Saturday, February 4, 2012

What is Chlamydomonas reinhardtii?


The Organism


Chlamydomonas reinhardtii is the best-researched member of the green algal genus Chlamydomonas (Greek chlamys, a cloak, plus monas, solitary). Chlamydomonas reinhardtii is unicellular with a definite cell wall that consists of glycoproteins rich in the amino acid hydroxyproline. A large, solitary chloroplast folded into a cup shape dominates most of the cytoplasm. The presence of this chloroplast allows autotrophic growth, although C. reinhardtii is capable of using acetate as an external carbon source. A circular body that is prominent within the chloroplast is referred to as the pyrenoid. It is the site of carbohydrate synthesis during the light-independent reactions of photosynthesis. The chloroplast also contains a red eyespot with a rhodopsin-like pigmented photoreceptor, called the stigma, that permits phototaxis. Chlamydomonas reinhardtii cells display positive phototaxis (that is, swimming toward light) in moderate light and negative phototaxis in intense light.















The cell nucleus is visible with light microscopy and predominates cross-sectional images in electron microscopy, along with the nucleolus. Electron microscopy also indicates sixteen or more chromosomes, which is consistent with the seventeen linkage groups defined by cytogenetic analysis. The cell’s anterior end consists of two contractile vacuoles, and mitochondria are dispersed throughout the cytosol. Two long, whip-like flagella extend from basal bodies, which are also located at the anterior end of the cell. Chlamydomonas reinhardtii swims using a breaststroke motion. Internally the flagella consist of a central pair of microtubules surrounded by nine doublets. Each doublet consists of arms made of the protein dynein. The dynein interacts with adjacent doublets by pressing and sliding against the neighboring microtubule when adenosine triphosphate (ATP) is hydrolyzed. This brings about the flagellar beat and allows the organism to swim.



Chlamydomonas reinhardtii reproduces asexually by mitotic divisions. Parental cells can produce as many as sixteen progeny cells by successive divisions within the cell wall. Each progeny cell secretes a cell wall and generates flagella. The new cells escape by secreting autolytic enzymes that digest the parental cell wall.




Mating and Laboratory Analysis

The vegetative form of C. reinhardtii is haploid and exists as one of two genetically distinct mating types (mt+) and (mt-). When deprived of nitrogen, cells of each mating type differentiate into gametes. Gametes of opposite mating types come into contact with each other by way of their flagella. The gametes fuse, thereby forming a zygote. The zygote secretes a heavy wall and becomes a zygospore. Zygospores can remain dormant and viable in soils for several years. Light and nitrogen can bring about zygospore germination. Four biflagellated cells, known as zoospores, are released. In some strains, meiosis occurs prior to the release of zoospores, followed by a mitotic division. The result is the release of eight zoospores rather than four.


Cells of C. reinhardtii are easy to culture. They grow copiously in defined culture media under varying environmental conditions. Mating can be induced when cells of opposite mating types are placed in a nitrogen-free medium. The zygote formed from such a mating can produce four unordered tetrads on appropriate media. Sometimes an additional mitotic event generates eight haploid products that are easy to recover. These features have made C. reinhardtii extremely useful as an experimental organism.




Mutagenesis and Transmission Genetics

Research in the 1950s led to the isolation of mutants displaying defects in the ability to photosynthesize. Since then mutants have been developed that affect every structure, function, and behavior of C. reinhardtii. Ultraviolet or chemical methods can be used to induce mutants. One of the first mutants isolated was resistant to the antibiotic streptomycin (designated sr). These cells are able to grow on media supplemented with streptomycin as well as media free of streptomycin. Wild-type cells (designated ss) are unable to grow on media containing the antibiotic. Reciprocal crosses with cells of these distinct phenotypes resulted in segregation patterns that departed significantly from Mendelian expectations. The sr phenotype was clearly transmitted only through mt+ cells. Further study has shown that resistance passed through the mt+ chloroplast. The chloroplast contains more than fifty copies of a circular, double-stranded DNA molecule. Uniparental inheritance has been demonstrated for the mitochondrial genome, too. This genome contains fewer genes than the chloroplast, but antibiotic resistant mutations have been generated, along with other types. It is interesting to note that mitochondrial inheritance of antibiotic resistance appears to be transmitted by way of mt- cells.


Mutational analysis has elucidated aspects of nuclear inheritance, also. The mating type phenotype segregates in a 1:1 ratio in accordance with Mendelian principles. With the advent of molecular techniques, insertional mutagenesis has resulted in a wide array of mutants, including nonphotosynthetic, nonmotile, antibiotic resistant, herbicide resistant, and many more. This type of analysis has resulted in mapping nearly two hundred nuclear loci.




Molecular Analysis

Transformation of C. reinhardtii is relatively easy and can be carried out by mixing with DNA-coated glass beads or electroporation, that is, using a current to introduce the DNA into a cell. The frequency of transformation success is highest in wall-less mutants or cells whose walls have been removed prior to transformation. Both nuclear, mitochondrial, and chloroplast transformation studies have been performed, leading to the development of several molecular constructs that have been used to study gene expression. Cosmids and BAC libraries have been created for several markers in order to make the current molecular map of about 240 markers, each having an average spacing of 400 to 500 kb. These markers have been placed on the seventeen linkage groups mentioned previously.


Thus far, the greatest impact these molecular markers are having is in the study of photosynthesis. A chloroplast gene known as Stt7
has been characterized using these methods. Stt7 is required for activation of the major light-harvesting protein and interactions between photosystem I and photosystem II when light conditions change. Chloroplast and nuclear transformations have been used in conjunction with developmental mutants to study chloroplast biogenesis. This has increased researchers’ understanding of the expression and regulation of many chloroplast genes. A cDNA library composed of many unique chloroplast genes is being constructed and their coding regions sequenced. These cDNAs are called expressed sequence tags (ESTs) and have proven extremely useful for identifying protein-coding genes in other organisms. Thousands of these cDNAs could be placed on pieces of glass the size of a microscope slide using microarray technology to monitor changes in gene expression of virtually the entire genome at the same time. Interactions between the nuclear genome and the chloroplast genome can be assessed in this manner as well.




Key Terms



bacterial artificial chromosome (BAC)

:

a vector used to clone large fragments of DNA (up to 500 kb) that can be readily inserted in a bacterium, such as
Escherichia coli






complementary DNA (cDNA)

:

a DNA molecule that is synthesized using messenger RNA (mRNA) as a template and the enzyme reverse transcriptase; these molecules correspond to genes but lack introns that are present in the actual genome




cosmid

:

a cloning vector, a hybrid of bacterial plasmid and bacteriophage vectors, that relies on bacteriophage capsules to infect bacteria; these are constructed with selectable markers from plasmids and two regions of lambda phage DNA known as cos (for cohesive end) sites




insertional mutagenesis

:

the generation of a mutant by inserting several nucleotides into a genome




microarray

:

a flat surface on which 10,000 to 100,000 tiny spots of DNA molecules fixed on glass or another solid surface are used for hybridization with a probe of fluorescent DNA or RNA





model organism


:

an organism well suited for genetic research because it has a well-known genetic history, a short life cycle, and genetic variation between individuals in the population




transformation

:

a change in both genotype and phenotype resulting from the uptake of exogenous DNA





Bibliography


Graham, Linda E., and Lee W. Wilcox. Algae. 2d ed. San Francisco: Cummings, 2009. Print.



Harris, Elizabeth H. “Chlamydomonas as a Model Organism.” Annual Review of Plant Physiology 52.1 (2001): 363–406. Print.



Harris, Elizabeth H., David Stern, and George Witman. The Chlamydomonas Sourcebook. 2d ed. 3 vols. Boston: Academic, 2009. Print.



Katsaros, Christos, and Kirsten Heimann. Advances in Algal Cell Biology. Berlin: De Gruyter, 2013. Digital file.



Merchant, S. S., et al. “The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions.” Science 318.5848 (2007): 245–250. Print.



Perrineau, Marie-Mathilde, et al. "Using Natural Selection to Explore the Adaptive Potential of Chlamydomonas reinhardtii." PLoS ONE 9.3 (2014): 1–9. Print.



Walter, Christian, and Clemens Posten. Microalgal Biotechnology. Berlin: De Gruyter, 2012. Digital file.

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