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The silence of the genes

New technique deactivates stretches of DNA, blocks production of protein in bees, and beckons with more wide- ranging applications

With the assistance of a technique that is gaining more credibility every day in molecular biology, baptized as RNA (ribonucleic acid) interference, or simply RNAi, researchers from the University of São Paulo in Ribeirão Preto have managed to deactivate a gene in adult worker honeybees of the Apis mellifera species. For 15 days, the gene responsible for making vitellogenin – the main protein for reproducing insects, with a probable influence on the immune system – was silenced in 96% of the 300 workers that received injections of RNAi. Silenced is the term coined by scientists to describe the neutralizing effect brought about on a gene by RNAi, a molecule created in the laboratory that differs from the conventional RNA for showing two strands of base pairs (chemical units) instead of just one.

This extra strand, present in the artificial molecule, was capable of destroying the chemical recipe that commanded the production of vitellogenin before this command reached the ribosome, the cell organelle charged with synthesizing the proteins.Without any orders, the ribosome stopped making vitellogenin. That is, this protein’s gene continued to exist and did not undergo any alteration in its sequence of base pairs. It was just switched off or deactivated, as its message is intercepted and sabotaged before reaching its destination. “We still cannot assert for sure whether the silencing is temporary or definitive”, says Zilá Paulino Luz Simões, of USP in Ribeirão Preto, the main Brazilian author of the work. The study, which had scientists from the Agricultural University of Norway taking part, was published on January 20 in the BMC Biotechnology electronic scientific magazine.

In another experiment, still under way, Zilá’s team succeeded in annulling the same gene in Apis mellifera queen bees. In this case, though, the technique was less efficient. Only half of the leaders of the swarm did not synthesize vitellogenin. “Perhaps it’s necessary to increase the quantity of RNAi that we are giving the queens”, Zilá explains. In eggs that would give rise workers, they injected a solution of 1 microliter (a millionth part of one liter) in which there were 5 micrograms of RNAi. In the queen bees, in which the vitellogenin gene is expressed (activated) with twice the intensity as in the workers, the same liquid medium was administered, though with 10 micrograms of RNAi. To try to improve the efficiency of the silencing technique amongst the queen bees that rule the colony, new dosages of RNAi will be inoculated into more queens.

In spite of still being in the initial stage, the work with the queens is supplying some good tidings: it is hoped that the bees whose gene was silenced may be capable of passing on this characteristic to their first generation of descendants. Their daughters would also not be able to produce the protein. As far as everything indicates, the order not to synthesize vitellogenin is stored in the memory of the maternal cells and afterwards transmitted to the offspring. The story of the bees is impressive, but it is not the first time that an animal shows the capacity for inheriting silenced genes. An article by researchers from Cold Spring Harbor Laboratory, of the United States, published in February in Nature Structural Biology , showed that mice with a gene deactivated by the RNAi technique passed this genetic modification to their descendants, apparently creating a stable lineage of animals with this characteristic.

The vitellogenin gene was chosen to be the target for the experiment with RNAi because honeybees have one characteristic that intrigued the researchers from USP. For being a gene that is profoundly involved in the reproductive process ofApis mellifera , it was to be expected that its expression would be exclusive to the queens, or at least far greater in the bees that are the leaders of the swarm than in the workers.

After all, the reproductive functions are practically exclusive to the queens, which lay the absolute majority of the eggs that guarantee the continuity of the swarm. It so happens that the expression of the gene is very high both in the queens and in the workers, although the former make twice the vitellogenin than the latter. “The quantity of vitellogenin produced by the queens may exceed 50% of all the proteins synthesized by this kind of bee”, Zilá comments. In the workers, this level comes to a noteworthy 30%, an indication that these subaltern bees, besides working, must also take part in reproduction and other important functions.

Against Aids and cancer
Since it was discovered in 1997, the technique of using the double-stranded RNA to interfere in the functioning of genes started to be used, with different degrees of success and for different purposes, in plants, animals, fungi, and even in human cells. Sometimes, the RNAi would not manage to silence a gene totally, but would lead to a considerable reduction in the production of the protein derived from the gene in question. The more optimistic believe that when – and if – it is fully controlled, the use of RNAi can turn out to be a therapeutic tool of great utility, capable of deactivating genes connected with a series of diseases, including cancer, and of originating some potent medicines.

In an article in last July’s issue of the Nature Medicine magazine, the American Philip Sharp, who won the Nobel Prize for Medicine in 1993 and is a researcher with the Massachusetts Institute of Technology (MIT), showed that the mechanism of RNA interference could be used to combat the infection caused by the HIV virus of Aids. Sharp was successful insilencing genes of HIV itself – a virus whose central molecule is RNA – and of human cells in a culture medium. Science magazine regarded the studies with RNAi as last year’s most important research line.

Mastering the stage of making in the laboratory a ribonucleic acid molecule with a double strand capable of silencing one gene specifically – and only one, without interfering with the other components of the genome – is possibly the determinant factor for the success or the failure of the use of the RNAi technique in an organism. This is because, for each gene that one wants to deactivate, one has to know the complete sequence of its base pairs that code the respective protein and, from this information on the molecule, derive and construct a very particular double stranded RNA.

It was, for example, only possible to stop the synthesis of vitellogenin in bees after the researchers from Ribeirão Preto had managed to produce precisely a double stranded RNA that was complementary to the sequence of a little more than 500 base pairs that make up the codifying part of this protein’s gene. “Any carelessness at this stage means that the RNAi technique does not work”, says Zilá. After getting the double-stranded RNA, the next step is to inject this material into the organism that has the gene to be deactivated (see the illustration).

Once this is done, this modified molecule of ribonucleic acid is, inside the cell, cut into pieces of some 25 base pairs, by an enzyme called Dicer. These small stretches of perforated double-stranded RNA are then joined to a protein complex, and, in conjunction, attack the messenger RNA, cutting it up into non-functional portions. As its name indicates, messenger RNA – a conventional ribonucleic acid molecule, derived from a gene and with just one strand of base pairs – is the postman that carries the chemical order coming from its respective gene to the ribosome, the cell organelle responsible for the production of proteins. In fragments, the messenger RNA cannot take forward the recipe supplied by the gene that originated it. Accordingly, the production of the protein that would be produced from that gene is halted. Broadly speaking, this is how the RNAi technique works.

Role reviewed
To boot, the apparent success of the method for silencing genes- apparent, because nobody knows yet for sure how long this deactivation lasts for, neither is there absolute certainty that the technique does not cause side effects – ended up raising the status of the ribonucleic acid molecule. RNA was always looked on as a sort of poor cousin of deoxyribonucleic acid, popularly known as DNA, the bearer of the genetic code, needed for the production of all the proteins of an organism.

It used to be thought that RNA basically played the role of go-between stretches of DNA (the genes) and the proteins. Ribonucleic acid was looked upon as a faithful and passive deliverer, a loyal messenger, of the formula dictated by the stretches of DNA for the ribosome. No doubt, its role was indispensable, but of a more bureaucratic than creative nature. In the same way as a translator should not stray far from the original content of the work, when turning it into another language, RNA should not interfere in the meaning of the message that is entrusted to it by the DNA.

It could even make the necessary adaptations for the ribosome to understand better the chemical text dictated by the genes, but no more than that. By adding an extra strand to the conventional RNA, this notion that ribonucleic acid would never interfere with the message from the gene has crashed to earth. It not only interferes, but it may even destroy it completely, as attested by the RNAi technique.

The Project
Africanized Bees: An Integrated Analysis of the Process of Apis mellifera with a Focus on Determinants of the Fertility of Drones, Queens and Workers (nº 99/00719-6); Modality Thematic project; Coordinator
Zilá Luz Paulino Simões – Faculty of Philosophy, Sciences and Literature at USP in Ribeirão Preto; Investment R$ 967,157.42