MiCROBIO QUIZ 3 QUESTIONS
MICROBIAL GENETICS P2: What are the four genes, and their corresponding proteins, which are involved with lactose catabolism? What is the function of the four proteins? (Note that one gene comprises one operon, while three genes comprise another operon.)
- lacI/LacI(corresponding protein): transcriptional repressor. In a monocistronic (single gene) operon by itself and NOT part of the polycistronic lacZYA operon. It's transcription is always active and is not regulated. - lacZ/LacZ(corresponding protein): beta (β)-galactosidase enzyme, which hydrolyzes lactose into galactose and glucose - lacY/LacY(corresponding protein): lactose permease, a cytoplasmic membrane protein responsible for transporting lactose into the cell - lacA/LacA(corresponding protein): surprisingly, no direct role in lactose catabolism is known. A postulated role for the detoxification of lactose structural analogs which can be harmful to E. coli *(lac A, lac Z, and lac Y are involved in lactose catabolism.)*
MICROBIAL GENETICS P3: Distinguish among the terms mutation, mutant, and mutagenesis.
- Mutation: changes to genome nucleic acid base sequence. Most mutations have some effect on the biology of the organism. - Mutant: an organism whose genome carries a mutation - Mutagenesis: the process of making mutations
MICROBIAL GENETICS P3: Describe the following types of mutations and their effects on expression of a protein-encoding gene: -Silent point mutation -Missense point mutation -Nonsense point mutation -Insertion mutation -Deletion mutation
-Silent point mutation: codon change to encode the same amino acid; no effect on the protein (Point mutation: single base pair change) -Missense point mutation: codon change produces an amino acid substitution. C to T base change at asterisk, giving an amino acid substitution. -Nonsense point mutation: Codon change to translational termination codon, truncating the protein at the carboxyl terminus.(point mutation) -Insertion mutation: in protein coding genes that are not multiples of 3 base pairs will cause translational frameshifts and often premature translational termination to produce an altered and truncated protein. -Deletion mutation: translational frameshifting
MICROBIAL GENETICS P2: What is quorum sensing, and how does it function to regulate gene expression in bacteria?
A cell density dependent, self induction of gene expression which relies on molecular diffusion. Genes are transcriptionally activated when the bacteria are growing at high cell density in a closed system.
BIOTECHNOLOGY P1: What is a complementary DNA(cDNA) library of molecular clones, and what advantages does it offer over a genomic DNA library?
A collection of only the genes that are encoded into proteins by an organism. Made from RNA cDNA is created through RNA is reverse transcribed into cDNA using reverse transcriptase, an enzyme of viral origin used to make RNA into DNA. Benefits: the library represents expressed genes, and clones made from mRNA are smaller than gene clones and therefore more easily maintained in microbial cells. Helpful because bacteria doesn't have introns.
BIOTECHNOLOGY P2: Provide an overview of the technique of transgenic animal engineering.
A. tumefaciens is a plant pathogen that causes a disease known as crown gall tumor, which is an overgrowth of cancerous plant cells. DNA of bacterial origin called T (tumor) DNA is transferred to the plant cell genome, where it stably integrates and initiates plant cell tumor formation. T DNA can be modified to both inactivate tumor production and to include any plant gene of interest, thereby genetically transforming the entire plant. A single cell is transformed first, and then it is treated to grow into a full plant.
BIOTECHNOLOGY P2: Provide an overview of the technique of transgenic plant engineering.
A. tumefaciens is a plant pathogen that causes a disease known as crown gall tumor, which is an overgrowth of cancerous plant cells. DNA of bacterial origin called T (tumor) DNA is transferred to the plant cell genome, where it stably integrates and initiates plant cell tumor formation. T DNA can be modified to both inactivate tumor production and to include any plant gene of interest, thereby genetically transforming the entire plant. A single cell is transformed first, and then it is treated to grow into a full plant.
BIOTECHNOLOGY P1: What are restriction enzymes? How do they work, and what role do they have in bacterial biology?
An enzyme that cleave DNA at short, defined base sequences. They are a defense against bacterial virus infection through cleavage of incoming viral DNA. Named after bacteria that produce them.
BIOTECHNOLOGY P1: What is the polymerase chain reaction, and how does it work?
An in vitro method for the production of nucleic acid molecules. The technique uses repeated cycles of DNA denaturation (strand separation) at temperatures near boiling, then primer-target base pairing at c. 55 C, and then 72C to allow synthesis of a new strand by a DNA polymerase.
MICROBIAL GENETICS P2: What is an operon, and why is gene expression in operons beneficial for bacterial cells?
An operon is a set of genes whose expression is coordinated through common transcription. Operons allow the cell to efficiently express sets of genes whose products are needed at the same time. EX- Lac Operon contains genes that encode proteins involved in uptake and metabolism of a particular sugar, lactose
BIOTECHNOLOGY P2: What advantages does the CRISPR-Cas system have over previously-discussed methods of modifying a genome (for example, artificial transformation)?
CRISPR-Cas systems are unique to prokaryotes and function by recognizing and degrading sequence-specific foreign DNAs. The Cas enzyme uses a "guide" RNA molecule complementary to a target DNA base sequence which promotes Cas recognition of the target DNA and double stranded DNA cleavage at a particular site.
MICROBIAL GENETICS P3: What is a plasmid, and what does plasmid DNA do for those cells which have plasmids?
Circular DNA molecules found in the bacterial cytoplasm that replicate independently of the bacterial chromosome.
MICROBIAL GENETICS P2: Discuss the functions of small RNA molecules and riboswitches in the regulation of gene expression.
Complementary RNAs base pair to a target mRNA and either interfere with the translation of mRNA or promote mRNA degradation. RNA sequences which function as riboswitches can bind metal ions or other compounds to promote premature transcriptional termination. (Riboswitches- regions of RNA secondary structure (intramolecular base pairing) which influence transcription or translation. )
BIOTECHNOLOGY P2: What chemical properties of double-stranded (ds) DNA molecules enable their separation by electrophoresis?
DNA is an acid. At pH values >3, DNA molecules are polyanions with one negative charge contributed by each phosphate group along the sugar-phosphate backbone or two negative charge per base pair.
MICROBIAL GENETICS P3: What is meant by "lateral DNA transfer"? -List and briefly define the three major mechanisms for lateral DNA transfer which occur in bacteria.
DNA transfer between cells unrelated by direct parent/ daughter ancestry is called "lateral" or horizontal" gene transfer Conjugation: Replicated DNA is transferred between cells by means of pilus protein tube. Natural transformation: DNA from lysed, dead cells is taken up and stably maintained by live ones. The acquired DNA can integrate into the genome of the living recipient cell by homologous recombination Transduction: DNA is transferred from a host cell genome to a recipient genome by means of a virus. This usually involves homologous recombination into the genome of the recipient cell.
MICROBIAL GENETICS P3: Discuss the process of F plasmid conjugation.
Direct replicative transfer of DNA from a living donor cell to a living recipient cell. When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F-) the F- cell is converted to a F+ cell.
BIOTECHNOLOGY P2: How may the technique of RNA interference (RNAi) be used to control gene expression?
Double- stranded RNAs are recognized by cellular enzymes and degraded, thereby regulating gene expression at the posttranscriptional level. Engineered, controlled expression of siRNA transcripts may be used to selectively turn off expression of certain genes without permanently mutating that gene.
MICROBIAL GENETICS P2: trp operon transcriptional regulation reduces tryptophan levels by controlling gene expression. Compare this reduction to the control of enzyme activity via feedback inhibition(Ch. 5).
Feedback inhibition
MICROBIAL GENETICS P2: Explain the three levels at which gene expression can be regulated.
Gene expression can be regulated in 3 levels: 1.DNA level- DNA recombination or other changes such as covalent modification can activate or deactivate genes 2.Transcriptional level- control of transcriptional initiation or stability of the messenger RNA (This is the most commonly used mechanism for the control of gene expression; regulation here saves the most cellular energy). 3.Translational level- control of translation initiation; protein stability; control of protein activity by the covalent modification.
MICROBIAL GENETICS P2: Why is it advantageous for cells to regulate gene expression so that only genes whose products are needed are expressed?
Gene expression expands much cellular energy. High-energy phosphate bonds are hydrolyzed to make mRNA, and GTO is hydrolyzed by ribosomes during translation to form the peptide bonds between the amino acids in proteins.
MICROBIAL GENETICS P2: What is meant by the phrase, "gene expression"? What are the two processes which comprise gene expression?
Gene expression is the genetic information and RNA needed for the cell to function. The two processes involved are transcription and translation. -Transcription: (genetics) the organic process whereby the DNA sequence in a gene is copied into mRNA -Translation: the process whereby genetic information coded in messenger RNA directs the formation of a specific protein at a ribosome in the cytoplasm
BIOTECHNOLOGY P2: Discuss two approaches for modifying the human genome: gene therapy and vaccination with DNA.
Gene therapy is the permanent insertion of DNA into a patient's cells to provide a medical benefit. DNA vaccines is the foreign protein in a somatic cell being put in a host cell that is dead or attenuated to produce an immunity by the memory immune cells being produced.
BIOTECHNOLOGY P1: What is a genomic DNA library?
Genomic library is collections of various cloned DNA fragments representing an entire genome. A population of microbes containing random molecular clones representative of a complete (or nearly complete) genome is called a genomic library.
MICROBIAL GENETICS P2: How does glucose exhaustion induce lac operon transcription? Recall that "induction" means canceling transcriptional repression.
Glucose exhaustion causes a buildup of the phosphorylated form of the glucose import protein -> relief of inducer exclusion
MICROBIAL GENETICS P2: Distinguish the transcriptional regulatory processes of an inducible operon (e.g., the lac operon) from those of a repressible operon (e.g., the tryptophan or trp operon).
In contrast to inducible operons, repressible operons are normally transcriptionally "on" but are turned "off" by repressor proteins when their products accumulate to optimum levels.
MICROBIAL GENETICS P2: What is signal transduction? What is a two-component signal transduction system, and how does it function to regulate gene expression in bacteria?
In two-component signal transduction, a membrane-bound sensor kinase binds a chemical and, in response, phosphorylates (adds a phosphate group to) a response regulator, which binds DNA or otherwise alters gene expression. About 30 such systems are known for E.coli, and they primarily regulate non-carbon nutrient uptake and metabolism. (Signal transduction-the transmission of molecular signals from a cell's exterior to its interior)
BIOTECHNOLOGY P1: How are the in vitro techniques of recombinant DNA technology different than the in vivo DNA repair process of homologous recombination?
In vitro means "in the test tube" or "in the laboratory" In vivo means "in a living organism" Recombinant DNA technology involves in vitro recombination: the joining and modification of unrelated genetic molecules by laboratory methods. In vivo DNA recombination, there is no requirement for homology between two joined sequences.
BIOTECHNOLOGY P1: Define and discuss the process of molecular cloning. -What additional steps must be taken to ensure expression from eukaryotic genes which have been cloned in bacteria?
Molecular cloning is the production of many copies of a nucleic acid as DNA molecules. 1. A gene of interest from any virus organism is isolated as a DNA fragment. 2. Enzymes are used to splice the DNA fragment (insert) into a carrier molecule (a vector) 3. The recombinant molecule from step 2 is introduced into a host microorganism. 4. The microbe containing the recombinant DNA is propagated to high numbers. Additional steps that must be taken is removing introns 5' caps and 3' poly-A tails because bacteria does not have these. to express eukaryotic genes in bacteria.
MICROBIAL GENETICS P3: What is a spontaneous mutation?
Mutations that occur as a result of natural biological and/or chemical processes are considered spontaneous. Can occur as a result of random genome DNA damage that is not repaired and as mistakes during DNA replication.
MICROBIAL GENETICS P3: How is genetic change different from changes in gene expression?
Phenotypic changes due to gene expression is after exposure to different environmental conditions (e.g different transcripts and proteins are made when cells switch from glucose to lactose catabolism). Genetic changes happen alot less and are much more stable once they occur. They are more difficult or impossible to reverse. REMEMBER THAT genetic changes indirectly affect cell physiology through the two processes of gene expression: transcription and translation.
MICROBIAL GENETICS P2: Explain the mechanism of transcriptional repression and how it is overcome by induction.
Protein binding to DNA physically excludes RNA polymerase from binding to a transcriptional promoter so no transcription. Cell negatively controls gene expression inhibits the production of a messenger RNA. An inducer molecule cancels the repression, enabling transcription and turning the operon "on".
BIOTECHNOLOGY P1: Discuss the artificial transformation techniques of protoplast fusion, DNA microinjection and the gene gun.
Protoplast fusion: 1. Bacterial cell walls are enzymatically digested, producing protoplasts. 2. In solution, protoplasts are treated with polyethylene glycol. 3. Protoplasts fuse. 4. Segments of the two chromosomes recombine. 5. recombinant cell grows new cell wall. DNA Microinjection: A small fraction of the injected DNA can be integrated into the genome and stably replicated during subsequent cell multiplications. If the DNA contains the right sequences for gene expression in the host cell, the gene will direct the synthesis of RNA and protein. The Gene Gun: Microscopic metal particles are coated with transforming DNA and propelled into plant cells using pressurized gas.
BIOTECHNOLOGY P2: Describe the process of detection of a particular DNA sequence by blotting.
Purified DNA is cleaved by restriction enzymes, and the fragments are separated according to size by electrophoresis through a gel. Fragments are transferred ("blotted") to a nitrocellulose membrane so that they retain the same relative positions they had in the gel. The membrane containing transferred DNA fragments is exposed to a radioactive or otherwise labeled molecular probe for detection of a target sequence. Base pairing of the probe to a target, creating a hybrid, base-paired molecule, means that the probe sequence is present in the genome.
MICROBIAL GENETICS P3: What are common types of DNA damage, and how is that damage repaired?
RADIATION DAMAGE: - Ultraviolet light: Light absorption can create covalent bonds between bases - Ionizing radiation (wavelength < 1 nm) can hydrolyze sugar-phosphate covalent bonds. it can lead to double stranded breaks in chromosomes. Example: X rays. CHEMICAL MUTAGENS: - Nitrous acid (HNO2): chemically oxidizes adenine bases so they pair with cytosine instead of thymine. Two rounds of DNA replication are needed for a stable AT -> GC base pair change. - Nucleoside analogs: nucleosides with slightly different chemical structures. These can be incorporated into replicating DNA by nucleotide polymerases (e.g., azidothymidine, AZT; 3' N3) and cause termination of DNA synthesis. - Benzpyrene: a component of smoke and soot (frameshift mutations) - Aflatoxin: a wheat mold metabolite (frameshift mutations) DNA REPAIR: - DNA polymerase enzyme has so-called "proofreading" activities which remove incorrectly-incorporated bases during DNA replication using the parent strand as the correct template. - Excision repair: enzymes can remove and replace damaged or incorrectly-paired DNA bases exclusive of replication using the parental or template strand as the template. - Repair of thymine dimers: DNA damage due to UV light can produce covalent bonds between two adjacent T bases on the same DNA strand. Some cellular enzymes can specifically break these bonds and repair the DNA, while other enzymes can excise damaged DNA and replace it.
BIOTECHNOLOGY P2: Describe the technique of restriction fragment length polymorphism (RFLP) and its intended purpose. - Why has this technique largely been replaced by the polymerase chain reaction (PCR)?
RFLP analysis associates the presence of a probe-specific restriction enzyme fragment of certain size with a phenotype of interest. RFLP is labor intensive so it has been largely replaced by the more rapid and technically much simpler procedure of PCR application of DNA sequences which are unique to microbial strains or disease genes.
MICROBIAL GENETICS P3: Homologous recombination is used as a mechanism for cellular DNA repair. What is meant by" homologous recombination"?
Recombination means that separate DNA base sequences are joined together or exchange pieces either inside the cell by cellular DNA repair process or in a test tube by recombinant DNA techniques. Homologous recombination can delete or reposition segments of DNA, creating mutations, which activate ore deactivate genes.
BIOTECHNOLOGY P2: What chemical properties of single-stranded (ss) DNA and RNA molecules enables their separation by electrophoresis?
Secondary structures can form by intramolecular base pairing on a single strand, altering molecular shape. A denaturing agent is added to prevent intramolecular base pairing and consequent molecular shape-induced changes in migration rate during electrophoresis.
BIOTECHNOLOGY P2: Describe the process of detection of a particular RNA sequence by blotting.
Similar to DNA blotting, but the entire population of RNA molecules made under a given set of conditions or made by a certain tissue type is resolved by electrophoresis and probed using nucleic acids which can base pair to a specific target sequence.
BIOTECHNOLOGY P1: Compare and contrast natural transformation (discussed in Chapter 8) with the recombinant DNA technique of artificial transformation.
Some bacteria such as E.coli do not readily take up foreign DNA by natural transformation. These bacteria must be chemically treated to make them competent for artificial transformation. More recent methods use either chemicals which increase membrane permeability or high voltage electric fields(electroporation) to create pores in the cell walls and membranes. Natural transformation is a bacterial adaptation for DNA transfer.
BIOTECHNOLOGY P2: In the context of all this scientific humor, there actually is such a thing as a "southwestern" blot. What do you suppose this is?
Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography.
MICROBIAL GENETICS P3: What is the Ames test? Include the importance of a positive selection for mutants. What assumptions enable its use to identify potential cancer-causing (carcinogenic) compounds?
The Ames test exposes bacteria to potential DNA mutagens and employs a positive selection to quantify mutant organisms which have experienced genetic damage.
BIOTECHNOLOGY P2: Describe the process of detection of a particular protein by blotting. What type of molecular probe is required?
The entire protein profile expressed in a certain body tissue or under certain microbial cell growth conditions is separated by electrophoresis and blotted to a membrane. The probe in this case is an antibody molecule, a large protein made by the animal immune system, which specifically recognizes a target protein on the blot.
MICROBIAL GENETICS P2: Discuss the transcriptional regulation of the trp operon.
The five proteins (TrpA through TrpE) are the enzymes of a metabolic pathway for synthesis of the amino acid tryptophan from simple carbon and nitrogen compounds, such as glucose and ammonia. trpR is a repressor gene. Transcription of this operon is normally "on" since E.coli grows in an environment naturally low in tryptophan and needs to synthesize its own.
MICROBIAL GENETICS P2: Explain how the process of inducer exclusion prohibits induction of the lactose (lac)operon when both glucose and lactose are available to E. coli.
The lac operon E.coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent. Two regulators turn the operon "on" and "off" in response to lactose and glucose levels: the lac repressor and catabolite activator protein (CAP). The lac repressor acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting as a repressor when lactose is present. The lac repressor senses lactose indirectly, through its isomer allolactose. Catabolite activator protein (CAP) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule cAMP.
BIOTECHNOLOGY P1: What is site-directed mutagenesis?
The modification of target DNA base pair(s) at a specific site(s) in the genome.
MICROBIAL GENETICS P2: How does glucose exhaustion positively regulate or activate lac operon transcription?
The phosphorylated form of the glucose transporting protein accumulates as glucose is exhausted and glucose import declines. The phosphorylated form allosterically stimulates the activity of an enzyme called adenylate cyclase, which converts ATP to cyclic adenosine monophosphate; a/k/a, cAMP. cAMP binds a protein called CAP and this complex binds to the lac operon promoter, enhancing RNA polymerase binding --> more transcription -->more LacY and LacZ--> enhanced ability to grow on lactose.
BIOTECHNOLOGY P2: What chemical properties of proteins enables their separation by electrophoresis?
The variety of net molecular charges on different protein molecules presents special problems for electrophoresis of protein molecules. Addition of the negatively-charged detergent sodium dodecyl sulfate(SDS) to a mixture of different-size protein molecules both denatures the molecules, producing the same shape, and binds in a constant ratio.
MICROBIAL GENETICS P3: What is a transposable element, and how do these elements influence the function of cells?
These are small segments of genomic DNA which can insert into new places in the genome. Such insertion into a protein- coding gene creates a frameshift mutation, completely inactivating the gene in almost all cases. Transposable elements encode an enzyme, transposable elements encode an enzyme, transposase, which catalyzes the insertion reaction.
MICROBIAL GENETICS P3: Define and discuss the DNA transfer process of transduction.
Transduction is the process by which a virus transfers genetic material from one bacterium to another. Viruses called bacteriophages are able to infect bacterial cells and use them as hosts to make more viruses. After multiplying, these viruses assemble and occasionally remove a portion of the host cell's bacterial DNA. Later, when one of these bacteriophages infects a new host cell, this piece of bacterial DNA may be incorporated into the genome of the new host.
MICROBIAL GENETICS P3: Discuss the process of Hfr conjugation. -As a bonus, describe how this process was used to map genes along the E. coli chromosome.
Unlike the F plasmid Hfr- transferred chromosome fragments remain linear and cannot replicate themselves .To initiate conjugation, the Hfr cell makes a physical bridge to the F-cell. A break in the donor DNA initiates a process by which single stranded DNA is synthesized and moved into the recipient (F-) cell. The amount of DNA transported is determined largely by how long the transporter bridge remains intact
BIOTECHNOLOGY P1: What properties do molecular cloning vectors have which enable them to function?
Vectors carry a positive selection genetic marker such as a gene conferring antibiotic resistance. This enables killing of all cells which do not take up the vector following artificial transformation. Vector= Carrier molecule.
BIOTECHNOLOGY P1: Describe how a researcher would isolate a molecular clone of the gene which encodes any desired protein using colony hybridization technology.
We will often start with a protein amino acid sequence and predict its DNA sequence from that. Chemically synthesized and labeled DNA probes are used to screen thousands of molecular clones in a genomic library to hopefully identify a few containing the DNA sequence of interest. A probe is a labeled molecule that is complementary to a target nucleic acid in the genomic library. Probe-target nucleic acid base pairing is called hybridization and this identifies the target clone.
MICROBIAL GENETICS P2: Does it make good sense (from acellular energy perspective) to preferentially use glucose over lactose?
Yes, glucose requires fewer steps and less energy to break down than lactose. To use lactose, the bacteria must express the lac operon genes.