The University of Arizona (PI Soderlund, Wing), UGA (Bennetzen) and Purdue (San Miguel) were awarded NSF grant #0321724 to construct, sequence and analyze MSLL and HMPR libraries for maize. Overview of HMPR and MSLL HMPR and MSLL libraries References Overview of HMPR and MSLL The maize genome comprises approximately 2500 million base-pairs. Over 60% of these base-pairs compose Long Terminal Repeat (LTR)-retrotransposons and 20% compose other repetitive sequences (Whitelaw et al. 2003). In adult tissues, most LTR-retrotransposons appear to be 100% cytosine 5-methylated at all 5'-CG-3' and 5'-CNG-3' sites (Yuan et al. 2003). Recent library construction technologies attempt to take advantage of these facts to (1) make gene-enriched libraries so that sequencing projects can largely exclude highly reiterated segments of a genome and (2) create bridge libraries of clones that span the repetitive segments and therefore anchor and link the gene containing sequence contigs. Hypo-Methylated Partial Restriction (HMPR, Emberton et al.) clones provide gene-enriched sequences and Methylation-Spanning Linker Library (MSLL, Yuan et al. 2003) clones span large methylated regions; both techniques use methylation sensitive enzymes that will not cut sites containing a 5-methyl cytosine. These two techniques complement the Methyl Filtration (MF, Rabinowicz et al. 1999) and High Cot (HC, Yuan et al. 2003) approaches. The following provides a brief description of the four types of clones: HMPR - clones are constructed from 2-4 kb fragments from partial digestion of the methylation-sensitive enzymes HpaII (5'-CCGG-3') or HpyCH4IV (5'-ACGT-3'). MF - clones are constructed from sheared genomic DNA and transformed into methylation-restrictive strains of E. coli. DNA fragments that contain methylated cytosines will be degraded and hence, "filtered" from the library. HC - clones are constructed from low copy DNA, where, after an initial denaturation, sheared DNA has been allowed to anneal for a long enough time to renature (repetitive) high copy DNA. After fractionating away the renatured DNA, the remaining single stranded (low copy) DNA is cloned. MSLL - clones are constructed from >35kb fragments from complete SalI (5'-GTCGAC-3') or HpaII (5'-CCGG-3') digestion of genomic DNA. The HMPR sequences are a good complement to the MF and HC, as (1) they can tolerate methylated bases within the clone; in constrast, MF clones cannot tolerate methylation, (2) they can tolerate repetitve sequences; in contrast, HC clones will under-represent gene families, etc and (3) clones >2kb are easily constructed; in constrast, MF and HC are <2kb. The disadvantage of HMPR is that is requires two restrictions sites within 2-4kb. As can be seen on our Browsers, these 3 types of clones provide a good range of coverage of the gene rich regions. The MSLL clones range from 35kb to over 100kb. Any clone that is this size resulting from a full digestion is more than likely spanning a region containing a number of methylated sites. That is, few >35 kb segments of a genome will lack SalI or HpaII sites; hence any fragments of this size generated by a complete digest with one of those enzymes will probably contain additional methylated sites. Methylated blocks of DNA will frequently be composed of repetitive DNA. Mate-pairs from MSLL clones can link gene-rich contigs across these long segments of repetitive sequence (such as retrotransposon clusters). The Consortium for Maize Genomics generated over 450k MF sequences and over 455k HC sequences, which were assembled into AZMs (Assembled Zea Mays, Whitelaw et al. 2003, see details.). This grant has generated HMPR and MSLL sequences, as described below. The results of both sets of sequences can be viewed on the Maize Mini-BAC Browser pages. HMPR and MSLL libraries HMPR: A total of 40,299 end sequences from clones with size range of 2-4kb. The following libraries are available from the AGI BAC/EST Center. The reads are available from genbank, query ZMMBH. AGI Enzyme Digestion Reads ZMMBHa HpyCHIV 10U/ug 171 ZMMBHb HpyCHIV 0.1U/ug 181 ZMMBHc HpaII 10U/ug 734 ZMMBHd HpaII 0.5U/ug 1482 ZMMBHe HpaII 0.25U/ug 5189 ZMMBHf HpaII 0.125U/ug 10428 ZMMBHg HpaII 0.05U/ug 1488 ZMMBHh HpaII 0.025U/ug 177 ZMMBHi HpaII 0.0125U/ug 174 ZMMBHj HpaII 0.0005U/ug 177 ZMMBHk HpaII 0.00025U/ug 183 ZMMBHl HpyCHIV 0.00025U/ug 1441 ZMMBHm HpyCHIV 0.00025U/ug 2239 ZMMBHn HpyCHIV 0.00025U/ug 1492 ZMMBHo HpyCHIV 0.00025U/ug 2550 ZMMBHp HpyCHIV 0.00025U/ug 8218 ZMMBHq HpyCHIV 0.00025U/ug 1782 ZMMBHr HpyCHIV 0.00025U/ug 1544 ZMMBHs HpyCHIV 0.00025U/ug 649 MSLL Libraries A total of 80732 end sequences from clones with size ranges listed below. Libraries a-d,f,h,i,j are currently available from the AGI BAC/EST Center. Sequences are available from Genbank, query the library name, e.g. "ZMMBLa". AGI Enzyme Length Sequences ZMMBLa SalI 35-60 9984 ZMMBLb HpaII 35-60 9984 ZMMBLc HpaII 20-35 9984 ZMMBLd SalI 20-35 13824 ZMMBLe HpaII 12-20 9206 ZMMBLf SalI 60-100 5436 ZMMBLh SalI >100 6158 ZMMBLi HpaII 60-100 8495 ZMMBLj HpaII >100 7715 ZMMBLz HpaII 7-12 8448 The libraries are BAC libraries with the exception of z, which is plasmid. References Emberton, J., Ma, J., Yuan, Y., SanMiguel, P., and Bennetzen, J. Gene Enrichment in Maize with Hypomethylated Partial Restriction (HMPR) libraries. Genome Research 15, 1441-1446. Rabinowicz, P.D., Schutz, K., Dedhia, N., Yordan, C., Parnell, L.D., Stein, L., McCombie, W.R., and Martienssen, R.A. (1999). Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nat Genet 23, 305-308. Whitelaw, C.A., Barbazuk, W.B., Pertea, G., Chan, A.P., Cheung, F., Lee, Y., Zheng, L., van Heering en, S., Karamycheva, S., Bennetzen, J.L., SanMiguel, P., Lakey, N., Bedell, J., Yuan, Y., Budiman, M.A., Resnick, A., Van Aken, S., Utterback, T., Riedmuller, S., Williams, M., Feldblyum, T., Schubert, K., Beachy, R., Fraser, C.M., and Quackenbush, J. (2003). Enrichment of gene-coding sequences in maize by genome filtration. Science 302, 2118-2120. Yuan, Y., SanMiguel, P.J., and Bennetzen, J.L. (2002). Methylation-spanning linker libraries link gene-rich regions and identify epigenetic boundaries in Zea mays. Genome Res 12, 1345-1349. Yuan, Y., SanMiguel, P.J., and Bennetzen, J.L. (2003). High-Cot sequence analysis of the maize genome. Plant J 34, 249-255.