Exam 3- Venk Flashcards
Describe eukaryotic genome, genes, and its overall features
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).
Describe the features of mitochondrial genome
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
Describe human karyotype, its use in genetic counseling and disease prognosis
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
What is trisomy 21?
Down syndrome
Due to an extra chromosome 21
Describe the process of MicroRNA (MiRNA) synthesis, its transport and it’s role in gene silencing.
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
M1 and M2 macrophages, and how it is derived from a common progenitor?
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
Properties of M1 and M2 macrophages
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
Describe morula and blastula of embryo during embryogenesis
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
Define Totipotent, Pluripotent and Multipotent with examples
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
Describe the 3 germ layers of blastocyst and explain the types of cell that can be derived from each.
- 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
Describe general spliceosome (splice complex) of an mRNA transcript.
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
Describe in detail the mechanism of alternate splicing and its advantage
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
What are some diseases that can manifest due to splicing defects of mRNA?
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)
What are splice regulator proteins?
Silencer or enhancers
- binding to splice complex either preventing or promoting splice complex to bind to DNA
What are the functions of splice regulators?
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
Describe the role of MiRNA in various diseases?
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
What are the two mechanisms to modify histones at the epigenetic level?
Methylation and acetylation
- methylation in general suppresses gene expression
- acetylation activates gene expression
What are CpG islands?
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
Describe the mechanism of epigenetic modification versus the chemical basis of chromatin remodeling
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
What are DNA repair enzymes?
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
Describe the mechanism of Lig4 versus cancer
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
Describe ELISA (Enzyme Linked ImmunoSorbent Assay)
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
Flow cytometry
Technique:
- Cells are reacted with a primary antibody conjugated to a fluorescents labeled dye (green or red)
- The cells are then sorted through a cell sorter and shined with a laser beam to see the dyes
- 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
Karyotyping
Technique:
- Specific stains are used for metaphase chromosomes
- Chromosome 1 (the longest) to chromosome 22 (the shortest) can be arranged in order of its length
- In abnormal cases the karyotype would be different compared to normal
- 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
Fluorescence In Situ Hybridization (FISH)
Technique:
- Fluorescent dye tagged onto a DNA or RNA probe specific to each chromosome is allowed to hybridize with each chromosome
- Different colors can be used to separate each chromosome type
Medical use:
- For example, if the probe is specific for chromosome 21 and karyotyping reveals 3 copies of chromosome, then it indicates Trisomy21
- 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
- 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
Microarray
Technique:
- A DNA chip corresponding to thousands of genes is arranged in a grid of glass or silicon
- cDNA (copy DNA) samples corresponding to mRNA are made using reverse transcriptase
- The cDNA is then labeled with fluorescent dye and hybridized with the chips
- 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
SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Technique:
- Protein samples are treated with SDS, which linearizes all protein into different size fragments
- Since all proteins are bound to SDS, it is all negatively charged bc of the sulfate anion bound to it
- Proteins are then ran on an SDS-PAGE gel buffer system that allows all proteins to move from cathode (-) to anode (+) poles
- Proteins are separated on the based of their sizes
Medical use: molecules are separated on the basis of molecular weight
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
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
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
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
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
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
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
The nucleotide analog that is used in DNA sequencing is ___________
Dideoxynucleotides (chain elongating inhibitors of DNA polymerase)
The DNA polymerase used in the PCR reaction is obtained form the organism ___________
Thermus aquaticus
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
What is the role of restriction enzymes in molecular cloning?
Restriction enzymes cut DNA at a certain
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)
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)
Southern Blot
- Genomic DNA is chopped into small pieces by restriction enzyme digestion
- DNA fragments of different sizes are separated on an agarose gel (gel electrophoresis helps us separated based on size)
- Separated DNA is transferred onto a filter paper that is positively charged
- 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
- Filter paper is exposed to film (X-ray)
- 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
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
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
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
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
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)
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
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
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
Describe the regions of tryptophan mRNA involved in tryptophan biosynthesis.
5’leader sequence-
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
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)
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
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
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
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
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
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
