Gene distribution and isochore organization in the nuclear genome of plants.

The genomic distribution of 23 nuclear genes from three dicotyledons (pea, sunflower, tobacco) and five monocotyledons of the Gramineae family (barley, maize, rice, oat, wheat) was studied by localizing these genes in DNA fractions obtained by preparative centrifugation in Cs2SO4/BAMD density gradients. Each one of these genes (and of many other related genes and pseudogenes) was found to be located in DNA fragments (50-100 Kb in size) that were less than 1-2% GC apart from each other. This definitively demonstrates the existence of isochores in plant genomes, namely of compositionally homogeneous DNA regions at least 100-200 Kb in size. Moreover, the GC levels of the 23 coding sequences studied, of their first, second and third codon positions, and of the corresponding introns were found to be linearly correlated with the GC levels of the isochores harboring those genes. Compositional correlations displayed increasing slopes when going from second to first to third codon position with obvious effects on codon usage. Coding sequences for seed storage proteins and phytochrome of Gramineae deviate from the compositional correlations just described. Finally, CpG doublets of coding sequences were characterized by a shortage that decreased and vanished with increasing GC levels of the sequences. A number of these findings bear a striking similarity with results previously obtained for vertebrate genes.     

Widespread occurrence of the homologues of the early nodulin (ENOD) genes in Oryza species and related grasses

Eighty accessions representing 23 species from the genus Oryza were examined for the presence of homologues of early nodulin (ENOD) genes. Southern analyses indicated a widespread distribution of homologues of ENOD genes across all the genomes of rice as well as other monocots. The degree of cross-hybridization of the legume ENOD genes with sequences in the genomes of various species, as revealed by hybridization differentials measured in terms of signal intensities, however, suggests that the homologues of ENOD genes are conserved to varied extents in different Oryza species. The presence of homologues of ENOD genes in a wide variety of plant species denotes that the biological functions of early nodulins may be diverse, and not restricted to nodule organogenesis alone. The fact that ENOD gene homologues exist widely both in dicots and monocots provides evidence that these homologues have arisen from a common ancestral plant.     

Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron

Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice 10 kD Prolamin

Using PCR technique, two prolamin genes from Oryza sativa var. indica (cv. Guanglu′ ai) and O. sativa var. japonica (cv. Zhonghua 8) were amplified and cloned. The prolamin gene contained 525 base pairs and encoded 134 amino acid residues. The two genes cloned from two different rice cultivars exhibited 100% homology and were highly homologous with the 10 kD prolamin gene in other rice species amountin an homology ranging from 96.6% to 100%. The deduced amino acid sequence shared 34.2% homology with that of maize 10 kD prolamin. As for dicots, only two types of storage protein shared some homology with rice 10 kD prolamin. One was from Brazil nut and the other from castor bean. Analysis on the signal peptide of rice 10 kD prolamin showed that it shared higher homology with that of storage proteins in some monocots such as maize, sorghum and oat. No similar sequence was found in dicots. The gene sequences of "Guangluai” and "Zhonghua 8” 10 kD prolamin would appear in EMBL data-base under the accession number L36604 and L36605 respectively.     

Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium

Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice) and dicots (Arabidopsis, Medicago and Populus) was performed. A total of 797,863 simple sequence repeats (SSRs) were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database 'BraMi' (Brachypodium microsatellite markers) of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium and related grass species, especially for the map based cloning of the candidate gene(s).     

The isochore organization and the compositional distribution of homologous coding sequences in the nuclear genome of plants.

The isochore structure of the nuclear genome of angiosperms described by Salinas et al. (1) was confirmed by using a different experimental approach, namely by showing that the levels of coding sequences from both dicots and Gramineae are linearly correlated with GC levels of the corresponding flanking sequences. The compositional distribution of homologous coding sequences from several orders of dicots and from Gramineae were also studied and shown to mimick the compositional distributions previously seen (1) for coding sequences in general, most coding sequences from Gramineae being much higher than those of the dicots explored. These differences were even stronger for third codon positions and led to striking codon usages for many coding sequences especially in the case of Gramineae.     

Compositional Properties of Homologous Coding Sequences from Plants

In this work, we investigated (1) the compositional distributions of all available nuclear coding sequences (and of their three codon positions) of six dicots and four Gramineae; this considerably expanded our knowledge about the differences previously seen between these two groups of plants; (2) the compositional correlations of homologous genes from dicots and from Gramineae, as well as from both groups; all correlations were characterized by very good coefficients, with slopes close to unity in the former two cases and very high in the last; (3) the compositional transition that accompanied the emergence of Gramineae from an ancestral monocot; (4) the compositional correlations between exons and introns, which were very good in Gramineae, but only poor to good in dicots; and (5) the compositional profiles of homologous genes from angiosperms, which were characterized by a series of peaks (exons) and valleys (introns) separated by 15–20% GC. The conservative and transitional modes of compositional evolution in plant genes and their general implications are discussed. Received: 24 June 1997 / Accepted: 20 August 1997     

Wheat EST sequence assembly facilitates comparison of gene contents among plant species and discovery of novel genes.

Using a strategy requiring only modest computational resources, wheat expressed sequence tag (EST) sequences from various sources were assembled into contigs and compared with a nonredundant barley sequence assembly, with ESTs, with complete draft genome sequences of rice and Arabidopsis thaliana, and with ESTs from other plant species. These comparisons indicate that (i) wheat sequences available from public sources represent a substantial proportion of the diversity of wheat coding sequences, (ii) prediction of open reading frames in the whole genome sequence improves when supplemented with EST information from other species, (iii) a substantial number of candidates for novel genes that are unique to wheat or related species can be identified, and (iv) a smaller number of genes can be identified that are common to monocots and dicots but absent from Arabidopsis. The sequences in the last group may have been lost from Arabidopsis after descendance from a common ancestor. Examples of potential novel wheat genes and Triticeae-specific genes are presented.     

Nucleotide distribution in gymnosperm nuclear sequences suggests a model for GC-content change in land-plant nuclear genomes

Nuclear protein coding sequences from gymnosperms are currently scarce. We have determined 4 kb of nuclear protein coding sequences from gymnosperms and have collected and analyzed >60 kb of nuclear sequences from gymnosperms and nonspermatophytes in order to better understand processes influencing genome evolution in plants. We show that conifers possess both biased and nonbiased genes with respect to GC content, as found in monocots, suggesting that the common ancestor of conifers and monocots may have possessed both biased and nonbiased genes. The lack of biased genes in dicots is suggested to be a derived character for this lineage. We present a simple but speculative model of land-plant genome evolution which considers changes in GC bias and CpG frequency, respectively, as independent processes and which can account for several puzzling aspects of observed nucleotide frequencies in plant genes.Abbreviations GC guanosine plus cytosine - GapC glycolytic glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 - GapA Calvin cycle glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.13 - O/E ratio of observed-to-expected dinucleotide frequencies Correspondence to: W. Martin     

Novel Structural and Functional Motifs in cellulose synthase (CesA) Genes of Bread Wheat (Triticum aestivum,L.)

Cellulose is the primary determinant of mechanical strength in plant tissues. Late-season lodging is inversely related to the amount of cellulose in a unit length of the stem. Wheat is the most widely grown of all the crops globally, yet information on its CesA gene family is limited. We have identified 22 CesA genes from bread wheat, which include homoeologs from each of the three genomes, and named them as TaCesAXA, TaCesAXB or TaCesAXD, where X denotes the gene number and the last suffix stands for the respective genome. Sequence analyses of the CESA proteins from wheat and their orthologs from barley, maize, rice, and several dicot species (Arabidopsis, beet, cotton, poplar, potato, rose gum and soybean) revealed motifs unique to monocots (Poales) or dicots. Novel structural motifs CQIC and SVICEXWFA were identified, which distinguished the CESAs involved in the formation of primary and secondary cell wall (PCW and SCW) in all the species. We also identified several new motifs specific to monocots or dicots. The conserved motifs identified in this study possibly play functional roles specific to PCW or SCW formation. The new insights from this study advance our knowledge about the structure, function and evolution of the CesA family in plants in general and wheat in particular. This information will be useful in improving culm strength to reduce lodging or alter wall composition to improve biofuel production.     

Evolutionary relationship of plant catalase genes inferred from exon-intron structures: isozyme divergence after the separation of monocots and dicots

In order to understand the molecular evolution of catalase genes in higher plants, we compared the exon-intron structures of 12 genomic sequences from six plant species. It was assumed that the putative single primordial catalase gene had seven introns, because only those catalase genes having this structure are found in the monocotyledonae and dicotyledonae classes. After the evolutionary divergence of monocots from dicots, consecutive duplication of the primordial gene followed by the differential loss of introns occurred in each class to form three (or possibly four in dicots) diverse isozyme genes. In monocots, three ancestral isozyme genes were formed before the divergence of ancestral rice and maize. One of the rice genes, CatA, has an entirely new short intron which was not found in any other plant catalase gene examined. We have investigated the existence of the intron in the CatA homolog in other rice species by polymerase chain reaction (PCR) analysis. One major PCR product was found with the genomic DNAs from O. sativa (indica and japonica types), O. rufipogon and O. glaberrima. DNAs from several accessions of O. longistaminata showed variation in both the number and size of the DNA fragments amplified. PCR analyses and sequencing of the PCR products revealed that there are several CatA homologs having different sequences in some accessions of O. longistaminata. We have extended our study to other species in the Poaceae. The results suggest that the gain of the intron, most likely by insertion of a retroposon, took place in the ancestral genome of rice after its evolutionary divergence from other ancestral cereals such as barley, wheat and oat. Received: 20 November 1997 / Accepted: 5 January 1998     

Genomewide computational analysis of nitrate response elements in rice and Arabidopsis

Nitrate response element (NRE) was originally reported to be comprised of an Ag/cTCA core sequence motif preceded by a 7-bp AT rich region, based on promoter deletion analyses in nitrate and nitrite reductases from Arabidopsis thaliana and birch. In view of hundreds of new nitrate responsive genes discovered recently, we sought to computationally verify whether the above motif indeed qualifies to be the cis-acting NRE for all the responsive genes. We searched for the specific occurrence of at least two copies of the above motif in and around the nitrate responsive genes and elsewhere in the Arabidopsis and rice (Oryza sativa) genomes, with respect to their positional, orientational and strand-specific bias. This is the first comprehensive analysis of NREs for 625 nitrate responsive genes of Arabidopsis and their rice homologs, representing dicots and monocots, respectively. We report that the above motifs are present almost randomly throughout these genomes and do not reveal any specificity or bias towards nitrate responsive genes. This also seems to be true for smaller subsets of nitrate responsive genes in Arabidopsis, such as the 21 early responsive genes, 261 and 90 genes for root-specific and shoot-specific response, respectively, and 25 housekeeping genes. This necessitates a fresh search for candidate sequences that qualify to be NREs in these and other plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and characterization of granule bound starch synthase (GBSS-I) from common buckwheat (Fagopyrum esculentum Moench)

Starch grains present in the endosperm of grains of common buckwheat (Fagopyrum esculentum Moench) show a monomodal distribution with size ranging from 4 to 10 μm. SDS-PAGE analysis of starch granule bound proteins revealed the presence of a single band corresponding to molecular mass of 59.7 kDa. The protein is localized within the central core of the starch grains. Antisera raised against the 59.7 kDa protein cross reacted with the 61 kDa GBSS-I from endosperm starches of maize and the 60 kDa GBSS-I from endosperm starches of rice and wheat, thereby indicating serological homology between the 59.7 kDa buckwheat starch granule bound protein and GBSS-I of wheat, maize and rice. 2D-PAGE of starch granule bound proteins of common buckwheat resolved the fraction into 7 spots with pI ranging from 5.2 to 5.6. N-terminal amino acid sequence for 25 residues of two immunoreactive proteins separated by 2D PAGE showed 94 % homology with N-terminal amino acid sequence of GBSS-I from Hordeum vulgare, Triticum spp. and Phaseolus vulgaris. Even though analysis of the sequence alignment revealed a clear diversification into monocotyledonous and dicotyledonous groups, the protein from buckwheat showed similarities with GBSS-I from both dicots as well as monocots. As is the case with dicots, the sequence of GBSS-I from buckwheat has valine as the 11th residue. GBSS-I from majority of monocots has methionine at this position. The sequence also showed similarities with monocots with valine at P’5 from the N-terminus. GBSS-I from majority of dicots has isoleucine at this position. The significance of these substitution remains to be ascertained.     

Genome-wide analysis of WOX gene family in rice, sorghum, maize, Arabidopsis and poplar

WUSCHEL-related homeobox(WOX)genes form a large gene family specifically expressed in plants.They are known to play important roles in regulating the development of plant tissues and organs by determining cell fate.Recent available whole genome sequences allow us to do more comprehensive phylogenetic analysis of the WOX genes in plants.In the present study,we identified 11 and 21 WOXs from sorghum(Sorghum bicolor)and maize(Zea mays),respectively.The 72 WOX genes from rice(Oryza sativa),sorghum,maize,Arabidopsis(Arabidopsis thaliana)and poplar(Populus trichocarpa)were grouped into three well supported clades with nine subgroups according to the amino acid sequences of their homodomains.Their phylogenetic relationship was also supported by the observation of the motifs outside the homodomain.We observed the variation of duplication events among the nine sub-groups between monocots and eudicots,for instance,more gene duplication events of WOXs within subgroup A for monocots,while,less for dicots in this subgroup.Furthermore,we observed the conserved intron/exon structural patterns of WOX genes in rice,sorghum and Arabidopsis.In addition,WUS(Wuschel)-box and EAR(the ERF-associated amphiphilic repression)-like motif were observed to be conserved among several WOX subgroups in these five plants.Comparative analysis of expression patterns of WOX genes in rice and Arabidopsis suggest that the WOX genes play conserved and various roles in plants.This work provides insights into the evolution of the WOX gene family and is useful for future research.     

Compositional properties of nuclear genes from cold-blooded vertebrates

Summary We have investigated the compositional properties of coding sequences from cold-blooded vertebrates and we have compared them with those from warm-blooded vertebrates. Moreover, we have studied the compositional correlations of coding sequences with the genomes in which they are contained, as well as the compositional correlations among the codon positions of the genes analyzed.The distribution of GC levels of the third codon positions of genes from cold-blooded vertebrates are distinctly different from those of warm-blooded vertebrates in that they do not reach the high values attained by the latter. Moreover, coding sequences from cold-blooded vertebrates are either equal, or, in most cases, lower in GC (not only in third, but also in first and second codon positions) than homologous coding sequences from warm-blooded vertebrates; higher values are exceptional. These results at the gene level are in agreement with the compositional differences between cold-blooded and warm-blooded vertebrates previously found at the whole genome (DNA) level (Bernardi and Bernardi 1990a,b).Two linear correlations were found: one between the GC levels of coding sequences (or of their third codon positions) and the GC levels of the genomes of cold-blooded vertebrates containing them; and another between the GC levels of third and first+ second codon positions of genes from cold-blooded vertebrates. The first correlation applies to the genomes (or genome compartments) of all vertebrates and the second to the genes of all living organisms. These correlations are tantamount to a genomic code.     

Compositional compartmentalization and gene composition in the genome of vertebrates

Summary The compositional distribution of coding sequences from five vertebrates (Xenopus, chicken, mouse, rat, and human) is shifted toward higher GC values compared to that of the DNA molecules (in the 35–85-kb size range) isolated from the corresponding genomes. This shift is due to the lower GC levels of intergenic sequences compared to coding sequences. In the cold-blooded vertebrate, the two distributions are similar in that GC-poor genes and GC-poor DNA molecules are largely predominant. In contrast, in the warm-blooded vertebrates, GC-rich genes are largely predominant over GC-poor genes, whereas GC-poor DNA molecules are largely predominant over GC-rich DNA molecules. As a consequence, the genomes of warm-blooded vertebrates show a compositional gradient of gene concentration. The compositional distributions of coding sequences (as well as of DNA molecules) showed remarkable differences between chicken and mammals, and between mouse (or rat) and human. Differences were also detected in the compositional distribution of housekeeping and tissue-specific genes, the former being more abundant among GC-rich genes.