Celebrating the Pioneering Experiments in Genetics

It seems that lately everything in genetics, which has morphed into genomics, is big, big, big.

• Data on half a million people represented in the UK Biobank are highlighting genome regions associated with difficult-to-study traits, like sexuality and handedness.
• The All of Us initiative at the National Institutes of Health, which plans to capture info on a million or more people, is ever-expanding, from embracing Native communities to welcoming health care providers.
• A pair of articles in the latest Nature Genetics describes algorithms that shift the mindset from “the” human genome to the many variations on the theme. Capturing how we differ will speed diagnoses, ease the finding of relatives, and fill in our evolutionary trees.

Another new Nature Genetics report, “A reference genome for pea provides insight into legume genome evolution ,” took me back to the origins of genetics and Gregor Mendel, who deduced the two basic laws of heredity by breeding pea plants with a handful of distinctive characteristics. A way for the general public to better understand what science is and how scientists think would be to set aside the mega studies for a moment and look back at the brilliant experiments that built the field of genetics, from those of Mendel to the beginnings of molecular biology.

So here are six of my favorite experiments in genetics, from Mendel’s peas to double helices, chosen for their insight and creativity. These are in addition to my DNA Science post about the 19-year-old college student who invented gene mapping, paving the way to genome sequencing and consumer DNA ancestry tests.

The Two Laws of Inheritance: Mendel

I’m harvesting my vegetable garden now, and I can see how observing variation in traits like bean color and pod length could inspire thoughts of heredity. Like other farmers and gardeners, Mendel (1822–1884) wondered why some traits disappeared in one generation, only to reappear in the next.

As a child, Mendel tended fruit trees. He developed an interest in statistics, and eventually taught natural science at a monastery in Brno in the Czech Republic. He did experiments, too. By pollinating pea plants by hand, Mendel controlled the breeding to observe the comings and goings of 7 traits, cataloging them in 24,034 plants, from 1857 to 1863.

He noted the ratios of phenotypes (visible traits) and imagined factors, which he called “elementen,” with two manifestations that were inherited, such as tall versus short plants and green versus yellow peas. Elementen combined at each generation, one from the male parent and one from the female. Mendel also deduced, using crosses and scoring phenotypes of progeny, how elementen on two different chromosomes are inherited – brilliant, because chromosomes and DNA had yet to be described.

Mendel’s laws are universal. What works for peas also underlies the inheritance of single-gene conditions, such as cystic fibrosis or sickle cell disease, in people.

A Transforming Principle: Griffith

World War I had just ended. Frederick Griffith (1871–1941), a medical officer at the British Ministry of Health, was investigating people who’d died during the great influenza pandemic of 1918, but from an especially fierce bacterial pneumonia. He noticed that some patients had more than one strain of Streptococcus pneumonia in their sputum.

To investigate how the severe pneumonia arose, Griffith recreated the scenario in mice infected with two types of the bacteria: one with smooth surfaces that the rodent immune system couldn’t destroy and that killed the mice swiftly, and rough bacteria that the immune system recognized and removed, so the mice lived. Griffith showed that instructions for the trait of killing ability passed from smooth to rough bacteria.

He “heat-killed” smooth bacteria, and injected alone, they couldn’t sicken mice. But when he injected heat-killed smooth bacteria along with live rough bacteria, the mice died, their blood swarming with bacteria wrapped in smooth, sugary coats! Something in the smooth bacteria, Griffith deduced, turned the normally tame rough bacteria into killers, and he named that something the “transforming principle.” The works of Griffith, published in 1928, and that of Mendel, were discovered and appreciated years after their deaths. Griffith was killed during the London blitz, in his lab.

The Transforming Principle: Avery, Macleod, and McCarty

At first people thought protein was the stuff of heredity, perhaps because we knew more about proteins. In 1944, three physician/geneticists at Rockefeller University, Oswald Avery (1877–1955), Colin MacLeod (1909–1972), and Maclyn McCarty (1911–2005), showed that DNA is the transforming principle.

They repeated Griffith’s experiments, but with a twist. They added either an enzyme that destroys protein or an enzyme that destroys DNA. Smooth bacteria could transform rough bacteria into killers when their protein was destroyed, but not when their DNA was destroyed.

DNA, Not Protein: Hershey and Chase

Next, Alfred Hershey (1908–1997) and Martha Chase (1927–2003), at the Cold Spring Harbor Laboratory on Long Island, provided more direct evidence of the primacy of DNA. They infected bacteria (E. coli) with viruses, called T2. Viruses aren’t cells, they’re just DNA or RNA in protein coats, and they inject their genetic material into host cells, leaving the protein coats outside. Viral DNA (or RNA copied into DNA) takes over protein synthesis in the bacteria, which mass-produce the viruses.

Hershey and Chase grew viruses on two types of growth medium. One contained radioactive phosphorus, which is found in nucleic acids but not in amino acids (the building blocks of protein). The other medium contained radioactive sulfur, which is found in amino acids but not in nucleic acids. Whichever marked molecule prevailed from bacterial generation to generation would indicate the nature of the genetic material.

So Hershey and Chase infected bacteria with the viruses, then poured the preparations into small tubes. The tubes then were spun in a centrifuge, which is a little like a clothing dryer, although the protocol became known as “blender experiments.” The oscillation shook off the viral protein coats clinging to the infected bacteria, easily scooped from the top, while the viral DNA entered the cells, which sunk to the bottom of the tubes.

When the radioactive sulfur ended up in the floating protein coats while the radioactive phosphorus in bacteria settled down, Hershey and Chase had their answer. The genetic material is DNA, and not protein.

How DNA Replicates: Meselson and Stahl

Watson and Crick’s famous 1953 paper introducing DNA ended with a tantalizing statement that the double helical structure “immediately suggests a possible copying mechanism for the genetic material.” A second paper fleshed out that idea: the double helix separates and each exposed half pulls in new nucleotide building blocks to build a new double helix, like a row of dance partners parting and pulling in new mates.

In 1954, two researchers met at a summer course that Watson and Crick were teaching at Woods Hole, and became intrigued with the idea of “semi-conservative” DNA replication, the “semi” referring to the fact that each DNA generation preserves half the helix from the parent strands. Matthew Meselson and Franklin Stahl met again at the California Institute of Technology, where they carried out what many have called “the most beautiful experiment in biology” (I agree). Their work not only demonstrated semi-conservative DNA replication, but ruled out the other two possibilities—that one double helix creates another (conservative replication), or that a double helix shatters and rebuilds itself into two using spare parts (dispersive replication).

Meselson and Stahl grew E. coli on medium containing radioactive nitrogen, which would become incorporated into DNA and make it heavier (denser). The heavy label would make DNA sink faster and further in a tube than non-radioactive DNA. By growing bacteria on radioactive medium, then switching the cells to non-radioactive medium, Meselson and Stahl deduced, from the patterns of heavy and light cells over a few generations of DNA replication, that the process is semi-conservative, and not due to either of the other two mechanisms. That is, newly-replicated DNA was half as heavy as it would have been if a double helix just doubled itself in its entirety. Further experiments ruled out the dispersive explanation. Their findings were repeated in other species, with DNA marked in different ways.

The Genetic Code: Crick, Nirenberg, and Matthaei

Marshall Nirenberg and postdoctoral fellow Johann Matthaei, at the National Cancer Institute, deciphered the genetic code: how a DNA sequence of four types of nitrogen-containing bases, A (adenine), G (guanine), C (cytosine), and T (thymine) tells a cell to string together a specific sequence of amino acids.

Crick had already deduced, in experiments that added or removed one, two, or three DNA bases at a time from a virus with known DNA sequence, that the genetic code was non-overlapping and that a 3-letter “code word” was the simplest to specify the 20 amino acids of biological proteins.

Nirenberg and Matthaei actually cracked the code, working with a soup of ribosomes (the cell structures on which proteins are assembled), enzymes, various RNAs, and amino acids. To this “cell-free system” they added short, simple RNAs made only of a single base, like alphabet soup that contained all one type of letter. Then they checked which amino acids the cell-free system knit together.

First, the RNA base uracil (U) (chemically similar to the DNA base thymine), led to protein snippets made solely of the amino acid phenylalanine. Therefore, UUU = phenylalanine.

The puzzle pieces emerged. AAA RNA encoded lysine, and CCC proline. GGG fell apart, but “co-polymers,” such as UGUGUG . . ., filled in the blanks.

UGUGUG specified alternating cysteines and valines; UGUUUUUGUUUU specified phenylalanines alternating with cysteines. Since UUU encodes phenylalanine, then UGU encodes cysteine and therefore GUG encodes valine. By 1961, the code was revealed in this stepwise, logical manner.

The genetic code is universal; all species use the same DNA and RNA code words to specify the same amino acids. Phrases like “the human genetic code” actually mean DNA or genome sequence. The universality of the genetic code is both the basis of biotechnology and provides profound evidence that all life descends from shared ancestors.

Coda

These elegant experiments share a few things that illustrate how science-to-explain-nature works – as opposed to uploading data and deploying an algorithm to fuel discovery.

• All of the experiments arose out of curiosity about something in nature, such as disappearing traits in plants and pneumonia in flu patients.
• Scientists invented words to describe their deductions and conclusions as findings unfolded, and sometimes the terminology didn’t last: Mendel’s elementem became genes by 1909, thanks to Danish botanist Wilhelm Johannsen. Watson and Crick’s assembling of the findings of others finally assigned a structure to Griffith’s transforming principle: the DNA double helix.
• The researchers devised ways to figure out how their observations happen – and just as importantly, how they don’t. Science disproves hypotheses to focus in on the most likely explanation for evidence. It doesn’t prove and may not be the final word.
• Observations and hypotheses rise to the level of theory or law as other researchers confirm findings in additional species and/or in different ways. Thus, inherited traits follow Mendel’s laws, the genetic material is a nucleic acid, DNA replicates semi-conservatively, and all creatures great and small use the same genetic code.