Microbial Recombination and Gene Transfer Questions and Answers

Answer: D

Correct answer: amplify a specific DNA sequence into many copies

The polymerase chain reaction amplifies a chosen DNA sequence exponentially, producing millions of copies from a very small starting quantity. It was devised by Kary Mullis in 1983, for which he received the Nobel Prize in 1993. Each cycle has three steps at different temperatures: denaturation at about 95°C separates the strands; annealing at 50 to 65°C lets short primers bind to the flanking sequences; and extension at 72°C lets the polymerase synthesise the new strands. Each cycle doubles the target, so \( n \) cycles give \( 2^{n} \) copies - thirty cycles yield over a billion. The reaction became practical with Taq polymerase, from the thermophile Thermus aquaticus, which survives the denaturation step and so need not be replenished each cycle. Its applications run from diagnosis and forensic identification to cloning and sequencing. Variants include reverse transcription PCR for RNA and quantitative PCR for measuring amount.

Answer: C

Correct answer: Electroporation

Electroporation applies a brief high-voltage pulse that transiently opens pores in the cell membrane, allowing DNA to enter. The membrane reseals within moments, and the cell recovers. It is one of the standard artificial transformation methods, and it achieves considerably higher efficiency than chemical treatment. It also works on a wide range of cell types - bacteria, yeast, plant protoplasts and mammalian cells - where the chemical methods are more restricted. The alternative for bacteria is the calcium chloride method: incubating cells in cold calcium chloride and then applying a brief heat shock at 42°C, which is simpler and needs no equipment but gives lower yields. Other delivery methods have their own niches: the gene gun or biolistic method fires DNA-coated particles into plant cells; microinjection introduces DNA directly with a needle; Agrobacterium transfers DNA naturally into plants; and viral vectors are used for gene therapy. Conjugation and transduction are natural processes rather than techniques.

Answer: B

Correct answer: only those adjacent to the prophage integration site

In specialised transduction only the host genes adjacent to the prophage integration site can be transferred. The mechanism explains the restriction. A temperate phage integrates at a specific site, and when it excises it occasionally does so imprecisely - cutting a little to one side, so that it leaves some phage DNA behind and carries an equivalent length of neighbouring host DNA away. Only genes physically next to the integration site can be picked up this way. The classic example is phage lambda in E. coli, which integrates between the gal and bio genes and can therefore transduce only those. The contrast with generalised transduction is the point examined. There the phage packages a random fragment of fragmented host DNA during the lytic cycle, so any gene may be carried with roughly equal probability. The resulting particles are usually defective, having exchanged phage genes for host ones.

Answer: A

Correct answer: curing

Curing is the loss of a plasmid from a bacterial cell. Because a plasmid is dispensable, its replication can be disturbed without killing the host - by growth at elevated temperature, or with acridine dyes, ethidium bromide or other agents - until daughter cells are produced that no longer carry it.

Curing is a standard tool for deciding whether a trait is plasmid-borne: if the cured strain has lost the antibiotic resistance or toxin production along with the plasmid, the genes lay on the plasmid rather than on the chromosome. The other three words have no defined meaning in this context.

Answer: C

Correct answer: Binary fission

Binary fission is how a bacterium divides - it passes genes vertically, from parent to offspring, and is therefore not horizontal transfer at all. The three mechanisms of horizontal gene transfer, which move DNA between contemporaries rather than down generations, are: conjugation, by direct contact through a sex pilus; transformation, by uptake of free DNA from the environment; and transduction, by a bacteriophage. The distinction matters greatly. Vertical transfer alone would confine a new gene to one lineage, and adaptation would depend on mutation. Horizontal transfer allows a gene to spread across a population and even between species, which is why antibiotic resistance appears far faster than mutation rates would predict, and why resistance to several drugs is often acquired together on a single plasmid. It also complicates bacterial taxonomy and phylogeny, since genes need not share the history of the organisms carrying them.

Answer: C

Correct answer: An antibiotic resistance gene

A selectable marker is a gene that allows cells carrying the vector to be identified and grown while those without it are killed, and an antibiotic resistance gene is the standard example. Plating transformed cells on medium containing ampicillin, for instance, means only those that took up an ampicillin-resistance plasmid survive. It solves a real problem: transformation is inefficient, and only a small fraction of cells take up the vector, so without selection the successful ones would be lost among the rest. A second stage is often needed to distinguish cells carrying a vector with an insert from those carrying an empty one. Blue-white screening does this: the cloning site sits within the lacZ gene, so an insert disrupts it, and colonies that cannot cleave the substrate X-gal remain white while empty vectors give blue. The other options are also essential parts of a vector - a restriction site to accept the insert, an origin to replicate, a ribosome binding site to express - but none of them selects.

Answer: D

Correct answer: a small circular DNA molecule that replicates independently of the chromosome

A plasmid is an extrachromosomal DNA molecule, usually circular and double-stranded, carrying its own origin of replication so that it replicates independently of the host chromosome. It is called a replicon for that reason. Plasmids are generally dispensable for growth but carry genes conferring advantage in particular conditions: R plasmids confer antibiotic resistance, F plasmids confer the ability to conjugate, Col plasmids encode bacteriocins, and virulence plasmids encode toxins. Their spread by conjugation is the principal route by which antibiotic resistance moves between bacteria, including between species, which is why resistance can appear far faster than mutation alone would allow. In the laboratory they are the standard cloning vectors: a plasmid with a selectable marker, an origin of replication and a multiple cloning site accepts foreign DNA and carries it into a host for replication and expression. A plasmid that can also integrate into the chromosome is an episome.

Answer: C

Correct answer: DNA from an RNA template

Reverse transcriptase synthesises DNA using RNA as the template - the reverse of the usual direction of information flow, which is what gives it its name. It was discovered independently by Temin and Baltimore in 1970. Its discovery required an amendment to the central dogma, which had held that information passes from DNA to RNA to protein. Reverse transcription showed that the first arrow can run backwards. It is the defining enzyme of the retroviruses, including HIV, whose RNA genome is copied into DNA and integrated into the host chromosome as a provirus. That step is the target of the antiretroviral drugs zidovudine and nevirapine. In the laboratory it is used to make complementary DNA from messenger RNA. Because mature mRNA has had its introns removed, cDNA contains only the coding sequence - which is what allows a eukaryotic gene to be expressed in a bacterium, and what makes cDNA libraries a record of which genes a tissue was actually transcribing.

Answer: C

Correct answer: McClintock

Barbara McClintock discovered transposable elements in maize in the 1940s, working on the unstable pigmentation patterns of kernels. She showed that certain genetic elements - the Ac/Ds system - could move to new chromosomal positions and alter the expression of nearby genes, and she named them controlling elements.

The work was decades ahead of its acceptance and she received the Nobel Prize in Physiology or Medicine in 1983. Griffith is remembered for the 1928 transformation experiment in pneumococcus, and neither Abelson nor Harvey is associated with transposons.

Answer: D

Correct answer: Episome

An episome is a plasmid that can either replicate autonomously in the cytoplasm or integrate into the host chromosome and be replicated as part of it. The F factor of Escherichia coli is the classic example: free in an F⁺ cell, integrated in an Hfr cell.

A lysogen is a bacterium carrying a prophage, which is a state of the cell rather than a type of plasmid. "Medisome" is not a term in use, and a lysosome is a eukaryotic organelle unrelated to bacterial genetics.