Molecular Genetics and Biotechnology Practice Test (page 3)
Review the following concepts if needed:
- History of Molecular Genetics and Biotechnology for Genetics
- Recombinant DNA Technology for Genetics
- DNA Sequencing for Genetics
- Production of Recombinant Gene Products in Industry for Genetics
- Bioinformatics for Genetics
Molecular Genetics and Biotechnology Practice Test
For each of the following definitions, give the appropriate term and spell it correctly. Terms are single words unless indicated otherwise.
- A technique that separates molecules according to their net charge in an electric field, usually on solid or semisolid support media such as paper or agarose.
- Separation of complementary chains of a DNA molecule, usually by heating.
- Reassociation of complementary single-stranded regions of DNA with DNA, or DNA with RNA.
- Exposure of a photographic film to DNA labeled with a radioactive isotope.
- Symmetrical sequences of nucleotide base pairs in double-stranded DNA that read the same on each strand from 5' to 3'.
- Bacterial enzymes that break phosphodiester bonds in DNA at specific base sequences. (Two words.)
- The random collection of a sufficiently large sample of cloned fragments of the DNA of an organism to ensure that all of that organism's DNA is represented in the collection. (Two words.)
- An enzyme used to add deoxyribonucleotides to the 3' ends of DNA chains without a template. (Two or three words.)
- An in vitro technique for copying the complementary strands of a target DNA sequence simultaneously for a series of cycles until the desired amount is obtained. (Three words.)
- The name of the product produced by reverse transcriptase enzyme from an mRNA template. (One or two words.)
Choose the one best answer.
- A radioisotope used to label proteins differentially from nucleic acids is (a) 32P (b) 14C (c) 35S (d) 15N
- Which of the following single strands would be part of a palindrome in double-stranded DNA? (a) GAATTC (b) ATGATG (c) CTAATC (d) CCCTTT (e) none of the above
- Which of the following is an enzyme used to form a phosphodiester bond in a nick between a 3' end of one DNA chain and a 5' end of another? (a) DNA polymerase (b) restriction endonuclease (c) DNA ligase (d) S1 nuclease (e) phosphodiesterase
- Bacterial cells are rendered more permeable to uptake of plasmids by treatment with (a) heat (b) calcium chloride (c) alkali (d) a blender (e) ultrasound
- The melting temperature of a DNA molecule is determined by using (a) electrophoresis (b) change in electrical conductivity (c) column chromatography (d) density-gradient ultracentrifugation (e) change in optical density
- Which of the following is a desirable characteristic for a cloning plasmid? (a) a site at which replication can be initiated (b) one or more unique restriction endonuclease sites (c) one or more antibiotic-resistance or drug resistance genes (d) a highly active promoter (e) all of the above
- The classical 1957 experiment of Meselson and Stahl was concerned with (a) mode of DNA replication (b) polymerase chain reaction (c) in vitro production of recombinant DNA molecules (d) synthesis of hybrid proteins (e) transduction via lambda phage
- Many of the genes in lambda phage are clustered according to similarity of function. Which of these gene clusters could most likely be deleted and replaced with foreign DNA, making the recombinant phage a useful cloning vector? (a) nucleases to destroy host DNA (b) head capsomeres (c) phage-specific RNA polymerase (d) establishment and maintenance of lysogeny (e) tail proteins
- Eukaryotic genes may not function properly when cloned into bacteria because of (a) inability to excise introns (b) destruction by native endonucleases (c) failure of promoter to be recognized by bacterial RNA polymerase (d) different ribosome binding sites (e) all of the above
- The DNA fingerprinting process involves (a) chain terminators (b) degenerate oligonucleotides (c) VNTR loci (d) RFLPs (e) cDNA
Molecular Genetics and Biotechnology Questions
- The buoyant density (ρ) of DNA molecules in 6M CsCl solution increases with the molar content of G + C nucleotides according to the following formula:
- Given two dsDNA molecules, the overall composition of which is represented by the segments shown below, determine which molecule would have the highest melting temperature. Explain.
- The primary mRNA transcript for chicken ovalbumin contains seven introns (light, A–G) and eight exons (dark) as shown below.
- About half the weight of RNA synthesized at any given time within a bacterial cell is rRNA. The 30S subunit of bacterial ribosomes contains one 16S rRNA molecule (1.5 kb); the 50S subunit contains one 23S rRNA (3 kb) and one small 5S rRNA (0.1 kb). Hybridization tests of 16S and 23S rRNAs with complementary single strands of DNA reveal that about 0.14% of DNA is coding for 16S rRNA and about 0.18% for 23S rRNA. Estimate the relative activity of rRNA genes as transcription templates compared with the average gene of the bacterial genome that gives rise to mRNA. Note: Assume that the amount of DNA allocated to 5S rRNA synthesis is negligible; likewise for all kinds of tRNAs.
- Some bacterial proteins are normally secreted from the cell. If transgenic rat insulin protein could be attached by genetic engineering to such a secreted bacterial protein, it too might be secreted from the cell. Suppose that you are given an agar plate containing several recombinant bacterial clones known to contain the gene for insulin. Propose an autoradiographic method for identifying those clones that are secreting this protein. (Hint: antibodies can be attached to certain kinds of plastic in a way that leaves their antigen-combining sites free to react.)
- Restriction endonuclease EcoRI makes staggered cuts in a 6-nucleotide DNA palindrome; restriction endonuclease HaeIII cleaves at one point in the middle of a 4-nucleotide palindrome. If different aliquots of a purified DNA preparation are treated with these enzymes, which one would be expected to contain more restriction fragments? Explain (give the rationale for) your choice.
- Only about 200 molecules of phage lambda repressor are made by bacteria when lambda is integrated at its specific attachment site between E. coli genes gal and bio. Some bacterial genes such as lac can be induced to produce more than 20,000 molecules of an enzyme per cell. If you could cut and splice genes and regulatory regions at your discretion, how would you design a bacterial cell for maximum synthesis of lambda repressor protein?
- The goal is to clone a specific human gene. Human DNA is isolated and cut into ~ 15-kb fragments that are spliced into phage vectors (shotgun method). The phage are introduced into recipient bacterial cells. (a) How many kinds of cells exist? List their characteristics. (b) How many recombinant phage would have to be screened in order to find the DNA fragment of interest (the human genome is ~ 3 × 106 kb)? (Hint: See Solved Problem 12.3.)
- The polymerase chain reaction (PCR) was originally performed with a DNA polymerase from the bacterium E. coli, a common inhabitant of the human gut (37°C). Each cycle of heating denatured the enzyme added during the previous cycle. In order to reduce costs and automate the PCR, another source of the enzyme had to be found. Where is the most likely place to find this alternative source?
- Protein P is synthesized in relatively high amounts in the mouse pancreas. This protein has been isolated and purified and the sequence of 6 amino acids from the N-terminal end has been determined. If the gene for protein P is desired to be cloned for recombinant expression in a bacterial host system:
- How can a probe be prepared to identify the gene for protein P?
- Which is the best type of library (genomic, cDNA or expression) to construct for cloning this gene?
- A cDNA library is being constructed from mouse pancreas tissue in order to clone the protein P gene.
- Outline the steps in cDNA production.
- Which type of vector is most appropriate to use in this case: plasmid pBR322 or phage lambda. Why?
- One method for cloning these cDNAs into a vector is to ligate short linker molecules on to the ends of each cDNA clone. The linkers each contain the recognition sequence for a particular restriction enzyme. For example, the linker sequence, 5' GCTGCAGC-3' contains the PstI restriction site (underlined). Now all the cDNA clones constructed contain PstI sites on each end. Outline the steps involved in ligating these clones into a vector, such as plasmid pBR322 (assume that pBR322 has a unique PstI site in the ampR gene).
- After transforming E. coli with the plasmids, how can the cells that contain a recombinant plasmid (i.e., one containing any DNA insert) be identified?
- If one million ampicillin-sensitive, tetracycline-resistant clones are grown on nutrient agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P?
- After selecting several clones identified as carrying the gene for protein P, recombinant cells from each clone are propagated to high density in nutrient broth. The recombinant plasmids (presumably containing a fragment of the gene for protein P) are then extracted and purified from the rest of the cellular DNA. How can the gene be isolated from the plasmid?
- How can we demonstrate that the gene we have isolated is indeed the one for protein P?
- The gene for protein P is isolated and its DNA sequence is determined. It is found to contain 1000 bp of coding sequence and two EcoRI restriction enzyme sites, -GAATTC-, one at 150 bp internal to the start and one at 150 bp internal to the stop codon of the normal gene (see illustration below). A defective protein P has been discovered and its gene has also been cloned a nd sequenced. In the abnormal gene, one of these sequences has been changed to - GCATTC-.
- Diagram the electrophoretic pattern expected from a triple digest of the plasmid in Solved Problem 12.2 by restriction enzymes A + B + C.
ρ = 1:660 + 0:00098 (G + C)
Find the molar percentage of (G + C) in DNA from the following sources: (a) Escherichia coli: ρ = 1.710 (b) Streptococcus pneumoniae: ρ = 1.700 (c) Mycobacterium phlei: ρ = 1.732.
If the DNA for ovalbumin is isolated, denatured to single strands, and hybridized with cytoplasmic mRNA for ovalbumin, how would the hybrid structure generally be expected to appear in an electron micrograph? Note: Double-stranded regions appear thicker than single-stranded regions.
We want to find out if fetal cells contain the normal or the abnormal gene. So DNA from fetal cells is cleaved with EcoRI and the fragments are separated on an agarose gel and then transferred to a nylon membrane. A probe for the normal gene hybidizes to the internal 700-bp EcoRI fragment and will also hybridize with the abnormal gene sequence in this same region. The size of the fetal DNA fragments can be estimated by running DNA fragments of known sizes on the same gel. What band pattern is expected if the fetal cells contain the abnormal gene?
- denaturation or melting
- renaturation or annealing
- restriction endonucleases
- gene (DNA) library or genomic library
- terminal (deoxyribonucleotidal) transferase
- polymerase chain reaction
- complementary DNA (cDNA)
Molecular Genetics and Biotechnology
- (a) 51.02 (b) 40.82 (c) 73.47
- (b), because it has a higher (G + C) / (A + T) ratio
- Of all the RNAs that hybridize with DNA, the 16S and 23S rRNAs account for only 0.0014 + 0.0018 = 0.0032 or 0.32%. Since these rRNAs and mRNAs are about equally represented in a cell, the ratio 1/0.0032 = 312.5 expresses how much more active in transcription are the genes for the rRNAs.
- Attach anti-rat insulin antibodies to a plastic disk about the size of an agar plate. Impress the disk onto the plate and allow any secreted insulin to be specifically bound by the antibodies. Remove the plastic disk and expose it to radioactive anti-insulin antibodies, forming an "immunological sandwich" with the antigen (insulin) between two antibody molecules. Wash away any unattached radioactive antibodies and then make an autoradiograph of the disk. Images on the film can be used to identify the locations of insulin-secreting clones on the agar plate.
- More fragments are expected from HaeIII because the probability of a specific four-base sequence is greater than the probability of a specific six-base sequence if the nucleotides are distributed along a chain in essentially a random order.
- Insert the gene for lambda repressor protein immediately adjacent to the lac promoter in a plasmid. With no operator locus between these two genes, thousands of repressor molecules should be made per cell constitutively.
- (a) Five kinds of cells: (1) bacteria that do not contain any plasmid DNA, (2) bacteria that took up the plasmid DNA, but do not contain any human DNA, (3) bacteria that contain the plasmid DNA with human DNA spliced in, but not the desired human gene sequence, (4) bacteria that contain plasmid DNA and a portion of the desired human gene, but not all of it, (5) bacteria that contain plasmid DNA and all of the desired human gene. (b) Approximately 600,000 recombinant phage, determined as follows.
- Organisms that live in hot springs must have heat-stable enzymes. The first thermostable DNA polymerase used in automating PCR was isolated from a bacterium called Thermus aquaticus that normally lives in hot springs at a temperature of 70-80°C. Consequently, its polymerase is stable at temperatures near this point.
- (a) The amino acid sequence can be used to construct a degenerateDNAprobe (see Example 12.5) that can be labeled with radioactivity. (b) A cDNA library is most appropriate since this is a mammalian gene and it may contain introns. Agenomic library will most likely allow the isolation of gene fragments, many of which may contain the introns. An expression library is more difficult to make, and useful if an antibody "probe" is being used.
- Steps in cDNAproduction: (1) isolateRNAfrom cells, (2) add reverse transcriptase enzyme, dNTPs and oligo-dT primers to make a first strand of cDNA, (3) degrade RNA, (4) add DNA polymerase I to make a second strand of cDNA, (5) clip the hairpin using S1 nuclease.
- Since the gene for protein P is 1000 bp or less in length, it is too small to clone into phage lambda, which requires fragments of approximately 15,000 bp. Thus, we should choose plasmid pBR322 and clone DNA fragments ranging from 2000 to 3000 bp. This will ensure that the entire gene will be found in one fragment, as well as its associated regulatory sequences.
- Cut the cDNAs and the pBR322 vector with PstI enzyme, then ligate the cDNAs with the cut plasmid.
- By employing the replica plating technique, we can select cells that are ampicillin-sensitive and tetracyclineresistant. Only those cells that have incorporated the plasmid are tetracycline-resistant, and of these only the cells with an insert in the ampR gene are ampicillin-sensitive.
- Blot each Petri plate with a nitrocellulose filter paper, thereby transferring some cells of each clone onto the filter for in situ hybridization. Lyse the cells and denature the DNA with a dilute sodium hydroxide solution. Single strands of the denaturedDNA thus will stick to the filter. Flood the filter with the radioactive probe. After washing to remove any of the unhybridized probe, subject the filter to autoradiography. Select cells from the plate that correspond in position to the radioactive loci on the filter.
- Cut the plasmids with the PstI enzyme. This should liberate the gene fragment, assuming that there are no PstI sites within the gene fragment.
- The gene for protein P should hybridize specifically with the probe on a Southern blot. Also, by sequencing the gene fragment, it will be possible to ascertain the genetic code and then compare this to the original amino acid sequence obtained and any other previously cloned relatives of protein P using bioinformatics. If protein P has a measurable activity (e.g., enzymatic conversion of clear substrate into a color), the protein might be synthesized in an in vitro translation system and its activity checked. Finally, the gene might be cloned into an expression vector and the protein produced in E. coli (or another appropriate system). This protein could also be tested for activity
- After digestion with EcoRI, the normal gene for protein P should produce one band, 700 bp long. The mutant gene for an abnormal protein P should produce one band of at least 850 bp due to the loss of one of the EcoRI sites. The size of the band is dependent on where the next EcoRI site may reside in the neighboring flanking genomic DNA.
- Note: The migration distance is not linear with increasing fragment length, but more like a log function.
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