Mol Gen Exam 4 Extended Response

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. Separate and clearly label your answers for parts a-b in the box below.

(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.

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 the F1 progeny are wild type, complementation has occurred and the two mutations must lie in two separate genes.

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.

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.

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.

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

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.

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.

What is Nucleotide Excision Repair?

Pathway that repairs bulky lesions in DNA that alter or distort the double helix e.g. uv-induced pyrimidine dimers, and DNA adducts. Different enzymes, that recognize and cut a specific number of nucleotides around the lesion e.g. in E. coli, the uvr gene products cut 13 nucleotides, including the lesion

Provide a brief description of the variable gene activity hypothesis as it relates to development. What information is often provided in support of this hypothesis?

Since all cells apparently have the same genetic information, yet may differ structurally and functionally, the variable gene activity hypothesis suggests that differential gene transcription (genetic regulation) accounts for such cellular diversity. Evidence in support of this model includes the following: chromosome puffs, isozymes, and the presence of genes but no gene products in some tissues (hemoglobin, for example).

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.

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.

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. 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.

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.

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

1. Alteration/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 post translational modification of the synthesized protein

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 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.

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. Proflavin adds or removes single bases from DNA, thus causing frameshift mutations. Ultraviolet light causes thymine dimers.

Describe the process of transcriptional networking and the use of CRM's.

A signal inputs activate gene A, which directs synthesis of transcription factor A, which binds to regulatory sites within B gene region. Interspersed upstream, downstream, and within the introns of gene B, CRMs that contain binding sites for transcription factors. Poduct of the A gene binds to one of the two CRMs inducing transcription of gene B. Product of B is also a transcription factor which activates gene C. Which produces an activator for gene D and feeds back to regulate. The output of D will determine the final developmental outcome.

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 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).

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.

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.

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

For the Drosophila experiment, one could do nuclear transplantation (similar to the classic experiments in amphibians and recent experiments in mammals) or transplant a cell from embryos of one genotype to the posterior end of the early embryos of another genotype. The transplanted cells would be reprogrammed into germ cells by the posterior P granules. The transplant can be detected in the next generation by the recognizable different phenotypes due to different genotypes of the donor and recipient.

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.

What is functional complementation?

Functional complementation is a process whereby plasmids or other vectors containing all, or at least a high percentage, of the genes from an organism are individually transformed into a mutant strain until one clone restores the mutant phenotype to wild type. This process usually allows the investigators an opportunity to isolate a functional stretch of DNA about which some knowledge of function is already available.

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," 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.

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. 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?

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.

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. Operons that exhibit catabolite repression are under the positive control of catabolic activator protein (CAP). For CAP to be active, it must form a complex with cAMP. Glucose affects the level of cAMP. The levels of glucose and cAMP are inversely proportional—as glucose levels increase, the level of cAMP decreases. Thus, CAP is not activated.

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

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.

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

KNOW THESE -Direct repair. DNA damage is repaired by directly changing the damaged nucleotide back to its original structure. -Base excision repair. The damaged base is excised, and then the entire nucleotide is replaced. -Photo reactive repair - reversal of pyrimidine dimers formed by uv light exposure. Requires the photoactivation enzyme

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

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 -

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 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.

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

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 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

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 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?

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).

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 the mutation repair mechanisms?

Reversal of Damaged DNA General Excision Specific Excision Mismatch Repair Postreplication Repair Double-Strand Break Repair

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.

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.

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?

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.

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.)

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

The table below 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. (Note: you are analyzing whether or not you see functional β-galactosidase enzyme or not). Please label your answer No Lactose #1-4 and With Lactose #1-4. β-galactosidase production No Lactose With Lactose 1. I+O+Z+ /F' I-O+Z+ 2. I-O+Z+ /F' I-O+Z+ 3. I-OCZ+ /F' I-O+Z- 4. ISOCZ+ /F' I+O+Z+

β-galactosidase production No Lactose With Lactose 1. I+O+Z+ /F' I-O+Z+ - + 2. I-O+Z+ /F' I-O+Z+ + + 3. I-OCZ+ /F' I-O+Z- + +