Compilation of mapping/sequencing results for randomly selected maize
--Tim Helentjaris, Ivone Torres-Jerez, Bo Shen, Newton Carneiro, Becky Stevenson, Tom McCreery,
Jeff Habben, Brian Larkins, Rob Ferl, Ernie Almira and Chris Baysdorfer
As first described in an article in the Newsletter last year, we have continued our efforts at gene identification/isolation through the analysis of randomly selected cDNAs. The bulk of the effort over the last year has concentrated upon two libraries prepared at Tucson, one from etiolated seedlings and the second from membrane-free polysomes from endosperm. These libraries consist of size-selected cDNAs which are directionally cloned into the ZipLox vector from Gibco-BRL.
After construction, each of the clones is screened for expression pattern by hybridizing a colony lift of several hundred clones at a time with a probe prepared from 1st strand cDNA from each of the two tissues. Those colonies not hybridizing with either probe are characterized as "rarely expressed". Those hybridizing with only one of the probes are denoted as "abundantly expressed and tissue-specific", while those which hybridize with both tissue-derived probes are characterized as "abundantly and generally expressed".
Clones are then submitted to "single-pass" sequencing from the presumed 5' end of the original mRNA. The data is submitted to GenBank by BlastX analysis and subsequently by BlastN if no homologies are identified. Strong homologies indicative of conserved function are usually indicated by BlastX scores of more than 180 and related functions are usually indicated by scores of more than 100. Some clones were also sequenced from the presumed 3' end but the data did not prove useful in identifying putative matches in GenBank.
Probes are then prepared from clones and hybridized to genomic DNA from the Brookhaven RI parents digested with one of three restriction enzymes. Informative clone:enzyme:cross combinations are noted and then all clones with putative identifications from sequencing and others with simple hybridization patterns are also applied to the RI progeny to determine map positions for these cloned sequences.
The results from this analysis to date are presented in the accompanying table, which lists only those clones with sequences indicative of some homology or function. In the future this table will be regularly updated, and will become part of the maize database at Columbia, MO, from which it can be easily accessed. All sequences are also being deposited in GenBank and can be accessed from there. All mapping data are being forwarded to Ben Burr to be included in the Brookhaven database and will be published annually in the MNL. All clones and data are currently available from Tucson upon request and without restriction. If investigators have requests, such as sequence types or probes in particular genomic regions, they can be communicated to Tucson and we will track the developing database for those requests and forward them as they are discovered. We would like to thank the following companies for providing unrestricted funding to help support this effort along with that from the USDA Plant Genome Program: CIBA-GEIGY (D. Alexander), Monsanto (M. Fromm), Pioneer Hi-Bred International (J. Howard), Rhone-Poulenc (G. Freyssinet), and Sandoz Crop Protection (K. Brunke).
Annotations to the Accompanying Table:
In the second column, the asterisk defines this sequence as having been first isolated and identified here in maize. Homologies are detected either by BlastX searches of the GenBank at the amino acid level, or if unsuccessful with this approach by BlastN searches at the nucleic acid level. The sequences are grouped roughly according to the function of their putative homologies.
In the third column, a GenBank accession number for one of the high scoring matches is given.
In the fourth column, clones are described as either "abundantly expressed" or "rarely expressed" depending upon whether they exhibit significant signal in a colony hybridization with a 1st strand cDNA probe. The probes they hybridize to are also indicated in this column (i.e. either "Endosperm" or "Seedling").
In the fifth column, a "Complex" pattern indicates somewhat more than three significant hybridizing fragments on a genomic Southern with more than one restriction enzyme. Those clones with "Simple" patterns possess three or less fragments. The designation in the sixth column refers to the map name for the locus(i) detected by this clone. Map locations in the last column are denoted as chromosome(s) and either short arm ("s"), long arm ("L"), or centromeric region ("c").
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