of the foreign DNA are "saved" into the chromosome.
Published	04/10/2024	{{c1::Immunity}}: the next time the bacteria encounters the same pathogen it is ready and uses {{c2::Cas/CRISPR}} to cut the offending DNA up into lit…
Published	04/10/2024	The {{c1::CRISPR RNAs}} target cleavage enzymes to specific DNA sequences in viruses
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Published	04/10/2024	Cas + CRISPR RNA (crRNA) + trans-acting crRNA (tracRNA) = {{c1::functional nuclease that cleaves dsDNA}}
Published	04/10/2024	PAM: {{c1::protospacer adjacent motif}}
Published	04/10/2024	{{c1::Cas + sgRNA}} = functional nuclease that cleaves dsDNA
Published	04/10/2024	{{c1::Cas + sgRNA (crRNA/tracrRNA)}} is a functional artificial CRISPR/Cas9 nuclease
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Published	04/10/2024	Cas9/CRISPR is so efficient you often get {{c1::biallelic}} modifications.
Published	04/10/2024	Gene targeting with ZFN and TALENs require {{c1::multiple rounds}} of editing for multiple genes.
Published	04/10/2024	CRISP/Cas9 can be {{c1::multiplexed}}
Published	04/10/2024	{{c1::Multiplexing}} is simply adding multiple sgRNAs
Published	04/10/2024	DNA recognition depends on {{c1::sgRNA}} instead of protein domains.
Published	04/10/2024	Advantages of CRISPR/Cas9DNA recognition depends on {{c1::sgRNA}} instead of protein domains Very easy to design/clone sgRNAs Highly {{c2::s…
Published	04/10/2024	CRISPRi: {{c1::Gene expression repression (inhibition)}}
Published	04/10/2024	CRISPRa: {{c1::Gene expression activation}}
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Published	04/10/2024	{{c1::CCR5}} is a protein that allows HIV to enter cells.
Published	04/10/2024	CRISPR/Cas9 has been used for deletion of {{c1::CCR5}}, {{c2::Leber congenital amaurosis (LCA)}}, and {{c3::sickle-cell disease }}
Published	04/10/2024	{{c1::Leber congenital amaurosis}} causes loss of eyesight.
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Published	04/10/2024	Various targeting/cutting methods produce DSBs at specific sites in DNA. Those need to be repaired, and typically {{c1::NHEJ}} dominates.
Published	04/10/2024	NHEJ can disrupt a gene, but {{c1::not incorporate specific mutations}}.
Published	04/10/2024	HR can use a provided template to {{c1::incorporate specific mutations.}}
Published	04/10/2024	{{c1::~10,000}} fold compaction of DNA is required to fit DNA into the nucleus.
Published	04/10/2024	Condensation of DNA is achieved through the action of {{c1::histone}} and {{c2::nonhistone chromosomal}} proteins.
Published	04/10/2024	The {{c2::DNA-histone}} complex is called {{c1::chromatin}}.
Published	04/10/2024	The most basic unit of chromatin is the {{c1::nucleosome}}, in which DNA associates with a core particle composed of {{c2::8}} histone proteins.
Published	04/10/2024	Nucleosomes are composed of a {{c1::histone core}} and a {{c1::linker DNA}}.
Published	04/10/2024	{{c1::Histones}} are small, basic, and highly conserved.
Published	04/10/2024	The core histones are {{c1::H2A}}, {{c1::H2B}}, {{c1::H3}}, and {{c1::H4}}. 2 copies of each core histone associate to create a histone {{c2::octamer}…
Published	04/10/2024	The core histones all share a common structural motif called a {{c2::histone fold}}, which has {{c1::three alpha helices and two loops}}.
Published	04/10/2024	The {{c1::N-terminal tails}} on histones are highly {{c2::covalently modified}} and regulate {{c2::nucleosomes}}. The modifications depend on what the…
Published	04/10/2024	{{c1::H3 and H4}} bind to each other to make a dimer, two {{c1::H3-H4}} dimers associate to form an {{c1::H3-H4}} tetramer. {{c2::H2A and H2B}} form d…
Published	04/10/2024	The DNA wraps around the {{c1::outside}} of the histone octamer.
Published	04/10/2024	{{c2::Head to tail}} dimer of the histone fold motifs in H2A-H2B / H3-H4 dimer creates {{c1::3}} DNA binding sites on each dimer
Published	04/10/2024	The path of the DNA around the octamer is not perfectly {{c1::smooth}}.
Published	04/10/2024	Histones interact with the DNA in many places: there are {{c1::142}} H-bonds between the DNA and the proteins of the octamer.
Published	04/10/2024	{{c1::Half}} the H-bonds between DNA and octamer are between the {{c2::AA backbone and the sugar-phosphate backbone}}.
Published	04/10/2024	A few H-bonds between DNA and octamer are to {{c1::minor groove}} atoms. There are also lots of {{c2::hydrophobic interactions and salt bridges}}. Any…
Published	04/10/2024	The DNA is in the {{c2::B-form}} in a nucleosome. It takes {{c1::80}} nucleotides to go around the core once. The DNA contacts a protein of the octame…
Published	04/10/2024	Histone {{c1::H1}} binds the octamer.
Published	04/10/2024	To study nucleosomes biochemically: Digest chromatin with a {{c2::non-specific endonuclease}} like {{c3::DNAse I}} to cleave between the nucleoso…
Published	04/10/2024	By digesting chromatin with a {{c1::low concentration}} of DNAse I, we can see a ladder of nucleosomes.
Published	04/10/2024	{{c1::Light digestion}} leaves {{c2::monomers (200 nte)}}, {{c2::dimers (400 nte)}}, {{c2::trimers (600 nte)}}, etc.
Published	04/10/2024	A little more digestion leaves only {{c1::200 nte}} monomers. A little more digestion trims off the {{c2::spacer}}, leaving {{c2::147 nte}} of DNA. He…
Published	04/10/2024	At very high concentrations, there is enough nuclease to cut the DNA where it is {{c1::least protected}}.
Published	04/10/2024	There are preferred sequences for nucleosome formation. {{c1::AT}} rich sequences bind to octamers better than {{c1::GC}} rich.
Published	04/10/2024	N{{c1::ucleosome phasing}}: one preferred region can guide the positions of neighboring nucleosomes.Once a nucleosome forms on the preferred sequence,…
Published	04/10/2024	{{c1::Non-histone proteins}} can also guide nucleosomes to specific positions. Some transcriptional regulators work this way.
Published	04/10/2024	How can we tell if a particular DNA sequence binds nucleosomes?
Published	04/10/2024	The N-terminal tail of an {{c1::H4}} subunit can interact with the tails of the {{c1::H2A and H2B}} subunits of the next nucelosome.
Published	04/10/2024	N-terminal tail interactions promote {{c1::30 nm fiber formation}} as well as {{c2::histone H1}}.
Published	04/10/2024	{{c1::Histone H1}} binds at where the DNA enters and exits.
Published	04/10/2024	H1 is not part of the octamer, the {{c1::N-and C-terminal tails}} interact extensively with DNA to help compact the 10 nm fiber into the 30 nm fiber.&…
Published	04/10/2024	H1 covers {{c1::linker DNA}} and sets up an angle that is important for further condensation.
Published	04/10/2024	The DNA can unravel fromt he octamer core for a sufficient amount of tiem to permit a {{c1::non-histone protein}} to bind.
Published	04/10/2024	Non-histone proteins can create '{{c1::nucleosome-free zones}}'
Published	04/10/2024	Some nonhistone proteins that recognize specific DNA sequences bind {{c1::more tightly}} than octamer cores.
Published	04/10/2024	Nonhistone proteins may {{c1::phase}} nucleosomes, which can help with optimal compaction; also might generate {{c2::recognition regions}} for other p…
Published	04/10/2024	{{c1::Euchromatin}}: active state{{c1::Heterochromatin}}: silent, highly compacted DNA
Published	04/10/2024	The chromatin is locally {{c1::decondensed}} as the chromatin is {{c1::remodelled}}.
Published	04/10/2024	Areas of decondensed chromatin are called {{c1::hypersensitive sites}}.
Published	04/10/2024	{{c2::Nucleosomes}} don't always stay bound to DNA, and they don't always stay in the same place. They must move during processes like {{c1::DNA repli…
Published	04/10/2024	During transcription, octamer cores appear to be {{c1::repositioned}} without coming off the DNA.
Published	04/10/2024	During DNA replication, {{c1::histones}} are not segregated in any particular way.
Published	04/10/2024	Chromatin is remodelled by large protein complexes called {{c1::Chromatin Remodelling Complex}}.
Published	04/10/2024	{{c2::Histone}} modifying enzymes: {{c1::methylase, acetylase}}
Published	04/10/2024	{{c1::SWI/SNF}}: a remodelling complex first identified in yeast, a multiprotein complex ({{c2::11}} subunits), requries ATP.
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Published	04/10/2024	Cells can change the position and composition of {{c2::octamer cores}}. Some remodelling complexes use the energy of ATP hydrolysis to {{c1::slide}} t…
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Published	04/10/2024	{{c1::H3.3}} has transcriptional activation function.
Published	04/10/2024	{{c1::CENP-A}} has centromere function and kinetochore assembly.
Published	04/10/2024	{{c1::H2AX}} can do DNA repair and recombination.
Published	04/10/2024	{{c1::H2AZ}} can alter gene expression, chromosome segregation
Published	04/10/2024	{{c1::macroH2A}} functions in transcriptional repression, X-chromosome inactivation.
Published	04/10/2024	The major reasons for {{c1::exchanging cores}} are to: introduce {{c2::variant forms}} of core histones {{c2::remove cores}} during transcri…
Published	04/10/2024	The domain that associates with histones in {{c1::SWI/SWF}} is the {{c2::bromodomain}} and {{c2::activates}} gene expression.
Published	04/10/2024	The domain that associates with histones in {{c1::Mi2/NURD}} is the {{c2::chromodomain}} and {{c3::represses}} transcription.
Published	04/10/2024	{{c1::Covalent modificatio}}n of N-terminal tails of the core histones can change their properties. They can change the state of {{c2::condensation}} …
Published	04/10/2024	{{c1::Acetylation}}: occurs on the \(\epsilon\)-amino group of {{c2::lys}}, {{c3::neutralizes}} the positive charge{{c3::weakens interaction}} wi…
Published	04/10/2024	{{c1::HDAC}} enzymes: histone deacetylases, remove acetate groups to turn off transcription
Published	04/10/2024	{{c1::Phosphorylation}}, {{c1::methylation}}, and {{c1::ubiquitination}} can also occur on the tails.
Published	04/10/2024	{{c1::Methylation}} is a mark of inactive chromatin
Published	04/10/2024	{{c1::Acetylation}} is a mark of active chromatin, i.e., DNA that is being transcribed.
Published	04/10/2024	Most histone modifications are thought to be {{c1::reversible}}.
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Published	04/10/2024	The protein domain that binds {{c1::methylated}} lysine is calld a {{c2::chromodomain}} or {{c2::PHD fingers/domains.}}
Published	04/10/2024	The binding of other proteins besides histones to the chromatin may act to set up physical placeholders that have the effect of {{c1::phasing}} the nu…
Published	04/10/2024	Chromodomains generally have a {{c1::pocket}} wherein the methylated lysine can fit.
Published	04/10/2024	The protein domain that binds {{c2::acetylated}} lysine is called a {{c1::bromodomain}}. There are 46 {{c1::bromodomain}}-containing proteins encoded …
Published	04/10/2024	Genes that are fully covered with nucelosomes are relatively {{c1::protected}}. Genes that are actively transcribed are relatively {{c2::sensitive to …
Published	04/10/2024	{{c1::DNAse I hypersensitive sites (DHSs)}} are chromatin regions sensitive to cleavage by the DNAse I.
Published	04/10/2024	DHSs are nucleosome free and are usually associated to {{c1::functional regulatory sequences}}.
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Published	04/10/2024	Transcription is similar to replication: Both processes use a DNA template to incorporate {{c1::nucelotide triphosphates}} into a {{c1::polynucle…
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