Deck 17: Applications of Recombinant DNA Technology and Reverse Genetics

Question 1

If genes are introduced into a yeast plasmid to serve as a shuttle vector,the plasmid has the ability to

A)move genes from human to yeast.
B)replicate in yeast or in bacteria.
C)degrade selected yeast genes.
D)recombine with yeast nuclear genes.
E)degrade other yeast plasmids.

Answer 1

B

Question 2

In nature,what is the order of events in processing the product of the gene for insulin?

A)cleavage of 35 amino acids from the middle of the polypeptide to make two chains that are then joined by disulfide bonds
B)cleavage of 24 amino acids from the N terminus to form proinsulin that is subsequently cleaved to make A and B chains
C)removal of 5′ leader sequence from the mRNA,and splicing out of a large central intron to produce the mRNA that codes for the final form of polypeptide
D)removal of large mRNA sequences by RNA editing,followed by splicing of the products of the two insulin genes
E)transcription of the two insulin genes A and B,translation,and posttranslational joining of the two polypeptides

Answer 2

B

Question 3

Suppose you have engineered a plasmid vector in order to produce a functional animal protein in E.coli.But the product you collect,while it has the correct amino acid sequence,is in fact nonfunctional.What might you try that would have a greater chance of success?

A)a eukaryotic expression vector that has sequences for regulating transcription
B)using a host other than E.coli that does not have the same codon bias
C)a eukaryotic expression vector that includes sequences for controlling posttranslational effects
D)treatment of the posttranslation proteins to add signal sequences
E)using yeast artificial chromosomes that can hold longer sequences

Answer 3

D

Question 4

Besides the obvious fact that chemical mutagens are dangerous to handle,another major disadvantage to their use is that

A)most of these mutations are immediately lethal.
B)most chemically induced changes are detectable only by sequencing.
C)chemical mutagens activate transposons.
D)two or more generations of further matings are required to isolate them.
E)they alter all A-T and C-G base pairing.

Question 5

In Drosophila,mutation screening usually involves use of a balancer chromosome that includes three elements: a set of overlapping inversions,an easily recognized dominant mutation,and a recessive lethal mutation that prevents balancer homozygotes from surviving.Which one,or combination,of these elements is necessary and sufficient to suppress crossovers?

A)inversions
B)dominants
C)lethal recessive
D)inversions plus dominant
E)lethal plus inversions

Question 6

P element transposition requires the enzyme transposase,which acts at the ends of the P element.To find transposons in a germ line,which of these is best?

A)use of a "crippled" P element that cannot transpose itself but can produce the enzyme
B)use of a "crippled" P element that can be transposed if there is another source of transposase
C)use of a P element that does not share the genome of the germ line
D)use of restriction sites that flank the inserted P element
E)use of DNA ligase to incorporate the P element

Question 7

The reasons for using transposon-based mutagenesis include all of the following except

A)disrupting the coding sequence.
B)disrupting a regulatory sequence.
C)creating improper splice sequences.
D)addition of alkyl groups to bases.
E)blocking of enhancer-promoter interactions.

Question 8

Hedgehog (Hh),and many other signaling pathways,were originally discovered in Drosophila.Hedgehog was so named because of bristles all over the early embryo,resembling a hedgehog.It works,with other genes,to develop the anterior-posterior axis of the embryo. The mutant develops bristles all over the embryo instead of just in a stripe toward the anterior.Thus Hh is a segment polarity gene.The hedgehog gene family in vertebrates is involved in development of central and peripheral nervous system,skeleton limbs,lungs,skin and germ cells.Shh (Sonic hedgehog)is responsible for major development of brain,spinal cord,and skeleton and is received at the membrane via the patched receptor (PTCH).In the absence of Shh,PTCH inhibits the activity of the pathway,while binding of Shh allows signal transduction to go forward.
In humans,holoprosencephaly is a developmental disorder involving incomplete cleavage of the forebrain,leading to the abnormalities of brain and facial structures.From the above,which abnormalities of genes must be involved?

A)some other member of the Hh gene family
B)Shh only
C)PTCH only
D)Shh in combination with PTCH
E)either Shh or PTCH

Question 9

Suppose you have just discovered a Drosophila gene that has a sequence similar to a gene of interest in humans.You need to have mutants of the Drosophila gene because

A)you need to see whether fly mutants have a defect in the same system.
B)you need to see whether the mutant produces the same phenotype.
C)you need to see whether the Drosophila mutant disrupts the same metabolic pathway.
D)you want to compare the mutant Drosophila sequence to each mutant human sequence.
E)you need to have as many alleles in Drosophila as you have found in humans.

Question 10

You have evidence that a gene,VTE4,of known sequence and well-understood transcriptional regulation,is available from another lab.The product of this gene enables seeds to produce a significantly increased amount of vitamin E from a substrate already produced in Brassica napus,the plant that gives us canola oil.To produce canola oil with increased vitamin E,how would you proceed?

A)Select Brassica plants with the highest titer of the vitamin,isolate the genes responsible,and cause these genes to duplicate.
B)Use a yeast system to engineer yeasts that produce the vitamin,and get manufacturers to add this to the canola oil during bottling.
C)Introduce the gene and necessary cis elements into Brassica,along with a reporter gene,then measure the vitamin E produced.
D)Clone VTE4 in A.tumefaciens and use these bacteria to produce transgenic Brassica.
E)Introduce VTE4 into tomato plants or another food that humans eat more than Brassica.

Question 11

EMS (ethylmethane sulfonate)is mutagenic because

A)it breaks phosphodiester binds.
B)it adds alkyl groups to bases.
C)it forms cross-links between DNA strands.
D)it makes DNA more susceptible to radiation.
E)it causes chromosomal deletions.

Question 12

If a gene in Drosophila is expressed only when an imaginal disc (A)begins to differentiate,it is referred to as a homeotic gene.Recessive mutations in such genes often result in loss of function so that the structure does not develop.If the gene is expressed,but in a different imaginal disc (B),what is the result?

A)loss of function of disc B's primary gene
B)gain of function of several inappropriate genes in disc A
C)gain of function in disc B at the "wrong" location
D)deregulation of gene expression throughout disc A
E)deregulation of gene expression throughout disc B

Question 13

In using Agrobacterium tumefaciens to transfer genes into plants,what is transferred from bacterium to plant?

A)Ti plasmid
B)T-DNA
C)opines
D)only the recombining sequence
E)the entire bacterium

Question 14

When a transgene is used in mice to knock out a specific function,two problems that might occur are the unintended introduction of multiple copies,or abnormal gene expression.The second of these problems is related to

A)position effect.
B)homologous recombination.
C)vector insufficiency.
D)mitotic recombination.
E)unequal crossing over.

Question 15

In reverse genetics,what is the correct order in which the experimenter proceeds?

A)silencing the genes in question using RNAi,followed by generating gain-of-function alleles
B)screening genes via PCR,followed by deletion of the flanking sequences
C)selection of a phenotypic alternative for the gene in question and sequencing its DNA
D)induction of and selection for a mutant allele with a known sequence,followed by screening for mutations of interest
E)random bombardment of the DNA with a known mutagen,followed by observation of offspring for newly acquired traits

Question 16

Enhancer trapping might occur if you create a library of insertions bearing enhancer-less GAL4 genes.Their expression will be controlled by

A)also inserting a viral enhancer.
B)also inserting a GAL4 reporter.
C)including a bacterial origin sequence.
D)enhancers mapping near the insertion site.
E)including an enhancer activator.

Question 17

If you wish to rid an experimental organism of the function of a gene,you can use RNAi.What would you inject into the organism?

A)co-suppressor with double-stranded (ds)DNA
B)ssRNA complementary to cDNA
C)ssRNA complementary to mRNA
D)dsRNA homologous to the gene
E)dsRNA including transposase

Question 18

To introduce dsRNA into C.elegans,it is enough just to dip the worms into media that includes E.coli containing the dsRNA because

A)E.coli infect C.elegans and then the RNA is released.
B)E.coli break up into pieces due to the presence of the worms.
C)C.elegans feed on the transgenic bacteria.
D)C.elegans emit toxins that paralyze the bacteria.
E)E.coli emit chemicals that cause pores to open in the nematode body wall.

Question 19

Which of the following is a possible advantage for the environment of having farmers use plants that genetically produce Bt toxin?

A)Plants with genes for the toxin are eaten less often by insects,and therefore less insecticide is needed.
B)Plants that produce Bt toxin must be grown from new seeds each season,and therefore fewer of them are planted.
C)Bt toxin is advantageous to many species of invertebrates that grow in the same fields.
D)Harvesting plants with these genes is less disturbing to the soil layers where they are planted.
E)Plants with these genes must be sprayed with the organism,but need much less than other plants.

Question 20

Positional cloning identified the human homologue of the Drosophila patched (PTCH)gene as a candidate gene for the human disorder NBCCS or Gorlin syndrome.Screening of the patched coding region revealed a wide spectrum of mutations in NBCCS patients.Most of these mutations were predicted to result in premature protein truncation.What phenomenon would the researchers look for in the patients?

A)haploinsufficiency for the protein
B)recessive familial pattern of inheritance
C)predisposition to developmental abnormalities of the brain
D)incomplete formation of the forebrain
E)Shh mutations

Question 21

Knockout libraries are available already prepared for some organisms such as Drosophila.These libraries are originally made by what method?

Question 22

In an organism,X,it is found to be impossible to generate loss-of-function mutants for most genes.There is an alternative method of introducing random mutations that can later be screened for the gene(s)in question.This can be accomplished by using insertions due to what structures?

Question 23

One way to genetically engineer S.cerevisiae is by homologous recombination.If a given sequence is thus introduced and is integrated via two regions of homology,is it more probably introduced as a linear or a circular entity?

Question 24

If a gene is replaced with a heterologous sequence such that the gene is then nonfunctional,what is the result usually called?

Question 25

If a mutagen such as EMS is used to produce numerous mutagenized cell lines for an organism,PCR can be used to select for a particular gene.However,what feature of this selected sequence is used to find the ones with mutations in that gene?

Question 26

In the formation of transgenic animals,DNA is usually inserted into eggs or embryos.If some cells of the embryo do not receive the introduced DNA,the result is an organism with cells of different genotypes;the organism is known as what?

Question 27

In nature,RNAi protects cells against infection by what kind of pathogen?

Question 28

If a researcher is cloning a sequence by using pUC and uses the plasmid to transform bacteria,which are then plated on X-gal medium,would the recombinant plasmids be found in blue or white colonies or both?

Question 29

For a transgenic animal line to be produced from a chimera,the transgene must be carried by what?

Question 30

What is the function of imaginal disc cells during the larval stage of Drosophila development?

Question 31

By what method is T-DNA integrated into the plant genome?

Question 32

The GFP gene is useful as a reporter gene because the cells and tissues with it emit bioluminescence when treated with what,that functions as a substrate?

Question 33

If an experimenter wants to use the GFP method but needs to detect the presence or absence of several proteins at the same time,he can take advantage of mutational variants of GFP that emit what?

Question 34

If a plant species cannot readily have a new gene integrated into it via a Ti plasmid,it must then be subjected to different methods.These often include removing the cell wall and inserting DNA,after treating the membrane with heat or electricity to do what to the membrane?

Question 35

In producing an E.coli plasmid to be an expression vector,an experimenter includes genes for selectable markers,a promoter for the expression of the introduced genes.She uses a cDNA sequence to ensure the insert contains no introns.The gene inserted is transcribed,but not translated.What sequence did she omit from the plasmid?

Question 36

In grape cultivation,growers often find that heterozygous forms are more desirable.To propagate the heterozygotes without having to interbreed in each generation,what method can they use?

Question 37

Agrobacterium tumefaciens is often used in the production of transgenic plants because of its Ti plasmid.This plasmid is usually "disarmed" by deleting its tumor genes along with the genes for producing what products that would have been advantageous to the bacterium?

Question 38

In the pUC plasmids,a polylinker region that includes multiple restriction sites is embedded in the lacZ component.If a sequence of interest is inserted into this region,what is the effect on lacZ?

Question 39

In the case of Jesse Gelsinger,a young man who died during gene therapy,the therapy involved delivery via a viral vector.Was this germ-line or somatic therapy?

Question 40

What kind of gene silencing is induced by short double-stranded RNAs?

Question 41

Design an experiment that uses enhancer trapping to drive the expression of a gene of interest.

Question 42

Suppose a mutagen is used on an inbred population of mice to establish cell lines,each including a different mutant allele,which can then be tested for function.This technique is known as ________.

Question 43

Suppose a gene from a cow is introduced into a frog.This results in a ________ transgene.

Question 44

What characteristics of plants allow them to be cloned relatively easily,whereas cloning in animals is rare and difficult to achieve?

Question 45

In species X,the UUU codon for phenylalanine is used significantly more frequently than other phenylalanine codons,and knowing this allows the scientist to manipulate translation efficiency.This unequal frequency is known as ________.

Question 46

In the 1990s,gene therapy was seen as a probable cure for many genetic diseases.List three or four reasons that it has so far been of limited success.

Question 47

Transgenic fusions between a gene and a reporter are used to determine whether a gene is expressed.Which of these components allows the researcher to work with living specimens,and why?

Question 48

If the process used to investigate a genetic condition begins with knowing a DNA sequence and aims at finding the phenotype it causes,this process is known as ________.

Question 49

If recombination results in random integration of a DNA sequence,so that it produces a nonhomologous region,the recombination is ________.

Question 50

Discuss the ethical and societal reasons for and against germ-line gene therapy.