Prokaryotes have an adaptive strategy to identify and respond to viral infections. This strategy uses segments of the cyclic DNA called CRISPRs and genes encoding CRISPR-associated (cas) proteins. When a virus enters the cell, a strand of viral DNA is excised by a cas protein and inserted into the bacterial DNA in a CRISPR region. When the same viral DNA is encountered subsequently, this foreign DNA is targeted by cas proteins that carry RNA markers transcribed from the inserted segment. The cas proteins cleave the viral DNA. The bacteria “remember” the infectious agent, providing a form of immunity.
- Use the diagram above to identify the components of a transcript-based response of bacteria to the presence of viral DNA by placing the corresponding number next to each feature of the diagram:
___ viral DNA ___ degraded viral DNA ___ cell membrane
___ cellular DNA ___ cas protein ___ stored viral DNA template
___ excised viral DNA ___ cas protein-RNA complex
___ cas protein-RNA-viral DNA complex
The CRISPR system was discovered in cultures of yogurt in 2002. Subsequently, researchers developed a technology based on the manipulation of this system. The code for the prokaryotic CRISPR/cas system is highly conserved and is found in the human genome. DNA sequences are known to encode proteins responsible for many heritable diseases. CRISPR/cas is a technology that allows DNA to be cleaved at the boundaries of a nucleotide sequence, making the protein dysfunctional. The break in the strand is then recognized and replaced with the code for the functional protein. If the editing is done with zygote-forming cells, the change is inherited. Not only the patient, but all progeny of the patient, are cured. This technology is the first to easily make genomic modifications of a germ line. In the words of a prominent molecular biologist, this technology, which was recognized as the Breakthrough of 2015 in the journal Science, “democratizes genetic engineering.” Just as PCR became a standard, widely used tool, any molecular biology lab is now able to apply this technology.
- Pose three questions—whose pursuit would require an understanding of genetics—regarding the ethical and social issues that accompany the use of this medical technology.
- Explain the value of genetic variation within a population. Predict a possible effect that this technology could have, if unregulated, on human genetic variation.
Gel electrophoresis of polymers and polymer fragments is an important element in many investigations. Samples of a solution are pipetted into the wells of a gel. The gel is placed in a solution that maintains a constant pH, and an electric field is applied over the length of the gel. Separated components are transferred to a substrate where they can be visualized and identified by comparison with samples of standards. Application of this method to DNA is called a Southern blot, named for the inventor of the technology. The method’s application to RNA is called a northern blot, another demonstration that biologist have fun (there are also western, eastern, and far-eastern blots, but these techniques are not named for their inventors).
- Consider the three amino acids shown below and explain how, when pipetted into a gel and subjected to an electric field, the amino acids move; how the amino acids are separated as they move; and which amino acid moves furthest.
- A biologist wants to determine whether a new protocol is successful in constructing and amplifying a molecular clone of a segment of DNA introduced as a plasmid. After the procedure is complete, the bacterial cells containing the plasmid with the inserted segment are lysed, and a gel is run into which samples of the lysate and the sequences to be cloned have been pipetted. Use the data displayed in the developed gel shown below to evaluate the question of whether the protocol was successful.
- Design a plan to answer the question of whether the new DNA has been incorporated into the DNA of the host organism.
17.3 Whole-Genome Sequencing
Genetic engineering can be applied to heritable information to produce what is referred to as a “knockdown organism.” Biotechnology also can be applied to produce nonheritable changes in a “knockdown gene.” Post-transcriptional strategies target the mRNA product of a gene. One such strategy uses the conserved genes that encode RNA interference (RNAi) proteins for the regulation of levels of mRNA transcription.
Some viral RNA is double stranded (dsRNA). A cell responds to the presence of double-stranded RNA by the attachment of the enzyme DICER, which cuts dsRNA into short fragments. One strand of the fragment is transferred to the RNA-induced silencing complex (RISC), which searches for an mRNA with a sequence matching that of the fragment strand. When detected, this mRNA is degraded.
- Common in cancer cells is a mutation of the gene that encodes the protein p53, whose role is to detect and repair errors in DNA; if repairs cannot be made, p53 initiates apoptosis. Create a visual representation to explain how the DICER-RISC system within the cell can be used to suppress the translation of a mutated form of the gene encoding p53, potentially destroying a tumor.
- Whole-genome sequences provide a library of potentially expressed proteins, but they do not provide information on the functions of each protein. In an approach called reverse genetics, investigations attempt to determine the function of the gene, often by silencing the gene using RNAi technology. Assume that you have the ability to synthesize dsRNA from a DNA segment taken from an organism whose whole genome has been determined. Design a plan for collecting data that could be used to assign a function to the protein encoded by this sequence. (Hint: Don’t worry about the number of experiments that might need to be conducted to implement your plan. An automated technique called high-throughput screening robotically supports thousands of simultaneous experiments.)