Exam 2 Flashcards
Haplosufficient
Promote a wild type function or full expression in only one copy
Dominant negative
As a heterozygote wipes out a gene function by a spoiler effect on the protein
Incomplete or partial dominance
Intermediate phenotype
Temperature Sensitive mutants
Allele can be expressed as both permissive and restrictive temperature, protein only encoded if permissive
Pleiotropic
When a single gene has multiple effects
Archibald Garrod
Discovered that many diseases show defects in metabolism
Beadle and Tatum
Used Neurospora to discover that one gene will lead to the production of one protein (one-gene-one-polypeptide)
Inferring Gene Interactions
1) Obtain and select a mutant, confirm single gene inheritance and asses phenotype
2) Test mutant for allelism and confirm the total number of genes involved
3) Combine the mutants into pairs to form double mutants to see if the genes interact
Complementation Test
Distinguishes if two mutant alleles are on the same gene/enzyme
Recessive Epistasis
A mutant allele of one gene masks the expression of the mutant allele of another gene and expresses its own phenotype
Suppressor Screens
Mutate the mutant and look for WT phenotype, looks for a return to an original state
Penetrance
Percentage of individuals with a given allele who shows the phenotype
Expressivity
Degree to which an allele is expressed, the intensity of the phenotype
Fredrick Griffith
1928, worked with pneumonia in mice to assume there had to be some biological matter transferring
Oswald Avery
1944, continued Griffith’s experiment with mice and found evidence of DNA
Hershey-Chase
1952, confirmed evidence of DNA by using phage and T2 virus
Key Properties of DNA
1) Makes copies of itself
2) Encodes information
3) Is able to mutate
Structure of DNA
Phosphate, deoxyribose, and the four nitrogenous bases
Purines
Adenine and Guanine
Pyrimidines
Cytosine and Thymine
Chargaff’s Rules
Amount of A = T and amount of C = G
Double Helix
Discovered by Watson and Crick with the help of x-ray differentiation images done by Rosalind Franklin and Maurice Wilkins
Semi-Conservative Replication
Some parental and some new DNA
Conservative Replication
Parental is used as a template
Dispersive Replication
Two copies of two different mixtures of parental DNA and the new strand
Meselso-Stahl
Confirmed semi-conservative replication in 1958 using E.Coli
Arthur Kornberg
Discovered DNA polymerase I in 1955 using E.Coli
DNA Polymerase I
Grows the DNA from 5’ to 3’ and removes mismatched bases from both ends (exonuclease) and removes RNA primers and fills gaps on lagging strand
DNA Polymerase III
Acts as a replication fork and elongates the RNA primers with new DNA
Lagging Strand
When synthesized it fragments the new DNA. Need RNA primase, DNA pol III & pol I, and ligase
Ligase
Connects the adjacent Okazaki fragments on a lagging strand
Replisome
Coordinates the activities of the replication fork, has helicases and topoisomerases. Copies the parental strands and disassembles/reassembles the nucleosomes
Beta Clamp
On the replisome, where is encircles the DNA like a donut and keeps pol II attached to the DNA molecue
Chromatin
Makes up chromosomes.
Basic unit is the nucleosome, which is DNA wrapped around histone proteins
Telomeres
Two ends of the linear DNA molecules that form caps. Tends to be shortened during the replication process, so non-coding repeats are added to the end
Telomerase
Adds the short non-coding repeats to the 3’ ends of the DNA molecules, plus it carries a small RNA molecules that acts a template for the synthesis of the telomeric unit
Exons
Encode proteins
Introns
Non-coding sequence that separates exons
Spliceosome
Removes introns and joins the exons together
Properties of RNA
1) usually single stranded
2) has a ribosome sugar in its nucleotides
3) uses uracil instead of thymine
mRNA
Messenger RNA, template for protein synthesis
tRNA
Transfer RNA, brings amino acids to the site of protein synthesis and has an anticodon. Is the adapter between amino acids and RNA template
rRNA
Ribosomal RNA, site of protein synthesis
snRNA
Small Nuclear RNA, spliceosome, removes introns
miRNA
MicroRNA, regulates gene expression
siRNA
Small Interfering RNAs, protects the integrity of the genome and against pathogens
Transcription Initiation
RNA polymerase binds to a specific promoter
Transcription Elongation
RNA polymerase unwinds the DNA and creates a transcription bubble, where bases are paired
Transcription Termination
1) Rho factor recognizes the termination signals for RNA polymerase
2) Stop codon is reached in a hairpin loop
Differences in Transcription
1) Eukaryotes have more genes, so the job is divided among three RNA polymerases
2) General Transcription Factors are needed
3) Prokaryotic DNA is naked and circular
RNA Polymerases
I transcribes rRNA
II transcribes protein-encoding genes
III transcribes small functional RNAs
Modifications of mRNA in Eukaryotes
1) Addition of a 5’ cap
2) Splicing of the introns
3) Polyadenylation and the poly tail
Carboxyl Tail Domain
Initiation phase ends and the elongation phase begins after the tail has phosphorylated. Has a sequence of repeats that can serve as binding sites.
Ends Required for Splicing
GU at the 5’ and AG at 3’ end
Cech
Won the Nobel Prize in 1989 for discovering self-splicing introns
RNA Interference
Co-Suppression = Transgene Silencing
DICER
Binds to long dsRNAs and cleaves them into small dsRNA fragments, they are unwound so that RISC to chop up into mRNA
Primary Structure
Where amino acids are located
Secondary Structure
Alpha and Beta sheets, in 2D
Tertiary Structure
3D structure
Quaternary Structure
Groups of 3D structures and polypeptides
Stop Codons
UGA, UAA, and UAG
Niernberg
Made synthetic RNA of UUUUUU and inserted it into E.COli to determine that nucleotides were read in triplets
tRNA Synthetase
Recognizes tRNA and corresponding amino acid
Wobble
Allows one tRNA to recognize multiple codons
Silent Mutation
Changes one amino acid into another for codon for the same amino acid
Missense Mutation
Nucleotide-Pair substitution leads to a replacement of an amino acid
Nonsense Mutation
Changes a codon into a stop codon
Frameshift Mutation
Insertion or deletion of nucleotide pairs, disrupting the reading frame
Ribosomes
Work with rRNA and mRNA to make protein. Has A, P, and E sites
Small Subunit
Binds to the mRNA and the large subunit attaches
Translation Initiation
Begins at AUG, methionine, the first aminoacyl tRNA is laced into the P site
Shine-Dalgarno Sequence
Is needed for prokaryotes to start translation because it indicated what AUG to use
Translation Elongation
mRNA acts as a blueprint to specify the delivery of the tRNAs, each amino acid is added to the polypeptide chain. Eventually tRNA is transferred to the A site
Translation Termiantion
Happens once a stop codon is reached and the subunits will separate. A water molecule in the peptidyl transferase center leads to the release of the peptide chain.
Posttranslational Folding
Happens inside the cell if the protein still needs to be in its correct 3D shape, with the help of chaperones
Phosphorylation
Kinases attached phosphate groups to the hydroxyl group of the amino acids to change protein conformation after translation
Ubiquitination
A protein is marked for degradation after translation
Polymerase Chain Reaction
A specific gene or DNA region is isolated and amplified by DNA polymerase. Target DNA can be inserted into a plasma or use vectors
PCR Process
1) Add primers
2) Heat to separate the strands, 95* C
3) Cool, the primers will anneal, 55-65* C
4) Heat to 72*C to allow for synthesis (Taq)
Restriction Enzymes
Cut the DNA into fragments, creating sticky ends
cDNA
Complementary DNA is a DNA version of mRNA made with the use of reverse transcriptase. Represent only the coding region of the genome
Reverse Transcriptase
Catalyzes the synthesis and copies the cDNA
Probes
Used to find and mark the desired clone. (homologous genes and protein products)
Gel Electrophoresis
Separates the molecules of the mixture. Molecules head towards the positive charge.
Southern Blotting
Imprints DNA molecules
Northern Blotting
Imprints RNA molecules
Western Blotthing
Imprints proteins
Dideoxy Sequencing
Sanger Sequencing
1) Primers are created that hybridize to one location on the cloned segment
2) Added to a tube with DNA polymerase, dNTPs, and ddATP
If bound to ddATP the chain will stop growin
Gene Gun
Blasts gold or tungsten coated DNA particles, where it then travels to the nucleus and inserts itself into a chromosome
Agrobacterium
Has the TI plasmid, which causes tumors to grow that transfer some of its DNA into the host plant’s genome (T-DNA)
Ectopic Insertions
Transgenes are inserted randomly into a genome using multicopy arrays
Gene Targeting
Sequence is inserted into a location occupied by a homologous sequence in the genome, which is replaced
