Introduction
SMC (Structural Maintenance of Chromosomes) proteins are a family of evolutionarily conserved ATPases that are involved in higher order chromatin structure. The eukaryotic SMC proteins can be subdivided into five distinct subfamilies (SMC1-4 and Rad 18), some of which form heterodimers to foster events of chromosome condensation, segregation, recombination repair, and dosage compensation (Hirano, 1999; Cobbe and Heck, 2000). The SMC2 and SMC4 members form heterodimers and assemble with three other non-SMC proteins to form the ‘condensin’ complex, which is involved in mitotic chromosome condensation (Kimura et al., 2001). In different organisms, members of the SMC1 and SMC3 families form heterodimers to give riseto two different protein complexes with different functions. The ‘cohesin’ complex is involved in sister chromatid cohesion (Michaelis et al., 1997; Losada et al., 1998) while the RC-1 complex is involved in recombination (Stursberg et al., 1999). Members of the Rad18 family play a role in recombination repair (Fousteri and Lehmann, 2000).

Results from our lab
We cloned an Arabidopsis SMC2 gene which we termed AtCAP-E1 (Arabidopsis thaliana Chromosome Associated Protein), and during the course of this work a second SMC2 gene (AtCAP-E2) was identified by the Arabidopsis genome initiative. The two proteins are 90% similar, and both are expressed in mitotically active tissues, but E1 is expressed at much higher levels than E2.

RT- PCR/CAPS analysis of SMC2 gene expression in :

Top: E1 products cleaved by CAPS enzyme

Bottom: E2 products cleaved by CAPS enzyme

Summary: Approximately 85% of transcripts are due to E1 transcription.

AtCAP-E1::GUS transgenics reveal expression in tissues that are actively undergoing mitosis such as the lateral root primordia (left) and the shoot apex (right).

Transgenic plants harboring an AtSMC2-1 antisense gene exhibited dramatic phenotypic defects during postembryonic growth and development.

The growth rate of these plants was retarded and some developed a grossly enlarged and dysfunctional primary shoot apical meristem (SAM). The tunica/corpus organization of theprimary SAM was compromised and cells of the SAM were highly vacuolated. Using a histone H4 probe as an S-phase-specific marker, in situ hybridization revealed that cells of the SAM had ceased cycling. Interestingly, axillary meristems were morphologically normal and functional, but aspects of fasciation (bifurcation of inflorescence stems and altered phyllotaxy) were observed.

The root apical meristem (RAM) also exhibited disorganization, and precocious tissue maturation occurred just above it, as indicated by the formation of mature root hairs and xylem tracheary elements adjacent to the RAM in the confocal image on the right. We suggest that improper chromosome condensation leads to cell cycle arrest and premature differentiation of cells in the apical meristems.

We identified T-DNA mutants in both E1 and E2 and found that although both single mutants are relatively normal, heterozygosity for insertion into one gene in a null background for the second gene confers two distinct phenotypes: lethality during embryogenesis (E1-/-E2+/-) or fasciation of relatively normal plants (E1+/-E2-/-).

These findings were reported in Development (Siddiqui et al., 2003; get PDF)

SMC 4

In Arabidopsis, there is one SMC4 gene. We have analysed mutants in it, and find phenotypes similar to that of the SMC2 double mutant (embryo defects/lethality). As a heterozygote, some aspects of fasciation are observed, suggesting a semi-dominant effect. These findings will be published in Planta in 2005.

Current and future work:

1. Examination of SMC2 localization and modification during the cell cycle. For this we are using E1 antibodies and synchronized suspension cultured cells.

2. Examination of SMC2 localization during meioisis and identification of novel meiotic factors which interact with SMC2. For this work we are using E1 antibodies and the naturally synchronous floral buds of Lilium longiflorum.





 

References:

• Cobbe, N. and Heck, M.M.S. (2000). SMCs in the world of chromosome biology- From prokaryotes to higher eukaryotes. J. Struct. Biol. 129, 123-143.
• Fousteri, M.I. and Lehmann, A.R. (2000). A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein. EMBO J. 19, 1691-1702.
• Hirano, T. (1999). SMC-mediated chromosome mechanics: a conserved scheme from bacteria to vertebrates? Genes Dev. 13, 11-19.
• Kimura, K., Cuvier, O., and Hirano, T. (2001). Chromosome condensation by a human condensin complex in Xenopus egg extracts. J. Biol. Chem. 276, 5417-5420.
• Losada, A., Hirano, M., and Hirano, T. (1998). Identification of Xenopus SMC protein complexes required for sister chromatid cohesion. Genes Dev. 12, 1986-1997.
• Siddiqui, N.U., Stronghill, P.E., Dengler, R.E., Hasenkampf, C.A. and Riggs, C.D. (2003) Mutations in Arabidopsis condensin genes disrupt embryogenesis, meristem organization and segregation of homologous chromosomes during meiosis. Development 130:3283-3295.
• Stursberg, S., Riwar, B., and Jessberger, R. (1999). Cloning and characterization of mammalian SMC1 and SMC3 genes and proteins, components of the DNA recombination complex RC-1. Gene 228, 1-12.