Visualization of fine-scale genomic structure by oligonucleotide-based high-resolution FISH.

The discovery of complex structural variations that exist within individual genomes has prompted a need to visualize chromosomes at a higher resolution than previously possible. To address this concern, we established a robust, high-resolution fluorescence in situ hybridization (FISH) method that utilizes probes derived from high complexity libraries of long oligonucleotides (>150 mers) synthesized in massively parallel reactions. In silico selected oligonucleotides, targeted to only the most informative elements in 18 genomic regions of interest, eliminated the need for suppressive hybridization reagents. Because of the inherent flexibility in our probe design methods, we readily visualized regions as small as 6.7 kb with high specificity on human metaphase chromosomes, resulting in an overall success rate of 94%. Two-color FISH over a 479-kb duplication, initially reported as being identical in 2 individuals, revealed distinct 2-color patterns representing direct and inverted duplicons, demonstrating that visualization by high-resolution FISH provides further insight in the fine-scale complexity of genomic structures. The ability to design FISH probes for any sequenced genome along with the ease, reproducibility, and high level of accuracy of this technique suggests that it will be powerful for routine analysis of previously difficult genomic regions and structures.

A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and animals.

In mammalian cells, the retinoblastoma (RB) protein regulates G1 progression and functions through its association with various cellular proteins. Two closely related mammalian RB binding proteins, RbAp48 and RbAp46, share sequence homology with the Msi1 protein of yeast. MSI1 is a multicopy suppressor of a mutation in the IRA1 gene involved in the Ras-cAMP pathway that regulates cellular growth. Human RbAp48 is present in protein complexes involved in histone acetylation and chromatin assembly. We report the cloning of cDNAs encoding four plant RbAp48- and Msi1-like proteins: one from tomato, LeMSI1, and three from Arabidopsis. Complementation studies confirm that LeMSI1 can function as a multicopy suppressor of the yeast ira1 mutant phenotype. The LeMSI1 protein localizes to the nucleus and binds to a 65-kD protein in wild-type as well as ripening inhibitor (rin) and Neverripe (Nr) tomato fruit. LeMSI1 also binds to the human RB protein and the RB-like RRB1 protein from maize, indicating that this interaction is conserved between plants and animals.

RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant.

Ran is a 25-kDa Ras-related nuclear GTP-binding protein which is very highly conserved in humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Ran has been found to form a stable, noncovalent complex with the chromatin-associated protein RCC1, a negative regulator of mitosis. In Sch. pombe, a temperature-sensitive mutation in the RCC1 homolog encoded by the pim1 gene causes premature induction of mitosis, and this mutation can be suppressed by overexpression of the Ran homolog encoded by spi1. We report here the cloning of three Ran cDNAs from tomato. The Ran protein is very highly conserved among plants, animals, and fungi. In tomato, Ran mRNA is expressed in all tissues examined, even those with little or no cell division, indicating that Ran in plants may have functions other than just control of mitosis. We have found that the tomato Ran protein can direct a beta-glucuronidase reporter protein to the plant cell nucleus, confirming that Ran is a nuclear protein in plants. We show that the tomato Ran protein can suppress the Sch. pombe pim1 mutation, indicating that the tomato Ran protein and the Sch. pombe spi1 protein are functionally homologous.

Cooperation between CCAAT and octamer motifs in the distal sequence element of the rat U3 small nucleolar RNA promoter.

Mammalian U3 small nucleolar RNA promoters possess a highly conserved distal sequence element (DSE) consisting of CCAAT and octamer motifs separated by 11-12 base pairs. We show here that both motifs are required for transcription of a rat U3D gene in Xenopus oocytes. Deletion of the CCAAT motif leaves residual DSE activity, while removal of the octamer motif does not. Changing the conserved spacing between the two motifs generally inhibits transcription less than deletion of either motif, but increasing the spacing between the motifs by one helical turn of DNA preserves normal levels of transcription. We also show that the rat U3D DSE is functionally equivalent to the human U2 snRNA DSE, which consists of adjacent GC and octamer motifs, and that elements from the Herpes Simplex Virus thymidine kinase promoter can replace part or all of the U3D DSE. These data are apparently paradoxical; despite high evolutionary conservation, the U3 DSE is relatively insensitive to mutation, and other upstream motifs are also able to drive transcription from the U3 basal promoter. We suggest that the conserved structure of the U3 DSE may be required for regulation rather than efficiency of U3 transcription.

The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation.

Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation. To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box. The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation. We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent.