AP Bio Unit 6 - Barron's 2020

Transformation experiment

Frederick Griffith discovered bacteria can make harmless cells virulent by transferring genetic factor from one bacterium to another (bacterial transformation)

Transformation factor experiment

Avery, MacLeod, McCarty discovered that transformation factor is DNA (not protein) through direct experimental evidence

Transformation factor supporting experiment

Hershet and Chase tagged bacteriophages w/ radioactive P and S; proteins sulfur not phosphorus, DNA phosphorus not sulfur; infected bacteria contained only tagged phosphorus (only bacteriophage DNA)

DNA shape photo experiment

Rosalind Franklin performed X-ray crystallography analysis of DNA showing it to be a helix (famous Photo 51); critical to Watson and Crick, but not credited

DNA structure proof

Watson and Crick used Franklin Photo 51 and biochemical analysis of DNA to predict double helix structure of DNA

DNA replication experiment

Meselson and Stahl proved that DNA replicates semiconservatively; bacteria used heavy N in DNA then transferred to light N and strands had mid density (showing that one strand heavy and one strand light)

DNA

deoxyribonucleic acid; double helix; two ANTIPARALLEL strands; one strand 5’ to 3’ and one 3’ to 5’ (upside down); polymer of repeating units of nucleotides (5-C sugar deoxyribose, phosphate, nitrogenous base); purine adenine, purine guanine, pyrimidine thymine, pyrimidine cytosine); bases bound by hydrogen bonds (2AT 3CG)

Purine

two rings, adenine and guanine (Pur(ine)e Guardian Angel)

Pyrimidine

one ring, thymine, uracil, and cytosine

Adenosine-thymine bond

double hydrogen bond

Cytosine-guanine bond

triple hydrogen bond

Histone

protein around which DNA is “wrapped” twice to form nucleosome structures/chromatin all the time except replication

RNA

ribonucleic acid; single-stranded helix; 5-C sugar is ribose; purine adenine, purine guanine, pyrmidine URACIL, pyrimidine cytosine)

DNA replication

semiconservative; starts at origin of replication (ORI) where two strands DNA separate to form replication bubble expanding BOTH DIRECTIONS AT ONCE; at end of each bubble is replication fork; DNA polymerase builds new strand 5’ to 3’ off preexisting RNA primer chain made by primase; single stranded binding proteins hold strands apart, helicases unwind, topoisomerases lessen tension by breaking rejoining

DNA ligase

enzyme that “glues together” Okazaki fragments

Helicase

enzyme that untwists double helix at replication fork

Single-stranded binding proteins

scaffolding that hold DNA strands apart during replication

Topoisomerase

lessens tension on tightly wound DNA helix by breaking and rejoining

Mismatch repair

proofreading carried out by DNA polymerase that corrects errors

DNA polymerase

enzyme that builds out (catalyzes) DNA 5’ to 3’ but CANNOT INITIATE SYNTHESIS (must attach to RNA primer) at about 50 nucleotides/sec in humans

Primase

creates RNA primer for DNA replication by joining RNA nucleotides

DNA nuclease

enzyme that excises damaged regions of DNA

Telomeres

nonsense nucleotide sequences found at the end of eukaryotic chromosomes that repeat thousands of times to guard against possible loss of genes during replication; created and maintained by telomerase; gets shorter with age, “clock” for end of cell life/division

Telomerase

enzyme that creates and maintains telomeres

How DNA makes proteins

triplet code in DNA transcribed into codon sequence in mRNA (preRNA) which is processed/modified in nucleus and translated into amino acid sequence (polypeptide) in ribosome in cytoplasm

Transcription

DNA copied into mRNA; 3 stages: initiation (RNA polymerase binds to DNA at promoter region and transcription factors recognize TATA box for RNA polymerase to bind to), elongation (RNA polymerase adds nucleotides TO 3’ END of chain to copy transcription unit), termination (continues for a bit after termination sequence AAUAAA then cut free)

RNA polymerase

binds to DNA at promoter region, then to TATA box as forms transcription initiation complex with transcription factors; adds nucleotides to 3’ end of chain, copying transcription unit of DNA

Transcription initiation complex

transcription factors and RNA polymerase bound to promoter on DNA

RNA processing

5’ cap added to help ribosome bind; poly (A) tail added to 3’ to protect from enzymes and release from nucleus; INTRONS SPLICED OUT BY snRNPs within SPLICEOSOMES

snRNPs

small nuclear ribonucleoproteins that act within SPLICEOSOMES in nucleus to SPLICE OUT introns/intervening sequences from mRNA

Alternative RNA splicing

process by which DIFFERENT RNA molecules are produced from the SAME TRANSCRIPT depending on what is treated as EXON (expressed) vs. INTRON (removed)

Translation

amino acids carried by tRNA (amino acid + anticodon) to mRNA (codon) and joined by aminoacyl-tRNA synthetase; uses GTP (guanosine triphosphate) AS ENERGY; 3 stages: initiation (mRNA attaches to ribosome subunit, AUG), elongation (tRNA brings AAs, polypeptide formed simultaneously by several ribosomes POLYRIBOSOME clusters), termination (ribosome reaches 1 of 3 stop codons and release factor breaks bond)

rRNA

involved in transLATION and makes up ribosome along with proteins; has 1 mRNA binding sites and 3 tRNA binding sites (A, P, E)

Start codon

AUG (also methionine)

Stop codons

UAA UGA UAG

Wobble

base pairing for 3rd base of codon not as strict, tRNA can bind with multiple codons (ex. UCU UCC UCA UCG all code for SERINE)

Somatic cell

body cell

Gamete

sex cell

Mutations

SPONTANEOUS and RANDOM permanent changes in genetic material caused by MUTAGENIC AGENTS (toxic chemicals, radiation); RAW MATERIAL for NAT SELECTION

Point mutation

base-pair substitution, simplest (ex. Sickle cell anemia); can be harmful, beneficial, or no effect (wobble)

Frameshift mutation

nucleotide insertion or deletion alters entire reading frame of DNA; causes MUTATED OR NO POLYPEPTIDE (nonsense mutation)

Virus

DNA or RNA enclosed in capsid (protein coat), sometimes with viral envelope cloaking capsid and aiding with infection; binds to SPECIFIC RECEPTORS on ONE CELL TYPE (ex. Cold only respiratory, AIDS only one type white blood cell)

Host range

the types of cells a virus can infect; expansion can lead to emergence of new viral disease

Bacteriophages

aka phages; reproduces via lytic cycle (break in, take control, replicate, burst) (if only lytic, VIRULENT PHAGE) or lysogenic cycle (replicate within host cell WITHOUT DESTROYING it by becoming incorporated into site in host’s DNA, remaining dormant) (called PROPHAGE, can SWITCH to lytic); viruses that can use lytic and lysogenic = temperate viruses

Lytic cycle

performed by virulent phages or temperate viruses; use cell to reproduce then burst it

Lysogenic cycle

performed by prophages or temperate viruses; incorporate into host DNA and remain dormant without killing it to replicate self, can become lytic based on environment

Retroviruses

viruses containing RNA instead of DNA which serves as template for complementary DNA (cDNA), REVERSING DNA->RNA flow; process under direction of reverse transcriptase; usually incorporates into host genome as prophage (ex. HIV, which causes AIDS)

Generalized transduction

moves random pieces of bacterial DNA as phage lyses one cell and infects another, leading to GENETIC RECOMBINATION

Restricted (specialized) transduction

transfers specific pieces of DNA; during lysogenic cycle, takes some of surrounding host genome when leaves and inserts into next host, INCREASING GENETIC DIVERSITY

Transposons

sections of DNA transferred by viruses via transduction

Nucleoid

area in prokaryotes that contains DNA with NO cell membrane

Bacterial DNA

circular, double-stranded DNA molecule tightly condensed into a structure with a small amount of protein; replicated in BOTH DIRECTIONS from SINGLE ORI

Binary fission

main form of bacterial reproduction; asexual, identical offspring with rare spontaneous mutations

Plasmid

FOREIGN, small, circular, self-replicating DNA molecule in a bacterium; bacterium carries many and expresses genes therein; how transformation works

F plasmid

Fertility plasmid; 1st plasmid discovered; contains genes for PILI, cytoplasmic bridges that connect to other cells and allow DNA to move between (conjugation)

R plasmid

makes cell resistant to specific antibiotics (ex. Ampicillin, tetracycline); can be passed via conjugation; ANTIBIOTIC RESISTANCE (gives advantage and can be passed, so big issue)

Operon

found only in bacteria; model of GENE REGULATION; set of genes and switches that control the expressions of those genes; 2 types: repressible (tryptophan) and inducible (lac)

Tryptophan operon

promoter and 5 adjacent structural genes (A B C D E) that code for 5 separate enzymes necessary for tryptophan synthesis; RNA polymerase bound to promoter = one long strand of mRNA w/ start and stop codons transcribed; if tryptophan present, it acts as COREPRESSOR activating REPRESSOR which binds to OPERATOR, PREVENTING RNA POLYMERASE FROM BINDING to promoter, ceasing transcription; called REPRESSIBLE because ALWAYS ON unless repressor activates

Repressor

protein that inhibits gene transcription; binds to operator to prevent binding to promoter (ex. Tryptophan is corepressor that activates repressor that binds to promoter, preventing RNA polymerase from binding and further tryptophan synthesis)

Lac operon

INDUCIVE; contains genes for enzymes for lactose breakdown, Z (beta-galactosidase), Y (permease), and A (transacetylase); usually repressed (NEGATIVE CONTROL), so repressor must be prevented from binding to operator and RNA polymerase must bind to promoter; ALLOLACTOSE INDUCER that binds to ACTIVE REPRESSOR TO INACTIVATE IT; drinking milk = ingesting allolactose = deactivate repressor = RNA polymerase binds to DNA promoter = transcription of lac genes = digestion of lactose

Gene regulation example

OPERON

Negative control

genes in operon ACTIVATED BY DEFAULT unless switched off by repressor (repressor inactive = turns on)

Positive control

genes in operon OFF BY DEFAULT unless ACTIVE REGULATOR PROTEIN PRESENT (inducer gone = turns off) (ex. Attachment of CAP to promoter)

CAP

catabolite activator protein; glucose sensor, activates transcription of lac operon only when glucose is low; senses INDIRECTLY THROUGH cAMP; attachment of CAP to lac promoter DIRECTLY STIMULATES GENE EXPRESSION so POSITIVE GENE REGULATION

RNA polymerase

enzyme that transcribes new RNA chain by linking ribonucleotides to DNA nucleotides

Operator

sequence of nucleotides near start of operon to which ACTIVE REPRESSOR CAN ATTACH to PREVENT RNA POLYMERASE from attaching to promoter and transcribing operon genes

Promoter

nucleotide sequence binding site of RNA polymerase, positioning it to transcribe at right position

Regulator gene

gene that codes for repressor; AWAY from operon; has OWN PROMOTER

Prions

misfolded proteins that cause normal proteins to misfold, too (ex. Scrapie, mad cow disease, Creutzfeldt-Jakob disease); all FATAL

Tandem repeats

back to back repetitive sequences; make up telomeres; cause diseases such as Huntington’s

nongene/noncoding DNA

gets transcribed into RNA into REGULATORY and REPETITIVE sequences that ALTER GENE EXPRESSION

Polymorphic regions

noncoding regions of DNA that are highly variable from one region to next

STRs

Short tandem repeats; units of 2-5 nucleotides (ex. GTTAC); quantity unique, so used for DNA TESTING

Nucleosome

basic unit of chromatin

Acetylation

adding acetyl groups; of HISTONE TAILS, LOOSENS CHROMATIN STRUCTURE for TRANSLATION; removing acetyl groups BLOCKS TRANSCRIPTION

Metyhlation

adding methyl groups; ADDING SILENCES DNA temporarily or long-term; REMOVING TURNS ON GENES (responsible for X-CHROMOSOME DEACTIVATION)

Epigenetic inheritance

alterations in genome that DON’T DIRECTLY INVOLVE NUCLEOTIDE SEQUENCE; REVERSIBLE; caused by diet, stress, prenatal nutrition (ex. One identical twin develops schizophrenia)

Degradation of mRNA

type of GENE REGULATION; some mRNA (bacterial) degraded within minutes, so are adaptable to changes; humans continually translate protein for hours or weeks (less adaptable); mRNA in developing blood cells STABLE and translate hemoglobin for long time

ncRNA

non-protein-coding DNA transcribed into RNA; bind to and assisted by ARGONAUTE PROTEINS; REGULATE DNA; miRNA, siRNA, piRNA

miRNA

microRNA; type of ncRNA about 22 nucleotides long that DEGRADES OR BLOCKS mRNA TRANSLATION

siRNA

small interfering RNA; similar to miRNA, BINDS AND DESTROYS mRNA; blocking of gene expression called RNA interference (RNAi)

RNAi

RNA interference; blocking of gene expression by non-coding RNA (small interfering RNA/siRNA); SILENCES GENES

piRNA

piwi-associated RNA; guide PIWI proteins to complementary RNAs derived from “jumping genes”; PROTECT GERM CELLS FROM TRANSPOSON ATTACKS

Protein activation

GENE EXPRESSION CONTROL; some proteins must be activated after translation (ex. Insulin must be cleaved by an enzyme)

Recombinant DNA

combining DNA from two or more sources into one molecule; viral transduction, bacterial transformation, conjugation, transposons (“jumping genes”)

Uses of recombinant DNA/gene cloning

protein product (insulin), replace nonfunctioning gene through gene therapy, copy gene for analysis, engineer bacteria to clean toxic waste

Gene cloning process

isolate gene of interest, insert gene into plasmid, make bacterium competent and insert plasmid to make bacterium into VECTOR, clone gene as bacteria reproduce by fission, select and harvest bacteria with gene

Restriction enzymes

cut DNA @ specific SYMMETRICAL recognition sequences/sites (ex. GAATTC); staggered to form sticky ends; resultant fragments called RESTRICTION FRAGMENTS

Restriction enzymes

EcoRI, BamHI, HINDIII; used in gene cloning

DNA probe

radioactively labeled single strand of nucleic acid molecule used to tag specific DNA sequence; can identify genetic defects like sickle cell, Tay-Sachs, Huntington’s

PCR

amplify DNA in tube w Taq polymerase, nucleotides, and primers; must know DNA to make primers, must be short, contamination issue

RFLPs

restriction fragment length polymorphisms; “DNA fingerprint” like barcodes; inherited Mendelian, unique except twins; used for PATERNITY TESTING

cDNA

complimentary DNA; produced by REVERSE TRANSCRIPTASE from retroviruses copying PROCESSED mRNA to get sequence WITHOUT INTRONS for use in bacteria cloning

Biotech ethics

safety (BGH); privacy (DNA probes->chips w genetic info)

Polymerase (RNA and DNA)

reads up (3’ to 5’) and writes down (5’ to 3’)