BIO 3070 exam 3

(a) What are maternal-effect genes? (b) When are gene products from these genes made, and where are they located? Mastering Genetics visit for instructor-assigned tutorials and problems. (c) What aspects of development do maternal-effect genes control? (d) What is the phenotype of maternal-effect mutations?

(a) Genes that control early development are often dependent on the deposition of their products (mRNA, transcription factors, various structural proteins, etc.) in the egg by the mother. When observable, these are maternal-effect genes. (b) They are made in the early oocyte or nurse cells during oogenesis and deposited in gradients throughout the egg. (c) Such maternal-effect genes control early developmental events such as defining anterior-posterior polarity. Such products are placed in eggs during oogenesis and are activated immediately after fertilization. (d) A variety of phenotypes are possible, including embryonic lethality, and they are often revealed in the offspring of females. Maternal effects reveal the genotype of the mother: for example, females homozygous for deleterious recessive mutations of maternal-effect genes are sterile.

Explain how the following mutations would affect transcription of the yeast GAL1 gene in the presence of galactose. (a) A deletion within the GAL4 gene that removes the region encoding amino acids 1 to 100. (b) A deletion of the entire GAL3 gene. (c) A mutation within the GAL80 gene that blocks the ability of Gal80 protein to interact with Gal3p. (d) A deletion of one of the four UASG elements upstream from the GAL1 gene. (e) A point mutation in the GAL1 core promoter that alters the sequence of the TATA box.

(a) This would remove the DNA-binding domain and not allow transcriptional activation. (b) With no Gal3p, there would be no disruption of the Gal4p/Gal80p complex and no transcription of the GAL1 gene. (c) If the Gal80p can't interact with Gal3p, the block of the Gal4p AD by Gal80p cannot be alleviated, so there would be no GAL1 transcription. (d) This would reduce transcription of the GAL1 gene. (e) Generally, mutations in the TATA box of a

Predict the effect on the inducibility of the lac operon of a muta- tion that disrupts the function of (a) the crp gene, which encodes the CAP protein, and (b) the CAP-binding site within the promoter.

(a) Because activated CAP is a component of the coopera- tive binding of RNA polymerase to the lac promoter, absence of a functional crp would compromise the positive control exhibited by CAP. (b) Without a CAP binding site there would be a reduction in the inducibility of the lac operon.

The SOS repair genes in E. coli (discussed in Chapter 15) are negatively regulated by the lexA gene product, called the LexA repressor. When a cell's DNA sustains extensive damage, the LexA repressor is inactivated by the recA gene product (RecA), and transcription of the SOS genes is increased dramatically. One of the SOS genes is the uvrA gene. You are a student studying the function of the uvrA gene product in DNA repair. You isolate a mutant strain that shows constitutive expression of the CUvrA protein. Naming this mutant strain uvrA , you construct the diagram shown above in the right-hand column showing the lexA and uvrA operons: (a) Describe two different mutations that would result in a uvrA constitutive phenotype. Indicate the actual genotypes involved. (b) Outline a series of genetic experiments that would use partial diploid strains to determine which of the two possible mutations you have isolated.

(a)Call one type of constitutive mutation lexA- (mutation in the repressor gene product) and the other OuvrA- (mutation in the operator). (b) One can make partial diploid strains using F′. OuvrA- will (given the other genes brought in by the F′ ele- ment) be dominant to OuvrA + and lexA- will be recessive to lexA+. OuvrA - will act in cis.

What features of eukaryotes provide additional opportunities for the regulation of gene expression compared to bacteria?

-Separated processes of transcription & translation-presence of chromatin-opportunity for posttranscriptional & pre translational regulation

Describe a tautomeric shift and how it may lead to a mutation.

-Tautomeric shift is an intramolecular proton shift that changes the bonding structure of the molecule -It allows hydrogen bonding of normally non complementary bases

Attenuation of the trp operon was viewed as a relatively inef- ficient way to achieve genetic regulation when it was first dis- covered in the 1970s. Since then, however, attenuation has been found to be a relatively common regulatory strategy. Assuming that attenuation is a relatively inefficient way to achieve genetic regulation, what might explain its widespread occurrence?

-provides control over gene expression and attests to complex measures organisms take for effective gene regulation-provides a direct route for amino acids to control gene expression

What are the major mechanisms of epigenetic genome modification?

1. DNA methylation, histone modification involving acetyl, methyl, and phosphate groups 2. chromatin remodeling 3. microRNAs, and long noncoding RNAs.It is likely that additional epigenetic

Explain how the expression of a single gene can be quickly, efficiently, and specifically shut down at the transcriptional, posttranscriptional, and posttranslational stages through the coordinated expression of a transcriptional repressor, an miRNA, and a ubiquitin ligase.

1. Ubiquitin ligase. Ubiquitin or E3 ubiquitin ligase transfer ubiquitin to target protein and directs it to be destructed by proteosomes. 2. mi RNA. mi RNA( micro RNA) are small non coding RNA Which binds to mRNA by complementary base pairing and function in RNA silencing and regulations of gene expression by many ways like degradation of mRNA, cutting of poly A tail of mRNA, etc. 3. A gene specific transcriptional repressor. Repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of mRNA. Comment

The homeotic mutation Antennapedia causes mutant Drosophila to have legs in place of antennae and is a dominant gain-of-function mutation. What are the properties of such mutations? How does the Antennapedia gene change antennae into legs?

A dominant gain-of-function mutation is one that changes the specificity or expression pattern of a gene or gene product. The "gain-of-function" Antp mutation causes the wild- type Antennapedia gene to be expressed ectopically in the eye-antenna disc and mutant flies have legs on the head in place of antenna.

Why is a random mutation more likely to be deleterious than beneficial?

A gene is likely to be the product of perhaps a billion or so years of evolution. Each gene and its product function in an environment that has also evolved, or coevolved. A coordinated output of each gene product is required for life. Deviations from the norm, caused by mutation, are likely to be disruptive because of the complex and inter- active environment in which each gene product must function. However, on occasion a beneficial variation occurs.

In principle, RNAi may be used to fight viral infection. How might this work?

A number of viruses use RNA as their genome rather than DNA. if the genome consists of SSRNA, DSRNA molecules could be generated directly from this genomic RNA using the virally encoded replicase. If the genome consists of SIRNA, cellular enzyme dicer could synthesize a complementary RNA strand that's making the viral genome available for degradation

Why would a mutation in a somatic cell of a multicellular organism not necessarily result in a detectable phenotype?

A recessive somatic mutation would not produce a vis- ible phenotype in a diploid organism. A dominant muta- tion would be more likely to be visible if it occurs early in development. If only a relatively small number of cells are affected, the effects of a dominant mutation could be masked by surrounding nonmutant cells. Similarly, if the mutation does not cause substantial alterations to the gene product or to regulation of expression of this product, an effect may not be visible. Those that occur in somatic cells are not transmitted to the next generation but may lead to altered cellular function or tumors.

The apterous gene in Drosophila encodes a protein required for wing patterning and growth. It is also known to function in nerve development, fertility, and viability. When human and mouse genes whose protein products closely resemble apterous were used to generate transgenic Drosophila [Rincon-Limas et al. (1999). Proc. Nat. Acad. Sci. (USA) 96:2165-2170], the apterous mutant phenotype was rescued. In addition, the whole-body expression patterns in the transgenic Drosophila were similar to normal apterous. (a) What is meant by the term rescued in this context? (b) What do these results indicate about the molecular nature of development?

A) It's when the introduction of a gene from an outside source restores the wild type phenotype of the organism B) they attest to the conservation from a distant common ancestor of fundamental molecular species development, they indicate the extreme conservation of proteins structure and function across phylogenetically distant organisms

Amino acids are classified as positively charged, negatively charged, or electrically neutral .(a) Which category includes lysine?(b) How does this property of lysine allow it to interact with DNA? (c) How does acetylation of lysine affect its interaction with DNA, and how is this related to the activation of gene expression?

A. Lysine is a positively charged amino acid. B. Positively charged lysine side chains can form ionic bonds with negatively charged phosphate groups in the DNADNA backbone. C. Acetylation of the lysine side chain removes its positive charge preventing the ionic interaction with phosphate and decreasing the interaction between the histone and the DNA. As a result, genes are more accessible and are able to be expressed.

Contrast and compare the mutagenic effects of deaminating agents, alkylating agents, and base analogs.

All three of the agents are mutagenic because they cause base substitutions. Deaminating agents oxidatively convert an amino group to a keto group such that cytosine is converted to uracil and adenine is converted to hypoxanthine. Uracil pairs with adenine, and hypoxanthine pairs with cytosine. Alkylating agents donate an alkyl group to the amino or keto groups of nucleotides, thus altering base-pairing affinities. 6-ethyl guanine acts like adenine, thereby pairing with thymine. Base analogs such as 5-bromouracil and 2-amino purine are incorporated as thymine and adenine, respectively, yet they base-pair with guanine and cytosine, respectively.

Distinguish between oncogenes and proto-oncogenes. In what ways can proto-oncogenes be converted to oncogenes?

An oncogene is a mutated gene that contributes to the development of a cancer. In their normal, unmutated state, oncogenes are called proto-oncogenes, and they play roles in the regulation of cell division.The conversion of a proto-oncogene to an oncogene is called activation. Proto-oncogenes can become activated by a variety of genetic mechanisms including transduction, insertional mutagenesis, amplification, point mutations, and chromosomal translocations.

What is apoptosis, and under what circumstances do cells undergo this process?

Apoptosis, or programmed cell death, is a genetically controlled process that leads to death of a cell. It is a natural process involved in morphogenesis and a protective mechanism against cancer formation. During apoptosis, nuclear DNA becomes fragmented, cellular structures are disrupted, and the cells are dissolved. Caspases are involved in the initiation and progress of apoptosis.

Provide a brief description of two different types of histone modification and how they impact transcription.

At least nine different types of histone modifications have been discovered. Acetylation, methylation, phosphorylation, and ubiquitylation. Methylation and demethylation of histones turns the genes in DNA "off" and "on," respectively, either by loosening their tails, thereby allowing transcription factors and other proteins to access the DNA, or by encompassing their tails around the DNA, thereby restricting access to the DNA. Histone acetylation therefore leads to transcriptional activation.

Describe the role of attenuation in the regulation of tryptophan biosynthesis.

Attenuation functions to reduce the synthesis of tryptophan when it is in full supply. It does so by reducing transcrip- tion of the tryptophan operon. The same phenomenon is observed when tryptophan activates the repressor to shut off transcription of the tryptophan operon.

Regulation of the lac operon in E. coli (see Chapter 16) and regu- lation of the GAL system in yeast are analogous in that they both serve to adapt cells to growth on different carbon sources. How- ever, the transcriptional changes are accomplished very differ- ently. Consider the conceptual similarities and differences as you address the following. (a) Compare and contrast the roles of the lac operon inducer in bacteria and Gal3p in eukaryotes in the regulation of their respec- tive systems.(b) Compare and contrast the cis-regulatory elements of the lac operon and GAL gene system. (c) Compare and contrast how these two systems are negatively regulated such that they are downregulated in the presence of glucose.

C. We know that the lac operon is an inducible operon. In lac operon, lactose and it's analogs are inducers. It binds to allosteric site of the lac repressor thereby inactivating the operator binding site of the lac repressor protein. Inactivation of lac repressor permits the induction of transcription of the structural genes of the lac operon. In case of eukaryotes galactose induces transcription switch which is regulated by the activator Gal4p, the repressor Gal80p and thetranducer Gal 3p. Gal3p function as the sensor and tranducer of galactose signal in the induction pathway, it is required only for the induction state and not for its maintenance. D. Cis regulatory elements are regions of non coding DNA, which regulates the transcription of neighbouring genes. In case of lac operon- operator is the cis acting element or sequence which is bound by the repressor which in turn prevent the transcription of the adjacent genes on the same DNA molecule. In GAl gene system upstream activating sequence is cis - acting regulatory sequence which increases the expression of neighbouring genes. It has a binding site for GAL4 and act as a positive regulator.

What functional information about a genome can be determined through applications of chromatin immunoprecipitation (ChIP)?

ChIP is used to investigate a particular protein-DNA interaction, several protein-DNA interactions, or interactions across the whole genome or a subset of genes.

Provide a definition of chromatin remodeling, and give two exam- ples of this phenomenon.

Chromatin remodeling is one method of nucleosome modi- fication, in which remodeling complexes reposition or reconfigure nucleosomes using ATP as an energy source. Examples are the replacement of histone H2A with the histone variant H2A.Z and the removal of nucleosomes bound to the GAL promoters by the chromatin remodeling complex, SWI/SNF.

Explain how a tissue-specific RNA-binding protein can lead to tissue-specific alternative splicing via splicing enhancers or splic- ing silencers.

Cis-acting sequences that promote (splicing enhancers) or inhibit (splicing silencers) splicing are recognized by differ- ent classes of RNA-binding proteins (RBPs). Since expres- sion of RBPs is often tissue-specific, alternative splicing can also be tissue-specific.

Explain the apparent paradox that both hypermethylation and hypomethylation of DNA are often found in the same cancer cell.

Complex regulatory systems are involved. for example Hypomethylation of oncogenes and hypermethylation of tumor- suppressor genes are expected in cancer cells. DNA methylation is a way by which a gene is turned off so that accidental transcription does not occur. But when, necessary genes are hypermethylated, they become inactive, such as tumor suppressor genes when hypermethylated cannot do its job, that is keep cell division at check leading to cancer. Similarly when genes that are supposed to be silent such as oncogenes, which are to be methylated due to some reason remain unmethylated or hypomethylated leads to their over expression, which ultimately leads to cancer.

DNA damage brought on by a variety of natural and artificial agents elicits a wide variety of cellular responses involving numer- ous signaling pathways. In addition to the activation of DNA repair mechanisms, there can be activation of pathways leading to apoptosis (programmed cell death) and cell-cycle arrest. Why would apoptosis and cell-cycle arrest often be part of a cellular response to DNA damage?

DNA repair mechanisms often cannot reduce the impact of mutations

The regulation of mRNA decay relies heavily upon deadenylases and decapping enzymes. Explain how these classes of enzymes are critical to initiating mRNA decay.

Deadenylases, which function in deadenylation-dependent decay, shorten the 3′3′-poly-AA tail and lead to the recruitment of either a degradative exosome complex or decapping enzymes.Decapping enzymes function in both deadenylation-dependent and -independent decay, by removing the 5′5′ cap and allowing XRN1 exonuclease degradation.

DNA supercoiling, which occurs when coiling tension is gener- ated ahead of the replication fork, is relieved by DNA gyrase. Supercoiling may also be involved in transcription regula- tion. Researchers discovered that enhancers operating over a long distance (2500 bp) are dependent on DNA supercoiling, while enhancers operating over shorter distances (110 bp) are not so dependent [Liu et al. (2001). Proc. Natl. Acad. Sci. USA 98:14,883-14,888]. Using a diagram, suggest a way in which supercoiling may positively influence enhancer activity over long distances.

Following is a diagram of a possible mechanism by which supercoiling may positively influence enhancer activity over a relatively long distance.

How do translocations such as the Philadelphia chromosome contribute to cancer?

Genetic mapping established that certain genes were combined to form a hybrid oncogene ( BCR/ABL ) that encodes a 200kDa protein that has been implicated in the formation of chronic myelogenous leukemia.

Mutations in tumor-suppressor genes are associated with many types of cancers. In addition, epigenetic changes (such as DNA methylation) of tumor-suppressor genes are also associated with tumorigenesis [Otani et al. (2013). Expert Rev Mol Diagn 13:445-455]. (a) How might hypermethylation of the TP53 gene promoter influence tumorigenesis? (b) Knowing that tumors release free DNA into certain sur- rounding body fluids through necrosis and apoptosis Kloten etal. [(2013). Breast Cancer Res. 15(1):R4] outline an experimental protocol for using human blood as a biomarker for cancer and as a method for monitoring the progression of cancer in an individual.

Hyper-methylation of TP53 promoter, influences tumorigenesis as - the concentration of p53 will be decreased, the process of tumorigenesis will be stimulated. An experimental protocol for using human blood as a biomarker for cancer and as a method for monitoring the progression of cancer in an individual - novel DNA should be looked for in blood.

While miRNA response elements (MREs) may be located any- where within an mRNA, they are most often found outside the coding region in the 5′ or 3′ UTR. Explain why this is likely the case given that miRNAs often target more than one mRNA.

If the MREs were located in a coding region, the sequence would be translated. This would require either that a unique MRE exist for each targeted mRNA or that mRNAs targeted by the same miRNA encode a conserved protein domain. As it is, the location of MREs in noncoding sequences allows miRNAs to be more versatile in their targeting.

Contrast the role of the repressor in an inducible system and in a repressible system.

In an inducible system, the repressor that normally interacts with the operator to inhibit transcription is inactivated by an inducer, thus permitting transcription. In a repressible system, a normally inactive repressor is activated by binding a co-repressor. The activated repressor will then bind to the operator to inhibit transcription. Because the interaction of the protein (repressor) has a negative influence on transcrip- tion, the systems described here are forms of negative control.

Present an overview of RNA interference (RNAi). How does the silencing process begin, and what major components participate?

In general terms, a cytoplasmic protein, Dicer, processes double-stranded small noncoding RNA (sncRNA) mol- ecules to produce shorter dsRNAs. These associate with RISC, where an Argonaut-family protein cleaves and dis- cards one of the two strands. The retained strand guides RISC to the complementary target message, where the com- plex acts to prevent expression. The specific mechanism of silencing depends on the type of sncRNA used.

What parts of the genome are reversibly methylated? How does this affect gene expression?

In general, periodic methylation occurs at CpG-rich regions and promoter sequences. When a gene is imprinted by methylation, it remains transcriptionally silent. In a mammalian embryo, imprinting may silence only the paternal set of chromosomes, for example.

Annotation of the human genome sequence reveals a discrepancy between the number of protein-coding genes and the number of predicted proteins actually expressed by the genome. Proteomic analysis indicates that human cells are capable of synthesizing more than 100,000 different proteins and perhaps three times this number. What is the discrepancy, and how can it be reconciled?

Increased protein production from approximately 20,000 genes is probably related to alternative splicing and various post translational processing schemes. In addition, a particular DNA segment may be read in a variety of ways and in two directions

List the functions of kinases and cyclins, and describe how they interact to cause cells to move through the cell cycle.

Kinases regulate other proteins by adding phosphate groups. Cyclins bind to the kinases, switching them on and off. CDK4 (cyclin-dependent kinase) binds to cyclin D, moving cells from G1 to S. At the G2/mitosis border, CDK1 combines with another cyclin (cyclin B). The resulting phosphorylation brings about a series of changes in the nuclear membrane via caldesmon, cytoskeleton, and histone H1.

From the data in Table 19.3, draw up a list of histone H3 modifications associated with gene activation. Then draw up a list of H3 modifications associated with repression.(a) Are there any overlaps on the lists? (b) Are these overlaps explained by different modifications? (c) If not, how can you reconcile these differences?

Modifications of lysine 4, lysine 9, lysine 14, lysine 27, lysine 36, and lysine 79 cause activation of gene expression. Modifications of lysine 9, serine 10, lysine 27, and lysine 79 cause repression of gene expression. (a) Yes, modification of lysine residues 9, 27, and 79 can cause either activation or repression of expression. (b) This can be resolved, for the most part, by taking into account the type of modification performed. The exception to this is third methylation of lysine 79, which can result in either activation or repression. (c) The consequences of the third methylation of lysine 79 may depend on the interaction of this modified amino acid with other modified residue.

What are DNA microarrays? How are they used?

Most microarrays, known also as gene chips, consist of a glass slide that is coated, using a robotic system, with single-stranded DNA molecules. Some microarrays are coated with single-stranded sequences of expressed sequenced tags or DNA sequences that are complementary to gene transcripts. A single microarray can have as many as 20,000 different spots of DNA (or as many as 1 million for exon-specific arrays), each containing a unique sequence. Researchers use microarrays to compare patterns of gene expression in tissues under different conditions or to com- pare gene-expression patterns in normal and diseased tissues. In addition, microarrays can be used to identify pathogens.

Of the two classes of genes associated with cancer, tumor-suppressor genes and oncogenes, mutations in which group can be considered gain-of-function mutations? In which group are the loss-of-function mutations? Explain.

Mutations that produce oncogenes alter gene expression either directly or indirectly and act in a dominant capacity. Proto-oncogenes are those that normally function to promote or maintain cell division. In the mutant state (oncogenes), they induce or maintain uncontrolled cell division; that is, there is a gain of function. Generally, this gain of function takes the form of increased or abnormally continuous gene output. On the other hand, loss of function is generally attributed to mutations in tumor-suppressor genes, which function to halt passage through the cell cycle. When such genes are mutant, they have lost their capacity to halt the cell cycle. Such mutations are generally recessive.

Describe the steps by which the TP53 gene responds to DNA damage and/or cellular stress to promote cell-cycle arrest and apoptosis. Given that TP53 is a recessive gene and is not located on the X chromosome, why would people who inherit just one mutant copy of a recessive tumor-suppressor gene be at higher risk of developing cancer than those without the recessive gene?

Normally, the p53 protein is bound by the MDM2 protein, which marks p53 for degradation and prevents its activation. Upon DNA damage or cellular stress, MDM2 dissociates from p53, which both stabilizes the protein and activates it. Once activated, p53 serves as a transcription factor, upregulating genes that lead to cell-cycle arrest (p21) and apoptosis (BAX). Individuals with two copies of a tumor suppressor gene would need to experience sepa- rate mutations in both copies to develop cancer, whereas individuals with only one functional copy (plus one mutant copy) would only need a single mutation. Therefore, it make sense that those who inherit one mutant copy of a recessive tumor-suppressor gene will have the higher risk of developing cancer.

Early development depends on the temporal and spatial inter- play between maternally supplied material and mRNA and the onset of zygotic gene expression. Maternally encoded mRNAs must be produced, positioned, and degraded [Surdej and Jacobs-Lorena (1998). Mol. Cell Biol. 18:2892-2900]. For example, transcription of the bicoid gene that determines anterior-posterior polarity in Drosophila is maternal. The mRNA is synthesized in the ovary by nurse cells and then transported to the oocyte, where it localizes to the anterior ends of oocytes. After egg deposition, bicoid mRNA is translated and unstable bicoid protein forms a decreasing concentration gradient from the anterior end of the embryo. At the start of gastrulation, bicoid mRNA has been degraded. Consider two models to explain the degradation of bicoid mRNA: (1) degradation may result from signals within the mRNA (intrinsic model), or (2) degradation may result from the mRNA's position within the egg (extrinsic model). Experimentally, how could one distinguish between these two models?

One could make transgenic flies that contain a series of deletions spanning ( Special segment) of the bicoid mRNA: the coding region and 5' and 3' untranslated regions, and compare (tRNA) of individual, deleted mRNA s with controls. If a degradation-sensitive region or signal sequence is located by(DNA) that same intact region, when ligated to a non involved, non degraded (mTNA)should (stabilise) degradation in a manner similar to the bicoid mRNA If the bicoid mRNA from the (anterior) end of the egg is placed on the (posterior) another egg, one could ask if the degradation process is comparable in both locations. The modulation of mRNA is very important for the development of various tissues and that play an important role and the development of fertilized egg into a a mature embryo . The differential mRNA formation and mRNA degradation both processes are important in the development of molecular programs controlling experiment. Transgenic flies are are made by the alteration of genes the code for different proteins and leads to emergence of different phenotypic character In the Bicoid RNA , anterior and posterior location of the gene transfer are comparable because of the complementarity of the RNA sequences .

BLAST searches and related applications are essential for analyzing gene and protein sequences. Define BLAST, describe basic features of this bioinformatics tool, and give an example of information provided by a BLAST search.

One initial approach to annotating a sequence is to compare the newly sequenced genomic DNA to known sequences already stored in various databases. The National Center for Biotechnology Information (NCBI) provides access to BLAST (Basic Local Alignment Search Tool) software, which directs searches through databanks of DNA and protein sequences. A segment of DNA can be compared to sequences in major databases such as GenBank to identify matches that align in whole or in part. For example, using a query sequence from mouse chromosome 11, one might find identical or similar sequences in a number of taxa. BLAST will compute a similarity score or identity value to indicate the degree to which two sequences are simi- lar, as well as an expect value (E-value, the likelihood that the sequence matches by chance) that indicates the level of significance of a match (a value close to 1 indicates the match may be random). BLAST is one of many sequence alignment algorithms (RNA-RNA, protein-protein, etc.) that may sacrifice sensitivity for speed.

Annotation involves identifying genes and gene-regulatory sequences in a genome. List and describe characteristics of a genome that are hallmarks for identifying genes in an unknown sequence. What characteristics would you look for in a bacterial genome? A eukaryotic genome?

One usually begins to annotate a sequence by compar- ing it to known sequences. Similarity to other annotated sequences often provides insight as to a sequence's func- tion. Hallmarks to annotation are the identification ofgene regulatory sequences found upstream of genes (such as promoters), downstream elements (termination sequences), and triplet nucleotides that are part of the coding region of the gene. Bacterial genes do not contain a number of the elements found in eukaryotic genes, so their annotation is sometimes less complicated. In eukaryotes, upstream elements would also include enhancers and silencers and downstream elements would also include a polyadenylation signal sequence. In addition, 5′ and 3′ splice sites that distinguish exons from introns are also used in annotation.

How may the covalent modification of a protein with a phosphate group alter its function?

Phosphorylation of a protein will change the conformation of binding site of a protein, that in turn would cause activation, inactivation or modification of the function of that protein

Explain how the addition of acetyl groups to histones leads to a weaker association of DNA in nucleosomes.

Positively charged histones are able to interact with nega- tively charged phosphate groups of the DNA backbone. Addition of acetyl groups to histone tails weakens this interaction by reducing the positive charge on histones.

Present an overview of the manner in which chromatin can be remodeled. Describe the manner in which these remodeling pro- cesses influence transcription.

Remodeling complexes are recruited to promoters by acti- vators or repressors to either open or close the promoter. Chromatin remodeling complexes alter chromatin con- formation by swapping histone variants, repositioning nucleosomes along the DNA, or pulling the DNA from the core nucleosome. Alterations that create a more open chromatin conformation will allow transcription, whereas downregulation of transcription will result if a more closed conformation is created

RNAi may be directed by small interfering RNAs (siRNAs) or microRNAs (miRNAs); how are these similar, and how are they different?

Silencing of homologous gene expression triggered by double stranded RNA is called RNA interference (RNAi). Introduction of long double stranded RNA into the cell of plants, animals leads to a sequence specific degradation of the homologous gene transcript. The long dsRNA molecules are cleaved by an RNase lll enzyme called Dicer. This generate small 21-23 nucleotide long dsRNA molecules called small interfering RNAs(siRNAs). miRNA are micro RNAs.. Similarities: association with RISC the source are double-stranded sncRNAs the result of cleavage. Differences the source of dsRNA processing of longer RNAs secondary structure mechanism of silencing

Compare the control of gene regulation in eukaryotes and bacteria at the level of initiation of transcription. How do the regulatory mechanisms work? What are the similarities and differences in these two types of organisms in terms of the specific components of the regulatory mechanisms?

Similarities: Transcription initiation requires interaction between cis-acting elements and the trans-acting factors. Promoters are required elements and are located upstream of the transcribed gene. Activators and repressors can influ- ence promoter recognition. The formation of DNA loops (although of different structure) contributes to regulation of transcription initiation. Differences: In bacteria, the promoter is recognized by the s subunit of the RNA polymerase holoenzyme. Differ- ent s subunits recognize different promoter sequences, thereby regulating transcriptional specificity. Repres- sor proteins that induce DNA conformational changes (repression loops) can prevent promoter binding by RNA polymerase. In addition, RNAs (attenuators, riboswitches, sRNAs) can either allow or repress initiation, making tran- scription responsive to environmental or cellular condi- tions. In eukaryotes, chromatin structure may need to be modulated to make the promoter more or less accessible to the transcriptional machinery. RNAP II is recruited to promoters by general transcription factors. In addition, activators and repressors bind to enhancers and silencers, respectively. It has been proposed that large DNA loops are induced, bringing promoters and enhancers (or silencers) close to each other.

miRNAs target endogenous mRNAs in a sequence-specific man- ner. Explain, conceptually, how one might identify potential mRNA targets for a given miRNA if you only know the sequence of the miRNA and the sequence of all mRNAs in a cell or tissue of interest.

Since miRNA must base-pair with its potential target to direct RISC the correct message, you can determine the sequence that is complementary to the miRNA and search for that sequence among the cellular mRNAs, whose sequences are also known.

Most mutations in a diploid organism are recessive. Why?

Since the diploid organism has two alleles of a single gene, in most of the cases one functionally active gene of each pair is enough to produce functional product to manifest the normal phenotype. Hence, the correct option is "In most cases, the amount of product from one gene of each pair is sufficient for production of a normal phenotype."

Many transcriptional activators are proteins with a DNA-binding domain (DBD) and an activation domain (AD). Explain how each domain contributes to transcriptional initiation. Would you expect repressors to also have each of these domains?

The DBD allows the activator to bind to enhancer elements, to correctly position the protein to interact with the correct gene sequence. The AD allows the activator to interact with other proteins, such as coactivators, important to the for- mation of the pre-initiation complex. It would make sense for the same types of domains to exist in repressor proteins as well.

Hereditary spherocytosis (HS) is a disorder characterized by sphere-shaped red blood cells, anemia, and other abnormal traits. Ankyrin-1 (ANK1) is a protein that links membrane proteins to the cytoskeleton. Loss of this activity is associated biochemically to HS. However, Gallagher et al. (2010) (J. Clin. Invest. 120:4453- 4465) show that HS can also be caused by mutations within a region from - 282 to - 101 relative to the transcriptional start site, which lead to constitutive transcriptional repression in erythroid cells due to local chromatin condensation. Propose a hypothesis for the function of the - 282 to - 101 region of the ANK1 gene.

The description of the regulatory element (location, effect on transcription upon mutation) suggests that it might be an enhancer. Interestingly, Gallagher et al. describe a differ- ent element—a barrier insulator—in this region. The insu- lators described in the text were those that prevented an enhancer from upregulating expression from a non-target gene. A barrier insulator prevents the spreading of hetero- chromatin into regions of euchromatin so that silencing of non-target genes does not occur. A mutation in a bar- rier insulator would allow heterochromatin to spread and would result in inappropriate gene silencing, as was seen for the ANK1 gene.

Enhancers can influence the transcription of genes far away on the same chromosome. How are the effects of enhancers restricted so that they do not exert inappropriate transcriptional activation of non-target genes?

The effects of enhancers are limited by insulators—protein- binding sites found between the enhancer and promoter of a non-target gene. Insulators likely work by binding pro- teins that induce DNA looping that favors enhancer inter- actions with target promoter and blocks interactions with non-target promoters.

RNAi is currently being tested as a therapeutic tool for genetic dis- eases and other conditions. Consider the following: cystic fibrosis caused by loss of function of the CFTR gene, HIV infection, and cancer caused by hyperactivity of a growth factor receptor. Which of these may be treatable by RNAi, and which not? Explain your reasoning.

The function of RNAi is to downregulate gene expression. However they cannot upregulate gene expression directly. Therapeutically, logical diseases would be those in which gene products are overexpressed (such as cancer) or those in which RNA targets are unique to the disease (such as viral infections). Diseases arising from mutations in a structural gene (such as cystic fibrosis) would not be candidates for therapeutic RNA

What is the histone code?

The histone code describes the patterns of reversible modifications of histones and the interactions between and among them that contribute to the regulation of gene expression.

Marine stickleback fish have pelvic fins with long spines that provide protection from larger predatory fish. Some stickleback fish were trapped in lakes and have adapted to life in a different environment. Many lake populations of stickleback fish lack pel- vic fins. Shapiro et al. (2004) (Nature 428:717.723) mapped the mutation associated with the loss of pelvic fins to the Pitx1 locus, a gene expressed in pelvic fins, the pituitary gland, and the jaw. However, the coding sequence of the Pitx1 gene is identi- cal in marine and lake stickleback [Chan et al. (2010). Science 327:5963,302-305]. Moreover, when the Pitx1 coding region is deleted, the fish die with defects in the pituitary gland and the jaw, and they lack pelvic fins. Explain how a mutation near, but outside of, the coding region of Pitx1 may cause a loss of pelvic fins without pleiotropic effects on the pituitary gland and jaw.

The mutation is likely in a tissue-specific enhancer ele- ment. Fish with pelvic fins possess a wild-type copy of the enhancer and can upregulate Pitx1 expression in the pelvic region. Fish without pelvic fins possess the mutated enhancer. While these fish are unable to upregulate expres- sion in the pelvic area, expression in the jaw and pituitary gland remains normal.

A bacterial operon is responsible for the production of the biosynthetic enzymes needed to make the hypothetical amino acid tisophane (tis). The operon is regulated by a separate gene, R. The deletion of R causes the loss of enzyme synthe- sis. In the wild-type condition, when tis is present, no enzymes are made; in the absence of tis, the enzymes are made. Muta- tions in the operator gene (O-) result in repression regardless of the presence of tis. Is the operon under positive or negative control? Propose a model for (a) repression of the genes in the presence of tis in wild-type cells and (b) the mutations.

The regulatory gene product is exerting positive control. (a) In wild-type cells, when tis is present, no enzymes are made; therefore, tis must inactivate the positive regulatory protein. When tis is absent, the regula- tory protein is free to exert its positive influence on transcription. (b) Mutations in the operator negate the positive action of the regulator

How can you determine whether a particular gene is being transcribed in different cell types?

There are several somewhat indirect methods for determining the transcriptional activity of a given gene in different cell types. First, if protein products of a given gene are present in different cell types, it can be assumed that the responsible gene is being transcribed. Second, if one is able to actually observe, microscopically, gene activity, as is the case in some specialized chromosomes (polytene chromosomes), gene activity can be inferred by the presence of localized chromosomal puffs. Third, a more direct and common practice to assess transcription of particular genes is to use labeled probes. If a labeled probe can be obtained that contains base sequences that are complementary to the transcribed RNA, then such probes will hybridize to that RNA if present in different tissues. This technique is called in situ hybridization and is a powerful tool.

Explain why many oncogenic viruses contain genes whose products interact with tumor-suppressor proteins.

To encourage infected cells to undergo growth and division, viruses often encode genes that stimulate growth and divi- sion. Many viruses either inactivate tumor-suppressor genes of the host or bring in genes that stimulate cell growth and division. By inactivating tumor-suppressor genes, the normal braking mechanism of the cell cycle is destroyed

What is the difference between saying that cancer is inherited and saying that the predisposition to cancer is inherited?

To say that a particular trait is inherited conveys the assumption that when a particular genetic circumstance is present, it will be revealed in the phenotype. When one discusses an inherited predisposition, one usually refers to situations where a particular phenotype is expressed in families in some consistent pattern. However, the phenotype may not always be expressed or may manifest itself in different ways.

How are mRNAs stored within the cell in a translationally inactive state, and how can their translation be stimulated?

Translation is often inactivated by preventing the interac- tions between the 5′ and 3′ ends necessary for initiation. For messages that contain the CPE (cytoplasmic polyade- nylation element) cis-regulatory sequence, the cytoplasmic polyadenylation element binding protein (CPEB) recruits poly-A ribonuclease (PARN) to the mRNA tail. PARN drasti- cally reduces the length of the poly-A tail, resulting in the binding of an insufficient number of poly-A binding pro- teins (PABPs) to support a stable interaction with initiation factors. Second, CPEB recruits the protein Maskin, which prevents interactions between initiation factors at the 5′ end of the message. Messages inactivated in this way are stored in complexes known as ribonucleoprotein particles. Upon receiving a signal to resume translation, CPEB is phosphorylated, resulting in a conformational change that releases PARN. A cytoplasmic poly-A polymerase lengthens the message tail, which is bound by multiple PABPs. These are able to displace Maskin and allow the formation of an initiation complex.

Compare DNA transposons and retrotransposons. What properties do they share?

Transposons: move using a "Cut-and-Paste" mechanism. Creates short direct repeats in the target site.Retrotransposons: Not removed from the original integration site.Also use "Copy-and-Paste" mechanism.Both: Autonomous, non-autonomous.

Define tumor-suppressor genes. Why is a mutated single copy of a tumor-suppressor gene expected to behave as a recessive gene?

Tumor suppressor genes make proteins that regulate the growth of cells, and they play an important role in preventing the development of cancer cells. Tumor suppressor genes are also known as antioncogenes or loss-of-function genes. Both copies of a specific tumor suppressor gene pair need to be mutated to cause a change in cell growth and tumor formation to happen. For this reason, tumor suppressor genes are said to be recessive at the cellular level.

What role do ubiquitin ligases play in the regulation of gene expression?

Ubiquitin ligases function to mark proteins for degradation. They do this by processive addition of the protein ubiqui- tin to lysine residues in target proteins as well as to lysine residues in the ubiquitin itself. Proteosomes recognize poly- ubiquinated pro

Contrast positive versus negative control of gene expression.

Under negative control, the regulatory molecule interferes with transcription, while in positive control, the regulatory molecule stimulates transcription.

A number of experiments have demonstrated that areas of the genome that are transcriptionally inactive are also resistant to DNase I digestion. However, transcriptionally active areas are DNase I sensitive. Describe how DNase I resistance or sensitivity might indicate transcriptional activity.

When DNA is transcriptionally active, it is in a less condensed state and as such, more susceptible to DNase digestion.

Explain differences between whole-genome sequencing (WGS) and whole-exome sequencing (WES), and describe advantages and disadvantages of each approach for identifying disease- causing mutations in a genome. Which approach was used for the Human Genome Project?

Whole genome sequencing (the strategy used for the HGP) provides sequencing results for entire genomes, includ- ing noncoding regions, whereas whole-exome sequencing provides sequence information only for exons. Since there are more disease-related variations in the exome than in other regions of the genome, WES is more likely to iden- tify these mutations than is WGS. However, only WGS is able to identify mutations in regulatory regions that lead to disease.

Compare and contrast WGS to a map-based cloning approach.

Whole-genome shotgun sequencing involves randomly cutting the genome into numerous smaller segments. Overlapping sequences are used to identify segments that were once contiguous, eventually producing the entire sequence. Difficulties in alignment often occur in repetitive regions of the genome. Map-based sequencing relies on creation of restriction maps of a chromosome, cloning into BACs or YACs, and further subcloning into smaller vectors prior to sequencing. After sequencing, similarly to the WGS method, alignment overlaps are identified and used to assemble a chromosome. Compared to whole-genome sequencing, the map-based approach is somewhat cumbersome and time consuming. Whole-genome sequencing has become the most common method for assembling genomes, with map-based cloning being used to resolve the problems often encountered during whole-genome sequencing.

Why are X rays more potent mutagens than UV radiation?

X rays are of higher energy and shorter wavelength than UV light. They have greater penetrating ability and can create more disruption of DNA.

Presented here are hypothetical findings from studies of hetero- karyons formed from seven human xeroderma pigmentosum cell strains: Note: + = complementation; - = no complementation These data are measurements of the occurrence or nonoccurrence of unscheduled DNA synthesis in the fused heterokaryon. None of the strains alone shows any unscheduled DNA synthesis. Which strains fall into the same complementation groups? How many different groups are revealed based on these data? What can we conclude about the genetic basis of XP from these data?

XP1 XP2 XP3 XP4 XP5 XP6 XP7 These groupings (complementation groups) indicate that at least three "genes" form products necessary for unscheduled DNA synthesis. All of the cell lines that are in the same complementation group are defective in the same product.

What genetic defects result in the disorder xeroderma pigmentosum (XP) in humans? How do these defects create the phenotypes associated with the disorder?

Xeroderma pigmentosum is a rare recessive disorder in which affected individuals are highly sensitive to UV radiation and have a 2000-fold higher incidence of cancer than unaffected individuals. Cells from XP patients are unable to undergo unscheduled DNA synthesis, which is a step in the nucleotide excision repair system. Studies with het- erokaryons provided evidence for at least seven different genes involved in the NER pathway. Since cancer is caused by mutations in several types of genes, interfering with DNA repair can enhance the occurrence of these types of mutations.

Skin cancer carries a lifetime risk nearly equal to that of all other cancers combined. Following is a graph [modified from K. H. Kraemer (1997). Proc. Natl. Acad. Sci. (USA) 94:11-14] depicting the age of onset of skin cancers in patients with or without XP, where the cumulative percentage of skin cancer is plotted against age. The non-XP curve is based on 29,757 cancers surveyed by the National Cancer Institute, and the curve representing those with XP is based on 63 skin cancers from the Xeroderma Pigmentosum Registry. (a) Provide an overview of the information contained in the graph. (b) Explain why individuals with XP show such an early age of onset.

a) . For non-XP individuals, the chance of skin cancer increases as they age. By age 20, approximately 80% of the XP population has skin cancer. . Individuals with XP are more likely to develop skin cancer in their youth than non-XP individuals. XP individuals almost always develop skin cancer by age 40. b) XP individuals lack one or more genes involved in DNA repair.

Dominguez et al. (2004) suggest that by studying genes that determine growth and tissue specification in the eye of Drosophila, much can be learned about human eye development. (a) What evidence suggests that genetic eye determinants in Drosophila are also found in humans? Include a discussion of orthologous genes in your answer. (b) What evidence indicates that the eyeless gene is part of a developmental network?

a) A number of studies have shown that similar genes influence eye development in both insects and vertebrates.Such genes, descended from common ancestral genes that have the same function in different species,are called orthologs. Orthologs are genes in different species that evolved from a common ancestral gene by speciation, and, in general, orthologs retain the same function during the course of evolution. b) In developmental pathways that normally specify the formation of other organs such as legs, wings, and antennae, abnormal expression of the eyeless gene results in eye formation on legs and other body parts.

What evidence indicates that mutations in human DNA mismatch repair genes are related to certain forms of cancer?

any condition that causes mutations in genes or DNA repair systems increases rate of cancer. The link between mutant mismatch repair system and cancer is seen in mice engineered to have defect in mismatch genes. Such organisms are cancer prone

Why are frameshift mutations likely to be more detrimental than point mutations, in which a single pyrimidine or purine has been substituted?

because they are like to change more than one aminoacid

In maize, a Ds or Ac transposon can alter the function of genes at or near the site of transposon insertion. It is possible for these ele- ments to transpose away from their original insertion site, caus- ing a reversion of the mutant phenotype. In some cases, however, even more severe phenotypes appear, due to events at or near the mutant allele. What might be happening to the transposon or the nearby gene to create more severe mutations?

deletions in a nearby gene, chromosome breakage, translocation of the gene into a heterochromatic region (the gene gets silenced).

A particular type of anemia in humans, called b-thalassemia, results from a severe reduction or absence of the normal y-globin chain of hemoglobin. However, the y-globin chain, normally only expressed during fetal development, can functionally substitute for y-globin. A variety of studies have explored the use of the nucleoside 5-azacytidine for the expression of y-globin in adult patients with b-thalassemia. (a) How might 5-azacytidine lead to expression of y-globin in adult patients? (b) Explain why this drug may also have some adverse side effects.

g globin is expressed during fetal development, but becomes silenced, which can be accomplished by methylation of cyti- dine in a CpG island in or near the promoter. 5-azacytidine is an analog of cytidine that cannot be methylated. When incorporated into DNA, it stimulates the expression of genes.(b) 5-azacytidine is not gene specific, so it is likely to have widespread influence on the genome, which could con- stitute a considerable health hazard.

How and why are eukaryotic mRNAs transported and localized to discrete regions of the cell?

mRNA localization is dependent on the binding with RBPs.- mRNA transport is dependent on motor proteins, which "walk" along microtubules of the cytoskeleton.- Because of mRNA transportation each component of a cell is unique.

Keeping in mind the life cycle of bacteriophages discussed earlier in the text (see Chapter 6), consider the following problem: During the reproductive cycle of a temperate bacteriophage, the viral DNA inserts into the bacterial chromosome where the resultant prophage behaves much like a Trojan horse. It can remain quiescent, or it can become lytic and initiate a burst of progeny viruses. Several operons maintain the prophage state by interacting with a repressor that keeps the lytic cycle in check. Insults (ultraviolet light, for example) to the bacterial cell lead to a partial breakdown of the repressor, which in turn causes the production of enzymes involved in the lytic cycle. As stated in this simple form, would you consider this system of regulation to be operating under positive or negative control?

negative

Where are the major regulatory points in the cell cycle?

one near the end of G1, a second at the G2/M transition, and the third during metaphase.

Distinguish between the cis-acting regulatory elements referred to as promoters and enhancers.

promoters -conserved DNA sequences that influence transcription from the "upstream" side (5') of mRNA coding genes.-usually fixed into position and within 100 base pairs of the initiation site for mRNA-i.e.: TATA, CAAT, and GC boxes enhancers -cis-acting sequences of DNA that stimulate the transcription from most promoters.-position need not be fixed-may be significant upstream, downstream, or within that gene being regulated-may be inverted without significantly influencing its action-can work on different genes, meaning they are not gene specific

Describe the human genome in terms of genome size, the percentage of the genome that codes for proteins, how much is composed of repetitive sequences, and how many genes it contains. Describe two other features of the human genome.

the nuclear genome comprises approximately 3 200 000 000 nucleotides of DNA, divided into 24 linear molecules. about 1% codes for proteins.about 25% make up genes and their regulatory elements.

Describe how the Ames test screens for potential environmental mutagens. Why is it thought that a compound that tests positively in the Ames test may also be carcinogenic?

• In the Ames assay, the compound to be tested is incubated with a mammalian Liver extract to simulate an in vivo environment.• This solution then placed on culture plates with an indicator microorganism Salmonelle typhimurium which is defective in it's normal repair processes.• The frequency of mutation in the tester strains is an indication of the mutagenicity of the compound. More than 80% of known carcinogen in 1970s were shown to be strong mutagen.