The distribution of genes in the genomes of Gramineae (chromosomesyisochoresyplants)

Recent investigations showed that most maize genes are present in compositional fractions of nuclear DNA that cover only a 1–2% GC (molar fraction of guanosine plus cytosine in DNA) range and represent only 10–20% of the genome. These fractions, which correspond to compositional genome compartments that are distributed on all chromosomes, were collectively called the ‘‘gene space.’’ Outside the gene space, the maize genome appears to contain no genes, except for some zein genes and for ribosomal genes. Here, we investigated the distribution of genes in the genomes of two other Gramineae, rice and barley, and used a new set of probes to study further the gene distribution of maize. We found that the distribution of genes in these three genomes is basically similar in that all genes, except for ribosomal genes and some storage protein genes, were located in gene spaces that (i) cover GC ranges of 0.8%, 1.0%, and 1.6% and represent 12%, 17%, and 24% of the genomes of barley, maize, and rice, respectively; (ii) are due to a remarkably uniform base composition in the sequences surrounding the genes, which are now known to consist mainly of transposons; (iii) have sizes approximately proportional to genome sizes, suggesting that expansion–contraction phenomena proceed in parallel in the gene space and in the gene-empty regions of the genome; and (iv) only hybridize on the gene spaces (and not on the other DNA fractions) of other Gramineae. Previous investigations on angiosperms showed that their nuclear genomes are characterized by a compositional compartmentalization (1, 2) and that their nuclear genes may be contained in compositional compartments that cover only a narrow GC (molar fraction of guanosine plus cytosine in DNA) range (3). Recent work on the distribution of genes in the nuclear genome of maize (4) showed that the 20 probes used localized genes in compositional compartments (collectively called the gene space) that covered a 1–2% GC range and represented 10–20% of total nuclear DNA. Some zein genes detected by the zein probe used and ribosomal genes were, however, located in compartments lower and higher in GC, respectively, compared with all other protein-encoding genes tested. The gene distribution of the maize genome is very different from that found in the human genome. Indeed, the latter, which has a comparable haploid size and a similar compositional distribution of DNA molecules and coding sequences, has its genes distributed over its entire 30% GC range, although in a remarkably nonuniform way, most genes being located in the GC-rich regions of the genomes (5–7). A question raised by the existence of the gene space of the maize genome is whether this particular gene distribution is shared by other plants of the family Gramineae (or Poaceae) and by other angiosperms. Here, we report results obtained for rice (from the Oryza group of the subfamily Bambooideae) and for barley (from the Triticum group of the subfamily Pooideae) and show that they are similar to those found in maize (from the Zea group of the subfamily Panicoideae). These results show, therefore, that gene spaces characterized by a remarkably uniform base composition exist in the genomes of all subfamilies of Gramineae. Moreover, the gene space of one Gramineae cross-hybridizes only with the DNA fractions corresponding to the gene spaces of other Gramineae and not with other fractions, a result which could be explained by the greater divergence of repeated vs. single copy sequences (8–10). These observations open the way to the in situ localization of the gene space on the chromosomes of Gramineae. In addition, gene enriched DNA fractions corresponding to the gene space can be prepared and used for cloning, mapping, and sequencing. MATERIALS AND METHODS Plants and DNA Preparations. Maize cv. F7 3 F2 and wheat cv. Cidéral were from the Institut National de la Recherche Agronomique, Versailles, France, and barley cv. Alexis, irrigated rice cv. Cigalon, and pluvial rice cv. Erat104 were from the Centre de Coopération International en Recherche Agronomique pour le Développement, Montpellier, France. Nuclear DNA was prepared from etiolated seedlings according to ref. 11. The sizes of DNA molecules were estimated by pulse field gel electrophoresis (PFGE) to be comprised between 50 and 100 kb. DNA Fractionation and Gene Localization. The nuclear DNAs of the four Gramineae were fractionated by preparative centrifugation in Cs2SO4 density gradients in the presence of 3,6-bis (acetatomercurimethyl)-1,4-dioxane (BAMD) as described (12). DNA fractions (usually 12–13) were then dialyzed, digested with EcoRI (Boehringer Mannheim), submitted to electrophoresis in 0.8% agarose gels, transferred to Hybond N1 filters (Amersham), hybridized with probes that were radioactively labeled (13) by use of a random priming kit (Amersham), and purified with use of microspin S-200 HR columns (Pharmacia). The intensities of the hybridization signals were quantitated with use of the software of Wayne Raband (National Institutes of Health, Bethesda) to assess the distribution of sequences probed in the DNA fractions. As in previous work (1–4), proportional loading of DNA fractions on the gels was used (see legend of Fig. 1). Although the principle of proportional loading is well established, in practice there may be errors associated with estimating DNA amounts in fractions. These errors were minimized, however, by using large initial DNA samples (500 mg) and by checking the amounts of DNA loaded on the gels. GC levels of DNA fractions were determined by analytical centrifugation in CsCl gradients. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y946857-5$2.00y0 Abbreviations: BAMD, 3,6-bis(acetatomercurimethyl)-1,4-dioxane; GC, molar fraction of guanosine plus cytosine in DNA; GC3, average GC level of third codon positions of genes. *To whom reprint requests should be addressed. e-mail: [email protected].