AP biology Gene expression
20. Biotechnology, nucleic acid hybridization, genetic engineering, and DNA sequencing
Biotechnology: process of manipulating organisms or their components for the purpose of making useful products. nucleic acid hybridization: Base pairing of one strand of a nucleic acid to a complementary sequence from another nucleic acid strand, either DNA or RNA. genetic engineering: The direct manipulation of genes for practical purposes DNA Sequencing: When researchers exploit the principle of complementary base pairing to determine the complete nucleotide sequence of a DNA molecule
Ribozyme
Catalytic RNA molecules that function as enzymes and can splice RNAs a. Because RNA is single stranded, a region of an RNA molecule may baseppair, in an antiparallel arrangement, with a complementary region elsewhere in the same molecule; this gives the molecule a particular three dimensional structure. b. Some bases in RNA contain functional groups that may participate in catalysis c. The ability of RNA to hydrogen bond with other nucleic acid molecules adds specificity to its catalytic activity
Why are cancer cells able to persist by continuing to divide while most body cells have a limited life span?
Cells from large tumors often have unusually short telomeres. Further shortening would presumably lead to self destruction of the tumor cells. Telomerase activity is abnormally high in cancerous somatic cells, suggesting that its ability to stabilize telomere length may allow these cancer cells to persist. Many cancer cells do seem capable of unlimited cell division. -The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions
Describe at least three types of post-translational modifications.
Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions. Enzymes may remove one or more amino acids from the leading end of the polypeptide chain. In some cases, a polypeptide chain may be enzymatically cleaved into two or more pieces. In other cases, two or more polypeptides that are synthesized separately may come together, if the protein has quaternary structure.
DNA technology has been used in the development of certain drugs such as Gleevec that have been highly effective in cancer treatment. In many cases, persons appear to be in nearly complete remission only to have a relapse. Explain what occurs in these cases.
Certain tumors have a random mutation that allows them to survive in the presence of a particular drug, and as a consequence of natural selection in the presence of the drug, these are the cells that survive and reproduce
Steps of elongation
Codon recognition: The anticodon of an incoming aminoacyl tRNA base pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. Peptide bond formation: An rRNA molecule of the large ribosomal subunit catalyzes the formation of a peptide bond between the carboxyl end of the growing polypeptide in the P site and the amino group of the new amino acid in the A site. This step removes the polypeptides from the tRNA in the P site and attaches to the amino acid on the tRNA in the A site. Translocation: The ribosome translocates the tRNA in the A site to the P site. At the same time, the empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site.
What are the components of a viral envelope?
Components of a viral envelope: -phospholipids and membrane protein viral envelope component derived from host -proteins and glycoprotein viral envelope component derived virus -Rod shaped, polyhedral, flue virus, bacteriophage (Virus that infects bacterial cells) (picture)
Spliceosomes
Consist of a variety of proteins and several small RNAs that recognize the splice sites -This complex binds to several short nucleotide sequences along an intron, including key sequences at each end. The intron is then released and the spliceosome joins together the two exons that flanked the intron. The small RNAs in the spliceosome not only participate in spliceosome assembly and splice site recognition, but also catalyze the splicing reaction; like proteins, RNAs can act as catalysts.
The eukaryotic gene and its transcript
Control elements: noncoding DNA that help regulate transcription by binding certain proteins -Proximal control elements are close to the promoter. Distal control elements are more distant groupings (far upstream, downstream, or in an intron) called enhancers. Enhancers bound by specific transcription factors may form activators or repressors. A given gene may have multiple enhancers, each active at a different time, cell type, or location in the organism. Each enhancer, however, is generally associated with only that gene.
16. What are the two chemical components of chromosomes?
DNA and protein
Explain the roles of each of the following enzymes in DNA proofreading and repair.
DNA polymerase: Proofread each nucleotide against its template as soon as it is covalently bonded in the growing strand. Upon finding an incorrectly paired nucleotide, the polymerase removes the nucleotide and then resumes synthesis. Enzymes that catalyze the synthesis of new DNA at a replication fork. Most DNA polymerases require a primer and a DNA template strand Nuclease: Most cellular systems for repairing incorrectly paired nucleotides, whether they are due to DNA damage or to replication eros, use a mechanism that takes advantage of the base-paired structure of DNA. In many cases, a segment of the strand containing the damage is cut out by a DNA cutting enzyme - a nuclease - and the resulting gap is then filled with nucleotides, using undamaged strand as a template. Ligase: Seals the free end of the new DNA to the old DNA, making the strand complete Repair enzymes: Teams of enzymes detect and repair damaged DNA, such as thymine dimer, which distorts the DNA molecule.
DNA technology and Recombinant DNA
DNA technology: DNA Sequencing and other techniques for manipulating DNA Recombinant DNA: DNA that has been artificially made, using DNA from different sources eg. Human gene inserted into E.coli
polymerase chain reaction
Denaturation: heat briefly to separate DNA strands Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3' end of each primer -Taq polymerasecan functions at high temperatures, which is key to automated PCR. -16 molecules will be produced by four PCR cycles
Combinational control of gene activation
Despite a large number of genes, there are only about 12 different control elements -An enhancer is about 10 control elements The availability of different activators (or repressors) in each cell dictates differential gene activity as they each can only bind to certain combinations of control elements
Determination
Determination is the point at which an embryonic cell is irreversibly committed to becoming a particular cell type. Once it has undergone determination, an embryonic cell can be experimentally placed in another location in the embryo and it will still differentiate into the cell type that is its normal fate. Differentiation, then, is the process by which a cell obtains its determined fate. As the tissues and organs of an embryo develop and their cells differentiate, the cells become more noticeably different in structure and function.
How can alternative RNA splicing result in different proteins derived from the same initial RNA transcript?
Different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. Regulatory proteins specific to s cell type control intron/exon choices by binding to regulatory sequences within te primary transcript.
induced pluripotent stem cells
Differentiated cells transformed into a type of embryonic stem cell by using a modified retrovirus to introduce extra, cloned copies of four stem cell master regulatory genes. -iPS cells could eventually provide tailor made replacement cells for patients without using any human eggs of embryos, thus circumventing most ethical objections. -Cells from patients with diseases have been reprogrammed to become iPS cells, which acts as a model for studying the diseases and potential treatments. In the field of regenerative medicine, a patient's own cells could be reprogrammed into iPS cells and then used to replace nonfunctional tissues.
Prophage
During a lysogenic cycle, the DNA molecule is incorporated into a specific site on the E coli chromosome by viral proteins that break down both circular DNA molecules and join them to each other.
Elongation of the polypeptide chain
During elongation, amino acids are added one by one to the C-terminus of the growing chain by elongation factors along the mRNA in a 5' → 3' direction -Empty tRNAs released from the E site return to the cytoplasm, where they will be reloaded with the appropriate amino acid
What are two techniques besides use of cloning vectors that can be used to introduce recombinant DNA into eukaryotic cells?
Electroporation and the injection of DNA directly into single eukaryotic cells using microscopically thin needles.
Termination of translation
Elongation continues until a stop codon in the mRNA reaches the A site Release factor: A protein that binds directly to the stop codon in the A site. The release factor causes the addition of a water molecule instead of an amino acid to the polypeptide chain. This reaction hydrolyzes the bond between the completed polypeptide and the tRNA in the P site, releasing the polypeptide through the exit tunnel of the ribosome's large subunit. Breakdown of the translation assembly requires the hydrolysis of two mre GTP molecules
RNA processing
Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm. During RNA processing, both ends of the primary transcript are altered. Also, in most cases, certain interior sections of the molecules are cut out and the remaining parts spliced together -The 5' cap is a modified form of a guanine nucleotide that is added onto the 5' end after transcription of the first 20-40 nucleotides has been transcribed -The 3' end is synthesized first and receives a 5' cap. The 3' end of the premRNA molecule is also modified before the mRNA exits the nucleus and a poly-A tail. The poly A tail facilitates the export of the mature mRNA from the nucleus, protect the mRNA from degradation by hydrolytic enzyme, and help ribosomes attach to the 5' end of the mRNA once it reaches the cytoplasm.
What are three ways bacteria may win the battle against the phages?
First, natural selection favors bacterial mutants with surface proteins that are no longer recognized as receptors by a particular type of phage. Second, when phage DNA does enter a bacterium, the DNA often is identified as foreign and cut up by cellular enzymes called restriction enzymes. A third defense is a system present in both bacteria and archaea called the CRISPR-Cas system. -When a phage infects a bacterial cell with the CRISPR-Cas system, the DNA of the invading phage is stored, integrated into the genome between two repeat sequences.
Wobble
Flexible pairing at the third base of a codon. Allows some tRNAs to bind to more than one codon. Wobble explains why the synonymous codons for a given amino acid most often differs in their third nucleotide base. Accordingly, a tRNA with the anticodon 3'-UCU-5' can base pair with either the mRNA codon 5'-UCU-3' or 5'-AGG-3', both of which code for arginine.
Replicating the ends of DNA molecules
For linear DNA, the usual replication machinery cannot complete the 5' ends of daughter DNA strands. There is no 3' end of a preexisting polynucleotide for DNA polymerase to add onto. Thus, repeated rounds of replication produce shorter DNA molecules with uneven ends -This is not a problem for prokaryotes, most of which have circular chromosomes Telomeres: Special nucleotide sequences that eukaryotic chromosomal DNA molecules have at their ends. Specific proteins associated with telomeric DNA prevent the staggered ends of the daughter molecule from activating the cell's systems for monitoring DNA damage. Telomeric DNA acts as a kind of buffer zone that provides some protection against the organism's genes shortening. Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules(i
Explain the idea of gene therapy and discuss the problems with this technique as demonstrated in the treatment of SCID.
Gene therapy is the introduction of genes into an afflicted individual for therapeutic purposes. For SCID, three patients subsequently developed leukemia, and one of them died. It is likely that the insertion of the retroviral vector occurred near a gene that triggers the proliferation of blood cells.
General vs specific transcription factors
General transcription factors: Transcription factors that are essential for the transcription of all protein coding genes. They bind to a DNA sequence or proteins. Result is a low rate of transcription Association of proteins on control elements result in specific transcription factors and result in high rates of transcription -The rate of gene expression can be strongly increased or decreased by the binding of specific transcription factors, either activators or repressors, to the control elements of enhancers.
What are maternal effect genes? Describe some effects they may control.
Genes of the mother that code for cytoplasmic determinants in the egg. A gene that, when mutated in the mother, results in a mutant phenotype in the offspring, regardless of the offspring's own genotype. The mRNA or protein products of maternal effect genes are laced in the egg while it is still in the mother's ovary. When the mother has a mutation in such a gene, she makes a defective gene product or none at all, and her eggs are abnormal; when these eggs are fertilized, they fail to develop properly. They control the orientation of the egg.
Genomic imprinting
Genomic imprinting is where methylation permanently regulates expression of either the maternal or paternal allele of particular genes at the start of development. A methylation pattern is maintained by being passed onto daughter cells, with cells forming specialized tissues that keep a chemical record of what occurred during embryonic development, accounting for genomic imprinting. Epigenetic inheritance: The inheritance of traits transmitted by mechanisms not involving the nucleotide sequence itself. For example, DNA methylation patterns are largely erased during gamete formation and reestablished during embryonic development.
telomerase
Germ cells produce telomerase. Telomerase catalyzes the lengthening of telomeres in eukaryotic cells, thus restoring their original length and compensating for the shortening that occurs during DNA replication. -If chromosomes of germ cells become shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce
Describe the relationship between glucose supply, cAMP, and CRP in the lac operon
Given access to both glucose and lactose, E. coli will use the glucose. The mechanism of E. coli sensing the glucose concentration and relaying this information to the lac operon depends on the interaction of an allosteric regulatory protein with a small organic molecule, cAMP, which accumulates when glucose is scarce. When cAMP binds to CRP, CRP attaches to the DNA strand and activates transcription, so beta-galactosidase will be produced. This is positive regulation (Occurs when an activator molecule interacts directly with the genome to switch transcription on) because it induces transcription to happen -cAMP acts as a volume switch. The level of cAMP vs glucose determines how fast transcription is happening
Histone acetylation and DNA methylation
Histone tail acetylation: The addition of an acetyl group to an amino acid in a histone tail is histone acetylation, which promotes transcription by opening up chromatin structure. This opens the nucleosome, turning the genes on DNA methylation: When a set of enzymes methylate the DNA itself on certain bass, usually cytosine. It adds methyl groups to histones, which leads to the condensation of chromatin and reduced transcription, turning genes off -The inactive mammalian X chromosome is heavily methylated. Once methylated, genes usually stay that way through successive cell divisions in a given individual. At DNA sites where one strand is already methylated, enzymes methylate the correct daughter strand after each round of DNA replication. Methylation patterns are thus passed onto daughter cells, and cells forming specialized tissues keep a chemical record of what occurred during embryonic development.
18. All genes are not "on" all the time. Using the metabolic needs of E. coli, explain how this conserves energy resources.
If the environment is lacing in tryptophan, which the bacterium needs to survive, the cell responds by activating a metabolic pathway that makes tryptophan from another compound. If the human host later eats tryptophan rich meal, the bacteria cell stops producing tryptophan, thus avoiding wasting resources to produce a substance that is readily available from the surrounding solution
Inducible (lac operon)
In E coli., lactose metabolism - Lactose not present: repressor active (regulatory gene), operone off; no transcription for lactose enzymes -Lactose present: Repressor inactive, operone on; inducer molecule (lactose) inactivates protein repressor Transcription is stimulated when inducer binds to a regulatory protein -Usually function in catabolic pathways (break down larger molecules), digesting nutrients to simpler molecules --Beta-galactosidase is the enzyme that is created, which breaks down lactose into glucose and galactose
Regulation of Chromatin Structure
In euchromatin, whether or not a gene is transcribed is affected by the location of nucleosomes along a gene's promoter and also the sites where the DNA attaches to the protein scaffolding of the chromosome. -Euchromatin is DNA that is active -Nucleosomes may hide promoter sites Although each chromosome has its own territory, loops of chromatin may extend into other sites in the nucleus. Some of these sites are transcription factors that are occupied by multiple chromatin loops from the same chromosome or other chromosomes.
Termination of transcription
In eukaryotes, RNA polymerase II transcribes polyadenylation signal sequence; the RNA transcript is released 10-35 nucleotides past this polyadenylation sequence
Briefly describe two ways genes may be edited or silenced.
In in vitro mutagenesis, specific mutations are introduced into a cloned gene, and the mutated gene is returned to a cell in such a way that it disables the normal cellular copies of the same gene.CRISPR-Cas9 is a highly effective way for researchers to knock out a given gene in order to study what that gene does.
How are coordinately controlled genes in eukaryotes expressed at the same time even when the genes may be on different chromosomes?
In prokaryotes, functionally related genes are usually clustered in a single operon, which is regulated by a single promoter and transcribed into a single mRNA molecule. Thus, the genes are expressed together, and the encoded proteins are produced at the same time. Eukaryotic genes: 1. Specific control element combinations 2. Clustered near each other on the same chromosome. Each gene has its own promoter. Change in chromatin structure allows access to all, allowing them to be transcribed, genes simultaneously
Synthesis of the leading strand during DNA replication
(ignore the top. Leading strand (top) is continuous while lagging strand (bottom) is in fragments) Replication fork: A Y-shaped region at the end of each replication bubble where parental DNA strands are being unwound
Restriction ezymes
(restriction endonucleases) used to cut strands of DNA at specific locations (restriction sites: A particular short DNA sequence) Restriction Fragments: have at least 1 sticky end (single-stranded end) -The most useful restriction enzymes cleave the sugar phosphate backbones in the two DNA strands in a staggered manner. The resulting double stranded restriction fragments have at least one single stranded end, called a sticky end. These short extensions can form hydrogen bonded base pairs with complementary sticky ends on any other DNA molecule cut with the same restriction enzyme.
Accurate translation requires two instances of molecular recognition:
--First, a correct match between a tRNA and an amino acid, done by aminoacyl-tRNA synthase. There are 20 different synthetases, one for each amino acid. --Second, a correct match between the tRNA anticodon and an mRNA codon
Okazaki fragments
-A series of segments that the lagging strand is synthesized as, which are joined together by DNA ligase Whereas only one primer is required on the leading strand, each Okazaki fragment on the lagging strand must be primed separately. After DNA polymerase III forms an Okazaki fragment, DNA polymerase I replaces the RNA nucleotides of the adjacent primer with the DNA nucleotides one at a time. DNA ligase joins the final nucleotide of this replacement DNA segment to the first DNA nucleotide of the adjacent Okazaki fragment.
RT-PCR
-Because the sequence of RNA genome of HIV is known, RT-PCR can be used to amplify and therefore detect and quantity even a small amount of HIV RNA in blood or tissue samples. -Medical scientists can now diagnose hundreds of human genetic disorders by using PCR with primers that target the genes associated with these disorders The amplified DNA product is then sequenced to reveal the presence or absence of the disease causing mutation.
What three processes lead to the transformation of a zygote into the organism?
-Cell division -cell differentiation: cells become specialized in structure and function. Differential gene expression results from different activators in different cells. -morphogenesis: the physical process that gives an organism its shape. It is the development of the form of an organism and its structures.
leading and lagging strand
-DNA polymerase III: synthesizes DNA, creating Okazaki fragments -DNA polymerase I: removes RNA and puts in DNA -DNA Ligase: joins DNA
Examples of virus
-Herpes: DNA virus -COVID: RNA virus
Repressible trp operon
-In E coli., As tryptophan accumulates, it inhibits its own production by activating and binding allosterically to the repressor protein, which binds to the operator, blocking transcription. Enzymes for tryptophan synthesis are not made -Generally function in anabolic pathways (make larger molecules), synthesizing end products. When the end product is present in sufficient quantities, the cell can allocate its resources to other uses
What property of a virus determines its attachment to a host cell membrane?
-Limited host range T even phages use their elaborate tall apparatus to inject DNA into a bacterium. Other viruses are taken up by endocytosis or, in the case of enveloped viruses, by fusion of the viral envelope with the host's plasma membrane. Entry = attach to host cell membrane receptors through capsid proteins or glycoproteins on viral envelope (An envelope is an accessory that helps them infect their hosts in animal cells) -Reproduce quickly within host cells and Can mutate easily
Proofreading and repairing DNA
-Mismatch repair: Repair enzymes replace incorrectly paired nucleotides that have evaded the proofreading process -Nucleotide excision repair: A nuclease cuts out and replaces damaged stretches of DNA -Sequence changes may become permanent and can be passed onto the next generation. These changes (mutations) are the source of the genetic variations upon which natural selection operates and are ultimately responsible for the appearance of new species
RNA splicing
-Most eukaryotic genes have long noncoding stretches of nucleotides that lie between coding regions. These are removed through RNA splicing Introns: Intervening sequences, the non coding segments of a nucleic acid Exons: eventually expressed, usually by being translated into amino acid sequences, and usually translated into amino acid sequences -The removal of introns is accomplished by spliceosomes
How do viruses spread throughout plant bodies?
-Once a virus enters a plant cell and begins replicating, viral genomes and associated proteins can spread throughout the plant through the plasmodesmata. The passage of viral macromolecules from cell to cell is facilitated by virally encoded proteins that cause enlargement of plasmodesmata. In horizontal transmission, an external source infects the plant. Because the invading virus must get past the plant's outer protective layer of cells, a plant becomes more susceptible to viral infections if it has been damaged by wind, injury, or herbivores. In vertical transmission, a plant inherits a viral infection from a parent. It can occur in aseuxal propagation or in sexual reproduction via infected seeds. Viroids: Small, circular RNA molecules that infect plants that Cause errors in regulatory systems that control plant growth
GENETICALLY MODIFIED (GM) ORGANISMS
-Organisms altered through recombinant DNA technology -Insert foreign DNA into genome or combine DNA from different genomes
RNA sequencing
-PCR is active (producing mRNAs) in a particular tissue 1. mRNAs are isolated from the tissue being studied 2. mRNAs are cut into similar sized, small fragments 3. mRNAs are reverse transcribed into cDNAs of the same size 4. cDNAs are sequenced 5. The short sequences are mapped onto the genome sequence. The resulting data, including the number of times a sequence is present, indicate which genes are expressed in a given tissue and at what level -RNA seq is not based on hybridization with a labeled probe, so it doesn't depend on knowing genomic sequences. It can measure levels of expression over a very wide range.
Mutations
-Point mutations are changes in just one nucleotide pair of a gene, which can lead to the production of an abnormal protein -If a mutation has an adverse effect on the phenotype of the organism, the condition is referred to as a genetic disorder or heredity disease Point mutations within a gene can be divided into two general categories: Single nucleotide pair substitution or Nucleotide pair insertions or deletions -Nucleotide pair substitution: replaces one nucleotide and its partner with another pair of nucleotides -Silent mutations: Have no effect on the amino acid produced by a codon because of redundancy in the genetic code. A nucleotide pair substution may transform one codon into another that is translated into the same amino acid. -Missense mutations: Still code for an amino acid, but not the correct amino acid. a substation that changes one amino acid to another. -Nonsense mutations: Change an amino acid codon into a stop codon through a point mutation, most lead to a nonfunctional protein
RFLPS
-Restriction Fragment Length Polymorphism -Cut DNA with different restriction enzymes -Each person has different #s of -DNA fragments created -Analyze DNA samples on a gel for disease diagnosis -Outdated method of DNA profiling (required a quarter-sized sample of blood)
STR ANALYSIS
-STR = Short Tandem Repeats Non-coding DNA has regions with sequences (2-5 base length) that are repeated -Each person has different # of repeats at different locations (loci) -Current method of DNA fingerprinting used - only need 20 cells for analysis -PCR is used to amplify particular STRs, using sets of primers that are labeled with colored fluorescent tags; the length of the region, and thus the number of repeats, can then be determined by electrophoresis.
alternative splicing
-Some introns contain sequences that regulate gene expression and many affect gene products -Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing Consequently, the number of different proteins an organism can produce is much greater than its number of genes
Breast cancer
-The absence or excess expression of these receptors can cause aberrant cell signalling, leading in some cases to inappropriate cell division, which can contribute to cancer. -The basal-like subtype of breast cancer does not express ER alpha, PR, or HER2. Because it does not express PR at normal levels, the cells are unresponsive to therapies that target PR, such as herceptin. -Mutations in BRCA1 and BRAC2 are found in at least half of inherited breast cancers, and tests using DNA sequencing can detect these mutations. -Viruses can interfere with gene regulation in several ways if they integrate their genetic material into the DNA of a cell. Viral integration may donate an oncogene to the cell, disrupt a tumor suppressor gene, or convert a proto oncogene to an oncogene. Some viruses produce proteins that inactivate p53 and other tumor suppressor proteins, making the cell more prone to becoming cancerous.
conservative model
-The two parental strands reassociate after acting as templates for new strands, thus restoring the double helix Semiconservative model: The two strands of the parental molecule separate and each function as a template for synthesis of a complementary strands, matches the data Dispersive model: each strand of both daughter molecules contains of a mixture of old and newly synthesized DNA
Viruses What are the four forms of viral genomes?
-obligate intracellular parasites: They can replicate only within a host cell. -Components = nucleic acid + capsid --Some viruses also have viral envelopes that surround capsid RNA viruses: no error-checking mechanisms. RNA viruses use virally encoded RNA polymerases that can use RNA as a template. double stranded DNA, single stranded DNA, double stranded RNA, and single stranded RNA
The initiation of transcription at a eukaryotic promoter
1. A eukaryotic promoter commonly includes a TATA box ( A promoter that is crucial in forming the initiation complex in eukaryotes, a nucleotide sequence containing TATA.) about 25 nucleotides upstream from the transcription start point 2. Several transcription factors, one recognizing the TATA box, must bind to the DNA before RNA polymerase II can bind in correct position and orientation 3. Additional transcription factors bind to the DNA along with RNA polymerase II forming the transcription initiation complex. RNA polymerase II then unwinds the DNA double helix, and RNA synthesis begins at the start point on the template strand
A model for the action of enhancers and transcription activators
1. Activator proteins bind to distal control elements grouped as an enhancer in the DNA. 2. A DNA bending protein brings the bound activation closer to the promoter. General transcription factors, mediator proteins, and RNA polymerase II are nearby 3. The activators bind to certain mediator proteins and general transcription factors, helping them form an active transcription initiation complex on the promoter. Activator: Specific transcription factor binds an enhancer resulting in transcription. Activators have 2 domains: DNA binding portion and 1+ activation portion
How does a bacteriophage destroy a bacterial cell?
1. Attachment: The T4 phage uses its tail fibers to bind to specific surface proteins on an E coli cell that acts as receptors 2. Entry of phage DNA and degradation of host DNA: The sheath of the tail contract, injecting the phage DNA into the cell and leaving an empty capsid outside. The cell's DNA is hydrolyze. 3. Synthesis of viral genomes and protein: The phage DNA directs production of phage proteins and copies of the phage genome by host and viral enzymes, using components within the cell 4. Self assembly: Three separate sets of proteins self assemble to form phage heads, tails, and tail fibers. The phage genome is packed inside the capsid as the head forms 5. Release: The phage directs production of an enzyme that damages the cell wall, allowing fluid to enter. The cell swells and finally bursts, releasing 100 to 200 phage particles
What are the two main ways of controlling metabolism in bacterial cells? Which is a short-term response, and which is a long-term response?
1. Cells can adjust the activity of enzymes already present. This is a fairly rapid physiological response, which relies on the sensitivity of many enzymes to chemical cues that increase or decrease their catalytic activity. The activity of the first enzyme in the pathway is inhibited by the pathway's end product through feedback inhibition, which is typical of anabolic pathways and short term. 2. Cells can adjust the production level of certain enzymes via a genetic mechanism; that is, they can regulate the expression of the genes encoding the enzymes. The control of enzyme production occurs at the level of transcription. This is long term.
Gene cloning and some uses of cloned genes
1. Gene inserted into plasmid (a cloning vector) 2. Plasmid put into bacterial cell 3. Host cell grown in culture to form a clone of cells containing the cloned gene of interest 4. Basic research and various applications
The replicative cycle of an enveloped RNA virus
1. Glycoproteins on the viral envelope bind to receptors on the host cell. For some viruses, the envelope fuses with the plasma membrane; others enter by endocytosis 2. The capsid and viral genome enter the cell. Digestion of the capsid by cellular enzymes releases the viral genome 3. The viral genome functions as a template for synthesis of complementary RNA strands by a viral RNA polymerase 4. New copies of viral genome RNA are made using the complementary RNA strands as templates 5. Complementary RNA strands also function as mRNA, which is translated into both capsid proteins and glycoproteins for the viral envelope 6. Vesicles transport envelope glycoproteins to the plasma membrane 7. A capsid assembles around each viral genome molecule 8. Each new virus buds from the cell, its envelope studded with viral glycoproteins embedded in membrane derived from the host cell
GENE THERAPY USING A RETROVIRAL VECTOR
1. Insert RNA version of normal allele into virus 2. Let retrovirus infect bone marrow cells that have been removed from patient and cured 3. Viral DNA carrying the normal allele inserts into chromosome 4. Inject engineered cells into patient
multistep model of cancer development
1. Loss of tumor suppressor gene APC (or other) 2. Activation of ras oncogene 3. Loss of tumor suppressor gene SMAD4 4. Loss of tumor suppressor gene p53 5. Additional mutations
What are four important applications of PCR?
1. PCR is speedy and specific. Only a minuscule amount of DNA need be present in the starting material, and this DNA can be partially degraded, as long as there are a few copies of the complete target sequence. 2. PCR is used to provide the specific DNA fragment for cloning. PCR primers are synthesized to include a restriction site at the end of each of the DNA fragments that matches the site in the cloning vector, and the fragment and vector are cut and ligated together. 3. The resulting plasmids are sequenced so that those with error free inserts can be selected. 4. PCR has been used to amplify DNA from a wide variety of sources.
how proteins are targeted for the endoplasmic reticulum
1. Polypeptide synthesis begins on a free ribosome in the cytosol 2. An SRP binds to the signal peptide, halting synthesis momentarily 3. The SRP binds to a receptor protein in the ER membrane, part of a protein complex that forms a pore 4. The SRP leaves, and polypeptide synthesis resumes, with simultaneous translocation across the membrane 5. The signal peptide is cleaved by an enzyme in the receptor protein complex 6.The rest of the completed polypeptide leaves the ribosomes and folds into its final conformation
five major events in transcription and translation.
1. RNA is transcribed from a DNA template 2. In eukaryotes, the RNA transcript (pre-mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm 3. The mRNA leaves the nucleus and attaches to a ribosome 4. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP 5. A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. When completed, the polypeptide is released from the ribosom
Using a restriction enzyme and DNA ligase to make a recombinant DNA
1. Restriction enzyme cuts the sugar phosphate backbones at each arrow 2. A DNA fragment from another source is added. Base pairing of sticky ends produces various combinations 3. DNA catalyzes the formation of covalent bonds that close up the sugar phosphate backbones of DNA strands. It is the stable recombinant DNA molecule that was produced by the ligase catalyzed joining of DNA from two sources that forms a stable recombinant plasmid containing foreign DNA. -example of a gene of interest that might be engineered into a plasmid: Insulin
Making complementary DNA (cDNA) for a eukaryotic gene
1. Reverse transcriptase is added to a test tube containing mRNAs, isolated from a sample of cells 2. Reverse transcriptase makes the first DNA strand using the mRNA as a template and a short poly-dT as DNA primer 3. mRNA is degraded by another enzyme 4. DNA polymerase synthesizes the second DNA strand, using a primer in the reaction mixture 5. The result is cDNA, which carries the complete coding sequence of the gene but no introns -only complementary DNA (cDNA)can be used in engineering a plasmid that will be inserted into a bacterial cell because They include only exons
the process of a specific amino acid being joined to a tRNA.
1. The amino acid and the appropriate tRNA enter the active site of the specific synthetase 2. Using ATP, the synthetase catalyzes the covalent bonding of the amino acid to its specific tRNA 3. The tRNA charged with its amino acid is released by the synthetase
Chargaff's rules
1. The base composition of DNA varies between species. 2. In any species the number of A and T bases is equal and the number of G and bases is equal.
The replicative cycle of HIV, the retrovirus that causes AIDS
1. The envelope glycoproteins enable the virus to bind to specific receptors on certain white blood cells 2. The virus fuses with the host cell's plasma membrane 3. The capsid proteins are removed, releasing the viral proteins, RNA, and reverse transcriptase 4. Reverse transcriptase catalyzes the synthesis of a DNA strand, using the viral RNA as template, and then hydrolyzing it 5. Reverse transcriptase catalyzes the synthesis of a second DNA strand complementary to the first 6. The double stranded DNA is incorporated as a provirus into the cell's DNA 7. Proviral genes are transcribed into RNA molecules, which serve as genomes for progeny viruses and as mRNAs for translation into viral protein 8. Viral envelope glycoproteins are made in the ER 9. Viral capsid proteins and reverse transcriptase are made in the cytosol 10. Vesicles transport the glycoproteins to the cell's plasma membrane 11. Capsids are assembled around viral genomes and reverse transcriptase molecules 12 New viruses, with viral envelope
A simplified viral replicative cycle
1. The virus enters the cell and is uncoated, releasing viral DNA and capsid proteins 2. Host enzymes replicate the viral genome 3. Meanwhile, host enzymes transcribe the viral genome into viral mRNA, which host ribosomes use to make more capsid proteins 4. Viral genomes and capsid proteins self assemble into new virus particles, which exit the cell
How many nucleotide bases are there? How many amino acids? How many nucleotides are required to have a unique code for each of these 20 amino acids? The language of DNA is a triplet code. How many unique triplets exist?
4 20 3 64
transformation
A change in genotype and phenotype due to assimilation of foreign DNA
What is an expression vector?
A cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame
Why is the DNA sample to be separated by gel electrophoresis always loaded at the cathode or negative end of the power source?
A gel made of polymer acts as a molecular sieve to separate nucleic acids or proteins differing in size, electrical charge, or other physical properties as they move in an electrical field. -. Shorter molecules are slowed down less than longer molecules, so shorter molecules move faster through the gel.
Direction of synthesis of DNA
A new DNA strand can only be synthesized in one direction. The new strands formed during DNA replication must also be antiparallel to their template strands. The antiparallel arrangements of the double helix, together with a constraint on the function of DNA polymerase, has an important effect on how replication occurs. Because of their structure, DNA polymerase III can add nucleotides only to their free 3' end of a primer or growing DNA strand, never to the 5' end. Thus, a new DNa strand can elongate only in the 5' → 3' direction from the free OH
Explain what is meant by 5ʹ and 3ʹ ends of the nucleotide
A polynucleotide strand has directionality, from the 5' end (with the phosphate group) to the 3' end (with the -OH group of the sugar). 5' and 3' refer to the numbers assigned to the carbons in the sugar ring.
Explain how transgenic "pharm" animals might be able to produce human proteins.
A transgene can be inserted into the genome of an animal in such a way that the transgene is secreted from the animal
Compare and contrast a prophage and a provirus.
After a virus enters a host cell, its reverse transcriptase molecules are released into the cytoplasm, where they catalyze synthesis of viral DNA. The newly made viral DNA then enters the cell's nucleus and integrates into the DNA of a chromosome. The integrated viral DNA is the provirus and it never leaves the host's genome, remaining a permanent resident of the cell. MEanwhile, prophage leaves teh host's genome at the start of a lytic cycle.
Bicoid What important understandings about embryonic development resulted from the research into bicoid?
After the egg is fertilized, the mRNA is translated into protein. The Bicoid protein then diffuses from the anterior end toward the posterior, resulting in a gradient of protein within the early embryo, most highly concentrated at the anterior end. First, it led to the identification of a specific protein required for some of the earliest steps in pattern formation. It thus helped understand how different regions of the egg can give rise to cells that go down different developmental pathways. Second, it increased the understanding of the mother's critical role in the initial phases of embryonic development. Third, the principle that a gradient of morphogens can determine polarity and position has proved to be a key developmental concept for a number of species.
Distinguish between the leading and the lagging strands during DNA replication.
Along one template strand, DNA polymerase III can synthesize a complementary stand continuously by elongating the new DNA in the mandatory 5' → 3' direction. DNA polymerase II remains in the replication fork on that template stand and continuously adds nucleotides to the new complementary strand as the fork progresses. The DNA molecule made by this mechanism is called the leading strand. To elongate the other new strand of DNA in the mandatory 5' → 3' direction, DNA polymerase III must work along the other template stand in the direction away from the replication fork. The DNA strand elongating in this direction is called the lagging strand. In contrast to the leading strand, which elongates continuously and moves toward the replication fork the lagging strand is synthesized discontinuously, as a series of segments.
How has our understanding of the arrangement of chromosomes in the nucleus changed from the time when interphase chromosomes were thought to be a tangled mass like a bowl of spaghetti?
Although an interphase chromosome lacks an obvious scaffold, there are proteins that further organize the 10 nm fiber into larger compartments and smaller looped domains. Some of the looped domains appear to be attached to the nuclear lamina, on the inside of the nuclear envelope, and perhaps also the fibers of the nuclear matrix. These attachments may help organize regions of chromatin where genes are active. The chromatin of each chromosome occupies a specific restricted area within the interphase nucleus, and the chromatin fibers of different chromosomes do not appear to be entangled. As a cell prepares for mitosis, its chromatin becomes organized into loops and coils, eventually condensing into a characteristic number of short, thick metaphase chromosomes that are distinguishable from each other.
19. Bacteria vs. Viruses
Bacteria -Prokaryotic cell -Most are free-living (some parasitic) -Relatively large size -Antibiotics used to kill bacteria Virus -Not a living cell (genes packaged in protein shell) -Intracellular parasite -Vaccines used to prevent viral infection -Very small
What seems to be the reason for the high incidence of abnormalities?
In the nuclei of fully differentiated cells, a small subset of genes is turned on and expression of the rest of the genes is repressed. This regulation often is the result of epigenetic changes in chromatin, such as acetylation of histones or methylation of DNA. During the nuclear transfer procedure, many of these changes must be reversed in the later stage nucleus from a donor animal for genes to be expressed or repressed appropriately in earlier stages of development. Researchers have found that the DNA in cells from cloned embryos, like that of differentiated cells, often has abnormally high numbers of methyl groups. This finding suggests that the reprogramming of donor nuclei requires more accurate and complete chromatic restructuring than occurs during cloning procedures. Because DNA methylation helps regulate gene expression, misplaced or extra methyl groups in the DNA of donor nuclei may interfere with the pattern of gene expression necessary for normal embryonic development. In fact, the success of a cloning attempt may depend in large part on whether or not the chromatin in the donor nucleus can be artificially modified to resemble that of a newly fertilized egg
How does the bacterial cell identify the phage DNA when it is encountered? What happens to the phage DNA?
Infection by a phage triggers a transcription of the CRISPR region of the bacterial DNA. This region consists of DNA from phages that previously infected the cell, separated by repeats. The RNA transcript is processed into short RNA strands. Each short RNA strand binds to a Cas protein, forming a complex. Complementary RNA binds to DNA from the invading phage. The Cas protein then cuts the phage DNA. After being cut, the entire phage DNA molecule is degraded and can no longer be replicated.
Synthesis of an RNA transcript
Initiation: After RNA polymerase binds to the promoter, the polymerase unwinds the DNA strands and initiates RNA synthesis at teh start point on the template strand Elongation: The polymerase moves downstream as the mRNA is being created, unwinding the DNA and elongating the RNA transcript 5' → 3'. After the transcription has occurred, the DNA strands reform a double helix Termination: Transcription proceeds through a terminator sequence in the DNA. The transcribed terminator (an RNA sequence) functions as the termination signal, causing the polymerase to detach from the DNA and release the RNA transcript, which requires no further modification before translation.
Insertions and deletions
Insertions and deletions are additions or losses of nucleotide pairs in a gene. These mutations have a disastrous effect on the resulting protein more often than substitutions do -Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation. This occurs whenever the number of nucleotides inserted or deleted is not a multiple of three. -Insertions or deletions outside the coding part of a gene could affect how the gene is expressed
What are three applications of personal genome analysis?
It allow individuals to send in a swab containing cheek cells that the company will analyze genetically, it can compare person's DNA signments with those from reference populations around the world, and an analysis of the sequence on the Y chromosome on males can trace their maternal lines of descent
Viral replication
Lytic Cycle: -Use host machinery to replicate, assemble, and release copies of virus -Virulent phages: phage that replicates only by a lytic cycle that dies through lysis or apoptosis. Lysogenic (Latent) Cycle: -DNA incorporated into host DNA and replicated along with it -Bacteriophage DNA = prophage, Animal virus DNA = provirus A certain chemical or high energy radiation causes lysogenic --> lytic cycle Temperate Phage: uses both methods of replication.
Post transcriptional regulation
Micro miMRNA, RNAi interferes and small interfering siRNA -As a result in binding a complementary sequence of mRNA and blocking translation or leading to breakdown of mRNA -Essentially, these RNAs break down other RNA, determining how long DNA is available to be transcribed
Prions
Misfolded, infectious protiens that cause misfolding of normal proteins Prions act very slowly, with an incubation period of at least ten years before symptoms develop. The lengthy incubation period prevents sources of infection from being identified until long after the first cases appear, allowing more infections to occur. Prions are not destroyed or deactivated by heating to normal cooking temperatures. Prions are virtually indestructible
Domains
Molecular architecture of proteins consisting of discrete regions -One domain of an enzyme, might include the active site, while another might allow the enzyme to bind to a cellular membrane. In quite a few cases, different exons code for the different domains of a protein. -Exon shuffling may result in the evolution of new proteins by missing and matching exons between different genes
Making multiple polypeptides in bacteria and eukaryotes
Multiple ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) Polyribosome: A ribosome that attaches to the mRNA, eventually resulting in a number of ribosomes trailing along the mRNA once a ribosome is far enough past the start codon. They enable a cell to rapidly make many copies of a polypeptide. -With no nuclear envelope, a prokaryotic celll can simultaneously transcribe and translate the same gene and the newly made protein can quickly diffuse to its site of function.
Exploring chromatin packing in a eukaryotic chromosome
Nucleotide: A region of the cell in a bacterium where the DNA is supercoiled Nucleosome: The basic unit of DNA packing. A nucleosome consists of DNA wound twice around a protein core of eight histones. Histones: Proteins responsible for the main level of DNA packing in interphase chromatin. Histones can undergo chemical modifications that result in changes in chromatin condensation -The amino acid end of each histone (the histone tail) extends outward from the nucleosome and is involved in regulation of gene expression
Types of codons
Of the 64 triplets, 61 code for amino acids; 3 are "stop" signals to end translation (UAA, UAG, and UGA) Start codon: It signals the protein synthesizing machinery to begin translating the mRNA at that location -The genetic code is redundant (more than one codon may specify a particular amino acid) but not ambiguous; no codon specifies more than one amino acid
Protein folding and modifications
Often translation is not sufficient to make a functional protein. Polypeptide chains are modified after translation or targeted to specific sites in the cell -During synthesis, a polypeptide chain begins to coil and fold spontaneously into a specific shape: a three dimensional molecule with secondary and tertiary structure -A gene determines the primary structure, and the primary structure in the turn determines the shape -Post translocation modifications may be required before the protein can begin doing its particular job in the cell
Processing
Often, eukaryotic polypeptides must be processed to yield functional protein molecules. For instance, cleavage of the initial insulin polypeptide forms the active hormone.
How does a DNA virus reproduce its genome?
Once the viral genome is inside, the proteins it encodes can command the host, reprogramming the cell to copy the viral genome and manufacture viral proteins. The host provides the nucleotides for making viral nucleic acids, as well as enzymes, ribosomes, tRNAs, amino acids, ATP and other components needed for the making of viral proteins. Many DNA viruses use the DNA polymerases of the host cell to synthesize new genomes along the template provided by the viral DNA.
Compare oncogenes and proto-oncogenes.
Oncogenes are cancer causing genes in certain types of viruses. The normal versions of the cellular genes, called proto0oncegenes, code for proteins that stimulate normal cell growth and division. -four mechanism involved in converting a proto-oncogene to an oncogene: epigenetic modifications: Mutations in a gene for a chromatin modifying enzyme can lead to loosening of the chromatin and inappropriate expression of a proto-oncogene b. translocations: Gene moved to new locus, under new controls result in normal growth stimulating protein in excess c. gene amplification: Multiple copies of the gene result in normal growth stimulating protein in excess d.point mutations: Point mutation within a control element results in normal growth stimulating protein in excess. Point mutation within the gene results in hyperactive or degradation resistant protein.
What process ensures that all the tissues and organs of an organism are in their characteristic places? Where do the molecular cues that control this process arise?
Pattern formation in animals begins in the early embryo, when the major axes of an animal are established. In a bilaterally symmetrical animal, the relative positions of head and tail, right and left sides, and back and front - the three major body axes - are set up before the organs appear. The molecular cues that control pattern formation, collectively called positional information, provided by cytoplasmic determinants and inductive signals. These cues tell a cell its location relative to the body axes and to neighboring cells, and determine how the cell and its descendants will respond to future molecular signals. Homeotic genes: Regulatory genes that control pattern formation.
Nucleotide
Phosphate group, deoxyribose, and nitrogenous base -Adenine and guanine are purines, nitrogenous bases with two organic rings, while cytosine and thymine are pyrimidines, which have a single ring. Pairing a purine (adenine) with a pyrimidine (thymine) is the only combination that results in a uniform diameter for the double helix. Each base has a chemical side group that can form hydrogen bonds with its appropriate partner: Adenine can form two hydrogen bonds with thymine and only thymine; guanine forms three hydrogens with cytosine and only cytosine.
Describe the two possible sources of viral genomes.
Plasmids: Small, circular DNA molecule found in bacteria and in the unicellular fungi, yeasts. They exist apart from the genome, can replicate independently of the genome, and are occasionally transferred between cells. Transposons: DNA segments that can move from one location to another within a cell's genome.
Targeting polypeptides to the specific locations
Polypeptide synthesis can always begin in the cytosol and finishes in the cytosol unless the polypeptide signals the ribosome attached to the ER. Polypeptides destined for the ER or for secretion are marked by a signal peptide -The signal peptide is a sequence of about 20 amino acids at or near the leading end of the polypeptide Signal recognition particle (SRP): Binds to the signal peptide. The SRP escorts the ribosome to a receptor protein built into the ER membrane -The signal peptide is removed by an enzyme -Other kinds of signal peptides target polypeptides to other organelles
Coding initiation of translation
Proteins may bind 5' end of mRNA blocking ribosome attachment -Wholesale activation of stored mRNA Protein factor stimulates translation
Basic principles of transcription and translation
RNA is the bridge between genes and protein synthesis Ribosomes: The sites of translation -In prokaryotes, translation of mRNA can begin before transcription has finished. In a eukaryotic cell, the nuclear envelope separates transcription from translation -Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA ' -Central dogma: DNA →m RNA → protein (amino acids strung together by a peptide bond) Primary transcript: The initial RNA transcript (pre-mRNA) from any gene prior to processing
Retrovirus
RNA virus that uses reverse transcriptase (RNA --> DNA) -Newly made viral DNA inserted into chromosome of host (provirus) -Host transcribes provirus to make new virus parts Example: HIV (Human Immunodeficiency Virus) -Viral RNA --> viral DMA --> using RNA polymerase, viral mrNA ---> viral amino acids
stem cells
Relatively unspecialized cells that can both reproduce themselves indefinitely and, under appropriate conditions, differentiate into specialized cells of one or more types. -Zygote = totipotent (A mature cell with the potential to differentiate and then give rise to all the specialized cell types of the organism) -Embryonic stem cells = pluripotent (many cell types). Reproduce indefinitely, and depending on culture conditions, they can be made to differentiate into a wide variety of specialized cells, but not all Adult stem cells: multipotent (a few cell types) or induced pluripotent, iPS (forced to be pluripotent. ). are not able to give rise to all cell types in the organism.
The structure and function of ribosomes
Ribosomes facilitation specific coupling of tRNA anticodons with mRNA codons in protein synthesis -The two ribosomal subunits (large and small) are made of proteins and rRNAs -P site: Holds the tRNA that carries the growing polypeptide chain -A site: holds the tRNA carrying the next amino acid to be added to the chain. -E site: The exit site, where discharged tRNAs leave the ribosome
What are SNPs? How are they used to help screen for certain diseases? What are some examples of diseases for which there are genetic markers?
SNPs are a single base pair site where variation is found in at least 1% of the population. Once a SNP is identified that is found in all people affected by the disease being studied, researchers focus on that region and sequence it. Some examples are diabetes, heart disease, and several types of cancer.
origins of replication.
Short stretches of DNA that have specific nucleotides where the replication of chromosomal DNA begins -Replication proceeds in both directions from each origin, until the entire molecule is copied -Proteins that initiate DNA replication recognize their sequence and attach to the DNA, separating strands and opening up multiple replication bubbles, which fuse and speed up the copying of the very long DNA molecules.
The basic principle: base pairing to a template strand
Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication -This yields two exact replicas of the parental molecule
Bacterial DNA replication proteins and their functions (Helicase, single stranded binding protein, topoismerase, *primate*, DNA polymerase III, DNA polymerase I, and DNA ligase)
Single strand binding proteins: holds DNA strands apart DNA ligase: joins DNA fragments together DNA polymerase: joins the RNA primer with the template strand of DNA (nucleotides) to create a new DNA strand from the 5 prime to the 3 prime end
New mutations and mutagens
Spontaneous mutations can occur during errors in DNA replication or recombination Mutagens: Physical or chemical agents that can cause mutations. Most carcinogens (cancer causing chemicals) are mutagens, and most mutagens are carcinogens -Nucleotide analogs are chemicals similar to normal DNA nucleotides but that pair incorrectly during DNA replication. Other chemical mutagens interfere with correct DNA replication by inserting themselves into the DNA and distorting the double helix. Still other mutagens cause chemical changes in bases that change their pairing properties.
thymine dimer
Teams of enzymes detect and repair damaged DNA, such as thymine dimer (often caused by ultraviolet radiation), which distorts the DNA molecule. An important function of the DNA repair enzymes in our skin cells is to repair genetic damage caused by the UV rays of sunlight: for example, adjacent thymine bases on a DNA strand can become covalently linked into thymine dimers, causing the DNA to buckle and interfere with DNA replication. A nuclease enzyme cuts the damaged DNA stand at two points, and the damaged section is removed. Repair synthesis by a DNA polymerase fills in the missing nucleotides, using the undamaged strand as a template. DNA ligase seals the free end of the DNA to the old DNA, making the strand complete.
telomere erosion
Telomeres become shorter during every round of replication. Thus, as expected, telomeric DNA tends to be shorter in dividing somatic cells of older individuals and in cultured cells that have divided may times. It has been proposed that shortening of telomeres is somehow connected to the aging process of tissues and even to aging of the organism as a whole.
Transcription
The first stage of gene expression RNA polymerase: Pries the DNA strands apart and joins together the RNA nucleotides, catalyzes RNA synthesis. Does not need any primer. Uses the DNA template strand to transcribe a new mRNA strand. -The RNA is complementary to the DNA template strand. RNA synthesis follows the same base pairing rules as DNA, except that uracil substitutes for thymine Promoter: the DNA sequence where RNA polymerase attaches initiates transcription of a gene begins and ends. Terminator: In bacteria, the sequence signalling the end of transcription Transcription Unit: The stretch of DNA downstream from the promoter that is transcribed into an RNA molecule
What situation did Archibald Garrod suggest caused "inborn errors of metabolism"?
The inability to make a particular enzyme -Each mutation affects protein synthesis at a specific step by preventing production of the enzyme that catalyzes that step. -One gene one polypeptide hypothesis: the function of a gene is to dictate production of a specific enzyme.
Ribosome association and initiation of translation
The initiation of translation starts when the small ribosomal subunit binds with mRNA and a special initiator tRNA. In a bacterial cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. -An initiator tRNA, with the anticodon UAC, base pairs with the start codon, AUG, and caries methionine, the first amino acid in the new polypeptide. Then the small subunit moves along the mRNA until it reaches the start codon (AUG) -Proteins called initiation factors bring in the large subunit that completes the translation initiation complex. -Hydrolysis of GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid
Degradation
The length of time each protein functions in the cell is strictly regulated by selective degradation. Many proteins, such as the cyclins involved in regulating the cell cycle, must be relatively short-lived if the cell is to function appropriately. To mark a protein for destruction, the cell commonly attaches molecules of ubiquitin to the protein. Proteasomes then recognize the ubiquitin tagged proteins and degrade them after translation -Post-transcriptional control includes regulation of mRNA degradation. For some mRNAs, the initiation of translation can be blocked by regulatory proteins that bind to the specific sequences or structures within the untranslated (UTR) region at the 5' or 3' end, preventing the attachment of ribosomes.
Distinguish between heterochromatin and euchromatin. How is this important to gene expression?
The less compacted, more dispersed interphase chromatin is called euchromatin to distinguish it from the more compacted, denser appearing heterochromatin. For both types of chromatin, the basic organizing unit is the 10 nm fiber - the basic organizing unit is nucleosomes joined by linker DNA. In heterochromatin, this 10 nm fiber is folded and bent back onto itself to a much greater degree than in euchromatin, accounting for its denser appearance. Because heterochromatin is so compacted, it is largely inaccessible to the proteins responsible for transcribing the genetic information, a crucial early step in gene expression. In contrast, the looser packing of euchromatin makes its DNA accessible to those proteins, and the genes present in euchromatin are available for transcription. Interphase chromosomes occupy specific restricted regions of the nucleus, and the fibers of different chromosomes do not become entangled -As the cell prepares for mitosis, the chromatin is organized into loops and coiled, eventually condensing into short, thick metaphase chromosomes
Anticodon
The loop extending from the other end of the tRNA. It is a particular nucleotide triplet that pairs to a specific mRNA codon.
Synthesizing a new DNA strand
The new DNA strand will start from the 3' end of the RNA primer -Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate -dATP supplies adenine to DNA and is similar to the ATP of energy metabolism. The difference is in their sugars" dATP has deoxyribose while ATP has ribose -As each monomer joins the DNA strand, via a dehydration reaction, it loses two phosphate groups as a molecule of pyrophosphate. Water is lost.
The structure and function of tRNA
The presence of complementary stretches of nucleotide bases that can hydrogen bond to each other allows this single strand to fild back on itself and form a molecule with a particular 3-D structure. The tRNA actually twists and folds into a compact 3-D structure that is roughly L-shaped, with the 5' and 3' ends of the linear tRNA both located near one end of the structure. The protruding 3' end acts as the attachment site for an amino acid.
17. Gene expression
The process by which DNA directs protein (or in some cases, just RNA) synthesis, includes transcription and translation -The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins -Proteins are the links between genotype and phenotype Gene: A region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule.
What is the fundamental difference between the repressor protein in a repressible operon versus the repressor protein in an inducible operon?
The repressor protein is inactive by itself and requires tryptophan, for example, as a corepressor in order to bind to the operator in a repressible operon. In an inducible operon, the repressor is active by itself, binding to the operator and switching the lac operon off. An inducer inactivates the repressor. lac repressor: When lactose is present, it binds to a repressor that inactivates the repressor and allows the pathway to break down the lactose, which is negative regulation -Both repressible and inducible operons demonstrate negative control because active repressors can only have negative effects on transcription
Building a polypeptide
The three stages of translation: -Initiation -Elongation -Termination All three stages require protein factors that aid in the translation process. Energy is required for some steps
How do different sets of activators come to be present in two cells? Explain how each of these occurs: a. distribution of cytoplasmic determinants b. induction
The unfertilized egg has molecules in its cytoplasm, encoded by the mother's genes, that influence development. After fertilization and mitotic division, the cell nuclei of the embryo are exposed to different combinations of cytoplasmic determinants and, as a result, express different genes. Cells in the lowest tier of cells are releasing molecules that signal nearby cells to change their gene expression
explain how a ras mutation leads to cancer.
There seem to be two categories of genes involved in cancer: oncogenes. and tumor-suppressor genes, which work like the brakes on a car. The Ras protein, encoded by the Ras gene, is a G protein that relays a signal from a growth factor receptor on the plasma membrane to a cascade of protein kinases. The cellular response at the end of the pathway is the synthesis of a protein that stimulates the cell cycle. Normally, such a pathway will not operate unless triggered by the appropriate growth factor. But certain mutations in the ras gene can lead to the production of a hyperactive Ras protein that triggers the kinase cascade even in the absence of a growth factor, resulting in increased cell division.
Why are both the gene of interest and the plasmid cut with the same restriction enzyme?
To check the recombinant plasmids after they have copied many times in host cells to make sure the fragment has been inserted, a researcher might cut the products again using the same restriction enzyme. If the insert is there, there would be two DNA fragments, one the size of the plasmid and one the size of the inserted DNA.
Stages in gene expression that can be regulated in eukaryotic cells
Transcription control: DNA structure, RNA polymerase, promoter sites, transposable elements like transposons and retrotransposons (repetitive DNAs), pseudogenes Post Transcriptional control: Splicing of preRNA → mature RNA, RNAi Translational control: Polyribosomes, life expectancy of mRNA Post translational modification: Biotransformation of polypeptide → protein (ER to golgi)
Transcription factors and initiation complex Picture: Transcription elongation
Transcription factors: Help guide the binding of RNA polymerase and the initiation of transcription Transcription initiation complex: A promoter that the completed assembly of transcription factors and RNA polymerase III is bound to -Anything that's going to be created is upstream, anything that has been created is downstream
Transcription and translation
Transcription: The synthesis of RNA using information in the DNA in the nucleus, producing mRNA. Just as a DNA strand provides a template for making a new complementary strand during DNA replication, it can also serve as a template for assembling a complementary sequence of RNA nucleotides. Translation: The synthesis of a polypeptide in the ribosomes in the cytoplasm using the information in the mRNA.
TECHNIQUES OF GENETIC ENGINEERING
Transformation: bacteria takes up plasmid (w/gene of interest) PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells Gel electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA 🡪 + pole)
Activation
Translation of all the mRNAs in a cell may be regulated simultaneously. In a eukaryotic cell, such global control usually involves the activation of one or more protein factors required to initiate translation. This mechanism plays a role in starting translation of mRNAs that are stored in eggs. Just after fertilization, translation is triggered by the sudden activation of translation initiation factors. The response is a burst of synthesis of the proteins encoded by the stored mRNAs.
Composite transposons carry an entire host gene and could be duplicated and inserted by a retrotransposon pathway
Transposons: jumping genes -Jump from one gene to another -They can damage genes and lead to pseudogenes -Composite transposons carry with them one of the hosts genes -Retrotransposon increase their numbers by using reverse transcriptase
The triplet code and template strand
Triplet code: A series of nonoverlapping three-nucleotide words that the follow of information from gene to protein is based on. Only in mRNA, not DNA -The words of a gene are transcribed into complementary nonoverlapping three-nucleotide words of mRNA -These words are then translated into a chain of amino acids, forming a polypeptide Template strand: One of the two DNA strands that provides a template for ordering the sequence of complementary nucleotides in an RNA transcript. This is always on the same side of the template strand -The mRNA molecule produced is complementary to the template strand
mutation of p53.
Tumor-suppressor genes help prevent uncontrolled cell growth. One that is found mutated (and therefore nonfunctional) in more than 50% of human cancer is p53. Once the p53 gene is activated, it activates several other genes, such as p21. The p21 halts the cell cycle by binding to cyclin-dependent kinases, allowing time for the cell to repair the DNA. p53 also activates expression of a group of miRNAs that inhibit the cell cycle. The p53 protein can also turn on genes directly involved in DNA repair. If DNA damage is irreparable, p53 activates suicide genes, whose protein products brin about programmed cell death. Thus, p53 acts in several ways to prevent a cell from passing on mutations due to DNA damage.
Drugs for Prevention/Treatment
Vaccine: weakened virus or part of pathogen that triggers immune system response to prevent infection Ex. Coronavirus vaccines, HPV, MMR, HepA, Flu shot Antiviral Drugs: block viral replication after infection Ex. Tamiflu (influenza), AZT (HIV)
T2 bacteriophage
Virus: DNA (sometimes RNA) enclosed by a protective coat, often simply protein. -only one of the two components of T2 phage (DNA or protein) enter an E. coli during infection, providing the genetic information -the phage DNA entered the host cells, but the phage protein did not. DNA inside the cell played an ongoing role during the infection process. They concluded that the DNA injected by the phage must be the molecule carrying the genetic information that makes the cells produce more new viral DNA and proteins.
What are three ways that viruses make us ill? Why do we recover completely from a cold but not from polio?
Viruses may damage or kill cells by causing the release of hydrolytic enzymes from lysosomes. Some viruses cause infected cells to produce toxins that lead to disease symptoms, and some have molecular components that are toxic, such as envelope proteins. People usually recover completely from colds because the epithelium of the respiratory tract, which the viruses infect, can efficiently repair itself. In contrast, damage inflicted by poliovirus to mature nerve cells is permanent because these cells do not divide and usually cannot be replaced
restriction enzymes
When phage DNA does enter a bacterium, the DNA often is identified as foreign and cut up by cellular enzymes called restriction enzymes, which restrict a phage's ability to replicate within the bacterium -Restriction enzymes don't destroy the DNA of the bacterial cells that produce them because The bacterium's own DNA is methylated in a way that presents attack by its own restriction enzymes
What advantages does use of yeast have over bacterial cells for an expression system?
Yeasts have plasmids and are used by biologists to avoid eukaryotic bacterial incompatibility. Many eukaryotic proteins of yeast will not function unless they are modified after translation.
To demonstrate you understand how the lac and trp operon work, let's assume a human host has had a meal of turkey (rich in the amino acid tryptophan) and washed it down with milk. Explain your answer to each of the following: a. Will the trp operon of E. coli in the gut of the human be active?b. Will the lac operon of E. coli in the gut of the human be active?
Yes, the enzymes for glucose break down in glycolysis are continuously present. No, only when lactose is present and glucose is in short supply does E. coli use lactose as an energy source, and only then does it synthesize appreciable quantities of the enzymes for lactose breakdown.
Positive control of the lac operon by cAMP receptor protein
a. Lactose present glucose scarce (cAMP level high): abundant lac mRNA synthesized. If glucose is scarce, the high level of cAMP binds to cAMP repressor protein (activating CRP or CAP) which binds to the promoter and increases RNA polymerase binding there, activating transcription. The lac operon produces large amounts of mRNA coding for the enzymes that the cell needs for use of lactose b. Lactose present, glucose present (cAMP level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CRP or CAP is unable to stimulate transcription and bind upstream the lac promoter at a significant rate because it is inactive, even though no repressor is bound . The lac operon will be transcribed but at a low level
The lytic and lysogenic cycle of phage
a. lytic cycle: A phage replicative cycle the culminates in death of the host cell b. lysogenic cycle: Allows replication of that phage genome without destroying the host
What are the three tools of biotechnology that are employed in RT-PCR?
cDNA synthesis, PCR amplification, and gel electrophoresis
Codons and the coding strand
mRNA base triplets that are read in the 5' -->3' direction during translation Coding strand: The nontemplate strand. The nucleotides of this strand are identical to the codons, except that T is present in the DNA place of U in the RNA A goes with T, U goes with A, C goes with G, G goes with C
mRNA, tRNA, and rRNA
mRNA: For a protein coding gene, this resulting RNA molecule is a transcript of the gene's protein building instructions. It carries a genetic message from the DNA to the protein synthesizing machinery of the cell tRNA: Each tRNA molecule enables translation of a given mRNA codon into a certain amino acid. Each carries a specific amino acid on one end and has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA rRNA: Ribosomal RNA is the main constituent of the A and P sites and of the interface between the subunits; it also acts as the catalyst of peptide bond formation. Responsible for both the structure and the function of the ribosome. The proteins, which are largely on the exterior, support the shape changes of the rRNA molecules as they carry out catalysis during translation
noncoding RNAs
much of the RNA that is transcribed is not translated into protein. These RNAs are called noncoding RNAs -One of the noncoding RNAs that regulate gene expression is microRNA (miRNA). 1. The miRNA binds to a target mRNA with at least 7 complementary bases 2. If miRNA and mRNA bases are complementary all along their length, the mRNA is degraded. If the match is less complete, translation is blocked.
Nuclear transplantation
nucleus of egg is removed and replaced with nucleus of body cell
operator, operon, repressor, inducer, and corepressor
operator: A DNA segment that is the on-off switch. It controls the access of RNA polymerase to the genes. (tryptophan not present) operon: The operator, the promoter, and the genes they control - the entire stretch of DNA required for enzyme production for the tryptophan pathway - repressor: Binds to the operator, preventing RNA polymerase from transcribing the genes, often by preventing RNA polymerase from binding. Coded from a regulatory gene. inducer: It inactivates the repressor. corepressor: A small molecule that cooperates with a repressor protein to switch an operon off
Gene cloning
process by which scientists can product multiple copies of specific segments of DNA that they can then work with in the lab ex: To make many copies of, or amplify, a particular gene and to produce a protein product from it. Cloning vector: a DNA molecule that can carry foreign DNA into a host cell and be replicated there (eg. bacterial plasmid) -Plasmids are widely used as cloning vectors because they can be readily obtained from commercial suppliers, manipulated to form recombinant plasmids by insertion of foreign DNA in a test tube, and then easily introduced into bacterial cells.
DNA microarray assays:
study many genes at same time 1. Isolate mRNA 2. Make cDNA by reverse transcription, using fluorescently labeled nucleotides 3. Apply the cDNA mixture to a microarray, a differential gene in eachh spot. The cDNA hybridizes with any complementary DNA on the microarray 4. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample
