Molgen Module 4

Malignant tumors are dependent on one or a few genes to maintain cancer phenotype: _____________

"oncogene addiction"

Three major types of RNAs were discussed in some detail: mRNA, rRNA, and tRNA. For each of the conditions below predict the consequences in terms of the population of proteins being synthesized in a particular cell. What qualitative and quantitative changes, if any, are expected in the individual protein involved (if one is involved) and in the population of proteins produced in that cell? (a) An acridine dye-induced mutation in mRNA. The condition is heterozygous in the involved cell. (b) A deletion (homozygous) that removes approximately half of the rRNA genes.

(a) Population of proteins: Half of the protein products of that gene will be defective, and the other half will be normal. Individual protein: The protein should show multiple amino acid substitutions "downstream" from the point of the mutation. If a nonsense triplet is introduced, the protein would be shortened in the substituted region. (b) Population of proteins: There would be an overall reduction in protein synthesis. Individual protein: All of the proteins would be made in their normal form, but at reduced levels.

a) Describe the structural components of the lactose operon in E coli. (b) State the function of the lac regulator gene. (c) State the function of β-galactosidase in the lac system. Separate and clearly label your answer parts A-C in the box below.

(a) operator-physical component of DNA where repressor binds, repressor-trans-acting protein that binds operator when lactose is absent, lacZ-Bgal structural gene, lacY-Bgal permease, lacA-Bgal transacetylase (b) The regulator gene produces a repressor protein, which interacts with the operator to shut off transcription. In the presence of lactose, the repressor protein does not interact with the operator. (c) β-galactosidase cleaves the lactose sugar into its components glucose and galactose.

What are Drosophila P elements (eukaryotic Class II transposon)?

+500 to 3k base pairs +31 bp inverted terminal repeats +Encodes 2 proteins: Transposase and repressor +Insertion into genes causes MUTATIONS +Transposase normally only expressed in germ line, where transposition can cause mutation +Repressor protein inhibits movement at other times +Drosophila P elements are used to generate mutants as an experimental technique -Method: 2 lines crossed, one with P transposase, one with deleted P element that is unable to mobilize -Result: mobilized element produces insertional mutants in germ line of F1 flies

What are adduct forming agents (chemical mutagens)?

+A DNA adduct is a substance that covalently binds to DNA, altering its conformation -EX. Acetaldehyde (cigarette smoke) and heterocyclic amines (HCAs) which are chemicals created by cooking meats at high temp -At least 17 different HCAs have been linked to cancers including stomach, breast, and colon

What is a gene?

+A gene is a segment of DNA composed of a transcribed region and a regulatory sequence that makes transcription possible -The DNA sequence upstream (or 5') of a gene usually contains the regulatory elements that control its expression

What is Mismatch repair (DNA Repair Mechanisms)?

+After DNA is replicated, ADENINE METHYLASE adds METHYL GROUPS TO ADENINES in the following sequence: 5'-GATC-3' or 3'-CTAG-3' (Note the As) +Immediately after DNA synthesis, the synthesized DNA strand is temporarily unmethylated 1) Recognize the mismatch and bind to unmethylated DNA strand 2) An endonucleasethat creates a nick in the DNA backbone adjacent to the mismatch 3) An exonucleasethat unwinds and degrades the nicked DNA strand 4) DNA polymerase fills in the gap. 5) A DNA ligase then reseals the phosphodiesterbackbone +In humans, mutations in genes that code for the DNA mismatch repair machinery, are commonly found in a wide range of cancers, such as (ex. ovarian, prostate, leukemias)

What are tautomeric shifts (base isomers)?

+Another source of pre-mutational base mispairing +Tautomeric shift is a migration of a hydrogen atom or proton, resulting in switch of a single bond and adjacent double bond +Tautomer: Each of two or more isomers of a compound that exist together in equilibrium, and are readily interchanged by migration of an atom or group within the molecule +Normal (keto) form of base skewed to rare (enol) form OR normal (amino) to rare (imino) of base +Ionization of bases can also lead to shift to rare form +Can lead to mispairing of bases

What are examples of gene regulation using RNA molecules or proteins in prokaryotes?

+Antisense RNA control +Riboswitches -can be repressive (ex. vitamin B12) OR the presence of the regulatory protein can cause a secondary structure that permits translation (inductive) +Riboswitches that can act as Ribozymes

What is the Mutagen Summary?

+Background mutation rate +Agents act to increase this rate -Non-chemical -Chemical -Isomerization phenomena +Some sites more susceptible to mutation

What are the different types of Chemical Mutagens?

+Base analogs: 2 aminopurine - analog of adenine (Protonated 2-AP, pairs with cytosine), 5-bromouracil - analog of thymine (5-BU becomes ionized, pairs with guanine) +Base alteration chemicals: Alkylating agents (add alkyl group) -EX. EMS=ehtyl methanesulfonate, mustard gas +Intercalating agents: Insert in double helix -> Indel ->Frameshift mutation -EX. ethidium bromide - have shapes that allow them to wedge themselves into DNA double helix -> Insertion/deletion mutation -> Frameshift mutation

What are the Insertion sequences of Class II transposons?

+Both prokaryotic and eukaryotic types +Simple insertion sequence (IS) Prokaryotic -Flanked on ends by small inverted repeats -Transposable gene encoded in internal sequence -Jump from location to location ("cut and paste") -Can disrupt genes when inserted

What is a spontaneous mutation?

+Changes in nucleotide sequence that have no known cause +Often occur during DNA replication or result from oxidative damage

How are Transcriptional Networks involved in Developmental genetics and gene regulation?

+Control of gene expression +Understanding the many genes involved in development and how they interact with one another at the molecular level +Allows us to begin to unravel how development proceeds in space and time in a highly coordinated manner -Ex. signal inputs activate gene A, which directs synthesis of transcription factor A, which binds to REGULATORY SITES within B region, it binds to CRMs in these regions. Product of B gene is also a transcription factor, which activates gene C, which also produces a transcription factor that activates D & feeds back to regulate B

What are Transcription Enhancers?

+DNA sequences that bind specific DNA binding proteins, which influences transcription Enhancers are modular and contain several short DNA sequences Enhancers often include binding sites for positive AND negative regulatory factors

What do activators contain?

+DNA-binding domain that binds the enhancer DNA sequence +A trans-activating domina that activates (enhances) transcription through protein-protein interactions with the factors in the transcription complex

What is the Maize Ac-Ds System (Another Class II system of transposons)?

+Discovered by Barbara McClintock by mutation analysis in maize +Ds: Dissociation +Ac: Activator +Correlated genetic observations with cytological observations of maize chromosomes +Results: -Ds element located on Chromosome 9, -in absence of Ac, Ds is not transposable, W gene expressed=wild type -If Ac present of genome, Ds induced breakage adjacent to its location during SOMATIC cell development. -Chromosome breakage leads to phenotypic events = W gene not expressed = mutant effect) -In presence of Ac, Ds can "jump" into genes, disrupting (mutating) them = W gene not expressed=mutant effect -Can also "jump" out of genes, restoring function = W restored = Wild type

What are consequences of mutation?

+Genetic variation that can be acted upon by natural selections (if mutations are fixed in germ line) +Somatic disease phenotype

What did Platensimycin study results show?

+High specificity +High toxicity to many gram positive bacteria +Low toxicity to eukaryotic cells & wild type E. coli (gram negative) -Important because we are full of e.coli in fauna +Cured mice infected with disseminated S. aureus +Potential as new antibiotic targeting a different pathway than other antibiotics +Powerful method to "select" for potential new antibiotics

What is fatty acid biosynthesis (Fab)?

+Involved in producing lipids that make up bacterial membrane +Essential for survival of bacteria +No membrane -> No bacteria +Logical target site to disrupt bacterial life cycle

What is the prokaryotic gene regulation summary?

+Many levels of control +Repressive or Inducible, Positive or Negative +Attenuation (Premature transcription termination) trp operon +Proteins or RNA molecules involved in the regulation

What are common causes of spontaneous mutations?

+Oxidative compounds +Depurination +Errors in DNA replication +Spontaneous mutations occur at low rate

What is Nucleotide Excision Repair (General Excision) (Mutation Repair Mechanisms)?

+Pathway that repairs bulky lesions in DNA that alter or distort the double helix -EX. In E. coli, the uvr gene products cut 13 nucleotides, including the lesion -Different enzymes, that recognize and cut a specific number of nucleotides around the lesion

What are the three RNA polymerases Eukaryotes have that recognize different types of promoters to transcribe certain sets of genes?

+RNA polymerase II transcribes mRNAs +

Why is regeneration in mammals limited?

+Rb and ARF are both tumor suppressors +Rb in regenerative vertebrates and mammals. +ARFonly in mammals +The ARF(alternative reading frame) protein is produced from the CDKN2 locus. +Both products of this locus, ARF and INK4a have been shown to be involved in decline of regenerative potential of multiple tissues during aging by affecting stem cell self renewal.

What is the E.coli trp operon?

+Repressible operon +Normally ON, producing enzymes of the tryptophan synthesis pathway +Turned OFF when tryptophan is present

What are AREs: Adenine-uridine Rich Elements?

+Sequences found in 3' UTR of many mRNAs that play important roles in inflammation or cancer +Proteins that bind to AREs recruit deadenylases that promote therapid degradation of mRNA

What are examples of resistant bacteria?

+Shigella (bloody diarrhea) +Neisseria gonorrhea (gonorrhea) +Enterococcus sp. (hospital settings) +Streptococcus pneumonia +Staphylococcus aureus

What is the significance of RNAi?

+Short synthetic double-stranded RNA molecules are introduced into cells +Become part of the RNA-induced silencing complex (RISC) -triggers the RNA degradation pathway

What are Signaling Pathways in Developmental Genetics and gene regulation?

+Signaling Pathways: Regulate development, coordinate transcription, direct differentiation of tissues and organs: Notch is an example of a signaling pathway and cell-cell interaction

What is true of new antibiotic development?

+Slow process +Most new antibiotics are deriative of earlier antibiotics +Screen geenrally by trial and error of compounds

How do HATs target genes?

+Specific transcription factors target HATs to genes +INSULATOR elements (DNA regions) bind specific proteins to act as barriers and prevent spreading of the remodeling to neighboring genes

What are screened products of Streptomyces platensis?

+Streptomyces are gram-positive aerobic bacteria +Selectively screened 250k natural product extracts +Identified small,potent, selective inhibitor of the Fab pathway +Platensimycin (C24H27HO7)

What are Ac and Ds?

+They are MOBILE GENETIC ELEMENTS +Ac contains two 11bp IMPERFECT ITR (inverted terminal repeats) -Ac contains 2 open reading frames (ORFs) one of the ORFs encodes a transposase gene +Ds is identical to Ac except for deletions (there are a variety of Ds elements) in the TRANSPOSASE gene....this is what explains Ds dependence upon Ac for its transposition

How can modifications to chromatin influence gene expression?

+Through histone modification -Histone modification by Histone Acetyltransferase (HAT) lessens the attraction between histones and DNA by addition of acetyl groups to basic AAs on histone tails which reduces attraction between negatively charged DNA and basic AAs of histones -Histone deacetylases (HDACs) reverse this remodeling

What are the CAAT box and GC box?

+Transcription factor binding sites +They are elements that bind transcription factors

What do Rescue mutant experiments do?

+Transform mutant E. coli with a plasmid (Circular DNA molecule) called F' that carries various parts of the lac operon +Determine if we can rescue the mutant phenotype +System induced with IPTG in place of lactose +Creates "Partial diploid" bacteria, has 2 copies of some genes, thus "partial". AKA merozygote

What is significant of Frog (xenopus) rDNA?

+Xenopus: N=18, 2N=36 +1 chromosome has rDNA genes +Constriction observed in chromosome +Nucleolar organizer region (NOR) +Region organizes into nucleolus +Nucleolus becomes transcriptionally active +Extra nucleoli are made in xenopus, these are EXTRAchromasomal, up to 1500 extra copies of the NOR +400 rRNA genes X 1500 copies = 648k copies of rRNA gene = over 1 million ribosomes +Why so many ribosomes in oocyte? Tadpoles don't have enough ribosomes of their own and die at the tadpole stage as they run out of ribosomes from the mother

Are there Transposable elements in humans?

+YES +Our genome is full of transposon DNA +Some estimates of up to one half of our genome +EX: -LINE (long interspersed elements); 6kb in length, up to 800k+ copies -SINE elements (short interspersed repeats), 100-500 bases, and around 1.5 million copies -May move and cause mutation

What are characteristics of Fragile X Syndrome?

-1 in 4k males, 1 in 8k females, dominant trait -Repeat expansion in 5' UTR -H0: CGG repeats modified, base within & around repeat methylated -Gene is for FMRP (fragile X mental retardation protein) -Normally expressed in brain, binds & potentially transports mRNAs -FMRP Not present -> No mRNA transport -> no translation of those mRNAs -> improper brain development

How can Nucleosome remodeling complexes alter nucleosome structure?

-Altering contacts between DNA and histone (alteration of DNA protein contacts) -Altering path of DNA around the nucleosome -Altering the structure of the nucleosome core itself

What observations sugget that DNA methylation plays a role in gene regulation?

-An inverse relationship between degree of methylation and degree of gene expression -More methylation = decreased gene expression -Methylation patterns are tissue specific and heritable for all cells in that tissue -Incorporation of 5-azacytidine causes undermethylation of sites of incorporation and changes in pattern of gene expression

What are the consequences of mutagens?

-Base replacement -Base alteration leading to mis-pairing -Base alteration leading to non-pairing

What does the Notch/Delta Pathway control?

-Blood cell development -Neurogenesis -Retina Development

How can cytoplasmic mRNAs be degraded?

-Deadenylation (removal of the poly(A) tail) initiates the degradation of the large majority of mRNAs -Can lead to decapping and degraded in a 5'-3' direction (MAJOR mRNA degradation pathway) -Can lead to degradation in a 3'-5' direction (MINOR mRNA degradation pathway)

What does the Wnt Pathway control?

-Dorsalization of Body -Female reproductive development -Dorsal-ventral differences

What are the common chemical and non-chemical mutagens?

-EMS (150 = chemical -X-rays = non-chemical -UV Rays = non-chemical -ICR-170 = chemical

What is a repeat unit expansion mutation?

-EX. CGG, CAG, or CTG repeats -Consequence: Human diseases -Huntington disease results from repeat expansion in protein coding region of CAG = glutamine -Mytonic Dystrophy results from repeat expansion at 3' end of transcript (CTG) -Fragile X syndrome results from a repeat expansion in transcribed region (CGG)

What is the significant history of microRNA history?

-First demonstrated in C. elegans by Victor Ambros -Showed lin-4 doesn't encode protein, but instead a pair of small RNAs (22nt and 61nt) -61nt RNA was predicted to form stem-loop structure and serve as precursor for 22nt RNA -Ambros and Ruvken labs noticed COMPLEMENTARITY in sequence of the lin-4 RNA to 3'UTR sequences in lin-14 mRNA transcript

Where are gene-poor chromosomes and gene-rich chromosomes located in the nucleus?

-Gene poor chromosomes located peripherally -Gene rich chromosomes located more internally

What is epigenetic inheritance?

-Heritable alterations in gene expression in which DNA sequence is unchanged -EX. Paramutation in plants -In paramutation an allele in one generation heritably affects the other allele in future generations, even if the allele causing the change IS ITSELF NOT TRANSMiTTED -EX. Parental imprinting -Genes rendered inactive depending upon parental source -Imprinted genes usually methylated by special methylases, demethylated by demethylases -Active genes less methylated (hypomethylated) -EX. X-chromosome inactivation -Random X chromosome inactivation in placental females -Inactivation hereditary through cell division

Do both HpaII and MspI cut methylated sites?

-HpaII does NOT cut methylated sites, it cuts nonmethylated sites -MspI DOES cut metyhlated sites, cuts both nonmethylated and methylated sites

Why is Eukaryotic gene regulation more complex than prokaryotic gene regulation?

-Larger amount of DNA -Larger # of chromosomes -spatial separation of transcription and translation -mRNA processing, RNA stability, and cellular differentiation in eukaryotes

What is the order of developmental expression?

-Maternal genes (Anterior, posterior, terminal group genes) -Zygotic genes (Gap genes, pair rule genes, segment polarity genes) -Homeotic selector genes

What does the TGF-Beta Pathway control?

-Mesoderm induction -Left-right asymmetry -Bone development

What does the Receptor Tyrosine Kinase Pathway control?

-Mesoderm maintenance

What is true of Kinase enzymes?

-Modify other proteins by phosphorylating them (add a phosphate) -The addition of the phosphate can change target protein's localization, activity, or association with other proteins -Kinases involved in cell signaling pathways and cell division pathways -Mutated kinases associated with disease phenotypes including cancer -Kinases are involved in signal transduction

What are different types of Maternal effect genes?

-Morphogens are mRNA placed in the oocyte -Bicoid: protein gradient in embryo produced from maternal mRNA, directs head development, bicoid protein is a morphogen -Nanos: protein gradient in embryo produced from maternal mRNA, needed for posterior development -Oscar: affects cells that will become germ line in zygote -If any offspring are homozygous for missing bicoid or nanos genes then they are LETHAL -Therefore it's the mother's genotype that is important as mother has to have at least one bcd+ allele

What are the different categories of mutagens?

-Non-chemical mutagens -Chemical mutagens -Base isomers

What does the Hedgehog Pathway control?

-Notochord induction -Somitogenesis -Gut/visceral mesoderm

What are the 3 main categories of mutations?

-Replication slippage leading to increased # of repeat units -Point mutations -Insertion of foreign DNA element disrupting DNA

What is Double-Strand Break Repair (Mutation Repair Mechanisms)?

-Responsible for attaching two broken DNA strands -Uses homologous recombination repair -Requires the complimentary region on the sister chromatid to use as a template

What is Base Excision Repair (Specific Excision) (Mutation Repair Mechanism)?

-System that corrects DNA that contains a damaged base pair -Requires DNA GLYCOSYLASE - multiple types that each recognize a specific base -EX. A Uracil DNA glycosylase recognizes the presence of uracil in DNA -Glycosylases cut the glycosidic bond between the base and sugar to create an apyrimidinic or apurinic site -Sugar with the missing base is recognized by an AP endonuclease that cuts the phosphodiester backbone -DNA polymerase and DNA ligase then fill in and repair gap

What is the fate of the mRNA ?

-Trafficked to specific subcellular localizations -Translated -Stored (P bodies) -Degraded

How is all of this new information about RNAs used?

-a method to develop new drugs -RNAi (RNA interference) techniques

What does the DNA-binding DOMAIN of an activator typically have?

-helix-turn-helix motif -a zinc finger -or a leucine zipper -Conserved motifs that we previously saw in prokaryotic transcription activators

What observations provided the first evidence for miRNA functionality?

-lin-14 3' UTR had been shown to mediate translational repression of LIN-14 protein -Complementary sequences to lin-4 RNA in lin-14 3' UTR was shown to be critical for translational repression

What is the main difference between siRNAs and miRNAs?

-siRNA targets genes from which they were transcribed -miRNA targets genes other than those from which they were transcribed

What are the two important types of small RNAs involved in gene regulation?

-small Interfering RNAs (siRNA) -Micro RNAs (miRNA)

What are some characteristics of microRNA?

-some miRNA can act as "oncogenes" and cause cancer -Other miRNA can act as "tumor-suppressor" genes and prevent cancer -One miRNA (miR-21) found to be overexpressed in most tumor types -this makes miR-21 a potential therapeutic target +Overall takeaway is that miRNA can effect, directly or indirectly, the type of protein being made

What are three ways in which gene regulation is accomplished by modifying the structure of chromatin?

1 Modification of Histone proteins. 2. Chromatin remodeling 3. DNA methylation

Name six different levels at which gene expression might be controlled.

1. Alteration or modification of the gene structure at the DNA level 2. Transcriptional regulation 3. Regulation at the level of mRNA processing 4. Regulation of mRNA stability 5. Regulation of translation 6. Regulation by pos -translational modification of the synthesized protein

What are the stages of salamander regeneration?

1. Amputation and wound healing 2. Dedifferentiation 3. Early bud 4. Mid bud 5. Late bud 6. Early differentiation

Describe some important differences between bacterial and eukaryotic cells that affect the way in which genes are regulated.

1. Bacterial genes are often clustered in operons and are coordinately expressed through the synthesis of a single polygenic mRNA. Eukaryotic genes are typically separate, with each containing its own promoter and transcribed on individual mRNAs. Coordinate expression of multiple genes is accomplished through the presence of response elements. Genes sharing the same response element will be regulated by the same regulatory factors. 2. In eukaryotic cells, chromatin structure plays a role in gene regulation. Chromatin that is condensed inhibits transcription. So, for expression to occur, the chromatin must be altered to allow for changes in structure. Acetylation of histone proteins and DNA methylation are important in these changes. 3. At the level of transcription initiation, the process is more complex in eukaryotic cells. In eukaryotes, initiation requires a complex machine involving RNA polymerase, general transcription factors, and transcriptional activators. Bacterial RNA polymerase is either blocked or stimulated by the actions of regulatory proteins. 4. Finally, in eukaryotes the action of activator proteins binding to enhancers may take place at a great distance from the promoter and structural gene. These distant enhancers occur much less frequently in bacterial cells.

Over the past decade, the most significant finding in biology has been the identification of miRNAs and siRNAs and their role in regulating the development of many multicellular organisms. Briefly describe the four different ways these small RNAs influence gene expression.

1. Cleavage of mRNA 2. Inhibition of translation 3. Transcriptional silencing 4. Degradation of mRNA

What are the somatic mutations in cancer genomes?

1. Driver mutations: confer growth advantage on the cell in which they occur, therefore positively selected for 2. Passenger mutations: not subject to selection, present in progenitor cell prior to clonal expansion, biologically neutral, do NOT offer growth advantage

What is the mechanism of Antisense RNA control under high osmolarity?

1. Extracellular osmolarity high 2. micF gene is activated and micF RNA produced 3. micF RNA pairs with 5' end of ompF RNA, blocking the ribosome-binding site 4. No OmpF protein produced -lower OmpF protein = less diffusion across membrane

What are the different types of Zygotic genes in development?

1. Gap genes: diide embryo into broad regions -activated by maternal gradient products -mutants of these have large gaps in segmentation pattern ex. hunchback, giant, knirps 2. Pair rule genes: divide broad regions established by gap genes, establishes boundaries and cell fates in segments. expressed only in presence of transcription factors 1&2 -mutants: hairy, even-skipped 3. Segment polarity genes: divides segments into anterior and posterior ends, ex. engrailed, wingless 4. Selector genes: determine the adult structure to be formed from each segment -mutants are HOMEOTIC MUTANTS: cause one segment to be transformed to the fate of a neighboring segment

What is Post replication repair (Mutation Repair Mechanisms)?

1. Lesion present in DNA unwound prior to replication 2. Replication skips over lesion and continues 3. Undamaged complementary region of parental strand is recombined 4. New gap is filled by DNA polymerase and DNA ligase -After this repair the lesion is still present but it may be repaired before the next round of replication

What is the hierarchy of mutations?

1. Promoter minus (P-) mutations (disables RNA polymerase binding), supercedes all other mutations in cis: system OFF all the time 2. O^Cmutations in cis supercede any I mutation: system ON all the time 3. I^S dominant over I^+dominant over I^- (constitutive expresion, ON all the time, no regulation)

What is the mechanism of Riboswitch control of translation?

1. Regulatory protein binds to riboswitch and stabilizes a secondary structure 2. Masks a ribosome-binding site 3. No translation takes place IF no Regulatory protein 1. Riboswitch assumes alternative secondary structure 2. ribosome-binding site available 3. Translation occurs

What is the Insertion mechanism of Bacterial Insertion sequence of transposons?

1. Transposase makes staggered cuts in target DNA 2. Transposon DNA inserts at target site 3. Gaps in target site DNA are filled by host repair enzyme

What is the mechanism of antisense RNA control under low osmolarity?

1. When extracellular osmolarity is low 2. ompF mRNA is translated to produce OmpF protein -OmpF protein functions as diffusion pores on bacterial membrane

What do E.coli lac operon mutation studies determine?

1. Whether some parts of the lac operon are cis acting (able to control expression of genes ONLY when on the same piece of DNA) 2. are trans acting (Able to control expression of genes on other DNA molecules) 3. And what is the hierarchy of the different types of mutations

Explain two different ways that intragenic suppressors may reverse the effects of mutations.

1.Intragenic suppression is the result of second mutations within a gene that restore a wild-type phenotype. The suppressor mutations are located at different sites within the gene from the original mutation. One type of suppressor mutation restores the original phenotype by reverting the meaning of a previously mutated codon to that of the original codon. The suppressor mutation occurs at a different position than the first mutation, which is still present within the codon. Intragenic suppression may also occur at two different locations within the same protein. If two regions of a protein interact, a mutation in one of these regions could disrupt that interaction. The suppressor mutation in the other region would restore the interaction. Finally, a frameshift mutation due to an insertion or deletion could be suppressed by a second insertion or deletion that restores the proper reading frame.

What role does RNA stability play in gene regulation? List three types of RNA structural elements that can affect the stability of an RNA molecule in eukaryotic cells. Explain how these elements act to influence RNA stability.

1.The total amount of protein synthesized is dependent on how much mRNA is available for translation. The amount of mRNA present is dependent on the rates of mRNA synthesis and degradation. Less-stable mRNAs will be degraded and become unavailable as templates for translation. The presence of the 5' cap, 3' poly(A) tail, the 5' UTR, 3' UTR, and the coding region in the mRNA molecule affects stability. Poly(A) binding proteins (PABP) bind at the 3' poly(A) tail. These proteins contribute to the stability of the tail, and protect the 5' cap through direct interaction. Once a critical number of adenine nucleotides have been removed from the tail, the protection is lost and the 5' cap is removed. The removal of the 5' cap allows for 5' to 3' nucleases to degrade the mRNA.

In general, mutation rates in humans occur in the range of ________ (frequency) per gamete per generation.

10^-5 to 10^-6

List at least two different types of DNA repair and briefly explain how each is carried out.

2. Direct repair. DNA damage is repaired by directly changing the damaged nucleotide back to its original structure. 4. Photo reactive repair - reversal of pyrimidine dimers formed by uv light exposure. Requires the photoactivation enzyme

In a survey of 240,000 human births, six achondroplastic births were recorded to parents who were unaffected. Given that this form of dwarfism is caused by a fully penetrant, dominant, autosomal gene, what is the mutation rate? Enter your answer in scientific notation to three significant digits.

480,000 gamete would be required to produce 240,000 offspring. So, since 6 gametes contain the mutation, the mutation rate would be: 6/480000 = 1.25X10^-5

Eukaryotic mRNAs are modified prior to translation by splicing and addition of the _________ and __________

5' cap and the 3' poly-A tail

Present a description of the molecular mechanism of the mutagenic action of any two of the following mutagens: 5-bromouracil, proflavin, ultraviolet light.

5-bromouracil is an analog of thymine, which anomalously pairs with guanine. Ultraviolet light causes thymine dimers.

What is the repressor protein of the Lac operon?

A DNA binding protein

What is a null mutation?

A TOTAL loss of function mutation

What is the SWI/SNF complex?

A chromatin remodeling complex that can be directed to specific DNA sites by: -transcription factors -Acetylation of histones -Binding to methylated DNA

What determines transcription of the lac operon?

A combination of positive and negative elements Negative regulation: lac repressor inhibits transcription in absence of lactose Positive regulation: cAMP-CAP overrides transcription of the lac operon in presence of glucose

Complementation analysis can be used to determine whether two mutations are in the same gene. Explain how to test for complementation and how one would interpret the results.

A cross is made between the two homozygous strains. If F1 are WT then complementation has occurred and two mutations must be in separate genes.

It has been found that a particular "+ —" combination of frameshift mutations in gene X gives a wild-type phenotype, whereas a "— +" combination gives a mutant phenotype, even though the sites of insertion/deletion are the same. How might these results be explained?

A frameshift in the "+ -" direction may have introduced a nonsense triplet, which was not introduced, by chance, by the opposite "- +" combination.

What are trans-acting elements?

A gene product (usually a diffusible protein or RNA molecule) that acts to regulate expression of a target gene

How might one determine whether a particular suspected gene is capable of causing cancer in mammals when it is overly expressed?

A general method could take the normal gene and, through the use of genetic manipulations such as translocations, inversions, or insertions, place the gene in question next to relatively strong gene promoters and enhancers. One could then introduce the composite gene complex into a mouse to generate a transgenic strain. If the transgene is expressed at high levels, one could determine its influence on the induction of cancer.

Describe the structure of a helix-turn-helix (HTH) motif. What is the general function of such motifs?

A geometric conformation is formed by two adjacent α helices separated by a "turn" of several amino acids. Many HTH-containing proteins regulate gene expression.

Describe what is meant by a gratuitous inducer. Give an example.

A gratuitous inducer is a chemical analogue of a natural inducer. It serves as an inducer but is not a substrate for the reactions related to the natural inducer. Isopropylthiogalactoside(IPTG) is a gratuitous inducer of the lactose operon.

What does a nucleosome consist of?

A histone protein core with DNA wrapped around its surface

What is IPTG?

A molecule that is also able to induce lac operon Turns on system more quickly, thus USEFUL for experimental procedures -IPTG molecular structure similar to one-half of lactose -IPTG induces Lac operon but is NOT a substrate for Beta galactosidase so levels of IPTG remain constant in the experiment

What are point mutations and the classes of point mutations?

A mutation affecting only one or few nucleotides of a gene sequence Missense mutation: can lead to a different AA being incorporated Nonsense mutation: leads to the introduction of a STOP codon, this will lead to a premature termination of protein translation

What is a mutation?

A mutation is an alteration in DNA sequence Mutations are typically rare (they may be unique to an individual or family line) -Can be a single nucleotide substitution, deletion or insertion mutations -Altered # of copies of repeated sequences -A major alteration in the structure of a chromosome -Can occur in protein coding or non coding regions (introns and promoter regions) -May or may not lead to a detectable change in phenotype

What is a behavioral mutation?

A mutation that affects the behavioral patterns of an organism

What is a lethal mutation?

A mutation that disrupts a gene or process that is essential for life

What is an induced mutation?

A mutation that is a result of a natural or artificial agent (e.g. mineral, u, and cosmic radiation) -Induced by mutagens (agents that increase the rate of mutations)

What is a conditional mutation?

A mutation that is present in the genome but can only be detected under certain conditions -E.G. Temperature-sensitive mutation: A mutated gene that encodes a protein that is functional at the permissive temperature but not at the non-permissive mutation

What is a nutritional mutation?

A mutation that leads to the loss of ability to synthesize an AA or vitamin

What is a loss of function mutation?

A mutation that reduces or eliminates the function of a gene product -Mutations can have a wide range of phenotypic effects ranging from none to severe

What is a gain of function (GOF) mutation?

A mutation that results in a new gene product with enhanced or new functions -These types of mutations are typically dominant

What is a silent or synonymous mutation?

A mutation(likely point mutation) that alters a codon but does NOT result in an AA change. EX. TTA-UUA = leucine TTG-UUG = leucine still

What is a nonsense subsitution?

A substitution that results in termination codon

What is Mutant I^S?

A superrepressor mutation: repressor cannot bind lactose, therefore system is OFF -Cannot be rescued by I+ or I- because the operon will be bound by the diffusible I^S product

What is meant by a suppressor mutation?

A suppressor mutation is a second mutation that reverts the phenotypic effects of an already existing mutation in a process defined synthetic rescue. Genetic suppression therefore restores the phenotype seen prior to the original background mutation.

What is a Transposon?

A transposable element (TE or transposon) is a DNA sequence that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size.

What is the "Cut and Paste" movement mechanism of transposons?

AKA Nonreplicative transposition 1. Transposon excises from original site 2. Transposon integrates into new site 3. DNA double-stranded break is repaired

What is the "Copy and Paste" movement mechanism of transposons?

AKA Replicative transposition 1. DNA synthesis copies transposon 2. Integration of copy into new site

What happens in mammals following damage?

ARF represses Rb -> no proliferation -> no regeneration -ARF protein is preventing uncontrolled growth = tumor suppression -Simultaneous inactivation of ARF and Rb in mammalian cells -> Leads to muscle cell cycle reentry and dedifferentiation CONCLUSION: In mammals the continued maintenance of the differentiated state (and tumor suppression) has come at the cost of potential regeneration in mammals

What is the normal base pairing?

AT, CG

What modification neutralizes the charges on histones that promote ionic interaction with DNA?

Acetylation

_______ bind to enhancers and form the __________ , which interacts with the transcription enzymes/proteins

Activators; enhanceosome

What is an insertion or deletion (indel) mutation?

Addition or deletion of one or more bases

Three human disorders-fragile X syndrome, myotonic dystrophy, and Huntington disease-are conceptually linked by a common mode of molecular upset. Describe the phenomena that link these disorders.

All three are caused by separate genes, but each gene was found to contain repeats of a unique trinucleotide sequence. In addition, the number of repeats may increase in each subsequent generation (genetic anticipation).

________ RNA processing generates different mRNAs, which can direct the synthesis of different polypeptides.

Alternative

Why is the number of proteins that a cell can make (proteome) not directly related to the number of genes in the genome?

Alternative splicing allows for different combinations of mRNA to be produced, which creates different proteins

What is an Endonuclease?

An enzyme capable of cutting WITHIN a dsDNA molecule

What is an Exonuclease?

An enzyme capable of degrading a DNA molecule from a free end

What is DNA polymerase?

An enzyme that adds complementary bases to DNA

What is a Ligase?

An enzyme that seals the final gap, joins to parts of DNA

What does replication slippage lead to?

An increased # of repeat units

What is cell-cell interaction in developmental genetics?

As development proceeds cells influence other cells

How do we know if a chemical (preservative, herbicide, pesticide, artificial sweetener etc, etc) is a mutagen?

Assessment of Mutagenicity: AMES test -Utilizes 2 Salmonella typhimurium mutant strains, both his- (requires histidine to grow) -Assay measures REVERSION (mutation) to wild type his+

Assume that you wished to generate conditional mutations (such as temperature sensitive mutations) for the study of cell-cycle genes in yeast. Of the mutagens listed in the following, which would be the most likely to generate such mutations?

B and C

What is required for binding of RNA polymerase II to the promoter?

Basal (general) transcription factors -TFIID, the first general transcription factor to bind the promoter binds to TATA box through TATA binding protein (TBP)

Assembly of the ____ ________ _______ occurs at the promoter

Basal Transcription Complex

What are consequences of spontaneous mutations?

Base mispairing Nonpairing or base substitutions Fixed mutation

Why do insertions and deletions often have more drastic phenotypic effects than base substitutions do?

Because amino acid codons consist of three nucleotides, insertions and deletions that involve any multiple of three nucleotides will leave the reading frame intact. However, the addition or removal of one or more amino acids may still affect the phenotype. Insertions or deletions that do not alter the translation reading frame are called "in-frame" insertions and deletions.

In some cases, genes undergo amplification (local, multiple duplications) when additional gene products are in high demand. Would you consider gene amplification as a form of genetic regulation in eukaryotes?

Because amplification increases the copies of genes and thus possibly the amount of gene product it would be considered a form of genetic regulation.

What does a repressor do?

Binds to promoter site, blocks transcription, so no RNA which means no proteins -This is "Negative Control" of the operon

Compare and contrast positive and negative control of gene expression in bacteria.

Both forms of control result from an interaction of a molecule (usually considered to be a protein) with the genetic material (either RNA or DNA). Positive control results when the interaction stimulates transcription, whereas negative control occurs when the interaction inhibits transcription.

How do siRNAs and miRNAs act in similar ways?

Both regulate gene expression by annealing to mRNA sequences that are partially or fully complementary

Explain how one would use epistasis analysis to determine order of gene action in genetic networks?

By comparing the phenotype of double mutants to single mutants..

Regulatory proteins that bind DNA have common motifs that interact with sequences of DNA. How do amino acids in DNA-binding proteins interact with DNA?

By forming hydrogen bonds with DNA bases

How can methylation state of a gene be determined?

By restriction enzyme analysis with HpaII and MspI -Both recognize same restriction enzyme site: CCGG -Methylation most often occurs at C of CG doublets

Name three types of consensus sequences or modular DNA sequences that exist upstream from the coding regions of some eukaryotic genes.

CAAT, TATA, GC repeats

What are CAP?

Catabolite Activator Proteins

What is a the definition of zygote?

Cell generated by fusion of the sperm and egg

What is significant of Pachynema of Meiosis Prophase I?

Chromatids visible, rRNA genes amplified, rRNA made in Pachynema

What is the difference in control between cis and trans acting operons?

Cis acting operons can only control expression of genes on the same piece of DNA Trans acting operons are able to control expression of genes on other DNA molecules

What are control regions (cis-acting)?

Cis acting regulatory elements are DNA sequences that lie UPSTREAM of the gene cluster on the SAME STRAND of DNA

Enhancers are said to be cis-acting. What is meant by cis-acting, and what are enhancers?

Cis-acting means that the genes under control must be in the same chromosome as the cis-acting element. Enhancers are sections of DNA that regulate transcription of other sections of DNA.

What are Human LINEs and SINEs, and copia (in Drosophila) examples of?

Class I transposons: retrotransposons -Transpose through an RNA intermediate by Reverse Transcriptase polymerase that catalyzes creation of dsDNA from ssRNA -Resemble retroviruses in their sequence -May be the ancestral source of eukaryotic telomerase

What are Class I transposons?

Class I: Retrotransposons - "copy and paste"mechanism only -Transpose through RNA intermediate, RNA reverse transcribed to dsDNA by reverse transcriptase • Examples: human LINE and SINE elements, Drosophilacopiaelements

What are Class II Transposons?

Class II -Transpose through DNA intermediate - "copy and paste"or "cut and paste"movement mechanism -Have inverted terminal repeats -Has transposase enzyme gene -Examples: bacterial IS elements, bacterial transposons, DrosophilaP elements, Maize Ac/Ds

What can harbor genes and can be a source of multiple drug resistance transfer?

Class II transposons

lin-4 inhibits translation of lin-14 mRNA by ______ _____ to 3' UTR region of lin-14 mRNA

Complementary binding

What are nonsynonymous substitutions?

Consists of missense and nonsense substitutions

What is mutant O^C?

Constitutive cis mutant in the operator DNA: no repressor can bind, system ON all the time

What are the steps of the AMES Test?

Control experiment: His- strains mixed with liver enzymes, quantifies spontaneous revertants(mutations) to his+ Treatment experiment: His- strains mixed with liver enzymes AND chemical to be tested a potential mutagen Conclusion: Any chemical that significantly increases the # of colonies appearing on the treatment plate is mutagenic, and therefore potentially carcinogenic

What are the different operon controls and combinations?

Controls: -Negative: regulatory protein is a repressor binding to DNA inhibiting transcription -Positive: regulatory protein is an activator stimulating transcription -Inducible: transcription normally OFF, something happens to induce transcription -Repressible: transcription normally ON, something happens to turn it off Combinations: -Negative Inducible -Negative Repressible -Positive Inducible -Positive Repressible

What is the energy hierarchy of the Electro magnetic spectrum?

Cosmic rays>Gamma rays>X-rays>UV>Visible>Infrared>Microwaves>Radio waves

What is a glycosidic bond?

Covalent bond joining a carbohydrate to another group

Transcriptional repression by methylation of DNA is most common in sequences called____________ islands.

CpG

What is cytoplasmic localization?

Cytoplasm exerts influence

What are P bodies?

Cytoplasmic foci enriched for mRNA degradation factors, mRNA can also be sequestered (stored) here for later usage

How does the repair system know which one is the correct nucleotide?

DNA Polymerase III In bacteria DNA polymerase III makes an error 1 in 100,000 nucleotides If an error is introduced, the incorrect nucleotide is removed and the correct one is inserted

The region of a protein that is capable of holding on to a particular nucleotide sequence in order to affect proper gene regulation.

DNA binding domain

Which term most appropriately refers to a regulatory protein in prokaryotes?

DNA binding protein

What is true of DNA binding proteins?

DNA binding proteins typically contain a conserved DNA-binding domain with characteristics such as: -helix-turn-helix motif -zinc finger -leucine zipper

Strand discrimination during the process of _____________ is based on DNA methylation in E. coli.

DNA repair

Transcription factors appear to be important molecules relating to the regulation of gene activity. Regarding eukaryotes, two general classes of transcription factors exist. Briefly describe each.

DNA-binding domain: binds to specific DNA sequences present in the cis-acting regulatory site trans-activating domain: activates or represses transcription through interacting with other transcription factors or RNA polymerase

Transcription factors appear to be important molecules relating to the regulation of gene activity. Regarding eukaryotes, two general classes of transcription factors exist. Briefly describe each.

DNA-binding domain: binds to specific DNA sequences present in the cis-acting regulatory site trans-activating domain: activates or represses transcription through interacting with other transcription factors or RNA polymerase

When regions around genes become sensitive to the enzyme ______________ this is an indication that those regions are becoming ______________ active.

DNase I, transcriptionally

What do mutations in promoters do to transcription?

Decrease transcription

What is the difference between determination and differentiation in developmental genetics?

Determination: Development fate fixed Differentiation: Cell achieves its final form and function

What is a significant drawback to using the mouse as a model organism compared with Drosophila?

Development must take place in utero and only a small window exists for in-vitro culturing

Experiments involving nuclear transplantation in amphibians indicate that nuclei derived from blastula are more likely to support development of complete and normal adults compared with those derived from later stages of development. What do these experiments tell us about the process of development?

Development occurs as a series of cascades, with early genes influencing late genes, at times with stability. Such progressive determination, if stable, may be irreversible and fail to support development of an entire organism from a single cell. A number of epigenetic factors such as chromatin remodeling and DNA methylation are also likely to come into play.

What do Promoter minus (P-) mutations do?

Disables RNA polymerase binding, supercedes all other mutations, in cis: system OFF all the time

The process by which excessive numbers of a sex chromosome are corrected, often by methylating the X chromosome to form a Barr body.

Dosage compensation

How do osmolarity conditions affect movement of water across a membrane?

Due to differences in ion concentrations on inside and outside of membrane

Maternal effects are cases of extrachromosomal inheritance in which the genotype of the mother influences the phenotype of her immediate offspring in a non-Mendelian manner. Explain the general molecular basis of a maternal effect.

During development of the egg, females provide numerous nutritional and informational substances, which direct and support early embryonic development. These substances are often in the form of transcription factors, receptors, mRNA, and proteins, although other substances are also likely (substrates and products). In some cases, these maternally supplied substances override the actual genotype of the zygote and produce a phenotype much like the genotype of the mother.

DNA Methylation is an example of _______ inheritance

EPIGENETIC

What is the mechanism of riboswitches that act as ribozymes?

EX. glmS gene catalyzes reaction to produce a sugar glucosamine-6-phosphate from a precursor -However, if there is plenty of GlcN6P around, it binds to ribozyme region, altering its structure -This leads to active form of ribozyme which degrades mRNA so it doesn't make more enzyme when it's not needed

How is the accessibility of the DNA to potentially being transcribed examined?

EXPERIMENTALLY by treating chromatin with DNase, which degrades DNA if DNA is accessible to the enzyme -Therefore IF a region is INSENSITIVE to the DNase enyme THEN DNA must be bound up with the histone proteins -BUT IF a region is SENSITIVE to DNase THEN the chromatin has been remodeled in that region and is more accessible (to both DNase and presumably to transcription factors)

What is the end result of lac operon regulation?

Efficient energy use and production of lactose metabolism enzymes only when needed

How do enhancer elements differ from suppressor mutations?

Enhancers act in the opposite manner from suppressors; they increase the expression of a given phenotype

Parts of chromosomes that are not methylated for inactivation, but rather are less dense-staining and are likely active in transcription in the cell.

Euchromatin

Transcription in _______ requires several steps

Eukaryotes +Eukaryotic chromosomal DNA is complexed with histones to form chromatin +Nucleosomes in the chromatin can inhibit transcription +Chromatin remodeling is required for transcription to occur

Regulation of gene expression using siRNAs is found in

Eukaryotes ONLY

Describe what is meant by the term forward genetics.

Forward genetics involves the isolation of mutants that show differences in a particular phenotype of interest. Mutant isolation is usually followed by an analysis of gene pathways through epistasis and/or complementation analyses. Then the gene is usually mapped and cloned and studied by a variety of molecular and developmental approaches.

Alternative RNA splicing is a method that apparently evolved for the production of many different polypeptides from the same pre-mRNA. Provide an example of alternative splicing.

Fourteen exons occur in the alpha-tropomyosin gene, six of which make up three pairs that are alternatively spliced. Ten different forms of alpha-tropomyosin are expressed in a tissue-specific manner.

In the absence of glucose and in the presence of galactose, several genes are activated to enable yeast utilize galactose. The key regulator(s) of the yeast GAL system is/are the _______ protein(s).

GAL4, GAL3, GAL80

What are the approximate positions (in bp) of each type of consensus sequence (modular element) found at the 5' end of eukaryotic genes?

GC box (-110), CAAT box (-70), TATA box (-30)

Transcription Initiation is a major form of ______ _________

Gene regulation -Promoters contain several elements -The TATA box is the region to which RNA polymerase II binds

Define gene regulation. What are three ways gene regulation can be controlled in a cell?

Gene regulation is defined as the control of a gene's transcript and its protein product. It can be achieved by altering either the transcription of the gene (RNA level), the translation of the protein from that transcript or by altering the structure of DNA such that transcription cannot occur. For full credit: they must define gene regulation and three different mechanisms to affect gene regulation (1 point each).

What is the common influence of ultraviolet light on DNA? Note that simply stating "mutation" is not an acceptable answer.

Generation of pyrimidine dimers.

What is the role of maternal-effect genes?

Genes derived from the maternal genome are actively transcribed to support the zygote and are required for the proper patterning of the embryo. Mutant phenotypes of maternal-effect reflect the genotype of the mother.

What are Zygotic genes?

Genes expressed after fertilization by cells of the embryo

What are Maternal genes?

Genes expressed in the oocyte during oogenesis. Products stored until fertilization -Mother's genes particularly important as it could cause death of child

What is the mechanism of Positive Control of operon by CAP-cAMP binding?

Glucose NOT present -> Adenyl cyclase works -> get cAMP -> cAMP binds CAP -> CAP-cAMP complex binds CAP binding site -> RNA polymerase binds -> Transcription enhanced

The catabolite repression system in E. coli essentially represses the lac operon when glucose is present. What evolutionary advantage would favor the evolution of such a system?

Glucose can enter glycolysis as is, but lactose must be broken down into glucose and galactose which requires beta-galactosidase which requires energy to be made. Energy efficient to burn glucose

What are cAMP levels dependent upon?

Glucose levels -Glucose blocks adenylase cyclase function

What is the mechanism of glucose inhibition of the lac operon?

Glucose present -> inhibits adenyl cyclase -> Don't get cAMP -> cAMP doesn't bind CAP -> CAP can't bind CAP binding site efficiently without cAMP - CAP complex -> RNA polymerase doesn't bind -> Transcription diminished

What are imaginal discs?

Groups of cells in the larvae that go on to form adult body structures

How was it determined that X rays are mutagenic?

H. J. Muller determined that a direct relationship occurred between X-linked recessive lethals and X ray dose in Drosophila.

What is the take home point of Eukaryotic development?

Higher eukaryotic development is a complicated, highly regulated, closely coordinated pattern of gene expression over space and time

What are homeotic genes and what do they do?

Homeotic genes code for regulatory proteins that control the fate and development of body segments

What is the significance of the I locus in the E.coli lac operon?

I locus: upstream gene encoding a "repressor" protein, shuts down lac operon when there is no lactose present

Trans mutation produces a diffusible product, can be rescued with ____

I+

What symbols are used to describe constitutive mutations in the lac operon?

I-, O^C

What are Repeated or Redundant genes?

Identical copies of the same gene on one or more chromosomes

Assume that a new mutation occurs in the germ line of an individual. What finding would suggest that the new mutation is dominant rather than recessive?

If dominant and if passed to the next generation, it would be expressed.

Yanofsky's findings beg the question, how are we still getting regulation of transcription in the absence of the trp repressor?

In BACTERIA ribosomes may attach to mRNA molecules and initiate TRANSLATION before TRANSCRIPTION of a gene/operon is complete

When generating a series of mutations in an organism, how does a genetic screen differ from a mutation selection?

In a genetic screen, one mutagenizes the organism and then physically searches for the mutations. In a selection experiment, conditions are established in which only the relevant organisms survive.

What is catabolite repression? How does it allow a bacterial cell to use glucose in preference to other sugars?

In catabolite repression, the presence of glucose inhibits or represses the transcription of genes involved in the metabolism of other sugars. Because the gene expression necessary for utilizing other sugars is turned off, only enzymes involved in the metabolism of glucose will be synthesized. .

Which of the following is NOT true regarding the differences in mRNA between prokaryotic cells and eukaryotic cells?

In eukaryotic cells, the transcription to mRNA and the translation from mRNA are coupled, while in prokaryotic cells transcription and translation are not coupled.

How is RNAi used to reduce translation in prokaryotes?

In prokaryotes single stranded antisense RNA in RNAi experiments -> reduce translation -> reduce targeted protein -RNA interference (RNAi) allows the silencing of single genes to allow rapid analysis of gene function

How could the # of repeats increase?

In replication a hairpin formation on the newly synthesized strand causes part of the template strand to be replicated twice, and increases the # of repeats on the newly synthesized strand, the two strands of new DNA then separate and the strand with extra CAG copies serves as a template for replication and the resulting DNA molecule contains additional copies of the CAG repeat

Where is miRNA coded?

In your genome

Since the binding of the _________ to the _________ prevents it from binding to DNA by causing a conformational change in its structure, it is called an _________ protein.

Inducer,repressor,allosteric

Present a detailed description of the actions of the regulatory proteins in a) inducible and b) repressible enzyme systems. Separate and clearly label your answers for parts a-b in the box below.

Inducible system: The repressor is normally active, but the inducer inactivates the repressor. Repressible system: The repressor is normally inactive but is activated by the corepressor. Active repressors turn off transcription.

Describe the function of promoters in eukaryotes. Where are they usually located, relative to the corresponding gene (give a quantitative answer in base-pairs)? What two sequence elements do they normally possess?

Initiate transcription located within 100 bp upstream of a gene usually contain TATA box and CAAT box

What are examples of frameshift mutations?

Insertions or deletions

What are the non-chemical mutagens usually?

Ionizing radiation - X-rays, gamma rays, and cosmic rays -Are more energetic than UV radiation, will penetrate deeply into tissues ionizing molecules along the way -Sufficient UV radiation can induce thousands of DNA lesions/hour -Liner increase of mutations with increased dose

The molecular nature of transposable elements was first understood in E. coli due to the simplicity of the E. coli genome. Which of the following types of transposable element contribute significantly to the genome complexity of eukaryotic organisms?

LINES, SINES, Retrotransposons

What type of systems are the lac operon and trp operon?

Lac operon: Negative inducible, presence of lactose induces the system to turn on Trp operon: presence of tryptophan "represses" the system to turn OFF

The lac repressor binds to:

Lactose and DNA

What are the parts of an E.coli lac operon?

Lactose: sugar (energy source) broken into simpler sugars galactose and glucose, lactose or IPTG must be present for operon to work Beta Galactosidase: (Product of lacZ gene) breaks lactose into galactose and glucose Two other genes, lacY [lactose permease] and lacA [trans-acetylase] transcribed from this operon -Much of the genetic analysis leading to lac operon model was done by FRANCOIS JACOB and JACQUES MONOD in early 1960s

What is the difference between a missense mutation and a nonsense mutation?

Missense mutations alter the coding sequence so that one amino acid is substituted for another. Nonsense mutations change a codon originally specifying an amino acid into a translation termination codon.

Some mutagens cause genetic changes that can be "corrected" by re-exposing cells to the same mutagen. Other mutagens do not behave in this way. Provide one example of each of these two types of agents and describe the mutational changes caused in DNA. Explain why some mutagens behave in one way, while others do not.

Mutagens that cause base substitutions are "corrected" by mutagens of the same class (nitrous acid, 2-aminopurine, and 5-bromouracil). Frameshift mutations, caused by proflavin or acridine orange are "corrected" by the same class of frameshift mutagens, but not by mutagens that cause base substitutions. X rays cause major structural changes in chromosomes (deletions, translocations, etc.) and are not "corrected" by any mutagen, including X rays.

What are Nutritional Mutants?

Mutants that now require nutrients to survive EX. his- unable to synthesize histidine, will not be able to grow on media that lacks histidine

What are Mutation Hot Spots in Mutation Site Heterogeneity?

Mutation Hot Spots are when some positions/regions are more susceptible to mutational changes Causes: replication slippage leading to indels, 5-methylcytosine sites (greater # of mutations at these sites)

What are Autosomal mutations?

Mutations found within genes located on the autosomes

What are X-linked and Y-linked mutations?

Mutations that are located on the X or Y chromosomes

What are somatic mutations?

Mutations that occur in every cell EXCEPT germ cells

What does presence or lack of presence of Lactose or IPTG result in?

NO lactose (or IPTG) present: Operon repressed Lactose (or IPTG) present: Operon induced (Functional proteins produced)

What does a deletion mutation in I,Z, Y from E. coli genome result in?

NO regulation from the F', operon always ON

Can operon regulation be rescued with operon linked to an O+ operator?

NO, which means the O^C mutant is acting in cis (only affects DNA to which it's attached)

How are siRNAs and miRNAs distinguished?

NOT by size or function BY ORIGIN -siRNAs originate from long dsRNAs -long dsRNAs are cleaved into siRNAs by Dicer

Under a system of ______________, genetic expression occurs unless such expression is shut off by some form of regulator.

Negative control

Choose the type of control illustrated by each example. Reference: Ref 16-1 E. coli lac operon control by lac I

Negative inducible

What are the differences between neutral mutations and silent mutations?

Neutral mutations are changes in DNA sequence that alter the amino acid coding sequence of a polypeptide, but do not change its biological function. Silent mutations, on the other hand, are changes in DNA sequence that produce synonymous codons specifying the same amino acid as the original, non-mutated sequence.

What does NTS stand for?

Non-Transcribed Spacer

How might one generate a line of male mice that contain two X chromosomes?

Nondisjunction during meiosis to cause two X's and one Y

E. coli bacteria are placed into a medium containing glucose and lactose. Which of the genes below do you expect to be turned on? Lac I Lac P None of the above Permease f-galactosidase

None of the above

What is the treatment for xeroderma pigmentosum?

Not going outside, avoiding UV light

What is Notch signaling pathway (cell-cell interaction)?

Notch gene product is a transmembrane receptor protein found on cell B -Notch receptor binds Delta protein found on cell A -Note: this is a physical interaction between adjacent cells -Binding of Delta by Notch leads to proteolytic cleaage of cytoplasmic end of Notch -This cleaved cytoplasmic tail of Notch protein moves to nucleus actiating transcription of a gene set directing developmental pathway that determines cell fate

What is the process of SCNT?

Nucleus of differentiated cell transfered into enucleated egg cell and allowed to develop, clone can be received

What are null mutations? What classes of mutagens would most likely generate null mutations?

Null mutations are mutations which cause a total loss of function, typically recessive mutagens

What was the takeaway between mutational signatures and cancer types?

Observed mutations: "Mutational signatures differed between cancer types (Fig. 1). In the lung cancers, melanomas, and glioblastomas studied they may reflect exposure to tobacco carcinogens, UV light, and mutagenic alkylating chemotherapy, respectively

Transcription factors appear to be important molecules relating to the regulation of gene activity. Regarding eukaryotes, two general classes of transcription factors exist. Briefly describe each.

One class assembles at promoter regions adjacent to the site of transcription. Activator and Repressor. Recruitment of an activator to a promoter results in increased gene expression. Recruitment of a repressor leads to decreased gene expression. The other class binds at more distant regions enhancers, silencers

What are single copy (unique genes)?

Only one copy of the gene is in a haploid set of chromosomes

What are operons, control regions (cis-acting), and trans-acting elements of prokaryotes?

Operons: Multiple genes in one unit arranged for coordinated gene expression Control regions (Cis-acting): Elements upstream of operon that affect transcription (e.g. promoter, operator) Trans-acting elements: diffusible product that interacts with a control region

What type of system is the Lac operon?

Overall the Lac operon is a Negative Inducible system -Operon is induced by presence of lactose

_____________ are complexes where, among other activities, a great deal of RNA degradation takes place.

P bodies

P-element transposons provide a powerful tool for the study of Drosophila genetics. What are P elements, and why are they so useful?

P-element transposons are mobile genetic elements that can move in and out of the genome. P-elements can also be used to generate mutations by inserting into genes, thus disrupting their function.

What is the consequence of UV irradiation?

Photodimers -EX. Dimer formed between adjacent thymidine residues along a DNA strand

What is meant by the term photoreactivation repair?

Photoreactivation repair, discovered in 1949, is a process described in E. coli in which UV-induced DNA damage can be partially reversed if cells are briefly exposed to light in the blue range of the visible spectrum.

How does Platensimycin (antibiotic) function?

Platensimycin interferes with normal binding of enzymes in Fab pathway -> dead bacteria -Platensimycin was developed by Merck scientists

Degradation of a eukaryotic mRNA is generally preceded by shortening of the _____________.

Poly A tail

Enhancers have several structural and functional characteristics that distinguish them from promoters. Describe three such characteristics, focusing on how these traits differ between enhancers and promoters.

Position need not be fixed. Orientation may be inverted without significant effect. They can act at a great distance from the promoter.

What is the difference between positive and negative control? What is the difference between inducible and repressible operons?

Positive transcriptional control: activator protein stimulates transcription Negative control: Repressor inhibits transcription An inducible operon normally is not transcribed. It requires an inducer molecule to stimulate transcription either by inactivating a repressor protein in a negative inducible operon or by stimulating the activator protein in a positive inducible operon. Transcription normally occurs in a repressible operon. In a repressible operon, transcription is turned off either by the repressor becoming active in a negative repressible operon or by the activator becoming inactive in a positive repressible operon.

What does the presence of glucose do to lac operon?

Presence of glucose shuts down operon -Ex. of catabolite repression (Presence of "breakdown" product shuts down the system) -Catabolite: product of catabolism (breakdown of complex molecules into simpler ones)

What are some examples of post-transcriptional regulation in eukaryotic mRNA?

Prior to translation, eukaryotic mRNA transcript undergoes: 1. Addition of 5' CAP 2. Removal of noncoding introns (splicing), Alternative splicing 3. Addition of poly (A) tail 4. Export from the nucleus 5. Translation 6. Degradation

Which of these is a level of regulation in eukaryotes? Select all that apply: operon processing transcriptional post-translational transport

Processing Transcriptional Post-translational Transport

What are the basic regulatory elements in a strand of DNA that allow for genes to be regulated? Are these different in eukaryotes and prokaryotes? If so, how?

Promoters, operators in prokaryotes. Promoters, proximal promoter elements, enhancers, and silencers in eukaryotes. Eukaryotic transcription is different in a few ways, most notably in that prokaryotic genes are transcribed as operons, while eukaryotic genes are not. In addition, the role of the nucleosome is different in eukaryotic transcription.

What are allosteric proteins?

Proteins that change shape when binding another molecule -EX. Repressor protein changes shape upon binding of lactose

What is a transversion mutation?

Purine to pyrimidine or pyrimidine to purine -8 possibilities

What are the purines and pyrimidines?

Purines: Adenine, Guanine Pyrimidine: Cytosine, Thymine

What is another role for Dicer?

RITS (RNA-induced initiation of transcriptional silencing) complex -In this pathway short RNAs can also mediate RNA-directed DNA Methylation (RdDM)

_______________ is also known as RNA silencing and posttranscriptional gene silencing.

RNA interference

What are most miRNAs processed from?

RNA pol. II transcripts of 100s-1000s of nucleotides in length -These transcripts are called pri-miRNAs (for primary miRNA) -Pri-miRNAs often contain the sequence for many different miRNAs -miRNAs can also be processed from introns and from 3'UTR of some pre-mRNAs

Which of the following is TRUE for both prokaryotic and eukaryotic gene expression?

RNA polymerase binds to the promoter region to begin transcription.

What is the general mechanism by which gene expression in eukaryotes is influenced by the extracellular environment?

RNA signaling endocrines and paracrines

Define RNA silencing (or interference). Explain how siRNAs arise and how they potentially affect gene expression. How are siRNAs different from the antisense RNA mechanism?

RNA silencing, or RNA interference, occurs when double-stranded RNA molecules are cleaved and processed to produce small single-stranded interfering RNAs (siRNAs). These siRNAs bind to complementary sequences in mRNA molecules, stimulating cleavage and degradation of the mRNA. The siRNAs may also stimulate DNA methylation at DNA sequences complementary to the siRNAs. The paired mRNA-siRNA attracts a protein-RNA complex that cleaves the mRNA in an area bound by the siRNA. Following the initial cleavage, the mRNA is further degraded. The cleavage and subsequent degradation of the mRNA make it unavailable for translation. DNA methylation in the nucleus stimulated by siRNAs also affects transcription. Antisense RNA binds to mRNAs also, but physically interferes with translation.

What happens in amphibians following damage?

Rb inactivated -> proliferation induced -> regenerated tissue

Describe the positive control exerted by the catabolite activating protein (CAP). Include a description of catabolite repression.

Regarding regulation of the lac operon, in the absence of glucose, CAP (dependent on cAMP and adenyl cyclase) binds to the CAP site and facilitates transcription (positive control). Transcription of the operon is inhibited in the presence of glucose (catabolite repression).

What are CRMs?

Regions of a few hundred base pairs containing binding sites for transcription factors

Spontaneous Mutations are RARE, but what do they usually arise from?

Replication errors and base modifications -DNA polymerases occasionally insert the incorrect nucleotide

What are transposable elements in humans thought to have arisen from?

Reverse transcription of viral mRNAs followed by integration of dsDNA copies into the genome

What happens when tryptophan is high-high levels of tRNA^trp

Ribosomes TRANSLATE the leader region - this leads to a conformational change in the mRNA that terminates transcription

What happens when tryptophan is low - low levels of tRNA^trp?

Ribosomes stalls - at the UGG codons, this leads to a conformational change in the mRNA that ALLOWS TRANSCRIPTION

Regarding a gene's DNA sequence, what is meant by a sequence motif?

Sequence motifs are short recurring patterns in DNA that often indicate sequence specific binding sites, like for transcription factors. They are generally highly conserved.

What is an example of a missense mutation?

Sickle cell hemoglobin

In some cases, genes undergo amplification (local, multiple duplications) when additional gene products are in high demand. Would you consider gene amplification as a form of genetic regulation in eukaryotes?

Since gene amplification involves an increase in the number (copies) of genes, which, in turn, provides the potential for increasing the amount of a gene product because more copies of a given gene are present, one would probably consider gene amplification as a form of genetic regulation. Amplification of ribosomal genes during oogenesis in Xenopus and chorion genes in Drosophila are examples.

_____ _______ is essential for eye regeneration in planarians

Sine ocularis

What is the consequence of X-rays?

Single and double stranded breaks of DNA

Under certain conditions, the rate of mutation of a particular gene may be determined in humans. What properties of the mutation would favor the most direct determination of mutation rate in humans? Select all that apply:

Single locus Dominant 100% penetrance fully expressed

How do you inhibit mRNA translation?

Small complementary RNA binding to mRNA inhibits translation

What are Fixed mutations?

Some mutations that escape all of the repair mechanisms and become fixed +Synonymous mutations +Missense mutations +Nonsense mutations +Frameshift mutations

In the lactose operon, the product of structural gene lacZ is capable of

Splitting the Beta linkage of lactose

A low rate of ________ _______ occurs

Spontaneous Mutation -This is considered the engine of evolution -Many agents act to create DNA mutations, increase rate -Multiple repair mechanisms exist to repair mutations, decrease rate -Fixed mutations lead to altered gene product -> altered phenotype

DNA may be damaged from the by-products of normal cellular aerobic respiration. Name three of these electrophilic oxidants that are generally classified as reactive oxidants.

Superoxides (O2-), hydroxyl radicals (*OH) and hydrogen peroxide (H2O2).

What did Yanofsky find?

That in the ABSENCE of the trp repressor: -the presence of tryptophan halts transcription after -140 bp -in the absence of tryptophan transcription continues

In cytoplasm mRNAs are translated, what is translation dependent on?

The amount of mRNA present, the concentration of mRNA is dependent on its rate of synthesis and degradation

What is the approximate genome size in the mouse, and how many chromosomes are present?

The approximate genome size of mouse = 2.8 x 10^9 base pairs. Mouse contains 40 chromosomes (2n=40).

What did cutting the human genome with Alu1 lead to?

The discovery of Alu elements, transposable SINE element that has an AGCT site in the middle, thus the name of this transposable element

What is the significance of the homeodomain?

The homeodomain is a highly conserved protein of 60 amino acids which is thought to play a role in DNA binding and transcriptional activation.

How might in situ hybridization aid in determining the tissue-specific and/or temporal-specific pattern of gene expression? Describe the process of in situ hybridization in your answer.

The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. Since it uses RNA, it would show proteins expressed, not all genes, and so could show genes expressed in a specific tissue or genes that interact at certain times in development.

Explain why mutations in the lacI gene are trans in their effects, but mutations in the lacO gene are cis in their effects.

The lacI gene encodes the lac repressor protein, which can diffuse within the cell and attach to any operator. It can therefore affect the expression of genes on the same or different molecules of DNA. The lacO gene encodes the operator. It affects the binding of DNA polymerase (OR the lacI repressor) to the DNA, and therefore affects only the expression of genes on the same molecule of DNA.

What experimental results would indicate that the mutation lacIs is dominant to lacI+?

The observation that lacIs is a trans-acting superrepressor and represses the operator on both sides of the DNA sequence, while lacI+ still has variable regulation

Explain why lacOc mutations are cis-acting, whereas lacI mutations can be trans-acting?

The operator. region does not produce a diffusible product in lacOc, while it does in lacI.

Provide a general set of statements that describe enhancers.

The position of an enhancer need not be fixed it can be upstream, downstream, or within the gene it regulates. It is orientation independent and can act at large distances from the promoter.

Which of the below is not true about the location of enhancers?

The position of the enhancer has no effect on gene regulation

In what way is gene rearrangement related to gene regulation? Give an example.

The reshuffling of genes provides a way for certain gene segments to rearrange in order to produce new products or change their expression pattern. In the production of immunoglobulins such gene reshuffling occurs.

Approximately 5% of the cytosine residues are methylated in the genome of any given eukaryote. In what way is DNA methylation related to genetic regulation?

There is an inverse relationship between the degree of methylation of eukaryotic DNA and the degree of gene expression

Why are 3' Untranslated regions (UTRs) important?

They are sequences that control the stability of an mRNA and are often located in the 3' UTR

What is true of loss of function and null mutations?

They are typically recessive

Which of these is a level or type of genetic regulation in eukaryotes? Select all that apply: transcriptional RNA splicing post-translational DNA methylation co-transcriptional

Transcriptional Post-translational DNA Methylation

____________________act as intermediaries between ______________________________ and specific _____ sequences to modify chromatin structure and activate transcription.

Transcriptional activators,Chromatin remodeling complexes,DNA*

Design an experiment that would allow you to determine if a particular nucleus in a Drosophila embryo is capable of directing development of an entire new fly.

Transfer the nucleus into a donor egg of a very different phenotype. Observe development and if the resulting offspring is phenotypically similar to the fly from which the nucleus originated, then that particular nucleus was capable of directing development of an entire new fly.

One type of mutation involves the replacement of a purine with a purine, while another causes the replacement of a pyrimidine with a purine. What general terms are associated with these two mutational phenomena?

Transition, transversion

What is a transition mutation?

Transition: purine (A,G) to purine or pyrimidine (C,T) to pyrimidine Consequence: A to G, C to T and G to A, T to C -4 possibilities

State whether the following statement is true or false; then give your reasoning. The terminating "hairpin" loop occurs in the tryptophan operon when sufficient tryptophan is present.

True, the hairpin loop terminates transcription

State whether the following statement is true or false; then give your reasoning. The terminating "hairpin" loop occurs in the tryptophan operon when sufficient tryptophan is present

True; the "hairpin" loops terminates transcription.

Monod discovered that if tryptophan is present in relatively high quantities in the growth medium, the enzymes necessary for its synthesis are repressed. How does this occur?

Tryptophan acts as a corepressor to activate the tryptophan repressor protein; it is also involved in an attenuation system, which causes the termination of transcription.

Assume that you are working with a mutant, nutritionally deficient strain of Escherichia coli and that you isolate "revertants," which are nutritional-normal. Describe, at the molecular level, two possible causes for the "reversion to wild type."

Two general classes of events could be involved: true reversion or suppression. True reversion would involve a precise correction of the original mutation. Suppression could be intragenic or intergenic. In intragenic suppression, a change in some other part of the mutant gene product serves to "compensate" for the original mutation. In intergenic suppression, a mutation in some other gene, such as a transfer RNA, serves to "compensate" for the original mutation.

Assume that you are working with a mutant, nutritionally deficient strain of Escherichia coli and that you isolate "revertants," which are nutritional-normal. Describe, at the molecular level, two possible causes for the "reversion to wild type."

Two general classes of events could be involved: true reversion or suppression. True reversion would involve a precise correction of the original mutation. Suppression could be intragenic or intergenic. In intragenic suppression, a change in some other part of the mutant gene product serves to "compensate" for the original mutation. In intergenic suppression, a mutation in some other gene, such as a transfer RNA, serves to "compensate" for the original mutation.

In what way do upstream activator sequences (UASs), regulatory sequences in yeast, differ from enhancers and silencers?

UASs only function upstream

What is the UTR in splicing?

UTR= untranslated region, part of the transcribed RNA that is NOT translated

Describe the phenomenon of photoreactivation repair.

UV-induced DNA damage, specifically pyrimidine dimers, can be partially reversed if cells are briefly exposed to light in the blue range of the visible spectrum

What are new molecular genetic methods to screen for novel antibiotics?

Use antisense RNAi inhibition to screen compounds that target particular biosynthetic pathways -Example targeting bacterial fatty acid biosynthesis pathway Wang et al. Nature 2006 1. Use antisense RNA (RNAi) to downregulate expression of FabF (component of fatty acid biosynthesis pathway) 2. Therefore cells are even more sensitive to any compounds that target this point in the pathway

How can the expression of a gene be drastically altered by the presence of comparatively small numbers of RNA molecules? What is the term for this class of gene regulation?

Using RNAi (RNA interference), can knock out gene function or target mRNA for degradation

What is Photoreactivation Repair (Reversal of Damaged DNA) (Mutation Repair Mechanism)?

Using a photoactivation enzyme the bond between thiamine dimers can be cleaved and pyrimidine dimers formed from UV light exposure reversed

Describe the use of DNA databases to characterize a cloned and sequenced gene whose function is unknown.

Various databases allow investigators access to a variety of comparison programs that search for regions of homology to other known sequences. When a homologous gene is identified, its function may already be known from work on another organism, thus providing hints about function. BLAST searches allow researchers to search protein sequences for regions of homology in other proteins, which may indicate likely function.

If positive charges cause chromatin to condense - what will happen to chromatin when histone tails are acetylated?

Weakens interaction of tails of histone with DNA and permits for transcription factors to bind DNA

Are Kinase genes that carry a driver mutation and therefore function as a cancer gene possibly associated with FGF (Fibroblast Growth Factor) signaling pathway?

YES

Are Posttranslational Mechanisms involved in regulation of gene expression?

YES

Can DNA moving around within the genome be another source of mutations?

YES, Mobile genetic elements

The sequencing of the human genome now allows comparison with cancer genomes derived from malignant cells to determine location of somatic mutations and also to determine those that may be "driving" the malignant phenotype

YUH

Targeted gene knockouts involve the deletion or disruption of a given gene. What method might be effectively used to knock out a gene in yeast? What method might be used in mice? Why would the method used in yeast be ineffective in mice?

Yeast: New segments are attached to the ends of DNA; homologous recombination occurs and the resulting colonies lack the target gene to gain some insight on the function of that gene Mice: The homologous recombination part is identical to that of yeast, however embryonic stem cells are used after to further achieve a mutated mouse by allowing those mutated cells to grow and eventually reproduce offspring expressing the mutated phenotype in hopes to understand its effect The yeast method might be ineffective in mice because we do not want just one mutated cell, we want a whole mutant mouse, as only with the complete organism can we make a full assessment of the effect of the gene inactivation on the phenotype.

What are zinc fingers, and why are they frequently encountered in descriptions of genetic regulation in eukaryotes?

Zinc fingers consist of amino acid sequences containing two cysteine and two histidine residues at repeating intervals. Interspersed cysteine and histidine residues covalently bind zinc atoms, folding the amino acids into loops (zinc fingers). They are one of the major groups of eukaryotic transcription factors. This structural motif has been identified in a variety of significant regulatory circumstances, including proto-oncogenes and developmental control genes in Drosophila.

One speaks of UAS (upstream activating sequences) as being DNase hypersensitive. We would interpret this to mean that

a UAS is constitutively open.

Apurinic (and apyrimidinic) sites (AP sites) involve a spontaneous loss of ______________ in an intact double-helix DNA molecule

a purine

Match each number with the closest type of DNA: a. moderately repetitive DNA b. highly repetitive DNA c. short interspersed elements d. long interspersed elements e. unique-sequence DNA Reference: Ref 11-1 gene-encoding sequence Enter only the letter of your choice.

a. rRNA, tRNA b. telomeres c. d. e. gene encoding sequence

Assuming one mutational event in a gene, on average, which of the following mutagens would be expected to cause the most damage to a protein synthesized by such a mutagenized gene?

acridine orange

Name two chemical mutagens that are collectively called acridine dyes.

acridine orange, proflavin

DNA methylation may be a significant mode of genetic regulation in eukaryotes. Methylation refers to

addition of methyl groups to the cytosine of CG doublets

The _________ can be used to quickly screen chemicals for their ability to be mutagenic (and hence potentially carcinogenic).

ames test

The trp mRNA leader sequence contains ....

an attenuator region and two tryptophan codons

In what part of the mRNA does degradation generally begin?

at the 3' end with the removal of the poly(A) tail

Riboswitches were first discovered in 2002 and have been found in...

bacteria, fungi, archae, and plants

The _________________, which binds to a core promoter, consists of general transcription factors and RNA polymerase.

basal transcription apparatus

The lac repressor protein controls expression of the lac operon via ________________

binding to the lac operator site to repress expression

An insulator is also known as a(n) __________________ .

boundary element

Why does DNA have to be packaged?

cellular DNA is up to 100 thousand times a cell length, too large

An allosteric molecule is one in which ________.

changes conformation after binding

When cells are NOT dividing DNA exists as a nucleoprotein complex called _______

chromatin -60% of total weight of chromosomes is proteins

This process moves a nucleosome from the TATA box of a gene's promoter so that transcription can occur.

chromatin remodeling

Proteins that affect chromatin structure without altering histone chemical structure are called _______________.

chromatin remodeling complex

This term describes genetic elements that affect other elements only when they are located adjacent to them. For example, the operator has this effect on its structural genes.

cis-acting

Which of the following clusters of terms applies when addressing enhancers or silencers as elements associated with eukaryotic genetic regulation?

cis-acting, variable orientation, variable position

Many transcription factors also contain domains that bind ___________

coactivators -such as hormones or small metabolites that regulate their activity

Which of the following are general categories of mutations?

conditional, lethal , behavioral or morphological, induced, regulatory

The isolation of cell-cycle mutations in yeast was greatly facilitated by the use of _______________ mutations, which allow investigators to study the effects of mutations that would be lethal if expressed constantly

conditional/permissive

What is the P-loop:

conserved structural region of kinases involved in ATP-binding

A condition in which a gene or group of genes is expressed all the time.

constitutive

Mutations in the lacI and lacO genes in the lactose system often lead to full production of the three structural genes related to the lac operon even with no lactose available to the organism. Such mutations would be called ________.

constitutive

When considering the binding of cAMP-CAP and RNA polymerase to the lac operon, both bind more efficiently than either singly. What term is applied to this increased efficiency of binding?

cooperativity

Regarding regulation of the tryptophan operon, what type of regulatory molecule might one appropriately call the amino acid tryptophan?

corepressor

Considering the electromagnetic spectrum, identify likely mutagens from the following list: radio waves, microwaves, infrared, ultraviolet, X rays, gamma rays, cosmic rays.

cosmic, gamma, UV, x-ray

Mutations in the promoter region of the b-globin gene indicate that some areas are more sensitive than others. When mutations occur in consensus sequences (modular elements such as GC box, CAAT box, TATA box) transcription ________________.

decreases

LINES differ from retrotransposons in that LINES:

do not contain LTRs (Long terminal repeats)

What is the Kinase activation segment?

domain frequently shown to harbor oncogenic mutations in known kinase cancer genes

Transcription factors are proteins with at least two functional _________.

domains

To use RNAi (RNA interference) as a research tool, investigators introduce short synthetic __________ RNA into cells.

double stranded, small interfering

Considering the location of genes in the interphase nucleus, certain chromosomal territories appear to exist. Specifically,

each chromosome appears to occupy a discrete domain

What is the function of the lacY gene in the lactose operon?

encodes a lactose permease

Parts of chromosomes that are not methylated for inactivation, but rather are less dense-staining and are likely active in transcription in the cell.

euchromatin

Recent discoveries on causes of fragile-X syndrome, myotonic dystrophy, and Huntington disease indicate what type of genetic alteration?

expanding trinucleotide repeats

When X rays penetrate cells, electrons are ejected from atoms of molecules. Stable molecules can be transformed into what types of hazardous materials?

free radicals and reactive ions

Of the two cell lines that can contain a mutation in an organism, the __________ is most consequential to subsequent generations.

germ line

Regarding the lactose utilization system in E. coli, a ___________ inducer is a molecule that is chemically analogous to lactose, induces the operon, but is not a substrate for the enzymes of the lac operon.

gratuitous

A highly-methylated region of a chromosome that has become largely deactivated and can be seen as dense-regions when viewed using electron microscopy.

heterochromatin

The ____________________ consists of modifications to histone proteins that affect the expression of DNA sequences.

histone code

A __________ is a DNA stretch of 180 bp that specifies a 60 amino acid homeodomain

homeobox

When siRNAs or miRNAs are present, the rate of mRNA degradation_____________ , and the rate of protein production ___________.

increases; decreases

Channels between chromosomes in the interphase nucleus are called

interchromosomal compartments

What is the function of cAMP in regulation of the lac operon?

it activates an activator protein

A mutant E. coli strain, grown under conditions that normally induce the lac operon, produces high amounts of ß-galactosidase. What is a possible genotype of the cells?

lacI+ lacP+ lacOc lacZ+ lacY+ lacA+

A mutant E. coli strain, grown under conditions that normally induce the lac operon, does not produce ß-galactosidase. What is a possible genotype of the cells?

lacI+ lacP- lacO+ lacZ+ lacY+ lacA+

Constitutive mutations may occur in various components of the lac operon. Name two genes of the lac operon in which constitutive mutations could occur.

lacOc and lacI-

Within the control region of the tryptophan operon is a section of DNA that is sensitive to levels of tryptophan in the system. What is the name of this region?

leader or attenuator region

The trp operon regulatory region contains a _____ and ______ sequences

leader;attenuator -Attenuate means to reduce

Histone tails contain several _____ residues (positively charged AAs), these are required for chromatin to ______

lysine; condense -The positive charge of Lysine can be neutralized by addition of acetyl group -Positively chaged lysine residues within histone tails undergo reversible acetylation and deacetylation

What is meant by polycistronic mRNA? Give an example.

mRNA that can be coded into more than one protein product, lactose and tryptophan operons would be examples

What did Slack's studies suggest?

miR-21 is an oncogene mir-21 may be involved in "oncogene addiction" aka maintenance of cancer phenotype

What is the mechanism of miRNA processing?

miRNAs are processed from hairpin structures that form in pri-miRNAs -A nuclear RNase called DROSHA cleaves the hairpin region from the rest of the pri-miRNA - This region is transported from nucleus by exportin 5 -miRNAs are generated from pre-miRNA by cleavage with a second dsRNA specific RNase called Dicer -Once cleaved by dicer, miRNAs are 21-23 bp in length and contain two unpaired nucleotides at each end -One of the two strands selected for assembly into mature RNA-induced silencing complex (RISC) -This single-stranded mature miRNA is bound by member of the argonaute family of proteins in RISC

High-throughput technologies such as DNA and protein expression _________ are often used to provide a global picture of gene expression.

microarrays

Under what condition(s) might one have an amino acid substitution in a protein that does not result in an altered phenotype?

missense mutation that produces an amino acid that is similar chemically and structurally that does not affect the phenotype Silent mutation

Transposable elements are also known as: Transformers Jumping repeats Jumping genes Moving genes Mobile genetic elements

mobile genetic elements jumping genes

Conditional mutations are more likely to result from which of the following alterations to the coding region of a gene.

mutation caused by a tautomeric shift

What term would be applied to a regulatory condition that occurs when protein greatly reduces transcription when associated with a particular section of DNA?

negative control

An operon is controlled by a repressor. When the repressor binds to a small molecule, it is released from binding to DNA near the operon. The operon is never expressed if a mutation prevents the repressor from binding to the small molecule.

negative inducible

An operon is controlled by a repressor. When the repressor binds to a small molecule, it binds to DNA near the operon. The operon is constitutively expressed if a mutation prevents the repressor from binding to the small molecule

negative repressible

A _________ mutation changes a codon that specifies an amino acid into one that terminates translation.

nonsense

A constitutive gene is _______ regulated and is expressed ____________.

not, continually

Loss-of-function mutations that eliminate the function of a gene product are also known as _____ mutations or gene knockouts.

null

Indicate the level of activity of the lac operon under the medium conditions listed below: a) No lactose present, no glucose present b) Lactose present, no glucose present c) No lactose present, glucose present d) Lactose present, glucose present For a-d, enter either "high" or "off", separated by commas.

off, high, off, off

A conditional mutation is one that allows a mutant gene product to function normally under the ________ condition, but to function abnormally under the ________ condition. Such mutations are especially useful for the study of ________ mutations.

permissive; restrictive; lethal

The ____________________ stabilizes the 5' cap, which must be removed before the mRNA molecule can be degraded from the 5' end.

poly (A) tail

When transcription factors interact with DNA, is the resulting genetic control typically positive or negative?

positive

Which of the following terms best characterizes catabolite repression associated with the lactose operon in E. coli?

positive control

This is the region of a gene where RNA polymerase binds to initiate transcription.

promoter

What is the general position of the consensus sequence called the GC box? What is its sequence?

promoter; GGGCGG

The process of error correction of mismatched bases carried out by DNA polymerases is called ________.

proofreading

Apurinic sites (AP sites) involve a spontaneous loss of ________ in an intact double-helix DNA molecule.

purines

What different results can be expected in a mutagenesis experiment that uses radiation as compared with site-directed mutagenesis?

radiation causes mutations randomly within the sequence but site-directed mutagenesis causes a mutation at a specific site

The general term for a non-polymerase protein that binds to an operator.

repressor

An example of a gene product encoded by a regulatory gene is

repressor protein

mRNAs are degraded by enzymes called ____________.

ribonucleases

In general, a "model organism" used in genetics studies is one in which there is a large body of genetic knowledge that has been compiled over decades of genetic research. In addition, model organisms have available their DNA sequences and collections of ________ that make detailed genetic analysis possible and efficient.

strains with specific mutations

Briefly describe the structure and abundance of the lac repressor protein.

tetramer of identical units, with 5 to 10 tetramers present in each cell

siRNAs and miRNAs are produced by

the cutting and processing of double-stranded RNA by Dicer enzymes.

Alternative splicing is known to be important in the regulation of

the mammalian SV40 virus.

It is possible for a repressor to negatively regulate the expression of an operon because

the repressor binding site overlaps the promoter site of the operon allowing it to physically block the binding of RNA polymerase.

This question relates to the regulation of enzymes involved in the synthesis of tryptophan in E. coli. If tryptophan is present in the medium and available to the bacterium,

the repressor is bound to the operator, thus blocking transcription

Insulators can block the effects of enhancers only when

they lie between an enhancer and a promoter.

Where do miRNAs bind?

to complementary regions in the 3'UTR of mRNAs -The miRNA-RISC complexes associate with target mRNAs by binding to complementary regions in the 3'UTRs of mRNAs -Multiple miRNA-RISC complexes can bind to a 3'UTR to mediate repression

In what way might one use heat-shock genes in Drosophila to facilitate the use of RNAi as a research tool?

to reduce gene activity

These are factors that need not be adjacent to the genes they control. An example would be the lac operon's repressor protein.

trans-acting factors

Genetic regulation in eukaryotes can take place at a variety of levels from transcriptional to post-translational. At what level is genetic regulation considered most likely in prokaryotes?

transcriptional

Regarding eukaryotic and prokaryotic genetic regulation, what process seems to be the most similar between the two?

transcriptional regulation

When referring to attenuation in regulation of the tryptophan operon, it would be safe to say that when there are high levels of tryptophan available to the organism ____ ____ _ ____

transcriptional termination is likely

Chromosome structure is continuously rearranged so that _____________ ________ _______ are cycled to the edges of chromosome territories

transcriptionally active genes

What evidence indicates that steroid hormones exert their influence at the level of DNA?

translocation of the hormone-receptor complex from the cytoplasm to the nucleus, increase in the rate of transcription

One type of mutation involves the replacement of a pyrimidine with a purine. What general term is associated with this mutational phenomena?

transversion

Two formal terms used to describe categories of mutational nucleotide substitutions in DNA are called

transversions and transitions

The following may be caused by mobile genetic elements except ________________ activate a gene in which they reside disrupt a gene undergo mutation cause chromosome breaks all of the above

undergo mutation

A(n) _______________ can function at variable distances and in either orientation. However, it differs from an enhancer in that it cannot function downstream of the transcription starting point.

upstream activator sequence, UAS

Under the system of genetic control of the tryptophan operon,

when there is no tryptophan in the medium, transcription of the trp operon occurs at high levels.

Ultraviolet light causes pyrimidine dimers to form in DNA. Some individuals are genetically incapable of repairing some dimers at "normal" rates. Such individuals are likely to suffer from

xeroderma pigmentosum

What are characteristics of Drosophila eyeless?

• Binary switch, turn on eyeless, triggers differentiation of entire organ: eye • Eyeless protein acts early in development • Loss of eyelessleads to degeneration of cells that were to become eye • eyelesshomologs found in vertebrates • Expression of eyelessin other tissue results in eyes in the wrong location: Ectopic expression

What is true of Homeotic Genes?

• Contain a conserved amino acid region (homeodomain) encoded by conserved DNA region (homeobox) • Sequences found in other segmented organisms including chicken, Xenopus, humans, and mice • In mammals these genes are called Hox gene clusters

What does Vertebrate myoD do?

• Controls skeletal muscle formation in vertebrates • Genes conserved in frog, rodents, and humans • Express myoD gene in non-muscle tissue (eg. skin cells, fat cells, pigment cells)-----------> get muscle • Suggests role of myoD as switch starting cascade to produce muscle tissue

What is the Drosophila developmental summary?

• Development follows temporally and spatially ordered path • Maternally encoded genes produce anterior-posterior gradients • Maternal mRNA's and proteins activate early zygotic genes: gap genes • Gap genes activate pair-rule genes • Next, segmentation gene products divides segments into anterior/posterior • Segments are given identity by homeotic selector genes

What are some Drosophila homeotic selector gene characteristics?

• Found in 2 clusters on chromosome 3 • Antennapedia(Antp-C) complex, 5 genes for head and thorax formation • Bithorax (BX-C) complex, 3 genes specifying posterior structures

What are consequences of lack of nucleotide excision repair (NER)?

• Humans who lack the NER pathway • Unable to repair all damage due to UV radiation • Leads to xeroderma pigmentosum(XP) • Increased skin cancers and other skin abnormalities

How could mutation lead to disease?

• Large or multiple exposures to UV light leads to photodimers. • Repair mechanism is unable to repair all of the damage to DNA and some mutations become fixed. • Fixed mutations can lead to tumor phenotype (skin cancer).

What are developmental binary switches?

• Master Regulatory Genes= Binary Switch Genes • Have ability to initiate complete development of an organ or cell type • 2 examples: vertebrate myoD and Drosophilaeyeles

Are mutations in all cells heritable?

• Mutations that occur in germs cells are heritable. These mutations are the basis for the transmission of genetic diversity and evolution • Mutations that occur in somatic cells, are not transmitted to the next generation

What is true of the Wnt Pathway?

• Normal roles in control of cell proliferation, differentiation, and movements • Wnt signaling altered in cancer, bone density diseases, and other conditions • Active target of new and novel therapies +Wnt pathway is involved in tissue regeneration +Critical for correct early development +Overexpression in early dev. leads to serious developmental problems

What are different types of Mutation Repair Mechanisms?

• Reversal of Damaged DNA • General Excision • Specific Excision • Mismatch Repair • Postreplication Repair • Double-Strand Break Repair -Multiple cellular systems act to reverse or repair damage to DNA molecule

What is the Nucleolus?

• Structure that is produced by the transcriptional activity of the nucleolar organizer in eukaryotes • Composed of rDNA, rRNA, and protein • Site of production of ribosomes • Some nuclei have more than one nucleolus -visible throughout interphase of the cell cycle -NOT present in mitosis or meiosis except in Prophase I of meiosis -Site of RNA synthesis -Frogs: 2 chromosomes (1 pair) with nucleolar organizers -The nucleolar organizer is amplified in prophase I, results in abundance of rRNA, ribosomes for early development

What does it mean when Drosophila eyeless gene works in mice?

• Therefore gene is evolutionarily conserved • Also, downstream targets of eyeless must be conserved since you can produce eyes in other locations using the mouse eyeless as a trigger