Micro Ch 9 Q&A

differential screening

, to allow visual differentiation of those colonies that carry inserts from those that do not. This screening is done all on one medium

In E. coli, you have isolated four Hfr strains with the following gene order: 1. A B C D E2. Y Z F E D3. Z Y X R T4. B A T R XWhat is the predicted order of these markers on the circular chromosome? A A B C D E F R T X Y Z B A B C D E F Z Y X R T C X Y Z T R E D C B A F D F R E D A C B Z Y X T

A B C D E F Z Y X R T

What is a cloning vector?

A DNA molecule that can be genetically manipulated and replicates in cells

What is an auxotroph?

A mutant with a mutation in a metabolic pathway

Genetic variation can occur within a cell by moving transposable elements from _____. chromosome to plasmid plasmid to chromosome from one location to another within the same chromosome All of these choices

All of these choices

Bacteria naturally exchange DNA, a process referred to as *******, and scientists have exploited these mechanisms of exchange to develop *******

Bacteria naturally exchange DNA, a process referred to as horizontal gene transfer, and scientists have exploited these mechanisms of exchange to develop recombinant DNA techniques.

The mutation that causes sickle cell disease eliminates a restriction enzyme cut site within the hemoglobin gene. If a person is affected by sickle cell disease and has two copies of the disease allele, what would you expect to observe? A Because a restriction site is eliminated, you would expect to see one larger band after digest. B Because a restriction site is eliminated, you would expect to see several smaller bands after digest. C Despite eliminating a restriction site, you would predict no change in the restriction fragment patterns. D Because a restriction site is eliminated, you would expect to see no bands on the agarose gel.

Because a restriction site is eliminated, you would expect to see one larger band after digest.

Why use a cosmid over a phage vector?

Can maintain DNA as a plasmid and incorporate it into phage particles

Suppose you wish to generate antibodies against a particular antigen. How might you use recombinant DNA technology to ensure that an animal generates B cells specific to a particular antigen? A Clone the desired antigen sequence into a plasmid, grow it in bacteria, and inject the bacteria into the animal. B Use the protoplast fusion to fuse a B cell with a cancer cell, and inject the cell into an animal to encourage monoclonal antibody production. C Clone the desired antigen sequence into a bacteriophage, and allow the bacteriophage to infect the B cells. D Infect an animal with a pathogen, isolate B cells, and clone the antigenic fragments into plasmids to screen for positive clones.

Clone the desired antigen sequence into a plasmid, grow it in bacteria, and inject the bacteria into the animal.

What is the idea behind replica plating?

Colonies are plated on multiple different kinds of plates to determine phenotype

****** refers to a specialized method of DNA transfer that requires cell-to-cell contact.

Conjugation

***** which contain plasmid DNA linked to phage cos sites, can be packaged by phage λ, allowing more efficient cloning.

Cosmid vectors,

With recombinant DNA techniques, specific fragments of DNA can be amplified and modified, a process referred to as

DNA cloning or, simply, cloning.

You have an F- strain of bacteria and you want to introduce a new gene, A+ using conjugation. You noticed, however, that your F- cell also contains a mutation that prevents it from undergoing homologous recombination (rec-). Which strain of bacterium could still potentially introduce a new bacterial gene into your F- strain? A Hfr B F+ C None of these choices D F' with A+

F' with A+

Which of the following would not be produced by a cross between Hfr and F- bacteria? A F- recombinant bacteria B Hfr bacteria C F- nonrecombinant bacteria D F+ bacteria

F+ bacteria

Which of the following statements is INCORRECT? -F stands for fertility. -F+ refers to recipient strains. -The F plasmid is a very large, circular dsDNA plasmid. -When transferred by conjugation from an F+ cell, the F plasmid converts an F- cell lacking the plasmid to an F+ cell. -The F plasmid contains all the genes needed for conjugation and plasmid maintenance in the cell.

F+ refers to recipient strains.

________, from SW-184 when plated with SW-188 on minimal agar supplemented with proline, resulted in only proline independent colonies (prototrophs). Refer to Figure B9.3. A Boiled FA B SW-184 C FA D Lt-7

FA

Recombinant DNA technology can be used to clone eukaryotic DNA into bacteria, yielding large amounts of plasmid DNA. If you wish to generate functional proteins from these recombinant DNA constructs, what would you need to do? A Functional eukaryotic proteins can be expressed in bacterial cells. B Functional eukaryotic proteins can be expressed at high quantities in hybridoma cell lines. C Functional eukaryotic proteins cannot be expressed in bacterial cells because they do not undergo the same post-translational modifications as in prokaryotes. D The recombinant DNA can be inserted into a bacteriophage, which creates high expression levels in the cells.

Functional eukaryotic proteins cannot be expressed in bacterial cells because they do not undergo the same post-translational modifications as in prokaryotes.

What is TRUE of homologous recombination? -Homologous recombination is also called crossover. -It occurs when six segments of DNA with identical or very similar sequences match up and exchange or replace some portion of their DNA. -Homologous recombination is also called conservative duplication. -It occurs when four segments of DNA with identical sequences match up and exchange some portion of their DNA. -All of these choices are correct.

Homologous recombination is also called crossover.

*****, in which DNA moves between bacterial cells, contributes to genome changes and rearrangements.

Horizontal gene transfer

What is the movement of DNA between microbes called?

Horizontal gene transfer

What is copy number?

In cells containing plasmids, each cell has a controlled number of plasmid molecules.

What does it mean that a strain is naturally competent?

It can readily take up foreign DNA

What purpose in a plasmid vector does a selectable marker play?

It imparts a phenotype, usually antibiotic resistance, on cells carrying vector DNA.

A strain of bacteria that was missing RecA would be unable to Unwind invading DNA Line up homologous sequences Find a place to integrate Do non-homologous recombination

Line up homologous sequences

Frederick Griffith studied S-type and R-type pneumococci. Suppose you use genetic engineering to mutate the S-type bacteria so that it no longer produces a capsule. What would you expect to observe? A Mice injected with live mutant S-type and live R-type would die. B Mice injected with live mutant S-type and killed R-type would die. C Mice injected with live mutant S-type would live. D Mice injected with live mutant S-type and live wild-type S-type would live.

Mice injected with live mutant S-type would live.

****** or ***** generally requires a cloning vector. Commonly used cloning vectors include ****, *****, and *****.

Molecular cloning, or DNA cloning generally requires a cloning vector. Commonly used cloning vectors include plasmids, phages, and cosmids.

Which one of the following is not an advantage to studying genetics in bacteria? Each cell is a complete organism More than one copy of each gene on the chromosome Short division time Cell differentiation and specialization is a comparatively simple process

More than one copy of each gene on the chromosome

What does the phrase "bacterial genomes are genetically compact" mean?

Most of the bacterial genome codes for proteins

During WWII, the Soviet Union used phage therapy to treat diseases such as gangrene. What is a major advantage of phage therapy? A One type of phage is able to infect all types of bacteria, so it would be a method to kill many disease-causing bacteria at once. B Phages are always more effective than antibiotics. C Bacteria cannot become resistant to phages, whereas antibiotic resistance is a common occurrence. D Phages are specific to only certain types of bacteria, so they will not kill beneficial bacteria like antibiotics.

Phages are specific to only certain types of bacteria, so they will not kill beneficial bacteria like antibiotics.

What is the difference between phenotypic selection and screening?

Phenotypic selection only allows mutants with a particular phenotype to survive, in screening, everything survives but mutants have a different phenotype.

****** refers to plasmids that utilize the same copy number control mechanism and, generally, cannot co-exist stably within a cell.

Plasmid incompatibility

Recombination can be used for many purposes, including the inactivation of a specific gene ***** formed by a ******. Such an alteration is referred to as a ****** mutation.

Recombination can be used for many purposes, including the inactivation of a specific gene cointegrate formed by a suicide plasmid. Such an alteration is referred to as a knockout mutation.

******* can be used to isolate mutants with certain phenotypic properties. ****** of these mutants then can be analyzed.

Replica plating can be used to isolate mutants with certain phenotypic properties. Clones of these mutants then can be analyzed.

******* cleave DNA at specific restriction sites. **** can be used to link cleaved DNA fragments, creating ******

Restriction enzymes cleave DNA at specific restriction sites. DNA ligase can be used to link cleaved DNA fragments, creating recombinant DNA molecules.

What role does reversion play in studying auxotrophs?

Reversion changes the auxotroph so it can grow in a similar manner to wild type.

FA, from SW-184 when plated with ________ on minimal agar supplemented with proline, resulted in only proline independent colonies (prototrophs). Refer to Figure B9.3. A SW-184 B Lt-7 C SW-191 D SW-188

SW-188

Cosmids are plasmid/phage hybrid vectors.

T

How does blue/white selection work?

The DNA fragment of interest is cloned into the lacZ gene in a cloning vector resulting in loss of lacZ function and white colonies

The ******** contains gene-encoding proteins needed to form the *******, which forms a bridge between two cells.

The F plasmid contains gene-encoding proteins needed to form the sex pilus, which forms a bridge between two cells.

The bacterial ***** typically consists of a single circular chromosome and a variable number of *****. However, some bacteria have multiple chromosomes, and some chromosomes can be linear.

The bacterial genome typically consists of a single circular chromosome and a variable number of plasmids. However, some bacteria have multiple chromosomes, and some chromosomes can be linear.

You want to insert a gene for green fluorescent protein (GFP) into a plasmid and you have located the restriction site EcoRV, a blunt cutting enzyme. When you grow the colonies on LB media, you observe that only half of the clones that contain the insert actually express the GFP gene. Why might this be? A Blunt cutters allow the insert to ligate in either direction, so only half of the inserts would be in the correct orientation to be expressed. B Blunt cutters are highly inefficient at cutting the double-stranded DNA. C The blunt enzyme likely cut the insert as well, preventing it from being expressed as a full protein. D Ligase is unable to re-attach the ends of the DNA unless they are sticky ends.

The blunt enzyme likely cut the insert as well, preventing it from being expressed as a full protein.

The chromosome and each plasmid is a *****, and the plasmid ****** within the cell is closely regulated.

The chromosome and each plasmid is a replicon, and the plasmid copy number within the cell is closely regulated.

What is a wild-type strain?

The original or laboratory strain

Phage Display is a molecular tool that generates a library of many distinct phage particles. What advantage could you see in having a library of many different sequences cloned within phages? A The phage library could be used to see which phages confer antibiotic resistance to the bacteria. B The phage library could be used to identify which sequence is able to bind to a desired target sequence, revealing potential antigenic targets. C The phage library can be used to determine which bacteria are pathogenic and which are temperate. D The phage library can be used to study the order in which the genes will be transferred to the bacteria.

The phage library could be used to identify which sequence is able to bind to a desired target sequence, revealing potential antigenic targets.

You have identified a novel bacteriophage and want to characterize it further. You set up an infection, but when you isolate the bacterial chromosome, you cannot find the phage sequence within the bacterial chromosome. Which of the following might explain these results? A The phage was a temperate phage and did not incorporate into the bacterial chromosome. B The phage used generalized transduction and existed as a plasmid within the bacterial cell. C The phage used specialized transduction and was unable to incorporate into the bacterial chromosome. D All of these may be possible.

The phage used generalized transduction and existed as a plasmid within the bacterial cell.

You are attempting to clone a DNA fragment into a plasmid using two restriction sites, BamHI and HindIII. You are in a hurry, so you want to clone this fragment into the plasmid as quickly as possible. What would you expect to see if you added the plasmid, DNA insert, restriction enzymes, and ligase to the same tube simultaneously? A The restriction digest and ligation would be just as efficient when the reactions are done simultaneously rather than sequentially. B The restriction enzymes would cut the plasmid, allowing the DNA to insert and become quickly and permanently ligated, resulting in more positive clones in a shorter time frame. C The restriction enzymes would cut the plasmid and the DNA insert, but it would also re-cut them following ligation, resulting in fewer positive clones. D The restriction enzymes would not be able to cut the plasmid and the DNA insert in the presence of ligase, resulting in fewer positive clones.

The restriction enzymes would cut the plasmid and the DNA insert, but it would also re-cut them following ligation, resulting in fewer positive clones.

What role do restriction enzymes play in molecular biology?

They cut DNA at specific sequences, allowing researcher to precisely cleave and stitch together DNA fragments

******, ****, *****< and ******* have been used extensively in gene-mapping studies.

Transconjugants, Hfr strains, F-prime (F′) strains, and the formation of merodiploids

********** refers to the transfer of DNA by bacteriophages, leading to the formation of a ******

Transduction refers to the transfer of DNA by bacteriophages, leading to the formation of a transductant.

What is the role of bacteriophage in transduction?

Transfer bacterial DNA from one cell to another.

Suppose you have a sample of bacteria that contains several different strains of bacteria. If one strain of bacteria dies, releasing its nuclear contents into the environment, how might the surviving bacteria acquire genes from these dead donors? A None of these choices - bacterial gene transfer can only occur between two live cells. B Trandsuction C Transformation D Conjugation

Transformation

******** refers to the uptake of DNA by bacterial cells from the surrounding environment. In the laboratory, ******* cells can be transformed in various ways, including *******.

Transformation refers to the uptake of DNA by bacterial cells from the surrounding environment. In the laboratory, competent cells can be transformed in various ways, including electroporation.

Barbara McClintock looked at corn embryos with either a purple or yellow kernel color. She observed that in embryos with a transposable element inserted, the kernels exhibited a mottled appearance. What does this phenotype indicate about transposable elements? A Transposable elements allow for the protoplast fusion, creating multiple genomes within a single embryo. B Transposable elements can introduce a new gene for corn kernel color from one embryo to another. C Transposable elements can insert randomly into the genome, potentially disrupting genes such as the gene for kernel color. D Transposable elements are not efficient, so they result in leaky expression of their genes.

Transposable elements can insert randomly into the genome, potentially disrupting genes such as the gene for kernel color.

_____ is responsible for cutting transposable elements out of a genome and then pasting it back into the genome.

Transposase

***** refers to the movement of DNA by ****** like insertion sequences, or IS elements, and *****. Transposition typically requires two enzymes: **** and ******

Transposition refers to the movement of DNA by transposable elements like insertion sequences, or IS elements, and transposons. Transposition typically requires two enzymes: transposase and resolvase.

What are transposons?​​ -Transposons are simple transposable elements. -Transposons are insertion sequences or insertion elements. -Transposons facilitate efficient cloning and allow the production of high quantities of cloned DNA. -Transposons contain other genes such as antibiotic resistance genes. -All of these are correct.

Transposons contain other genes such as antibiotic resistance genes.

True or false: A bacterial cell controls the number of each plasmid present.

True: each type of plasmid is maintained at a specific number

f you want to introduce antibiotic resistance into a strain of bacteria, which of the following methods would be most likely to cause mutations in the original plasmid sequence? A Using an Hfr strain to introduce an R gene B Using a transposon to introduce an R gene C Using transformation to introduce an F factor D Using conjugation to introduce an R factor

Using a transposon to introduce an R gene

In bacteria a replicon is defined as: A all the enzymes involved in the replication process of a bacterial chromosome. B a bacterial plasmid and chromosome with its own origin of replication. C a plasmid without its own origin of replication. D all the enzymes involved in the replication process of the plasmid.

a bacterial plasmid and chromosome with its own origin of replication.

What does the term "mutant" refer to?

a strain that has a change in the DNA relative to wild-type

What kinds of genes are carried on plasmids?

accessory genes that provide non-essential functions

A desirable property for a good cloning vector is: a piece of DNA that is able to replicate autonomously within a bacterial cell. a piece of DNA that has been well characterized including known restriction sites. a piece of DNA that includes easily detectable marker genes. all of these choices.

all of these choices.

Mutations may result in loss of gene function. restoration of gene function lost previously. modification of gene function. all of these choices.

all of these choices.

By comparing wild-type strains and mutant strains of bacteria, scientists can identify ***** of genes.

alleles

cloning plasmids typically have the following features:

an origin of replication that functions in the host cell(s) a selectable marker that imparts a phenotype such as antibiotic resistance, so that only cells carrying vector DNA are able to grow under certain conditions a multiple cloning site that facilitates cloning and allows screening of cells containing cloned DNA a small size that maximizes transfer efficiency into host cells a high copy number so many copies of the cloned DNA exist in a small number of cells

When growing bacteria resistant to streptomycin in a culture tube, in order to count colony forming units (CFU), you would need to plate them on agar containing streptomycin. Before plating the bacterial cells, you make serial dilutions and then you spread the bacteria. Why would you make these dilutions? A because you want to be able to differentiate between different types of bacteria. B because you want to get rid of colonies that are streptomycin sensitive. C because you only want to select for bacteria that are streptomycin sensitive. D because you want to be able to count the colonies individually.

because you want to be able to count the colonies individually.

A functional lacZ gene codes for an enzyme known as ____________. This functional enzyme metabolizes the sugar X-gal present in the agar and causes a blue product to be produced. A beta-galactose. B gamma-galactosidase C alpha-galactosidase D beta-galactosidase

beta-galactosidase

To create recombinant DNA molecules, researchers must be able to

cleave and stitch together pieces of DNA very precisely. The discovery of restriction enzymes, bacterial proteins that cleave DNA at specific sequences, allowed microbiologists to fulfill both of these needs. Their in vitro use led to the development of molecular biology. restriction enzymes allow researchers to cleave DNA at specific sites and then join together the resulting DNA fragments, thereby creating recombinant DNA molecules.

Most cloning experiments include the following pattern: A transforming bacteria, ligating DNA fragments, growing bacteria on selective media, digesting DNA with restriction enzymes B digesting DNA with restriction enzymes, ligating DNA fragments, transforming bacteria, growing bacteria on selective media C transforming bacteria, digesting DNA with restriction enzymes, growing bacteria on selective media, ligating DNA fragments D growing bacteria on selective media, transforming bacteria, digesting DNA with restriction enzymes, ligating DNA fragments

digesting DNA with restriction enzymes, ligating DNA fragments, transforming bacteria, growing bacteria on selective media

the main advantage of phage vectors is their

efficient ability to deliver cloned DNA fragments into host cells. Because packaging of DNA into preassembled capsid heads and tails only requires the presence of cos sites, the rest of the phage sequence can be omitted.

Because of their ***** bacteria are ideal candidates for genetic studies

genomic structure,

The ***** describes the alleles of an organism.

genotype

Bacterial strains are described using specific *****, which ensures that their characteristics are known and understood.

genotypic notation

Auxotrophs are bacteria that: A do not exhibit altered metabolic requirements, when compared to prototrophs. B are never nutritional mutants. C can synthesize all amino acids and vitamins, just like the parental strains. D have disrupted metabolic pathways.

have disrupted metabolic pathways.

Phenotypuc selection would apply to what factors?

high temperature growth, antibiotic resistance, or phage resistance usually restricted to retrieving mutants with phenotypes that are distinguished by ability to grow or not under specific cultivation conditions.

Transferred DNA molecules can join together via

homologous or non-homologous recombination.

A plasmid that shares a similar control mechanism and origin of replication with another plasmid inside of the same cell and is unable to replicate is a(n) A a mutant. B incompatible plasmid. C acosmid. D a phage.

incompatible plasmid.

You are sequencing a genome and find evidence of a transposon. What might this indicate about the DNA contained in the transposon?

it likely contains antibiotic resistance genes

Plasmid-cloning vectors often contain a ******, a short section of DNA that contains cleavage sites for several restriction enzymes.

multiple cloning site

By studying *****, microbiologists can learn much about the normal workings of bacterial cells.

mutants

Nutritional mutations are defined as ________________________. A mutations that allow organisms to grow on minimal medium. B mutations found in prototrophs. C mutations that do not allow an organism to grow on minimal medium, but allow them to grow on complete medium. D mutation not found in auxotrophs.

mutations that do not allow an organism to grow on minimal medium, but allow them to grow on complete medium.

Mutations associated with phenotypic screening

non-motility, pigment production, or antibiotic sensitivity mutations

Researchers used ****, also known as auxotrophic mutants or auxotrophs, in some of the earliest genetic studies of bacteria.

nutritional mutants

FA, from SW-184 when plated with SW-188 on minimal agar supplemented with proline, resulted in ________. Refer to Figure B9.3. A a mixture of proline and methionine colonies B a 50/50 mixture of leucine and proline colonies C a mixture of proline, leucine and methionine colonies D only proline independent colonies (prototrophs)

only proline independent colonies (prototrophs)

One of the first widely used cloning vectors was

pBR322 (Figure 9.13). This relatively small plasmid (4,361 base pairs) has unique restriction sites that can be used for inserting fragments of DNA following cleavage with the corresponding restriction enzymes. It also has two different antibiotic resistance genes, one for ampicillin resistance and one for tetracycline resistance. Both of these antibiotic resistance genes contain restriction sites within them.

Because phages normally transfer genetic material into host cells, ***** facilitate efficient cloning and allow the production of high quantities of cloned DNA.

phage vectors

Mutants often can be distinguished from wild-type strains by

phenotypic selection and phenotypic screening.

different types of cloning vectors have been developed, *****, *******, and ****** have been used most extensively for DNA cloning.

plasmids, phages, and cosmids Cloning vector: a DNA molecule that can be genetically manipulated and replicates within cells

Blue-white screening

probably represents the most commonly used of these screening approaches. Plasmids used for blue-white screening combine antibiotic resistance selection, to eliminate colonies without plasmid Transformants with inserts therefore appear white when grown in the presence of X-gal, whereas transformants without inserts appear blue.

To avoid problems associated with *****, researchers used double and triple mutants, thereby decreasing the probability that spontaneous revertants would arise.

reversion

Conjugation involves a special structure called a _____ which allows exchange of genetic material between a donor cell and recipient cell.

sex pilus

Whereas many plasmid vectors have a narrow host range, ***** can be used in a wider range of organisms.

shuttle vectors

Restriction endonuclease enzymes are used in recombinant DNA technology, especially if they produce pieces of DNA with staggered (sticky) ends. What causes a fragment of DNA to become staggered? A blunt ends B a mixture of poly-A sequences C single stranded tails which are complementary to other tails D improperly cut pieces of DNA

single stranded tails which are complementary to other tails

Non-homologous recombination may occur via site-specific recombination.

site-specific recombination.

Several lines of experimental evidence have indicated that mutations arise

spontaneously, in the absence of selective pressure, and provide the phenotypic variety on which natural selection acts.

vertical gene transfer,

the inheritance of a gene from a direct ancestor.

What does a gradient of transfer tell you?

the relative location of a gene on a chromosome

​Viruses help transfer DNA between bacteria using a process called _____.

transduction

The uptake of free DNA is referred to as _____.

transformation