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Exam 3- Venk Flashcards

1
Q

Describe eukaryotic genome, genes, and its overall features

A

Human genome is packaged in 22 pairs of different chromosomes (Autosomes) and 2 sex chromosomes. Sex in male XY and female XX

Homologous chromosomes are pairs of which one comes from father and one comes from mother

Chromosome 1 is the longest and decreases in size so that chromosome 22 is the smallest

  • Total size of genomic DNA is ~ 3 billion bp’s. ~ 23,000 protein coding genes (less than 1.5% of total genome)
  • Bulk of DNA corresponds to introns (some are regulatory), some are RNA genes: (non-protein coding tRNA, rRNA, MiRNA, SnRNA).
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2
Q

Describe the features of mitochondrial genome

A

Circular DNA
Consists of ~16,569 base pairs comprising only 37 genes
These 37 genes encode 13 different proteins, 22 tRNAs, and 2 rRNAs
Mitochondrial transcriptional and translational machinery are similar to prokaryotic system

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3
Q

Describe human karyotype, its use in genetic counseling and disease prognosis

A

A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. Visualize them in metaphase of mitosis. It is a simple way to identify gross chromosomal irregularities. Down syndrome, for example, is very simple to see as an extra 21st chromosome

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4
Q

What is trisomy 21?

A

Down syndrome

Due to an extra chromosome 21

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5
Q

Describe the process of MicroRNA (MiRNA) synthesis, its transport and it’s role in gene silencing.

A

1- primary MiRNA is cleaved into pre-MiRNA by RNAases called Drosha and Pasha inside the nucleus
2- the pre-MiRNA is then transported by a nuclear membrane bound protein called RAN-GTP.exportin5
3- once in the cytoplasm, the pre-MiRNA is diced up into short miRNA duplex by the Dicer protein, a type of RNAase
4- From the duplex, the single stranded anti-sense strand of the MiRNA associates with RNA induced silencing complex (RISC)
5- MiRNA along with RISC can silence the expression of the protein by complementarily binding to either 5’-UTR or 3’-UTR of mRNA
- if it binds to 5’UTR of the mRNA it can directly suppress the translation initiation process
- if it binds to 3’-UTR it can indirectly bend over and affect the translation process

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6
Q

M1 and M2 macrophages, and how it is derived from a common progenitor?

A

M1 macrophages: activated classically, typically by IFN-gamma or lipopolysaccharide (LPS)
- MiR125b, MiR155, MiR223

M2 macrophages: alternatively activated, by exposure to few cytokines such as IL-4, IL-10, or IL-13
- MiRNA223

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7
Q

Properties of M1 and M2 macrophages

A

M1 macrophages: Cytotoxicity, Tissue Injury
- M1 macrophage produce pro-inflammatory cytokines and phagocytize microbes. M1 initiate an immune response. M1 macrophages produce NO or ROS to kill the engulfed bacteria that got phagocytosed

M2 macrophages: Tissue Repair
- M2 macrophages produce either polyamines to induce proliferation or proline to induce collagen production. These macrophages are associated with wound healing and tissue repair. There are 3 subtypes of M2 macrophages: M2a, M2b, and M2c

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8
Q

Describe morula and blastula of embryo during embryogenesis

A

Morula —> Blastocyst —> tissue/organ development

Morula: is an early stage of embryonic cell mass that can differentiate into any cell type (Totipotent)

Blasocyst: a pluripotent cell mass which contains ectoderm, mesoderm, and endoderm which differentiate into specific organs

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9
Q

Define Totipotent, Pluripotent and Multipotent with examples

A

Totipotent: can differentiate into any cell type

  • can differentiate into all cell types in the body and placenta
  • ex. Morula

Pluripotent: differentiates into specific organs

  • can differentiate into cell types in the body
  • 3 germ layers ex. Blastula

Multipotent: capable of differentiating into different tissue types (within a specific organ)
- Ex. Layers of ectoderm, mesoderm, and endoderm

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10
Q

Describe the 3 germ layers of blastocyst and explain the types of cell that can be derived from each.

A
  • Ectoderm into skin and CNS
  • Mesoderm into blood and muscle cells
  • Endoderm into tissues like pancreas and liver

The ectoderm, mesoderm, and endoderm cells are multi-potent that are capable of differentiating into specific organs

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11
Q

Describe general spliceosome (splice complex) of an mRNA transcript.

A

A spliceosome is a large and complex molecular machine found primarily within the nucleus of eukaryotic cells.
- Ribonucleoprotein (RNP) with several SnRNAs and a splice regulator protein (SR-protein)

The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript

Splice Regulator can be a silencer or Enhancer and is part of 3’ of Exon 1 or 5’ of Exon 2. Depending on whether to have splicing or not the enhancer or repressor of splicing will takeover

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12
Q

Describe in detail the mechanism of alternate splicing and its advantage

A

Alternative splicing: alternative patterns of pre-mRNA splicing that produced different mature mRNAs containing various combinations of Exon’s from a single precursor of mRNA.

DNA is transcribed into a messenger mRNA template by a process called transcription. However, in eukaryotes, before the mRNA can be translated into proteins, non-coding portions of the sequence, called introns, must be removed by protein-coding parts, called exons, joined by RNA splicing to produce a mature mRNA

Benefit: allows many different transcripts to be made from same stretch of genes. Plays a role in the regulation of normal physiological functions such as immunity

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13
Q

What are some diseases that can manifest due to splicing defects of mRNA?

A

implicated in certain diseases

Lung Cancer: Let-7; RAS (small GTPase, controls growth)
B-cell Lymphoma: MiR-17-92; E2F1 (transcription factor)
B-cell Lymphoma: MiR-155; hAT1R
Diabetes: MiR-375; myotrophin
CLL (chronic lymphocytic leukemia): MiR-15 & MiR-16; BCL-2 (controls apoptosis)

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14
Q

What are splice regulator proteins?

A

Silencer or enhancers

- binding to splice complex either preventing or promoting splice complex to bind to DNA

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15
Q

What are the functions of splice regulators?

A

Regulate alternative splicing
Splice regulators can be a silencer or enhancer and is part of 3’ of Exon 1 or 5’ of exon 2. Depending on whether to have splicing or not the enhancer or repressor of splicing will takeover

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16
Q

Describe the role of MiRNA in various diseases?

A

MiRNA inhibits translation of specific genes. Defect in MiRNA either will have upregulation of that gene which can cause diseases like cancer

MiRNAs with Hypermethylated promoters —> decreased activity of MiRNAs —> overexpression of genes in a cancer type

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17
Q

What are the two mechanisms to modify histones at the epigenetic level?

A

Methylation and acetylation

  • methylation in general suppresses gene expression
  • acetylation activates gene expression
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18
Q

What are CpG islands?

A

In addition to histone methylation, upstream to DNA promoter element there are CpG repeats (cytosine followed by a guanine) called CpG islands that are often heavily methylated which silences the gene expression

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19
Q

Describe the mechanism of epigenetic modification versus the chemical basis of chromatin remodeling

A

Epigenetic modifications - methylation and acetylation

Chromatin remodeling - at the level of epigenetic. Heterochromatins heavily methylated and in highly condensed form. Euchromatin acetylated making neg charges of phosphodiester backbone of DNA and histone acetyl to repel each other.
- this allows the euchromatin to become loose for easy access for TF’s and RNA polymerase II

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20
Q

What are DNA repair enzymes?

A

DNA repair enzymes are enzymes that recognize and correct physical damage in DNA, caused by exposure to radiation, UV light or radioactive oxygen species
- recognition and repair of damaged DNA

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21
Q

Describe the mechanism of Lig4 versus cancer

A

In cancer, DNA repair genes have frequently hypermethylated promoters which allows overexpression of hundreds of thousands of genes in a cancer type. And microRNA’s with hypermethylated promoters may be allowing overexpression of hundreds of thousands of genes in a cancer type.

Promoter hypemethylation of LIG4 occurs in 82% of colorectal cancers. Lig4 is an ATP dependent DNA ligase that joins double stranded breaks during non-homologous end joining of the DNA double-strand breaks as a part of the repair mechanism. If it is hypermethylated, wont be made and repair won’t occur

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22
Q

Describe ELISA (Enzyme Linked ImmunoSorbent Assay)

A

Technique:
1. Cell extracts are placed in a well plate and are allowed to bind tightly to the plate (coating) by overnight incubation with coating buffer
2. Unattached samples are removed using phosphate buffered saline (PBS)
3. Samples reacted with primary antibody selective to the protein of interest (raised in rabbit for ex.)
4. Plates washed with PBS to remove unbound primary antibody
5. Secondary antibody is then added and conjugated to the enzyme (raised in goat for example)
6. Plates washed with PBS afer several hours to remove unbound primary antibody
7. Protein-primary antibody-secondary antibody complex is reacted with a substrate specific to the enzyme of the secondary antibody
A. Substrate is PNP and is cleaved into a yellow colored product (PN)
8. Intensity of color is proportional to amount of protein (antigen) bound to plate
Medical use: Several samples from different patients can be analyzed for particular protein of interest. Hapten like steroid or small peptides can also be detected and quantified
1. Hapten- small protein conjugated molecule

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23
Q

Flow cytometry

A

Technique:

  1. Cells are reacted with a primary antibody conjugated to a fluorescents labeled dye (green or red)
  2. The cells are then sorted through a cell sorter and shined with a laser beam to see the dyes
  3. Red and green cells are separated

Medical use: separation and sorting of cell types and its quantification
Ex. If sample is from a normal and an HIV infected pt one can sort the CD4 and CD8 positive cells

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24
Q

Karyotyping

A

Technique:

  1. Specific stains are used for metaphase chromosomes
  2. Chromosome 1 (the longest) to chromosome 22 (the shortest) can be arranged in order of its length
  3. In abnormal cases the karyotype would be different compared to normal
  4. Samples from placenta, blood, amniotic fluid, bone marrow can be used for karyotyping

Medical Use:
Chromosomal abnormalities like trisomies, deletion, insertion, and translocation can be located

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25
Fluorescence In Situ Hybridization (FISH)
Technique: 1. Fluorescent dye tagged onto a DNA or RNA probe specific to each chromosome is allowed to hybridize with each chromosome 2. Different colors can be used to separate each chromosome type Medical use: 1. For example, if the probe is specific for chromosome 21 and karyotyping reveals 3 copies of chromosome, then it indicates Trisomy21 2. If there is a translocation of a chromosome then the homologous chromosome will appear as a different size bc the chromosome that lost a piece would appear smaller and the chromosome that gained a piece would appear longer 3. If there is a deletion corresponding to a known gene of a chromosome then it would show no fluorescence with probe specific to that region
26
Microarray
Technique: 1. A DNA chip corresponding to thousands of genes is arranged in a grid of glass or silicon 2. cDNA (copy DNA) samples corresponding to mRNA are made using reverse transcriptase 3. The cDNA is then labeled with fluorescent dye and hybridized with the chips 4. Hybridization can be detected by fluorescence measurements Medical use: Gene expression of different cells or a single cell can be identified 1. Reed (for cancer cell cDNA) and green (for normal cell cDNA) colors can be used for dual color microarray to test the expression of mRNA that is low or high in cancer cells relative to normal gene expression
27
SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Technique: 1. Protein samples are treated with SDS, which linearizes all protein into different size fragments 2. Since all proteins are bound to SDS, it is all negatively charged bc of the sulfate anion bound to it 3. Proteins are then ran on an SDS-PAGE gel buffer system that allows all proteins to move from cathode (-) to anode (+) poles 4. Proteins are separated on the based of their sizes Medical use: molecules are separated on the basis of molecular weight
28
Isoelectric focusing below
Technique: 1. Non-denaturing/native gel made of pH gradient 2. Samples are loaded into gel and under the electric field they move until their charge is 0 (isoelectric point) A. At low pH most proteins are positively charged and at high pH most proteins are negatively charged 3. Proteins with acidic side chains are negatively charged and will migrate closer to anode (+) 4. Proteins with basic residues are positively charged and will migrate closer to cathode (-) Medical use: proteins can be separated based on their acidity and basic its of side chains
29
For trisomy21 detection what technique mighty be of immediate use for detection?
Karyotyping | - fluorescence in situ hybridizidation (FISH) can also detect but will take longer
30
A patient was suspected to have sickle cell anemia. What diagnostic test can be used to test the normal (HbA) versus the mutant HbS Hb?
Flow cytometry
31
A scientist speculates that there might be 2 genes for the same protein PAPSS. What technique from the list above would he use to test this hypothesis?
FISH
32
Population A was detected to have a higher preponderance for type I diabetes. Blood samples were collected from 92 individuals with the hope of measuring the insulin levels of the serum. What is the convenient test one can perform?
ELISA
33
A patient was diagnosed with a cancer based on physical exam. The patients sibling was normal. There are several proteins that are down regulated (up or down) in a cancer situation. Whole cell extracts of the cancer patient and the correspond tissue extract from the normal siblings was available. What technique might analyze/ diagnose for the cancer occurrence?
Microarray
34
Name the two types of drug resistance and the mechanism of resistance.
Specific drug resistance: A) marked amplification of dihydrofolate reductase gene B) MTX resistance can arise due to alterations in the folate transporters C) Generation of mutant dihydrofolate reductase, that is insensitive to MTX. - combinations of the above are possible Multi-drug resistance: Synthesis of proteins MDR1 (glycoproteins) or MRP (multi drug resistance protein) - these membrane bound proteins are pumps that remove the drugs from the cells, resulting in the reduction of effective cytotoxic concentrations of the drugs
35
The nucleotide analog that is used in DNA sequencing is ___________
Dideoxynucleotides (chain elongating inhibitors of DNA polymerase)
36
The DNA polymerase used in the PCR reaction is obtained form the organism ___________
Thermus aquaticus
37
Name the components that are required for gene cloning
A cloning vector and DNA of interest - a small piece of DNA (cloning vector), taken from a virus, a plasmid or a cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment is inserted for cloning purposes
38
What is the role of restriction enzymes in molecular cloning?
Restriction enzymes cut DNA at a certain
39
Describe the process involved in molecular cloning.
- Restriction enzymes cut DNA at a certain sequence - DNA ligase re-binds the strands of DNA. The ligated vector is then ready for transformation into bacteria. - Transformation and Selection: the ligated vector is transformed into bacteria by CaCl2 and/or heat shock method. The recombinant bacteria are selected by antibiotic sensitivity toward ampicillin or kanamycin (bacterial specific antibodies)
40
Name few recombinant screening techniques.
In blue/white color selection method, beta-galactosidase assay is used - white colony- recombinant bacteria - blue colony- nonrecombinant bacteria Restriction enzyme Fragment Length Polymorphism (RFLP)
41
Southern Blot
1. Genomic DNA is chopped into small pieces by restriction enzyme digestion 2. DNA fragments of different sizes are separated on an agarose gel (gel electrophoresis helps us separated based on size) 3. Separated DNA is transferred onto a filter paper that is positively charged 4. Filters is exposed to denaturant to separate the strands and hybridized with a known single strand labeled probe (radioactively labeled or bases tagged onto a fluorescent dye) that would recognize and bind to the immobilized DNA/gene 5. Filter paper is exposed to film (X-ray) 6. Band size is identified for the gene that was probed. - Use of the Technique: Genetic Screening for Mutation. If a mutation had altered the restriction enzyme site then the DNA would be uncut, that would result in a larger fragment
42
Northern blot
Instead of DNA, isolated mRNA are separated and probed with labeled oligonucleotide for an mRNA of interest. For example, mRNA corresponding to acetyl CoA carboxylase can be measured - Use: The band intensity gives an idea about the level of expression. It can test the levels of gene expression in the presence and absence of hormone that control a particular gene
43
Western Blot
In this technique proteins are separated on a SDS-PAGE gels. Transferred on to a filter paper that is positively charged. The transferred immobilized protein is then reacted with primary antibody (10Ab) for a protein of interest to be tested (ex. ACC antibody). Then the 10Ab is reacted with a secondary antibody (20Ab) conjugated to a fluorescent dye and the blot is exposed to a film. The exposed band size and intensity are measured for a protein of interest. This give an idea about the level of expressed protein
44
Southwestern Blot
This technique is used for identifying the transcription factors that binds to a corresponding gene promoter element. Labeled oligonucleotides of the promoter element DNA is mixed with and without nuclear protein extracts and then separated on a gel, transferred onto a nylon filter paper and then the filter exposed to film. The exposed band that bound to the transcription factor would have shifted in terms of its mobility in contrast to unbound DNA
45
Describe in detail the control of lactose metabolism in E. Coli through Lac operon system.
When lactose is not present in the cell - Lac operon is shut off by lac repressor protein - Lac Repressor binds to DNA region of the lac operon to shut off transcription/translation of lactose metabolizing genes by inhibiting RNA polymerase from transcribing downstream genes ZYA When lactose levels are high - Allolactose (a form of lactose) serves as inducer of the lac operon by binding to the lac repressor protein, preventing it from binding to the operator - RNA polymerase is then free to bind to promotor and continue downstream to transcribe genes Z-Y-A When glucose is low - cAMP levels increase, which allows Catabolize Activating Protein (CAP) to bind to cAMP - the complex then binds upstream to the RNA polymerase binding region of the lac operon - this stimulates the increase of Z-Y-A gene transcription by 50 fold
46
Describe the genes that are involved in metabolizing lactose
In order to metabolize lactose the gene products Z (beta galactosidase), Y (permease), and A (transacetylase) need to be coordinately controlled - Beta galactosidase (Z gene product) cleaves lactose into glucose and galactose - Permease (Y gene products) transports lactose from external milieu to inside - Transacetylase (A gene product) transfers an acetyl group to Beta-galactosidase (role remains unclear)
47
Describe the secondary structure properties of Lac repressor.
- Helix-turn-helix motif that allows it to bind to DNA (usually as a dimer) - amino terminus of lac repressor protein forms the DNA binding domain - Larger carboxy domain forms the dimeric protein interaction
48
What is a catabolite activating protein (CAP)? Describe its role with reference to Lac operon.
Catabolite activating protein (CAP) binds to cAMP and the complex then binds upstream to RNA polymerase binding region of the lac operon This increases the transcription of Z-Y-A genes by 50 fold
49
Describe the regulatory molecules involved in purine biosynthesis of prokaryotes.
Purine repressor binds to hypoxanthine or guanine of many operator sites located throughout the E. Coli genome to control purine biosynthesis
50
Describe the regions of tryptophan mRNA involved in tryptophan biosynthesis.
5’leader sequence-
51
Describe the different mRNA secondary structures that are involved in tryptophan biosynthetic regulation
When tryptophan levels are high: - stem loop secondary structure —> when translation reaches trp codon, trp is incorporated into the protein very rapidly. A messenger RNA secondary structure will then form and block further transcription/translation When tryptophan levels are low: - stem only secondary structure —> when translation reaches trp codon, trp is incorporated into the protein slowly. A secondary structure of mRNA will then form allowing the formation of full length transcript to be produced and the translation of all 5 genes occurs
52
Why is it that proper secondary structure upstream to the start codon is crucial in the regulation of tryptophan biosynthetic regulation?
The type of secondary structure upstream to the start codon will ensure that translation of the trp operon genes either happens (if trp levels are low) or does not happen (if trp levels are high)
53
How can amino acid excess or scarcity in a bacteria be sensed to regulate its own biosynthesis at the level of mRNA transcription/translation (Hint: think of mRNA secondary structures)
- stem loop secondary structures form in the mRNA under high levels of respective amino acids and arrest transcription/translation - stem only secondary structures form int he leader mRNA when levels of respective amino acids are low and allow for the complete mRNA transcript to be synthesized and its corresponding proteins produced through translation
54
Control of gene expression and protein levels in eukaryotes
- inactivation of specific chromosome (X chromosome inactivation in women) or chromosome region during development - local chromatin modification (acetylation) activates, methylation inactivates genes - Gene amplification: many oncogenes are present in every cell. However erbB is amplified in breast cancer. DHFR is amplified in certain cancer. - specific transcription factor: steroid hormone receptors, CREB, HOX, etc - processing of mRNA: alternate splicing (soluble form vs membrane bound form) - rate of translation: heme increases the initiation of beta-globin genes - protein modification: pro insulin to insulin cleavage - protein degradation: ALA synthetase has a half life of one hour in hepatocytes - post translational modifications: phosphorylation, acetylation, hydroxylation, methylation, etc
55
Some enzymes/proteins unique to PANCREAS as opposed to liver
Pancreas specialized in making digestive enzymes: ``` Procarboxypeptidase A1 Pancreatic trypsinogen 2 Chymotripsinogen Pancreatic trypsin1 Elastase III B Pancreatic lipase Procarboxypeptidase B Pancreatic amylase ```
56
What are some enzymes/proteins unique to LIVER as opposed to pancreas
Liver cells make pretty much everything else: ``` Albumin Apolipoprotein A1, C1, C3, and E ATPase 6/B Cytochrome oxidase 2 and 3 Alpha-antitrypsin ```
57
Describe the differential mechanism of regulation that can result in tissue specificity?
Specificity of these tissues is in part due to the CpG methylation differences of the specialized cells
58
What are HAT’s and HADC’s?
HAT’s: histone trans acetylases that add an acetyl group to histones. They are transcription factors/coactivators that activate gene expression HADC’s: histone deacetylases that remove the acetyl group from histones. Have the opposite effects to HAT’s, deactivate gene expression
59
Describe how HAT’s and HDAC’s regulate gene expression?
HAT’s: reduce the affinity of histone for DNA. Allows for recruitment of other TF’s to transcriptional machinery and initiates the remodeling of chromatin structure HDAC’s: removes acetyl group to produce opposite effect The fine control of HAT’s and HDAC’s allow for certain genes to be expressed and certain genes to be suppressed. - dysregulation of this process can lead to cancer
60
Deamination of 5-methylcytosine would result in the formation of ___________. Describe how it can affect the chromatin landscape?
TpG | This alters the promoter landscape dramatically.
61
Describe the properties of bromodomain.
- four helix bundle domain that specifically binds to acetyl-lysine of histone proteins - part of transcription factors - present in some chromatin remodeling engines similar to helicases that use ATP to induce conformational changes in chromatin
62
With reference to mammalian system describe the roles of transferrin, transferrin receptor and ferritin
Transferrin- transports iron Transferrin receptor- binds to transferrin in the liver and kidney cells and delivers iron into the cell. Levels increase when iron is scarce (regulation at the translational level) Ferritin- stores iron in the liver cell - 24 ferritin form a shell and 2,400 iron atoms are stored per shell
63
Describe the role iron response protein (IRP) and its role in translation of ferritin
Binds to 5’-UTR of the iron response element (IRE) and blocks the translation initiation step - IRE is a stem loop structure in Ferritin mRNA In the presence of high iron, IRP has iron bound (4Fe-4S cluster) - IRP bound to iron cannot bind the 5’-UTR of ferritin mRNA - the free ferritin mRNA can then be translated into ferritin protein - ferritin protein then ties up the excess iron present (becomes iron oxide-hydroxide) and stores it in the liver cell
64
Describe the role of 3’UTR in transferrin receptor mRNA.
3’UTR of transferrin receptor protein - has IRE like regions Under low iron- IRP binds to the IRE regions. However, translation still occurs since it binds in the 3’UTR and not the 5’ Under high iron- IRP no longer binds to transferrin receptor mRNA. The free transferrin receptor mRNA is then degraded
65
Describe the role of 5’UTR of ferritin mRNA
Is the region that IRP binds to in the stem loop structure (IRE) in order to prevent translation
66
Describe how ferritin and transferrin are reciprocally regulated
Ferritin and transferrin receptor mRNA translation is reciprocally regulated Under low iron: 1. IRP binds to 5’ UTR in ferritin mRNA and prevents translation 2. IRP binds to 3’ UTR in transferrin receptor mRNA and translation continues as normal High iron: 1. IRP can no longer bind to ferritin mRNA and translation continues 2. IRP can no longer bind to transferrin receptor mRNA, which is then degraded
67
Describe Mammalian Target of Rapamycin Complex 1 (mTORC1)
Is a nutrient/energy sensing mechanism in mammals that control protein synthesis - when cellular levels of ATP are high, this complex signals protein synthesis High AMP levels is indicative of low energy levels - high AMP levels activates AMP-kinase (AMPK), which phosphorylates many proteins and shuts off translation/protein synthesis
68
Describe how nutrient availability can be sensed by mTORC1?
Carbohydrate consumption activates mTORC1 through insulin growth factor dependent pathway Amino acid consumption activates mTORC1 through branched chain amino acids and amino acid derivatives (Rag pathway)
69
What are the 2 compounds that can control mTORC1 pathway?
- the branched chain amino acid Leucine - amino acid derivatives: Beta hydroxylase, beta-methyl butyric acid Both signal the phosphorylation of mTOR to its active form mTORC1 - dietary restriction inhibits mTORC1 signaling - in resistance exercise (strength building), mTORC1 signals the production of myosin, titin, and actin resulting in muscle hypertrophy
70
Describe autophagy.
Autophagy is a degradation pathway present in eukaryotes that removes damaged organelles through a process called macroautophagy - involves formation of pre-autophagosome that encloses cellular debris and/or damaged proteins, which then fuses with a lysosomes to degrade the contents - individual amino acids and raw material that results from this degradation can be recycled to form new proteins and new organelles
71
Micro and macro autophagy
Macroautophagy: the primary pathway of autophagy that removes damaged organelles. Pre-autophagosome encloses cellular debris in membrane and then fuses with lysosomes to degrade its contents Macroautophagy: aggregation of protein and removal of cellular debris from the cytoplasm through engulfment by lysosome
72
Describe the relationship between mTORC1, autophagy and cancer
mTORC1 has the ability to inhibit autophagy and stimulate protein synthesis at the same time This can result in the accumulation of damaged proteins and organelles that can cause damage at the cellular level
73
Describe the relationship between mTORC1, autophagy and neurodegenerative diseases.
Autophagy slows as an individual ages In conditions like cancer, neurodegenerative disorder, and diabetes autophagy is dysregulated In neurodegenerative disorders, autophagy is slowed to the point where cell debris/cell waste aggregates within the brain. Activating autophagy can help alleviate problems associated with clearing cell aggregates and cell debris.
74
Describe various levels of eukaryotic gene regulations.
In eukaryotes, genes of the same biosynthetic pathways are located in several different chromosomes/genes Homeotic proteins (HOX, PAX-2, etc) are similar to prokaryotic helix-turn helix where one helix would insert into major groove of the DNA Basic leucine zippers has pair of long alpha helices B-zip has coiled-coiled structure made of leucine that makes contacts with DNA
75
Name 3 androgens.
Testosterone, dehydroepiandrosterone, dihydrotestosterone
76
Name an estrogen.
17-beta-estradiol
77
Name the hormone that is required for embryo implantation
Progesterone
78
What are the functions of DHEA?
It is a weak androgen that can be converted into a more potent androgen in extra-adrenal tissue In postmenopausal women, DHEA is an important precursor of estrogen Inhibits G6P dehydrogenase dehydrogenase and regulates NAD+
79
Functions of aldosterone
Causes secretion of K+ and retention of Na+ which results in increased blood pressure and fluid retention Produced in high levels during stress
80
Functions of cortisol
Stimulates gluconeogenesis in the liver, protein degradation in muscle and mobilizes fatty acids in adipose tissue.(Generally helps increase blood glucose)
81
What is the role of StAR?
Steroidogenic acute regulatory protein It mediates acute regulation of steroid hormone biosynthesis (in order of seconds to minutes)
82
What is the RLS in steroid biosynthesis?
Side chain cleavage of cholesterol to pregnenolone by desmolase
83
What is the role of 5-alpha-reductase?
An enzyme in the ER which converts testosterone to dihydrotestosterone (a more potent androgen)
84
Name the enzyme that converts testosterone into estradiol
Aromatase
85
Name the organ in which aldosterone and cortisol are produced
Adrenal steroids are made in the adrenal gland. Aldosterone: zona glomerulosa Cortisol: zona fasciculata and reticularis
86
Name 4 types of tissue within the adrenal gland
Adrenal gland has 4 types of tissues:zone fasciculata, zone glomerulosa, zone reticularis, and medulla
87
Name the most common enzyme in steroid biosynthesis
Cytochrome P450
88
Conversion of cholesterol into prenenolone occurs in
The mitochondria
89
Describe the signaling relay for the synthesis and secretion of cortisol.
When the hypothalamus is stimulated, it releases corticotropin releasing factor, which stimulates the anterior pituitary to release adrenocorticotropic hormone (ACTH). ACT stimulates the adrenal cortex to release cortisol. The presence of Cortisol acts as feedback inhibition to the hypothalamus
90
Describe the signaling relay for the synthesis and secretion of aldosterone.
Production of aldosterone in the zona glomerulosa of the adrenal cortex is regulated by the renin-angiotensin system - renin secreted from kidneys in response to variations in blood pressure (& volume, Na, K) - renin converts angiotensinogen to angiotensin I - ACE converts Ang I to Ang II - ACE converts Ang II to Ang III —> the effect of Ang II and III on the zona glomerulosa causes the release/secreation of aldosterone
91
Describe the signaling relay for the synthesis and secretion of estradiol and progesterone.
Hypothalamus secretes Gonadotropin releasing hormone (GnRH) which stimulates the anterior pituitary to secrete FSH and LH. In females, LH and FSH target the ovary to release estradiol and progesterone. In males, LH stimulates testis to release testosterone
92
Describe the role of cholesterol as a precursor for various metabolism
Biosynthesis of vitamin D3 (cholecalciferol), cholesterol sulfate (gene regulator of skin differentiation), membrane biosynthesis, bile acid/salt (cholic acid/salt) synthesis, and steroid biosynthesis (pregnenolone the mandatory precursor) Cholesterol is a precursor for several steroid hormones that affect different types of metabolism. For example, cortisol causes GNG in the liver and protein degradation in muscle
93
What is the second messenger that stimulates the synthesis and secretion of cortisol?
cAMP
94
What is the second messenger that stimulates the synthesis and secretion of aldosterone?
IP3 (inositol 1,4,5-triphosphate)
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Name the second messenger that is produced as a result of atrial natriuretic factor binding to kidney cells.
cGMP (antagonizes the synthesis and secretion of aldosterone)
96
Guanylate cyclase catalyzes the formation of ____________ from _____________
cGMP from GTP
97
Cholesterol esterase catalyzes the formation of
Cholesterol by cleaving a cholesterol-ester into cholesterol and a fatty acid
98
The hormone that triggers synthesis and secretion of adrenal steroids is ____________
Adrenocorticotropic hormone (ACTH)
99
Desmolase (20,22-lyase) catalyzes the formation of _________ from ___________ in the ____________
Pregnenolone from cholesterol in the mitochondria Pregnenolone —-> cortisol
100
CYP is defined as
Genes that encode for cytochrome p450 hydroxylase enzymes
101
The conversion of angiotensin I to angiotensin II is catalyzes by
Angiotensin converting enzyme (ACE)
102
In congenital adrenal hyperplasia the enzyme that is deficient is __________.
Cytochrome p450 steroid 21-hydroxylase
103
Adrenal insufficiency causes ____________
Addison’s disease
104
Name two reasons for Cushing’s syndrome
Prolonged exposure to glucocorticoid such as use of cortisol as an anti-inflammatory drug or tumors of adrenal cortex
105
Name an adrenal steroid that causes hypertension upon its production
Mineralocorticoids like aldosterone
106
Name an aldosterone antagonist.
Spironolactone
107
Name competitive inhibitors of ACE
Captopril, lisinopril, eanpril
108
Pregnancy is maintained by continuous secretion of progesterone, which is stimulated by the hormone _____________.
Luteinizing hormone (LH)
109
Describe how apoptosis is stimulated during the ovarian cycle.
The absence of LH and HCG causes the corpus luteum to atrophy. Oxytocin and PGF2alpha causes luteolysis (destruction of corpus luteum)
110
Testosterone to estradiol conversion is catalyzed by _________
CYP19 (aromatase)
111
Two hormones that stimulate luteolysis are _______
Oxytocin and PGF2 alpha
112
Human placenta lacks the hydroxylase enzyme _______
CYP17 therefore it cannot synthesize estrogen from DHEA (circumvented by enzymes of fetus)
113
Testicular feminization is the result of deficiency of __________.
5alpha-reductase Causes decreased ability to reduce testosterone into the more potent DHT. DHT in utero exerts androgenic effects causing male sexual differentiation
114
Cortisol is transported from site of synthesis (adrenal gland) to the target tissue by
Transcortin, aka corticosteroid binding globulin (CBG)
115
Transcortin binds to
Cortisol
116
Testosterone is transported by
Testosterone-estrogen binding protein (TEBP)
117
A plasma protein that binds nonspecifically to steroids is
Albumin | - aldosterone binds albumin
118
Name two compounds that inactivate steroids upon conjugation
Glucouronides or sulfates - UDP Glucouronate - 3’-phosphoadenosine 5’-phosphosulfater (PAPS)
119
Receptors for FSH, LH, GnRH, Angiotensin II and III are located
On the plasma membranes of receptor cells
120
Receptors for cortisol and aldosterone are located
In the cytosol
121
Receptors for testosterone, estrogen, and progesterone are located
In the nucleus
122
What are heat shock proteins
Heat shock proteins are chaperone proteins Receptors for cortisol (glucocorticoid) and aldosterone (mineralocorticoid) are usually bound to a cytosolic protein called heat shock protein (hsp). While bound to hsp, the receptor is in an inactive state, since the DNA binding region of the receptor is occluded. Upon binding of the steroid to the receptor, the hsp dissociates, and the receptor assumes an active conformation, whereby the hormone receptor complex can enter the nucleus, bind to DNA and active transcription
123
Define hormone response element.
A region of the DNA that binds a hormone-receptor complex
124
Glucocorticoid causes the repression of _________ gene
ACTH and ultimately cortisol
125
True or False. The active conformation of the steroid hormone receptor is dimer.
True
126
Describe the DNA binding domain of a hormone receptor
• In the center of the receptor moiety between the ◦ N terminal: variable (antigenic domain) ◦ C terminal: steroid binding domain • 2 Zinc finger domains associated with the the DNA binding domain which facilitate binding to DNA near the transcription initiation complex
127
What are zinc fingers?
Secondary structures of four cysteine residues coordinated with zinc which facilitate binding to DNA
128
What are the secretion signals for aldosterone, cortisol, testosterone, 17beta-estradiol and progesterone respectively?
``` Aldosterone: Angiotensin II/III Cortisol: ACTH Testosterone: LH Progesterone: LH 17beta-estradiol: FSH ```
129
Name the bone forming and bone resorbing cells.
Osteoblasts | Osteoclasts
130
Name some bone antiresorptive compounds.
Biphosphanates Alendronate (Fosamax) Risedronate (Actonel)
131
Name a catabolic product of estradiol that induces breast cancer.
16-hydroxy estrogen (16-OHEN) is a potent breast cancer inducer
132
What are the compounds that are being used in hormone replacement therapy (HRT)
Current hormone replacement therapy (HRT) that involves equine estrogen/medroxy progesterone (progestin) Osteoporosis is a major concern amount post menopausal women. Treatment - current hormone replacement therapy. Side effects include breast cancer, uterine cancer, cardiovascular problem
133
Name the universal sulfonate donor compound
3’-phosphoadenosine 5’-phosphosulfate (PAPS)
134
What happens if there is a deficiency in adrenal isoform of desmolase activity? Describe the clinical problems associated with this condition.
Cannot convert cholesterol → pregnenolone If there was a deficiency in desmolase activity, cholesterol would not be cleaved into pregnenolone and isocaproaldehyde. Without pregnenolone, none of the steroid hormones would be formed.
135
Congenital adrenal hyperplasia - biochemical basis
A genetic defect that results in insufficient cortisol production. Due to decreased cortisol production. ACTH production is not feedback inhibited by cortisol, therefore levels of ACTH are very high and cause adrenal hyperplasia. Progesterone accumulates and is channeled into the production of androgens, DHA and androstenedione. Increased production of androgenic steroids causes virilization (masculinization) of females, precocious sex organ development in prepubertal males or diseases related to salt imbalance because of decreased levels of aldosterone
136
Addison’s Disease
An overall deficiency of the adrenal steroid hormones (also known as adrenal insufficiency) ACTH levels are always high. Patients are hypoglycemic due to decreased levels of glucogeogenesis as a result of low cortisol levels. Low blood pressure due to low levels of mineralocorticoids (aldosterone). Tx: Hormone supplementation
137
Cushing’s syndrome
Prolonged exposure to glucocorticoid (either due to the usage of cortisol as an anti-inflammatory or due to tumors of adrenal cortex) results in elevated blood glucose levels. Sx include truncal obesity but have thin arms and legs. In addition, these patients may have HTN
138
Hypertension due to a tumor and Tx
Excessive secretion of mineralocorticoids resulting from adrenal tumor results in hypertension Tx: aldosterone antagonists (spironolactone) which form an inactive complex with the aldosterone receptors. Other drugs captopril, lisinopril, and enalapril (usually used to treat HTN and CHF) which are small peptides that are competitive inhibitors of ACE
139
Osteoporosis
Estrogens induce the synthesis of osteoprotegrin, which causes osteoblasts (bone forming cells) differentiation, and suppress osteoclasts (bone resorbing) Low estrogen mainly in post-menopausal women can cause osteoporosis
140
Testicular Feminization
A genetic deficiency in 5-alpha reductase results in decreased ability to reduce testosterone into the more potent DHT. DHT in utero exerts androgenic effects causing male sexual differentiation. Therefore, individuals with XY genotype (genotypically male) are born phenotypically females lacking ovaries and uterus.
141
Describe the role of fetal adrenal gland and fetal liver with reference steroid metabolism during pregnancy
Fetal adrenal gland converts cholesterol to DHEA (important precursor for estrogen) which is transported to the fetal liver to form 16-hydroxy-DHEA. In placenta 16-hydroxy-DHEA is converted to estrogen by aromatization. Thus, there is cooperation between maternal and fetal tissue
142
Describe the process of estradiol mediated programmed cell death.
Blood levels of estradiol and progesterone drop during the ovarian cycle, resulting in the sloughing of the endometrium ie menstruation. These cells undergo apoptosis
143
Describe atrial natriuretic factor (ANF) mediated cell signaling.
Signals opposite to those that activate the formation of angiotensin (for example hypervolemia) generate ANF in heart atria. ANF binds to a specific zona glomerulosa cell membrane receptor and activates guanylate cyclase, which catalyzes the formation of a second messenger cGMP from GTP. cGMP antagonizes the synthesis and secretion of aldosterone
144
Describe the key enzyme of steroid metabolism
p450 hydroxylase
145
Describe the mechanism of p-450 hydroxylase catalyzed reaction.
P-450 hydroxylases that use molecular oxygen (O2) as a substrate, where one oxygen atom is incorporated into the steroidal substrate (as OH) and the second atom reduced to water molecule. Electrons are donated by NADPH Cholesterol —> pregnenolone & isoccaproaldehyde (desmolase = P450 hydroxylase) Pregnenolone —> cortisol (series of P450 hydroxylases)

M1 Biochemistry (17 decks)

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