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FA13 Biochemistry > Biochem-01-Molec_Cell_Lab > Flashcards

Biochem-01-Molec_Cell_Lab Flashcards

1
Q

Chromatin structure

A

Beads on a string: DNA loops twice around nucleosome (positively-charged histone octamer)

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2
Q

Histones and Nucleosomes

A
  • Positively-charged: Rich with lysine and arginine
  • Nucleosome: Two each of H2A, H2B, H3, H4
  • H1 ties nucleosome beads together; it’s not part of nucleosome core; it helps keep the DNA around the bead
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3
Q

Heterochromatin

A

Highly condensed, transcriptionally inactive, sterically inaccessible

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4
Q

Euchromatin

A

Less condensed, transcriptionally active, sterically accessible

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5
Q

DNA methylation

A
  • Cytosine and adenine are methylated after DNA replication; it allows mismatch repair enzymes to distinguish between old and new strants
  • Methylating a DNA sequence also silences it
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6
Q

Histone methylation

A
  • Inactivates transcritpion of DNA

* {Methylation makes DNA Mute}

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7
Q

Histone acetylation

A
  • Relaxes DNA coiling by reducing the positive charge of histones, allowing for transcription
  • {Acetylation makes DNA Active}
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8
Q

Purines and their general structure

A
  • Adenine and guanine {PURe As Gold}

* 2 rings

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9
Q

Amino acids necessary for purine synthesis

A
  • Glycine
  • Aspartate
  • Glutamine
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10
Q

Pyrimidines and their general structure

A
  • Cytosine, Thymine, Uracil {CUT the PY}

* 1 ring

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11
Q

Structural features of guanine

A

Guanine has a ketone and an amine

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12
Q

Structural features of thymine

A
  • Thymine has a methyl and two ketones

* found only in DNA

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13
Q

Structural features of adenine

A

Adenine has an amine group

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14
Q

Structural features of cytosine

A

Cytosine has a ketone and an amine

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15
Q

Structural features of uracyl

A
  • Deamination of cytosine makes uracyl; [it has two ketones]

* found only in RNA

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16
Q

Hydrogen bonding of bases

A
  • G-C has 3 hydrogen bonds and is thus stronger

* A-T (or A-U) has 2 hydrogen bonds and is thus weaker

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17
Q

Nucleoside vs. Nucleotide

A
  • Nucleoside: base bound to sugar

* Nucleotide: phosphorylated nucleoside

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18
Q

Bond that links nucleotides with other nucleotides

A

3’-5’ phosphodiester bond

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19
Q

Role and synthesis of PRPP (5-phosphoribosyl-1-pyrophosphate)

A
  • It’s an activated pentose that participates in the synthesis and salvage of purines and pyrimidines
  • formed from Ribose 5-P
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20
Q

De novo pyrimidine synthesis

A

• General: make temporary base (orotic acid) and then add sugar and phosphate (PRPP)
• Requires aspartate
1. Carbamoyl phosphate made from glutamine and CO2
2. Aspartate added to carbamoyl phosphate and after a few steps, ortic acid is made
3. Orotic acid is combined with PRPP to make UMP (Uridine 5’-monophosphate); it is phosphorylated to make UDP (or UTP)
4. CTP is made by aminating UTP; glutamine provides the amine
5. UDP is converted to dUDP by ribonucleotide reductase and then converted to dUMP
6. dUMP is converted to dTMP by thymidylate synthase; N5,N10-Methylene THF provides the methyl group

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21
Q

Role of folate in pyrimidine synthesis

A
  1. N5,N10-Methylene THF provides the methyl group that is needed to convert dUMP to dTMP
  2. The product of this reaction is DHF; DHF is reduced to THF by dihydrofolate reductase
  3. THF is converted to N5,N10-Methyliene THF once again
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22
Q

Action of hydroxyurea

A

Inhibits ribonucleotide reductase

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23
Q

Action of 5-FU (5-fluorouracil)

A

Permanently inhibits thymidylate synthase by acting as its substrate and then permanently binding to it (thymidylate synthase converts dUMP to dTMP)

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24
Q

Action of methotrexate (MTX)

A

Inhibits dihydrofolate reductase (which converts DHF to THF)

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25
Q

Action of trimethoprim (TMP)

A

Inhibits bacterial dihydrofolate reductase (which converts DHF to THF)

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26
Q

De novo purine synthesis

A

• General: start with sugar and phosphate (PRPP) and then build the base on top
• Requires aspartate, glycine, glutamine, and THF for de novo synthesis
1. IMP is made from PRPP through a series of steps requiring glutamine, glycine, N10-Formyl-THF, and Aspartate
2. IMP can then be converted to AMP and GMP

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27
Q

Action of 6-MP (6-mercaptopurine)

A

Blocks de novo purine synthesis by inhibiting the first enzyme that adds an amine group to PRPP

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28
Q

Deoyribonucleotide reductase

A

Ribonucleotides are synthesized first and then converted to deoyribonucleotides by this enzyme

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29
Q

Ornithine transcarbamoylase deficiency

A
  • OTC is a key enzyme in the urea cycle to help get rid of ammonia; it requires carbamoyl phosphate and ornithine
  • OTC deficiency leads to an accumulation of carbamoyl phosphate, which is then converted to orotic acid
  • There will also be a hyperammonemia
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30
Q

Orotic aciduria

A
  • Inability to convert orotic acid to UMP due to defect in UMP synthase
  • autosomal recessive
  • Findings: increased orotic acid in urine, megaloblastic anemia (does not improve with administration of vitamin B12 or folic acid), failure to thrive
  • Does not lead to hyperammonemia (OTC eficiency does lead to hyperammonemia with increase orotic acid)
  • treatment: Oral administration of uridine
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31
Q

Adenosine metabolism

A
  1. AMP is converted to Adenosine (no phosphate)
  2. Adenosine is converted to inosine by adenosine deaminase
  3. Inosine gets its sugar removed and become Hypoxanthine
  4. Hypoxanthine is converted to xanthine by Xanthine oxidase
  5. Xanthine is converted to Uric acid by xanthine oxidase
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32
Q

Guanosine metabolism

A
  1. GMP is converted to Guanosine (no phosphate)
  2. Guanosine gets its sugar removed and becomes Guanine
  3. Guanine gets converted to Xanthine
  4. Xanthine is converted to uric acid by xanthine oxidase
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33
Q

Purine salvage

A
  • Adenine can become AMP with the addition of PRPP; this is catalyzed by APRT (Adenine phosphoribosyltransferase)
  • Guanine can become GMP with the addition of PRPP; this is catalyzed by HGPRT (Hypoxanthin-guanine phosphoribosyltransferase)
  • Hypoxanthine can become IMP with the addition of PRPP; this is catalyzed by HGPRT
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34
Q

Adenosine deaminase deficiency

A
  • One of the major causes of SCID (Severe Combined Immunodeficiency Disease), which affects kids
  • autosomal recessive
  • Leads to an excess of ATP and dATP, which then inhibit ribonucleotide reductase activity
  • reduced DNA synthesis disproportinately affects lymphocytes, leading to decreased lymphocyte count
  • first disease to be treated by experimental human gene therapy
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35
Q

Lesch-Nyhan syndrome

A
  • Absence of HGPRT leads to defective purine salvage, excess uric acid production, and de novo purine synthesis {He’s Got Purine Recovery Trouble}
  • X-linked recessive
  • findings: retardation, self-mutilation, aggression, hyperuricemia, gout, choreoathetosis
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36
Q

Features of the gentic code

A
  • Unambiguous
  • Redundant/degenerate (exceptions: Methionine and tryptophan encoded by only one codon)
  • Comaless/nonoverlapping (exception: some viruses)
  • Universal: genetic code is conserved throughout evolution (exception of mitochondria in humans)
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37
Q

Silent mutation

A

Same amino acid (often due to change in 3rd position–tRNA wobble)

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38
Q

Missense mutation

A
  • Changed amino acid

* Conservative mutation: new amino acid is similar chemically

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39
Q

Nonsense mutation

A

Results in early stop codon

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40
Q

Frameshift mutation

A

Results in misreading of all nucleotides downstream; can lead to truncated, nonfunctional protein

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41
Q

Origin of replication

A
  • Consensus sequence where DNA replication begins

* may be single (prokaryotes) or multiple (eukaryotes)

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42
Q

Replication fork

A

Y-shaped region on DNA template where leading and lagging strands are synthesized

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43
Q

Helicase

A

Enzume that unwinds DNA template at replication fork

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44
Q

Single-stranded binding proteins

A

Prevent strands from reannealing

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45
Q

DNA topoisomerases

A
  • Cerate a nick in the helix to relieve supercoils created during replication
  • Fluoroquinolones inhibit DNA gyrase (prokaryotic topoisomerase II)
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46
Q

Enzyme inhibited by Fluoroquinolones

A

DNA gyrase (prokaryotic topoisomerase II)

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47
Q

Primase

A

Makes an RNA primer on which DNA polymerase III can initiate replication

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48
Q

DNA polymerase III

A
  • Prokaryotic only
  • Elongates leading strand by adding deoxynucleotides to the 3’ end
  • Has 3’-5’ exonuclease activity for proofeading newly-added nucleotides
  • Elongates the lagging strand until reaching a primer
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49
Q

DNA polymerase I

A
  • Prokaryotic only

* Degrades the RNA primer (in 5’-3’ fashion) and replaces it with DNA

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50
Q

DNA ligase

A

Catalyzes phophodiester bond of Okazaki fragments

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51
Q

Telomerase

A
  • Adds DNA to 3’ ends of chromosomes

* This prevents the loss of genetic material with every duplication

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52
Q

Nucleotide excision repair

A
  • for bulky helix-distorting lesions, usually UV DNA damaged (thymine dimers, etc.)
  • Endonucleases remove the oligonucletide containing the damaged bases and then DNA Polymerase and ligase fill and release the gap
  • mutated in xeroderma pigmentosum; this prevents the repair of pyrimidine dimers due to UV light exposure
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53
Q

Enzymes that are mutated in xeroderma pigmentosum

A

Nucleatide excision repair enzymes

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54
Q

Base excision repair

A
  • For small, non-bulky lesions
  • Glycosylases recognize and remove damaged bases themselves, leaving an apurinic/apyrimidinic site; Apurinic/apyrimidinic endonuclease cuts DNA at these sites, and then the empty sugar is replaced by DNA polymerase
  • This is an important mechanism in the repair of spontaneous/toxic deamination
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55
Q

Mismatch repair

A
  • Newly-synthesized mismatched pairs are recognized, removed, and the gap is filled and resealed
  • This mechanism is mutated in hereditary nonpolyposis colorectal cancer (HNPCC)
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56
Q

DNA repair mechanism affected in hereditary nonpolyposis colorectal cancer (HNPCC)

A

Mismatch repair

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57
Q

Nonhomologous end joining

A
  • Joins double-stranded breaks of DNA fragments; no homology is required
  • mutated in ataxia telangiectasia
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58
Q

Process mutated in ataxia telangiectasia

A

nonhomologous end joining

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59
Q

Direction of DNA and RNA synthesis

A
  • 5’ to 3’

* a 3’ OH is required in the chain to attack the triphosphate bond is in the 5’ end of the incoming base

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60
Q

Direction of protein synthesis

A
  • N-terminus to C-terminus

* mRNA is read 5’ to 3’

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61
Q

Types of RNA

A
  • rRNA is the most abundant {rampant}
  • mRNA is the longest {massive}
  • tRNA is the smallest {tiny}
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62
Q

mRNA start codon

A
  • AUG (rarely GUG or others in mitochondria or bacteria) {AUG inAUGurates protein synthesis}
  • in eukaryotes the start codon coes for methionine, and it may be removed before translation is completed
  • in prokaryotes it codes for formylthemionine (f-met)
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63
Q

mRNA stop codons

A
  • UGA {U Go Away}
  • UAA {U Are Away}
  • UAG {U Are Gone}
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64
Q

Promoter

A
  • Site upstream of gene location where RNA polymerase and other transcritpion factors bind to DNA
  • AT-rich sequence with TATA and CAAT boxes
  • mutations here lead to dramatic decrease in gene transcription
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65
Q

Enhancer

A
  • Site of DNA that alters gene expression by binding transcritpion factors
  • may be located close, far, or within (intron) a gene
66
Q

Silencer

A
  • Site of DNA where negative regulators (repressors bind)

* may be located close, far, or within (intron) a gene

67
Q

RNA polymerases in eukaryotes

A
  • RNA pol I makes rRNA
  • RNA pol II makes mRNA
  • RNA pol III makes tRNA
  • They have no proofreading function, but they can initiate chains
  • RNA pol II opens DNA at promoter site
  • alpha-amanitin, found in Amanita phalloides (death cap mushrooms) inhibits RNA polymerase II and causes severe hepatotoxicity
68
Q

What does Amanita phlloides (deathcap mushrooms) inhibit

A

• alpha-amanitin inhibits RNA polymerase II and causes severe hepatotoxicity

69
Q

RNA polymerase in prokaryotes

A

• A single RNA polymerase mlutisubunit complex makes all 3 types of RNA

70
Q

Initial mRNA transcript (before any processing)

A
  • heterogeneus nuclear RNA, hnRNA

* hnRNA that is destined for translation is called pre-mRNA

71
Q

RNA processing

A
  • occurs in eukaryotes
  • occurs in the nucleus after transcription
  • Capping on 5’ end with 7-methylguanosine cap
  • Polyadenylation on 3’ end (~ 200 A’s); does not require a transcript
  • Splicing of introns
  • Processed transcript is called mRNA
  • only processed mRNA is exported out of th enucleus
72
Q

Steps of RNA splicing

A
  1. Primary transcript combines with snRNPs and other proteins to form the spliceosome
  2. Lariat (looped) intermediate is generated by binding one end of intron to the branch point
  3. Lariat is released and the two exons are joined
    • Lupus patients make antibodies to spliceosomal snRNPs
73
Q

Introns vs. Exons

A
  • Introns are intervening sequences that stay in the nucleus
  • Exons are expressed and exit the nucleus
  • Alternative splicing can occur
74
Q

tRNA structure

A
  • 75-90 nucleotides, secondary structure, cloverleaf form, 3’ aminoacyl end and anticodon end
  • CCA at 3’ end binds the amino acid {CCA: Can Carry Aminoacids}
75
Q

tRNA charging

A
  • aminoacyl-tRNA synthetase scrutinizes amino acid before and after binding
  • ATP is required for binding
76
Q

tRNA wobble

A

• due to degeneracy of genetic code, acute base pairing is usually only required for the first 2 nucleotides of a codon, and the “wobble” (third) position may differ

77
Q

Initiation of protein translation

A
  1. initiation factors (eukaryotic Ifs) and GTP help assemble the 40S ribosomal subunit with the initiator tRNA (which goes to the P site)
  2. the Ifs are released after GDP hydrolysis when the mRNA and the ribosomal unit assemble with th ecomplex
78
Q

Elongation of protein translation

A
  1. New tRNA binds to A site (requires EF-tu and GTP)
  2. Peptidyl transferase (an rRNA ribozyme) ctalyzes the formation of the peptide bond formation and the growing peptide is now on the tRNA at the A site
  3. Ribosome translocates 3 nucleotides toward 3’ end, and the peptidyl tRNA is now at the P site (requires GTP)
79
Q

Termination of protein translation

A

Stop codon is recognized by release factor and the completed protein is released from the ribosome

80
Q

Eukaryote ribosome size

A

40S + 60S -> 80 S {Eukaryotic is Even}

81
Q

Prokaryotic ribosome size

A

30S + 50S -> 70S {PrOkaryotic is Odd}

82
Q

roles of ATP and GTP in protein synthesis

A
  • ATP is important for charging the tRNA {ATP is for Activation}
  • GTP is important for initiation and elongation (translocation) of polypeptide {GTP is for Gripping and Going places}
83
Q

Action of aminoglycosides

A

Bind to 30S unit and inhibit formation of the initiation complex; cause misreading of RNA

84
Q

Tetracyclines

A

Bind 30S and block aminoacyl tRNA from entering the acceptor site

85
Q

Chloramphenicol

A

Binds 50S and inhibits peptidyl transferase

86
Q

Macrolides

A

Bind 50S and prevent release of uncharged tRNA after it has donated its amino acid

87
Q

Posttranslational modifications of polypeptides

A
  • Trimming: removal of N- or C0terminal propeptides from zymogens to generate mature proteins
  • Covalent alterations such as phosphorylation, glycosylation, hydroxylation, methylation, and acetylation
  • Proteasomal degradation: Attachment of ubiquitin to defective proteins to tag them for breakdown
88
Q

Phases of the cell cycle

A
  1. G1 G0 {G for Gap or Growth}
  2. S Phase
  3. G2 {S for Synthesis}
  4. Mitosis: Prophase, metaphase, anaphase, telophase
    • Interphase: G1, S, G2
89
Q

CDKs (Cyclin dependent kinases)

A
  • When activated by cyclins, they phosphorylate other proteins necessary to proceed through the cell cycle
  • They are constitutively found in the cell
90
Q

Cyclins

A
  • Regulatory proteins that control cell cycle events by activating CDKs
  • They are phase-specific
91
Q

Cyclin-CDK complexes

A

Must be both activated and inactivated for cell cycle to progress

92
Q

p53

A
  • Tumor suppressor that inhibits the G1-to-S progression
  • activated by various stressors and leads to cell cycle arrest, DNA repair, or apoptosis
  • kept sequestered by MDM2 protein, and a conformational change and phosphorylation activate it
  • Mutations result in unrestrained cell division
93
Q

Rb

A
  • Tumor supressor that inhibits the G1-toS progression
  • In its hypophosphorylated state, it binds transcription factors, preventing DNA replication
  • When phosphorylated, it becomes inactive and the cell can go through S phase
  • mutations result in unrestrained cell division
94
Q

Permanent cell type

A
  • Cell remain in G0 and regenerate from stem cells

* examples: neurons, skeletal and cardiac muscle, RBCs

95
Q

Stable/quiescent cell type

A
  • Cells enter G1 from G0 when stimulated

* examples: hepatocytes, lymphocytes

96
Q

Labile cell type

A
  • Cells never go to G0; they divide rapidly with a short G1

* examples: Bone marow, gut epithelum, skin, hair follicles, germ cells

97
Q

Role of rough endoplasmic reticulum

A
  • Site of synthesis of secretory protein
  • site of N-linked oligosaccharide addition to proteins
  • mucus-secreting goblet cells of small intestine and antibody-secreting plasma cells are rich in RER
98
Q

Nissle bodies

A
  • Rough endoplasmic reticulum in neurons

* synthesizes enzymes (e.g., choline acetyltransferase, which makes Ach) and peptide neurotransmitters

99
Q

Free ribosomes

A

synthesize cytosolic and organellar proteins

100
Q

Smooth endoplasmic reticulum

A
  • site of steroid synthesis
  • site of detoxification of drugs and poisons
  • liver hepatocytes and steroid-producing cells of adrenal cortex are rich in SER
101
Q

Role of golgi

A
  • distribution center for proteins and lipids from the ER to the vesicles and plasma membrane
  • modifies N-oligosaccharides on asparagine
  • adds O-oligosaccharides on serine and threonine
  • adds mannose-6-phosphate to proteins, which tags them for delivery to lysosomes
  • cis face receives vesicles from ER; trans face sends vesicles to endosomes
102
Q

Role of endosomes

A
  • sorting centers for material from outside the cell or from the golgi
  • they send things to lysosomes, membrane, or golgi
103
Q

I-cell disease (inclusion cell disease)

A
  • Lysosomal storage disorder due to a failure of addition of mannose-6-phosphate to lysosomal proteins (enzymes are instead secreted)
  • results in coarse facial features, clouded corneas, restrited joint movement, and high plasma levels of lysosomal enzymes
  • often fatal in childhood
104
Q

COPI

A
  • vesicular trafficking protein (lines outside of membrane buds)
  • lines vesicles going retrograde (backwards; trans to cis) on golgi
  • lines vesicles going from Golgi to ER
105
Q

COPII

A
  • vesicular trafficking protein (lines outside of membrane buds)
  • lines vesicles going anterograde (forwards cis to trans) on golgi
  • lines vesicles going from ER to golgi
106
Q

Clathrin

A
  • vesicular trafficking protein (lines outside of membrane buds)
  • lines vesicles going from trans-golgi to lysosomes
  • lines vesicles going from plasma membrane to endosomes (receptor-mediated endocytosis)
107
Q

Peroxisome

A

membrane-enclosed organelle involved in ctabolism of very long fatty acids and amino acids

108
Q

Proteasome

A

Barrel-shaped protein complex that degrades (damaged or unnecessary) proteins tagged with ubiquitin

109
Q

Microtubule structure and function

A
  • largest component of cytoskeleton; they are “support beams”
  • Cylindrical structure composed of polymerized dimers of alpha- and beta- tubulin; these dimers make protofilaments and 13 of these make a cylinder
  • tubulin dimers have GTP bound (become unstable when it is GDP) and build from the negative end to positive end of microtubule; they grow slowly and collapse quickly
  • centrosomes are the main microtubule organizing center
  • Incorporated into flagella, cilia, mitotic spindles
  • involved in cellular transport: Dynein motors go retrograde (toward negative end), and Kinesin motors go anterograde (toward positive end)
110
Q

Mebendazole/thiabendazole

A

Antihelminthic drug that acts on microtubules

111
Q

Griseofulvin

A

Antifulgan drug that acts on microtubules

112
Q

Vincristine/vinblastine

A

Anticancer drug that acts on microtubules

113
Q

Paclitaxel

A

Anti-breast cancer drug that acts on microtubules

114
Q

Colchicine

A

Anti-gout drug that acts on microtubules

115
Q

Chediak-Higashi syndrome

A
  • mutation in the lysosomal trafficking regulator gene (LYST), whose product is required for microtubule-dependent sorting of endosomal proteins into late multivesicular endosomes
  • reesuts in recurrent pyogenic infections, partial albinism, and peripheral neuropathy
116
Q

Cilia

A
  • Cilia cytoskeleton (axoneme) is composed of 9 + 2 arrangement of paired microtubules (9 + 0 for nonmotie cilia)
  • Axonemal dynein: ATPase that links peripheral 9 doublets and causes bending by sliding of doublets
117
Q

Kartagener’s syndrome (primary ciliary dyskinesia)

A
  • Immotile cilia due to a dynein arm defect
  • results in: male infertility (sperm are immotile), decreased female fertility, bronchiectasis, recurrent sinusitis (bacteria not pushed out)
  • also associated with situs inversus
118
Q

Role of Actin and myosin (microfilaments) in cytoskeleton

A
  • Present in microvili
  • Are responsible for muscle contraction
  • Important in cytokinesis (contractile ring)
  • Present in adherens junctions
  • are the “beams” in cytoskeleton
119
Q

Microfilament structure

A
  • Actin present as a monomer is known as G-actin
  • when G-actin binds ATP, it can form filaments (double helix) which is termed F-actin; ATP hydrolysis destabilizes the polymer
  • actin monomers point the same way, and the side with the ATP-binding site exposed is termed the (-) end
  • actin is usually nucleated at the plasma membrane (unlike microtubules)
  • Myosin’s head domain can bind to actin filaments and use ATP hydrolysis to walk along it
120
Q

Role of microtubules incytoskeleton

A
  • Movements of the cell cytoskeleton: cilia, flagella

* Trafficking inside the cell: mitotic spindle, axonal trafficking, centrioles

121
Q

Role of intermediate filaments in cytoskeleton

A
  • Provide structure; are the “ropes” that provide tension to the cytoskeleton
  • Present in desmosomes
  • Many different types: vimentin, desmin, cytokeratin, lamins, glial fibrillary acid proteins (GFAP), neurofilaments; Specific intermediate filaments vary by tissue type
  • Lamins make the fibrous protein network tha tlines the inside of the nuclear membrane
  • We can stain for intermediate filaments and determine the type of cell
122
Q

Intermediate filament structure

A
  • Monomers are an elongated fibers
  • Two monomers formed a coiled dimer
  • two dimers aggregate in an anti-paralel fashion to make a tetramer
  • 8 tetramers aggregate laterally to form a sheet that is then supercoiled into a tight bundle
123
Q

Cell type that has vimentin intermediate filament (and can be stained for it)

A

Connective tissue

124
Q

Cell type that has desmin intermediate filament (and can be stained for it)

A

Muscle

125
Q

Cell type that has cytokeratin intermediate filament (and can be stained for it)

A

Epithelial cels

126
Q

Cell type that has GFPAP intermediate filament (and can be stained for it)

A

Neuroglia

127
Q

Cell type that has neurofilaments intermediate filament (and can be stained for it)

A

Neurons

128
Q

Sodium potassium pump

A

For every ATP: 3 Na+ pumped out, 2 K+ pumped in

129
Q

Ouabain

A

inhibits the sodium potassiump pump by binding to the K+ site

130
Q

Mechanism of action of cardiac glycosides (e.g., digoxin)

A
  1. Directly inhibit the sodium potassium pump
  2. This indirectly inhibits Na+/Ca++ exchange
  3. More calcium stays intracellularly, leading to increased cardiac contractility
131
Q

Collagen function

A
  • Most abundant protein in the human body
  • It organizes and strengthens extracellular matrix
  • is extensively modified posttranslationally
  • 4 types {Be (So Totally) Cool, Read Books}
132
Q

Collagen type I

A
  • Most common type of collagen (90%)
  • found in Bone, skin, tendon, dentin, fascia, cornea, late wound repair
  • Defective in osteogenesis imperfecta
  • {type I: bONE}
133
Q

Collagen type II

A
  • Found in cartilage (including hyaline), vitreus body, nucleus pulposus
  • {Type II: carTWOlage}
134
Q

Collagen type III

A
  • Found in reticulin - skin, blood vessels, uterus, fetal tissue, granulation tissue
  • deffective in Ehlers-Danlos syndrome, vascular type {ThreE D)
135
Q

Collagen type IV

A
  • Found in basement membrane or basal lamina
  • defective in alport syndrome
  • {Type IV: under the floor (basementmembrane)
136
Q

Collagen type VII

A

Forms anchoring fibrils in dermoepithelial junction

137
Q

Collagen synthesis

A
  1. Synthesis (RER): Translation of collagen alpha chains (preprocollagen); alpha chains are usually Gly-X-Y (X and Y are proline or lysine)
  2. Hydroxylation (ER): Hydroxilation of proline and lysine residues requires vitamin C (deficiency leads to scurvy)
  3. Glycosylation (ER): Glycosylation of hydroxylysine residues residues and formation of procollagen triple helix via hydrogen and disulfide bonds (problems forming triple helix lead to osteogenesis imperfecta)
  4. Exocytosis of procollagen into intracellular space
  5. Proteolytic processing: cleavage of disulfide-rich terminal regions, transforming procollagen into insoluble tropocollagen
  6. Cross-linking: Staggered tropocollagen molecules are reinforced by covalent cross-linkage of lysine and hydroxylysine (mediated by lysyl oxidase, which conatains Cu++); this makes collagen fibrils (problems with cross-linking lead to Ehlers-Danlos syndrome)
138
Q

Osteogenesis imperfecta

A
  • Genetic bone disorder (brittle bone disease) caused by a variety of gene defects
  • Most commonly it’s autosomal dominant; incidence is 1:10,000
  • Defect of type I collagen that prevents the formation of procollagen triple helix
  • Multiple fractures with minimal trauma (even during birth)
  • Blue sclera (due to translucency of connective tissue over the choroidal veins)
  • Hearing loss (abnormal middle ear bones)
  • Dental imperfections due to lack of dentin
  • May be confused with child abuse
139
Q

Ehlers-Danlos syndrome

A
  • Defect in collagen synthesis; can be due to problems cross-linking collagen fibrils
  • Leads to: hyperextensible skin, tendency to bleed (easy bruising), and hypermobile joints
  • can be associated with joint dislocation, berry aneurysms, organ ruputure
  • There are 6 types, with variable severity and variable inheritance (autosomal dominant or recessive)
  • Type III collagen is affected in vascular type (type 4 ehlers-danlos)
  • Type I and V collagen affected in classical type (type 1 and 2 ehlers danlos)
  • May be confused with Marfan’s syndrome, which is a defect in fibrillin, not collagen
140
Q

Alport syndrome

A
  • due to a variety of Defects that result in abnormal type IV collagen
  • common form is x-linked recessive
  • Characterized by progressive hereditary nephritis, deafness, ocular disturbances (Type IV collagen is an important structural component of the basement membrane of kidney, ears, and eyes)
141
Q

Scurvy

A

Vitamin C deficiency leads to problems hydroxylating residues on preprocollagen alpha chains

142
Q

Structure and function of elastin

A
  • Stretchy protein within skin, lungs, large arteries, eastic ligaments, vocal cords, ligamenta flava (which connect vertebral lamina)
  • Rich in proline and glycine in their nonhydroxylated forms
  • Elastin is made by cross-linking tropoelastin extracellularly (which is what gives it its elastic properties)
  • Elastic fiber is made up of elastin supported by a fibrillin scaffolding
  • It is broken down by elastase, which is inhibited by alpha-1-antitrypsin
143
Q

Marfan’s syndrome

A

Caused by a defect in fibrillin

144
Q

Emphysema

A

Can be caused by an alpha1-antitripsin deficiency, which results in excess elastase activity

145
Q

Cause of wrinkles

A

Reduced collagen and elastin production with aging

146
Q

Steps of PCR

A
  1. Denaturation of DNA
  2. Annealing of primers
  3. Elongation by heat-stable DNA polymerase
147
Q

Agaraose gel electrohporesis

A

Used for size separation of PCR products and comparison against DNA ladder; smaller molecules travel further

148
Q

Blotting procedure

A
  1. Run sample on gel
  2. transfer sample from gel to filter
  3. Used probe to assess presence of specific sample
149
Q

Southern blot

A

DNA

150
Q

Northern blot

A

RNA

151
Q

Western blot

A

Protein

152
Q

Southwestern blot

A

Identified Dna-binding proteins using oligonucleotide probles

153
Q

Microarrays

A
  1. Nucleic acid sequences arranged in grids
  2. DNA or RNA probes are hybridized
  3. Scanner detects amount of complementary binding
    • Used to profile gene expression levels of thousdands of genes simultaneously
    • able to detect single nucleotide polymorphisms (SNPs) for genotyping, forensic analysis, disease predisposition, cancer mutations, genetic linkage analysis
154
Q

Enzyme-linked immunosorbent assay (ELISA)

A
  • Indirect ELISA: use a test antigen to test for the presence of an antibody in the patient’s blood; a second antibody coupled to a color-generating enzyme is used to detect the first antibody
  • Direct ELISA: Uses a test antibody coupled to a color-generating enzyme to see if a specific antigen is present in the patient’s blood
  • Sensitivity and specificity approach 100%, but false-positive and false-negative results can still occur
155
Q

Fluorescence in situe hibrydization (FISH)

A
  • Fluorescent DNA or RNA probe binds to specific gene site on chromosomes
  • Used for specific localization of genes and direct visualization of anomalies (e.g., microdeletions)
156
Q

Cloning method

A
  1. Isolate eukaryotic mRNA
  2. use reverse transcriptase to make cDNA
  3. Insert cDNA into bacterial plasmids (containing antibiotic resistance genes)
  4. Surviving bacteria on antibiotic mediom produce cDNA library
157
Q

Transgenic strategies in mice

A
  • random insertion of gene into mouse genome
  • targeted insertion or deletion of a gene through homologous recombination with mosue gene
  • knock-out: removing a gene
  • knock-in: inserting a gene
158
Q

Cre-lox system

A

Can inducibly manipulate genes using a controlled expression of Cre, which then acts on loxP sites

159
Q

RNA interference (RNAi)

A

dsRNA complementary to the mRNA of interest promotes the degradation of target mRNA, knocking down gene expression

160
Q

Karyotyping

A
  • Chromosomes in metaphase are stained, ordered,and numbered according to morhpology, size, arm-length ratio, and banding pattern
  • Can be performed on blood, bone marrow, amniotic fluid, or placental tissue
  • Used to diagnose chromosomal imbalances (autosomal trisomies, sex chromosome disorders, etc.)

FA13 Biochemistry (3 decks)

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