Cell Bio Exam 3 Study Guide

State the amount of variation that typically distinguishes one human genome from another and describe the most common form of genetic variation.

0.1%, the most common form is ones that developed early on in our evolution when the population was still really small and this variation has been reshuffled through the generations, also single nucleotide polymorphisms (point mutations) and silent mutations - both cause differences in people

Summarize how the length and saturation of lipids' hydrophobic tails affect the fluidity of a cell membrane.

A higher-saturated tail means that the tail is straighter and can be more compact (more rigid), versus unsaturated has a kink and cannot be compressed as tightly(more fluid). The fluidity of the bilayer depends on its composition. The closer and more regular the packing of the tails, the more viscous and less fluid the bilayer will be. Two major properties of hydrocarbon tails affect how tightly they pack in the bilayer: their length and the number of double bonds they contain. For instance, A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another and therefore increases the fluidity of the bilayer. The chain that harbors a double bond does not contain the maximum number of hydrogen atoms that could, in principle be attached to its carbon backbone; it is thus said to be unsaturated with respect to hydrogen. The carbon tail w/ no double bond, has all of its hydrogen atoms and is said to be saturated. Each double bond in an unsaturated tail creates a small kink in the tail which makes it more difficult for the tails to pack against one another. Lipid bilayers that contain a large proportion of unsaturated hydrocarbon tails are more fluid than those with lower proportions.

Articulate why cloning in bacteria involves the generation of recombinant DNA.

After the DNA is broken into fragments to prepare for cloning, the fragments are recombined by ligase It needs to be tagged by something that will proliferate like the replication origin (that's the reason for recombination). Recombinant DNA is DNA that has been artificially formed by combining constituents from different organisms or sources. Some ways this is perform is through restriction endonuclease, DNA ligases, polymerase. Once, it has been broken into smaller pieces, the fragments can be cloned by inserting the DNA fragments into a carrier or vector (another piece of DNA that can be copied inside cells - this can also be done w/ plasmids, multi cloning sites). The resulting molecules are called recombinant DNA. This process is also how antibiotic resistance is formed

Describe how membranes retain their orientation during transfer between cell compartments.

All of the previous orientations remain the same and the vesicles break in one spot and open up, the vesicles are shaped such that the extracellular part of the membrane is the inside of the vesicle and then it opens outward. Membranes retain their orientation during transfer between cell compartments. Membranes are transported by a processor vesicle budding and fusing. Here, a vesicle is shown budding from the Golgi apparatus and fusing with the plasma membrane. Notes that the orientations of both the membrane lipids and proteins are preserved during the process: the original cytosolic surface of the lipid bilayer remains facing the cytosol, and the noncytosolic surface continues to face away from the cytosol, toward the lumen of the Golgi and the transport vesicle or toward the extracellular fluid. Similarly, the glycoprotein shown here remains in the same orientation, with its attached sugar facing the noncytosolic side.

Illustrate how homologous recombination can lead to gene duplication.

Allows for the resealing of a broken double helix by using an intact chromosome duplicate, but it can also catalyze crossovers that leads to the two genes being joined together, usually occur on homologous chromosomes because genetic sequence has to be nearly identical Since they are nearly identical sequences they can misalign and possibly join in prophase I. Gene duplication was thought to occur by homologous recombination via. short repetitive sequence. This occurs when crossover occurs between short DNA sequences which causes them to be not aligned and have a lopsided exchange. Such unequence crossovers can generate one chromosome that has an extra copy of the gene and another with no copy. This shorter chromosome will eventually be lost

Differentiate between a symport, an antiport, and a uniport.

Antiport- two different ions moving different directions (potassium and calcium pumps) Symport- move together down the port- sodium glucose transport in gut lumen, uses the concentration of Na to increase the concentration of glucose against its concentration gradient Uniport- single molecule transport (ex. glucose transporter, aquaporins)

Relate the structure and function of the membrane protein bacteriorhodopsin.

Bacteriorhodopsin is a bacterial protein formed by seven alpha helices that cross the plasma membrane. The center contains a molecule called retinal that, when activated by light, takes a proton from the residue next to it and causes a conformational change in the protein that leads to a proton being pumped out of the cell. This can be used to create a proton gradient that can further be used to generate ATP for the cell. (Passes through 7 times). Bacteriorhodopsin revealed how alpha helices cross the lipid bilayer. It acts as a proton pump. The polypeptide chain of this small protein crosses the lipid bilayer as seven alpha helices. Strategically placed polar amino acid side chains guide the movement of the proton (H+) across the bilayer, allowing it to avoid contact with the lipid environment. The retinal is then regenerated by taking up a H+ from the cytosol, retiring the protein to its original conformation. Retinal is also used to detect light in our own eyes, where it is attached to a protein with a structure very similar to that of bacteriorhodopsin.

Explain how the CRISPR system can be used to produce a transgenic organism in which a target gene is inactivated or replaced.

CRISPR can be used to genetically modify organisms by implementing target sequences into host cells that will be expressed Cas9 is the piece that cuts the DNA. Cas9 isn't specific. It can cut anywhere as long as you have the guide RNA to tell where to cut. Then you use ligase to seal it into another sequence. The Cas9 protein, along with a guide RNA designed by the experimenter, are both artificially expressed in the cell or species of interest. One portion of the guide RNA associates with Cas9, and another segment is designed to match a particular target sequence in the genome. Once Cas9 has made a double-strand break in the target gene, that gene can be replaced with an experimentally altered gene by the enzymes that repair double-strand breaks through homologous recombination. In this way, the CRISPR system promotes the precise and rapid replacement of a target gene. By using a mutant form of Cas9 that can no longer cleave DNA, Cas9 can be used to activate a normally dormant gene or turn off an actively expressed gene.

Outline how a comparison of the nucleotide sequences from two closely related organisms can be used to reconstruct the amino acid sequence of a protein from their extinct, common ancestor.

Can look at the sequence of the evolutionary-older species and determine what the original sequence was based on that and other organisms, whichever the amino acid is present in more of the organisms is probably the original and the others are divergence You can track point mutations that would change the amino acid and that allows you to determine what could change the sequence and which ancestors are most related based on what the more ancestral organisms demonstrated

Compare how transporters and channels discriminate among solutes, moving only a select subset across the membrane.

Channels- provide a hydrophilic opening that allows specific molecules to move Controls based on a concentration gradient, and discriminate based on charge and size of the molecule and specific ions have their own channels (don't change confirmation) Transporters- slower because much more specific, move single ions at one time, selective based on shape of the transporters, ion binds, transporter undergoes a conformational change, and ion is allowed through, still passive (can change confirmation - like a bridge). Channels discriminate based on size and electric charge: when the channel isoprene only ions of an appropriate size and charge can pass through. A transporter transfers only those molecules or ions that fit into specific binding sites on the protein.

State how the inclusion of cholesterol affects the permeability of animal cell membranes.

Cholesterol serves to prevent phospholipids from being loosely compacted and help the membrane control its fluidity, makes the membrane more rigid and less flexible. Cholesterols fills gaps between phospholipids, stabilizes membrane structure, reduces membrane fluidity, and provides 'suitable working environment' for membrane proteins, make its less permeable

Review how reporter genes can be used to track the location of proteins within cells.

Code for something that can be tracked and is promoted through the regulatory sequence of the gene. Reporter gene encodes a protein that can be easily monitored by its fluorescence or enzymatic activity.

Contrast the types of DNA sequences that can accommodate mutations with those that cannot.

Coding sequences that accumulate mutations will likely not reproduce and will accumulate less mutations overtime versus non coding or regulatory can have more mutations and the organism will probably be able to reproduce and gain more mutations over time Coding sequences can accumulate mutations, but will eventually be wiped out unless it is selectively beneficial (purifying selection)

Review how hybridization allows the detection of any DNA fragment of interest.

Complementary. You get probed DNA and then you get complementary. Hybridization is when you make your own dna that is probed to detect sequence in DNA and based on complementary binding that is how you find your dna fragment of interest. For instance if you have a glowing sequence if where you are sequencing, you follow that glow - you follow the probed or tracked DNA. For two single-stranded molecules to hybridize, they must have complementary nucleotide sequences that allow base-pairing. In this example, the red and orange strands are complementary to each other. and the blue and green strands are complementary to each other. Although denaturation by heating is shown, DNA can also be denatured by alkali treatment. Hybridization (renaturation) can also occur between complementary strands of DNA and RNA or between two RNAs. Hybridization can be employed for detecting any nucleotide sequence of interest, whether DNA or RNA. One simply designs a short, single stranded DNA probe that is complementary to that sequence. Because the nucleotide sequences of so many genomes are known - and are stored in publicly accessible databases - designing such a probe is straightforward. The desired probe can then be synthesized in the laboratory usually by a commercial organization or a centralized academic facility.

Review the forces that govern the passive transport of charged and uncharged solutes across a cell membrane.

Concentration determines which direction the solute will travel The other is membrane potential (also called electrochemical gradient), maintaining the needed charges. Passive transporters move a solute along its electrochemical gradient.

State how bands of DNA fragments on a gel can be visualized.

DNA is labeled or stained (CBB) and sometimes UV light is used with a dye. You can also label gels with fluorescent dyes. One way to perform this method is to expose the gel to a dye that fluorescence under ultraviolet (UV) high when it is bound to DNA. The the gel is placed on a UV light box, the individual bands glow bright orange or white when in a black and white photograph.

Review the purpose of each of the three basic steps involved in each cycle of PCR.

Denaturing- heating strands denatures the hydrogen bonding and strands separate Annealing- cooling allows primers to attach to the 3' end of the parent strands Elongation- Taq polymerase synthesizes the new DNA strand using dNTPs onto the 3' end of the new strand moving 5-3

Review how RNA interference (RNAi) can be used to inhibit the activity of a target gene.

Double stranded RNA molecule with a nucleotide sequence that is the same as the one to be inactivated and results in the destruction of any generated RNAs with similar sequence through turing the double stranded RNA into smaller fragments that hybridize with the target gene's mRNA and direct their degradation This is the defense mechanism of the mRNAi. One of the fastest and easiest ways to silence genes in cells and organisms is via RNA interference (RNAi). One example of this is double-stranded RNA (dsRNA) can be introduced into C. elegans by feeding the worms E.coli that express the dsRNA. Gene function is reduced in all tissues, including the reproductive tissues where embryos are produced by self-fertilization. In a wild-type worm embryo, the egg and sperm pronuclei come together in the posterior half of the embryo shortly after fertilization. In an embryo in which a particular gene has been silenced by RNAi, the pronuclei fail to migrate. This experiment revealed an important but previously unknown function of this gene in embryonic development.

Articulate how gene duplication can lead to the evolution of genes with new functions or to the generation of pseudogenes.

Duplications of genes allows for the mutation of the duplicate and mutations can occur there without disrupting function of the original gene Thus you get one original gene and then you can do whatever you want to the second copy. It becomes more specialized Example of pseudogene: we have a hemoglobin pseudogene. They have been duplicated, but they haven't been tinkered enough to be functional. Repeated rounds of this cause gene duplication and divergence. You get pseudogenes when you a DNA sequence has been disabled by the accumulation of many inactivating mutations. One example of his is hemoglobin. There are specialized globing genes where there are several duplicated DNA sequences in alpha and beta globing gene clusters that are not functional genes. They are similar in DNA sequence to the functional globin genes, but they have been disabled by the accumulation of many inactivating mutations. The existence of such pseudogenes make it clear that not every DNA duplication leads to a new functional gene.

Contrast the actions of flippases and scramblases.

Flippases help to maintain the asymmetric aspect of the bilayer versus scramblases move random phospholipids around and maintain the distribution of molecules and new lipids Flippases remove specific phospholipids from the exterior side of the bilayer and move to the other side. Scamblases catalyzes transfer of random phospholipids from one monolayer to another

Identify the lipids that show the most consistent or dramatic differences in distribution and indicate which monolayer they favor and why.

Glycolipids go on outside because they functions require that they are there, but they stay there because flippases can't move glycolipids because they don't recognize them. Phospholipids and glycolipids are distributed asymmetrically in the lipid bilayer of an animal cell plasma membrane. Phosphatidylcholine and sphingomyelin are concentrated in the noncytosolic monolayer, whereas phosphatidylserine and phosphatidylethanolamine are found mainly on the cytosolic side. In addition to these phospholipids, phosphatidylinositols, a minor constituent of the plasma membrane, are shown in the cytosolic monolayer, where they participate in cell signaling. Glycolipids are drawn with hexagonal head groups to represent sugars; these are found exclusively in the noncytosolic monolayer of the membrane. Within the bilayer, cholesterol is distributed almost equally in both monolayers.

List three sources of energy used by transmembrane pumps to actively transport a solute against its concentration gradient.

Gradient driven - where you use energy for one pump and that pump creates a gradient that allows for the other molecule to move with the created gradient ATP driven Light driven- energy stored in light. (I) Gradient-drive pumps link the uphill transport of one solute across a membrane to the downhill transport of another. (II) ATP-driven pumps use the energy released by hydrolysis of ATP to drive uphill transport; (III) light driven pumps which are found mainly in bacterial cells, use energy derived from sunlight to drive uphill transport ex. Bacteriorhodopsin

Compare the number of protein-coding genes present in humans versus Drosophila, C. elegans, and Arabidopsis, and analyze how these values relate to the complexity of the organism.

Human- 19,000 Flies- 14,000 C. elegans- 22,000 Arabidopsis- 28,000

Distinguish between integral membrane proteins and peripheral membrane proteins.

Integral are either in the bilayer or covalently linked to the bilayer. They are proteins that are directly attached to the lipid bilayer - whether they are transmembrane, associated with the lipid monolayer, or lipid-linked - can be removed only be disrupting the bilayer with detergents. The remaining membrane proteins are peripheral membrane proteins. They are non covalently linked through other proteins. They can be released from the membrane through more gentle extraction procedure that interfere with protein-protein interactions but leave the lipid bilayer intact

Explain how PCR can be used to produce either genomic or cDNA clones.

Just depends on what segment of DNA you select and prepare?? And also what primers are added. To use PCR to clone a segment of chromosomal DNA, total DNA is first purified from cells. PCR primers that flank the stretch of DNA to be cloned are added, and many cycles of PCR are completed. Because only the DNA between (and including) the primers is amplified, PCR provides a way to obtain selectively any short stretch of chromosomal DNA in an effectively pure form. To use PCR to obtain a cDNA clone of a gene, total mRNA is first purified from cells. The first primer is added to a population of single-stranded mRNAs, and reverse transcriptase is used to make a DNA strand complementary to the specific RNA sequence of interest. A second primer is then added, and the DNA molecule is amplified through many cycles of PCR.

Indicate how the concentration of ions in the cell differs from that outside the cell.

K+ is high inside the cell and Na+ is high outside the cell, higher concentration of Cl- inside the cell to counteract the + charge from the Na and has a higher negative potential

Differentiate between L1 elements and Alu sequences.

L1 is a common human retrotransposon, constitutes 15% of the human genome, reverse transcriptase is encoded in the gene and helps it spread Alu makes up 10%, do not encode for reverse transcriptase and depends on enzymes

Explain how mobile genetic elements can alter the activity of regulation of a gene or promote gene duplication and exon shuffling.

Mobile genetic elements - DNA sequence with the ability (and machinery) to move from one chromosome to another can cause 'spontaneous' mutations that can disrupt gene activity (direct insertion) or expression (regulatory sequences). Mobile elements can move between chromosomes and location on chromosomes, have been found to have dramatically affected the shape of genomes, often disrupt the function of other regulatory genes or create novel genes Transposons can also turn on or turn off expression of a gene by being inserted into regulatory sequences. When the mobile genetic element is put into the promoter it can stop it or overexpress it. When two mobile genetic elements are in close proximity to each other you can move entire segments even if another exon is present in the middle of the two segments. The mobile elements are prone to moving around and are coding so it would be considered exon shuffling Through exon shuffling you can make novel genes. Retrovirus' creates duplicated sequences, which increase likelihood of homologous recombination events (they require similar sequences). One example of mobile gene elements is the development of modern corn from a wild, grassy plant called teosinte which required only a small number of genetic alterations. Mobile genetic elements provide opportunities for genome re-arrangements by serving as targets of homologous recombination. For instance, the duplications that gave rise to the beta-globing gene cluster are thought to have occurred by crossovers between the abundant mobile genetic elements sprinkled throughout the human genome.

Summarize the main differences between cloning in bacteria and cloning by PCR.

PCR can be done without bacteria and is highly efficient and easier to work with, and a much more straightforward approach PCR is more specific. PCR uses DNA polymerase and specific DNA primers to amplify DNA sequence in a test tube eliminating the need for bacteria. The benefit to PCR is that it can clone any piece of DNA or RNA without the time and effort needed to construct a DNA library. DNA cloning involves isolating a specific fragment of DNA and then inserting that fragment into a plasmid so bacteria can replicate the DNA. PCR is using two specific primers in order to replicate and isolate a specific DNA sequence.

Distinguish between active and passive transport, and indicate which type of membrane transport protein carries out each.

Passive transport- channel proteins or no proteins at all, does not require energy, involves a solute traveling along its concentration gradient. It is determined by permeability and concentration. Two types are free diffusion (net movement of a substance from a region of high concentration to lower concentration) and facilitated diffusion (material diffuse across the plasma membrane from area of high to low concentration with the help of membrane proteins), and Osmosis (movement of water molecules). Active transport- requires input of energy to move a solute against the concentration gradient, maintaining an imbalance, requires some type of transporter protein. It is the movement of molecule across the cell membrane against the concentration gradient and requiring cellular energy.

Express how exon shuffling can facilitate the evolution of new proteins.

Piecing small parts together in different combinations can lead to new wholes Nearly all proteins come from exon shuffling. Each domain in on one exon, so by rearranging you are making a new function

Contrast how the plasma membrane is mechanically stabilized in animal cells versus plant cells.

Plant cells have a rigid cell wall- a layer of proteins, sugars, and other macromolecules that encase a plasma membrane Animal cells are stabilized by a network of filamentous proteins called the cell cortex that is attached to the underside of the membrane and stabilizes the membrane. It is rich in actin filaments and drives shape and movement of the cell

Express how changes in regulatory DNA sequences can contribute to the evolution of species.

Point mutation in the inhibitor of neuronal proliferation in humans. Lead to the divergence of humans as an individual species Affect how much of a protein the species produces and dictate the organism's developmental program. RNA-seq can also code for noncoding RNAs by detecting genes that do not code for proteins, but instead encode functional or regulatory RNAs

Define "resting membrane potential.

Potential gradient that forces ions to move in one direction or the other unstimulated and movement of ions is balanced- between -20 to -200mV of charge in the cell maintained by these concentrations This membrane potential is important for allowing action potentials to fire in neurons. When a cell is "unstimulated' the movement of anions and cations across the membrane will be precisely balanced. In such steady-state conditions, the voltage difference across the cell membrane - called the rest membrane potential - hold steady. It is not zero. In animals it is the value above. It is expressed in negative numbers because the interior of the cell is more negatively charged than the exterior.

Compare the relative mass and number of protein and lipid molecules in a typical animal cell membrane.

Protein - constitutes about half of the membrane Lipids- make up most of the other half (more lipids by weight within the membrane)

Differentiate between the amount of the human genome that codes for proteins versus the amount that consists of mobile genetic elements.

Protein coding is 19,000, less than 2% Mobile genetic elements- half of the DNA of the human genome- about 50%

Outline the ways that cells restrict the lateral movement of their membrane proteins.

Proteins can be tethered to the cell cortex inside the cell, to the extracellular matrix molecules outside the cells, or to proteins on the surface of another cell, diffusion barriers which restricts a cell to a particular domain

Contrast how plant cells, animal cells, and protozoa maintain their osmotic equilibrium.

Protozoan- vacuoles that remove the excess, discharge that water Plant- vacuoles, they need the pressure, cell wall prevents osmotic lysis, Animal- ion movement, movement in water on both ways, can pump ions out and decrease the water going in. A freshwater amoeba avoids swelling by periodically ejecting the water that moves into the cell and accumulates in contractile vacuoles. The contractile vacuole first accumulates solutes, which cause water to follow by osmosis; it then pumps most of the solutes back into the cytosol before emptying its contents at the cell surface. The plant cell's tough cell wall prevents swelling. The animal cell reduces its intracellular solute concentration by pumping out ions.

Describe the techniques of RNA-Seq and in situ hybridization and compare what each can reveal about gene expression in a particular cell type or tissue.

Provides an analysis of the transcriptome (the collection of RNAs produced by a genome) and can reveal the number of times a sequence appears in a sample You are going to look at the number of RNA produced by the cell for specific gene and that will tell you the expression rate and how it is being used. It is the mRNA produced at a certain set of conditions. It can show the number of mRNA produced between phases. Only uses cDNA. In most cases, a collection of RNAs is converted into complementary DNA (cDNA) by reverse transcriptase, and these cDNAs are then sequenced. This method, called RNA-seq or deep RNA sequencing provides a quantitative analysis of the transcriptome - the complete collection of RNAs produced by a cell under a certain set of conditions. It reveals the number of times a particular sequence appears in a sample and can detect rare mRNAs that harbor sequence variations, and noncoding RNAs. This technology has led to dramatic new insights into the genes expressed in a variety of cells and tissues at different times in development, during different stages of the cell-division cycle, in response to treatment with different drugs, or as a result of different mutations. In situ hybridization can reveal when and where a gene is expressed. In situ hybridization uses single-stranded DNA or RNA probes, labels with either fluorescent dyes or radioactive isotopes, to detect complementary nucleic acid sequences within a tissue or even on an isolated chromosome. The latter application is used in clinic to determine for example whether fetuses carry abnormal chromosomes. It can also be used to study the expression patterns of a particular gene or collection of genes in an adult or developing tissue, providing important clues about when and where these genes carry out their functions.

Recall the role played by restriction enzymes in bacteria and describe how these enzymes are employed in the laboratory.

Restrict the transfer of DNA sequences between the strains of viruses, can cleave a DNA sequence at a certain smaller sequence, used in lab to cut large pieces of DNA into smaller pieces and will reliably generate the same fragments based in the cutting sequences (determines what it cuts) They are used to cut the DNA - synonym is endonuclease Originated from bacterial enzymes. Restriction enzymes cut DNA molecules at specific sites. They can be used to restrict the transfer of DNA between strains of bacteria. Each bacterial species produce different restriction enzymes each cutting at a different, specific nucleotide sequence. A particular restriction enzyme will reliably generate the same set of DNA fragments.

Compare the movement of DNA-only transposons and retrotransposons.

Retrotransposons are moved by first being transcribed from DNA to mRNA and then back into DNA and then converted back to DNA by a reverse transcriptase and inserted elsewhere DNA only move as DNA pieces (Goes DNA to DNA - they are still replicative - bacteria are DNA only they do not have retrotransposons or retroviruses). Retrotransposons move from place to place by reverse transcription mediated RNA intermediate. One example of a retrotransposon is L1 element (long interspersed nuclear elements). They make up about 15% of human genome and they encode for reverse transcriptase.

Assess how retroviruses and retrotransposons are similar.

Retroviruses are made up of RNA that replicate by first making a double stranded DNA intermediate that becomes integrated into the cell's chromosome Retrotransposons are ancestry to retroviruses. They both need reverse transcriptase

Review the types of information that can be gathered from comparative genome analyses.

Revealed a large amount of sequence conservation across species but noncoding regions tend to diverge more and rapidly, useful when a piece of DNA is extracted and unknown, can compare the sequence to genes in the public domain with known functions and can determine from which organism a piece of DNA was derived, related species, and whether the sequence contains a gene and what that function could be let's you look at what is conserved b/t species and if you have a coding sequence that you don't know what is, you can put in website and compare it with known sequences to determine a possible function

Differentiate between the classical genetic approach to studying gene function and the methods referred to as reverse genetics.

Reverse is more targeted versus classical is more generic Reverse goes from mutation to genes, example the knockout mice Regular tries to modify a gene and see what the outcome will be Studies what happens to an organism if a gene is turned off through mutation which through technology can be done intentionally. One of the best ways to determine a gene's function is to see what happens to an organism when the gene is inactivated by a mutation. A classic genetic approach is when you add more selective pressure to create more random mutations to see what phenotype is produced. Reverse genetics is when you know the gene, but you want to know if that is changed, what phenotype will result. For this approach, you need knowledge of the sequence.

Outline how DNA fragments can be separated by size using gel electrophoresis.

Separates based on length size and overall net charge which becomes more negative the larger the molecule is, gel is semi solid and porous It is because of pores in the gels; small molecules travel the farthest. Gel electrophoresis separates molecules based on length. It works by when a voltage is applied across the gel, the negatively charged DNA fragments migrate toward the positive electrode; larger fragments migrate more slowly. In the end, you get a ladder of discrete bands.

Contrast the structure and potential function of single-pass and multipass transmembrane Proteins.

Single pass means the protein only passes through the membrane one time Many are extracellular receptors or small ion channels Multipass means that multiple helices of the same protein are in the membrane Can be arranged so that all of the hydrophobic side chains all fall on one side and then the hydrophilic side chains on the other which forms a hydrophilic pore and allows for polar molecules to pass through Multiple alpha helices pass through

Review the properties that govern the rate at which a given solute can cross a protein-free lipid bilayer.

Size, polarity, charge, and concentration on the inside and outside of the cell, cells work to maintain concentration differences because it allows work Also have to balance the charge formed by the gradients, using chloride ions to balance the positive charge Membrane potential (electrical imbalances generate a voltage difference across the membrane)

Contrast the fate of mutations that arise in somatic cells versus germ-line cells.

Somatic mutations are not passed down but germ line cells are (he used skin cancer gene example)

Explain how the structure of ion channels leads to their ion selectivity.

Specific to each ion based on size and shape and charge to only allow a single kind of ion to fit through Specific means of moving one ion from one side to the other, slower and gated by need of a stimulus (which makes it open more frequently) Cells only need a small fraction of displaced ions to generate a potential. Ion channels are highly selective be their selectively depends on the diameter and shape of the ion channel and on the distribution fo the charged amino acids that line it. Because the ion channel is narrow it forces ions into contact with the channel wall, so that only those ions of appropriate size and charge are able to pass

Outline how gene duplication and divergence gave rise to the globin gene family.

The alpha and beta both originated from a duplication and mutation, and then other members of this family originated from other subsequent mutations (likely point mutations) and regulation changes There are also several duplicated genes within the alpha and beta genes that serve no function and have probably been deactivated by mutations. Globin arose as a single copy gene in ancestral organism found in marine worms, insects, and primitive fish. In this globin, cooperative O2 bound very efficiently. Comparative genomics suggest that 500 million years ago duplication for alpha and beta occurred. From here, as mammals developed, another duplication of the beta gene resulted in special fetal hemoglobin.

Review how lipids move within the lipid bilayer.

The bilayer is flexible and semifluid, some types of movement are rare and other types are frequent and rapid Proteins (flipase) facilitate the movement of lipids from one side of the membrane layer to the other But they continuously exchange places with their neighbors due to thermal energy Rotation and flexion Rotation is rotation about a singular phospholipid Flexion is exchanging position with neighbors(weaving through a crowd). The fluidity of the bilayer depends on its composition. The closer and more regular the packing of the tails, the more viscous and less fluid the bilayer will be. Two major properties of hydrocarbon tails affect how tightly they pack in the bilayer: their length and the number of double bonds they contain. For instance, A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another and therefore increases the fluidity of the bilayer. The chain that harbors a double bond does not contain the maximum number of hydrogen atoms that could, in principle be attached to its carbon backbone; it is thus said to be unsaturated with respect to hydrogen. The carbon tail w/ no double bond, has all of its hydrogen atoms and is said to be saturated. Each double bond in an unsaturated tail creates a small kink in the tail which makes it more difficult for the tails to pack against one another. Lipid bilayers that contain a large proportion of unsaturated hydrocarbon tails are more fluid than those with lower proportions.

Review the structure of genes in the genomes of human, fly, and yeast, and compare how densely genes are distributed in the chromosomes of each organism.

The human genome is less dense, yeast has more genes on average, fly has lots of big exons and not many introns Human genome is more dispersed and introns are more prominent, exons are more dispersed. 4.5% of the human genome is highly conserved.

Recall how detergent molecules can extract proteins from a cell membrane.

The hydrophobic and hydrophilic parts of the detergent can interact with the same parts of the plasma membrane phospholipids to disrupt the plasma membrane. The hydrophilic end of the detergent draw the membrane proteins into the aqueous solution and forms detergent-protein complexes.The detergent also solubilizes the phospholipids, preventing the membrane from reforming. Membrane proteins can be solubilized by a mild detergent such as Triton X-100. The detergent disrupts the lipid bilayer and interacts with the membrane-spanning hydrophobic portion of the protein. These actions bring the proteins into solution as protein-detergent complexes. The phospholipids in the membrane are also solubilized by the detergents, forming lipid-detergent micelles.

Review how a DNA fragment is inserted into a plasmid.

The plasmid is cleaved with a restriction enzyme and then the plasmid can be inserted at the sticky ends and sealed into the plasmid by Ligase. To insert of piece of DNA into a plasmid vector, the purified plasmid DNA is opened up by a restriction enzyme that cleaves it at a single site, and the DNA fragment to be cloned is then spliced into that site using DNA ligase. This recombinant DNA molecules is now ready to be introduced into.a bacterium, where it will be copied and amplified. In other words, the plasmid is first cut open at a single site with a restriction enzyme. It is then mixed with the DNA fragment to be closed, which has been cut with the same restriction enzyme. The staggered ends base-pair, and when DNA ligase and ATP are added, the nicks in the DNA backbone are sealed to produce a complete recombinant DNA molecule.

Describe the process of horizontal gene transfer and state how this form of genetic exchange has significantly impacted human health.

The process of sharing plasmids or specialty genes with other species that often gives them new function and resistance to other bacterial metabolites and always contains information to continue spreading genetic material, through the process of conjugation, rare in eukaryotes This is how antibiotic resistance spreads (ex. Neisseria gonorrhoeae - resistant to penicillin). Horizontal gene transfer is the exchange of genes or portions of genomes between members of different species. It is common in pros. Some methods of transfer are conjugation, transformation, and transduction.

Outline how the asymmetric distribution of phospholipids, characteristic of different membrane types, is established and maintained.

The top layer is more heavily compacted than the bottom layer, meaning that the top layer curves over the bottom. Flippases help to establish and maintain the asymmetric destruction of phospholipids characteristic of animal cell membrane. When membranes leave the ER and are incorporated in the Golgi, they encounter a different set of transporters called flippases, which selectively remove phosphatidylserine, and phosphatidylethanolamine from the noncytosolic monolayer and flip them to the cytosolic side. This transfer leaves phosphatidylcholine and sphingomyelin concentrated in the noncysolic monolayer. The resulting curvature of the membrane may help drive the subsequent vesicle budding.

State how expression vectors direct the production of large quantities of a selected protein.

There are transcription and translation signals that direct the gene to be produced in high levels Example of this is bacterially produced insulin to aid diabetic patients because bacteria are engineered to produce a lot of it when you insert this insulin into it to make multiple copies. Insulin has to be cut twice before it can be active. Large amounts of a proteins can be produced from a protein-coding DNA sequence inserted into an expression vector and introduced into cells. For this to work, a plasmid vector has been engineered to contain a highly active promoter, which causes unusually large amounts of mRNA to be produced from the inserted protein-coding gene. Depending on the characteristics of the cloning vector, the plasmid is introduced into bacterial, yeast, insect, or mammalian cells, where the inserted gene is efficiently transcribed and translated into protein.

Express why fats such as triacylglycerol coalesce into fat droplets in water.

They are completely hydrophobic, meaning that when in water, they force the water molecules to essentially make a cage around them, which causes free energy usage because is going to a more ordered state, and less energy is used when the hydrophobic molecules are forced together and form droplets Micelles. They cannot form favorable interactions with water, so water forms cavelike structures around them to maximize their hydrogen bonds with each other and reduce the energy needed to get in proper confirmation. Thus, you get the droplets of fat.

Recall why water-soluble molecules and ions have difficulty crossing a lipid bilayer.

They are too large or Charged and polar molecules have trouble moving across because it's energetically unfavorable

Review how and why viruses use the host's biochemical machinery to reproduce themselves.

They are too small to house the enzymes needed for independent replication so they hijack the host's enzymes and proteins In order to be classified as a virus you have to use some part of host. Retroviruses use the enzyme reverse transcriptase to convert RNA into DNA. When the single-stranded RAN genome of the retrovirus enters a cell, the reverse transcriptase makes a complementary DNA strand and forms a DNA/RNA hybrid double helix. The RNA strand is removed and reverse transcriptase (which can use either DNA or RNA as a template) now synthesize a complementary DNA strand to produce DNA double helix. This DNA is inserted, or integrated into a random-selected site in the host genome by a virally encoded integrase enzyme. In this integrated state, the virus is latent: each time the host cell divides, it passes on a copy of the integrated viral genome, which is known as a provirus, to its progeny. From here, the integrated viral DNA is copied into RNA by the host-cell RNA polymerase which produces large number of single-strands RNAs identical to the original infectious genome. These viral RNAs are then translated by the host cell ribosomes to produce the viral shell proteins, the envelope proteins, and reverse transcriptase - all of which are assembled with the RNA genome into new virus particles.

Explain how purifying selection leads to the conservation of functionally important DNA sequences and characterize the roles that these conserved sequences might play.

They eliminate any sequence that interacts with gene function, allow for the most genetically efficient protein to be conserved without interference. Purifying selection is the elimination of individuals carrying mutations that interfere with important functions. When you compare these to other organisms, you can see which areas are regions of high conservation (maybe called conserved synteny)

Evaluate how RNA sequencing can provide a more accurate estimate of the number of genes in a genome than DNA sequencing.

This technique detects only genes that are actively transcribed

Categorize membrane proteins based on the way they interact with the lipid bilayer.

Transmembrane proteins are amphipathic and extend through the membrane with parts of their mass on either side Monolayer associated- anchored in the membrane by an amphipathic alpha helix Lipid linked- covalently attached to a lipid and are either completely extracellular or cytosolic Protein attached- weak noncovalently attached to the membrane through interaction with other proteins, peripheral membrane proteins are this

List some functions of plasma membrane proteins.

Transport nutrients, metabolites, and ions across the membrane Pretty much anything that the cell needs that cannot readily diffuse through the membrane quickly. Anchor the membrane to macromolecules on either side. Others function as receptors that detect chemical signals in the cel's environment and relay them into the cell interior, or work as enzymes to catalyze specific reactions at the membrane.

Recall the main constituents and functions of the plasma membrane.

Two ply sheet of lipids and studded with different types of proteins Lipids form the primary barrier and proteins function in controlled transport through the membrane and cell to cell communication and recognition It serves as a semi permeable membrane that only allows certain things to enter and exit. Is made up of a phospholipid bilayer that also contains membrane proteins and cholesterol. A plasma membrane is a protein-studded, fatty film. It is used to separate and protect its chemical components from the outside environment. Without membranes there would be no cells. The structure of the plasma membrane is a two-ply sheet of lipid molecules about 5nm - or 50 atoms - thick, into which proteins have been inserted. It serves as a barrier to prevent the contents of the cell from escaping and mixing with molecules in the surrounding environment, it passes nutrients across the plasma membrane and moves waste out, the membrane is self-healing (reseals itself if nicked)

Outline how recombinant DNA techniques make it possible to analyze the activity of a protein starting from its gene or to assess gene function starting with its encoded protein.

Use the mutant to determine the original genotype and then after can manipulate the original Exon shuffling as well. Recombinant DNA techniques make it possible to move experimentally from gene to protein or from protein to gene. A small quality of a purified protein or peptide fragment is used to obtain a partial amino acid sequence, which is used to search a DNA database for the corresponding nucleotide sequence. This sequence is used to synthesize DNA primers, which can be used to clone the gene by PCR from a sequenced genome. Once the gene has been isolated and sequenced, its protein-coding sequence can be inserted into an expression vector to produce large quantities of the protein which can then be studied biochemically or structurally. IN addition to producing protein, the gene or DNA can also be manipulated and introduced into cells or organisms to study its function.

Outline the construction of a reporter gene and review how such reporters can be used to assess patterns of gene expression.

Using the regulatory gene of a protein to also initiate the coding of a reporter gene that can be seen through fluorescence or enzymatic activity Fused to the regulatory sequence of the gene in question. A recombinant gene usually mimics the expression of the gene of interest, producing the reporter protein when, where, and in the same amounts as the normal protein would be made. This approach can also be used to study the regulatory DNA sequences that control the gene's expression. This is how reporter genes work, you what to know which cell types express protein X, but it is difficult to detect the protein directly with antibodies. Using recombinant DNA techniques, the coding sequence for protein X can be replaced with the coding sequence for the reporter protein Y, which can be easily monitored visually; two commonly used reporter proteins are enzyme beta-galactosidase and green fluorescent protein (GFP). The expression fo the reporter protein Y will now be controlled by the regulatory sequences that control the expression of the normal protein X. To determine which regulatory sequences normally control expression of gene X in particular cell types, reporters with various combinations of the regulatory regions associated with gene X can be constructed. These recombinant DNA molecules are then tested for expression after their introduction into the different cell types.

Articulate how viruses differ from mobile genetic elements.

Viruses are also mobile but can escape from host cells and move to other cells, essentially small genomes enclosed by a coating and need a host cell's enzymes to reproduce and causes cell death to the host in most cases Mobile genetic elements can't leave the cell. Whereas, viruses can. Few viruses actually cause genetic alterations in Eukaryotes as well. Lysogenic phage in prokaryotes is common means of moving genetic material. Lysogenic phage are phage the insert virus into eukaryotes. When it movs onto the next eukaryote, it accidnetly sucks up some of the DNA of the eukaryotes

Compare the uniport glucose transporter and the glucose-sodium symport in terms of their activities and roles in glucose transport in intestinal epithelial cells.

sodium glucose transport in gut lumen, uses the concentration of Na to increase the concentration of glucose (to facilitate the movement of the glucose one) against its concentration gradient, but can also occur passively (they are symport). The binding of sodium and glucose is cooperative. The binding of one enhance the binding of the other. If one of the two solutes is missing, the other fails to bind; therefore both molecules must be present for this gradient-driven transport to occur and Na+ will not leak into the cell w/o doing useful work.

Review how chain-terminating dideoxynucleotides are used to determine the sequence of a piece of DNA.

Allows for the sequencing of small specific sequences of DNA, makes small partial copies of DNA that can be sequenced and eventually produce enough fragments to have a termination at every point in the original sequence When the dideoxynucleotides bind they don't have the OH group on the 3' so you end up with short fragment b/c it can't elongate all the way. You put them in w/ the regular nucleotides and it can help you create short fragments. To determine the complete sequence of single-stranded fragment of DNA, the DNA is first hybridized with a short DNA primer. The DNA is then mixed with DNA polymerase, an excess amount of normal dNTPs, and a mixture containing smalls amounts of all four chain-terminating ddNTPs, each of which is labeled with a fluorescent tag of a different color. Because the chain-terminating ddNTPs will be incorporated only occasionally, each reaction produces a diverse set of DNA copies that terminate at different points in the sequence. Thereaciton products are loaded onto a long, thin capillary gel and separated by electrophoresis. A camera reads the color of each band on the gel and feeds the data to a computer that assembles the sequence. The sequence read from the gel will be complementary to the sequence of the original DNA molecule. A tiny part of the data from such an automated sequencing run. Each colored peak represents a nucleotide in the DNA sequence.

State where new phospholipids are produced and explain how membranes are able to grow evenly.

Assembly begins in the ER through using free fatty acids as substrates and these enzymes deposit new lipids onto the cytosolic half of the bilayer, then transfers between the bilayers are catalyzed by scramblase which is a transporter protein that moves randomly selected lipids from one side to the other, which leads to the even distribution of the new lipids. So you don't have one clump of molecules here and another one there. Flipase moves the phospholipids between the cytosolic and extracellular layers of the membrane, is specific, and is important in putting the correct lipids on the correct side of the plasma membrane

Express how the sodium−proton exchanger in the plasma membrane allows animal cells to control the pH of their cytosol.

Common in other cell types and how cells generate energy Allows cells to control presence of H+ ions Pumps protons out and that raises the pH (antiport). A type of ATP-dependent H+ pump found in the membrane of some intracellular organelle such as the lysosomes of animal cells and the central vacuole of plant and fungal cells. In these pumps - which resemble the turbine-like enzymetaht synthesizes ATP in mitochondria and choloroplast- actively transport H+ out of the cytosol into the organelle, thereby helping to keep the pH of the cytosol neutral and the pH of the interior of the organelle acidic.

Review the reasons why cells carefully regulate their membrane fluidity.

Fluidity enables many proteins to diffuse rapidly in the plane of the membrane and interact with each other and ensures that all molecules are evenly distributed between daughter cells during mitosis and allows newly synthesized molecules to join the plasma membrane. Because it changes how permeable the membrane is, the membrane is crucial for cell signaling

Compare a genomic library and a cDNA library in terms of what each contains, how each is made, and how each might be used.

Genomic is everything, made through DNA cloning by putting fragments into plasmids and allowing them to be reproduced cDNA is coding DNA made by mRNAs, DNA copied from mRNAs by reverse transcriptase and polymerase, then these DNA fragments are introduced into a plasmid and reproduced. It allows you to see what is in each cell (time, place, and environment. It has only exons. It is only RNA therefore it would require reverse transcriptase). A genomic library represents the entire genome of that organism. Whereas, cDNA (complementary DNA) is the DNA that is copied from the mRNA present in a particular type of cell. cDNA is made from mRNA that is extracted from double-strand DNA copies. Some differences between genomic DNA clones and cDNA clones is that genomic clones represent a random sample of all DNA sequences found in an organism's genome and, with very rare exceptions, will contain the same sequences regardless of the cell type from which the DNA came. Also, genomic clones from eukaryotes contain large amounts of noncoding DNA, repetitive DNA sequences, introns, regulatory DNA, and spacer DNA; sequences that code for proteins will make up only a few percent of the library. By contrast, cDNA clones contain predominantly protein-coding sequences, and only those sequences that have been transcribed into mRNA in the cells from which the cDNA was made.

Compare the fate of germ-line mutations that are deleterious, selectively neutral, or that provide a selective advantage.

Germ-line mutations will be passed down to offspring but somatic cells won't, selective advantage will favor the survival of the gamete that has the best "features" Selectively neural ones are - introns and point mutations Deleterious - lethal - the gametes won't survive Selective advantage - helps survive (sickle cell - helps survive malaria) The whole sequence (intron and exons) will pass down as long as it is in the sex cells

Review how the sodium pump in animal cells uses the energy supplied by ATP hydrolysis to maintain the concentration gradients of sodium and potassium ions.

How a cell maintains the concentration of sodium outside the cell through coupling the energy of ATP and using potassium too High concentration of Na outside, and low inside Opposite for K Pump uses ATP to take sodium from the interior (3 ions) uses ATP to flip to the outside and brings in 2 K ions Each piece has to happen for the proper function of the gradient Binding of the Na allows the protein to by phosphorylated gives energy, phos triggers a change and allows the protein to open and release Na, then K binds at the binding site, desphs, and pump returns to original conformation. Na+ pump plays a central part in the energy economy of animal cells it is typically accounts for 30% or more of their total ATP consumption. This pump uses the energy derived from ATP hydrolysis to transport Na+ out of the cell as it carries K+ in. The pump is therefore sometimes called the Na+ K+ ATPase or the Na+K+ pump. During the pumping process, the energy from aTP hydrolysis fuels a stepwise series of protein conformational changes the drives the exchange of Na+ and K+ ions. As part of the process, he phosphate group removed from ATP gets transferred to the pump itself. The transport of Na+ ions out, and K+ ions in, takes place in a cycle in which each step depends on the one before. If any of the individual steps is prevented from occurring, the entire cycle halt. Ex. The toxin ouabain for example inhibits the Na+ pump by preventing the binding of extracellular K+ arresting the cycle. The Na+ pump undergoes a series of conformational changes as it exchanges Na+ ions for K+. The binding of cytosolic Na+ and the subsequent phosphorylation by ATP of the cytosolic face of the pump induce the protein to undergo conformational changes that transfer the Na+ across the membrane and release it outside the cell. The high-energy linkage of the phosphate to the protein provides the energy to drive the conformational changes. The binding of K+ from the extracellular space and the subsequence dephospho rylation allow the protein to return to its original conformation, which transfer the K+ across the membrane and releases it into the cytosol. The change in conformation are analogous to those shown for the glucose transporter, except that here the Na+ dependent phosphorylation and K+dependent dephospho rylation of the protein cause the conformational changes to occur in an orderly fashion,e enabling the protein to do useful work. In mammals, there are three binding sites for Na+ and two for K+.

Compare how conformational changes of ion channels and transporters with respect to the passage of individual solutes across the membrane.

Ion channels- tend to be passive, allow ions to travel into the cell and may not have a conformational change. Their structure is what determines the ion that can pass through. Transporters- tend to be active, but also can be active depending on the situation (if ion goes against gradient), can undergo a conformational change that results from the solute binding to the transporter No movement across the membrane- membrane potential is 0. Differences b/t ion channels and transporters: ion channels are selective, ion channels are not continuous open, most ion channels are gates: a specific stimulus triggers then to switch between a closed and an open state by inducing a change in their conformation, ion channels do not need to undergo conformational changes, ion channels transport molecules faster than a transporter.

Recall why the DNA polymerase (Taq) used in PCR is ideally suited for this application.

Isolated from extreme thermophilic archaea and can elongate at high enough temperatures that the hydrogen bonds can't reanneal

Recall why a ribosomal RNA gene was selected to construct a phylogenetic "tree of life."

It is conserved within all living species since translation is fundamental to all living cells. It has also accumulated small mutations over time that allow it to be tracked It created the domains b/c its highly conserved. Archaea are more closely related to eukaryotes more than bacteria

Compare the mechanisms of exon shuffling and gene duplication.

Mechanism of exon shuffling- promoted by the same type of recombination as duplication but recombination occurs within the introns surrounding the gene It just depends on location of where it is cut and where you are putting it back in. Exon shuffling is two or more genes 'broken' and rejoined to make a new hybrid gene. Recombination occurs between introns, so recombination doesn't interrupt coding sequence. Because gene duplication interrupts the coding sequence, you get divergence and new species. Exon shuffling is a mechanism for generating new genes. Exon from different genes (or duplications) are brought together to form a new exon-intron structure. Shuffling allows the addition of new parts of a gene. Probably common for functional protein domains. Duplication and movement of exon is promoted by the same type of recombination that gives rise to gene duplications. In this case, recombination occurs within the introns that surround the exons. If the introns in question are from two different genes, this recombination can generate a hybrid gene that includes complete exons from both. The results of such exon shuffling are seen in many present-day proteins, which contain a patchwork of many different protein domains.

State the function of membrane transport proteins and differentiate between transporters and channels.

Membrane transport proteins are how proteins are are too large or charged to pass through the membrane get across. Two types of membrane transport proteins are transporters and channels. Transporters are much more specific but they both allow ions through the membrane based on size and charge. They shift small organic molecules or inorganic ions from one side to the other by changing shape. They are very selective. Thus, their transfer rate is slower than channels. Channels mainly involve facilitated diffusion while transporters often require energy. They are hydrophilic pore across the bilayer that permits a specific molecule to diffuse according to its concentration gradient. They mainly discriminate based on size and charge of molecule.

Contrast cut-and-paste and replicative transposition for DNA-only transposons.

Mobile genetic elements are also called transposons. They encode the components they need for movement. Cut and paste does not replicate the mobile element like replicative transposition does. Replicative transposition replicates the DNA and then inserts Cut and paste removes itself and replication replicates the entire sequence. In cut-and-paste transposition, the element is cut out of the donor DNA and inserted into the target DNA, leaving behind a broken donor DNA molecule, which is subsequently repaired. In replicative transposition, the mobile genetic element is copied by DNA replication. The donor molecule remains unchanged, and the target molecule receives a copy of the mobile genetic element. In general, a particular type of transposon moves by only one of these mechanisms. However, the two mechanisms have many enzymatic similarities and a few transposons can move by either mechanism. The donor and target DNAs can be part of the same DNA molecule or reside on different DNA molecules. The advantage of this, make viruses more fit

Outline how the glucose transporter in the plasma membrane of mammalian cells imports glucose after a meal and exports glucose to provide fuel for other tissues in the body.

Move along the concentration gradient, protein spans the membrane 12 different times and allows for conformational change at the entrance and exit, binding site is active and causes a change, deposits the glucose molecule, moving with a concentration gradient, passive transport. The glucose transporter is a passive transporter. It moves glucose along its electrochemical gradient because glucose is uncharged and the electrical component of its electrochemical gradient is zero. When glucose is plentiful outside of cells, as it is after a meal, the sugar binds to the transporter's externally displayed binding sites; if the protein the switches conformation - spontaneously and at random - it will carry the bound sugar inward and release it into the cytosol, where the glucose concentration is low. Conversely, when blood glucose levels are low - as they are when you are hungry - the hormone glucagon stimulates liver cells to produce large amounts of glucose by the breakdown fo glycogen. As a result, the glucose concentration is higher inside liver cells than outside. This glucose can bind to the internally displayed binding sites on the transporter. When the protein then switches conformation in the opposite direction - again spontaneously and randomly - the glucose will be transported out of the cells and made available for import by other, energy-requiring cells. The glucose will go inward if more glucose is binding to the transporter's externally displayed sites, and outward if the opposite is true. Glucose transporter only binds to D-glucose and not L-glucose

Summarize six basic mechanisms that generate genetic change.

Mutation within a gene- an existing gene is modified by a mutation that changes a single nucleotide or delete or duplicates multiple nucleotides, alters splicing and RNA transcript, and changes protein/ RNA product Mutation within a regulatory sequence- changes in regulation sequences change phenotypic expression Gene duplication and divergence- existing gene is duplicated and the additional gene can acquire mutations and assume new functions and expression patterns Exon shuffling- two or more existing genes are broken and rejoined to make a hybrid gene containing DNA segments that originally belonged to separate genes, in eukaryotes this often happens in introns and are removed by splicing (whole exons move) Transposition of mobile genetic elements- specialized sequences can move from one chromosome to another, alters activity and can promote duplication, shuffling, and other rearrangements (moves intracellular) (Mobile genetic elements are ancestors of viruses) Horizontal gene transfer- piece of DNA is passed from parent to daughter or to another species, plasmid transfer (in prokaryotes) (single celled eukaryotes)

Recall how potassium leak channels participate in establishing the cell's resting membrane potential.

Passive transport down concentration gradient and trying to equilibrate from the inside to the outside While the concentration gradient seems to be getting better, you build up a positive charge outside of the cell which in turn puts a positive back into the cell Nernst equation to calculate membrane resting potential (but you don't need to calculate it). The K+ concentration gradient and K+ leak channels play major parts in generating the resting membrane potential across the plasma membrane in animal cells. A hypothetical situation in which the K+ leak channels are closed and the membrane potential is zero. As soon as the channels open, K+ will tend to leave the cell, moving down its concentration gradient. Assumingthe membrane contains no open channels permeable to other ions, K+ will cross the membrane but negative ions will be unable to follow. The resulting charge imbalance gives rise to a membrane potential that tends to drive K+ back into the cell. At equilibrium, the effect of the K+ concentration gradient is exactly balanced by the effect of the membrane potential, and there is no net movement of K+ across the membrane. The Na+ pump also contributes to the resting potential - both by helping to establish the K+ gradient and by pumping 3 Na+ ions out of the cell for every 3K+ ions it pump in. Moving one more positively charged ion out of the cell with each pumping cycle helps to keep the inside of the cell more negative than the outside.

Recall the structures of phospholipids, glycolipids, triacylglycerol, and cholesterol.

Phospholipids- hydrophobic tails and polar hydrophilic head so forms a bilayer Glycolipids- two hydrophobic tails with sugars on the head instead of a phosphate and cannot be transferred by a flippase, so it remains the way that it was formed and trapped in the monolayer and ends up with the sugars being extracellular Triacylglycerols- completely hydrophobic with hydrocarbon tails (3) and a glycerol connecting the tails in the middle Cholesterol - 4 six membered rings with a 5 membered ring and the structure allows it to fill spaces between the phospholipid fatty acid tails and makes the membrane more rigid, making it less permeable and less flexible

Recall how point mutations typically arise and evaluate how the locations of these mutations can dictate their effects on an organism's appearance or fitness.

Point mutations are caused by failures of normal mechanisms for copying and repairing. A point mutation is a change that affect a single nucleotide pair. they usually arise from euros in DNA replication. Usually failure of replication proofreading or repair Some mutations can lead to selective advantage and progression of the mutation (usually polymerase errors) They can appear in regulatory sequences, within exons, within noncoding regions, and within introns. The mutations in the noncoding regions do not have an effect on the actual phenotype of a gene. The non-coding regions are where a lot of mutations accumulate. The mutations in exons are most likely to affect the phenotype of the organism of the mutated gene. These point mutations lead to genetic variation. Mutations occur randomly, but can be increased by adding pressures. Even though pt mutations seem like a bad thing, more often then not, they have neutral effects where they don't influence an organism's appearance, viability, or ability to reproduce. This occurs when the mutation occurs in the third nucleotide of the sequence. Example of a pt mutation is malaria, lactose gene,

Distinguish between the shotgun method for assembling a genome sequence and the clone-by- clone approach, which uses bacterial artificial chromosomes

Shotgun breaks into random fragments separate the fragments and sequence the single stranded fragment and then uses a computer to overlay the overlapping fragments, works best for small genomes, can lead to errors in assembly (random breaks) Clone by clone- broke human genome into overlapping fragments, put the segments into bacterial artificial chromosomes (BAC) and inserted them into E. coli which reproduced the genome and then researchers determined where the fragments fit into the existing map of the genome by using different restriction enzymes to generate a unique restriction site signature, and the locations of those allowed for the mapping of the whole genome (kind of used the restriction sites as a label) (specific cuts by restriction nucleases - makes consistent cuts). There are two main strategies for accomplishing genomic breakage and reassembly: the shotgun method and clone-by-clone approach. In shotgun sequencing, the genome is first broken into much smaller, overalapping fragments. Each fragment is then sequenced, and the genome is assembled based on overlapping sequences. The trouble with shotgun sequencing is that the reassembly process can be derailed by repetitive nucleotide sequences. In clone by clone approach, clones are digested with five different restriction enzymes, and the sites at which the different enzymes cut each clone are recorded. The distinctive pattern of restriction sites allows investigators to order the fragments and place them on a restriction map of a human genome that had been previously generated using the same nucleases.

Explain why—and how—cells keep the cytosolic concentration of calcium ions low.

Signaling molecule Important to keep cytosolic concentration low to receive signals properly Calcium pumps move from the cytosol to the ER to be stored for later (and so it doesn't signal something it shouldn't). Two ions bind to an active site, ATP driven, transition and conformational change that moves calcium to the other side of the lumen of the ER. Calcium can bind tightly to a variety of proteins in the cell, altering their activities. An influx of Ca2+ into the cytosol through Ca2+ channels, for instance is used by different cells an intracellular signal to trigger various complex processes, such as muscle contraction; fertilization, and nerve cell communication. When a muscle cell is stimulated, Ca2+ floods into the cytosol from the sarcoplasmic reticulum - a specialized form of endoplasmic reticulum. The influx of calcium stimulates the cell to contract; to recover from the contraction, calcium must be pumped back into the sarcoplasmic reticulum by this calcium pump. The calcium pump uses ATP to phosphorylate itself, inducing series of conformational changes when the pump pis open to the lump fo the sarcoplasmic reticulum, the calcium binding site are eliminated, ejecting the two calcium ions into the organelle.

Define homologous genes and state the percentage of human genes that have clear homologs in species such as the fruit fly and nematode.

Similar in nucleotide sequence because of their common ancestry, can also refer to similarities of proteins and nucleic acid sequences Non-coding sections of genes have the most mutations because it doesn't actually affect the phenotype of the organism. When looking at species that are closely related you look at non-coding, but if they are far away on the phylogenetic tree you look at coding sequences. Homologous genes are genes with similar in sequence and function due to common ancestry. We have 50% homologs w/ fruit flies, nematodes, and us.

Describe the structure of a membrane-spanning β-barrel transmembrane protein.

Similar to alpha helices in that the inside of the beta barrel has hydrophilic residues and the outside has hydrophobic residues. Forms a barrel or keg-shaped channel. The beta barrel is found in porin proteins in mitochondrial and bacterial outer membranes. It consists of 16-stranded Beta sheet curved around on itself to form a transmembrane water filled channel.

Distinguish between simple diffusion and facilitated transport.

Simple diffusion is the ability to diffuse through the membrane without help, facilitated involves a protein or helper/channel to get through (Doesn't need energy) Example: glucose too big to pass through membrane, so needs to use one of those to pass through

Outline how patch-clamp recording can be used to study the activity of ion channels.

Take a tiny micro electrode and put it down on a membrane, and apply a bit of suction to it, and can pull that tiny little patch off of the membrane and take the ion channels with you Can measure the change in voltage through generation your own system Resting potential and when initiator binds, pops open and gate opens, then closes and get a wave graph Experimentally determining how quickly that channel can let signals pass through. Patch-clamp recording is used to monitor ion channel activity. First, a micro electrode is filled with an aqueous conducting solution, and its tip is pressed against the surface of the cell. With gentle function, a tight seal is formed where the cell membrane contact the mouth of the micro electrode. Because of the extremely tight seal, current can enter or leave the microelectrode only by passing through the ion channel or channels in the patch of membrane coving its tip. It expose the cytosolic face of the membrane, the patch of membrane held in the micro electrode can be torn for the cell. This technique make it easy to alter the composition fo the solute noneither side of the membrane to test the effect of various solutes on channel activity. The circuitry for patch=clamp recording. AT the open end of the mciroelectrode, a metal wire is inserted. Current that enter the micro electrode through ion channels in the small patch of membrane covering its tip passes via the wire, through measuring instruments, back into the bath of medium surrounding the cell or the detached patch. The Patch clamp recording was the first technique used to detect conformational changes. The activity of an ion channel is very much "all or none" when an ion channel is open, it is fully open, when it is closed, it is fully closed.

Explain how hybrid cells allowed investigators to monitor the movement of membrane proteins within the plane of the lipid bilayer.

The hybrid cells are a fusion of two different cell types with different sets of membrane proteins, the movement of the different protein types can be monitored overtime Example is the fusion of a human and mouse cell- at first they form two distinct halves, but over time the proteins mix and become evenly dispersed. Formation of mouse-human hybrid cells shows that some plasma membrane proteins can move laterally in the lipid bilayer. The the mouse and human cells are first fused, their proteins are confined to their own halves of the newly formed hybrid-cell plasma membrane. Within a short time however, the membrane proteins - and lipids - completely intermix. To monitor the movement of a selected sampling of the proteins, the cells are labeled with antibodies that bind to either human or mouse proteins; the antibodies are coupled to tow different fluorescent tags so they can be distinguished in a fluoresce microscope.

Articulate how the amphipathic nature of membrane lipids drives the assembly of a lipid bilayer, as well as the formation of closed vesicles, organelles, and even whole cells.

The hydrophilic polar end can interact with water molecules on the outside and inside of the membrane and the hydrophobic areas are placed together in the middle of the membrane, and naturally take the shape of a sphere to fully close of the different layers The hydrophobic and hydrophilic interactions cause/force the phospholipids into a confirmation where the hydrophobic ends are in the middle and the hydrophilic ends face the water. Amphipathic nature of membrane lipids drives the assembly of lipid bilayer because hydrophilic molecules dissolve readily in water because they contain either charged group or uncharged polar groups that can form electrostatic attraction or hydrogen bonds with water molecules. Hydrophobic molecules, by contrast, are insoluble in water because almost all of their atoms are uncharged and non polar; therefore they cannot interact favorably with water. Instead, they force adjacent water molecules to reorganize into cagelike structures around them. However, this formation is highly ordered and requires free energy. The energy cost is lowered when the hydrophobic molecules cluster together, limiting their contact with the surrounding water molecules. As a result, you get fat droplet when dispersed in water

Illustrate how selectively neutral mutations, combined with analysis of the fossil record, can be used to construct a phylogenetic tree

The mutations accumulate overtime steadily, and the differences accumulate at such a rate that they can be measured and the selective neutrality means that all of the organisms live at the same selective success Selectively neutral mutations could be changes in the three nucleotide, so you can see the differences by looking at the dna in the fossils and comparing them The majority of the DNA sequencing that has occurred from fossils has confirmed and reaffirmed phylogenetic tree placement. For closely related species, neutral mutations are useful to gauge divergence. Because they accumulate at a steady rate unconstrained by selection pressures and these tiny differences help determine common ancestry. For instance, Chimps and humans share 99% of the same DNA.

State what distinguishes third-generation sequencing methods from older approaches and describe a third-generation method.

Third generation allows for the sequencing of a single DNA molecule- the fluorescent thing that allows the bases to be determined one at a time based on color using a single DNA poly and a DNA template with an attached primer anchored together in a tiny little compartment, which reveals the sequence of the template (allows for less errors; doesn't require amplification) Older approaches cannot do one molecule at a time (less efficient and takes longer and they require amplification of the gene via PCR or cloning through bacteria plasmid. Third generation doesn't require this). An example of a third-generation sequencing is Illumina sequencing. In this type of sequencing, it is based on the use of chain-terminating nucleotides uniquely colored fluorescent tags. However, in the method, the fluorescent tags and the chemical group that blocks elongation are removable. One DNA polymerase has added the labeled, chain-terminating nucleotide, a photo of the slide is taken and the identity of the nucleotide added at each cluster is recorded; the lab and the chain-terminator are then stripped away allowing polymerase to add the next nucleotide. Another example and probably the better example is Single Molecule Real Time sequencing it employs a special apparatus I which a single DNA polymerase and a DNA template with an attached primer are anchored together in a tiny compartment with differently colored fluorescent dNTPs. As DNA synthesis proceeds, the attachment of each nucleotide to the growing DNA strand is determined one base at a time, reveling the sequence of the template; as in other sequencing methods, large number of reactions are measured in parallel in separate compartments.

Summarize how PCR can be used to produce a DNA fingerprint.

Uses primers to target DNA areas that are known to be highly varied among humans and establishes what the sample's variations are and can compare to known samples based on certain loci. PCR can be used to detect an infection at its earliest stages. In the case, short sequences complementary to the suspected pathogen's genome are used as primers, and follow many cycle of amplification, even a few copies of invading bacterial or viral genome in a patient sample can be detected. PCR can also be used to track epidemics, detect bioterrotist attacks, and test products for the presence of potentially harmful microbes. In forensic medicine, it can be used to isolate DNA from even the smallest traces of human blood or other tissue to obtain a DNA fingerprint of a person who left the sample behind. Using primer pairs targeted at genome sequences that are known to be highly variable in the human population, PCR makes it possible to generate a distinctive DNA fingerprint for any individual.

Explain how the polypeptide chain of a transmembrane protein, with its hydrophilic backbone, is able to span the hydrophobic interior of the lipid bilayer.

Usually crosses the bilayer as an alpha helix that have different charges The hydrophobic lipid tails interact with the hydrophobic side chains of the alpha helix and the thing moving through the channel can interact with the polar interior of the helix Hydrophobic on outside; hydrophilic inside. The backbone of a polypeptide chain is hydrophilic. The atoms on either side of a peptide bond are polar and carry partial positive or negative charges. These charges are allow these atoms to hydrogen-bond with one another when the polypeptide folds into an alpha helix that spans the lipid bilayer.

Describe the ways in which water can move across cell membranes, and articulate what governs whether water will enter or exit a cell.

Very selective movement Eukaryotes have aquaporins even though water molecules sometimes get through (semipermeable) Pore is large enough to allow a single water molecule to pass through but no other ions and protons are limited too Whether it will enter or exit is governed by the concentration of solutes inside or outside of the cell. Water moves across the cell through osmosis. Water molecules diffuse rapidly through aquaporin channels in the plasma membrane of some cells. Shaped like an hourglass, each aquaporin channel forms a pore across the bilayer, allowing the selective passage of water molecules.