1. Department of Genetics, Harvard Medical School and
2. Department of Molecular Biology, Massachusetts General Hospital,
Boston, MA 02114 USA
email: Howard
Goodman.
This laboratory has previously presented data used for the generation of a yeast artificial chromosome (YAC) physical map of Arabidopsis thaliana chromosome 2 (1) consisting of a minimal tiling path of 34 CIC (2) YACs. The average insert size of these YACs is ~420Kb (2). Since publication one of the gaps in the YAC tiling path, Gap 3, has been closed using three additional YACs (3). As a first step toward developing a higher resolution map consisting of clones suitable for direct use as sequencing substrates we have hybridized probes derived mainly from the YACs comprising the minimal tiling path (38 in all) to the Texas A&M University Bacterial Artificial Chromosome (BAC) library (4).
Methods
The YAC probes were produced by the "Sequence Independent Amplification" (SIA) method (5), with the modification that TAQ polymerase was used for both the Primer A and Primer B reactions. Amplified products were labeled by PCR, and hybridized overnight at 65 oC to filter sets (3) of the Texas A & M University (TAMU) BAC clones (4). The filters were washed once in 40 mM Church buffer (6) + 0.5% SDS for 30 min. at 65 oC and for an additional 30 min. in 40 mM Church buffer + 1% SDS at 65 oC. Filters were exposed to Reflection film (Dupont NEN) with one intensifying screen at -80 oC for 2-24 hrs. The BAC hybridization signals were then scored for each YAC probe. A 'repetitive probe' was made from YAC EG03G01 (EG03G01 contains both chloroplast and ribosomal DNA) and also hybridized to the BAC filters (3).
Results and Discussion
The BAC map (Figure 1a-h and Table 1) has been constructed from those BACs which were scored as hybridizing to one or two of the 38 SIA probes. Note: these assignments have not been confirmed by Southern hybridization. Additional BACs fell into four other catagories that are not readily explainable. The first group contains BACs which hybridized to three or more probes from contiguous YACs (Table 2). These may indicate that these YAC(s) are completely (or almost completely) overlapping and only have ~100Kb of unique sequence among them. The second group contains BACs which hybridized to probes from two non-overlapping YACs(Table 3). BACs in the third group hybridized to probes from three or more non-overlapping YACs (Table 4). Note that a majority of the BACs in this class were from the 18 YACs at the "top" (short arm) of the chromosome (1). The reason for this is unknown, but may be the result of similar sequences at disparate regions of the chromosome. The fourth group consisted of BACs containing repetitive DNA as identified by hybridization to the EG03G01 probe (Table 5) (3).
Examination of the BAC distribution beneath the YAC physical map in Figure 1 indicates that: First, there are "gaps" in the map where no BACs are listed. Second, there is an uneven distribution of BACs hybridizing to each YAC probe along the chromosome. Third, the number of probe positive BACs is not a function of YAC size. These inconsistencies may be explainable in several ways. One possibility is a non-random distribution of Arabidopsis DNA within the BAC library. Secondly, the nature of the amplification procedure in developing YAC probes, may result in a loss, or under representation, in the probe of certain regions of the YAC and the resultant loss of hybridization signal. Thirdly, repeated use of the filters as the project progressed, from chromosome bottom to top, may have resulted in signal loss. Fourly, the presence of repetitive or homologous regions scattered through out the chromosome may have impeded our ability to assign positive signals to one YAC probe or in the case of overlaping YACs, two YAC probes.
References:
1. Zachgo, E.A., M.L. Wang, J. Dewdney, D. Bouchez, C. Comilleri, S. Belmonte, L. Huang, M. Dolan and H.M. Goodman (1996) A physical map of chromosome 2 of Arabidopsis thaliana. Genome Research. 6: 19-25.
2. Creusot, F., E. Fouilloux, M. Dron, J. Lafleuriel, G. Picard, A. Billault, D. Le Paslier, C. D., M.E. Chaboute, A. Durr, J. Fleck, C. Gigot,C. Camilleri, C. Bellini, M. Caboche and D. Bouchez (1995) The CIC library :a large insert YAC library for genome mapping in Arabidopsis thaliana. PlantJ. 8: 763-770.
3. Wang M.L., L. Huang, D.K. Bongard-Pierce, S. Belmonte, E.A. Zachgo, J.W. Morris, M. Dolan, and H.M. Goodman (1997) Construction of an ~2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2. PlantJ. 12: in press.
4. Choi, S.D., R. Creelman, J. Mullet and R.A. Wing (1995) Construction and characterization of a bacterial artificial chromosome library from Arabidopsis thaliana. Weeds World. 2: 17-20.
5. Bohlander, S.K., R. Espinosa, M.M. Le Beau, J.D. Rowley and M.O. Diaz(1992) A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics. 13: 1322-1328.
6. Church, G.M. and W. Gilbert (1984) Genomic Sequencing. PNAS 81: 1991-1995
Acknowledgments. This work was supported by a grant from Hoechst AG to HMG.