BIO201 Exam 1

in general, a negative result needs to be supported by what kind of control?

positive control

which type of graph is a better choice for categorical rather than continuous data, line or bar graph?

bar graph

True or false: circular DNA molecules are only found in prokaryotic cells.

false: circular DNA can also be found in the mitochondria and chloroplasts

2 structural differences and 2 similarities between plant and animal cells

plant cells have a cell wall and chloroplasts, animal cells do not, many small Golgi units for plants, usually one prominent Golgi complex for animals, plant cells are often larger than animal cells

both cells have a plasma membrane, membrane-bound organelles, linear chromosomes that are packages with histones, DNA inside membrane-bound nucleus, cytoplasm, ribosomes, rough and smooth ER, Golgi apparatus, lysosomes, same genetic code, cytoskeleton (nucleus and cell membrane main)

Is it possible to predict whether a large plant, such as an oak tree, will have a bigger genome than a small plant, such as a daffodil? (can we predict genome size?)

no — very broad generalizations can be made (eukaryotes generally have bigger genomes that prokaryotes, vertebrates generally have bigger genomes than fungi) but cannot be more specific

5 themes of life

1. structure and function

2. transformation of matter and energy

3. information storage, transmission, and flow

4. interactions between and within systems

5. evolution

The six most commonly used model organisms in biology are Arabidopsis (a plant), fruit flies, mice, C. elegans (a nematode), yeast, and E. coli. E. coli is a prokaryote. What processes would you be unable to study using E. coli?

transport of necessary proteins into and out of the nucleus

mechanisms by which cells differentiate (specialize into different tissue types)

eubacteria are single-celled organisms and do not differentiate into multiple types of tissue, also do not have a nucleus

what are some processes we can use to study E. coli?

transport of necessary ions into and out of the cell

transcription of DNA into RNA

metabolism of food sources into chemical energy

what is the experimental group, positive control, and negative control?

finding out whether the protein YFP1 is found in the cell membrane — experiment where you fluorescently tag the protein, induce its production, and monitor fluorescent over time in different parts of the cell, also similarly tag and monitor the CFTR protein (found in the cell membrane) and TUBA1C protein (found throughout the cytoplasm)

experimental group: YFP1

positive control: CFTR

negative control: TUBA1C

Independent and dependent variable in the graph

did the controls work as expected?

what can we conclude about YFP1 localization in the cell?

independent: which protein was tagged and observed

dependent: signal strength/amount of fluorescence observed

controls did work as expected because CFTR was found in the Golgi and then in the plasma membrane, and TUBA1C was not

YFP1 is in the cell membrane

which graph would have been different if YFP1 were a secreted protein and why?

secreted = protein reaches cell membrane, crosses it, and diffuses away in extracellular fluid

if secreted, would not accumulate in the plasma membrane and the curve on the plasma membrane graph would look more similar to the one for the Golgi but just shifted to the right on the X axis

observations of RER and cytoplasm — would would the graphs look like?

CFTR & YFP1: RER graph would show signal present at the start and then none; TUBA1C would not be seen

CFTR & YFP1: cytoplasm graph would show signal very close to zero at all the points observed; TUBA1C should show a rapid increase to some max level followed by slow decrease as the proteins produced naturally degrade over time

arrange the following in order from the one that makes the fewest bonds to most: carbon, hydrogen, nitrogen, oxygen

hydrogen < oxygen < nitrogen < carbon

difference between a non polar covalent bond and a polar covalent bond

non polar covalent bond is between atoms that have the same/similar electronegativity (difference ≤ 0.4)

polar covalent bond is between atoms with greater difference in electronegativity (difference 0.4-1.8)

most polar covalent bonds that will be seen in this course is going to involve either oxygen or nitrogen

how many carbons and hydrogens does benzene have?

6 carbons, 6 hydrogens

water-soluble or not?

yes

has hydrocarbons but also nitrogens and oxygens around its perimeter which makes it fairly water-soluble

water soluble or not?

no

almost entirely hydrocarbons, with only one H-bond donor and one acceptor → insoluble in water, but soluble in lipids

water soluble or not?

yes

has hydrocarbons but also nitrogens and oxygens around the perimeter

water soluble or not?

yes

has carbonyls and hydroxyls (more than enough) to compensate for a few carbon atoms

water soluble or not?

no

the benzene ring, three more hydrocarbons, single amino group - non polar

possible interactions between molecule and water

OH₂ — O bonds with the H from NH₂ group, H₂O — H bonds to N

features of prokaryotes (bacteria, e.g., E. coli)

no nucleus, DNA located in a nucleotide region, usually one main circular chromosome, may contain plasmids, no membrane-bound organelle, ribosomes are present, transcription and translation can occur in the same general cellular region, usually smaller and structurally simpler

eukaryotes (yeast, plants, animals, and fungi)

nucleus surrounded by a nuclear envelope, multiple linear chromosomes, membrane-bound organelles, ribosomes, more extensive internal compartmentalization, transcription occurs in the nucleus, translation occurs in the cytoplasm or on the RERg

what is a genome?

complete genetic material of an organism

what does a bacterial genome commonly include

a main circular chromosome, genes arranged in a compact region (nucleoid), optional plasmids, relatively little noncoding DNA compared compared with many eukaryotes, genes sometimes arranged in operons

what does a eukaryotic genome generally include

multiple linear chromosomes, DNA associated with histone proteins, nucleus, noncoding regions, regulatory DNA, introns and exons in many genes, mitochondrial DNA

intron

non-coding DNA sequences, any nucleotide sequence within a gene that is not expressive/operative in the final RNA product, removed by RNA splicing, found only in eukaryotes

exons

protein-coding DNA sequences that require the necessary codons/information necessary for protein synthesis, found in eukaryotes and prokaryotes

yeast as a eukaryotic model organism

good because it has nucleus and can be used to study eukaryotic processes while remaining relatively easy to grow and genetically manipulate, able to perform many eukaryotic cellular processes, and normal and mutant forms

genotype

the genetic information an organism possesses

change genotype can cause a change in phenotype

phenotype

observable characteristics: colony color, growth rate, shape, metabolism

phenotype can also change without a change in genotype

cytoplasmic proteins

protein synthesized on free ribosomes generally remains in the cytoplasm unless it contains a targeting signal

secreted proteins

ribosome on the RER → RER lumen → transport vesicle → Golgi apparatus → secretory vesicle → plasma membrane → extracellular space

what type of proteins are antibodies?

antibodies are secreted proteins and so they are synthesized on ribosomes associated with the RER and then pass through the Golgi apparatus

membrane proteins

synthesized on ribosomes associated with the RER — inserted into the ER membrane, transported through the Golgi, and delivered to the plasma membrane

what groups can water form hydrogen bonds with

hydroxyl groups, amino groups, carbonyl groups, charged groups

phospholipid bilayer

hydrophobic, so molecules that are small and nonpolar/hydrophobic will cross relatively easily

examples of molecules that can cross across a phospholipid bilayer easily

O2, CO2, steroid hormones, some weak acids in their uncharged form

molecules that cross poorly across a phospholipid bilayer

ions, large polar molecules, strongly charged molecules, large proteins, nucleic acidswh

what do molecules that cross the phospholipid bilayer usually require

channels, transporters, pumps, endocytosis/exocytosis

what does every amino acid have?

an amino group, carboxyl group, central alpha carbon, hydrogen, R groupw

what determines the amino acid’s chemical properties?

R group — polar, nonpolar, acidic, basic

nonpolar amino acids

R groups are mostly hydrocarbon and tend to be buried inside water-soluble proteins

polar uncharged amino acids

R groups can form hydrogen bonds but do not usually carry a full charge at physiological pH

acidic amino acids

R groups are usually negatively charged in cellsb

basic amino acids

R groups are usually positively charged in cells

primary structure of a protein

amino acid sequence that is held together by peptide bonds, which is a covalent bond between the carboxyl group of one amino acid and the amino group of another

secondary structure of a protein

alpha helices, beta sheets

stabilized mainly by hydrogen bonds between backbone groups: carbonyl oxygen, N-H hydrogen

tertiary structure of a protein

the overall 3-D shape of one polypeptide chain

involves hydrophobic interactions, hydrogen bonds between R groups, ionic interactions, disulfide bonds, van der Waals interactions

quaternary structure of proteins

how multiple polypeptide chains interact

stabilized by hydrophobic interactions, ionic interactions, hydrogen bonds, disulfide bonds

quaternary vs tertiary protein structure

a protein does not need multiple chains to have tertiary structure, but it must have multiple chains to have quaternary structure

protein variation

if a protein has n amino acids and each position can contain any of the 20 amino acids, possible sequences = 20^n

structure of an antibody

typically contains

• 2 identical heavy chains

• 2 identical light chains

• disulfide bonds connecting chains

• constant regions

• variable antigen-binding regions

variable regions of antibodies and antibody-antigen binding

variable regions recognize antigens, and antibody-antigen binding can involve hydrogen bonds, ionic interactions, hydrophobic interactions, van der Waals interactions

exposed binding surfaces of antibodies

because they function in blood (aqueous environment), the exposed binding surfaces of antibodies often contain polar or charged groupsb

4 major classes of biomolecules

proteins, lipids, polysaccharides, nucleic acidspr

proteins — examples and common cellular locations

enzymes, antibodies, channels

found everywhere in the cell

lipids

phospholipids, steroids

found in membranes, droplets, signaling

polysaccharides

cell walls, glycogen

found in cell walls, storage

nucleic acids

DNA, RNA

found in nucleus, cytoplasm, ribosomes

how does yeast DNA extraction separates biomolecules

separation is based on properties such as solubility:

lyticase breaks down the yeast cells wall → SDS disrupts membranes and denatures proteins → potassium acetate helps precipitate denatured proteins and debris → centrifugation separates debris from nucleic acid containing supernatant → isopropanol precipitates nucleic acids → ethanol washes away excess salt → TE buffer redissolves the nucleic acids

solubility

substance is soluble when it interacts favorable with the solvent

hydrophilic molecules have

charged groups, many exposed polar groups, hydrogen-bond donors, hydrogen-bond acceptors

hydrophobic molecules generally have

large hydrocarbon regions, few polar groups, few charged groups

best test of prediction of solubility

place the substance in water and observe whether it dissolves

positive control is known soluble substance, known insoluble substance is negative control

yeast RNA types

mRNA, rRNA, tRNA, other noncoding RNAs

mRNA

carries coding information from DNA, used to make proteins

rRNA

forms part of ribosomes, helps catalyze peptide-bond formation

tRNA

carries amino acids to the ribosome, matches anticodons with mRNA codons

other noncoding RNAs

helps with RNA processing, gene regulation, ribosome production, splicing

RNase

enzyme that degrade RNA — if a sample has both DNA and RNA, RNase treatment reduces/removes DNA, and DNA should remain mostly intact

RNA signal may disappear/become much less visible after RNase treatment on agarose gel

EcoRI

restriction enzyme that cuts DNA at a specific recognition sequence

if DNA contains several EcoRI sites, it is cut into multiple fragments (# of fragments depends on the number and arrangement of cutting sites), smaller fragments travel farther through an agarose gel, larger fragments remain closer to the wells

restriction-enzyme cutting frequency

expected frequency = 1/4^n

ELISA

used to detect or quantify a specific protein, antibody, or antigen

antisense oligonucleotide technology

uses short nucleic-acid sequence that binds a target mRNA and interferes with translation/promotes mRNA degradation; used to reduce expression of a specific gene

SDS-PAGE

separates proteins mainly by size after sis gives them similar charge-to-mass ratios

smaller proteins migrate farther through the gelW

Western blot

uses antibodies to detect a specific protein after gel electrophoresis

• is the protein present? is the size correct? is its abundance changed?

immunohistochemistry

uses antibodies to determine where a protein is located in cells or tissues

useful for testing localization (e.g., is a receptor in the nucleus?)

x-ray crystallography

determines the 3-D structure of crystallized molecules

useful for high-resolution structural information

cryogenic electron microscopy

determines structures of large molecules or complexes frozen in near-relative conditions

useful for when crystallization is difficult

mutational analysis

changes a gene/protein sequence and observes the effect

useful for identifying functional regions, binding sites, structural residues, localization signals, catalytic residues

what DNA change causes a missense mutation

usually caused by a base substitution that changes a codon into a different codon specifying a different amino acid

resulting protein then has a changed primary structure

silent mutation

base substitution, but same amino acid

nonsense mutation

base substitution has led to a new amino acid that is a stop codon

single-base insertion

frameshift mutation

codons are read in groups of 3, so inserting one base shifts the reading frame for every codon after the insertion

possible effects of frameshift mutation

many amino acids change, premature stop codon appears, protein becomes shortened, protein folds incorrectly, protein loses its function

three-base insertion

does not shift the reading frame, but adds one amino acid

how can a missense mutation affect tertiary structure?

missense mutation changes one amino acid in the primary sequence — effect depends on location and chemical difference

conservative change from missense mutation

one amino acid is replaced by another with similar properties → little or no effect on folding

nonconservative change

nonpolar amino acid is replaced by a charged/polar amino acid or vice versa

change in hydrophobic interactions and bonding, ionic interactions change, buried hydrophobic region becomes unstable, new charge appears, protein folds differently, active site changes, protein is degraded

key chain of reasoning for mutation

DNA mutation → altered codon → altered amino acid sequence → altered chemical interactions → altered tertiary structure → altered function

predicting interactions of molecules — nonpolar molecules

hydrophobic and van der Waals interactions

predicting interactions - charged molecule

ionic interactions with oppositely charged groups

predicting interactions - polar molecule

hydrogen bonds

enzymes in metabolic pathways

enzymatic pathway is a sequence of reactions where each enzyme usually catalyzes one particular step

interpreting pathway mutations: A → B → C → D

mutation disables enzyme that converts B into C

less C and D, more B, possibly normal amounts of A

feedback inhibition

in many pathways, final product inhibits an early enzyme where A → B → C → final product

if there is already plenty of final product, the pathway is slowed or stopped, which conserves energy and raw materials

what does SDS do?

2 major effects: unfolds proteins, making them more linear; coats proteins with negative charge

proteins have similar charge-to-mass ratios after SDS treatment, main factor affecting their movement through the gel is size

separates proteins primarily by size

what can SDS-PAGE tell you

how many major protein bands are present, approximate protein sizes, whether a protein sample is relatively pure, whether total protein patterns differ between samples

limitation of SDS-PAGE

SDS-PAGE alone usually cannot identify a specific protein because many different proteins can have similar molecular masses