MolGen 4500 Module 4

What is the significance of the homeodomain? (SA)

The homeodomain is a highly conserved protein of 60 amino acids found in a variety of organisms, which, in conjunction with other factors, is thought to play a role in DNA binding and transcriptional activation.

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

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+? (SA)

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 (for example: transcription is off when lactose is not present, but transcriptionis on when lactose is present.)

Which of the below is not true about the location of enhancers? (MC)

The position of the enhancer has no effect on gene regulation.

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

The reshuffling of genes provides a way for certain gene segments to rearrange in order to produce new products or change their expression pattern. The relative positions of promoters and/or enhancers with respect to structural genes may influence transcription. 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? (SA)

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

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 (SA)

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? (LA)

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." (LA)

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? (MC)

UASs only function upstream.

Describe the phenomenon of photoreactivation repair. (SA)

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

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 (SA)

Upstream Activator Sequence (UAS)

Regarding the tryptophan operon, trpR- maps a considerable distance from the structural genes. The mutation either inhibits the interaction with tryptophan or inhibits repressor formation entirely. In the presence of tryptophan in the medium, would you expect the tryptophan operon to be transcriptionally active in this mutant? Explain. (LA)

With either of the two scenarios mentioned in the problem, absence of repressor function in a repressible system means that there would be no repression of the operon. The operon would be transcriptionally active.

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

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. They were originally discovered in the Xenopus transcription factor TFIIA, and 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 (MC)

a UAS is constitutively open

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

a mutation caused by a tautomeric shift

The following table lists several genotypes associated with the lac operon in E. coli. For each, indicate with a "+" or a "—" whether β-galactosidase would be expected to be produced at induced levels. (Assume that glucose is not present in the medium.) Genotype No lactose With lactose a I + O+ Z+/ F' I + O+ Z+ _________ _________ b I - Oc Z -/ F' I - Oc Z- __________ _________ c I - Oc Z +/ F' I - O+ Z+ __________ _________ d I s Oc Z -/ F' I s O+ Z+ __________ _________

a) -,+ (b) -,- (c) +,+ (d) -,-

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

acetylation

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? (MC)

acridine orange

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

addition of methyl groups to the cytosine of CG doublets

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

addition of methyl groups to the cytosine of CG doublets.

After translation, eukaryotic proteins can be modified by (MC)

all of the above: acetylation, addt of methyl groups, addt of phosphate groups, removal of amino acids

________ RNA processing generates different mRNAs, which can direct the synthesis of different polypeptides (SA)

alternative

In what part of the mRNA does degradation generally begin? (MC)

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

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

basal transcription apparatus

What is meant by the term reverse genetics? (SA)

begins w gene of unknown function, first inducing mutations then checking the effect of the mutation on phenotype.

The lac repressor protein controls expression of the lac operon via ________________ (MC)

binding to the lac operator site to repress expression

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

boundary element

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

chromatin remodeling

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

chromatin-remodeling complex

A promoter that affects only genes that are on the same piece of DNA is ____________-acting. (SA)

cis

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

cis-acting, variable orientation, variable position

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. (SA)

conditional

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________. (SA)

constitutive mutations

Mutations in the promoter region of the β-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 usually ________. (SA)

decreases

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

dimer formation

LINES differ from retrotransposons in that LINES: (MC)

do not contain LTRs

Transcription factors are proteins with at least two functional _________ (SA)

domains

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

dosage compensation

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

each chromosome appears to occupy a discrete domain

A eukaryotic DNA sequence that affects transcription at distant promoters is called a(n) ________________. (SA)

enhancer

Regulation of gene expression using siRNAs is found in (MC)

eukaryotes only

A class of mutations that results in multiple contiguous amino acid changes in proteins is likely to be the following: (MC)

frameshift

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

free radicals

__________ mutations produce new traits and are usually dominant. (MC)

gain-of-function

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

gene-encoding sequence: e DNA, rRNA, tRNA: a telomeres: b

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

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 (SA)

gratuitous

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

gratuitous inducer is a chemical analog to a natural inducer but it does not serves as a substrate. Example is IPTG in the lactose operon.

Which of these are major structural classifications of DNA-binding domains that are found in eukaryotic transcription factors? Please select all that apply:

helix-turn-helix, zinc fingers & leucine zipper

The ____________________ consists of modifications to histone proteins that affect the expression of DNA sequences (SA)

histone code

A __________ is a DNA stretch of 180 bp that specifies a 60 amino acid homeodomain. (SA)

homeobox

Which of the following are general categories of mutations? Select all that apply.

induced, morphological & regulatory

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. (SA)

inducer, repressor, allosteric

Channels between chromosomes in the interphase nucleus are called (MC)

interchromosomal compartments

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+ lacO+ lacZ- lacY+ lacA+ lacI- lacP+ lacO+ lacZ- lacY+ lacA+ lacI+ lacP- lacO+ lacZ+ lacY+ lacA+ lacI+ lacP+ lacOc lacZ+ lacY+ lacA+ (MC)

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? (MC)

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. (SA)

lacI- and lacOc

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

lacOc, lacI-

The lac repressor binds to: (MC)

lactose & DNA

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? (SA)

leader/attenuator

Which of the following are general categories of mutations? Select all that apply.

lethal, conditional, behavioral

Attenuation involves the termination of ______ synthesis (SA)

mRNA

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

microarrays

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

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 (MC)

negative inducible

Choose the type of control illustrated by each example E. coli lac operon control by lac I (MC)

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. (MC)

negative repressible

E. coli bacteria are placed into a medium containing glucose and lactose. Which of the genes below do you expect to be turned on? (MC)

none of the above

A _________ mutation changes a codon that specifies an amino acid into one that terminates translation (MC)

nonsense

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

null

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

null mutations cause complete lack of function and no production of functional gene product. frameshift mutations

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

off, high, off, off

What term refers to a contiguous genetic complex that is under coordinate control? (MC)

operon

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. (MC)

permissive; restrictive; lethal

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

poly (A) tail

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

poly A tail

A catabolite-activating protein (CAP) exerts _____________control over the lac operon. (SA)

positive

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

positive

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

positive control

This is the region of a gene where RNA polymerase binds to initiate transcription (SA)

promoter

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

promoter; GGGCGG

The process of error correction of mismatched bases carried out by DNA polymerases is called (SA)

proofreading

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

purine

Recombinational repair is activated when damaged DNA has escaped repair and the distortion disrupts the process of replication; which is dependent on the product of the ________ gene (SA)

recA

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

repressor

An example of a gene product encoded by a regulatory gene is (MC)

repressor protein

The ________ the wavelength of a radiation source, the greater its likelihood of causing damage. (SA)

shorter

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, fully expressed, dominant & 100% penetrant

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

splitting the β-linkage of lactose

A mutation that occurs naturally, without exposure to a known mutagen, is called a (SA)

spontaneous mutation

Mutations that arise in nature, from no particular artificial agent, are called (MC)

spontaneous mutations

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. (MC)

strains with specific mutations

What is meant by a suppressor mutation? (SA)

suppressor mutation inhibits transcription of genes

siRNAs and miRNAs are produced by (MC)

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

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

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, (MC)

the repressor is bound to the operator, thus blocking transcription

Insulators can block the effects of enhancers only when (MC)

they lie between an enhancer and a promoter.

Nutritional mutations can be defined as (MC)

those mutations which do not allow an organism to grow on minimal medium, but do allow the organism to grow on complete medium.

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? (MC)

transcriptional

____________________act as intermediaries between ______________________________ and specific _____ sequences to modify chromatin structure and activate transcription (SA)

transcriptional activators, chromatin remodeling complexes, DNA

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

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, (MC)

transcriptional termination is likely.

Which of these is a level or type of genetic regulation in eukaryotes? Select all that apply:

transcriptional, DNA methylation, post-translational

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

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

Which of these is a level of regulation in eukaryotes? Select all that apply:

transport, post-translational, transcriptional, & processing

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

transversion

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

transversions and transitions

The ___________ operon is typically characterized by the following two terms: negative control and repressible. (SA)

tryptophan

The following may be caused by mobile genetic elements except ________________ (MC)

undergo mutation

The difference between a genetic screening experiment and a selection experiment is that a screening experiment involves ________, whereas a selection experiment creates conditions that ________ irrelevant organisms (MC)

visual examination; eliminate

Briefly describe the structure and abundance of the lac repressor protein. (SA)

when lactose is absent, the lac repressor protein binds to the operator inhibiting the transcription of the genes.

Under the system of genetic control of the tryptophan operon, (MC)

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

Present a detailed description of the actions of the regulatory proteins in a) inducible and b) repressible enzyme systems. (LA)

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

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? (LA)

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.

Frameshift mutations are caused by the __________ or __________ of one or more nucleotides in DNA. a. chemical modification b. removal c. reversion d. insertion e. suppression

b-removal & d-insertion

A condition in which a gene or group of genes is expressed all the time (SA)

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? (SA)

cooperative binding

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

x-rays, ultraviolet, gamma rays & cosmic rays NOT: radio, microwaves & infrared

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 (MC)

xeroderma pigmentosum

What human condition is caused by the inability to repair UV-induced DNA lesions? (SA)

xeroderma pigmentosum

The following table lists several genotypes associated with the lac operon in E. coli. For each, indicate with a "+" or a "—" whether β-galactosidase would be expected to be produced at induced levels. (Assume that glucose is not present in the medium.) Genotype No lactose With lactose a I + O+ Z+/ F' I - O+ Z+ _________ _________ b I - Oc Z +/ F' I - O+ Z- __________ _________ c I s Oc Z +/ F' I + O+ Z+ __________ _________ d I - O+ Z +/ F' I - O+ Z+ __________ _________

(a) -, + (b) +,+ (c) +,+ (d) +,+

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. (LA)

(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. (LA)

(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

Use the following table for the questions below: Supplements: 1 2 3 4 Strain A + - + - Strain B + + + - Strain C + + + + Strain D - - + - Determine a possible metabolic pathway that would give the results seen for the four mutant strains, A through D. (If necessary, use a separate piece of paper to diagram the pathway). Then, fill-in the blanks below with Strain A-D or Supplement 1-4, using this generic metabolic pathway as a guide: (X) --> + --> 1a --> + --> 1b --> + --> 1c--> + --> 1d (supp.) | | | | 1e 1f 1g 1h (mut.strains)

-Which supplement (1-4) is needed at position 1a? -Which supplement (1-4) is needed at position 1b? -Which supplement (1-4) is needed at position 1c? -Which supplement (1-4) is needed at position 1d? -Which strain's (A-D) gene product would function at position 1e if it were not mutant? -Which strain's (A-D) gene product would function at position 1f if it were not mutant? -Which strain's (A-D) gene product would function at position 1g if it were not mutant? -Which strain's (A-D) gene product would function at position 1h if it were not mutant?

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. (LA)

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

Fill in the blanks in the "strain genotype" column of the following table. Write chromosomal genotypes with no partial diploidy. (+) means transcription of the lac operon. (-) means no transcription of the operon. The first line is filled in for reference. Please label your answers 1-3. Include the strain genotype for lacI, lacP, lacO, lacZ, and lacY Strain Lac absent Lac present 1 - + 2 + + 3 - -

1. lacI+ lacP+ lacO+ lacZ+ lacY+ 2. lacI+ lacP+ lacOc lacZ+ lacY+ OR lacI- lacP+ lacO+ lacZ+ lacY+ 3. lacI+ lacP- lacO+ lacZ+ lacY+

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

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

Explain two different ways that intragenic suppressors may reverse the effects of mutations (LA)

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. (LA)

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. (SA)

10^-5 to 10^-6

Fill in the blanks in the "level of transcription" column of this table with: + for high levels of transcription, and - for minimal levels of transcription of the lac operon. Consider regulation by both the lac repressor and CAP (catabolite activator protein). The strain is wild type, with no partial diploidy. Please label your answer with numbers 1-4 based on the chart below. Medium conditions level of transcription 1-high glucose, no lactose 2-no glucose, high lactose 3-high glucose, high lactose 4-no glucose, no lactose

1= - 2= + 3= - 4= -

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

23,000, 20 chromosomes

In what way can 5'-azacytosine influence transcription? (SA)

5-azacytosine causes undermethylation of sites of incorporation and changes in the pattern of gene expression

Name two chemical mutagens that are collectively called acridine dyes (SA)

5-bormouracil & 2-aminopurine

Name two mutagens that would be classified as base analogs (MC)

5-bromouracil and 2-aminopurine

It is estimated that transposable elements compose approximately what percent of the human genome? (MC)

50

What is a mutation? (MC)

A change in the DNA sequence

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? (SA)

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

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

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.

What is a homeobox, and what is its significance? (SA)

A homeobox is a DNA sequence, around 180 base pairs long, found within genes that are involved in the regulation of patterns of anatomical development (morphogenesis) in animals, fungi and plants.

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. (LA)

All three are caused by disparate 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).

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

Ames test

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? (MC)

B & C: -nitrosoguanidine & ethyl methane sulfonate (EMS)

This structure forms when an extra X chromosome is methylated and largely inactivated. It is most commonly found in females (SA)

Barr body

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

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.

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? (MC)

By forming hydrogen bonds with DNA bases

The _____ box sequence appears to be critical to the ability of many eukaryotic promoters to facilitate transcription (SA)

CAAT

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

CAAT, TATA, GC repeats

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

DNA binding domain

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

DNA binding protein

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

DNA repair

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

DNAse I, transcriptionally

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. (LA)

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.

What is the function of the lacY gene in the lactose operon? (SA)

Encodes a lactose permease.

How do enhancer elements differ from suppressor mutations? (SA)

Enhancer elements enable transcription of a gene to be increased. Suppressor mutations cause transcription to be inhibited

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

Epistasis is the masking of the phenotype of one mutant by the phenotype of a mutant in another locus. Hence, epistasis analysis can be used to determine a functional order of action of two genes by seeing if the expected phenotype occurs by masking certain genes.

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

Expansion of trinucleotide repeats

Under certain circumstances, one can make use of expression libraries to identify and clone a gene of interest. Describe an expression library and how one could be used to clone a gene. (LA)

Expression libraries contain cDNA clones inserted into vectors that have a promoter that will drive the expression of the cDNA within E. coli host cells. Antibodies specific for a purified protein will recognize the protein produced in bacteria from the cDNA vector. This allows the investigator an opportunity to select the relevant clone from the library.

Describe what is meant by the term forward genetics. (LA)

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 (SA)

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., The drosophila protein Dscam has many (~38000) sites that could be alternatively spliced. In theory this leads to more polypeptide products than there are genes in the Drosophila genome, Genetic mutations in the human SMN2 gene can lead to alternatively spliced protein products that can cause spinal muscle atrophy

What is the role of maternal-effect genes? (LA)

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.

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? (LA)

Glucose can enter glycolysis "as is," while lactose must first be split into glucose and galactose. To do so, the energy requiring synthesis of β-galactosidase is needed. It is energy-efficient to burn glucose rather than lactose.

How was it determined that X rays are mutagenic? (LA)

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

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. (MC)

H2O2, O2- & OH-

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

HTH domains were the first DNA-binding motifs to be identified. They are found in the cro, lac, and Trp repressors. A geometric conformation is formed by two adjacent α helices separated by a "turn" of several amino acids. Such motifs bind to the major grooves of DNA and interact with the DNA backbone. Many HTH-containing proteins regulate gene expression.

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

Heat shock proteins are activated upon exposure to heat. The heat shock protein could be associated with RNAi such that RNA knockdown is only activated with heat

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? (LA)

If dominant and if passed to the next generation, it would be expressed. New recessive mutations are not normally expressed in the next generation unless, through a combination with a like mutation from the other parent, they are homozygous

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

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. Selection is usually accomplished by killing or inhibiting the growth of irrelevant organisms.

Riboswitches were first discovered in 2002 and have been found in... (MC)

In all of the listed organisms: fungi, plants, archaea, bacteria

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

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

What is the general scheme for generating a transgenic mouse strain? (LA)

In short, generating transgenic mice involves five basic steps: 1. purification of transgenic construct, 2. harvesting donor zygotes, 3. microinjection of transgenic construct, 4. implantation of microinjected zygotes into the pseudo-pregnant recipient mice, 5. genotyping and analysis of transgene expression in founder mice

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. (SA)

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue

Describe how acridine dyes cause frameshift mutations. (SA)

Intercalation b/t bases of intact DNA causes DNA polymerase to add or skip a base during replication

What is the function of cAMP in regulation of the lac operon? (MC)

It activates an activator protein.

What is the difference between a missense mutation and a nonsense mutation? (LA)

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. (LA)

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 the differences between neutral mutations and silent mutations? (LA)

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.

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

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

Regarding the nature of the ABO blood groups, dysfunction in what process leads to the O blood type? (SA)

O blood type lacks the A and B antigens on the surface of the red blood cells. This happens when the person with the O type blood group inherits only the alleles which don't code for any antigens from either parents.

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. (SA)

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

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. (SA)

One class of activator or repressor assemble at promoter regions adjacent to transcription site. Activator increases expression & repressor decreases expression. Other class binds at more distant regions, ehancers & silencers.

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

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? (LA)

P-element transposons are mobile genetic elements that can move in and out of the genome. A transposase enzyme recognizes and acts on 31 bp inverted repeats at each end of the P element. Genetically engineered P elements can be injected into eggs, which enables the P-element bearing gene to be inserted into the embryo's DNA. With proper markers, the flies bearing the modified element can be recognized and transformed and mosaic flies may pass the element in the germ cells. P-elements can also be used to generate mutations by inserting into genes, thus disrupting their function.

What is meant by polycistronic mRNA? Give an example. (SA)

Polycistronic mRNA can be translated into more than one protein. An example of this is the Trp operon

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

Positive transcriptional control requires an activator protein to stimulate transcription at the operon. In negative control, a repressor protein inhibits or turns off transcription at the operon. 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.

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? (LA)

Promoter regions are necessary for the initiation of transcription. Promoters that interact with RNA polymerase II are usually located within 100 bp upstream of a gene and usually contain a TATA box (-25 to -30) and a CAAT box (-70 to -80).

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? (LA)

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.

_______________ is also known as RNA silencing and posttranscriptional gene silencing. (SA)

RNA interference

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

RNA polymerase binds to the promoter region to begin transcription

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? (LA)

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.

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? (SA)

SINES, LTR retrotransposons, LINES

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

Site-directed mutagenesis is not random and uses technology in which it is directly inserted by transpon, radiation can alter DNA base structure and is not localized

Two modular elements that appear as consensus sequences upstream from RNA polymerase II transcription start sites are (MC)

TATA & CAAT