CHEMICAL & ENGINEERING

NEWS volume 38. NUMBER 23 The Chemical World This Week JUNE 6, 1960

Scientists Dig Deeper into Nucleic Acids Hundreds of workers probe their structure and composition, gain insight into their biochemical role in life processes

A revolution in biochemistry, biol­ogy, and medicine is in the making. And at the bottom of this is the in­creasingly intensive research in nucleic acids—those huge molecules which, in one way or another, direct the func­tions of all living cells.

Results in this kind of research-being done by hundreds of workers all over the world—come very slowly. To get them, scientists must use the most painstaking techniques and all the ingenuity at their command. But the dividends promise to more than justify the efforts. As the picture of the structure and functions of nucleic acids becomes clearer, so also will un­derstanding of all basic biochemical processes. Among them: protein synthesis, genetics, cell differentiation, viruses, diseases, immunology, and cancer.

Structure a Big Problem. The problem of the structure of nucleic acids is common to all the areas in which work is going on. Both de­oxyribonucleic acid (DNA) and ribo­nucleic acid (RNA) are among the most complex materials known. Each has a backbone of sugar units linked by phosphate groups-deoxyribose for DNA and ribose for RNA. A purine or pyrimidine base is attached to each sugar fragment. In DNA, the bases are adenine, guanine, cytosine, and thymine. In RNA, the fourth base is generally uracil instead of thymine. This part of the picture is fairly simple.

What complicates the structure problem is the tremendous size of the nucleic acids. The simplest natural ones contain thousands of sugar-base units. Those from higher organisms may have a thousand times as many. For each nucleic acid, the order of

the four bases strung out along each chain is different. The order of the bases is the code that determines the species of an organism, differences be­tween individuals in a species, differ­ences between cells of an individual, and the biochemical processes that go on in every cell. To do all this, there must be a staggering number of differ­ent DNA's and RNA's.

Watson and Crick. One break­through in understanding DNA struc­ture came when Watson and Crick of Cambridge University proposed their model for its general structure. They proposed that it consists of two long strands wound in a double helix around a common axis. The two strands are held together by hydrogen bonds between the bases along each strand.

The Watson-Crick theory has held up well over the past decade. Most research men in the field acclaim it as a significant development, and evi­dence generally supports their picture. But exceptions are beginning to show up. Dr. R. L. Sinsheimer at Caltech has found a single-stranded DNA, and Dr. Aaron Bendich of Sloan-Kettering Institute believes that polyoma DNA may also be single-stranded. He ex­pects to have an answer on this in a few months. Also, RNA is now be­lieved to be single-stranded, but Dr. Wendell Stanley of the University of California points out that single strands may fold back on themselves.

Dr. Paul Doty and his group at Har­vard recently showed that DNA could be separated into its two strands by heating, then put back together again (C&EN, May 9, page 38) . He even succeeded in combining single strands from two different strains of bacteria

to make a hybrid DNA. Although this is strong evidence for the Watson-Crick structure itself (all that the Doty group claims), there is some disagreement as to whether it bears out Watson and Crick's ideas on DNA replication, since the Harvard results were obtained under conditions that don't exist in the cell.

Watson and Crick envisioned the separation of the strands of a helix, during which each one directs the syn­thesis of its complementary partner. The result: two helixes, each with two strands. Dr. Liebe Cavalieri of Sloan-Kettering believes that the strands do not separate and that a Watson-Crick helix replicates in toto. He isolates DNA as a double (two-stranded) helix, and later as a dimeric helix (four-stranded). The latter one may be two helixes stuck together, immediately after replication of the DNA.

Finding the Sequence. Most inves­tigators agree that the most difficult of the structure problems is the sequence of purine and pyrimidine bases along a given nucleic acid chain. Many sci­entists are putting a great deal of ef­fort into getting this information, bit by bit. Among them: Dr. Leon Hep-pel and coworkers at the National In­stitutes of Health.

These investigators use enzymes that hydrolyze RNA at specific bonds or in a definite order. Pancreatic ribonuclease, for example, has long been very valuable for this type of research, Dr. Heppel says. This enzyme hydrolyzes the phosphate ester bond that connects a pyrimi­dine nucleotide portion of RNA to the next nucleotide fragment of the chain. It will not touch similarly bonded

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ULTHACENTRIFUGE YIELDS NUCLEIC ACID FACTS. Dr. R. L Sinsheimer of Caltech used the ultracentrifuge to confirm that he had found a single-stranded DNA. Ultracentrifugation is an important technique in research on nucleic acids

purine nucleotides. By analyzing all the fragments, Dr. Heppel gets some information on the order of the bases. His group is using other enzymes, t o o -some discovered at NIH, some by other investigators. One enzyme-snake venom diesterase—hydrolyzes RNA stepwise, as determined in the laboratories of Dr. H. G. Khorana and Dr. Heppel. Although this process can't be stopped at will, it does yield some useful information, Dr. Heppel says.

Another group, headed by Dr. David Lipkin at Washington Univer­sity in St. Louis, has a method based on alkaline degradation of RNA. They isolate the nucleotides to determine the end groups in nucleic acid chains.

Also using controlled degradation, Dr. K. Burton of Oxford University, England, finds evidence that some complex polynucleotides have blocks of purines and pyrimidines in their chains, rather than a more random arrangement.

No Pure DNA. Scientists hope that these bits of information will some day form a complete map of the base sequence in a particular nucleic acid. But one major obstacle remains be­fore this can ever be done—isolation of a pure nucleic acid. Scientists are obtaining increasingly pure samples of DNA and RNA, Dr. Heppel says, but no one can say that he has a pure one in the sense that there is exactly

the same order of bases in all of the molecules of a sample. In this sense, he adds, knowledge of RNA is in an even more primitive state than that of DNA.

Dr. G. L. Brown of King's College, London, believes that the fractiona­tion problem is such a big one it is not likely to be solved soon, if ever. Even if the molecules were separated into fractions of the same molecular weight and composition, he says, there would be many isomeric molecules that dif­fered only in base sequence. The most likely way to get around this, he adds, is to study DNA and RNA from cer­tain viruses or bacteriophages that seem to have only one type of mole­cule. Example: tobacco mosaic virus, which consists of one strand of RNA, coiled or folded, surrounded by a pro­tein.

Many research workers are attacking the structure problem by studying small polynucleotides. Dr. Heppel at NIH uses nucleases to make polyribo­nucleotides of various sizes, then studies their properties in enzyme sys­tems, as well as certain physical prop­erties. At first he investigated mole­cules with only two to five units; later, work by NIH's Dr. H. A. Sober made it possible to fractionate molecules with as many as 12 units. Dr. Heppel hopes to get even larger ones.

Dr. A. M. Michelson at the Guinness

Laboratory in Dublin has studied the properties of some synthetic, small polynucleotides. He has shown that stacking of bases—essential for the macromolecular configuration—occurs even at the dinucleotide stage and that the interaction that causes this isn't due only to hydrogen bonding, but probably involves pi electron interac­tion.

Ochoa and Kornberg. Among the top scientists in nucleic acid research there seems to be agreement on one point: The work of Dr. Severo Ochoa and Dr. Arthur Kornberg is one of the most important developments of recent years. World recognition came in the form of the 1959 Nobel Prize in Medi­cine, which these two men shared (C&EN, Oct. 26, 1959, page 19). The accomplishments: Dr. Ochoa's synthesis of RNA and Dr. Kornberg's synthesis of DNA.

Dr. Ochoa's work is based on the discovery of polynucleotide phos-phorylase by his associate Dr. Mari­anne Grunberg-Manago. Using this enzyme, he has been able to polymer­ize single nucleotides (adenylic, guanylic, uridylic, and cytidylic acids) into RNA strands. The method will make homopolymers (poly A, poly U) or copolymers with varying propor­tions of different nucleotides (poly AU,polyAGUC).

In their synthesis of DNA, Dr. Korn­berg and his coworkers started with the four nucleotides that make up DNA and converted them to their triphos­phates. The key step takes place in a neutral phosphate buffer containing the four substrates, a small amount of natural DNA to act as a "template," and the enzyme polymerase, extracted from E. coli. The enzyme polymerizes the nucleotides in the same order as they are in the template. The new DNA seems to be identical with the natural material, but its biological ac­tivity remains to be demonstrated.

Another important development in nucleic acid synthesis came from a re­search team headed by Dr. H. G. Khorana at the British Columbia Re­search Council in Vancouver. Their work differs in an important way from that of Ochoa and Kornberg. No en­zyme is used in the synthesis. With straight organic chemical techniques the Khorana group synthesized small polynucleotides and determined their structure. Many workers point to this as a good example of the contribu­tion that organic chemistry' can make in nucleic acid research.

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