[MOLECULAR BIOLOGY] / DNA

translation (RNA → Protein) — Ribosomes — Protein

RNA Dependent RNA Polymerase

• cDNA
  • Definition
    • reverse transcription에 의해 만들어지는 DNA
  • STRUcture
    • Doesn’t have introns
    • cDNA also does not contain any other gDNA that does not directly code for a protein (referred to as non coding DNA).
    • cDNA will only contain genes that are actively being used by a specific cell or tissue at a point in time

ASO

	Enter cell	Enter nucleus	Bind m RNA	Degraded by RNase H
mechanism	adsorptive endocytosis and fluid phase pinocytosis

MOE: 2’-O-methoxyethyl Questions:

• Dose our HDO bind pre-m RNA and degraded by RNAse H inside the nucleus, or does it bind mature m RNA in the cytoplasm?
• Route:
• 0.2 ug/g 되는 dose 를 이미 짖았니?

HDO Structure

		'Parent ASO' (LNA+DNA+LNA)
	cRNA	Ligand

DNA

structure

• neuclotides 들의 long sequence 인 macromolecule , genetic material
  • 항상 5’ end → 3’ end ( i.e. a free 3’ phosphate group)
  • 3’ 의미 : sugar 내에서 carbon atom 의 위치가 세번째

Nucleic acid	Nucleotide	Base	Eg - Purine: A, G - Pyrimidine: T, C
		DEOXYRIBOSE (Sugar)
		phosphate
	Nucleotide	Base	- Purine: A, G Pyrimidine: T, C
		DEOXYRIBOSE (Sugar)
		phosphate
	Nucleotide	Base	- Purine: A, G Pyrimidine: T, C
		DEOXYRIBOSE (Sugar)
		phosphate

DNA	RNA
almost always double-stranded,	almost always single-stranded,

• Reverse Transcription (cDNA Synthesis): The synthesis of DNA from an RNA template

DNA / RNA paired diagram visible: phosphate-deoxyribose/ribose-base nucleotides with 5', 4', 3', 2', 1', OH, H carbon labels; Bases panel Pyrimidines T C / Purines A G for DNA and Pyrimidines U C / Purines A G for RNA; Polynucleotides panel showing two paired strands with T-A, G-C, C-G, A-T rungs (DNA) and a single strand with U, G, C, A (RNA); 5’ end and 3’ end labels.

• Reverse Translation: inferring DNA sequence from the amino acid sequence of a protein.

DNA binding proteins

Histone

		원래 역할	Histone에 chemical modificiation (methylation, phosphorylation acetylation) → the strength of the interaction between DNA and the histones변화 → DNA의 transcription factor에 대한 accessibility 가 변화 → transcription속도가 변화
non-specific DNA-binding proteins	Histone	DNA를 붙잡으면서 Chromatin 의 compact한 구조를 유지
	High-mobility group proteins	bent or distorted DNA에 결합하면서 역시 chromatin 구조 유지
specific DNA-binding proteins	replication protein A	Single stranded DNA에만 붙어 STABILIZE시킴
	Transcription factor	DNA의 특정 Sequence에만 결합	① TF가 RNA polymerase에 결합 → 그 polymerase를 promoter 옆에 붙어 → ↑ transcription ② TF가 Enzyme에 결합 → promoter 근처의 histone을 modify → DNA의 polymerase에 대한 accessibility 가 변화

DNA modifying enzymes

nuclease
ligase
topoisomerase
helicase
polymerase

Disulfide bonds is a covalent bond

Protein a dimer is a macromolecular complex formed by two, usually non-covalently bound

[western blot] http://www.researchgate.net/post/ls_it_possible_to_do_IP_in_non_reducing_conditions Boiling in SDS simply denatures the proteins and breaks any non-covalent bonds such as antigen-antibody bonds. It does not destroy the -ss-bond. For that you need to boil the sample in SDS with DTT or 2-ME. , 요약하면 WB하느라 boil 하면 covalent bond (eg disulfide bond) 는 안 깨지고, non-covalent bond는 깨신다.

1. 만약 activated GBA 가 GBA-GBA dimer라면, 대개 dimer 는 non-covalent bond니까, 깨졌을 거고, 그럼 activated GBA 가 보이는 게 말이 안 됨.
2. 만약 GBA-sapC complex가 covalent bond라면 150kda 에서 sapC 항체에 보이는 게 말이 되고, non-covalent bond 라면 깨졌을 거니까 sapC 항체에 보이는 게 말이 안 됨.

CG islands

The CG island is a short stretch of DNA in which the frequency of the CG sequence is higher than other regions. It is also called the CpG island, where “p” simply indicates that “C” and “G” are connected by a phosphodiester bond.

CpG islands are usually defined as regions with 1) a length greater than 200bp, 2) a G+C content greater than 50%, 3) a ratio of observed to expected CpG greater than 0.6, although other definitions are sometimes used.^[18]

Excluding repeated sequences, there are around 25,000 CpG islands in the human genome, 75% of which being less than 850pb long.^[19] They are major regulatory units and around 50% of CpG islands are located in gene promoter regions, while another 25% lie in gene bodies, often serving as alternative promoters. Reciprocally, around 60-70% of human genes have a CpG island in their promoter region.^[20][21] The majority of CpG islands are constitutively unmethylated and enriched for permissive chromatin modification such as H3K4 methylation. In somatic tissues, only 10% of CpG islands are methylated (?), the majority of them being located in intergenic and intragenic regions.

DNA methylation

Definition

• DNA methylation entails the conversion of cytosine to 5-methylcytosine (5mC), predominantly due to a DNA methyltransferase (DNMT) enzyme transferring a methyl group from S-adenosyl-methionine (SAM) to the 5^th carbon of the ring of cytosine.

Where?

• This conversion is usually found within CpG dinucleotide sites
• occur in intronic, exonic and intergenic regions [12

FUNCTION

(continues below visible crop)

Uncertain Spans

location	text/status	reason
Top central-dogma figure	partial labels at top edge	Only the bottom edge of the figure is in this crop; labels translation (RNA -> Protein), Ribosomes, Protein, RNA Dependent RNA Polymerase are visible but not the upstream transcription portion.
HDO Structure table	row count	Only one populated row (cRNA / Parent ASO (LNA+DNA+LNA) / Ligand) is visible plus several empty rows; full structure of the table requires an adjacent photo.
Nucleic acid table cells	empty Eg cells under the second/third Nucleotide	The right column under the second and third Nucleotide is visually blank; the first nucleotide carries the Purine/Pyrimidine examples.
CpG islands percentages	200bp, 50%, 0.6, 25,000, 850pb, 60-70%, 10%	All values are legible from the body_r05 crops; the citation markers [18] [19] [20] [21] are tiny but consistent with both OCR engines.