Who would have thought that a Scottish sheep named Dolly could cause an international uproar? But she has, and it's all because of her unorthodox beginnings. Dolly is not an ordinary sheep, produced through the mating of a ewe and a ram. She is a clone, an exact genetic replica of her donor "mother," a six-year-old female sheep. And that fact, together with another recent announcement that scientists in Oregon have cloned monkeys from embryonic cells, has triggered debates among researchers, ethicists, and politicians all over the world. (See: Making The Monkeys)

The ethical controversy over any future experiments that involve the cloning of human beings -- which has NOT occurred -- is likely to dominate news coverage of the subject for months to come. Before engaging in such a debate, it is important to understand what the process of cloning involves, and why Dolly is so special. It is also important to understand some of the key questions that Dolly raises.

WHAT IS A CLONE?

The noun "clone" and the verb "to clone" are not used consistently. In biology, a clone is a cell or an organism that is genetically identical to another cell or organism. Many simple organisms such as bacteria reproduce themselves by copying their DNA and splitting in half. The two bacteria that result from this form of asexual reproduction are genetically identical; they are clones of each other. In contrast, during the process of sexual reproduction, the nucleus of a sperm cell, which carries the father's DNA, fuses with the nucleus of the egg cell, which contains the mother's DNA. The resulting offspring carry genetic material from both parents and are not identical to either parent. (See: More Ideas About Clones...And Differentiation)

The verb "to clone" refers to the process of creating cloned cells or organisms. The process differs, depending on the kinds of cells used in the cloning procedure and the desired result. Usually, when scientists clone an animal, they take the nucleus of a cell -- which contains chromosomes made of deoxyribonucleic acid (DNA) and proteins -- and place it into an egg cell (also called an oocyte) from which the nucleus has been removed. The egg cell then divides to produce an embryo that develops into an animal, if the procedures work as planned.

In previous cloning experiments with cattle and mice, the "donor" chromosomes came from very early embryos or, in the case of cloned frogs, from young tadpoles. Very early embryos from mammals are called blastocysts, balls of immature cells that are produced after a fertilized egg divides several times, but before any specialized tissues begin to develop. Recently, two Rhesus monkeys were produced from embryonic cells in blastocysts by a method called nuclear transfer

HOW WAS DOLLY CREATED?

Dolly is different. She was generated from a specialized adult cell, not from an unspecialized embryonic cell.

To create Dolly, Ian Wilmut of the Roslin Institute in Edinburgh, Scotland, and his colleagues used a cell derived from the udder of a six-year-old sheep in the final stage of pregnancy. The researchers fused the adult udder cell with an oocyte that was ready to be fertilized, but taken from a different sheep. The scientists had previously removed the nucleus from the oocyte, and they used an electrical current to fuse it with the udder cell. The key to Dolly's success was to make the nucleus of the donor cell "quiet" so that it stopped behaving like an udder cell and could be reprogrammed to become an embryo. (See: "Why Was Dolly A Success?" below.)

The resulting embryo -- which became Dolly -- carried all of the chromosomes from the donor udder cell and none of the nuclear chromosomes from the host egg cell. Therefore, Dolly is an exact genetic copy -- a clone -- of her donor-cell "mother." Wilmut and his colleagues published their findings in a scientific paper in the 27 February, 1997, issue of Nature.

WHY DID RESEARCHERS CREATE DOLLY?

Wilmut and his collaborators created Dolly because they are trying to find ways to produce livestock that carry specific genetic traits. Two biotechnology companies funded the research. One, called PPL Therapeutics, is trying to find ways to produce animals that carry certain proteins in their milk. The theory is that if researchers can develop animals with desirable characteristics, they can then clone those animals to produce entire herds that carry the same traits.

WHY IS DOLLY SO SPECIAL?

While some people worry about cloning humans, most scientists celebrate Dolly. Her very existence contradicts a long-standing idea about embryonic development, and scientists enjoy research results that turn an old theory upside down. However, it is important to remember that the researchers in Scotland tried 277 times to create cloned sheep, and they succeeded only once. They are still trying to improve the techniques used to create Dolly.

Dolly is special because she disproves the notion that cells from an adult animal are too specialized to generate a new organism. Researchers have known for a long time that during embryonic development, cells become specialized because some of their genes are turned off (inactivation) and others are turned on (activation). Before Dolly, scientists thought that many genes in adult cells are permanently turned off. They believed that only the genes in the fertilized egg, embryo, or very young animal are totipotent -- all-powerful, fully activated, and capable of generating a new organism. But Dolly, it seems, proves the old theory wrong.

"Dolly is remarkable because she was cloned from an adult, somatic cell," says Alan Wolffe of the laboratory of molecular embryology at the National Institute of Child Health and Human Development in Bethesda. A somatic cell is a cell of the body that carries the full number of chromosomes for that species -- 54 chromosomes for sheep and 46 for humans. Somatic cells such as muscle cells, nerve cells, and skin cells are usually specialized to perform certain functions.

Despite the enthusiasm about Dolly, not all scientists are convinced that she was generated from an adult cell that was fully specialized or differentiated. (See: More Ideas About Clones...And Differentiation) Some researchers think that Dolly may have resulted from an udder cell that retained some of the characteristics of embryonic cells. The skeptics point out that some of the cells in the udder of a pregnant sheep (such as Dolly's genetic "mother") are not fully differentiated. This is because the udder tissue in a sheep or cow is mammary tissue -- similar to that in the breast of a human. During pregnancy, cells in mammary tissue make the glands and ducts that produce and carry milk. So, some scientists reason, certain cells in the mammary tissue of a pregnant female sheep such as Dolly's donor "mother" are capable of developing into different kinds of cells -- possibly making it easier to reprogram them to an embryo-like state.

Despite some lingering questions about the properties of the cell used to generate Dolly, she remains an international phenomenon. Dolly proves that it is possible to clone an apparently healthy animal from the nucleus of an adult cell.

WHY WAS DOLLY A SUCCESS?

The key to Dolly's success was to make the nucleus of the donor udder cell behave like the nucleus of a normal, fertilized egg that could develop into a baby sheep. The process involved several key steps that involved the preparation of the donor udder cell and the preparation of the host oocyte.

To prepare the donor udder cell, Wilmut and his colleagues removed cells from the udder of a pregnant sheep, and grew them in plastic culture dishes in an incubator. Then, after the cells had divided several times, the researchers changed the growth medium -- the nutrient-rich fluid used to grow cells in culture. Instead of containing 10 percent blood serum, the new growth medium for the udder cells contained only 0.5 percent serum, which literally "starved" the cells of nutrients. And, in the case of generating Dolly, serum-starvation blocked the udder cells from dividing further, a phenomenon called cell-cycle arrest.

To understand how Dolly was created, it is necessary to understand the normal cell cycle. The cell cycle is a series of molecular events that allows a cell to reproduce itself. In normally dividing cells, the cell cycle progresses through four phases -- mitosis or cell division (M), a cell-growth phase called "gap" 1 (G₁), a period of DNA synthesis in which the DNA replicates itself (S), and a second "gap" phase (G₂) in which the cell, which now carries twice its normal amount of genetic material, prepares to divide. But serum-starved cells depart from the cell cycle and arrest in a phase called G_0. The nucleus of the udder cell used to generate Dolly was arrested in G₀, which allowed it to be reprogrammed to form an embryo.

Meanwhile, the Scottish researchers also had to prepare the host oocyte, which they obtained from a different sheep. They collected oocytes that were ready to be fertilized, meaning that the nuclei of the oocytes carried only half the normal number of chromosomes. The researchers removed the nucleus from each egg cell and used a mild electrical current to fuse it with a nucleus from an udder cell. At that point, a series of events occurred that is still not well understood.

Researchers know that the cytoplasm of an oocyte contains proteins that encourage embryonic development. They do not know what all of those proteins are. In the case of Dolly, some of the proteins in the egg cell cytoplasm reprogrammed the nucleus of the donor udder cell so that it could divide to form an embryo. This series of events involved stripping away proteins that are normally bound to the DNA of adult cells and replacing them with proteins that are normally bound to the DNA of embryonic cells. (Different kinds of proteins are bound to DNA; a major group is called histones.)

After the DNA-linked proteins were removed and replaced, other proteins in the oocyte cytoplasm began to turn on genes in the udder cell nucleus. These events normally occur after an egg cell is fertilized by a sperm cell. At this point in the case of Dolly, the nucleus of the udder cell began to behave like the nucleus of a fertilized egg. The gene-activating proteins are called transcription factors, because they allow the DNA that makes up the genes to be transcribed into a related molecule called messenger RNA (ribonucleic acid). The conversion -- or transcription -- of DNA into messenger RNA is the first step toward making the new proteins that a growing embryo (or any other kind of cell) requires.

Once the donor, udder-cell nucleus and the host, egg-cell cytoplasm interacted successfully, the embryo that became Dolly began to develop normally. After it divided several times and became a tiny ball of cells called a blastocyst, the researchers placed it into the uterus of an adult female sheep -- Dolly's surrogate mother. The sheep carried Dolly through a normal pregnancy and gave birth to the lamb that has caused so much controversy. When Wilmut and his collaborators analyzed the DNA of Dolly's cells, they found that it was identical to the DNA of the donor udder cell. That is why Dolly is a clone.