Study Guide 13 - Winter 2015

Figure 1 shows the results of a site directed mutagenesis experiment analyzing the upstream region of the mouse Beta-globin gene. Specific point mutations were introduced into upstream region and then the ability of the mutated upstream region to promote transcription was measured, with the transcriptional activity of the wild-type sequence designated as 1.0.

What conclusion about the ATATAA sequence marked by the open box is warranted by the data?

Figure 1 shows the results of a site directed mutagenesis experiment analyzing the upstream region of the mouse Beta-globin gene. Specific point mutations were introduced into upstream region and then the ability of the mutated upstream region to promote transcription was measured, with the transcriptional activity of the wild-type sequence designated as 1.0.

The molecule being mutated in this experiment is:

Figure 1 shows the results of a site directed mutagenesis experiment analyzing the upstream region of the mouse Beta-globin gene. Specific point mutations were introduced into upstream region and then the ability of the mutated upstream region to promote transcription was measured, with the transcriptional activity of the wild-type sequence designated as 1.0.

The right angle arrow above the location labeled as the “Cap site” indicates

Figure 1 shows the results of a site directed mutagenesis experiment analyzing the upstream region of the mouse Beta-globin gene. Specific point mutations were introduced into upstream region and then the ability of the mutated upstream region to promote transcription was measured, with the transcriptional activity of the wild-type sequence designated as 1.0.

Transcription begins ______________ the GGCCAATC sequence.

cDNA for twelve different liver specific genes was spotted onto a membrane as shown in the bottom left corner. cDNA for the actin, the alpha subunit of tubulin (used to make microtubules), and the beta subunit of tubulin genes were spotted as shown in Figure 2. The DNA from the plasmid pB (used as a vector to carry the cDNA) was spotted on the membrane as shown. DNA for the methionine tRNA gene was spotted on the membrane as shown. The DNA was denatured (single stranded) and remained tightly bound to the membrane. Three replicate spotted membranes were produced. A rat was fed 32P-labeled UTP for several days. The rat was sacrificed and the total RNA from the rat liver tissue, the rat kidney tissue, and the rat brain tissue were separately isolated. The radioactive RNA from each
tissue type was used as a probe to hybridize to the DNA targets on the membrane. The autoradiograms shown in Figure 2 are for the liver, kidney, and brain probe conditions.

This experiment is most like which of the following?

cDNA for twelve different liver specific genes was spotted onto a membrane as shown in the bottom left corner. cDNA for the actin, the alpha subunit of tubulin (used to make microtubules), and the beta subunit of tubulin genes were spotted as shown in Figure 2. The DNA from the plasmid pB (used as a vector to carry the cDNA) was spotted on the membrane as shown. DNA for the methionine tRNA gene was spotted on the membrane as shown. The DNA was denatured (single stranded) and remained tightly bound to the membrane. Three replicate spotted membranes were produced.
A rat was fed 32P-labeled UTP for several days. The rat was sacrificed and the total RNA from the rat liver tissue, the rat kidney tissue, and the rat brain tissue were separately isolated. The radioactive RNA from each tissue type was used as a probe to hybridize to the DNA targets on the membrane. The autoradiograms shown in Figure 2 are for the liver, kidney, and brain probe conditions.

Why was there no hybridization to pB?

cDNA for twelve different liver specific genes was spotted onto a membrane as shown in the bottom left corner. cDNA for the actin, the alpha subunit of tubulin (used to make microtubules), and the beta subunit of tubulin genes were spotted as shown in Figure 2. The DNA from the plasmid pB (used as a vector to carry the cDNA) was spotted on the membrane as shown. DNA for the methionine tRNA gene was spotted on the membrane as shown. The DNA was denatured (single stranded) and remained tightly bound to the membrane. Three replicate spotted membranes were produced.
A rat was fed 32P-labeled UTP for several days. The rat was sacrificed and the total RNA from the rat liver tissue, the rat kidney tissue, and the rat brain tissue were separately isolated. The radioactive RNA from each tissue type was used as a probe to hybridize to the DNA targets on the membrane. The autoradiograms shown in Figure 2 are for the liver, kidney, and brain probe conditions.

Why was the methionine tRNA gene included in the experiment?

cDNA for twelve different liver specific genes was spotted onto a membrane as shown in the bottom left corner. cDNA for the actin, the alpha subunit of tubulin (used to make microtubules), and the beta subunit of tubulin genes were spotted as shown in Figure 2. The DNA from the plasmid pB (used as a vector to carry the cDNA) was spotted on the membrane as shown. DNA for the methionine tRNA gene was spotted on the membrane as shown. The DNA was denatured (single stranded) and remained tightly bound to the membrane. Three replicate spotted membranes were produced.
A rat was fed 32P-labeled UTP for several days. The rat was sacrificed and the total RNA from the rat liver tissue, the rat kidney tissue, and the rat brain tissue were separately isolated. The radioactive RNA from each tissue type was used as a probe to hybridize to the DNA targets on the membrane. The autoradiograms shown in Figure 2 are for the liver, kidney, and brain probe conditions.

Which one of the following conclusions is best supported by this data?

In 1969, Bob Roeder published the results of experiments that were designed to resolve yeast RNA polymerase enzymes using chromatography. The results of this work are shown in Figure 4. Fractions 1-36 are presented from left to right on the X-axis. An aliquot of each fractions was assayed for RNA polymerase activity using radioactive UMP (*UMP).

Which of the following fractions contained the most total protein?

In 1969, Bob Roeder published the results of experiments that were designed to resolve yeast RNA polymerase enzymes using chromatography. The results of this work are shown in Figure 4. Fractions 1-36 are presented from left to right on the X-axis. An aliquot of each fractions was assayed for RNA polymerase activity using radioactive UMP (*UMP).

If this had been performed using a negatively charged ion exchange chromatography column, these results would suggest that the RNA polymerases are…

In 1969, Bob Roeder published the results of experiments that were designed to resolve yeast RNA polymerase enzymes using chromatography. The results of this work are shown in Figure 4. Fractions 1-36 are presented from left to right on the X-axis. An aliquot of each fractions was assayed for RNA polymerase activity using radioactive UMP (*UMP).

The solid line labeled CPM (*UMP) reflects…

In 1969, Bob Roeder published the results of experiments that were designed to resolve yeast RNA polymerase enzymes using chromatography. The results of this work are shown in Figure 4. Fractions 1-36 are presented from left to right on the X-axis. An aliquot of each fractions was assayed for RNA polymerase activity using radioactive UMP (*UMP).

The results represented above demonstrated that…

In order to examine the role that certain gene sequences in the H2A gene play in transcription, you make mutant versions of the H2A gene containing deletions in those regions. You then inject the DNA containing those deletion mutations into frog eggs and examine the RNA produced using electrophoresis. The location of deletions is shown in Figure 5. The agarose gel electrophoresis results are shown in Figure 6. H2B mRNA and three other large mRNA’s were added as a control in each lane.

Which conclusion is validated by the delta-A sample in lane 2?

In order to examine the role that certain gene sequences in the H2A gene play in transcription, you make mutant versions of the H2A gene containing deletions in those regions. You then inject the DNA containing those deletion mutations into frog eggs and examine the RNA produced using electrophoresis. The location of deletions is shown in Figure 5. The agarose gel electrophoresis results are shown in Figure 6. H2B mRNA and three other large mRNA’s were added as a control in each lane.

Which conclusion is best validated by the B sample in lane 3?

The concept that eukaryotic regulatory proteins have multiple functional domains was arrived at in stages. However, a few experiments stand out, such as the study by Roger Brent and Mark Ptashne published in 1985. When the study began, one known mechanism for activation of transcription by an activator protein was simply direct interaction with RNA polymerase. The investigators also considered an alternative mechanism: that the transcription activator functioned by altering the structure of the DNA to which it bound, facilitating the binding of RNA polymerase.

This study focused on two different regulatory proteins. The first was a well-characterized bacterial repressor called LexA. The LexA repressor controls a regulon in E. coli, the SOS response, that is activated when cellular DNA is subjected to extensive damage. The sequence of its binding site on DNA was known, and the protein had been studied by the Ptashne group and others. The second regulatory protein was a eukaryotic gene activator protein from yeast, Gal4p, which activates transcription of the GAL1 gene when yeast cells are grown on galactose. Ptashne and his coworkers knew that the DNA-binding element of Gal4p was located in the N-terminal 74 amino acids of the protein. They deleted these amino acids and replaced them with the first 87 amino acids of the LexA protein, which they knew contained the DNA-binding elements of that protein. They then expressed this fusion protein, LexA-Gal4, in both yeast and E. coli. They separately expressed the native LexA protein by itself. To monitor the effects of the fusion protein in yeast, the researchers needed to construct several variants of a second plasmid containing the ß-galactosidase gene (the lacZ gene, encoding an enzyme activity that is easy to measure) fused to an unrelated yeast gene, CYC1. The different constructs contained a variety of regulatory sequences upstream from the fusion genes.

Next, they carried out a series of measurements of ß-galactosidase activity with the two-plasmid system in yeast cells, with the results shown in Table 1 (adapted from their published table). In the table, ß-galactosidase activity is given in units of blue color produced in conversion of substrate to product. UAS is upstream activator sequence; UASC1 and UASC2 are binding sites for activator proteins that function at the CYC1 gene; and UASG is the normal binding site for Gal4p. UASG consists of four separate binding sites for Gal4p, each 17 bp long. The 17mer is a site with just one of these sequences. The abbreviation “op” means operator, which is the LexA-binding site; -178 and -577 indicate the distance in base pairs between the operator and the transcription start site. In the yeast cells used for the study, the genes encoding the endogenous Gal4p and the CYC1 gene activators were all present and functional.

The transcription activated by the LexA-Gal4 fusion protein acting at the LexA operator

The concept that eukaryotic regulatory proteins have multiple functional domains was arrived at in stages. However, a few experiments stand out, such as the study by Roger Brent and Mark Ptashne published in 1985. When the study began, one known mechanism for activation of transcription by an activator protein was simply direct interaction with RNA polymerase. The investigators also considered an alternative mechanism: that the transcription activator functioned by altering the structure of the DNA to which it bound, facilitating the binding of RNA polymerase.

This study focused on two different regulatory proteins. The first was a well-characterized bacterial repressor called LexA. The LexA repressor controls a regulon in E. coli, the SOS response, that is activated when cellular DNA is subjected to extensive damage. The sequence of its binding site on DNA was known, and the protein had been studied by the Ptashne group and others. The second regulatory protein was a eukaryotic gene activator protein from yeast, Gal4p, which activates transcription of the GAL1 gene when yeast cells are grown on galactose. Ptashne and his coworkers knew that the DNA-binding element of Gal4p was located in the N-terminal 74 amino acids of the protein. They deleted these amino acids and replaced them with the first 87 amino acids of the LexA protein, which they knew contained the DNA-binding elements of that protein. They then expressed this fusion protein, LexA-Gal4, in both yeast and E. coli. They separately expressed the native LexA protein by itself. To monitor the effects of the fusion protein in yeast, the researchers needed to construct several variants of a second plasmid containing the ß-galactosidase gene (the lacZ gene, encoding an enzyme activity that is easy to measure) fused to an unrelated yeast gene, CYC1. The different constructs contained a variety of regulatory sequences upstream from the fusion genes.

Next, they carried out a series of measurements of ß-galactosidase activity with the two-plasmid system in yeast cells, with the results shown in Table 1 (adapted from their published table). In the table, ß-galactosidase activity is given in units of blue color produced in conversion of substrate to product. UAS is upstream activator sequence; UASC1 and UASC2 are binding sites for activator proteins that function at the CYC1 gene; and UASG is the normal binding site for Gal4p. UASG consists of four separate binding sites for Gal4p, each 17 bp long. The 17mer is a site with just one of these sequences. The abbreviation “op” means operator, which is the LexA-binding site; -178 and -577 indicate the distance in base pairs between the operator and the transcription start site. In the yeast cells used for the study, the genes encoding the endogenous Gal4p and the CYC1 gene activators were all present and functional.

Positioning of the LexA operator 577 bp rather than 178 bp away from the transcription start site lowers transcription activation by about