5. DNA: The Substance of the Genes

1

Genetic Recombination in Bacteria

Index to this page

Bacteria have no sexual reproduction in the sense that eukaryotes do. The have

But the essence of sex is genetic recombination, and bacteria do have three mechanisms to accomplish that:

Transformation

Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from their surroundings [View]. The ability to deliberately transform the bacterium E. coli has made possible the cloning of many genes — including human genes — and the development of the biotechnology industry.

Link to a discussion of cloning genes by transforming E. coli with recombinant DNA molecules.

The first demonstration of bacterial transformation was done with Streptococcus pneumoniae and led to the discovery that DNA is the substance of the genes. The path leading to this epoch-making discovery began in 1928 with the work of an English bacteriologist, Fred Griffith.

The cells of S. pneumoniae (also known as the pneumococcus) are usually surrounded by a gummy capsule made of a polysaccharide. When grown on the surface of a solid culture medium, the capsule causes the colonies to have a glistening, smooth appearance. These cells are called “S” cells.

Streptococcus pneumoniae (pneumococci) growing as colonies on the surface of a culture medium. Left: The presence of a capsule around the bacterial cells gives the colonies a glistening, smooth (S) appearance. Right: Pneumococci lacking capsules have produced these rough (R) colonies. (Courtesy of Robert Austrian, J. Exp. Med. 98:21, 1953.)

However, after prolonged cultivation on artificial medium, some cells lose the ability to form the capsule, and the surface of their colonies is wrinkled and rough (“R”). With the loss of their capsule, the bacteria also lose their virulence. Injection of a single S pneumococcus into a mouse will kill the mouse in 24 hours or so. But an injection of over 100 million (100 x 106) R cells is entirely harmless.

Encapsulated (left) and nonencapsulated (right) pneumococci. The encapsulated forms produce smooth colonies (above). (Courtesy of Robert Austrian, J. Exp. Med. 98:21, 1953.)

The reason? The capsule prevents the pneumococci from being engulfed and destroyed by scavenging cells — neutrophils and macrophages — in the body [View]. The R forms are completely at the mercy of phagocytes.

Pneumococci also occur in over 90 different types: I, II, III and so on. The types differ in the chemistry of their polysaccharide capsule.

Unlike the occasional shift of S -> R, the type of the organism is constant. Mice injected with a few S cells of, say, Type II pneumococci, will soon have their bodies teeming with descendant cells of the same type.

However, Griffith found that when living R cells (which should have been harmless) and dead S cells (which also should have been harmless) were injected together, the mouse became ill and living S cells could be recovered from its body. Furthermore, the typeof the cells recovered from the mouse’s body was determined by the type of the dead S cells. In the experiment shown, injection of

  • living R-I cells and
  • dead S-II cells

produced a dying mouse with its body filled with living S-II pneumococci.

The S-II cells remained true to their new type. Something in the dead S-II cells had made a permanent change in the phenotype of the R-I cells. The process was named transformation.

Oswald Avery and his colleagues at The Rockefeller Institute in New York City eventually showed that the “something” was DNA.

In pursuing Griffith’s discovery, they found that they could bring about the same kind of transformation in vitro using an extract of the bacterial cells.

Treating this extract with

  • enzymes to destroy all polysaccharides (including the polysaccharide of the capsule)
  • a lipase to destroy any lipids
  • proteases to destroy all proteins
  • RNase to destroy RNA

did notdestroy the ability of their extracts to transform the bacteria.

But treating the extracts with DNase to destroy the DNA in them did abolish their transforming activity. So DNA was the only material in the dead cells capable of transforming cells from one type to another. DNA was the substance of genes.

View an electron micrograph showing DNA entering a pneumococcus.

Although the chemical composition of the capsule is determined by genes, the relationship is indirect. DNA is transcribed into RNA and RNA is translated into proteins. The phenotype of the pneumococci — the chemical composition of the polysaccharide capsule — is determined by the particular enzymes (proteins) used in polysaccharide synthesis.

Conjugation

Some bacteria, E. coli is an example, can transfer a portion of their chromosome to a recipient with which they are in direct contact. As the donor replicates its chromosome, the copy is injected into the recipient. At any time that the donor and recipient become separated, the transfer of genes stops. Those genes that successfully made the trip replace their equivalents in the recipient’s chromosome.

Features:

  • Can only occur between cells of opposite mating types.
    • The donor (or “male”) carries a fertility factor (F+).
    • The recipient (“female”) does not (F−).
  • F
    • is a set of genes originally acquired from a plasmid and now integrated into the bacterial chromosome;
    • establishes the origin of replication for the chromosome.
    • A portion of F is the “locomotive” that pulls the chromosome into the recipient cell.
    • The rest of it is the “caboose”.
  • In E. coli, about one gene gets across each second that the cells remain together. (So, it takes about 100 min for the entire genome (4377 genes) to make it. But,
  • the process is easily interrupted so
    • it is more likely that host genes close behind the leading F genes (“locomotive”) will make it than those farther back
    • The “caboose” seldom makes it so failing to receive a complete F factor, the recipient cell continues to be “female”
  • The DNA that makes it across finds the homologous region on the female chromosome and replaces it (by a double crossover).
  • By deliberately separating the cells (in a kitchen blender) at different times, the order and relative spacing of the genes can be determined. In this way, a genetic map — equivalent to the genetic maps of eukaryotes — can be made. But here the map intervals are seconds, not centimorgans (cM).
    Discussion of genetic mapping in eukaryotes.

Demonstration

The figure shows the mechanism of conjugation in E. colicells where

  • The “male” lacks functional genes needed to synthesize the vitamin biotin and the amino acid methionine (Bio−, Met−) so these must be added to its culture medium.
  • The “female” has those genes (Bio+, Met+) but has nonfunctional (mutant) genes that prevent it from being able to synthesize the amino acids threonine and leucine (Thr−, Leu−) so these must be added to its culture medium).
  • When cultured together, some female cells receive the functional Thr and Leu genes from the male donor.
  • A double crossover enables them to replace the nonfunctional alleles.
  • Now the cells now can grow on a “minimal” medium containing only glucose and salts.

Transduction

Bacteriophagesare viruses that infect bacteria. In the process of assembling new virus particles, some host DNA may be incorporated in them.

Illustrated discussion

The virion head can hold only so much DNA so these viruses

  • while still able to infect new host cells
  • may be unable to lyze them.

Instead the hitchhiker bacterial gene (or genes) may be inserted into the DNA of the new host, replacing those already there and giving the host an altered phenotype. This phenomenon is called transduction.

Significance of genetic recombination in bacteria.

Transformation, conjugation, and transduction were discovered in the laboratory. How important are these mechanisms of genetic recombination in nature? We don’t really know, but

Some thoughts:

  • The completion of the sequence of the entire genome of a variety of different bacteria (and archaea) suggest that genes have in the past moved from one species to another. This phenomenon is called lateral gene transfer (LGT).
  • The remarkable spread of resistance to multiple antibiotics may have been aided by the transfer of resistance genes within populations and even between species.
  • Many bacteria have enzymes that enable them to destroy foreign DNA that gets into their cells. It seem unlikely that these would be needed if that did not occur in nature. In any case, these restriction enzymes have provided the tools upon which the advances of molecular biology and the biotechnology industry depend.

Reductionism

The understanding of complex systems almost always has to await unraveling the details of some simpler system. You may feel that trying to find out how one type of pneumococcus could be converted into another was an exceedingly specialized and esoteric pursuit. But Avery and his coworkers realized the broader significance of what they were observing and, in due course, the rest of the scientific world did as well. By electing to work with a well-defined system: the conversion of R forms of one type into S forms of a different type, these researchers made a discovery that has revolutionized biology and medicine.

Attempting to understand the workings of complex systems by first understanding the workings of their parts is called reductionism. Some scientists (and many nonscientists) question the value of reductionism. They favor a holistic approach emphasizing the workings of the complete system.

But the record speaks for itself. From skyscrapers to moon walks, to computer chips to the advances of modern medicine, progress comes from first understanding the properties of the parts that make up the whole.

The late George Wald, who won the 1967 Nobel Prize in Physiology or Medicine for his discoveries of the molecular basis of detecting light [Link], once worried that his work was overly specialized — studying not vision, not the eye, not the whole retina, not even their rods and cones, but just the chemical reactions of their rhodopsins. But he came to realize “it is as though this were a very narrow window through which at a distance one can see only a crack of light. As one comes closer, the view grows wider and wider, until finally through this same window one is looking at the universe. I think this is the way it always goes in science, because science is all one. It hardly matters where one enters, provided one can come closer….”.

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26 June 2011

The Hershey and Chase Experiments

In 1952 (7 years after Avery’s demonstrationthat genes were DNA), two geneticists:

  • A. D. Hershey and
  • Martha Chase

provided further proof. They worked with a DNA virus, called T2, which infects E. coli (and so is a bacteriophage).

The figure shows the essential elements of the infective cycle of DNA bacteriophages like T2.

  • The virions attach to the surface of their host cell (a).
  • The proteins of the capsid inject the DNA core into the cell (b).
  • Once within the cell, some of the bacteriophage genes (the “early” genes) are transcribed (by the host’s RNA polymerase) and translated (by the host’s ribosomes, tRNA, etc.) to produce enzymes that will make many copies of the phage DNA and will turn off (even destroy) the host’s DNA.
  • As fresh copies of phage DNA accumulate, other genes (the “late” genes) are transcribed and translated to form the proteins of the capsid (c).
  • The stockpile of DNA cores and capsid proteins are assembled into complete virions (d).
  • Another “late” gene is transcribed and translated into molecules of lysozyme. The lysozyme attacks the peptidoglycan wall (from the inside, of course).
    Link to illustrated description of the action of lysozyme on the bacterial cell wall.
  • Eventually the cell ruptures and releases its content of virions ready to spread the infection to new host cells (e).

Bacteriophages produced within bacteria growing in radioactive culture medium will themselves be radioactive.

  • If radioactive sulfur atoms (35S) are present, they will be incorporated into the protein coats of the bacteriophages since two of the amino acids — cysteine and methionine — contain sulfur. But the DNA will be nonradioactive because there are no sulfur atoms in DNA.
  • If radioactive phosphorus (32P) is used instead, the DNA become radioactive — because of its many phosphorus atoms — but not the proteins.

Hershey and Chase found that

  • When bacteriophages containing 32P (radioactive), were allowed to infect nonradioactive bacteria, all the infected cells became radioactive and, in fact, much of the radioactivity was passed on to the next generation of bacteriophages.
  • However, when the bacteria were infected with bacteriophages labeled with 35S, and then the virus coats removed (by whirling them in an electric blender), practically no radioactivity could be detected in the infected cells.

From these experiments, it was clear that

  • the DNA component of the bacteriophages is injected into the bacterial cell while the protein component remains outside.
  • However, it is the injected component — DNA — that is able to direct the formation of new virus particles complete with protein coats.

So here is further proof that genes are DNA.

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2 April 2008

The Double Helix

The double helix of DNA has these features:

  • It contains two polynucleotide strands wound around each other.
  • The backbone of each consists of alternating deoxyribose and phosphate groups.
  • The phosphate group bonded to the 5′ carbon atom of one deoxyribose is covalently bonded to the 3′ carbon of the next.
  • The two strands are “antiparallel”; that is, one strand runs 5′ to 3′ while the other runs 3′ to 5′.
  • The DNA strands are assembled in the 5′ to 3′ direction [More] and, by convention, we “read” them the same way.
  • The purine or pyrimidine attached to each deoxyribose projects in toward the axis of the helix.
  • Each base forms hydrogen bonds with the one directly opposite it, forming base pairs(also called nucleotide pairs).
    Discussion of base pairing in DNA
  • 3.4 Å separate the planes in which adjacent base pairs are located.
  • The double helix makes a complete turn in just over 10 nucleotide pairs, so each turn takes a little more (35.7 Å to be exact) than the 34 Å shown in the diagram.
  • There is an average of 25 hydrogen bonds within each complete turn of the double helix providing a stability of binding about as strong as what a covalent bond would provide.
  • The diameter of the helix is 20 Å.
  • The helix can be virtually any length; when fully stretched, some DNA molecules are as much as 5 cm (2 inches!) long.
  • The path taken by the two backbones forms a major (wider) groove (from “34 A” to the top of the arrow) and a minor (narrower) groove (the one below).

This structure of DNA was worked out by Francis Crick and James D. Watson in 1953. It revealed how DNA — the molecule that Avery had shown was the physical substance of the genes [Link] — could be replicated and so passed on from generation to generation. For this epochal work, they shared a Nobel Prize in 1962.

External Link
Link to John Kyrk’s animations showing the structure of DNA.
Please let me know by e-mail if you find a broken link in my pages.)

 

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21 February 2011

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