Cumulative Genetics
Environmental agents that can alter gene expression by affecting the epigenome
- Nutrition - Chemicals - Medical or recreational drugs - Social interactions and exercise
5' Capping (transcription)
- Occurs immediately after transcriptional initiation - Protects transcript from 5ʹ-exonucleases - Facilitates transport of mRNA to cytoplasm; mRNA splicing; translation - Can occur post-transcriptionally in nucleus and cytoplasm - 5' cap is a guanosine triphosphate that does 5' to 5' linkage
Exons
- Protein coding sequences that are retained and expressed
Anaphase II (meiosis)
Centromeres divide; sister chromatids are pulled to opposite poles
Phosphatases
Enzymes that remove phosphates
Gene targeting
target specific allele, locus, or base sequence and learn its function on gene of interest.
3 major Cell-Cycle Checkpoints
- "checked" by master control molecules (kinases) before next stage (G1, G2, and M) - If checkpoints fail to recognize and stop errors, cell division proceeds and tumor results. - If cell cycle is arrested at checkpoints, cell is removed from population of dividing cells, preventing potential malignancy.
Histone structure
- DNA is wrapped around nucleosomes - 48 bp per nucleosome - Nucleosome = histone octamer - 2 copies of H2A, H2B, H3, and H4 - > 70 Histone modifications
Recruitment model
- DNA looping delivers activators and GTFs to promoter region - Increases rate of PIC assembly - Enhancers and repressors and TFIID close DNA loops
Cytokinesis
- Follows karyokinesis - Cytoplasmic division - Partitions cellular volume into two parts and encloses each cell with a plasma membrane
variable number tandem repeats (VNTR)
- Found between genes - Also called minisatellites
Substitution RNA editing
- Identities of individual nucleotide bases are altered - Prevalent in mitochondrial and chloroplast RNAs transcribed in plants
Single nucleotide polymorphism (SNP)
- If the freq. of the polymorphism is >1% of the population it is an SNP - Not considered a SNV
Restriction enzymes vs. Cas9
- Restriction enzymes: cut specific seq. - Cas9: does not cut specific seq., needs NGG site + relies on a guide RNA
Frameshift Mutations
- Result from insertions or deletions of nucleotide (INDEL). - Loss or addition of nucleotide causes shift in the open reading frame. - Frame of triplet reading during translation is altered. - Altered triplets may code for stop codon
Induced mutation
- Result from the influence of an extraneous factor, either natural or artificial - Ex: Radiation, UV light, Natural and synthetic chemicals
Dominant mutation
- Results in mutant phenotype in diploid organism - Typically, only one pair of the gene is mutated - but it is sufficient to cause the protein to malfunction
Missense mutation
- Results in new triplet code for different amino acid - oThese are usually single bp substitutions that change what the amino acid codes for.
Nonsense mutation
- Results in triplet code for stop codon (translation terminated prematurely)
Mitochondrial DNA & the genetic code exceptions
- Revealed exceptions to the universal genetic code -- Codon UGA normally specifies termination but in MtDNA UGA codon encodes tryptophan in yeast and humans -- Codon AUA normally specifies isoleucine but in Human mitochondria encode internal insertion of methionine
Methods used to develop a model organism
- Reverse genetics - RNA interference - Spontaneous mutation - Forward genetics
Reverse genetics vs. Forward genetics
- Reverse genetics: You know the gene - Forward genetics: you don't know the gene, you know the phenotype
DNA Hypomethylation
- Reverses silencing of genes - Leads to unrestricted transcription - Relaxes control over imprinted genes
Chain elongation (transcription)
- Ribosomes added to RNA chain - σ subunit dissociates from holoenzyme. - Elongation proceeds under direction of core enzyme.
Anticodon (tRNA)
- tRNA has anticodon that complementarily bp with codon in mRNA. - Corresponding amino acid is covalently linked at 3′end of all tRNAs.
Genetic Variant
- term used to describe a different DNA sequence between two individuals or among individuals in a population - Includes Mutations, SNP, SNV, INDEL, CNV
cell coat (glycocalyx)
- the outer covering that most animal cells have over the plasma membrane - Made of glycoproteins and polysaccharides. - Provides biochemical identity at cell surface
What is the final product of mitosis?
-- 2 cells, each with the same number of chromosomes as parent cell (2n). (2 daughter cells)
Where will reduction and crossing over occur?
Meiosis I
Meiosis I & II
Meiosis I: reductional division Meiosis II: equational division (just mitosis) - each have prophase, metaphase, anaphase, and telophase stages
What is the accepted model for replication in viruses, prokaryotes, and eukaryotes?
Semiconservative replication
DNA topoisomerases
enzymes that participate in the overwinding or underwinding of DNA
Whole Genome Sequencing (WGS)
WGS of maternal plasma cfDNA used to sequence entire exome of fetus.
Satellite DNA
- Differs in its density compared to rest of DNA. - Highly repetitive DNA. - Represents a variable proportion of total DNA. - Found in heterochromatic centromeric regions of chromosomes. - Not found in prokaryotes.
Gene-expression microarrays
- Detect gene-expression patterns in specific genes. - Contain probes for specific genes thought to expressed differently
Preimplantation Genetic Diagnosis (PGD)
- Genetic analysis of cells from embryo created by IVF - Expected to expand to include epigenetic analyses in the future
Mechanisms of mRNA decay (image)
- RNA susceptible to decay by exoribonucleases - Eukaryotic mRNAs degraded by deadenylation-dependent decay
Transcription
- RNA synthesized on DNA template - Produces mRNA - Each triplet codon complementary to anticodon of tRNA
Retroviruses
- RNA-containing viruses (transmit their genetic information by passing on RNA not DNA) - RNA serves as template in reverse transcription process using enzyme reverse transcriptase.
Polyploidy
More than 2 sets of chromosomes are present
What is responsible for promoter recognition in transcription?
RNA polymerase σ factor
telocentric centromere
end of chromosome
George Beadle and Edward Tatum
"one gene, one enzyme hypothesis"
What diseases is human chromosome 15q11-q13 associated with?
- Angelman syndrome - Prader-Willi Syndrome - Chromosome 15q Duplication Syndrome
Polymerase switching
- Pol α dissociates from template and ε or δ (which have higher processivity) replaces Pol α for elongation
Nondisjunction during meiosis I
Produces 2 gametes that contain 2 copies of a particular chromosome & 2 gametes that are deficient for that chromosome
Monosomy
The loss of a single chromosome from an otherwise diploid genome
Who always passes on any mitochondrial disorders?
The mother
47, XXY Syndrome
The only consistently shared characteristic found so far in the 47, XYY karyotype is that such males are over 6 feet tall
Trisomy
The gain of one chromosome
metacentric centromere
centromere in middle of chromosome
What does the separation of homologs during mitosis and meiosis depend on?
centromeres
Null mutation
results in complete loss of function
Inheritance pattern of genomic diseases
- De novo - Inherited
Classification of Mutation Based on Effect of Coding Sequence (mRNA)
- Missense - Nonsense - Sense - Neutral
Structural differences between Thymine and Uracil
- same except extra Methyl group present on thymine
Western blot
- used for analyzing proteins
What type of research are mice the widely used model?
Biomedical research
Core promoter
Determines accurate initiation of transcription
What is the start (initiator) codon?
Methionine (AUG)
Ribosomal Structure (image)
Prokaryote: 70S (50S + 30S) Eukaryote: 80S (60S + 40S)
Mutations
Source of alleles
Who was the first to use the word "genetics"?
William Bateson
ex of polyploid (2+ genome) organisms
plants
What is the outcome of a female with an extra X chromosome?
- 47, XXX - Most females go through their lives without being aware and are normal
Prognostic testing
Predicts likelihood of developing genetic disorder
Chromatin during cell division
Chromatin coils and condenses back into visible chromosomes
Chromatin during interphase
Chromatin is dispersed throughout nucleus and replicated
Mendel's Fourth postulate
Independent Assortment: During gamete formation, segregating pairs of unit factors assort independently of each other
Blunt ends
fragments produced with double-stranded ends
Where do autosomal mutations occur?
within genes located on the autosomes
What is the phenotype of an organism the product of?
· interactions between the genome and the epigenome
What DNA polymerase(s) has 3'-5' exonuclease activity?
DNA pol. I, II, III
cell-type specific gene expression
- Skin cells express keratin. - Muscle cells express myosin II
Autosomal Recessive Trait - Human Pedigree
- Typically skip generations - Appear equally in both sexes
Kerry Mullis
Discovered the development of PCR (while he was high)
The early stages of transcription in bacteria (image)
(a) the components of the process (b) template binding at the −10 site involving the σ factor of RNA polymerase and subsequent initiation of RNA synthesis (c) chain elongation, after the σ factor has dissociated from the transcription complex and the enzyme moves along the DNA template.
Robertsonian translocation
(centric fusion) - involves breaks at the extreme ends of the short arms of two nonhomologous acrocentric chromosomes
Gregor Mendel's first 3 postulates (principles of inheritance)
- "factors" governing traits exists in pairs - Independent assortment of factors - concepts of dominant and recessive traits
osteogenesis imperfecta
- 1 in every 12,000-15,000 births - Difficult to diagnose during infancy and childhood - Types I -IV: mild (bone dislocations to frequent bone fractures [lethal]) - Caused by mutations in collagen genes or in genes associated with collagen formation - Different genes causing the same phenotype = locus heterogeneity
How did Meselson and Stahl show that semiconservative replication was the mode used by bacteria for replication?
- 15N-labeled E. coli grown in medium containing 14N - Used sedimentation equilibrium centrifugation technique to distinguish between DNA containing 15N from 14N
Avery MacLeod, and McCarty -- Transforming Principle
- 1944 publication on chemical nature of transforming principle in bacteria - First direct experimental proof that DNA is biomolecule responsible for heredity - Demonstrated transforming principle was DNA not protein
Cystic Fibrosis
- 1:2,500 individuals affected with CF - An estimated 1 in 29 Caucasian Americans have a mutation in the CFTR gene. - More common among those of Northern or Central European descent
Trisomy
- 2n + 1 chromosomes - Trisomy for the sex chromosomes has a less dramatic phenotype than a trisomy of an autosome, which are often lethal - 3 copies of one chromosome are present, so pairing configurations are usually irregular. - At any particular region along the chromosome length, only two of the three homologs may synapse, though different regions of the trio may be paired - Largely occurs on chromosome 21
Important factors in translation of Eukaryotes
- 3' Poly-A tail plays an important role in translation initiation + protects it from degradation - Poly-A binding proteins protect mRNA from degradation and also bind to initiation factors. - Initiation factor eIf4G binds to m7cap resulting in required complex for translation initiation. - mRNA forms loop where poly-A tail and 5′-cap are brought together—closed loop translation.
9mers
- 5 repeating sequences of 9bp - A-T rich; enhance helical unwinding - Mark where the origin of replication is at
Pyrimidines
- 6-membered single ring - Cytosine (C) + Uracil (U) + Thymine (T)
Purines
- 9-membered double ring - Adenine (A) + Guanine (G)
Diploid number (2n)
- A condition in which each chromosome exists in pairs; having two of each chromosome - Ex: Homans: 2n number of 46 chromosomes
de novo mutation
- A new germ cell mutation that was not inherited from either parent (parent does not carry mutation) - Offspring can then transmit to its offspring, making it inherited
Aneuoploidy
- Abnormal number of chromosomes. - organism gains or loses one or more chromosomes but not a complete set
Analytic techniques that have been useful during the investigation of DNA & RNA
- Absorption of UV light: Hyperchromic shift. - Molecular hybridization. - Fluorescent in situ hybridization (FISH). - Electrophoresis of nucleic acids.
Post-transcriptional modifications of Eukaryotic mRNA
- Addition of 5′ cap (7-methylguanosine (m7G): Stabilizes and protects mRNA from nuclease attack. - Poly-A polymerase catalyzes addition of poly-A tail at 3'-OH (250 adenylic acid residues): mRNAs without AAUAAA signal seq. are protected from degradation by poly-A binding protein
Post-translational modifications (a.a.)
- Addition of a functional group to the protein structure - The N-terminus a.a. is usually removed or modified - Individual a.a. residues are sometimes modified - CHO side chains are sometimes attached - Polypeptide chains may be trimmed - Signal sequences are removed - Polypeptide chains are often complexed with metals
Autosomal Recessive
- Affected people are usually born to unaffected parents. - Parents of affected people are usually asymptomatic carriers. - There is an increased incidence of parental consanguinity. - Affects either sex. - After the birth of an affected child, each subsequent child has a 25% chance of being affected. - Ex: Cystic Fibrosis, Sickle cell, Phenylketouria
X-linked
- Affects either sex, but more females than males - Females are often more mildly and more variably affected than males. - The child of an affected female, regardless of its sex, has a 50% chance of being affected. - For an affected male, all his daughters but none of his sons are affected
X-linked recessive
- Affects mainly males. - Affected males are usually born to unaffected parents; the mother is normally an asymptomatic carrier and may have affected male relatives. - Females may be affected if the father is affected and the mother is a carrier, or occasionally as a result of non-random X-inactivation. - There is no male-to-male transmission in the pedigree (but mating of an affected male and carrier female can give the appearance of male-to-male transmission
Y-linked
- Affects only males. - Affected males always have an affected father (unless there is a new mutation). - All sons of an affected man are affected
Blue-white screening mechanism:
- Agar plates contain X-gal: Analog of lactose—substrate for β-galactosidase. - When X-gal is cleaved by enzyme, it turns blue. - Bacterial cells with functional lacZ gene carrying a nonrecombinant plasmid = blue. - Bacterial cells with recombinant plasmid = white
Evolutionary advantages of inversions
- Alleles with inversions may be preserved generation to generation. - Balancer chromosome: Used in lab studies—In organisms heterozygous for balancer chromosome, desired alleles are preserved
Punnett Squares
- Allows genotypes and phenotypes resulting from cross to be visualized easily. - Vertical columns represent female parent. - Horizontal columns represent male parent. - Provides all possible random fertilization events - Shows what might happen during meiosis
Functions of introns
- Alternative splicing: produces different mature mRNAs from same pre-mRNA - Evolutionary advantage: exon/intron gene structure allows for new genes to evolve - Contain functional noncoding RNA - Regulate transcription via cis- regulatory elements
What is the amino and carboxy end of a protein
- Amino end: start codon, or 5' end - Carbozy end: 3' end of gene
Chargaff's Rule
- Amount of A=T and C=G - Sum of A+G is equal to sum of C+T - Percentage of G+C is not necessarily equal to percentage of A+T
Steady-state level of mRNA
- Amount of mRNA in cell available for translation - Regulated by cell need - Molecular mechanism that plays important role in regulation of gene expression
Autosomal Dominant
- An affected person usually has at least one affected parent. - Affects either sex. - Transmitted by either sex. - A child of an affected × unaffected mating has a 50% chance of being affected (this assumes the affected parent is heterozygous, which is usually true for rare conditions).
Reverse Transcriptase
- An enzyme encoded by certain viruses (retroviruses) that uses RNA as a template for DNA synthesis. - RNA-dependent DNA polymerase enzyme - DNA can be incorporated into host-cell genome - When transcribed, copies of original retroviral RNA chromosomes also produced.
Audiography
- Applied cytologically—pinpoints location of radioisotope in cell. - Photographic emulsion placed over cellular material and stored in the dark. - Develops much like photographic film. - Result: Presence of dark grains identifies location of newly synthesized DNA
Taylor-Woods-Hughes experimental procedure (image)
- As it enters mitosis, both sister chromatids of the chromosome are labeled by autoradiography. - After a second round of replication (b), this time in the absence of 3H-thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. - Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld.
Messenger RNA (mRNA)
- Associate with ribosomes and ultimately produce proteins - serves as intermediate in transferring genetic information from DNA to proteins
Misregulation of genes
- Associated with developmental defects of disease - Uncontrolled proliferation—hallmark of cancer
Yeast ORI
- Autonomously replicating sequences (ARSs): Consist of 120 base pairs of consensus sequences (sequence that is the same/nearly the same in all yeast ARSs)
Huntington's disease
- Autosomal dominant mutation in the Huntington gene - Neurodegenerative disorder - Affects muscle coordination and leads to cognitive decline and dementia -Noticeable in middle age - Causes abnormal involuntary movement - Trinucleotide expansion repeats disease
TGS: Third-generation sequencers
- Based on sequencing a single molecule of ssDNA - SMRT—single molecule sequencing in real time: - Attaches single molecule of DNA polymerase anchored to substrate. - Visualizes in real time the syntheses of a strand of DNA by polymerase
One-Gene: One-Enzyme Hypothesis
- Beadle and Tatum showed genes are directly responsible for synthesis of enzymes - Hypothesized after they observed that nutritional mutations in bread mold Neurospora caused loss of enzymatic activity. - Eventually changed to the one-gene: one-protein hypothesis
Translation of mRNA
- Biological polymerization of amino acids into polypeptide chains - Requires Ribosomes + tRNA
M checkpoint
- Both the successful formation of the spindle-fiber system and the attachment of spindle fibers to the kinetochores associated with the centromeres are monitored. - If spindle fibers are not properly formed or attachment is inadequate, mitosis is arrested
R group (side chain) of a.a.
- Bound to central carbon atom - Determines type of amino acid - 4 classes of R groups
How did Francis Crick support the nature of the triplet code? What were his conclusions?
- By using a phage T4 study of frameshift mutations - Found that the gain or loss of one or two nucleotides caused a frameshift mutation, but when three nucleotides were involved, the frame of reading was reestablished - Work also suggested that most triplet codes are not blank, but rather encode amino acids, supporting the concept of a degenerate code.
Fragile X Syndrome: Non-coding Coding Trinucleotide Repeat
- CGG repeats - Normal allele = 29-31 units (not affected) - Pre-mutation allele = 55-200 units (not affected) - Full mutation allele = 200-4,000 units (affected) - Expansion requires maternal transmission - Expansion almost certain as TNR > 90 units - Somatic expansion - Large expansion arises in non-dividing oocytes (expansion increases with age) and terminally differentiated cells - Full mutation tends to contract in male spermatogonia - Loss of expression of FMR1 gene due to DNA methylation of expanded repeat
Core promoters classified in 2 ways
- Focused core promoters - Dispersed core promoters
Properties of Bacterial DNA Pol. I, II, III
- Can elongate existing DNA strand (primer) - All three possess 3′- 5′ exonuclease activity (proofread newly synthesized DNA) - Cannot initiate DNA synthesis
Types of Alternative Splicing
- Cassette exons - Alternative splice site - Intron retention - Mutually exclusive - Alternative promoters - Alternative polyadenylation
histone acetyltransferase enzymes (HATs)
- Catalyzes histone acetylation of the nucleosome - Associated with increase transcription
Peptidyl transferase
- Catalyzes peptide bond formation between a.a on tRNA at A site and growing peptide chain bound to tRNA in P site. - catalytic activity of 23S rRNA
23S rRNA
- Catalyzes peptide bond formation between amino acid on tRNA at A site and growing peptide chain bound to tRNA in P site. - Uncharged tRNA moves to E (exit) site. - tRNA bound to peptide chain moves to P site. - Sequence of elongation and translocation is repeated over and over. - Peptidyl transferase: catalytic activity of 23S rRNA
DNA ligase
- Catalyzes the formation of phosphodiester bonds - Seals nicks and joins fragments
Sex chromosome abnormalities
- Caused by meiotic non-disjunction - X chromosome is necessary for development, but the Y chromosome is not. - Kleinfelter syndrome + Turners
G1/S Checkpoint
- Cell monitors its size and determines whether its DNA has been damaged - If cell size and DNA integrity are normal, the G1/S checkpoint is traversed, and the cell proceeds to S phase.
Advantages of closed loop translation
- Cell saves energy by not translating partially degraded mRNA. - Closed loop structure allows for efficient ribosome recycling.
Amino acids structure
- Central carbon - Amino group (NH2) - Carboxyl group (COOH) - Hydrogen - "R" group
Anaphase (mitosis)
- Centromeres split and sister chromatids separate from each other (disjunction); They are no longer chromatids but daughter chromosomes
Mutation
- Change in the DNA sequence - Can occur anywhere in the genome - Typically arise in the gametes (i.e., sperm or egg), but they can also occur in somatic cells (e.g., skin) - Provide basis for genetic analysis + act as "markers" for genes
Spontaneous mutaiton
- Changes in nucleotide sequence of genes that occur naturally - Often occur during enzymatic process of DNA replication. - Rates of spontaneous mutations vary among loci in different organisms
Chromosome Rearrangements
- Changes that delete, add, or rearrange substantial portions of one or more chromosomes - Deletions, Duplications, Inversions, Translocations
MN blood group in humans
- Characterized by antigen glycoprotein: found on surface of RBC - 2 forms of glycoprotein exist: M and N. - Individual may exhibit either one or both. - MN system is under control of autosomal locus on chromosome 4 and two alleles LMLN
What is meant by saying that the genetic code shows order?
- Chemically similar amino acids share one or two middle bases in triplets encoding them. - End result of "ordered" code: buffers potential effect of mutation on protein function.
Chloroplast DNA (cpDNA)
- Chloroplast provides photosynthetic function for plants. - cpDNA shares similarities to DNA found in prokaryotes - Circular, double-stranded, and free of associated proteins found in eukaryotic DNA. - Larger than mtDNA—larger number of genes. - Has both introns and duplications.
Nondisjunction
- Chromosomal variation originating from random errors during gamete production. - Paired homologs fail to disjoin and move to opposite poles during meiosis I or II. - Leads to a variety of conditions in humans and other organisms.
Sutton and Boveri: Chromosome Theory of Inheritance
- Chromosomes are linear structures, which contain heritable "factors/genes" - Paternal and maternal chromosomes that separated during meiosis and that "may constitute the physical bases of the Mendelian law of heredity"
Nondisjunction
- Chromosomes fail to separate properly during meiosis - Results in gametes with too many or too few chromosomes
Homologous chromosomes
- Chromosomes in diploid cells exist in pairs - Have identical genetic similarities, identical gene sites along their lengths (locus)
Bacterial Chromosomes
- Circular, double-stranded DNA compacted into nucleoid. - E. coli most extensively studied -
Cis-acting DNA elements
- Cis = next to - Adjacent parts of the same DNA molecule - Regulatory element
Enhancers + Silencers are what type of regulatory element?
- Cis- acting transcription regulatory elements - Regulate transcription
Insertions of TEs in various locations and their effects:
- Coding region: Translation disruptions. - Intron: Termination signal can terminate transcription. - Intron: Splicing of RNA transcribed from a gene. - Gene's transcription regulator region: Effect's gene expression. - Identical TEs in genome: Potential for transposon recombination.
During prometaphase/metaphase
- Cohesin is degraded by enzyme separase - Sister chromatid arms disjoin except at centromere - Enzyme Shugoshin prevents degradation of cohesin at centromere
Sticky Ends
- Cohesive ends - fragments produced with overhangs
Epigenetics Traits are Heritable: Coat Color in Mice
- Color controlled by dominant allele Agouti (A) - Homozygous AA mice: Agouti phenotype - Mutant allele Avy: yellow phenotype - Allele result of insertion of transposable element near transcription start site of Agouti gene - Degree of methylation in transposon's promoter related to amount of yellow pigment deposited
Euploidy
- Complete haploid sets of chromosomes are present - Multiples of n
Spindle Fibers
- Composed of microtubules consisting of polymers of protein tubulin - Play important role in movement of chromosomes as they separate during cell division
Heterochromatin
- Condensed areas mostly inactive - Appears stained during interphase - Some chromosomes are entirely heterochromatin
RISC (RNA-induced silencing complex)
- Contain Argonaute family of proteins: Proteins bind RNA - have silencer activity - RISC cleaves and evicts one of two strands of double stranded siRNA - Single stranded siRNA "guide" is another strand: Recruits RISC to complementary mRNA
Mitochondria and chloroplast
- Contain their own DNA - Inherited through maternal cytoplasm in most organisms - The nature of their DNA is similar to viruses and bacteria
Complementary DNA (cDNA) library
- Contains complementary DNA copies (cDNA) made from mRNAs isolated from cultured cells or tissues. - Represents only genes that were expressed at the time library was made: - Useful for identifying genes involved in cancer formation - Genomic libraries contain all of the DNA
Genomic Library
- Contains of many overlapping fragments of the genome. - Has at least one copy of every DNA sequence in organism's chromosomes. - Constructed by cutting genomic DNA with restriction enzymes and ligating fragments into vectors. - Libraries built from eukaryotic cells will contain coding and noncoding segments such as introns
RNA Polymerase from E. coli
- Contains of subunits α, β, β', and ω. - Holoenzyme - more complex form of enzyme: has additional subunit called σ(sigma) factor + plays regulatory function in the initiation of RNA transcription
Histone Modificaiton
- Covalent bonding of functional groups onto N-terminal tails of histone proteins: - Most common: acetyl, methyl, phosphate - Acetylation decreases positive charge: reduces affinity of histone to DNA - May assist formation of chromatin conformations
Epigenome
- Created from the epigenetic changes - Regulates normal development or generates physiological responses to environmental signals
2 Functions of Alternative Splicing of mRNA
- Creates diversity in terms of the number of proteins they make - The way that gene function regulate their expression
Sutton and Boveri
- Credited with initiating chromosomal theory of inheritance. - Independently published works linking behavior of chromosomes to Mendelian principles of segregation and independent assortment
Mitosis
- Critical for all eukaryotic organisms. - Single-celled fungi, protozoa, and algae—mitosis serves as basis for asexual reproduction. - Multicellular organisms—responsible for wound healing, cell replacement, and growth.
Formation of Pre-Initiation Complex (PIC)
- Critical step in initiation of transcription - PIC consists of RNAP II and GTFs - First step in PIC formation: binding of TFIID to TATA Box via TATA binding protein (TBP) - GTFs help recruit RNAP II to promoter
Dihybrid cross (two traits)
- Cross involving two pairs of contrasting traits → generates unique F2 ratio. - F1 will show both dominant traits. - F2 will show 9:3:3:1 ratio
Monohybrid cross (one trait)
- Crosses of only one pair of contrasting traits. - Involves mating true-breeding individuals from two parent strains. - Reveals how one trait is transmitted from generation to generation.
Centrioles
- Cytoplasmic bodies located in centrosome: - Found in animal and plant cells - Centrioles organize spindle fibers that function in meiosis and mitosis
Gene duplication and Products
- DNA Commonality → products distinct - Related genes diverged sufficiently during evolution, and products became unique. - Ex: Trypsin, chymotrypsin, myoglobin, etc.
3 major epigenetic mechanisms
- DNA Methylation - Histone Modification - Regulatory RNAs
Hyperchromic Shift
- DNA absorbs light differently when we are talking about double stranded DNA and single stranded DNA - DNA with lower GC content requires lower heat - Technology is used to differentiate between one nucleotide
Eukaryotic Gene Regulation
- DNA associated with histones and other proteins - Chromatin compaction affects transcription. - Transcription and translation are separated spatially and temporally. - mRNA: spliced, capped and Poly-A tail
DNA Ligase
- DNA fragments will seal phosphodiester backbone. - Joins restriction fragments covalently to produce intact DNA molecules
Cloning vectors
- DNA molecules that accept DNA fragments - Can replicate cloned DNA fragments in host cell. - Must be able to replicate independent of host chromosome(s). - Have several restriction enzyme sites to allow insertion of DNA fragment. - Carry selectable gene marker/reporter gene to distinguish host cells that have taken them up from those that have not.
CpG islands
- DNA regions rich in CG residues (> 50%) - Combination called CpG dinucleotides—clustered in regions - Islands located adjacent to essential and cell-specific genes are unmethylated - available for transcription
Consensus sequences
- DNA sequences homologous in different genes of same organism - located within the promoter - usually (10-35 nucleotides) upstream (in the 5' direction) of the site of transcription, where the RNA polymerase starts making the RNA.
Pseudogenes
- DNA sequences representing evolutionary vestiges. - Have undergone significant mutational alterations (insertions and deletions) - Are not transcribed.
Concurrent DNA synthesis occurs on the leading and lagging strand
- DNA synthesized on both leading and lagging strands concurrently. - Lagging strand looped which inverts physical but not biochemical direction.
Plasma Membrane
- Defines cell boundary. - Delimits cell from its external environment. - Controls movement of material in and out of cell.
Change in DNA content mutation
- Deletion or duplication of large (100bp to millions of bp) region - Copy Number Variants (CNV)
Molecular Hybridizaiton
- Denaturation/Renaturation of nucleic acids - Possible between DNA strands of different organisms - RNA molecules will hybridize with segments of DNA from which it was transcribed. - Probes are used to identify complementary seq.
Dideoxynucleotides (ddNTPs)
- Deoxynucleotide with a hydrogen at 3′ instead of an hydroxyl group - Causes DNA synthesis to terminate (terminated by ddNTP).
Expression Vectors
- Designed to ensure mRNA expression of cloned gene—to produce many copies of encoded protein in host cell - Available for both prokaryotic and eukaryotic host cells: Ti plasmid and soil bacterium can be used for introducing genes in plants
Triplet Binding Assay
- Developed by Nirenberg and Leder to determine specific assignments of triplets. - Ribosomes bind to single codon of three nucleotides. - Complementary amino acid-charged tRNA can bind
Watson, Crick, and Maurice Wilkins
- Did NOT discover DNA - Discovered the structure of DNA & created the double helical model of DNA
Epigenetics Traits are Heritable: Diet (Mice study)
- Diet of pregnant mice was supplemented with methylation precursors - Variation in coat color in offspring was reduced and shifted toward pseudoagouti phenotype (highly methylated phenotype) - Less methylated = yellow
Isoforms of a protein
- Differ in a.a. encoded by differently included or excluded exons - Isoforms of the same gene may have different functions
Dispersed core promoters
- Direct initiation from several weak transcriptional start sites - Used by 70% of eukaryotes - specify weak transcription initiation at multiple start sites over an approximately 100-bp region
DNA Polymerase I
- Directs DNA synthesis - Req. DNA template and 4 deoxyribonucleoside triphosphates (dNTPs). - Reads template strand 3'-5' and synthesizes 5'-3' - Removes primers on the lagging strand
RNA polymerase
- Directs RNA synthesis using DNA template - Has same substrate requirements as DNA polymerase - ** No primer required - Catalyzes initiation
What proteins are involved with the unwinding of the DNA Helix?
- DnaA - DNA helicase - Single-stranded binding proteins
Non-LTR retrotransposons
- Do not have LTR sequences at their ends - SINEs- short interspersed elements; are transcribed but not translated - LINEs- long interspersed elements; some are transcribed and translated
Fragile X Syndrome
- Dominant - More severe in boys - Most common single gene disorder causing intellectual disability and autism in males - Elongated face - Low muscle tone - Nervous speech - Late onset deficits in working memory
PCR requirements
- Double stranded target DNA - DNA polymerase - Mg2+ (cofactor of DNA pol) - 4 deoxyribonucleoside triphosphates - Primers:Short, single-stranded sequences + one complementary to 5′ end and another complementary to 3′ end.
What features are shared in eukaryotic and bacterial DNA replication?
- Double-stranded DNA unwound at ORI - Replication forks formed - Bidirectional synthesis creates leading and lagging strands. - Eukaryotic polymerases require four deoxyribonucleoside triphosphates, template, and primer.
The role of gene duplication in evolution
- Duplicated copy can acquire many mutational changes over extended periods - Duplicated gene may change sufficiently; product assumes divergent role in cell. - New function may impart "adaptive" advantage to organisms, enhancing fitness
Bar mutation in Drosophila
- Duplications cause phenotypic variation. - Bar-eyed flies have narrow, slit-like eyes versus normal oval-shaped eyes. - 16A region on X chromosome duplicated and responsible for eye shape in Bar flies; triplicated in double Bar flies. - Phenotype inherited same way as X-linked dominant mutation.
Mendelian Genetics
- Each parent contributes an allele to their offspring - If the genotypes of the parents are known, Mendel's 4 postulates can be used to estimate the genotypes and phenotypes of the offspring
Semiconservative Replication
- Each replicated DNA molecule consists of one "old" and one new strand. - one parental strand one daughter strand
Diploid number (2n)
- Each species possesses a specific number of chromosomes. - During formation of gametes (meiosis), the number is halved (haploid, n). - During fertilization, two gametes combine; diploid is reestablished.
Nonsense-Mediated Decay (NMD)
- Eliminates mRNA with premature stop codon
Enhancer/silencer vs. activator/repressor
- Enhancers + silencers are regulatory elements: seq. of DNA - that interact with proteins that will regulate expression - Activators + repressors are proteins that will interact with regulatory elements to increase or decrease expression of a gene, they bind to enhancers + silencers
Telomerase
- Enzyme that is capable of adding several more repeats to 3' end of G-rich strand - TERT: catalytic unit of this enzyme - TERC: RNA component - ribonucleoprotein
DNA gyrase
- Enzyme that relieves coiled tension from unwinding of helix (DNA supercoiling) - Member of a larger enzyme group: DNA topoisomerases - Makes single or double stranded cuts undoing supercoiling knots and twists. - Driven by energy released during ATP hydrolysis.
Chromatin Remodeling: Histone modification?
- Epigenetic modification involving addition of acetyl, methyl and phosphate groups to alter chromatin structure: makes genes on nucleosomes with modified histones accessible or inaccessible for transcription + RNA pol. - Involves repositioning or removal of nucleosomes on DNA by chromatin remodeling complexes.
Haploid number (n)
- Equal to one-half of diploid (2n) number. - Genetic information in haploid set of chromosomes constitutes genome of species.
General transcription factors (GTFs)
- Essential RNAP II can't bind without them - TFIIA, TFIIB, etc. TFIID binds to TATA box
Replication
- Essential function of genetic material - Must be precise for genetic continuity between cells following cell division
Lethal alleles
- Essential genes - Req. for organism's survival. - Mutations resulting in nonfunctional gene product can be tolerated if heterozygous: One wild-type allele is sufficient for survival. - Homozygous recessive will not survive. - Mutation behaves as recessive lethal allele. - Recessive lethal alleles result in homozygous recessive individuals and do not survive.
regulation via ubiquitin-mediated protein degradation
- Eukaryotic cells target protein degradation by covalently modifying with ubiquitin. - Attached to target protein via ubiquitination - Poly-ubiquitinated proteins recognized by proteasome—unwinds and removes ubiquitin tags
Kozak Sequence
- Eukaryotic mRNAs contain purine (A or G) three bases upstream from AUG initiator codon, followed by G (Kozak sequence). - A/GNNAUGG - Considered to increase the efficiency of translation initiation in eukaryotes.
Reciprocal Translocation
- Exchange of segments between two nonhomologous chromosomes. - Genetic information not lost or gained just rearranged. - Does not directly alter viability of individual. - Homologs that are heterozygous for reciprocal translocation undergo unorthodox synapsis during meiosis. - Genetically unbalanced gametes are produced.
Pedigree
- Family tree with respect to given trait. - Pedigree analysis reveals patterns of inheritance of the trait being studied - used to study modes of inheritance of phenotypes in humans
Meiosis - Prophase 1
- First meiotic division - Diploid cell duplicates its genetic material - Meiosis is similar to mitotic prophase, except homologous chromosomes pair up (synapsis- aka crossing over) - At the end of prophase one, the centromeres are present on equatorial plate - Final phase: Nuclear envelope and nucleolus break down, and two centromeres of tetrad attach to spindle fibers.
Core-promoter elements
- Found in focused promoters - Usually located between −40 and +40 nucleotides, relative to the transcription start site - Initiator element - TATA box - TFIIB recognition element (BRE) - Motif ten element (MTE) - Downstream promoter element (DPE)
Mitochondria
- Found in most eukaryotic cells including animal and plant cells. - Site of ATP synthesis. - Site of oxidative phases of cell respiration - DNA distinct from that found in nucleus
Chloroplasts
- Found in plants, algae, and some protozoans. - Site of photosynthesis - DNA distinct from that found in nucleus
Polytene Chromosomes
- Found in various tissues. - Can be seen in nuclei of interphase cells. - Represent paired homologs. - Not usually found in eukaryotic cells: unique - Visualized by light microscopy - DNA of paired homologs undergoes many rounds of replication without strand separation or cytoplasmic division.
Thomas Hunt Morgan
- Founder of Drosophila genetics - "Genes" reside on chromosomes - Sex-linked traits - Discovered genetic linkage
Factors affecting the manifestation of a genetic/genomic disease
- Function of the mutated gene - Type of molecular defect - Mode of Inheritance - Penetrance - Expressivity - Gene by environment interactions
DSCAM gene (drosophila) as example for alternative splicing
- Gene can possibly produce 38,016 different proteins through alternative splicing
Drosophila Mhc gene as example of alternative splicing
- Gene encodes motor protein responsible for muscle contraction. - Expression of different isoforms result in differences in contractile properties: Results in flies beating their wings at lower frequency.
3 aspects of duplications
- Gene redundancy - Phenotypic variation - Source of genetic variability during evolution
Bacterial artificial chromosomes (BACs)
- Generally, very large but low copy number (one to two copies/bacterial cell) plasmids - DNA inserts 100-300 kb range
Unit factors
- Genes located on homologous pairs of chromosomes - During gamete formation—members of each pair of homologs separate/segregate
X-Linkage
- Genes present on X chromosome sex of animals and plants determined by unlike chromosomes X and Y. - Exhibit unique patterns of inheritance compared to autosomal genes. - Results in modification of Mendelian ratios.
2 major conclusions reached with the Triplet Binding Assay
- Genetic code is degenerate: One amino acid can be specified by more than one triplet; 20 amino acids,64 possible codons - Genetic code is unambiguous: Single triplet specifies only one amino acid
Genetic Anticipation
- Genetic disease has earlier onset and increased severity with each succeeding generation. - In trinucleotide expansion repeat diseases, anticipation is due to the repeat expanding from one generation to the next, which worsens the effect of the mutation
3 lines of evidence support role of epigenetic alterations in cancer
- Global hypomethylation may cause genome instability and large-scale changes characteristic of cancer. - Epigenetic mechanism can replace mutations to silence tumor suppressor genes or activate oncogenes. - Epigenetic modifications can silence multiple genes, making them more effective in transforming normal cells into malignant cells.
Multigene families
- Groups of contiguous genes whose products have similar function - EX: SRGAP2 gene in primates
Bacterial DNA associated DNA binding proteins
- HU and H-NS (Histone-like Nucleoid Structuring Protein). - Proteins fold and bend DNA creating coils for compaction of DNA.
RNA
- Has nucleotides linked with phosphate chains (like DNA) - Ribose sugar replaces deoxyribose of DNA - Uracil replaces Thymine - Can be single or double stranded (most are single, exception is animal viruses)
Yeast artificial chromosomes (YACs)
- Have telomeres at each end, origin of replication (ori), and centromere. - Up to 1000kb of DNA insert possible
Coherent Model Summarizing DNA Replication (image)
- Helicase: unwinding double helix - Single stranded binding proteins: associate with strands to prevent reformation of helix - DNA gyrase: diminishes the tension created as helix supercoils - DNA Pol. I: replaces RNA primers w DNA - DNA Ligase: join the okazaki fragments - Sliding clamp: Each half of the dimeric polymerase is a core enzyme bound to one of the template strands by sliding clamp
Autosomal Dominant Trait Pedigree
- Heterozygous for dominant allele - Appear in each generation - Affected individuals have an affected parent - Appear equally in both sexes
Chromatin Remodeling- Histone acetylation
- Histone acetylation opens up chromatin structure, making genes available for transcription - Removal of acetyl groups closes configuration—silencing genes by making them unavailable. (note: Acetyl groups have an -OH group on them, as well as a (-) charge so when placed together, they push each other away)
Southern Blot
- Hybridization between complementary DNA molecules - Used to identify which clones in library contain given DNA sequence - Identifies number of copies of particular sequence or gene present in genome. - Involves separation of DNA fragments by gel electrophoresis. - Transfer (blotting) of DNA binding membrane and hybridization of fragments labeled with probe. - Membrane is washed to remove excess probe. - Overlay of X-ray film for autoradiography - Only fragments hybridized to probe are visible
Alternative splicing of calcitonin/calcitonin gene-related peptide (CT/CGRP) in thyroid cells
- In thyroid cells, pre-mRNA of CT/CGRP is spliced. - Produces mRNA with first four exons only. - Exon 4 triggers termination (exon 5+ 6 omitted): 32 a.a. peptide hormone is made (CT)
Neurospora experiment from Beadel and Tatum (image)
- Induction, isolation, and characterization of a nutritional auxotrophic mutation in Neurospora: - (a) Most conidia are not affected, but one conidium (shown in red) contains a mutation. - In (b) and (c), the precise nature of the mutation is established and found to involve the biosynthesis of tyrosine.
Limitations of PCR
- Information about nucleotide sequence of target DNA is required to synthesize primer. - Minor contamination from other sources can cause problems (e.g., skin cells from researcher). - Cannot amplify long segments of DNA.
Biparental inheritance
- Inheritance from two parents (ovum and sperm). - Each pair of loci derived from each parent - Each diploid contains two copies of each gene.
Down Syndrome: Trisomy 21
- Inheritance pattern = de novo - Trisomy 21: Down syndrome (47, 21+) - Trisomy of chromosome 21.
Interphase (mitosis)
- Initial stage of cell cycle. - Interval between divisions. - Replication of DNA of each chromosome. - 2 phases: G1 and G2
Deadenylation-dependent decay of Eukaryotic mRNAs
- Initiated by deadenylase: enzymes that degrade poly-A tail - Decapping enzymes remove 5'-cap. - XRN1 exoribonuclease digests mRNA 5' to 3' direction. - mRNAs also cleaved internally by endoribonucleases
Initiation of translation in prokaryotes (bacteria)
- Initiation codon of mRNA (AUG) calls for modified amino acid formyl methionine (fMet). - 3 initiation factors bind to small ribosomal subunit—complex binds to mRNA. - Shine-Dalgarno sequence facilitates initiation
Introns
- Intervening sequences -Noncoding internal sequences removed during RNA processing. - Not represented in the final mRNA product - Important as they are often regulatory elements
Where do suppressor mutations occur?
- Intragenic - Intergenic
SRGAP2 gene in primates
- Involved in development of the brain. - Humans have at least 4 similar copies of gene. - Nonhuman primates have single copy of gene.
Polymerases α, δ, and ε
- Involved in initiation and elongation. - Each function differently
What are DNA polymerases II, IV, and V involved in?
- Involved in various aspects of repair of DNA damaged by external forces, such as UV light.
Fluorescent in Situ Hybridization (FISH)
- Involves hybridizing probe directly to chromosome or RNA without blotting. - Carried out with isolated chromosomes on slide or in situ in tissue sections or entire organisms. - Helpful when embryos are used for various studies in developmental genetics. - Can be used to produce special karyotypes.
Chromatin Remodeling
- Involves repositioning or removal of nucleosomes on DNA by chromatin remodeling complexes. - Repositioned nucleosomes make chromosome regions accessible to: Transcription regulatory proteins + RNA polymerase.
cDNA library is constructed by
- Isolating mRNA from cells. - Synthesizing complementary DNA using reverse transcriptases. - Cloning cDNA molecules into vector
Mechanism for RNA virus inside cell: HIV
- It works as the HIV particle binds to a cell & then it inserts the RNA into the cell. - The RNA then hijacks some of the enzymes inside the cell & it converts its RNA into DNA (reverse transcriptase) - The DNA is then inserted into the genome. - When it wants to make more virus, it uses the correct direction (DNA to RNA) and then the RNA is then packaged inside more viruses & then they are excreted from the cell etc.
Okazaki fragments
- Lagging strand synthesized as Okazaki fragments, each with RNA primer (discontinuous synthesis of DNA).
Genomic diseases
- Large mutations resulting from genome architecture - Meiotic dependent - Mutation leads to: Gain or loss of gene(s) + Disrupted coding sequence - Dosage sensitive genes - Usually affect a group of genes, but one gene is usually responsible for most of the symptoms of the condition.
Actin mRNA localized to lamellipodia
- Leading edge of cell where actin polymerization occurs - Localization dependent on zip code, a cis-regulatory element that serves as binding site for zip code binding protein 1 (ZBP1). - Actin translation and polymerization direct cell movement
Tetranucleotide Hypothesis
- Levene observed that DNA contains equal amounts of 4 nucleotides - Postulated identical groups and repeats of four components were the basis for DNA structure - Was later disproved by Chargaff
Consequences of inversions during gamete formation
- Linear synapse not possible if only one member of homologous pair has inverted segment. - Inversion heterozygotes: organisms with one inverted chromosome and one noninverted homolog. - Pairing between these two chromosomes requires inversion loop
Phosphodiester bonds**
- Linkage between 2 mononucleotides involves a phosphate group linked to two sugars - Phosphoric acid joins two alcohols by ester linkage on both sides - Nucleotides linked by phosphodiester bonds between phosphate group at C-5' position and OH group on C-3' position
Sanger Sequencing: Dideoxynucleotide chain-termination sequencing
- Most common method of DNA sequencing - Small amount of dideoxynucleotides are added
2 important tools used to construct and amplify DNA molecules
- Restriction enzymes: DNA-cutting enzymes - Cloning vectors
Charging tRNA
- Linking of a.a. to tRNA molecules. (a.a. converted to active form) - Catalyzed by Aminoacyl tRNA synthase + ATP used as energy source - a.a. reacts with ATP to create aminoacyl adenylic acid: Highly specific enzyme (isoaccepting tRNAs) - Rxn leaves the a.a. covalently bonded to the 3' end of the tRNA and returns the enzyme to its original state. - For each of the 20 amino acids there is a specific aminoacyl tRNA synthetases and one or more tRNAs (20 different synthetases and 30-55 different tRNAs).
Enhancers
- Located on either side of gene, some distance from gene, or even within gene - Increase rate of transcription - Confer time- and tissue-specific gene expression
Proximal-Promoter elements
- Located upstream of TATA and BRE motifs - Enhance levels of basal transcription - Examples: CAAT box and GC boxes
Translocations
- Location of genes altered within genome - Occurs between 2 chromosomes - Nonreciprocal: a piece of one chromosome is put on another - Reciprocal: translocation between 2 chromosomes
Nucleolus
- Location where rRNA is synthesized and initially assembled. - NOR (nucleolus organizer region)—portions of DNA that encode rRNA
Telomeres
- Long stretches of short repeating sequences that preserve the integrity/stability of chromosomes - protect the ends of linear chromosomes
small interfering RNAs (siRNAs)
- Longer then miRNAs - RNAi mechanism recognizes double stranded siRNAs and inactivates them + protects cell from viruses or transposon elements - Associate with RISC for additional cleavage - Usually come from viruses
Recessive mutation
- Loss of function - Apply to mutations where both alleles are affected
Angelman syndrome
- Loss of function of maternal UBE3A - Nonpenetrant to paternal allele - Rare disorder - Severe intellectual disability (IQ 30-50) - Ataxia: Motor incoordination - Seizures - Absent speech
Prader-Willi Syndrome
- Loss of paternal derived HBII-85 snoRNA - First human genetic disorder that is caused by non-protein coding RNA - Feeding difficulties - Failure to thrive - Mild developmental delay - Hypotonia - Severe obesity
Classification of Mutation Based on Effect of Function
- Loss-of-function - Null mutation - Recessive mutation - Dominant mutation - Dominant negative mutation
Cri Du Chat syndrome ("cry of the cat")
- Loss/deletion of small variable part of short arm on chromosome 5 (46, 5p-)—segmental deletion. - Not inherited, results from sporadic loss of chromosomal material in gametes. - Severity of syndrome varies with length of deletion. - Eerie cry similar to cat's meowing: Abnormalities in glottis and larynx - Intellectual disability - Delayed development - Small head size
DNA Helicase
- Made of DnaB polypeptides - Hexamer of subunits assembles around exposed ssDNA - Recruits holoenzyme to bind to replication fork and initiate replication - Helicases require energy supplied by hydrolysis of ATP—denatures hydrogen bonds and stabilizes double helix. - Will create supercoiling
Cassette exons (type of alternative splicing)
- May be excluded from mature mRNA by joining 3'-end of upstream exon to 5'-end of downstream exon. - Most prevalent type of alternative splicing in animals - 40% of alternative splice events
Prokaryotic splicing mechanism
- Mechanism for intron removal: 1. Endonuclease cuts each end of intron. 2. Intron removed and terminal ends of adjacent exons joined by ligase. - Occurs in tRNAs in bacteria.
RNAi interference (RNAi)
- Mechanisms by which ncRNAs guide post-transcriptional silencing - ncRNAs associate with mRNAs for complementary bp: Target specific mRNAs and block translation
FXN of fully formed PIC
- Mediates unwinding of promoter DNA at start site - Mediates transition of RNAP II from transcription initiation to elongation
lampbrush chromosomes
- Meiotic chromosomes first studied in oocytes of sharks. - Large with extensive DNA looping. - Found in most vertebrate oocytes and spermatocytes of some insects. - Easily isolated from oocytes in diplotene stage of prophase I of meiosis. - Contain large number of condensed areas (chromomeres).
Reverse transcription PCR (RT-PCR):
- Method for studying gene expression (mRNA production by cells or tissues) - Reversetranscriptase is used to generate cDNA
Agarose Gel Electrophoresis
- Method that separates DNA fragments by size - Smallest piece moving farthest through gel - Fragments can be visualized with staining and illuminating with UV
Middle Repetitive Multiple-Copy Genes
- Middle repetitive DNA includes functional genes present in multiple copies - Many copies exist of genes encoding ribosomal RNA.
Allelic Heterogeneity
- Monogenic trait caused by different variants/mutations in the same gene - Ex: Cystic Fibrosis
How is Eukaryotic DNA replication more complex than Prokaryotic DNA rep?
- More DNA than prokaryotic cells - Linear chromosomes - DNA complexed with nucleosomes
Intron retention (type of alternative splice site)
- Most common type of alternative splicing in plants, fungi and single celled eukaryotes - Introns in some cases are included in mature mRNAs and are translated: Produce isoforms - In other cases, intron retention serves to negatively regulate gene expression - 2 functions: Used to destabilize DNA + Keep RNA retained within nucleus
Applications of PCR
- Most widely used technique in genetics and molecular biology - Rapid technique—takes a few hours - Used in genetic testing, forensics, and molecular pathology
2 jobs of the zip code binding protein 1 (ZBP1)
- Moving mRNA in the cell to be translated - The interaction between the proteins will interfere with translation
Human metallothionein 2A gene (MT2A)
- Multiple cis-acting elements and transcription factors o Example of how a gene can be transcriptionally regulated due to interplay of the following: Promoters, Enhancers, + transcription factors that bind to them
Gene redundancy - rRNA
- Multiple copies of genes code for rRNA. - Single copy not sufficient. - DNA that codes for rRNA is called rDNA - Gene product essential in abundance in a cell to support protein synthesis
Loss-function mutation
- Mutation in gene causes change in conformation enzyme, thus, changing affinity: - If loss is complete → mutation results in null allele
single base substitution mutation
- Mutation that involves one nucleotide change - Ex: A changed to C
Oligogenic/Polygenic (Complex)
- Mutation/variant present in a few to many genes needed to manifest phenotypes associated with disease trait - Varying degrees of penetrance and expressivity - Inherited
Monogenic (one gene) Genetic disease
- Mutation/variant present in one gene is capable of manifesting phenotypes associated with disease trait - Inheritance Patterns: Autosomal recessive. Autosomal dominant, Sex chromosome linked, de novo
When is something considered a mutation or a polymorphism?
- Mutation: If the frequency of a mutation in a population is rare (<1%) - Polymorphism: If the frequency of a mutation in a population is common (>1%)
What 2 factors modified the one-gene:one-enzyme hypothesis?
- Nearly all enzymes are proteins—not all proteins are enzymes - Proteins have a subunit structure with two or more polypeptide chains
Sense mutation
- New triplet code still codes for same amino acid - aka silent mutation
Structural differences between Cytosine and Thymine
- Nitrogen on cytosine, while Thymine has carbonyl
What do nucleotides consist of?
- Nitrogenous base (2 kinds): Purines and pyrimidines - Pentose sugar - Phosphate group
Protein folding
- Not spontaneous - Dependent on chaperones: proteins that mediate folding process - Ubiquitins tag misfolded proteins and move them to proteasome for degradation.
Viral Chromosomes
- Nucleic acid, either DNA or RNA, single or double stranded - Circular (closed loops) or linear molecules.
Karyotype
- Number of chromosomes - Mitotic chromosomes have been photographed, cut out of the print, and matched up, creating a display, the karyotype
Copy Number Variants (CNVs)
- Number of copies in duplicated sequences vary among individuals in same species. - Have both positive and negative associations with disease. - Pathogenic CNVs associated with autism, CV disease, Type I diabetes
Mechanism of chain elongation by DNA pol. I
- Occurs in 5' to 3' direction by adding one nucleotide at a time to 3'- OH. - Nucleotide added → two terminal phosphates cleaved off, providing newly exposed 3'-- OH.
Transcription in Eukaryotes
- Occurs in nucleus (unlike bacteria) - mRNA leaves nucleus for translation - 3 forms RNA pol. needed - Chromatin remodeling: must uncoil to make DNA accessible to RNA pol. - RNA pol. rely on GTFs to bind DNA - Enhancers and silencers control transcription regulation - Transcription termination is complex: sequence-specific cleavage of transcript - Primary transcripts (pre-mRNAs) undergo alterations—processing: Addition of 5′-cap and 3′-tail + Removal of intervening sequences
Single-stranded binding proteins (SSBPs)
- Once helix is open, ssDNA is available for base pairing. - SSBPs bind specifically to single strands of DNA to prevent reformation of the helix - Supercoiling occurs as coiling tension is created at replication fork.
Dominant negative mutation
- One allele may encode inactive gene product that interferes with function. - Can result from haploinsufficiency.
3 major classes of MAE
- One class involves genes expressed in parent-of-origin pattern (genome imprinting) - 2 classes involve random monoallelic expression: - Random inactivation of X chromosome in females - Randomly generated allele inactivation
Inosine
- One of the modified bases found in tRNA, may pair with C, U, or A - It is a purine; hybrid between A & G
Cell wall
- Only in Plant Cells; supports and provides protection - composed of polysaccharide (cellulose)
Monoallelic expression (MAE)
- Only one allele is transcribed - Another allele is transcriptionally silent
Continuous DNA synthesis
- Only one strand can serve as template for continuous DNA synthesis (leading strand). - This strand is synthesized continuously toward the replication fork
EcoR1
- Restriction enzyme identified in E. coli: - Produces DNA fragments with cohesive ends. - ssDNA fragments from different sources can anneal (stick together) by H bonding.
Endoplasmic Reticulum: Smooth + ROugh
- Organelle compartmentalizes cytoplasm and increase surface area. - Smooth ER: Site of lipid (fatty acid) synthesis. - Rough ER: Studded with ribosomes: site of protein synthesis.
Aneuploidy
- Organism gains/losses one or more chromosomes but not a complete set - 2n +/- x
Dispersive Replication
- Parental strands are dispersed into two new double helices. - Idea here is that you have a "mixing" -sort of a cutting and pasting.
Examples of Trisomy
- Patau syndrome (trisomy 13) - Edwards syndrome (trisomy 18).
Sex-limited inheritance
- Patterns of gene expression affected by gender. - Expression of specific phenotype absolutely limited to one gender. - Patterns of gene expression may affect by gender even when not on X chromosome. - Expression of genes dependent on hormone constitution of individual - Ex: hen feathering
Penetrance
- Percentage of expression of mutant genotype in population.
Klinefelter syndrome (47, XXY)
- Persons with Klinefelter syndrome have: - male genitalia - Tall with large hands and feet - more than one X chromosome (usually XXY, or a 47, XXY karyotype
G2/M Checkpoint
- Physiological conditions in the cell monitored - If DNA replication or repair to any damaged DNA is not complete, the cell stays here - If cell conditions satisfied cell proceeds to mitosis
Bacterial Plasmid Vectors
- Plasmids used in DNA cloning: - Genetically modified bacterial plasmids—first vectors developed - Engineered to contain:Multiple cloning sites: Short sequence with restriction sites for common restriction enzymes
Proteins
- Polypeptides folded up into a functional 3D conformation - Fxn determined by structure + structure determined by a.a. composition, which is determined by genetic code
Agrose gel
- Porous matrix restricts migration of larger molecules more than it restricts smaller ones. - DNA can travel through this gel
Histones
- Positively charged proteins associated with chromosomal DNA in eukaryotes (note: DNA is neg. charged) - Play essential role in structure of proteins associated with DNA - 5 main types - Contain + charged lysine and arginine, which allows electrostatic bonding to phosphates
RNA editing
- Post Transcriptional RNA processing. - Insertion/deletion editing: Nucleotides are added/deleted from the total number of bases. - Substitution editing: Identities of individual nucleotide bases are altered..
Spliceosome
- Pre-mRNA introns are spliced out by spliceosome. - Consists of snRNAs that complex with proteins (small nuclear ribonucleoproteins (snRNP)): U1, U2, U3, U4, U5, U6
Dominant lethal allele
- Presence of one copy of allele results in death. - Mutation behaves as dominant lethal allele. - Ex: Huntington disease.
Intergenic mutations
- Present between genes - Upstream and downstream of genes - Regulatory elements - Suppressor mutations that occur elsewhere in the genome
Puff Regions
- Present on polytene chromosomes - Localized uncoiling during genetic activity. - Visible manifestations of high-level gene activity
miRNAs
- Primary-miRNAs (pri-miRNAs) form hairpin structures due to self-complementary sequences. - Enzyme Drosha removes non complementary 5'- and 3' ends to produce pre-miRNAs: - Exported to cytoplasm and cleaved by Dicer - Mature miRNAs associate with RISC to target complementary sequences on mRNAs. - Binding sites serve as miRNA response elements (MREs).
4 levels of protein structure
- Primary: sequence of amino acids - Secondary: α-helix and β-pleated sheets - Tertiary: 3-D conformation - Quaternary: Composed of more than one polypeptide chains
Non-Mendelian Genetics
- Principles of Mendelian inheritance apply, but the manifestation of the trait does not. - Traits do not segregate in accordance with Mendel's postulates
Restriction Enzymes
- Produced by bacteria as defense mechanism against infection by bacteriophage - Restrict or prevent infection by degrading DNA of invading virus. - Bind to DNA at specific recognition sequence (restriction site) and cuts DNA to produce restriction fragments - Enzyme cleaves both strands of DNA (digestion).
Nondisjunction during meiosis II
- Produces 2 normal gametes: - One gamete w 2 copies of a particular chromosome + One gamete deficient for that chromosome
Alternative splicing
- Produces different mature mRNAs from the same pre-mRNA by splicing out introns - Important regulation of gene expression - May generate sliceforms or isoforms of a protein - increases the # of proteins made from each gene
Alternative polyadenylation
- Produces spliceforms with different 3'-ends. - Exon containing signal is skipped. - Downstream signal is used. - Specifics different 3'- untranslated regions important for other post-translational regulatory events.
Transcription factors
- Promoters, enhancers and silences act as binding sites for transcription factors. - Serve to increase levels of transcription - Activators + Repressors
Product of MT2A gene
- Protein which binds to heavy metals and protects cells from toxic effects - Protects cells from oxidative stress - Expressed in low levels in all cells - Transcribed at high levels when exposed to Heavy metals + stress hormones
2 goals of NIH Roadmap Epigenomic Project
- Provide 1000 reference epigenomes of health and diseased individuals. - Delineate epigenetic differences in conditions such as Alzheimer disease, autism and schizophrenia. 2015: Study showed genetic variants associated with complex human disorders
Transversions (base substitution)
- Purine and Pyrimidine are interchange - Cytosine/Thymine to Guanine/Adenine and vice versa
Transitions (base substitution)
- Pyrimidine replaces pyrimidine, or purine replaces purine - C to T or G to A
Termination (transcription)
- RNA polymerase reaches a terminator sequence (Stop codon) - Causes newly formed transcript to fold back on itself—hairpin secondary structure - At times, termination depends on the rho(ρ) termination factor
Expressivity
- Range of expression of mutant phenotype. - Result of genetic background differences and/or environmental effects
polymerase chain reaction (PCR)
- Rapid method of DNA cloning - Extends power of recombinant DNA. - Eliminates need to use host cells for cloning. - Copies specific DNA sequence via in vitro reactions - Amplifies target DNA sequences present in very small quantities in population of molecules. - Sources include dried blood, semen, or hair
Telophase I (meiosis)
- Reappearance of nuclear membrane around dyads - Nucleus enters into short interphase period - Chromosomes do not replicate, since they already consist of sister chromatids
What was some direct evidence that supported the concept that DNA is the genetic material in Eukaryotes?
- Recombinant DNA technology (most convincing) - Genomics
X-Linked Diseases: Recessive
- Red, green color blindness - Duchenne muscular dystrophy - Severe combined immunodeficiency syndrome (SCIDS)
Loss-of-function mutation
- Reduces/eliminates function of gene product - Ex: Frameshift, Missense
Promoters
- Region of DNA recognized by RNAPII and transcription factors - Required for transcription initiation - Located immediately adjacent to regulatory genes - Specify site where transcription begins
Chiasma
- Region where chromatids are still intertwined - Point where nonsister chromatids have undergone genetic exchange through crossing over.
posttranslational modification
- Regulation by phosphorylation mediated by kinases and phosphatases - Regulation via ubiquitin-mediated protein degradation
Intragenic Mutation
- Regulatory elements: Promoters, enhancers, silencers, polyA signal - Exon - Intron - Splice sites and branch points - Suppressor mutation that can occur within the same gene that suffered the first mutation
Wobble Hypothesis
- Relaxed base pairing required of "wobble" -Initial two ribonucleotides of triplet codes often more critical than third - Third position: Less spatially constrained + need not adhere as strictly to established base-pairing rules
Cytoplasm
- Remainder of cell within plasma membrane, excluding nucleus - Includes extranuclear cellular organelles: Cytosol + Cytoskeleton
Histone deacetylases (HDACs)
- Remove acetyl groups from histone tails. - Recruited to genes by transcription repressor proteins
Repetitive DNA sequences
- Repeated many times within eukaryotic chromosomes. - Function genes present in more than one copy—multiple copy genes. - Majority do not encode for proteins. - Makes up most of our genome
Duplications
- Repeated segment of chromosome - Occur as a result of uneven crossing over btw synapsed chromosomes during prophase of meiosis - Pairing in heterozygotes produces compensation loop. - Arise through replication error prior to meiosis - Play an important role in evolution
What is the direction of DNA replication?
- Replication is bidirectional. - Two replication forks
Silencers
- Repress the level of transcription initiation - Acts as negative regulator of transcription - Acts in tissue- or temporal-specific ways in gene expression
Eukaryotic Transcription
- Requires binding of regulatory factors - Regulatory factors bind to specific DNA sequences called cis-acting DNA elements located in and around eukaryotic genes - trans-acting factors: DNA binding proteins that influence expression of genes
Requirements for the elongation process (translation)
- Requires several elongation factors. - Requires energy derived from hydrolysis of GTP
DNA Methylation and Cancer
- Researchers observed cancer cells had much lower levels of methylation than normal cells from the same tissue. - Genomic hypomethylation is a property of all cancers examined to date
Fxn of DNA pol. I
- Responsible for removing primer (on lagging strand) and synthesis that fills in gaps produced after removal.
Diseases of protein folding
- Scrapie (in sheep) - Bovine spongiform encephalopathy (mad cow disease) - Creutzfeldt—Jakob disease (in humans) - Transmittable brain disorder in mammals - Presence of prions (misfolded proteins) in brain - Huntington disease, Alzheimer disease, and Parkinson disease: (linked to formation of abnormal protein aggregates in brain)
3 Modes of DNA Replication proposed
- Semiconservative - Conservative - Dispersive
Nucleic Acid Electrophoresis
- Separates DNA and RNA fragments by size. - Smaller fragments migrate through gel at faster rate than large fragments. - Agrose gel used - DNA which is neg. charged, travels to pos. charge
noncoding RNAs (ncRNAs)
- Serve as post-transcriptional regulators of gene expression - Involved in RNAi (RNA interference) - Transcribed but not translated - Do not code for protein - 2 groups: Short ncRNAs Long ncRNAs
Telomeres in Disease, Aging, and Cancer
- Several human diseases associated with loss of telomerase activity and short telomeres - Connection between telomere length and aging topic of research and speculation. - Cells suffer chromosomal damage when telomeres become short—enter senescence—cell division ceases. - Human cancer cells retain telomerase activity—key to their immortality
Long noncoding RNAs (IncRNAs)
- Share many properties with mRNAs : 5' caps, 3' poly-A tails, Splicing - Lack extended open frame that codes for the insertion of amino acids into a polypeptide. - Do not have start and stop codons - Do not encode proteins. - Influence chromatin modifications - Alter patterns of gene expression - Function as competing endogenous RNAs (ceRNAs)
Shine-Dalgarno sequence
- Short sequence on mRNA. - bp with region on 16SrRNA. - Facilitates initiation of transcription in bacteria
Interspersed Retrotransposons
- Short, interspersed elements (SINEs) and long interspersed elements (LINEs) - Transposable mobile sequences—can relocate within genome. - Dispersed throughout genome rather than tandemly repeated.
Thomas Hunt Morgan
- Showed X-linked inheritance in studies of white-eyed mutation in Drosophila. - Inheritance pattern clearly related to sex of parent carrying mutant allele. - Study concluded that white locus is present on X chromosome and not autosomes
Taylor-Woods-Hughes experiment
- Showed semiconservative replication mode in eukaryotes - Used root tips of vicia faba - Monitored process of replication with labeled 3H-thymidine and performed autoradiography. - Similar to Meselson & Stahl, except that instead of looking at size of DNA, they looked at radioactivity.
When does complete disjunction occur?
- Shugoshin degrades - Cohesin complex is cleaved by separase - Sister chromatids are pulled toward opposite poles of cell
Termination (translation-prokaryotes)
- Signaled by stop codons UAA, UGA, UAG (do not specify any a.a.) - Release factors (RF1 or RF2): Stimulates hydrolysis of polypeptide form peptidyl tRNA - RF3 binds to ribosome and tRNA is released from P site of ribosome
Next-generation sequencing (NGS) technologies
- Simultaneous reactions synthesize DNA from tens of thousands of identical strands. - Use fluorescence imaging techniques to detect new strands. - Generate massive amounts of DNA sequence data rapidly and at reduced costs
Genetic Disease
- Single base mutations or small insertions/deletions - Replication dependent - Mutation leads to: Loss-of-function (null), Hypomorphic (reduced activity), Gain-of-function, Altered biochemical function
Viral and Bacterial Chromosomes
- Single nucleic acid molecule - Largely devoid of associated proteins - Much smaller than eukaryotic chromosomes - Contain less genetic information
Types of Mutations
- Single-base pair substitution - Insertion or deletion (INDEL) of one or more bp - Change in DNA content: deletion or duplication of large (100bp to million of bp) region - Copy Number Variant - Aneuploidy (change in chromosome number)
sliding clamp loader
- SlidingDNA clamp is a critical component of the DNA polymerase III holoenzyme consisting of five subunits that attach a circular protein complex to the polymerase in an ATP-dependent reaction. - Pair with core enzyme to facilitate function of sliding DNA clamp
What does initiation of translation require?
- Small + large ribosomal subunits (w 3 sites) - mRNA molecule, GTP, charged initiator tRNA, and three proteinaceous initiation factors (IFs).
Transfer RNA (tRNA)
- Small in size and very stable. - 75-90 nucleotides. - Transcribed from DNA as large precursors. - Contain modified bases. - Loops contain modified bases and do not form base pairs = clover leaf structure (hairpin) (this is a secondary structure dictated by bp) - Adapt specific triplet codons in mRNA to correct amino acid: - tRNA anticodons complement mRNAs - tRNAs carry correct amino acids
Fxn of the small and large subunit in elongation of translation
- Small subunit "decoded" triplets present in mRNA - Large subunit is responsible for peptide bond synthesis
Classifications of Mutations Based on their Location in the Body
- Somatic mutation - Germ-line mutation
Gain-of-function mutation
- Some mutations may enhance function of wild-type allelic function: - Increases quantity of gene product by affecting regulation of transcription of gene.
Promoters
- Specific DNA sequences in 5′ region upstream of the initial transcription point. - Promoter sequences govern efficiency of initiation of transcription - RNA polymerase σ factor responsible for promoter recognition
Focused core promoters
- Specific transcription initiation at start site - Major type used by lower eukaryotes - Have core-promoter elements - specify one specific transcript initiation site
Griffith Transformation Studies
- Speculated that transforming principle could be part of polysaccharide capsule or compound required for capsule synthesis. - Used Serotype IIR (rough, no capsule) which was avirulent + Serotype IIS (smooth, with capsule) was virulent to inject mice and study this.
Alternative splice site (type of alternative splicing)
- Splicing occurs at alternative splice site within exon. - May be upstream or downstream of usual splice site. - Some splice events may be errors. - Some instances are important regulatory events
7 visible features used by Mendel
- Stem height - Seed shape and color - Pod shape and color - Flower color and position
MT2A under stressful conditions
- Steroid hormone glucocorticoid is secreted. - Glucocorticoid receptor binds to glucocorticoid response element (GRE). - MT2A transcription enhanced - Transcription can be repressed by protein P Z120 which binds to transcription start site.
Histone code
- Sum of complex patterns and interactions of histone modifications. - Reversible enzymatic modifications of histone amino acids recruits nucleoplasmic proteins: further modify chromatin structure + regulate transcription
Classifications of Mutations Based on Location in Genome
- Suppressor: Intragenic + Intergenic - Autosomal mutation - X-linked and Y-linked mutation
Palindomes
- Symmetry exhibited by recognition sequences: - Nucleotide seq. reads the same on both strands. - Restriction enzymes cut DNA in characteristic cleavage pattern
Compensation loop
- Synapsis occur between normal homolog and chromosome with intercalary deletion. - Unpaired region of normal homolog must "buckleout" into deletion or compensation loop for synapsis to occur
What direction does RNA pol. synthesize the strand? What direction is it read?
- Synthesize in the 5'-3' direction - Reads it in the 3'-5' direction
DNA Methylation in mammals
- Takes place after replication and during differentiation - Involves addition of methyl group (-CH3) catalyzed by DNA methyltransferase (DNMTs)
5' Capping (image)
- The 5' cap is a guanosine triphosphate - It does a 5' to 5' linkage - By adding this cap, it no longer has a free 5' phosphate which could be recognized by a 5' exonuclease. - protects RNA from degradation + facilitates movement of RNA into cytoplasm
Mechanism of the Localization and translational regulation of actin mRNA.
- The RNA-binding protein ZBP1 associates with actin mRNA in the nucleus and escorts it to the cytoplasm. - ZBP1 blocks translation and binds cytoskeleton motor proteins (MP), which transport ZBP1 and actin mRNA to the cell periphery. - At the cell periphery, ZBP1 is phosphorylated by Src and dissociates from actin mRNA, allowing it to be translated by a ribosome (40S/60S). - Actin translation and polymerization at the leading-edge direct cell movement
47, XXX Syndrome
- The presence of three X chromosomes along with a normal set of autosomes (47, XXX) results in female differentiation. - Frequently, 47, XXX women are perfectly normal. - In other cases, underdeveloped secondary sex characteristics, sterility, and mental retardation may occur.
Discontinuous DNA synthesis
- The synthesis of DNA in discontinuous fragments on the lagging strand during replication. - The fragments, known as Okazaki fragments, are subsequently joined by DNA ligase to form a continuous strand.
Protein structure: Tertiary
- The tertiary level of protein structure in a respiratory pigment, myoglobin. - The bound oxygen atom is shown in red
What is the outcome of a male lacking an X chromosome?
- This condition in lethal. - Males do not develop
Trans-acting factors
- Trans = across from (on diff. chromosome) - bind to cis-acting DNA elements to influence gene expression - regulatory element
Transcription Factors (Eukaryotic Transcription)
- Trans-acting factors—facilitate RNAP II binding - Initiate transcription
RNAP II
- Transcribes protein-coding genes. - Highly regulated on gene-by-gene basis. - Activity dependent on cis-acting elements and trans-acting transcription factors - Core-promoter determines where RNAP II binds to DNA
Bacterial gene regulation
- Transcription and translation take place in cytoplasm - mRNA: translated directly + also degrades rapidly
Overview of transcription and translation in eukaryotes (image)
- Transcription occurs in nucleus: 5′ end of mRNA capped with 7-methylguanosine residue at maturation, which is essential for translation - Poly-A tail added at 3′ end of mRNA. - Translation occurs in the cytoplasm
What type of microscopy allowed scientists to see the cells in its highly varied organized structure?
- Transmission electron microscopy:
Retrotransposons
- Transposable elements generated via RNA intermediate (LINEs). - Note: there is some idea that these play a role in brain development
Codominance
- Two alleles of single gene produce two gene products. - Joint expression of both alleles in heterozygote. - No dominance or recessiveness
Conservative Replication
- Two newly synthesized strands come together' original helix is conserved. - The parental strand gives rise to an entirely new daughter strand, and parental strand remains together
Sister Chromatids
- Two parts of each chromosome - Genetically identical & visibly connected at centromere
Double Helical Model proposed by Watson + Crick **
- Two polynucleotide chains coiled around a central axis, forming a right-hand double helix - The two chains are antiparallel (5' --> 3' and 3' --> 5'). - The bases are flat structures lying perpendicular to the axis (stacked upon one another). - Each turn of the helix is 3.4 angstrom long (3.4 X10-10m) - Each turn includes 10 bases. - The helix consists of a larger major groove alternating with a smaller minor groove. - The double helix has a diameter of 20 Angstroms (2.0 nm)
Mutually exclusive (Alternative splicing)
- Type of cassette exon - Inclusion of one exon leads to exclusion of others in the same cluster. - Allows for swapping of protein domains encoded by different exons
DNA Methylation
- Type of chromatin modification - Plays role in gene regulation: Represses gene expression - Methylation occurs most often: at 5 position cytosine (5-methylcytosine) - On cytosine of CG doublets of DNA. o Methylatable CpG sequences concentrated on CpG islands - In mammals, methylation of DNA takes place after replication and during differentiation - Involves addition of methyl group (-CH3) catalyzed by DNA methyltransferase (DNMTs).
DNA Methylation
- Type of chromatin modification - Represses gene expression - Occurs most often at 5 pos. cytosine + on cytosine of CG doublets of DNA. - Methylatable CpG sequences concentrated on CpG islands - In mammals, takes place after replication and during differentiation - Occurs on cytosine bases adjacent to guanine base
miRNAs and siRNAs
- Type of short ncRNA - Transcribed as precursor molecules - Contain double stranded stem-loop and single-stranded regions. - RNAs act as repressors of gene expression - involved in RNAi - siRNA: exogenous (from outside the cell) - miRNA: endogenous (from inside the cell)
piRNAs (piwi-interacting RNA)
- Type of short ncRNAs - Form RNA-protein complexes that participate in epigenetic gene silencing in germ cells.
Ubiquitin-mediated protein degradation (image)
- Ubiquitin ligase enzymes recognize substrate proteins and catalyze the addition of ubiquitin (Ub) residues to create a long chain - Ubiquitinated proteins are then recognized by the proteasome, which removes ubiquitin tags, unfolds the protein, and proteolytically cleaves it into small polypeptides.
Euchromatin
- Uncoiled and active - Appears unstained during interphase
Complementarity of DNA strands
- Unwound double helix serves as template as each nucleotide along two parent strands has affinity for its complementarity nucleotide - Complementarity due to potential hydrogen bonds formed
Hershey and Chase
- Used E. coli and bacteriophage T2. - Demonstrated that DNA, not protein, is the genetic material - Used radioisotopes 32P and 35S - Demonstrated that DNA enters bacterial cell during infection and directs viral reproduction
Blue-White Screening
- Used to identify cells containing recombinant and nonrecombinant DNA - Plasmid contains lacZ gene, which encodes β-galactosidase
Norther Blot Analysis
- Used to study patterns of gene expression (RNA production) by cells and tissues - Both characterizes and quantifies transcriptional activity of genes
Fluorescent in situ Hybridization (FISH):
- Uses fluorescent probes to monitor hybridization. - Mitotic cells fixed to slides and subjected to hybridization. - Probe: ssDNA or RNA added, and hybridization monitored
reverse transcriptase
- Uses mRNA as template to synthesize cDNA. - Produces mRNA/cDNA duplex. - mRNA is partially digested with RNAse H: Produces gaps in RNA strand. - 3′- ends of mRNA serve as primers for DNA Poly I. - Results in double-stranded cDNA molecule—used for cloning
2 main transformation techniques
- Using calcium ions and brief heat shock to pulse DNA into cells - Electroporation: a brief but high-intensity pulse of electricity to move DNA into bacterial cells
Mutation rate
- Value that is calculated to estimate how likely a mutation will occur in a locus - Likelihood that gene will undergo mutation in single generation or single gamete. - Rate is low for all organisms. - Rate varies for gene to gene. - Viral and bacterial gene mutations are 1 in 100 million
What does Middle (or moderately) repetitive DNA include?
- Variable number tandem repeats (VNTRs) - Microsatellites (short tandem repeats, STRs)
Deletions
- When a chromosome breaks in one or more places + a piece is lost - Terminal: at end chromosome - Intercalary: within chromosome
Features of the genetic code
- Written in linear form using ribonucleotide bases that compose mRNA. - Each "word" consists of three ribonucleotide letters or triplet code. (Codon: every three ribonucleotides) - Unambiguous—each triplet specifies only one amino acid. - Degenerate: A given amino acid can be specified by more than one triplet codon - Contains "start:" and "stop" signals: triplets that initiate and terminate translation - Comma less: once translation begins, codons are read with no break - Nonoverlapping: Any single ribonucleotide within mRNA is part of one triplet - Nearly Universal: A single coding dictionary is used by viruses, prokaryotes, archaea, and eukaryotes.
Rosalind Franklin
- X-Ray diffraction analysis of DNA - DNA molecules are bombarded with X-Rays, which scatter based on atomic structure - Scatter pattern is captured and analyzed. - DNA showed 3.4 angstrom periodicity, a characteristic of helical structure.
Replication fork
- a Y-shaped point that occurs at the site of replication when the helix is unwound - 2 replication forks since DNA replication is bidirectional
Holoenzyme
- active form of DNA pol III - contains core enzyme complexes made up of subunits-provides catalytic function - each subunits have different functions
Repeating Copolymers
- chemically synthesized long RNAs - Short repeating sequences enzymatically joined short sequences together—produced long RNAs - Used for in vitro translation to determine more codon assignments
TATA box
- cis-acting DNA regulatory element - Binds TATA-binding protein (TBP) of transcription factor TFIID: determines transcription start site
Inversions
- class of structural variation - Rearrangement of linear seq. - No loss of genetic information. - Require two breaks in chromosome and subsequent reinsertion of inverted segment. - Chromosomal loop forms prior to breakage. - Creates "sticky ends"—close together and rejoin. - Inverted segment may not include centromere. - Genetic material is exchanged with segment of nonhomologous chromosome or transferred to another chromosome
Cell cycle
- continuous alternation between division and nondivision of cells: - Composed of interphase (G1, S, G2) and mitosis
What does UV radiation create?
- creates pyrimidine dimers :Two identical pyrimidines—distort DNA conformation
Eukaryotic closed-loop translation
- eIF4E binds to the 5'-cap on the mRNA and to a scaffold protein, eIF4G, which binds to poly-A-binding proteins (PABPs) on the poly-A tail of the mRNA - Ribosomes assemble at the cap, scan for the start codon, translate around the loop terminating at a stop codon, and may then reinitiate translation in a process called ribosome recycling.
Kinases (+ cyclins)
- enzymes products of mutated genes - serve as "master control" molecules - cyclins: proteins that bind with kinases → activating them at appropriate times during cell cycle.
Ribosomes
- essential role in expression of genetic information - Consist of ribosomal proteins and rRNAs - Contain 3 mRNA binding sites: A,E,P - Large + Small subunits - Prokaryotes = 70S - Eukaryotes = 80S
Alternative splicing of calcitonin/calcitonin gene-related peptide (CT/CGRP) in neurons
- exon 4 is spliced out—exon 5 and 6 are transcribed - 37-a.a. peptide hormone is made (CGRP). - Stimulates dilation of blood vessels.
What is the difference between Angelman's and Prader-Willi Syndrome?
- germ line it happens in - Fathers: Prader-Willi Syndrome - Mothers: Angelman's Syndrome
Viral Genetic Material
- inert until released into host cell - Able to package long DNA into small volume just like bacteria and eukaryotic cells.
DnaA
- initiator protein responsible for initiating replication by binding to a region of 9mers - encoded by DnaA gene - Binds to ORI, causing conformational change - causes helix to destabilize and open up & exposes single-stranded DNA
Ex of haploid (1 genome) organisms
- insects (male ants) - gametes (sperm and egg) - unicellular organisms (algae and fungi)
Pol α enzyme
- involved in RNA primer synthesis during initiation on leading and lagging strands - Possesses low processivity (the rate at which DNA is synthesized & strength of the association between enzyme and its substrate)
Monosomy
- loss of single chromosome from diploid genome. - produce a 2n - 1 complement - monosomy for any of the autosomes is usually not tolerated in humans and other animals.
Polycistronic mRNA
- mRNA produced that encodes more than one protein - Usually result from a group of bacterial genes that have products that function together + are clustered along a chromosome - All but the last gene lack termination sequence - rare in eukaryotes
Polyribosomes (polysomes)
- mRNAs with several ribosomes translating at once - mRNA passes through ribosome + is free to associate with another small subunit
Ubiquination
- major regulatory step - Ubiquitin ligases recognize, bind, and catalyze addition of ubiquitin residues
3 classes of Short ncRNAs
- miRNAs (microRNAs) - siRNAs (short interfering RNAs) - piRNAs (piwi-interacting RNAs)
Ex of diploid (2 genome) organisms
- most mammals -paternal and maternal genome
Mitochondrial DNA (mtDNA)
- mtDNA in most eukaryotes exists as double-stranded closed circle - Does not contain chromosomal proteins. - Introns mostly absent from mitochondrial genes. - Gene repetitions seldom present - Replication dependent on enzymes coded by nuclear DNA
Sex-determining chromosomes
- nonhomologous - Females are XX - Males are XY (Y is smaller)
rRNA
- rDNA: rRNA genes that are present in clusters at various chromosome sites - Each cluster consists of tandem repeats separated by noncoding spacer DNA sequence
Genetic Imprinting
- selective expression of either the maternal or paternal copy of a gene - Some genomic regions escape rounds of global demethylation and remethylation - Genes remain imprinted with methylation marks of maternal and/or paternal chromosomes. - Produces allele specific imprinting - remain transcriptionally silent (if methylated)
What snRNAs are involved in RNAi?
- siRNAs + miRNAs - Arise from different sources - Have similar mechanism of action
RNA interference pathway (image)
- siRNAs: Perfect matches affect translation + can lead to degradation - miRNAs: Not perfect match + inhibit translation
snRNA
- small nuclear RNA - 80-400 nucleotide RNAs rich with uridine residues
Probe (used in FISH)
- ssDNA or RNA added, and hybridization monitored - nucleic acids that will hybridize only with specific chromosomal areas.
Anti-codon base-pairing rules
- strands have to be antiparallel - So, the idea of the wobble hypothesis is that there is some wiggle room in the pairing that exists: (flexibility in the pairing of the system): The first two nucleotides are the most important Ex: AUG: A will pair with U, C will pair with G, however, G can pair with C or U (there is flexibility in the 3rd nucleotide). · Note: tRNA also has Inosine (I), which can pair with A, U, or C
Bivalent
- synapsed pair of homologous chromosomes - number of bivalents is equal to haploid number
Spermatogenesis (process)
-- Begins when undifferentiated spermatogonium germ cell enlarges to become primary spermatocyte -- Primary spermatocyte undergoes first mitotic division—produces haploid secondary spermatocytes. -- Secondary spermatocytes undergo meiosis II: Produce two haploid spermatids. -- Undergo developmental changes, spermiogenesis. -- Become motile spermatozoa or sperm.
Cell-Cycle Regulation
-- Cell cycle is genetically regulated and conserved throughout evolution. -- Disruption of regulation can lead to uncontrolled cell division characterizing malignancy. -- Mutations exert effect during cell cycle
Prokaryotic Organisms
-- Cells lack nuclear envelope and membranous organelles -- Long, circular DNA molecule compacted into nucleoid area -- Does not undergo extensive coiling like eukaryotic cells: DNA not as extensively associated with proteins. -- Prokaryotic cells lack distinct nucleolus but do contain genes for rRNA synthesis
Metaphase (mitosis)
-- Chromosome configuration following migration -- Centromeres align on metaphase plate
Metaphase I (Meiosis)
-- Chromosomes are at maximum shortness and thickness. -- Terminal chiasmata hold nonsister chromatids together. -- Tetrads bind to spindle fibers and moves chromatids to metaphase plate. -- Alignment of tetrads is random
Chromatin structure
-- Digestion of chromatin by endonucleases. -- Chromatin fibers are composed of linear array of spherical particles called nucleosomes -- Histones H2A, H2B, H3, and H4 occur as tetramers. -- Nucleosome core particle created when nuclease digestion is extended and 200bps of DNA is removed.
Telophase (mitosis)
-- Final stage of mitosis with two complete sets of chromosomes, one set at each pole -- Cytokinesis -- Chromosomes uncoil and become chromatin again -- Nuclear envelope reforms -- Spindle fibers disappear and nucleolus reforms -- Cell enters interphase
Oogenesis (process)
-- Formation of ova in ovaries -- Oogonium, undifferentiated germ cell, enlarges to become primary oocyte -- Two meiotic divisions produce daughter cells: receive equal amounts of genetic material, but do not receive equal amounts of cytoplasm - Almost all cytoplasm of primary oocyte is concentrated to one of the daughter cells. - Cell with little cytoplasm forms first polar body. - Secondary Oocyte: Receives bulk of cytoplasm. -- Undergoes secondary division -- Divides unequally: Produces two haploid cells: an ootid with bulk of cytoplasm and a second polar body -- Ootid differentiates into mature ovum -- Secondary division is completed after fertilization
Two phases of interphase
-- G1 (gap I) and G2 (gap II) occur in cytoplasm: -- No DNA synthesis occurs. -- Phase of metabolic activity and cell growth differentiation -- By end of G2, cell has doubled in size (DNA replicated and mitosis initiated)
Anaphase I (meiosis)
-- Homologous chromosomes separate -- Disjunction: Half of each tetrad (dyad) is randomly pulled to opposite poles. (Nondisjunction may occur—separation not achieved.) -- At the end of anaphase, I, number of dyads equals haploid number present at poles
Prophase (mitosis)
-- Period of chromosome movement -- Centrioles (in animal cells only) divide and move to opposite ends of cell. -- Nuclear envelope breaks down. -- Nucleolus disintegrates. -- Chromatin fibers condense and become visible chromosomes
Centromeres
-- Primary constrictions along eukaryotic chromosomes. -- It is largely made up of satellite DNA, the largely repetitive DNA. -- Play an important role in mitosis and meiosis -- Will be important for attaching to proteins -- Have minimal region that supports function of chromosomal segregation—CEN region. -- Location of centromere dictates appearance of chromosomes
Meiosis
-- Produces gametes or spores -- Only one haploid set of chromosomes -- Reduces amount of genetic material by ½ -- Fertilization restores diploid number -- Maintains genetic continuity from generation to generation -- Gives rise to genetic variation in gametes through: crossing over of homologous chromosomes
Cohesin
-- Protein complex that holds sister chromatids together. -- Complex formed during S phase. -- degrased by the enzyme separase
Kinetochore proteins
-- Region that binds to spindle fiber microtubules during cell division. -- an assembly of multilayered plates of proteins associated with the centromere
Primase (RNA polymerase)
-Recruited to replication form by helicase -Synthesizes RNA primer -Provides free 3'-OH required by DNA polymerase III for elongation
Eukaryotic splicing mechanism: self-excision
-Self-excision - Removal of introns from primary mRNA and tRNA transcripts produced in mitochondria and chloroplasts. - Referred to as group II introns - Group I introns occur in preliminary transcripts
Overlapping genes
-single mRNA has multiple initiation points -creates different reading frames -specifies more than one polypeptide
Stress induced epigenetic changes
-study on newborn rats raised with low levels of maternal nurturing --> rats did not adapt well to stress and anxiety-inducing situations in adulthood = increased methylation - Rats exposed to high levels of maternal nurturing resulted in adults that are stress adaptive = decreased methylation
How many ORI do bacteria have?
1
3 parts of the mRNA
1. 5' UTR 2. Reading frame: starts with AUG + ends with stop codon 3. 3' UTR
3 steps of elongation in translation (in a nutshell from video)
1. Binding of the tRNA to the ribosome 2. Formation of a peptide bond between adjacent a.a. 3. Movement of the ribosome to the next codon
Elongation (translation-prokaryotes) mechanism
1. Charged tRNAs transported into complex by elongation factors (EFs)—enter A site. 2. Terminal a.a. in P site links to a.a. in A site (energy supplies by peptidyl transferase) 3. Tripeptide forms and uncharged tRNA in P site moves to E site. 4. tRNA bound to peptide chain in A site moves to the P site, ejecting the uncharged tRNA in the E site 5. Polypeptide chain synthesized and exits ribosome.
4 cis-acting DNA elements that regulate transcription initiation
1. Core promoter (TATA box) 2. Proximal-promoter elements 3. Enhancers 4. Silencers (Note: these play a role in how the gene is transcribed)
Basic structure of chromatin
1. DNA 2. Nucleosome 3. Solenoid 4. Chromatin fiber 5. Chromatid
Sequence elements that are always present (RNA splicing)
1. DNA --> GT (dinucleotide sequence) 2. RNA --> GU ("") 3. Branch point: 7 nucleotides near the 3' AG splice site
PCR Steps
1. Denaturation—dsDNA is denatured into single strands 2. Hybridization/Annealing—Primers bind to ssDNA—starting point for DNA polymerase to synthesize new DNA strands 3. Extension—DNA polymerase synthesizes DNA strands
Determining mode of inheritance using a pedigree:
1. Determine whether the trait is dominant or recessive. If the trait is dominant, one of the parents must have the trait. Dominant traits will not skip a generation. If the trait is recessive, neither parent is required to have the trait since they can be heterozygous. 2. Determine if the chart shows an autosomal or sex-linked (usually X-linked) trait. For example, in X-linked recessive traits, males are much more commonly affected than females. In autosomal traits, both males and females are equally likely to be affected (usually in equal proportions).
2 main categories of transcription factors
1. General transcription factor (GTFs): Essential RNAP II can't bind without them + TFIIA, TFIIB, etc. TFIID binds to TATA box 2. Transcriptional activators and repressors: Bind to enhancer and silencer to regulate transcription.
3 Steps of Translation
1. Initiation 2. Elongation 3. Termination
RNA splicing group I Intron
1. Interaction between a free guanosine (G) + the primary transcript. 2. After G is positioned in the active site of the intron, its 3'-OH group attacks and breaks the phosphodiester bond (P) between the nucleotides at the 5'-end of the intron and the 3'-end of the left-hand exons. 3. The second reaction involves the interaction of the newly formed 3'-OH group on the left-hand exon and the phosphodiester bond at the right intron/exon boundary. 4. The exon is spliced out and the two exons are ligated, leading to a mature transcript
3 things present in Bacterial plasmid vectors
1. ORI - origin of replication 2. Antibiotic resistance gene 3. Multiple cloning site
Steps for cloning DNA with a plasmid vector
1. Plasmid DNA and DNA to be cloned are cut with the same restriction enzyme. 2. DNA restriction fragments from DNA to be cloned are added to linearized vector in presence of DNA ligase. 3. Sticky ends anneal. 4. Recombinant DNA is produced and introduced into bacterial host cells by transformation
What are the 2 kinds of nitrogenous bases?
1. Purines (AG) 2. Pyrimidines (CUT)
Transcription Initiation (image)
1. Que to transcribe gene 2. TFIID binds to promoter region of gene + looks for specific codes (TATA here) 3. Once code is found, TFIIA is recruited 4. 2 TFs assemble on promoter + recruit preinitiation complex (involves RNAP II + other TFs which will guide it to the promoter) 5. Elongation occurs after phosphorylation of the RNAPII 6. Leads to other TFs falling off and elongation takes place
Transcription Termination (Torpedo Model Image)
1. RNAP II searches for poly A cleavage enzyme 2. After cleavage, the poly A polymerase is going to add a bunch of adenines (A) to the end of the transcript that will protect RNA from 3' exonuclease 3. Because the RNA has been cut, there is a 3' -OH group and a 5' P available, which becomes exposed to be degraded by the cell 4. XRN2 is a 5' exonuclease, which recognizes a 5' P on RNA + degrades it
Polyadenylation signal sequence
1. RNAP II transcribes beyond 3'-end until it reaches Poly-A tail (AAUAAA) 2. Transcript is cleaved 3. RNAP II is destabilized 4. RNA and DNA are released - transcription terminated
4 major characteristics needed for a molecule to serve as genetic material
1. Replicate (cell cycle) 2. Storage of information (molecule acts as repository of genetic information) 3. Expression of information (information flow within the cell) 4. Allow variation by mutation (change in chemical composition of DNA)
2 groups of ncRNAs
1. Short ncRNAs (< 31 nucleotides) 2. Long ncRNAs (>200 nucleotide)
Mechanism of Termination (translation-prokaryotes)
1. Stop codon enters A site + RF1 or RF2 bind to codon, which triggers peptidyl transferase to cleave complete polypeptide from tRNA in P site 2. RF3 GDP binds to the ribosome, which stimulates release of RF from stop codon + ribosome 3. Deconstruction of complex
2 consensus sequences in E.coli promoters
1. TTGACA (pos. -35 with respect to transcription initiation site) 2. TATAAT (-10 region Pribnow box)
What 2 processes make up the central dogma of molecular genetics?
1. Transcription - synthesis of RNA from information in DNA 2. Translation - Uses information in mRNA to synthesize proteins
Types of base substitutions
1. Transitions 2. Transversion
Mendel's first 3 postulates
1. Unit Factors in Pairs: Genetic characters are controlled by unit factors (genes) existing in pairs in individual organisms. 2. Dominance/Recessiveness: Pair of two unlike unit factors for single characteristic in individual, one unit factor dominant, the other recessive. 3. Segregation: Paired unit factors segregate (separate) randomly during gamete formation (meiosis).
4 classes of R groups for a.a.
1.Nonpolar: hydrophobic 2.Polar: hydrophilic 3.Positively charged 4.Negatively charged
How many different polymerases does the human genome encode for?
14 different DNA polymerases, with only 3 being involved in DNA replication
What does a crossover within a paracentric inversion produce?
2 recombinants: - Dicentric chromatid: two centromeres - Acentric chromatid: lacking a centromere
What does a single crossover event within a heterozygous pericentric inversion give rise to?
2 viable + 2 inviable gametes
What portion of the eukaryotic genome constitutes protein-encoding genes?
2-10%
How many chromosomes are present in the human genome?
22 and an X and Y
What wavelength of UV light is absorbed by DNA and RNA? Protein?
260nm - DNA + RNA 280nm - Proteins (Note: molecule serving as genetic material is expected to absorb mutagenic wavelength - 260nm)
Function of the ε subunit of the DNA Pol. III Holoenzyme
3'-5' exonuclease
How many chromosomes are in a cell of the body?
46
Telomeric sequence in humans
5' - TTAGGG - 3' 3' - AATCCC - 5'
Function of the α(alpha) subunit of the DNA Pol. III Holoenzyme
5'-3' polymerization
Turner Syndrome (45, X)
= Persons with Turner syndrome usually have: - a single X chromosome - no Y chromosome (45, X karyotype) - female genitalia - webbed neck short stature - It is from these studies that we know that we only need one X chromosome - provide evidence that the Y chromosome determines maleness
What is expression?
A combination of how much of a gene is being transcribed and translated minus how much of the RNA is being degraded
Locus Heterogeneity
A monogenic trait caused by different variants/mutations in different genes
Suppressor mutation
A second mutation that either reverts or relieves the effects of a previous mutation
What is methionine encoded by?
AUG -- start codon
Wild-type (normal)
Allele that occurs more frequently in population
Allele
Alternative forms of a gene
Alleles
Alternative forms of the same gene
Peptide bond formation
Amino group of a.a. reacts with the carboxyl group of another amino acid during a dehydration reaction.
At what stage of meiosis does disjunction occur?
Anaphase I
What do introns usually begin and end with?
Begin with 5' GU End with 3' AG
submetacentric centromere
Between middle and end
Complex heterozygotes
Both alleles mutated but with different mutations on each allele
Nucleotides
Building blocks of nucleic acids
How is DNA packaged?
By histone proteins
How is DNA reproduced?
By semiconservative replication
Double stranded break
Can occur when a chromosome becomes fragmented internally as a result of DNA damage
Telomerase reverse transcriptase (TERT)
Catalytic unit of telomerase enzyme
Metaphase II (meiosis)
Centromeres are positioned at metaphase plate
What is the complementary strand called in transcription?
Coding strand
Proximal-promoter elements
Contain binding sites for sequence-specific DNA-binding proteins
Function of the θ subunit of the DNA Pol. III Holoenzyme
Core assembly
Phenotype
Physical expression of trait—genetic makeup (tall and dwarf).
Chromatin
DNA binding proteins
Avery, MacLeod, McCarty
DNA mediates transformation -- DNA is genetic information
What DNA polymerase(s) has 5'- 3' exonuclease activity?
DNA pol I
Which DNA polymerase has genes that activate for transcription when DNA synthesis is disrupted?
DNA pol II
Nitrogenous bases of DNA & RNA
DNA: A, C, T, G RNA: A, C, U, G
Marshall Nirenberg and Heinrich Matthei
Deciphered the first specific coding sequences, which served as a cornerstone for the complete analysis of the genetic code
Major types of chromosomal aberrations
Deletions Duplications Inversions Translocations
Fred Sanger
Developed synthetic DNA sequencing technique (Sanger Sequencing)
Synapsis
Each bivalent give rise to a unit - tetrad (two pairs of sister chromatids)
Prophase II (meiosis)
Each dyad composed of one pair of sister chromatids attached by common centromere
Fxn of DNA pol. III
Enzyme responsible for for 5′ to 3′ polymerization essential in vivo
polynucleotide phosphorylase
Enzyme that catalyzes production of synthetic mRNAs. - Does not require DNA template
RNA replicase
Enzyme that catalyzes the replication of RNA from an RNA template (needed for replication)
Kinases
Enzymes that catalyze addition of phosphate group to serine, tyrosine, or threonine amino acid chains
Where are the centromeres present at the end of prophase I of meiosis?
Equatorial plate
What is one of the main differences between bacterial and eukaryotic genome?
Eukaryotes have linear chromosomes, that are a lot bigger whereas Prokaryotes have a smaller circular genome
How many sites of origin replication are present in Eukaryotes?
Eukaryotic chromosomes contain multiple ORIs, which facilitate rapid synthesis of large quantity of DNA.
Chromatin at interphase
Eukaryotic chromosomes uncoil and decondense into chromatin
How do new genes arise?
Evolution by gene duplication
Alternative promoters (Alternative splicing type)
Exons that are not translated - do not code for protein
RNA Interference
Expression of a gene is "knocked-down" to study its function
What do the number of new mutations in a newborn depend on?
Fathers age at conception
· Encode - Encyclopedia of DNA Elements
Focused on collecting epigenome maps for cell lines grown in labs.
What is the most common genetic disorder to cause intellectual disability in humans?
Fragile X Syndrome
X-Linked Diseases: Dominant
Fragile X Syndrome Rett Syndrome
Genomics
Full set of DNA sequences in an organism
What technology can be used to generate gain-of-function mutations in flies?
GAL4-UAS system
Genotype
Genetic (allelic) makeup of individual for specific trait (DD, Dd, dd).
Karyokinesis
Genetic material evenly divided into two daughter cells during nuclear division (2 nuclei).
Chromosomal Theory of Inheritance
Genetic material is contained within chromosomes—transmitted from generation to generation.
What is the chemical affinity provided by in DNA?
H-bonds: - A=T: Double bond - C-G: Triple bond
5 main types of histones
H1, H2A, H2B, H3, H4
Copy number polymorphism
If population has more than 1% (deletion/duplication) it is called a polymorphism
Aneuploidy mutation
Involves a change in chromosome number (ex: monosomy or trisomy)
Meischner
Isolated a cell nuclei which contained a derived acid substance containing DNA -- nuclein (also present in chromosomes)
Daniel Nathans, Werner Arber, and Hamilton Smith
Isolation of restriction enzymes, the advent of recombinant DNA technology
Ex Question: The protein coding gene exon is 30 nucleotides, the coding part of the gene is 30 nucleotides, what do you think duplicating it will do to my copy of the gene?
It will maintain the frame (divisible by 3), but it will duplicate the amino acids that are coded for by the exon. So, the protein has 10 additional amino acids.
What is the outcome of a male with an extra X chromosome?
Klinefelter syndrome: These males may have large breasts & small testes & may be mentally retarded.
Elongation
Lengthening of growing polypeptide chain by one amino acid
Homology-directed repair (HDR)
Less error prone because it uses an undamaged homologous chromosome or sister chromatid as a template to correctly repair a broken chromosome
Locus (Loci plural)
Location of a gene on a chromosome
Locus
Location on chromosome where gene occurs
Where does oogenesis take place?
Ovaries
Cytoskeleton
Made of microtubules (which are made of the protein tubilin) and microfilaments (derived from protein actin), and provides lattice of support for structures within cell
Gene conservation
Many gene sequences are conserved (orthologs) from yeast to higher vertebrates and carry out similar functions in a wide range of organisms
DNA Methylation at Promoters (image)
Methylation patterns of CpG dinucleotides in promoters control activity of adjacent genes
What 2 processes are involved in genetic continuity of nucleated cells in eukaryotes?
Mitosis (daughter cells) + Meiosis (sex cells; gametes or spores)
What was some indirect evidence that supported the concept that DNA is the genetic material in Eukaryotes?
Mutagenesis -- Used UV Light (which can be a mutagen) to compare the action spectrum of UV and compare it to the absorption spectrum of molecules. (Note: UV light is most mutagenic at 260nm, which is the light DNA + RNA absorb)
insertion or deletion mutation
Mutation that involves insertion or deletion of one or more bp (1-100)
Inherited mutation
Mutation that is inherited from parent
Neutral mutation
Mutations in noncoding regions
Mutagen
Natural or artificial agents that induce mutations
Ex of question on exam: What are expected genotypes and phenotypes? Given the dominant and recessive traits
Note: To have recessive trait, both alleles have to be homozygous. To have the dominant, you can have one or the other
Insertion/deletion RNA editing
Nucleotides added/deleted from the total number of bases
Independent assortment leads to extensive genetic diversity
Number of different possible gametes: 2n, where n is haploid number
Proteome
Number of proteins organism can make, exceeds number of genes in genome.
In what direction does DNA pol. III synthesize DNA?
ONLY in the 5'-3' direction (toward replication fork)
Where does DNA replication begin?
ORI (origin of replication)
Somatic mutation
Occur in any cell except germ cells; not heritable
Germ-line mutation
Occur in gametes; inherited
Telophase II (meiosis)
One member of each homologous chromosome (now called monads) is at each pole
Paracentric vs pericentric inversion
Para = no centromere Peri = includes centromere
G0 (mitosis)
Point in G1 phase where cells withdraw from cell cycle and enter nondividing but metabolically active state
Polypeptides
Precursors of proteins
What are the defining characteristics of eukaryotes?
Presence of nucleus and membranous organelles
At what stage of meiosis does crossing (synapsis) over occur?
Prophase 1 of meiosis 1
Chaperons
Proteins that mediate folding process
Ubiquitins
Proteins that tag misfolded proteins and move them to proteasome for degradation
Structural difference between DNA and RNA backbone
RNA has hydroxyl group on the 2' Carbon
RNA homopolymers
RNA nucleotides with only one type of ribonucleotide (used in Nirenberg's study)
How does RNA polymerase differ from DNA polymerase?
RNA pol. does not require a primer for synthesis
3 RNA Pol. in Eukaryotes
RNAP I: transcribes rRNAs (involved in translation) RNAP II: uses to transcribe all protein coding genes + other regulatory RNAs RNAP III: used to transcribe rRNA and tRNA
What is the product of meiosis?
Reduces genetic content and leads to production of sex gametes, which contains half the number of chromosomes (n). -- 4 haploid cells
Genome editing
Removing, adding or changing specific DNA sequences in genome of living cells
Where is replication slippage more common in?
Repeat seq. (hot spots)
Frameshift mutations
Result from the addition or deletion of one or more nucleotides within a gene and subsequently the mRNA transcribed by it
Cytokinesis (Meiosis II)
Results in four haploid gametes from a single mitotic event
What does translation of mRNA require?
Ribosomes + tRNA
What is the most common method of DNA sequencing?
Sanger Sequencing
Disjunction
Separation of chromosomes during meiosis (occurs during anaphase)
Telomerase RNA component (TERC)
Serves as both a "guide" to proper attachment of the enzyme to the telomere and a "template" for synthesis of its DNA complement.
Microsatellites
Short tandem repeats (STRs)
Zygote
Single-celled fertilized egg
Ribosomes
Site where genetic information in mRNA is translated to protein.
Competing endogenous RNAs (ceRNAs):
Soak up miRNAs due to complementary binding sites MREs
Recombinant DNA Technology
Splicing together DNA sequences from different organisms (ex: Humane Hormone Insulin)
What studies were conducted by Nirenberg, Matthaei, and others in regards to the genetic code?
Studies to decipher the genetic code that involved synthesising synthetic polypeptides in a cell-free system using polynucleotide phosphorylase & RNA homopolymers + heteropolymers & radioactive tagging of amino acids
Fxn of Pol ε
Synthesized leading strand
Fxn of Pol δ
Synthesizes lagging strand
What do telomeres use to protect the ends of linear chromosomes?
T-loops & Shelterin complex that "tie off" the end of the chromosome & make them resistant to nuclease digestion and DNA fusions.
Activators
TFs that increase transcription initiation
Repressors
TFs that reduce transcription initiation
Neutral mutation
The gene product presents no change to phenotype
Deletions and Duplications
Total amount of genetic information in chromosome can change
Time spent in the cell cycle:
Total time: 16 hours (15 in interphase + 1 in mitosis)
Polyadenylation signal
Transcription of polyadenylation signal terminates transcription
RNA Sequencing (RNA-seq)
Transcriptome-wide analysis of genes expressed by cells within population
Which 2 amino acids are encoded by a single codon?
Tryptophan + Methionine
What is the outcome if a female only has one X chromosome?
Turners syndrome: sterile due to underdeveloped ovaries
RNA heteropolymers
Two or more different ribonucleosides were used to decipher relative proportion of each type of ribonucleotide diphosphate in synthetic mRNA (in nirenberg study)
What are the 3 codons that do not code for an amino acid (terminator codons)?
UAA UGA UAG - Are not recognized by tRNA - Translation terminates when these codons are encountered
What is tryptophan encoded by?
UGG -- only one codon
Genes
Units of inheritance—found in alternative versions called alleles.
What is the basis for DNA fingerprinting in forensics?
Variable number tandem repeats (VNTRs) & STRs
What do changes in DNA sequences result in?
Variations: - Phenotypic variability - Adaptation to environmental changes - Evolution
H-bonds
Weak bond between covalently bonded hydrogen and atom with unshared e- pair.
When is the MT2A gene transcribed at high levels?
When exposed to heavy metals + stress hormones
Who was the first to use the word "gene" "genotype" and "phenotype"?
Wilhelm Johannsen
Where do X-linked and Y-linked mutations occur?
Within genes located on X and Y chromosome, respectively
What are the mutagenic forms of ionizing radiation?
X rays, gamma rays, cosmic rays
Testcross
a way to determine whether an individual displaying the dominant phenotype is homozygous or heterozygous for that trait.
Quantitative real-time PCR (qPCR):
allows researchers to quantify amplification reactions as they occur in real time.
Where do the a.a. bing to the tRNA?
at the 3' end of the tRNA
Cytosol
colloidal material which surrounds cellular organelles
What is the largest source of genetic variation?
deletions/duplications
How many mutations does a newborn usually have compared to their parents?
genome contains average of 60 new mutations compared to parent
Homozygous
genotype has identical alleles (DD, dd).
Heterozygous
genotype has two different alleles (Dd).
what is regulation by phosphorylation mediated by?
kinases + phosphatases
Through what process is genetic material the source of variation amongst organisms?
mutation
Epimutations
mutations in imprinted genes caused by dysfunctional epigenetic changes
Spontaneous mutation
naturally occurring mutation
Acrocentric centromere
near the end
What is the misconception with mendelian genetics?
one gene, one trait
How many replicons constitute a bacterial chromosome?
one replicon
P arm and Q arm
p-shorter piece of chromosome q- longer piece of chromosome
Shugoshin enzyme
prevents degradation of cohesin at centromere
RNA splicing
process of removing introns and joining together exons
Gametogenesis
production of male & female gametes (spermatogenesis + oogenesis)
What does the variation in mutation rates among organisms reflect?
reflect efficiencies of DNA proofreading and repair.
Components of a nucleoside
sugar + Nitrogenous base
Components of a nucleotide
sugar + nitrogenous base + phosphate group (how DNA + RNA are put together)
How does transcription begin?
template strand binding by RNA pol. at the promoter
Where does spermatogenesis take place?
testes
What makes inversions and translocations unique from deletions and duplications?
that there is not a change in DNA content, rather the DNA is moved around the genome
Replicon
the length of DNA that is replicated
Proband
the person from whom the pedigree is initiated
Epigenetics
the study of environmental influences on gene expression that occur without a DNA change
How are plasmids introduced into bacteria?
transformation
Chromosome 15q Duplication Syndrome
· Autism spectrum disorder · Intellectual disability · Seizures · Hypotonia · Maternal (but not paternal) derived duplications of chromosome 15q11-13 encompassing UBE3A
Huntington's Disease: Coding Trinucleotide Repeat
· CAG repeat in exon (glutamine) · Normal allele = < 28 repeat units (not affected) · Pre-mutation allele = 29-35 repeat units (not affected) · Reduced penetrance/mutation allele = > 35 repeat units (partial penetrance) · Full mutation = > 40 repeat units (affected) · Expansion of CAG occurs entirely through paternal transmission · Expansion believed to occur late in germ cell (sperm) development · Somatic heterogeneity is extensive Expansion continues in somatic cells throughout lifetime
Transposable Elements (TEs)
- "jumping genes" - DNA sequences that move within and between chromosomes. - Insert themselves into various locations within genome. - Found in all organisms, precise function still unknown.
Whole Exome Sequencing (WES)
- Alternative to WGS - Patient's exome sequenced and compared to general population using bioinformatics - WGS and WES can be used together to identify mutations
Gene knockout
- An example of a loss of function mutation - Disrupt or eliminate specific gene/genes and see "what happens."
Large Animal Models: Spontaneous
- Animals acquire mutations through spontaneous processes, similar to the formation and inheritance of mutations in humans. - Animal is diagnosed with a condition and then the mutation is identified. - Dogs are a common large animal model - Population structure and high degree of inbreeding allow disease causing genes to be easily identified
What makes a good model organism?
- Be easy to grow - Short generation time - Produce abundant progeny - Be readily mutagenized and crossed - Faithfully mirror some aspect of the human condition
Nonhomologous end joining (NHEJ)
- CRISPR-Cas9 - Simply involves the ligation of broken DNA fragments. - This process is error prone and often results in small insertions or deletions (indels) at the repair site
Large Animal Models: CRISPR/Cas9
- CRISPR/Cas9 system is used to introduce frame-shift mutations or deletions in a gene of interest. - Gene of interest is targeted using a single-stranded guide RNA (sgRNA).
DNA microarray
- Can contain up to 500,000 different fields - Each field can represent different DNA sequence - Can test for mutations in single gene - Can include probes that detect SNPs
Limitations of CRISPR-Cas
- Cas9 can cut at off-target sites in genome. - sgRNA may have more than one perfect match. - sgRNA may direct Cas9 to sequences with one or few mismatches
Ex vivo gene therapy
- Cells from person with genetic condition removed - Treated in lab with therapeutic gene - Transplanted back into person - No immune system rejection
Nonviral delivery methods
- Chemically assisted transfer of genes across membranes - Nanoparticle delivery - Fusion of cells with artificial lipid vesicles (liposomes) - Short-term expression with "gene pills" is being explored.
Copia elements in Drosophila
- Class of DNA element transcribed into "copious" amounts of RNA. - Dispersed throughout genome and transposable to different chromosomal locations
Haplotypes
- Contiguous segments of DNA that do not undergo recombination - Distinguish which cfDNA segments are maternal and which are fetal
Applications of CRISPR/Cas9
- Creation of tomatoes that ripen quickly. - Fight off diseases in livestock. - Modify food crops for additional nutritional traits, pest, and drought resistance. - Most anticipated applications: Gene therapy + Clinical trials already underway for cancer
Replication Slippage
- DNA pol slop or sutters during rep. - Loop occurs in template strand - DNA polymerase misses looped out nucleotides—small insertions and deletions occur.
Direct-to-consumer (DTC) genetic testing
- DTC tests require saliva sample, cheek swab or hair sample to be mailed in - Testing based on SNP test such as ASO - Test is controversial: no involvement of health care professional - FDA does not regulates these tests
Intellectual property (IP) rights
- Debated as aspect of ethical implications of genetic engineering - Genes in their natural state cannot be patented - Genes cloned and patented by scientists could require licensing fees for its use.
Genome scanning
- Detect DNA mutations. - SNP sequences as probes on DNA microarray allow for screening of thousands of mutations. - Can analyze hundreds of disease alleles.
Genetic Testing
- Detect gene alterations associated with single-gene disorders - Gene tests used for prenatal, childhood and adult diagnosis of genetic disease
Consequences of free radicals
- Directly/indirectly affect genetic material. - Alter purines and pyrimidines. - Break phosphodiester bonds. - Produce deletions, translocations, and fragmentation
Common model organisms
- Drosophila melanogaster (fruit fly) - Danio rerio (zebrafish) - Mus musculus (mouse) - Large animal models: Dogs, pigs, non-human primates
Ethical, Legal, and Social Implications (ELSI) Research Program
- Established to address concerns when Human Genome Project was first discussed. - Focuses on four areas: - Privacy and fairness of genetic information - Transfer of genetic knowledge from lab to clinical practice - Participants understanding risks involved - Public and professional education.
Knock-in animals (Transgenic animals)
- Express or overexpress particular gene of interest (transgene): turning genes on instead of off - Vector with transgene undergoes homologous recombination into host genome. - Vector with transgene can also be put into ES cells and injected into embryos. - Allow for study of effects on appearance and function in mice.
CRISPR-Cas
- Fastest and most efficient approach - Discovered as genome editing tool by scientists studying how bacteria fight viral infections - uses nuclease Cas9
Restriction fragment lengthy polymorphism analysis
- First methods used for genetic testing - Primary method for detecting sickle-cell anemia - Only 5-10% of point mutations detected by RFLP analysis
Mutagen examples
- Fungal toxins - Cosmic rays - UV light - Industrial pollutants - Medical X rays - Chemicals in tobacco smoke
2 essentials/criteria for gene therapy
- Gene or genes involved in causing disease have been identified. - Gene can be cloned or synthesized in lab.
Genome Wide Association Studies (GWAS)
- Genomes from thousands of unrelated people with particular disease analyzed - Genomes compared to people without disease - Aim to identify genetic variations for developing disease
Diagnostic testing
- Identifies mutation or genetic change that causes condition - Usually used to confirm a disorder
Lentiviral vectors for gene therapy delivery
- Lentivirus: retrovirus that accepts large pieces of genetic material - Integrating virus: delivers therapeutic genes into the cytoplasm where reverse transcriptase converts RNA into DNA. DNA then integrates into the genome, ensuring that therapeutic DNA will be passed into daughter cells during cell division. - Can infect non dividing cells - Insertional mutation rate lower because virus does not gravitate toward gene-regulatory regions
Reverse Genetics
- Loss-of-function mutation induced in a particular gene to study its function - Ex: Homologous recombination + CRISPR/cas system
Tetracycline Inducible System
- Method of induced gene expression through the use of the antibiotic tetracycline or one of its analogs (e.g., doxycycline). - Bigenic system: requires 2 vectors (tTa, TRE) - The vectors are introduced into the genome via pronuclear transfer. Two transgenic mouse models are created. They are crossed together to make double transgenic mice (tTA/+; TRE/+).
Mouse (Mus musculus) models
- Mice are most widely used model in biomedical research. - Short generation times of 8-9 weeks and 8+ offspring per mating. - Similar body plans and stages of development as humans
Solutions to limitations of CRISPR-Cas in progress
- Modify Cas9 to improve specificity. - Improve sgRNA design via algorithms. - Find alternative enzymes from bacteria or archaea
White-apricot mutation in Drosophila
- Mutation changes wild-type eye color red to orange yellow. - Mutation of insertion of copia into the second intron of the white gene.
Forward genetics
- Mutations are randomly created to generate a desired phenotype
Structure of LTR retrotransposons
- Open reading frames (ORFs) encode the enzymes integrase and reverse transcriptase (RT). - Transcription promoters and polyadenylation sites are located within 5′ and 3′ long terminal repeats (LTRs).
What technologies can be used to generate loss-of-function mutations in flies?
- P element excision - The expression of a gene can also be "knocked-down" or reduced by RNA interference (RNAi). - CRISPR/Cas9
Ionizing radiation (as a mutagen)
- Penetrates deeply into tissues. - Causes ionization of molecules forming free radicals, which contain 1+ unpaired e-
Prenatal genetic tests
- Performed before baby is born - Fetal cells obtained by amniocentesis or chorionic villus sampling (CVS).
Primary goals for precision medicine
- Provide screening prior to treatment - Enables choice of drug and dosage to be tailored to patient's genomic profile.
Noninvasive procedures for genetic testing of fetal DNA
- Reduces risk to fetus - Uses cell-free DNA (cfDNA): DNA in bloodstream released from dead or dying cells - DNA cut into small fragments by enzymes in blood - Blood of pregnant woman has snippets of cfDNA from fetus. - Traces of fetal DNA tested for genetic conditions
ASO Testing
- Requires small amounts of DNA for analysis - Ideal for preimplantation genetic diagnosis (PGD): Genetic analysis of cells from embryo created by IVF - Single cell removed from early-stage embryo for analysis - Could kill embryo - DNA analyzed via FISH
The Newborn Sequencing in Genomic Medicine and Public Health (NSIGHT) research program
- Sequencing genome of 1,500 babies - Carrying out comparative genomic analyses of specific sequences - Aim is to identify genes involved in disease conditions
Single-Cell Sequencing (SCS)
- Sequencing sequence from a single cell - Isolate genomic DNA from single cell - WGA and PCR used to produce sufficient sequence - Valuable for analyzing somatic cell mutations and germ-line mutations. - Allows exploration of genetic variations from cell to cell.
Genetic Information Nondiscrimination Act (GINA)
- Signed into law 2008 - Designed to prohibit improper use of genetic information in health insurance and employment
Pharmacogenomics
- Study of how individual's genetic makeup determines body's response to drugs. - Development of drugs specifically targeted to patient's genetic profile.
Viral vector for gene therapy
- Successful gene therapy requires delivery system to transfer genes into patient's cells. - Genetically engineered viruses main tools used. - Viral vectors carry therapeutic DNA as payload. - Virus will infect target cells and successfully deliver the DNA.
Retrotransposons
- TEs that move within genome using RNA intermediate. - Copy and paste transposition method. (can be their own type of genetic variation) - Resemble retroviruses. - 2 types: LTR (long terminal repeat), non-LTR
CRISPR-Cas9 to disrupt gene fxn or correct mutation
- Takes advantage of eukaryotic cell's dsDNA break repair mechanisms: Nonhomologous end-joining (NHEJ) + Homology-directed repair (HDR) - Cas9 and sgRNA (single guide RNA) are introduced into cells in order to either disrupt gene function or create nonfunctional allele
Challenges to precision medicine
- Technology needs to be faster, cheaper and more accurate - Cost of genetic testing and counseling - Storage and interpretation of data - Need to develop automated health information technologies - Medical education for doctors
2 vectors of the Tetracycline Inducible System
- Tet Transactivator vector (tTA): Engineered with a specific promoter that expresses the transactivator protein, a transcription factor specific to the Tet Responsive Element. - Tet Responsive Element vector (TRE): Engineered with a specific gene that is expressed by the transactivator protein/tet responsive element.
2 methods to mutate the gene in large animal models using he CRISPR/Cas9 system
- The Cas9 enzyme and sgRNA are introduced into a zygote by pronuclear injection, generating a mutation in the developing animal. - The gene is mutated in a cell line (e.g., fibroblasts). The genetically engineered cell line is then cloned into an animal by somatic cell nuclear transfer (cloning).
What does gene conservation allow?
- The fundamentals of metabolism, development, and disease to be studied in simple lab organisms - The knowledge obtained to be applied to more complex eukaryotes
Cas9
- The most widely used CRISPR-Cas system for genome editing uses a nuclease called Cas9 - From bacterium Streptococcus pyogenes - Has two nuclease domains—creates double strand break in DNA. - Only cuts DNA near specific sequence protospacer adjacent motif (PAM) 5′-NGG-3′. - Cas9 requires single guide RNA (sgRNA) for activity and specificity.
In vivo gene therapy
- Therapeutic DNA introduced into affected cells - Challenge is restricting delivery to intended tissues and not all tissues throughout the body.
NIH National Center for Biotechnology Information (NCBI)
- To avoid misleading results based on genetic testing, ClinVar database was developed. - ClinVar integrates data from clinical genetic testing labs and research literature - Provides updated resources or researchers and physicians
Allele-specific oligonucleotides (ASOs)
- Unlike RFLP, ASOs detect single-nucleotide polymorphisms (SNPs). - ASO hybridizes with complementary DNA only - Genetic testing with ASOs and PCR available for many disorders. - ASO testing for sickle-cell anemia - SNPs can affect ASO probe binding leading to false positive or negative results.
Goal of personalized medicine
- Use patient's genome information to help diagnose disease. - Select treatment tailored to particular patient. - Gene-expression information derived from different tests
Embryonic Stem (ES) cells for the introduction of target vectors into cells
- Using ES cells, scientists introduce targeting vectors into cells via electroporation. - ES cell takes in targeting vector, and endogenous enzyme recombinase catalyzes homologous recombination. - Recombinant ES cells are injected into mouse embryo. - Results: chimeras
Single-cell RNA sequencing (scRNA-seq)
- We can now isolate DNA and RNA from same cells, sequence DNA and RNA. - Enables comparison of genes present in a cell - Comparison of relative levels of expression for each transcript encoded by genome.
When are large animal models developed?
- When small animal model does not recapitulate the condition - to use as preclinical models: - more physiological similar to humans - body size is comparable - complex phenotypes: behavioral and neurological
Duschenne Muscular Dystrophy (DMD)
- X linked recessive mutation in the DMD gene that causes progressive degeneration of skeletal and cardiac muscle. - Mouse model does not recapitulate condition in human. - Mutation in DMD identified in at least 8 breeds of dogs
Pronuclear injection
- You take a newly fertilized oocyte (zygote) + inject DNA into the actual zygote (fertilized egg) + the DNA would be incorporated into the genome to create a knockout moue - Limited because you couldn't target it into a specific site of a genome
Amniocentesis procedure
- involves placing a needle transabdominally through the uterus into the amniotic sac and withdrawing some of the fluid. - The fluid contains sloughed fetal cells that can be cultured. - These cultured cells can then be karyotyped and also utilized in DNA tests.
Adenovirus vectors
- non integrating virus - Used in early gene therapy trials - Capable of carrying large number of therapeutic genes - deliver therapeutic genes without integrating them into the genome of target cells. - Delivered DNA resides as minichromosomes (episomes), but over time as cells divide, these nonintegrating hoops of DNA are gradually lost - Disadvantage: Humans produce antibodies against adenoviruses that render therapeutic genes ineffective.
UV light (as a mutagen)
- one of most recognized mutagens - Short wave lengths can disrupt organic molecules. - Purines and pyrimidines absorb UV radiation at 260nm
Why is Drosophila amendable to genetic analysis?
- the ease with which it can be grown - the size of its genome - its short generation time of about 10 days from fertilized egg to adult - its ability to produce about 3000 offspring in a lifetime - only moderate crossing over in females and none in males, simplifying genetic analysis. - Flies are amendable to forward genetic screens
Random mutagenesis
- used to generate one mutation per genome so that only one gene product is disrupted in each organism - UV light, ethyl methane sulfonate (EMS), and nitrosoguanidine cause single bp changes or small indels
Preimplantation Genetic Diagnosis (PGD) procedure
1. A single cell from an early-stage human embryo created by in vitro fertilization can be removed and subjected to preimplantation genetic diagnosis (PGD) by ASO testing. 2. DNA from the cell is isolated, amplified by PCR with primers specific for the gene of interest, and then subjected to ASO analysis. 3. In this example, a region of the gene was amplified and analyzed by ASO testing to determine the sickle-cell genotype for this cell.
Mechanism of the CRISPR-Cas9 genome editing system
1. An sgRNA guides Cas9 to cleave a target site adjacent to a PAM sequence 2. The double-stranded DNA break can be repaired by: NHEJ, which introduces insertions or deletions (indels), or by HDR, which can make specific edits using an introduced donor template
Creating knockout mice
1. Construct targeting vector: Creates segment of DNA for introduction into cell 2. Targeting vector Undergoes homologous recombination with gene of interest and renders it nonfunctional. 3. Target vector has mutated copy of gene of interest 4. Foreign DNA disrupts reading frame and produces nonfunctional protein
3 methods to generate mouse models
1. Pronuclear injection 2. Homologous recombination in embryonic stem (EC) cells 3. CRISPR-Cas9
2 types of large animal models
1. Spontaneous models: naturally occurring genetic mutations 2. Genetically engineered models: Developed by homologous recombination in fibroblast cells or the CRISPR/Cas9 system
Gene Knockout: Homologous Recombination
1. Vector is engineered with gene of interest. 2. The open reading frame is disrupted by insertion of a selectable marker (e.g., neomycin resistances gene). 3. Vector is electroporated into ES cells, where neomycin gene is incorporated into the gene of interest by homologous recombination, creating a mutant/null allele of the gene. 4. The genetically engineered ES cells are injected into a mouse blastocyst and then implanted into a recipient female, generating chimeric mice. 5. The chimeric mice are bred to wild-type mice to transmit the null allele.
chorionic villus sampling (CVS)
A technique associated with prenatal diagnosis in which a small sample of the fetal portion of the placenta is removed for analysis to detect certain genetic and congenital defects in the fetus.
What allows for the knock in, knock out, and gene targeting in mice?
Properties associated with mouse embryonic stem cells
Pros and Cons of a Model Organism
Pros: - Amendable to genetic manipulation - Ethics - Costs - Human disease often affects animal in similar Cons: - Ethics - Costs - Human disease may not affect animal in similar way
What gene family are the genes involved in drug metabolism a part of?
cytochrome P450 gene family
Pharmacogenetics
study of how sequence variations within specific candidate genes affect individual's drug responses.
