BI 311 Genetics Achieve/Homework/Quizzes/Weeks 9-10

How does the Lac repressor protein inhibit transcription of the structural genes within the lac operon?

By binding to the operator, and physically obstructing RNA polymerase from binding to the promotor

How does comparative genomics aid in the search for factors that contribute to complex human conditions?

Conserved genetic sequences likely influence similar phenotypic traits across species.

Plasmids are small circular DNA molecules found in bacteria that replicate separately from chromosomes. Why are plasmids essential for recombinant DNA technology?

DNA from a gene of interest can be inserted into a plasmid, then the modified plasmid can be inserted into a bacterial cell to replicate a gene of interest many times.

What is the purpose of primers in a PCR reaction?

DNA polymerase requires an existing 3' hydroxyl group to which it can add free nucleotides.

Different types of environmental studies are listed. Select the metagenomic studies.

DNA sequencing is used to determine gut microbiota change after altering a primate's diet./ Genetic analyses of deep sea sediment samples characterize regional microbial composition.

DNA Sequencing: Assume that you are routinely sequencing a gene of a bacterial species obtained from different sources to detect the genotype diversity. Which of the following is the best sequencing method for your application?

Dideoxy sequencing

True or False. In multicellular eukaryotes, there is a correlation between the genome size and the number of genes.

False

True or False. Metagenomic studies depends on laboratory cultures of organisms that undergo genome sequencing and analysis.

False

True or False? Metagenomic studies depends on laboratory cultures of organisms that undergo genome sequencing and analysis.

False

True or False? Two different DNA molecules cut by HindIII enzyme can NOT spontaneously pair to connect the fragments.

False

Describe the construction of a recombinant plasmid containing the gene for luciferase and the insertion of the plasmid into a bacterial cell by placing the steps in order.

First step *Use PCR to amplify the gene for luciferase. *Perform a restriction digestion of the luciferase gene and the plasmid. *Ligate together the luciferase gene and the plasmid to generate a recombinant plasmid. *Transform bacteria with the recombinant plasmid using heat shock. *Plate the bacterial cells, and screen for positive transformants Last step

Why is it important to use restriction enzymes with "sticky ends" for cloning?

For easy insertion of foreign DNA segments into plasmids

You are performing Sanger sequencing on the DNA template 3' ATCCCAGAC 5′. In the reaction tube that contains ddGTP, how many fragments will be produced?

Four

Which is not found in a PCR reaction?

Helicase

Lactose metabolic activity in the lac operon is represented in the image. Determine the nature of the expression of the lacZYA genes, represented by L, and label each genotype as constitutive, inducible, or no transcription in the presence of lactose. Assume that all promoters are wild type. Symbol Representation I− lacI mutant cannot bind to the operator IS lacI mutant always binds to the operator OC Operator mutant that prevents repressor binding F' the wild‑type operator, O+, and lacI gene on a plasmid L lacZYA genes

I+ O+ L+ Inducible I− O+ L+ constitutive I+ OC L+, F' Constitutive I− O+ L+, F' Inducible IS O+ L+ no transcription IS O+ L+, F' no transcription

What is true about lac operon?

It includes a promotor, an operator, and 3 structural genes

Which statement is true of DNA cloning?

It is used to isolate specific sequences of DNA, and it requires the enzyme DNA ligase.

What is the purpose of the hot (usually about 95 °C) portion of the PCR temperature cycles?

It separates or denatures the template strands of DNA.

Removal of acetyl groups from the histones that make up nucleosomes in chromatin will cause what?

It will cause a decrease in gene expression

What is true about the lac operon with respect to the regulation of gene expression?

Lactose, through conversion into allolactose, will induce transcription of lac operon genes

Suppose a researcher studied gut tissue samples from patients with Crohn's disease, an inflammatory gut disorder, and normal controls. He found that the identity and relative abundance of gut bacterial species varied within both the Crohn's and the control populations. He also discovered general differences between the two populations. The guts of Crohn's patients had reduced levels and diversity of bacteria from the phylum Firmicutes compared to those of controls. The guts of Crohn's patients also had higher levels of bacteria from the phylum Proteobacteria than those of controls. Which type of study did the researcher perform?

Metagenomic

How does methylation of particular DNA sequences relate to the conversion of euchromatin into facultative heterochromatin?

Methyl groups that are added to the pyrimidine base cytosine block the binding of transcription factors

For the E. coli lactose operon genotypes listed below: + indicates a functional component, − indicates a nonfunctional component, superscript C indicates a constitutively active component, and superscript S indicates a component that is unresponsive to an inducer. The last three genotypes show a partially diploid cell that has an F' factor. No glucose is present. Strain: lacI+ lacP+ lacO+ lacY+ Condition: Lactose absent. Willβ‑gal be synthesized?

NO

For the E. coli lactose operon genotypes listed below: + indicates a functional component, − indicates a nonfunctional component, superscript C indicates a constitutively active component, and superscript S indicates a component that is unresponsive to an inducer. The last three genotypes show a partially diploid cell that has an F' factor. Strain: lacI+ lacP+ lacOC lacY- / F' lacI- lacP+ lacO+ lacY+ Condition: Lactose absent. Will β‑gal be synthesized?

NO

For the E. coli lactose operon genotypes listed below: + indicates a functional component, − indicates a nonfunctional component, superscript C indicates a constitutively active component, and superscript S indicates a component that is unresponsive to an inducer. The last three genotypes show a partially diploid cell that has an F' factor. Strain: lacI+ lacP- lacO+ lacY+ Condition: Lactose present. Will β‑gal be synthesized?

NO

For the E. coli lactose operon genotypes listed below: + indicates a functional component, − indicates a nonfunctional component, superscript C indicates a constitutively active component, and superscript S indicates a component that is unresponsive to an inducer. The last three genotypes show a partially diploid cell that has an F' factor. Strain: lacI- lacP+ lacO+ lacY+ / F' lacI+ lacP+ lacO+ lacY+ Condition: Lactose absent. Will β‑gal be synthesized?

NO

For the E. coli lactose operon genotypes listed below: + indicates a functional component, − indicates a nonfunctional component, superscript C indicates a constitutively active component, and superscript S indicates a component that is unresponsive to an inducer. The last three genotypes show a partially diploid cell that has an F' factor. Strain: lacI- lacP+ lacO+ lacY+ / F' lacIS lacP+ lacO+ lacY+ Condition: Lactose present. Will β‑gal be synthesized?

NO

How is a repressible operon different from an inducible operon?

Only a repressible operon involves a repressor protein that is inactive unless bound by a co-repressor

Consider that a typical PCR does not start with a single molecule of template DNA, but rather something in the range of 25 nmol of template DNA. What does this tell you about the potential of PCR to amplify DNA?

PCR is an efficient technique with the potential to produce a large amount of DNA

What is an example of gene regulation at the post-transcriptional level, prior to the initiation of translation?

Regulation of the half-life of RNA

As depicted in the table, certain plant genomes are significantly larger than mammal genomes, including the human genome. Genome Approximate Genome Size Human 3,200,000,000 Mouse 2,600,000,000 Norway Spruce 19,600,000,000 Loblolly pine 20,150,000,000

Repetitive DNA from transposons accounts for the majority of these plant genomes.

Complementary DNA (cDNA) is a double stranded molecule. In the laboratory, how is cDNA generated from a eukaryotic messenger RNA (mRNA)?

Reverse transcriptase generates a single stranded cDNA and then DNA polymerase synthesizes the complementary strand.

The diagram below shows how HindIII enzyme digests DNA molecules, with the dotted lines indicating strand cleavage sites. Which of the following terms best describes the ends of DNA molecules that result after digestion with HindIII?

Sticky-ended

DNA is the ____________ for in vitro synthesis.

Template

What'd be the effect on the PCR reaction if: there's no template DNA in the reaction, no primers in the reaction, no dNPTs in the reaction, or no Taq polymerase in the reaction?

The PCR reaction will not commence

What is true about a growth scenario under which the amino acid tryptophan becomes available?

The Trp repressor protein will bind to the trp operator, thereby greatly reducing transcription of trp structural genes

What is true about growth under the condition where the amino acid tryptophan is absent?

The Trp repressor protein will remain inactive and will not bind to the trp operator

James Noonan and his colleagues (2005. Science 309:597-599) set out to study genome sequences of an extinct species of cave bear. They extracted DNA from 40000 year old bones from a cave bear and used a metagenomic approach to isolate, identify, and sequence the cave bear DNA. Why did they use a metagenomic approach when their objective was to sequence the genome of one species, the cave bear?

The cave bear samples could be heavily contaminated so scientists used metagenomics to sequence all of the DNA. They then compared the sequences obtained with modern bear DNA to identify which DNA fragments were from the cave bear.

Which of the statements defines somatic gene therapy?

The insertion of functional genes in people with disease to cure or decrease the symptoms of the disease.

Early life experiences (diet, stress, toxicants) can affect chromatin structure to alter gene expression in a cell. What is true?

These changes in chromatin structure could be passed on from a parent to their F1 offspring

What is true about the cells in the multicellular organism?

They all have the same set of DNA molecules.

What is true about inducible operons?

They code for enzymes that help to break down substrates not frequency encountered by a cell, e. g. some types of sugars

Before the development of automated sequencing, dideoxy sequencing involved performing four separate sequencing reactions to get the sequence of a piece of DNA. How do these four reaction tubes differ from each other?

They contain different ddNTPs.

How do ddNTPs differ from dNTPs?

They do not have a 3′ hydroxyl group on the sugar.

How does lactose, through conversion into allolactose, ensure that the gene products necessary for its metabolic breakdown are present when needed?

Through binding to the Lac repressor protein; this makes the protein unable to bind to the operator to inhibit transcription

Researchers construct different types of genomic maps to gain information about genes and their locations in the genome. Why would a researcher choose to construct a physical map of genes rather than a genetic map?

To determine the locations of specific genes on a chromosome and the distance between them.

True or False? In multicellular eukaryotes, there is a correlation between the number of genes in the genome and the phenotypic complexity of the organism.

True

True or False? Using SNPs, genome-wide association studies could successfully locate the genes that contribute to complex diseases caused by genetic or environmental factors.

True

True or False. PCR is used for pathogens (such as viruses and bacteria) that already contain DNA while RT-PCR is used for pathogens containing RNA that needs to be transcribed to DNA before replication.

True.

What is true about negative control of gene expression?

Under negative control, a regulatory molecule negatively influences gene expression

What is true about positive control of gene expression?

Under positive control, gene expression does not occur very efficiently unless its positively influenced

You insert your gene of interest into the lac z gene in a plasmid, and plate transformed bacteria on media containing x-gal.______indicate bacteria with recombinant plasmids.

White colonies

For the E. coli lactose operon genotypes listed below: + indicates a functional component, − indicates a nonfunctional component, superscript C indicates a constitutively active component, and superscript S indicates a component that is unresponsive to an inducer. The last three genotypes show a partially diploid cell that has an F' factor. Strain: lacI+ lacP+ lacOC lacY+ Condition: Lactose absent. Will β‑gal be synthesized?

YES

A recombinant plasmid contains

a foreign piece of DNA and an origin of replication.

The vehicle that carries a piece of foreign DNA to be cloned into a bacterial cell is called

a vector.

Suppose a scientist wants to clone the gene for human insulin. He uses a plasmid that contains the lacZ gene, an origin of replication, and an ampicillin resistant gene. He inserts the human insulin gene into a restriction site located within the lacZ gene. Then he transforms this plasmid into bacteria without a functional lacZ gene and spreads the appropriate dilution onto bacterial plates. When the product of the lacZ gene, beta galactosidase, interacts with the subtrate X Gal on LB agar, colonies turn blue. Which plate indicates that the human insulin gene was successfully inserted in the plasmid?

an LB agar plate with ampicillin and X Gal that produces colorless colonies

A plasmid vector generally contains which item or items?

an antibiotic resistance gene, a cloning site, and a replication origin

Restriction enzymes are used during DNA cloning to

cut DNA and produce sticky ends on the vector and insert DNA.

The amount of target DNA

doubles after each cycle of PCR.

What is the role of guide RNA (gRNA) in CRISPR-Cas9 system?

gRNA ensures that the Cas9 enzyme cuts at the right point in the genome

The table below shows 4 plating conditions. Condition Lactose present Glucose present 1 No No 2 Yes Yes 3 No Yes 4 Yes No Under which condition would a lac operon produce the greatest amount of β‑galactosidase? Under which condition would a lac operon produce the least amount of β‑galactosidase?

greatest = Condition 4 least = Condition 3

The purpose of the polymerase chain reaction (PCR) is to

make many copies of a single DNA segment.

With current sequencing technology, contigs are integral to understanding the entire genome of an organism. What is a contig?

overlapping DNA fragments constructed into a continuous DNA sequence

Which is an example of gene therapy?

replacing a disease-causing allele for a gene with a normal allele

Many copies of a recombinant plasmid are produced by _________.

replication of the plasmid DNA, using the host enzymes and cell division of the host cell

Identify each feature in the diagram which depicts the generation of a recombinant plasmid from plasmid and donor DNA.

restriction enzyme=orange balls restriction site= B&W section DNA ligase=yellow balls Ligation=arrow recombinant plasmid=bottom

In DNA sequencing, gel electrophoresis is used to

separate the newly synthesized DNA molecules by size

In the dideoxy method of sequencing DNA, the term "dideoxy" refers to

special nucleotides.

In a dideoxy sequencing reaction, the incorporation of a ddNTP into the growing DNA strand

terminates the polymerization reaction.

The process by which a recombinant plasmid is introduced into a host bacterial cell is

transformation.

Suppose that archaeologists find a tomb in Egypt containing a mummy. The tomb has been looted and any identifying hieroglyphs on the walls have worn away. The mummy does not have any visible distinguishing features, but her tissues still contain intact DNA. What could scientists do to identify who this mummy was?

use genome analysis to compare her DNA with DNA from other identified mummies in the area

Suppose that you are studying an inducible operon that contains two genes required for the metabolism of compound xyz. Gene A encodes enzyme A, and gene B encodes enzyme B. When compound xyz is absent, the operon is repressed, but when compound xyz is present, the operon is induced. You have isolated four distinct mutants, numbered 1, 2, 3, and 4, which have the effects shown in the table, where + indicates that the enzyme is synthesized and − indicates that the enzyme is not synthesized. Identify the component of the operon that is mutated in each mutant xyz absent xyz present Enzyme A Enzyme B Enzyme A Enzyme B Component mutated Wildtype − − + + Mutant 1 − − + - Mutant 2 + + + + Mutant 3 − − - + Mutant 4 − − − −

xyz absent xyz present Enzyme A Enzyme B Enzyme A Enzyme B Component mutated Wildtype − − + + Mutant 1 − − + - Gene B Mutant 2 + + + + Repressor Mutant 3 − − - + Gene A Mutant 4 − − − − Promotor

The Venn diagram given below depicts genomes of three organisms: human, mouse, & Arabidopsis plant. It is used to identify the genes that code for neuron development in humans. The overlapping regions are used to represent the genes that are shared between these organisms. Which region contain the genes that code for neuron development? · Q · P · S · R

· R

A researcher treats a DNA sample and a plasmid with the ampR and lacZ (for the enzyme β galactosidase) genes with the restriction enzyme NotI with the goal of making recombinant DNA molecules. The researcher places the lacZ gene in the cloning region so that it is disrupted by DNA inserts. Place the remaining steps for this process in the correct order. Not all steps will be used.

*Treat DNA with the restriction enzyme. *All the fragments have identical sticky ends: 5′−GGCC−3′5′−GGCC−3′ . *Use DNA ligase to join the sticky ends of the DNA sample and the plasmid. *Grow cells containing the recombinant plasmid in a medium with ampicillin and X gal (a galactose derivative). *White colonies that survive on the culture medium contain the recombinant plasmid. *White colonies that survive on the culture medium contain the recombinant plasmid *Remove colonies containing the recombinant plasmid from the culture medium and continue growing.

Suppose a researcher studied gut tissue samples from patients with Crohn's disease, an inflammatory gut disorder, and normal controls. He found that the identity and relative abundance of gut bacterial species varied within both the Crohn's and the control populations. He also discovered general differences between the two populations. The guts of Crohn's patients had reduced levels and diversity of bacteria from the phylum Firmicutes compared to those of controls. The guts of Crohn's patients also had higher levels of bacteria from the phylum Proteobacteria than those of controls. Which conclusions might you be able to draw from the data?

*Two normal individuals may have different compositions of bacteria in their guts. *Bacteria from the phylum Proteobacteria may contribute to the development of Crohn's disease.

Polymerase chain reaction (PCR) is a technique used to amplify (copy) DNA. Suppose a single, linear molecule of double stranded DNA (dsDNA) is amplified by PCR. After 30 PCR cycles (a typical number of cycles), how many molecules of dsDNA will there be?

1 billion

Polymerase chain reaction (PCR) is a technique used to amplify (copy) DNA. Suppose a single, linear molecule of double stranded DNA (dsDNA) is amplified by PCR. After one PCR cycle, how many molecules of dsDNA will there be?

2

How many different ddNTPs are needed to get a complete sequence of a fragment of DNA?

4

The image represents a small portion of the flow cell for Illumina sequencing. Ten cycles of sequencing are shown. Write out the base sequence of the DNA fragment represented by the cluster of fragments enclosed in the white boxes within each cycle.

5' - AGCCATAGCA - 3'

Suppose a researcher decides to sequence a short fragment of DNA. She uses polymerase chain reaction (PCR) and a series of four dideoxy reactions. She then separates the products of the reactions using gel electrophoresis and obtains the banding pattern. Enter the sequence of the original template strand in the 5′ to 3′ direction.

5' - AGCTGCATC- 3'

You are given the gel electrophoresis results of a Sanger sequencing attempt. What is the sequence of the target (template) DNA?

5' - TCCTGATTTCGGGTAC - 3'

A linear piece of DNA was broken into random, overlapping fragments and each fragment was then sequenced. The sequence of each fragment is shown here, listed in random order. Using the overlapping regions shared between sequences, create a contig sequence of the original fragment by placing the sequence that is closest to the 5' end first and moving towards the 3' end.

5' end 5′−CGGGGTTGATCCC−3′ 5′−GGGGTTGATCC−3′ 5′−GGTTGATCCCATAACGCCA−3′ 5′−TTGATCCCATAACGCCA−3′ 5′−GATCCCATAACGCCA−3′ 5′−TAACGCCATTTA−3' 3' end

The chromatogram shows fluorescent peak data from a dye-terminating nucleotide-sequencing reaction. The peaks are shown with shortest fragment on the left to longer fragments on the right. Select the DNA sequence that matches the data.

5'-AGCGGGCTCAACCT-3'

Electrophoresis gels are used to separate DNA fragments. Match the DNA fragment size, in base pairs (bp), to each band on the electrophoresis gel. A DNA ladder with known fragment sizes is visible in lane 1. The sizes of the most intense bands in the ladder are provided to the left. Electrode charge is indicated to the right of the gel.

7,000 = 4,750 3,500 = 2,250 1,000 = 500

Polymerase chain reaction (PCR) is a technique used to amplify (copy) DNA. Suppose a single, linear molecule of double stranded DNA (dsDNA) is amplified by PCR. After three PCR cycles, how many molecules of dsDNA will there be?

8

The phenomenon of epigenesis relates to what?

A heritable change in gene expression in the absence of a change to the base sequence of DNA

What relates to facultative heterochromatin?

A reversible state that can be remodeled into euchromatin

Which of the following best describes a genome wide association study?

A study that tests the association between a trait and all genomic variants

Shown is the DNA sequencing chromatogram used by researchers to demonstrate that CRISPR‑Cas genome editing had precisely removed exon 23 in mdx mice and eliminated a stop codon responsible for muscular dystrophy in these mice1. Using the key, write out the bases found in the DNA that was sequenced. Start with the beginning of exon 22 and proceed through the end of exon 24.

AAATTACAGAATCACATA

Select all that apply: The main improvement(s) of next generation sequencing methods is/are: · Ability to use fluorescently labelled ddNTPs · All the given answers are correct · Ability to perform many sequencing reactions parallelly Ability to obtain information on the entire genome

Ability to perform many sequencing reactions parallelly Ability to obtain information on the entire genome

What would increase transcription of the trp operon( an example of a repressible operon)?

Absence of the biosynthetic end-product tryptophan

How do noncoding RNA's relate to the epigenetic conversion of euchromatin into facultative heterochromatin?

All of the above

Why is ampicillin added to the agar medium upon which transformed bacterial cells are grown?

Ampicillin selects for cells containing the plasmid.

At which level do prokaryotes primarily regulate gene expression?

At the level of transcription

A 2.6 kilobase linear fragment of DNA was digested with restriction enzyme "A" and produced a 2 kb fragment and a 0.6 kb fragment. When the same 2.6 kb fragment was cut with restriction enzyme "B", it yielded 0.3 kb, 0.5 kb and 1.8 kb fragments. When the 2.6 kb fragment was cut with a mixture of two enzymes, fragments of 0.1, 0.2, 0.5 and 1.8 kb were produced. The order of the restriction sites on this DNA fragment is:

BAB

Scientists have engineered CRISPR-Cas systems to serve as molecular editing tools. Which of the following helped scientists create CRISPR-Cas9?

Bacteria fighting off viruses

A geneticist uses a cloning plasmid that contains the lacZ gene and a gene that confers resistance to penicillin. She inserts a piece of foreign DNA into a restriction site located within the lacZ gene and uses the plasmid to transform bacteria. She then grows the bacteria on selective media containing penicillin and X gal. Explain how the geneticist can identify bacteria that contain a plasmid with the foreign DNA.

Bacteria with the desired plasmid produce white colonies.

Classify each characteristic as being commonly found in bacterial genomes, in eukaryotic genomes, or in both genomes.

Bacterial genomes only generally have one chromosome have very few noncoding regions in the DNA often contain plasmids Eukaryotic genomes only have a diploid chromosome number commonly have several linear chromosomes use histone proteins in the nucleus Characteristic of both have a DNA double‑helix structure must be replicated before cell division takes place contain genes that code for proteins

Samples of a plasmid containing a segment of unknown DNA are digested using the restriction enzymes EcoRI, BamHI, and a combination of EcoRI and BamHI. The digests are then run on an agarose gel in order to separate the resulting fragments by size. Use the results of the gel electrophoresis shown in the image to determine the sizes of the fragments and label the restriction map. Create a map of the DNA segment by dragging the enzyme name to the location it cuts (the vertical lines above the segment) and labeling the fragments with their lengths (numbers, in kilobases). You will not use all the numbers.

BamHI EcoRI BamHI 5' ----------------------------------- 3' 1kb 4kb 2kb 3kb

Why is the lac operon considered to be an inducible operon?

Because this operon is turned on in the presence of lactose