At first Chlamydomonas, a type of algae, seems to be little more than green goo in a petri dish. Plants can seem boring enough, let alone one-celled plants like algae. Algae do not have any flowers, and do not produce any fruit, yet Drs. John Boynton and Nicholas Gillham have found Chlamydomonas to be an incredibly interesting tool for genetic research.
Dr. Boynton, professor of botany, and Dr. Gillham, James B. Duke Professor of Zoology, have collaborated for the past 26 years on their research into the genetics of Chlamydomonas reinhardtii, a one-celled eucaryotic plant. Chlamydomonas, or Chlamy for short, is seemingly the ideal organism upon which to conduct research. Not only does it reproduce within a matter of hours, but it has qualities of both higher plants and simpler bacteria.
Modern-day eucaryotes like Chlamy are thought to have arisen through a process of endosymbiosis, in which a primitive eucaryotic cell incorporated procaryotes into itself. Such a process is probably responsible for certain organelles (membrane-bound vesicles) in cells. Mitochondria, responsible for generating energy in the cell, are believed to have come from the alpha-purple sulfur bacterium; chloroplasts, responsible for photosynthesis in plants, are thought to have begun as cyanobacteria. For this reason, the chloroplasts of Chlamydomonas offer a unique opportunity to the genetic researcher. The chloroplast has retained its own circular DNA and its own ribosomes, separate from those in the nucleus and cytoplasm of the cell. However, it is no longer genetically autonomous.
Drs. Boynton and Gillham have been able to further the use of Chlamy as a research tool by developing a technique for transforming, or altering, its chloroplast and mitochondrial DNA with a helium "gun" that shoots new DNA into the cells on small tungsten or gold bullets. Chloroplast transformation occurs by homologous recombination and has several advantages over nuclear transformation, where the insertion of new genes is random. First of all, one can introduce site-directed mutations into resident genes; that is, it is possible to replace a specific sequence of DNA at will. Second of all, one can target the insertion of foreign genes in the chloroplast genome by flanking them with resident sequences.
The chloroplast DNA is much smaller than nuclear DNA and so it is easier to identify and isolate specific genes. Most genetic research today is conducted with simple "model" organisms such as yeast and E. coli (a bacterium), since it is easier to manipulate their small DNA. The Chlamy chloroplast offers the same advantage.
The two scientists have made considerable advances in characterizing the ribosomes of the chloroplast. (Ribosomes are composed of both RNA and proteins and are responsible for using the genetic code in messenger RNA to create the proteins needed by the cell or the chloroplast.) Chloroplast ribosomes are sensitive to inhibition by the same spectrum of antibiotics as their procaryotic ancestor. As what was once a free-living bacterium came under the control of the cell, the chloroplast lost much of its genetic information. Both the chloroplast and the nucleus were initially able to synthesize amino acids, but the set of procaryotic genes coding for these enzymes was lost so that the process was not duplicated. Moreover, a number of the proteins that are necessary for the chloroplast to function are encoded in the nucleus, indicating that genes moved from the chloroplast to the nucleus during evolution.
Drs. Boynton and Gillham, however, have shown that over one-third of the ribosomal proteins that are necessary for the chloroplast ribosome to function are made by the chloroplast itself, in addition to the ribosomal RNA that was previously proven to be encoded in the DNA of the chloroplast. This is an important finding, as it shows that the chloroplast has retained many similarities to its cyanobacterial ancestors. The finding also suggests the chloroplast ribosome as a model system for research on interactions between nuclear and organelle genomes. By isolating and characterizing mutant varieties of Chlamy with altered chloroplast protein synthesis, the researchers are able to identify what genes, and thus what proteins, are responsible for how the chloroplast ribosome works.
Their studies have recently explored the "translational fidelity domain" of the chloroplast ribosome, which is responsible for checking that the correct amino acids are added to the protein being synthesized. Specifically they have used the drug streptomycin to cause the ribosome to misread the messenger RNA sequence. This causes the cell to create incorrect protein sequences; the resulting proteins are not functional for their intended purpose, which disrupts cell function.
It is possible to create mutant ribosomal RNA or protein and achieve a resistance to streptomycin. One interesting mutant, for example, became dependent on the streptomycin so that it was too slow at manufacturing the protein without the streptomycin present. The mutant ribosome rechecked the coding sequence and the corresponding amino acid sequence for mistakes so often that without the streptomycin present to create mistakes the process of translation was retarded.
The process by which gene expression is regulated in the chloroplast is also a field of interest for the two scientists. In order to make a protein a cell must first transcribe the gene into messenger RNA encoding that protein. This RNA message is then translated into protein by the ribosome. Unlike the nuclear genes of the cell, where transcription factors bind to the DNA to control the rate of gene expression, the chloroplast appears to control protein synthesis through what is referred to as translational regulation. In this process, the RNA message is transcribed from DNA without much regulation, but it is only translated into protein at certain times in response to particular signals.
The two scientists have identified what is referred to as the "leader region" of the RNA as responsible for translational regulation. The leader region is a two hundred-base region at the beginning of the messenger RNA that is transcribed into RNA but not translated into protein. Proteins binding to the leader region can control when the messenger RNA can associate with the chloroplast ribosome and be translated into protein. Experiments in which leader regions on two different messenger RNA's are exchanged show a change in when a specific protein is turned on.
Drs. Boynton and Gillham have been able to identify a number of proteins that bind to this leader region constitutively, as well as proteins that bind in response to specific environmental stimuli. Environmental factors include things such as bright light. The cell must know when light is present so that it can manufacture all the proteins that are necessary for photosynthesis. Light signals the chloroplast to translate a large number or all of the RNA messages present since many are necessary for photosynthesis. Such environmental factors are thus termed class-specific factors as opposed to gene-specific.
Drs. Boynton and Gilham believe that Chlamydomonas could become as good a tool as yeast for basic genetic research. Although its nuclear genome is five times the size of yeast's, it may be more easily manipulated than those of more complex eucaryotes. Moreover, knowledge gained from utilizing Chlamy as a model system for studying chloroplast genetics and molecular biology might make it possible someday to manipulate this organelle in crop plants. The chloroplasts of plants contain many copies of their genome per cell. Thus it may be possible to amplify proteins by inserting their genes in the chloroplast DNA rather than in the nuclear DNA. To do this, however, one must know how the chloroplast DNA and protein-synthesizing systems function. There are multiple factors that control the transcription and translation of a protein from the chloroplast that would need to be adjusted in order to use the chloroplast as a factory for the manufacture of foreign proteins.
The researchers hope that the tobacco plant, with genes inserted into its chloroplasts, might be used someday to manufacture mass quantities of commercially valuable proteins. In collaboration with a visiting Japanese scientist from Sumitomo Chemicals, Ltd., the Boynton and Gillham lab has isolated a gene encoding resistance to a new herbicide developed by Sumitomo that can be used in lower concentrations to kill weeds and is less toxic to mammals.
The two are associated with the Duke Chlamydomonas Genetics Center, directed by Dr. Elizabeth Harris. The Center, which houses more than 1,500 strains of Chlamy, is composed largely of different mutants identified and characterized by researchers around the world -- including many from the Boynton and Gillham laboratory.
At the time this article was written, Iain Cheeseman was a Trinity College sophomore planning on majoring in biology.
Fall 1994 contents
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