Exam 3 Chapter 11 Study Questions

In what way do instances of extensive horizontal gene transfer challenge the "branching tree" view of evolutionary lineages?

As discussed in Chapter 4, evolutionary trees are usually based on the accumulation of single nucleotide changes, or descent with modification. Horizontal gene transfer brings many nucleotide changes simultaneously into the genome via a single genetic event.

n the past few years, CRISPR has become a widely used method to "edit" the genomes of any organism. Very briefly describe how CRIPSR is done

CRISPR technology currently uses the Cas9 enzyme that can cleave DNA and a small RNA that is complementary to the region of the genome to be edited. Cas9 also requires a tracrRNA to guide the cellular ribonuclease to make a cut in the DNA. The simplest edit to make is a small deletion in part of the gene of interest. A small RNA of approximately 25 nucleotides is synthesized in vitro to be complementary to the region in which the edit is made. (Since Cas9 cuts both strands, the small RNA will be complementary to one strand and identical to the other, except for the replacement of T with U in the RNA.) The tracrRNA required by Cas9 is also synthesized so the RNA molecule includes both the small RNA sequence and the tracrRNA sequence as one longer RNA molecule. This is introduced into the cell along with the Cas9 protein or gene; it is common to have the Cas9 gene encoded on a plasmid, which is then transcribed and translated by the cell into an active enzyme. This combination of Cas9 and the guide RNA will make a double-stranded cut at the region targeted by the guide RNA. This double-stranded cut is repaired by the cell, usually by the process of non-homologous end-joining (NHEJ). Small deletions or insertions usually occur at the junction site where NHEJ has worked, which results in the small deletion at that site that knocks out the gene (say).

Define the following terms Competent F plasmid Ti plasmid Lytic and lysogenic phage Transposable element

Competent—cells that are able to incorporate DNA via transformation F plasmid—a circular DNA molecule that encodes about 28 genes that are directly needed for conjugation Ti plasmid—a circular DNA molecule in Agrobacterium that transfers sequences from the bacterium to the infected plant cell, where it is incorporated into the plant's genome. Lytic and lysogenic phage—Phage are viruses that infect bacteria. Lytic phage are those that replicate immediately upon infection and destroy or lyse the host bacteria. Lysogenic phage can incorporate their genome into the bacterial chromosome and remain quiescent with only minimal phage gene expression for some time. At some point, they can switch to a lytic phase and destroy the host cells. Transposable element A transposable element is a discrete (meaning that it has defined ends and sequence) segment of DNA that can move to other locations within a genome.

Transposable elements are found in the genomes of nearly every species. What sequence features can be used to distinguish RNA-based elements or retrotransposons from DNA based elements?

DNA-based elements encode transposase and have inverted repeats at their ends. RNA-based elements are more diverse in structure, but often encode reverse transcriptase (abbreviated pol in Figure 11-23) and include a long terminal repeat at the end that serves as the promoter for transcription.

Once DNA has been transferred to a recipient cell via conjugation, describe two ways in which the DNA might then be maintained in the recipient cell that would enable it to be subsequently transmitted vertically.

Either it is incorporated into the bacterial genome by crossing over with the chromosome or it is maintained extrachromosomally, as a plasmid.

Briefly describe the differences between generalized transduction and specialized transduction, including which genes are being transferred in each case.

Generalized transduction arises from phage that can incorporate any region of the bacterial genome into the phage particle; usually this occurs because the phage does not insert its own DNA into the bacterial chromosome. Thus, any region of the bacterial genome can be transferred. Specialized transduction occurs when a phage has a specific insertion site for its genome into the bacterial genome. Since the phage genome is always inserted at the same location, it will transfer those genes closest to its insertion site the most frequently.

Why is HGT particularly important in organisms that reproduce asexually?

In asexually reproducing organisms, each generation is genetically the same, other than the occasional mutation. Horizontal gene transfer introduces new alleles and new genes into an organism's genome, which increases the amount of genetic diversity.

Undergraduate researchers Daniel and Jenny were interested in identifying the bacteria that live on bamboo leaves and spruce tree needles. They collected samples of the leaves from both plants on their University campus and mixed them vigorously in a wash buffer containing some surfactant. Inevitably, some of the leaves were broken. They then collected the washes (which had a slight green tint) and extracted total DNA from them. When they screened their washes for bacterial ribosomal RNA genes, they did not find any ribosomal DNA that was identical to DNA from known terrestrial bacterial species. They did however find that each sample contained lots of DNA that was similar to ribosomal DNA from marine cyanobacteria. They considered it unlikely that their samples would harbor marine organisms. What is the most likely explanation for their finding? How might they confirm your hypothesis?

Recall that chloroplasts are endosymbionts in plant cells which contain their own DNA. Based on DNA sequence comparisons, chloroplasts are probably derived from cyanobacteria, as shown in Figure 3.11. It is likely that the sequences they recovered are not derived from the bacterial genome but from the chloroplast genome in the plant cell. To confirm this hypothesis, Jenny and Daniel could compare their sequences to database sequences from plants. Other potential approaches (which are more expensive and tedious) include extracting and purifying plant chloroplasts and obtaining sequence from them, or by sequencing the plant's genome including its chloroplast, and comparing that sequence to what was obtained by the students. They could also look for other genes that are known to be chloroplast-specific to confirm the presence of contaminating chloroplast DNA.

Streptococcus pneumoniae is the best-studied example of a naturally competent organism. Its genome consists of a 2MB chromosome and almost never includes independently-replicating extrachromosomal elements like plasmids. Over 250 genomes of independent isolates of this species have been sequenced. Considerable variation among S. pneumoniae is indicative of extensive horizontal transmission of DNA. The normal habitat for Streptococcus pneumoniae is in the nostrils of humans and other mammals, where it comes in contact with DNA from a broad range of organisms and diverse potential sources of new DNA. What, in your estimation, accounts for the fact that most of the DNA in S. pneumoniae genomes originates from other Streptococci?

Since Streptococcus pneumoniae is naturally competent, it can acquire DNA from its surroundings by transformation. In addition, since S. pneumoniae rarely harbors extrachromosomal DNA, an imported DNA must recombine with the recipient genome if it is going to be transmitted vertically. Because other Streptococci are more likely to share synteny and identical sequences with each other than with other genera, DNA from co-generic sequences is most likely able to recombine with the S. pneumoniae genome. It does not able to acquire DNA from other bacteria that colonize nostrils, so the habitat alone is not a sufficient explanation.

What is meant by the term "Genomic Island"?

Since horizontal gene transfer typically involves a contiguous block or segment of DNA, the sequence of the acquired DNA often has features that distinguish it from the surrounding parts of the genome. This defines it as a genomic island—a region of the genome that has been acquired via horizontal gene transfer.

Briefly explain the difference between the core genome and the pangenome of a species of bacterium.

The genes found in all natural isolates of a bacterium define its core genome. Genes and other genetic elements found in some isolates but not in all together make up its pangenome.

Define transformation, transduction and conjugation. Describe how these are different from one another and how they are similar to one another.

Transformation occurs via the uptake of DNA from the environment. While the DNA may originally be double-stranded, it is often made single-stranded during the transfer. Transduction occurs via the activity of different viruses and is usually involves double-stranded DNA. Conjugation occurs via direct contact between the donor and recipient with transfer mediated by a specific set of genes. While single-stranded DNA is transferred, it is replicated upon transfer to become double-stranded.

How does vertical gene transmission differ from horizontal gene transfer?

Vertical gene transmission occurs over generations, while horizontal gene transfer can occur within a generation. Vertical gene transmission involves transfer or transmission from parent or parents to daughter cells or offspring, while horizontal gene transfer is from a donor to a recipient. Vertical gene transmission occurs within a species, while horizontal gene transfer can occur between species. Genetic diversity arising from vertical gene transmission occurs by recombination and mutation, while genetic diversity arising from horizontal gene transfer involves transfer of blocks of DNA sequence.

In what way do CRISPR regions of bacterial genomes provide insights into the history of that bacterial strain?

When foreign DNA (such as from a virus) invades a bacterium, some of its DNA will be included as a spacer sequence and incorporated into the genome of the bacteria as part of the CRISPR array. Thus, the spacer sequences in the CRISPR array record the history of which viruses have invaded the bacteria in the past.

he following type of experiment was used to elucidate mechanisms of horizontal gene transfer early in the history of bacteriology. In the experiment, two strains of bacteria in liquid culture were placed in opposite arms of a U-shaped tube. Dividing the two compartments was a glass filter whose pore size was too small to allow bacteria through. However, when pressure was applied at either end, liquid from the media with any macromolecules it contained could pass from one arm to another. One of the strains was a donor and the other a recipient of DNA that can be transmitted horizontally. Predict if recombinant cells would occur if the process of transfer was each of the following. Explain your answer. a. Conjugation. b. Transformation. c. Transduction.

a. Conjugation. Conjugation requires direct contact between cells, so it could only occur if cells can come in contact with each other and form a pilus between them. This cannot happen when the filter is present. Applying pressure to the media to increase the liquid flow would push cell fragment and macromolecules across the filter but, since intact cells cannot pass, gene transfer would probably not occur. b. Transformation. This could definitely occur since DNA could pass through the pores of the filter and be acquired by the recipient. c. Transduction. This could occur if the virus can pass through the pores of the filter.

Crossing over is needed between the incoming DNA and the bacterial genome for stable maintenance and vertical transmission of DNA by transformation, conjugation, and transduction. Crossing over is not needed when plasmids are transferred by transformation. a. Why is crossing over not needed for stable maintenance of plasmids? b. What function must be encoded on a plasmid in order for it to be transferred vertically?

a. Crossing over integrates sequences from one DNA molecule into another DNA molecule. Since plasmids are maintained as self-replicating extrachromosomal pieces of DNA, they do not need to be integrated into the chromosome. b. It has to be replicated. It also typically includes some functions to ensure its stable transmission to the next generation, most notably the ability to segregate reliably between daughter cells.

Restriction enzymes are very widely used tools in molecular biology research. a. What properties of restriction enzymes have allowed them to be so useful in laboratory techniques? b. What is the natural role of restriction enzymes and how do bacteria protect themselves from this natural role?

a. Restriction enzymes make sequence-specific double-stranded breaks (DSB) or cuts in DNA. Because their activity is sequence-specific, the location of the DSB can be targeted. The DSB can be used as the site of an insertion of other sequences or to target other changes. Many restriction enzymes make a staggered cut at their target sequence, which results in short single-stranded "sticky" ends. These sticky ends can be used to join DNA molecules together in vitro and in vivo. b. The natural role of restriction enzymes is to detect and cut foreign or invading DNA by recognizing a specific sequence within the invading DNA molecule. Bacteria protect themselves against this activity by methylating the recognition site or target sequence when it occurs within their own genomes.