In Drosophila subobscura, the presence of a recessive gene called grandchildless $(\mathrm{gs})$ causes the offspring of homozygous females, but not those of homozygous males, to be sterile. Can you offer an explanation as to why females and not males are affected by the mutant gene?

By searching a human genomic database, you have found a gene that encodes a protein with weak homology to Argonaute, a factor present in complexes that bind to certain miRNAs and mediate the ability of these miRNAs to regulate the stability or translation of target mRNAs (review Figs. 16.25 and 16.26 on p. 563). a. How would you determine which specific miRNAs night be associated with the new protein you discovered? (Think about how you might use a variation of the ChIP-Seq technique described in Fig, 16.17 on p. 556 to explore this question.) b. If a mouse could be obtained that was homozygous for a null mutation of the mouse gene almost identical to the human gene you found, how could you use this mutant mouse to ask what mRNAs might be targeted by the miRNA-RISC complexes containing your Argonaute-like protein?

You are studying a transgenic mouse strain that expresses a GFP reporter gene under the control of cisacting regulatory elements that normally control a gene needed for the early development of mice. Previous evidence from transcriptome sequencing (RNA-seq) indicates that mRNA for the gene of interest can be identified between days 8.5 and 10.5 of gestation. In your strain, GFP fluorescence can be seen from about day 8.75 until at least day 12. a. Explain the discrepancy between mRNA and protein expression. b. Would you expect GFP protein expression to indicate more accurately the normal onset of activity for this gene or the normal cessation of this gene's activity? Explain.

The hunchback gene, a gene necessary for proper patterning of the Drosophila embryo, is translationally regulated. The position of the coding region within the transcipt is known. How could you determine if if the sequence within the 5' UTR or 3' UTR, or both, are necessary for proper regulation of translation?

Antibodies are currently available that will bind specifxcally to DNA fragments containing 5-methylcytosime but not to DNA lacking this modified nucleotide. How could you use these antibodies in conjunction with the ChlP-Seq technique outlined in Fig. 16.17 on p. 556 to look for inprinted genes in the human genome?

In a population of organisms. the frequency of the homozygous recessive genotype is $0.3$, the frequency of the heterozygous genotype is $0.6$, and the frequency of the homozygous dominant genotype is 0.1. Fill in the correct numbers for each variable below:
$p^2=$
$2 p q=$\

$$q^2=$$

Interestingly, imprinting can be tissue-specific For example, a gene that is maternally imprinted infetal placental tissue is not imprinted at all in the fetal heart Guided by the diagram in Fig. 16.22 a on P. 560 , suggesta mechanism that could explain the tissue specificity of in printing (Hint Remember that a genenay liave nultiple enhancers that allow expression in different tissues )

Follow the expression of a paternally imprinted gene through three generations. Indicate whether the copy of the gene from the male in generation I in the accompanying diagram is expressed in the germ cells and somatic cells of the individuals listed.

a. generation I male (I-2): germ cells

b. generation II daughter (II-2): somatic cells

c. generation II daughter (II-2): germ cells

d. generation II son (II-3): somatic cells

e. generation II son (II-3): germ cells

f. generation III grandson (III-1): somatic cells

g. generation III grandson (III-1): germ cells

A graduate student came up with the following idea for identifying insulators in the Drosophila genome: Perform an experiment like the one described in Problem 11, but instead of using an "enhancerless" construct, use one that contains an enhancer, and screen for lines that do not express GFP. a. What is wrong with this experimental design? b. Can you think of a different experiment that you could perform to identify insulators?

The modular nature of eukaryotic activator proteins gave scientists an idea for a way to find proteins that interact with any particular protein of interest. The idea is to use the protein-protein interaction to bring together a DNA-binding region with an activation region creating an artificial activator that consists of two polypeptides held together noncovalently by the interaction. The method is called the yeast two-hybrid system, and it has three components. First, the yeast contains a construct in which

$$UAS_G$$

(an enhancer-like sequence that binds the activator Gal4) drives the expression of an E. coli lacZ reporter (encoding the enzyme

$$\beta \text { -galactosidase }$$

) from a yeast promoter. Second, the yeast also expresses a fusion protein in which the DNA-binding domain of Gal4 is fused to the protein of interest; this fusion protein is called the "bait." The third component is a cDNA library made in plasmids, where each cDNA is fused in frame to the activation domain of Gal4, and these can be expressed in yeast cells as "prey" fusion proteins. How could you use a yeast strain containing the first two components, along with the plasmid cDNA expression library described, to identify prey proteins that bind to the bait protein? How is this procedure relevant to the goal of finding proteins that might interact with each other in the cell?

MyoD is a transcriptional activator that turns on the expression of several muscle-specific genes in human cells. The Id gene product inhibits MyoD action. How could you determine if Id acts by quenching MyoD or by blocking access to the enhancer? What differences would you expect to see experimentally?

Yeast genes have cis-acting elements upstream of their promoters, similar to enhancers, called upstream activating sequences or UASs. Several target genes involved in galactose utilization are regulated by one type of UAS called

$$UAS _ { G }$$

, which has four binding sites for an activator called GAL4. Two target genes regulated by

$$UAS _ { G }$$

are GAL7 and GAL10. The GAL80 protein is an indirect repressor of GAL7 and GAL10 transcription; at

$$UAS _ { G }$$

, GAL80 binds to GAL4 protein and blocks GAL4's activation domain. In the presence of galactose, GAL80 no longer binds GAL4. In which gene(s) (GAL4 and/or GAL80) should you be able to isolate mutations that allow the constitutive expression of the target genes GAL7 and GAL10 even in the absence of galactose. In each case, what characteristics of the protein would the mutation disrupt?

a. You want to create a genetic construct that will express GFP in Drosophila. In addition to the GFP coding sequence, what DNA element(s) must you include in order to express this protein in flies if the construct were integrated into the Drosophila genome? Where should such DNA element(s) be located? How would you ensure that GFP is expressed only in certain tissues of the fly, such as the wing? b. In making your construct, you insert the GFP coding region plus all of the DNA elements required by the answer to part (a) excepting the enhancer in between inverted repeats found at the end of a particular transposable element. Because all th DNA sequences located between these inverted repeats can move from place to place in the Drosophila genome, you can generate many different fly strains, each with the construct integrated at a different genomic location. You now examine animals from each strain for GFP fluorescence. Animals from different strains show different patterns: some glow green in the eyes. others in legs, some show no green fluorescence, etc. Explain these results and describe a potential use of your construct.

A male mouse from a true-breeding strain of hyperactive animals is crossed with a female mouse from a true-breeding strain of lethargic animals. (These are both hypothetical strains.) All the progeny are lethargic. In the $F_2$ generation, all offspring are lethargic. What is the best genetic explanation for these observations? Propose a cross to test your explanation.