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process, the right amino acid must pair up with the right RNA; mistakes may result in errors in protein synthesis, which cause cell damage and disease. The ligation of each amino acid to its matching tRNA is performed by a matching enzyme. Sheppard’s students are converting a tRNA that pairs with glutamine into a tRNA for pyroglutamate instead. Pyrogluta- mate has been implicated in brain diseases (some suspect it of being an “Alzheimer’s incendiary device”), but it’s complicated for researchers to introduce it into proteins for study, so they’d love to have a tRNA that would simplify the process for them. Making a tRNA for pyroglutamate in vitro (in a test tube) is quite doable. The researchers start by using enzymes as “molec- ular scissors” to dissolve particular linkages in strands of genetic material, isolating just the se- quence they want to manipu- late. On that segment, “we change just one base pair in the gene,” says Heal- ton, “and then we use standard reagents and an incubator to generate match- ing tRNAs in a test tube.” Doing this in vivo (in a living organism) is trickier. It en- tails placing the manipulated DNA segment into a plasmid— a circular piece of DNA used as a genetic Trojan horse—and in- serting the plas- mid into a re- search strain of Escherichia coli bacteria, whose cells serve as DNA factories for generating lots of the in- serted genetic material.


Base pairs. The four main chemicals that pair up and repeat in long sequences to encode genetic information. In DNA, cytosine usually pairs with guanine, and adenine with thymine; in RNAs, thymine is replaced by uracil, and other combinations can occur.


DNA, RNA, genes. Along the double chain of chemicals in a molecule of DNA (deoxyribonu- cleic acid), certain sequences are genes—that is, they contain instruction codes for making proteins. RNAs (ribonucleic acids) are single- stranded versions that carry and translate genetic instructions into the parts of the cell that actually synthesize proteins.


Electrophoresis. A chemical and electrical process that draws apart and sorts protein molecules (including genes) by size. The different proteins appear as blurry grey bars, or sometimes as dyed spots, arrayed on a small sheet of clear gel.


Escherichia coli. A family of common bacteria, some of which cause illness but many of which are used as “factories” in genetics and other labs.


Mathematical model. Equations or graphs that precisely describe, compute, simulate, or predict the behaviors and relationships in a system of variables.


Nucleotides. Component submolecules of DNA/RNA molecules, featuring the four base chemicals of genetics: cytosine, guanine, ade- nine, and thymine (except in RNAs, where thymine is replaced by uracil).


pH. A measure of acidity or alkalinity. Vine- gar’s pH is around 2 or 3, ammonia’s about 11; plain water and saltwater are close to neu- tral, at about 7.


Since the stu- dents are work- ing on a submi- croscopic scale, Healton ex- plains, the only


14 SCOPE WINTER 2012


way to see if the plasmid has suc- ceeded is to purify the genetic mole- cules from their yellowish bacterial soup and run a


ON A GEL THE RNAS ARE VISIBLE AS SHADOWS UNDER UV LIGHT. THEY EXCISE THESE DARK AREAS—BY HAND, WITH A RAZOR—AND RINSE THEM REPEATEDLY UNTIL THEY’VE RELEASED ALL THEIR RNA.


sample onto an electrophoresis gel, which separates DNA, RNA, and other proteins by size; an added indicator chemical makes the genetic material fluoresce bright orange. Even then, Walsh says, “the molecules are so close in size that we can’t be sure they’re the right ones. We can see if we have tRNA genes, but not whether they’re our altered versions. For that, we send samples out to Yale for genetic sequencing.” For several days, Walsh has trouble. He says, “Our culture medium includes an antibiotic, to kill off any bacteria lacking the resistance that comes with our inserted DNA, but it’s been killing everything. So either the plasmid doesn’t contain a func- tional DNA insert, or the E. coli aren’t accepting it.” His hunch: “It may be a kinetics issue. The insert DNA needs to collide with the plasmid’s DNA to bind with it correctly; maybe that’s not happening.” Even with a good plasmid, E. coli can have a bad reaction. As Sheppard puts it, “Biochemistry can be fickle, and E. coli don’t always want to cooperate.” New culture plates are ordered from the supplier, and Frank even has Sheppard watch over her shoulder as she makes plates herself. The plates seem OK. Still, the temperature, the chem- istry of the solution, or other conditions may need adjusting. “It’s frustrating,” Walsh admits. “Then again, it would be very surprising if it worked on the first try.” And their success in de- veloping the altered tRNA in vitro is a good step toward facilitat- ing more research into pyroglutamate’s role in disease. Also in Sheppard’s lab, Brittany Ulrich ’12 and Katie Stein ’13, with Stefani Mladenova ’14, are cloning certain genes from Staphylococcus and Bdellovibrio bacteria, using E. coli to express and reproduce them. From parts of that DNA, they’re making transfer RNA. Ulrich’s recipe: Use enzymes to cut out the desired portion of DNA. Mix with a polymerase and other re agents. Add nucleotides (available from retail lab-supply houses). Incubate mixture, swishing constantly, at 37 degrees Celsius (99 F) for about five hours. To test for doneness, look for precipitate gath- ering in the bottom of the vessel. To separate the freshly cooked tRNA from the other ingredi- ents, Ulrich and Stein run the solution onto a gel where the RNAs are visible as shadows under ultraviolet light. They excise the shadowed areas of the gel—by hand, with a razor—and rinse those pieces repeatedly, checking their light absorbency until it’s evident they’ve released all their RNA. Next they add ethanol and spin the solution in a centrifuge until “we get a tiny pellet at the bottom,” says Ulrich. That pellet is one batch of fairly pure tRNA. They need to add many more pellets to it before they have enough for their testing.


The standard procedure is to store the tRNAs in a freezer, to fend off the inevitable degradation that all biological flesh is heir to. Freezing also slows the action of enzymes whose job is


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