PHAR 737 Midterm 1 Flashcards
Describe the ENCODE project
ENCyclopedia Of DNA Elements
International collaboration of research groups funded by the National Human Genome Research Institute.
Goal is to build a list of functional elements in human genome
How many base pairs, genes and chromosomes comprise the human genome.
3x10^9 base pairs
20,000 genes
23 chromosomes
Define Genome
All the genetic material (DNA) of an organism
Define Genetics
Study of single genes and its effects
Define Genomics
Study of all genes in the genome, including their interactions with environmental factors
Define Pharmacogenetics?
The study of genetic influences on an individual’s response to drugs
Define Pharmacogenomics
The study of all genes collectively that influence drug responses, and how genome-wide analysis may be used to identify such genes in the search for novel drug targets or as key determinants for drug reactions.
Of the 3 billion base pairs in the human genome, how many are variable, how many capture the full human variation, and how many are pharmaceutically relevant?
3 million base pairs are variable
100,000 capture the full human variation.
Less than 10,000 may be relevant to pharmaceutical development.
How are drugs approved?
By trial and error
What do pharmaceutical companies need to specify when it comes to drug action?
A specific population, or else the drug will be abandoned (most likely)
How much does it cost to develop a new drug? How long will it take to launch the drug from patent and when will the patent expire?
Drugs development usually ranges in price from 500 million to 700 million dollars.
Patent to launch is usually around 12 years, and will last 7.
Define efficacy
Patients cured at a given dose .
Define therapeutic Index
Dose range at which drug shows highest efficacy and low toxicity
How prevalent is genetics when looking at US death rates?
9 of 10 deaths in the US have a genetic component
What factors influence drug response?
Gender Age Body mass Diet Presence of other drugs Disease Exposure to certain chemicals or toxins (alcohol, tobacco) Genetic factors
Most diseases are caused by this type of interaction
gene-environment
What main interventions are used for groups that are at high risk of gene-environment interactions caused disease(s)?
Diet
Physical activity
Smoking cessation
Alcohol avoidance
What is an SNP?
Single Nucleotide Polymorphism (or substitution) of one base for another every 1000 nucleotides. SNPs are evolutionarily stable (not changing much between generations)
What is the significance of Mendelian Inherited Diseases?
1200 genes that are currently identified as causing human diseases/traits exhibit inherited phenotype
What is a Transcriptome?
The full range of RNA molecules expressed by an organism or present in a cell at a given time.
Changes in transcriptional activity contributes to a disease.
What is a Proteome?
The entire set of proteins expressed by a genome, organism, cell or tissue at a certain time.
Constantly changing since proteins are continually being synthesized, modified and degraded. Also species and cell-state dependent.
What is the Epigenome?
The epigenome is a series of chemical compounds that can tell the genome what to do.
Epigenomics is the study of changes in the regulation of gene activity and expression that ARE NOT dependent on gene sequence
What is the purpose of Systems Biology?
To connect the molecular characteristics of a disease with pharmacogenomics to deliver a personalized therapy option to a patient.
What is the importance of Simple Viral and Bacterial Genomes?
This is an untapped resource of raw genomic material
What is the importance of Ancestral Genomics?
Can be used to track the evolution of genomes, duplication events and similarities
What is the importance of the interpretation of shared characteristics?
Determining traits that mammals have gained and lost through evolution
What are the five stages that drugs undergo in the body?
Absorption Distribution Target Interaction Metabolic Processing Excretion from the body
How prevalent are genetic factors when it comes to variation in drug response between individuals?
20-95 percent
What genes are important for pharmacokinetic properties of drugs? Pharmacodynamics?
Those for drug metbolizing enzymes and drug transporters.
Those for enzymes, receptors, and ion channels.
How common are SNPs?
Occur in at least 1 percent of the population
Define Haplotype
A group of alleles that are rarely separated by recombination (generally inherited together)
Human haplotypes are 60,000 base pairs in size and contain 60 SNPs that travel as a group
Haplotypes are better predictors of drug responsiveness than single, isolated SNPs
Describe a “Missence” SNP
Characterized by changes of amino acids, with about half of such changes occurring in coding sequences.
Can result in the alteration of protein function and is the cause of most monogenetic disease.
Describe “Nonsense” SNPs
Introduce a stop codon with the same consequences as Missence SNPs (alteration in protein function, cause of monogenetic disease)
How many SNPs does the human genome have? Which are most prevalent, where do they occur, and how do they effect us?
3 million, distributed randomly
Occur in both coding and non-coding regions, with 2 out of every 3 involving the replacement of cytosine (C) with thymine (T). Some have no effect on cell function but may predispose people to a disease or impact how they respond to certain drugs.
What is TSC and what do they do?
The SNP Consortium (HapMap Project)
A partnership of scientists from Canada, China, Japan, Nigeria, UK and US that seeks to determine the frequency of certain SNPs in three major world populations.
Goal is to make a public resource that researchers can use to find genes associated with disease and pharmaceutical response in humans.
How is pharmacogenetic testing prior to drug therapy limited?
Largely to specialized drugs such as chemotherapy agents
Explain GWAS
Genome Wide Associated Study is a study of many common genetic variants in individuals to determine if any variant is associated with a trait.
What are nucleotide subunits composed of?
Sugar-phosphate molecule with a nitrogen containing side group, or base, attached to it
What are the activated precursors in RNA synthesis?
Ribonucleotide triphosphates
What direction does the chain grow in RNA synthesis?
5’ to 3’
What are the stop and start sites in transcription?
Start = AUG Stop = TAA
List 4 important transcriptional co-regulators
- Mediator complexes
- Histone chaperone complexes
- Histone modifying enzyme complexes
- ATP-dependent chromatin remodeling complexes
Explain eu- and hetero-chromatin
Euchromatin is uncondensed and is considered “good” chromatin (ideal for replication) while heterochromatin is condensed and not favorable for replication (repressing or silencing)
Describe how transcription is modulated
DNA bound activator protein upstream from enhancer sequence attracts proteins to promoter region that activate RNA polymerase. The DNA can loop around its self to facilitate the interaction of proteins that activate RNA polymerase.
Active repressors can bind to operator sequences near the promoter and interfere with RNA polymermase binding to the promoter.
Describe the general process of Translation
Amino acids are transported to the ribosome by tRNA (20 different tRNA molecules total, one for each amino acid), tRNA anti-codons find their RNA complement and peptide bonds form between amino acids to form a peptide, with translation stopping once the stop codon has been reached.
What is Chromatin?
A nucleoprotein complex that causes condensation and organization of DNA. Strings of nucleosomes compose the primary structural unit while nucleosome interactions provide a secondary level of compaction.
Describe a histone octamer
The 8 protein coplex found at the center of a nucleosome, consisting of 2 copies of each core protein (H2A, H2B, H3, and H4)
What is epigenomics?
The study of the complete set of epigentic modifications on the genetic material of a cell
Describe CRMs and their impact on cells
Cis Regulatory modules are short sequences that regulate expression of nearby genes (on the same DNA strand) when transcriptional regulators bind to them.
As a results, only half of the 25,000 protein-coding genes of the mammalian genome are expressed in any given cell.
Describe Epigenetics.
Heritable traits that aren’t linked to changes in the DNA sequence (changes caused by external or environmental factors)
Describe DNA methylation of the human genome
Methyl groups added to DNA at CpG sites, with the pattern being determined during embryogenesis (and getting passed over to differentiating cells).
In humans, 3-5% of DNA cytosine is methylated, and this methylation occurs almost exclusively at cytosines that are followed by a Guanine-CpG dinucleotide (5’CpG3’). The process is performed by DNA methyltransferase, and is heritable, reversible, and often used as a therapeutic target. Methylation on the human genome is not uniform.
CpG is methylated in non-promoter regions and un-methylated in promoter region (methylation in promoter region correlates with silencing)
In histone modification, acetylation and deacetylation are regulated by…
Histone acetyltransferases (HATs) and Histone Deacetylases (HDACs)
How do HATs work?
HATs catalyze the transfer of the acetyl moiety from acetyl-CoA to the amino group of histone lysine residues, resulting in acetylated lysine and CoA.
Positively charged histones are neutralized and their interaction with the negatively charged DNA is decreased.
In general, this increases gene expression.
What can acetlyation regulate in DNA?
DNA replication, histone deposition and DNA repair
What is the result of lysine acetylation?
More opened euchromatin state which is more accessible to transcription factors
What are two primary disease examples in humans resulting from histone modifications? Describe each disease.
Coffin-Lowry Syndrome:
>Genetic disorder characterized by mental retardation and head/facial abnormalities
>X-linked dominant genetic trait
>results from mutations in the RSK2 gene (histone phosphorylation)
Rubinstein-Taybi Syndrome:
>Short stature, intellectual disability, distinctive facial features, and broad thumbs and first toes
>results from mutations in CREB-binding protein (histone acetylation)
What are the three main methods we use for DNA sequencing
Chain Termination, aka di-deoxy method (aka Sanger sequencing)
Shotgun Sequencing (fragments DNA into manageable chunks)
Pyrosequencing (fast and accurate)
Explain RT-PCR
Reverse Transcriptase-Polymerase Chain Reaction is used to determine whether a particular type of mRNA is present.
The process starts with mRNA. cDNA is formed in a reverse transcription process
Describe RNA-Seq
RNA-seq is simple, faster, and more reliable than other methods. Allows for a comprehensive view of the transcriptome (each transcript is sequenced)
What is CHIP-seq
Chromatin Immunoprecipitation Sequencing
DNA-bound protein is immunoprecipitated using a specific antibiody, followed by purification of that DNA and then synthesis of sequencing of those purified strands. This method is used to indicate DNA-protein interactions.
Explain why RNA expression odes not always reflect protein levels
Translational control, degradation and turnover
What does Functional Proteomics Encompass?
Post-translational modifications
Protein-Protein and Protein- Ligand Interactions
Sequence structure-function relationships
Describe Metabolomics
New field that uses systematic determination of metabolite levels in the metabolome and their changes over time as a consequence of stimuli
Define Metabolome
The complete set of small-molecule metabolites found within a biological sample
Define Metabolites
Intermediates and products of metabolism (antibiotics, pigments, carbs, fatty acids, amino acids, etc.)
Describe transgenic mice
Mice with germ-line transformations that involve the insertion of genes into the reproductive cells of an organism, which permanently alters the individual and all offspring (transgenic)
Used to study the functions of specific genes in development or disease processes
Define Allele
One of two or more alternative forms of a gene that arise by mutation and are found at the same place on a chromosome
Describe the structure/levels of DNA
DNA is wrapped around protein molecules called histones; the nucleosome is the combination of DNA and histone protein; the nucleosomes form a bead-like chain, called the chromatin; chromatin will form chromosomes when a cell is getting ready to divide
What base pairs are present in DNA? RNA?
CG AT
CG AU (uracil substituted for thymine in the case of RNA)
Define phenotype
The set of observable characteristics of an individual resulting from the interaction of the genotype with the environment
Describe PCR
Polymerase Chain Reaction, causes DNA amplification
Uses heat to denature DNA, separating the strands, followed by addition of primers that allow polymerase to synthesize the strands in question
Main applications of Metbolomics
Drug assessment, clinical toxicology, nutri-genomics, functional genomics
Challenges with Metabolomics
Database is still new and lacks content, there is still a need for standardization, efficiency in identification and better interpretation of the data
Integration with other ‘omics’ fields is also a current challenge
What methods are used to measure gene expression levels?
RT-qPCR
RNA Microarray Chips
RNA-seq
Explain RNA Microarray Chips
Reverse transcription used to create cDNA strands (1 infected + 1 normal) which are then combined. Infected is labeled fluorescently. cDNA strands are then applied to the CHIP, which is scanned to determine which genes are expressed in the infected and uninfected samples.
What are the 3 different stop codons?
UGA, UAA, UAG
Methylation of K27 results in…
Repression
Acetylation of K27 results in…
Activation
Methylation of K4 results in…
Activation
What are the steps that lead to protein quantification?
Sample extraction, protein fractionation, peptide fractionation, mass spectrometry, protein identification, and finally protein quantification.
