Artificial chromosomes have proven to be powerful transformation vectors. However, to create artificial chromosomes, intimate knowledge about the structure of the kinetochore is essential. In a recent review article published in the December, 2000 issue of Trends in Plant Science (5(12):543-47), Hong-Guo Yu, Evelyn Hiatt and RK Daw at the University of Georgia, have reviewed the current status of our knowledge on, “the Plant Kinetochore”. Their review encompasses the molecular composition of the plant kinetochore including constitutive and structural components, putative microtubule-organizing proteins, proteins involved in chromosome motility, spindle checkpoint proteins and proteins of unknown functions. The review concludes with a review of structural and functional conservation of the plant kinetochore.
They begin the article by defining that the kinetochore and the centromere are two different structures and that kinetochores are large protein complexes that bind to centromeres. They recount that only in 1980, a major breakthrough occurred in mammalian kinetochore research. The breakthrough was the discovery of antigens which could be used to identify plant kinetochores such as found in the monocots Haemanthus and Tradescantia. According to the authors, technological advancements such as the discovery of PCR and creation of genomic sequence databases have made it possible to identify several new plant kinetochore genes.
The authors then describe that recent studies have revealed that kinetochores generate as well as regulate chromosome movement by interacting with microtubules and their associated motor proteins; kinetochores have also been shown to function in the spindle checkpoint to ensure commencement of anaphase only after metaphase is complete. Most of our knowledge on the centromere is animal based except in budding yeast in which as many as 12 centromere- associated proteins have been reported.
Hu, Hiatt and Daw provide further information about the specific kinetochore genes which encode for proteins enabling a mitotic mother cell to divide accurately into two and a meiocyte into four new cells. In other words, these specific proteins serve as checkpoint guards that make sure all is in order before they permit cell division to proceed further. Using the Pioneer EST database, the above authors have identified two proteins that localize to maize kinetochores. One of the proteins is a maize homolog of yeast MAD2 known as the spindle checkpoint and the other is a maize homolog of mammalian CENP-C . The latter is considered to take part in the early stages of kinetochore assembly. In yeast mutants, Mad2, segregation of chromosomes is disturbed resulting in the formation of aneuploid cells (cells with an excess or deficient chromosome number), affecting cell growth and proliferation. Over a 23 amino acid region known as Region I, homology has been detected between the C-terminal portion of CPC and the mammalian CENP-C. Also, Hong-Guo Yu et al. observed that maize MAD2 , a checkpoint protein is located primarily on prometaphase chromosomes; and is conspicuously absent from kinetochores at later stages of division i.e. metaphase and anaphase. The same phenomenon occurs also when animal cells divide indicating that MAD2 acts in a similar fashion in both animals and plants.
The authors elaborate on the kinesin superfamily of protein motors, CNEP-F, a nuclear matrix protein of unknown function and finally Skp1p, a protein directly involved in linking kinetochore function with the cell cycle-regulatory machinery.
Recent studies show that chromosome movement occurs as a result of the force generated by polymerization and depolymerization of microtubules assisted by the action of motor proteins. CENP-E protein, a member in the kinesin superfamily of motors, is one among many motor proteins that acts at kinetochores. This protein binds kinetochores to the actively growing (plus) ends of microtubules, thereby inducing movement of chromosomes towards the metaphase plate. The fact that antisera against human CENP-E recognize Vicia faba and Hordeum vulgare kinetochores, leads the authors to suggest that a similar mechanism also operates in plant cells. It has also been suggested that kinetochores initiate the movement of chromosomes as a result of the action of spindle checkpoint proteins which transmit the necessary information to the anaphase-promoting complex (APC). So sensitive is the entire process that if any chromosome fails to align at the metaphase plate, the onset of anaphase is delayed for hours or even stopped terminating the cell cycle altogether. The spindle checkpoint is considered to be very important as it ensures maintenance of stability of chromosome numbers in a species by eliminating aneuploidy. In other words, species survival depends on the proper functioning of this and other proteins in the spindle checkpoint pathway.
Another recently discovered nuclear matrix protein is CNEP-F. It is detectable during G2 of interphase and it is also seen associated with the kinetochores during prophase, metaphase and early anaphase; thereafter it is lost from kinetochores. The protein may play a role in the early stages of kinetochore maturation., but the mechanism of its action is still not known. Recent studies have also revealed that one particular serum containing antibodies to meiotic histone of Lilium stains only the centromeric region of the chromosomes of this species, in contrast to other antibodies in which the whole chromosomes are stained. This differential staining is an indication that a unique histone H1 characterizes meiotic centromeres.
The final protein in this list is Skp1p, a component of the Skp1 (Saccharomyces kinetochore protein) reported first in yeast. It is an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery. Both H. vulgare and V. faba kinetochores contain putative homologs of yeast Skp1p. This protein has been recently shown to be involved in the segregation of homologous chromosomes in the pollen mother cells of Arabidopsis.
A large amount of data have accumulated to suggest that the plant kinetochore contains homologs of many of the proteins involved in the functioning of animal and fungal kinetochores; these findings also indicate that the kinetochore, besides being extremely plastic, has a redundant structure . Plasticity of the centromere/kinetochore is proven by the fact that each of the two parts of a divided maize centromere is still functional; more plant examples showing redundancy were reported subsequently. All these unique features indicate that there is a significant amount of DNA base sequence redundancy in plant centromeres.
Though a lot of ground has been covered in elucidating the role of at least a dozen plant kinetochore proteins, the authors conclude, that the study of the plant kinetochore must continue in order to complete functional analysis of this extremely vital part of the chromosome. Such findings will be instrumental in the development of artificial chromosomes as transformation vectors. The authors hope that the study of the Arabidopsiskinetochore, in which genetic analysis has become easy, will help in the accomplishment of this seemingly difficult task.