Arabidopsis and Brassica Comparative Genomics: Sequence, Structure and Gene Content in the ABI1-Rps2-Ck1 Chromosomal Segment and Related Regions

Corresponding author: C. F. Quiros, Department of Vegetable Crops, University of California, Davis, CA 95616., cfquiros{at}ucdavis.edu (E-mail)

Communicating editor: B. S. GILL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The region corresponding to the ABI1-Rps2-Ck1 segment on chromosome 4 of Arabidopsis thaliana was sequenced in Brassica oleracea. Similar to A. thaliana, the B. oleracea homolog BoRps2 is present in single copy. The B. oleracea orthologous segment was located on chromosome 4 and can be distinguished by the presence of an N-myristoyl transferase coding gene (N-myr) between the Rps2 and Ck1 (BoCk1a) genes. The N-myr homologs in Arabidopsis are on chromosomes 2 and 5. Additional homologs for Ck1 are located on these two chromosomes. A second Ck1 homolog found on B. oleracea (BoCk1b) chromosome 7 served to define another orthologous segment located in Arabidopsis chromosome 1. The two segments displayed identical gene content and order in both species, namely BoCK1b, a gene encoding a hypothetical protein (BohypothA) and transcription factor eiF4A. High levels of sequence identity were observed for the coding sequences of all genes examined. Although in general larger spacers were found in Brassica than in A. thaliana, this was not always the case. Promoters were poorly conserved, except for several sequence stretches of a few nucleotides. Comparative sequencing revealed microsyntenic changes resulting from chromosomal structural rearrangements, which are often undetectable by genetic mapping.

SINGLE mutations, transpositions, and chromosomal rearrangements, together with environmental selection pressure, over time have created the architecture of genomes and their functional integrity. Many of these changes can be readily detected by comparative microsynteny studies, which are based on actual DNA sequence comparisons. Because coding gene sequences are conserved not only among species or genera, but also across family boundaries, it has been suggested that comparative mapping and gene-specific sequencing will be useful for studying chromosome structure and gene organization among related species. Furthermore, such information might help in determining the ancestral relatives of cultivated species as well as their evolution. However, until recently most comparative studies were coarse and based on genetic maps and various kinds of markers.

Arabidopsis thaliana serves as a model for comparative microsynteny studies with Brassica species, considering that species containing genomes approaching the Brassica ancestral genome are unknown and probably extinct. Other great advantages of A. thaliana are its small genome size and the extensive sequence information readily available from this species. It must be kept in mind, however, that the crucifers A. thaliana and brassicas are classified taxonomically in different tribes (Arabidae and Brassiceae, respectively; PRICE et al. 1994 ), making a direct ancestral relationship between these two genera unlikely, as suggested by GALE and DEVOS 1998 and LAGERCRANTZ 1998 . Furthermore, on the basis of mtDNA data the lineages leading to these tribes are estimated to have split 14.5 to 20.4 million years ago (YANG et al. 1999 ). Earlier comparative mapping of Arabidopsis and Brassica species disclosed islands of conservation for the chromosome segments tested (KOWALSKI et al. 1994 ; SADOWSKI et al. 1994 , SADOWSKI et al. 1996 ; LAGERCRANTZ and LYDIATE 1996 ; CONNER et al. 1998 ). Furthermore, it has been estimated by restriction fragment length polymorphism mapping that only 20% of a chromosome maintains colinearity between A. thaliana and Brassica oleracea, but 90% of chromosomal regions of <5 cM remain conserved (PATERSON et al. 1996 ). Some of these segments conserving gene repertoire and order may represent orthologous segments between these species. Comprehensive comparative microsynteny studies between species of these two genera will allow the assignment and alignment of these segments across species in the near future.

We selected for this study the A. thaliana ABI1-Rps2-Ck1 segment on chromosome 4, which, according to previous comparative genetic mapping, is structurally conserved in Brassica species (SADOWSKI and QUIROS 1998 ). Rps2 is a single-copy locus in both A. thaliana (MINDRINOS et al. 1994 ) and B. oleracea (WROBLEWSKI et al. 2000 ), which facilitates assignment of orthology in both genomes. Genomic sequences corresponding to these two homologous chromosomal segments in A. thaliana and B. oleracea were analyzed and used to determine the structural changes distinguishing these two crucifers.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Library construction in B. oleracea and screening:
We used B. oleracea variety Purple Cauliflower (B0265) to construct a cosmid library. DNA was extracted from 6- to 8-week-old plants grown for 2 days in darkness before nuclei isolation (KIANIAN and QUIROS 1992 ). The isolated DNA was cloned in the SuperCos1 (Stratagene, La Jolla, CA) cosmid vector according to the manufacturer. The library was screened according to SAMBROOK et al. 1989 . Arabidopsis cDNA clones for the genes ABI1, Rps2, and Ck1 obtained from the Arabidopsis stock center were used as probes, which were labeled with [-³²P]dCTP using the Multiprime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ). After selection of the colonies, cosmids were isolated by alkaline lysis (SAMBROOK et al. 1989 ).

Physical mapping:
Cosmid clones were digested with NotI for incomplete vector trimming. A total of 300 ng of cosmid DNA was partially digested with 0.2–1.0 units of EcoRI or HindIII enzyme for 45 min in a volume of 20 µl. Samples were fractionated by electrophoresis in a 0.5% agarose gel at 1.5 V/cm for 40 hr. The gel was alkaline-blotted onto a ZetaProbe membrane (Bio-Rad, Hercules, CA). The membranes were hybridized with 5'-labeled 21-mer T3 and T7 oligonucleotides corresponding to the sequences flanking the inserts in the vector. The hybridization was conducted overnight at 50° according to Bio-Rad protocol with SDS as a blocking agent. Oligonucleotides used as probes were labeled with [-³²P]dATP by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Restriction maps for the cosmids were assembled manually.

Sequencing:
For sequencing, HindIII fragments of the digested cosmids were purified from agarose gels and subcloned into plasmid pUC19. After alkaline lysis (SAMBROOK et al. 1989 ) the clones were purified by PEG/NaCl precipitation. Sequencing was performed on an automated sequencer, ABI PRISM 377. To complete the 5' end sequences of two caseine kinase homologs (BoCk1a and BoCk1b), which were truncated in the cosmid clones, we isolated two BAC clones from a library constructed from a doubled-haploid broccoli line derived from the variety Early Big (G. LI and C. F. QUIROS, unpublished results). Primers were based on exon 5 of BoCk1a, intron 2 of BoCk1b, and a few hundred nucleotides upstream of the ATG codons of both genes.

Data analysis:
DNA Strider (MARCK 1988 ) was used to visualize open reading frames (ORF), restriction sites, and repeated sequences. Homology searches were carried out against nonredundant public libraries on the National Center for Biotechnology Information server using the BLAST program (ALTSCHUL et al. 1990 ). Predicted coding regions were analyzed using the programs Genemark (BORODOVSKY and MCININCH 1993 ), Genscan (BURGE and KARLIN 1998 ), NetPlantGene (HERBSGAARD et al. 1996 ), and Genefinder (ZHANG 1997 ). The PLACE database (HIGO et al. 1998 ) was used to analyze the putative gene promoter regions.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Clone identification and sequencing for the ABI1-Rps2-CK1 B. oleracea segment:
We screened a B. oleracea cosmid library with an A. thaliana Rps2 probe and obtained four clones, ranging in insert size from 32.1 to 42.0 kb, which contained homologs to this gene (AtRps2). Three of the clones also hybridized with a gene downstream of Rps2, Col-0 casein kinase-like protein (AtCk1), and the fourth clone hybridized to the protein phosphatase ABI1 gene (abscisic acid insensitive, AtABI1) upstream of Rps2. None of the B. oleracea cosmid clones carried all three genes. Restriction maps were constructed for each clone by digestion with HindIII and EcoRI. Two of the three Brassica clones containing the Rps2 (BoRps2) and Ck1 (BoCk1a) homologs were selected for sequencing on the basis of their restriction profiles. The insert of the first clone (Fig 1, clone 1) was 39.6 kb in length, whereas in the second one (Fig 1, clone 2) it was 38.3 kb. Comparison of their restriction maps matched profiles expected for the segment carrying AtRps2 (MINDRINOS et al. 1994 ). We sequenced two contiguous HindIII fragments for both clones 1 and 2 and proved that they were identical. On the other hand, the adjacent sequences in a third HindIII fragment downstream of BoRps2, containing the rest of the spacer and BoCK1a, were different in the two clones due to a rearrangement. This difference, however, did not affect the sequences targeted in the analyses of the clones (Fig 1).

View larger version (15K): In this window In a new window Download PPT slide   Figure 1. Schematic representation of the HindIII segments selected for sequencing in the inserts of four cosmid clones. The composite diagram depicted was constructed by overlapping common sequences from clones 1, 2, and 4. The direction of transcription is indicated by arrows above the name of each gene.

A 4306-bp HindIII segment of clone 4, hybridizing to both ABI1 and Rps2, was also sequenced (Fig 1, clone 4). After contig assembly of the B. oleracea ABI1-Rps2-Ck1 segment (AF180355), we found structural differences between the B. oleracea and Arabidopsis counterparts.

Microsyntenic changes for the ABI1-Rps2-CK1 segment distinguishing A. thaliana from B. oleracea:
Sequencing of the corresponding A. thaliana chromosome 4 segment ABI1-Rps2-Ck1 in the B. oleracea clones 1 and 2 revealed a major change in gene content. An N-myristoyl transferase (N-myr) gene was found between BoRps2 and BoCk1a (Fig 2A). This segment has been mapped on chromosome 4 of B. oleracea (J. SADOWSKI, D. BABULA and M. KACZMAREK, unpublished results). A homology search in GenBank disclosed two N-myr homologs in A. thaliana, one on chromosome 2 (AtN-myr/2, BAC F6E13) and another one on chromosome 5 (AtN-myr/5, P1 MHM17). The segment carrying this gene on chromosome 2 was annotated, showing that this chromosome contains homologs for two contiguous genes on chromosome 4, Ck1 (AtCk1/2), and NAP1 (AtNAP1/2). Unlike the B. oleracea arrangement, AtN-myr/2 and AtCk1/2 are not contiguous, but are a few megabases away from each other (Fig 2B). Although the A. thaliana clone P1 MHM17 on chromosome 5 carrying the homolog AtN-myr/5 is not annotated, we were able to construct a physical map for AtN-myr/5 and its flanking genes on the basis of similarity to various reported sequences. Similar to the arrangement in B. oleracea, in this A. thaliana segment a Ck1 homolog (AtCk1/5) was contiguous to AtN-myr/5. Additionally, an AtABI1 homolog (AtABI2) was on the same segment, separated from AtN-myr/5 by three other genes (Fig 2B). In the corresponding B. oleracea segment, BoN-myr is in between BoRps2 and BoCk1a instead (Fig 2A). In all segments, the orientation of the Ck1 and N-myr was the same. The only exception was the presence of a NAP1 homolog 36 kb upstream of AtCk1/5. Both are on the same strand, which is contrary to the arrangement observed when these genes are found to be contiguous in other chromosomes in both Arabidopsis and Brassica (Fig 2A and Fig C).

View larger version (23K): In this window In a new window Download PPT slide   Figure 2. Schematic representation of the homologous chromosome segments of B. oleracea and A. thaliana relevant to this study. Open reading frames (ORF) are depicted by boxes. Sizes of ORFs and spacers in base pairs are shown under each of these elements. Arrows show the direction of transcription. (A) B. oleracea chromosome 4 segment carrying BoRps2, BoN-myr, and BoCk1a and its Arabidopsis ortholog, also on chromosome 4. (B) A. thaliana segments on chromosomes 2 and 5 displaying homology to Brassica and Arabidopsis chromosomes 4. (C) B. oleracea chromosome 7 segment carrying BoeiF4A BohypothA and BoCk1b and its Arabidopsis ortholog on chromosome 1. This segment is partially duplicated on the same chromosome and on chromosome 3.

Structural characteristics and similarity of specific homologs in both species:
Overall, the various homologs in the compared B. oleracea chromosome 4 and A. thaliana chromosome 4 segments were highly similar, except for spacers and introns, as shown in Fig 3A.

View larger version (10K): In this window In a new window Download PPT slide   Figure 3. Dot plots for Arabidopsis and Brassica sequences. (A) Brassica sequence AF180355 against Arabidopsis BAC F20B18 aligning orthologous ABI1-Ck1 segments. The diagonal in the top left corner represents ABI1 followed by Rps2 and then by Ck1. The first gap represents the spacer between the first two genes. The second gap represents the absence of N-myr in the A. thaliana sequence and spacers. The trail of dots (arrow) represents the N-myr promoter and the Ck1 untranslated trailer with homology to Rps2. (B) Brassica sequence AF1800356 against Arabidopsis BAC F28P22 aligning orthologous eiF4A-Ck1 segments. The broken line in eiF4A (arrow) is due to the lack of intron 3 in the Brassica sequence. The short lines above represent short intergenic spacer sequences showing similarity. The larger breaks on Ck1 correspond to the largest introns (intron 2, bottom arrow; intron 6, top arrow). The short broken segments correspond to the rest of the introns.

BoRps2: This gene is structurally very similar to its homolog, AtRps2 (BACF20B18), with respect to orientation, size, and lack of introns. Furthermore, there is a single copy of this gene in both Arabidopsis and in at least two Brassica species, B. nigra and B. oleracea (SADOWSKI and QUIROS 1998 ; WROBLEWSKI et al. 2000 ). The amino acid identity of the ORFs of AtRps2 and BoRps2 is 87%. A detailed comparative analysis in the Brassiceae of the different Rps2 domains, such as the leucine zipper, nucleotide-binding site, and leucine-rich region, has been reported elsewhere (WROBLEWSKI et al. 2000 ). The upstream sequences carrying the promoters in both AtRps2 and BoRps2 could be aligned by two stretches of a few bases, showing the rest of the sequences' low similarity values. The only exception was a 59-nucleotide region upstream of the translation initiation signal, which has 78% nucleotide identity between the two homologs. Similarly, the trailer sequence of 33 bp downstream of the stop signal is also conserved (82%). Upstream of the conserved leader, the B. oleracea sequence contains putative promoter domains including CAAT and GC boxes and a TATA box (TATAT) at position 112. The promoter sequence was confirmed by sequencing this region in clones 1 and 2. These domains are also present in AtRps2, but at different positions. A putative TATA box (TATTAAA) is located in AtRps2 at nucleotide 91 from the initiation signal.

BoCk1a: Sequencing of the two clones carrying BoCk1a disclosed that the 5' end of this gene was truncated in both clones. We were able to sequence the missing portion of this gene from a BAC clone (as explained above) to complete all 14 exons and promoter (Table 1). BoCk1a and AtCk1/4 have the highest identity of all homologs, in spite of belonging to different species (Table 2). The majority of the Ck1 homologs have 14 exons, and the first 12 exons from the 5' end of the gene show size conservation. AtCk1/5 is the exception due to the fusion of exons 11 and 12 with the loss of a few bases, resulting in an exon of 150 bp. Exons 13 and 14 are variable in size, along with most of the introns in all homologs (Table 1). The promoter and transcribed but nontranslated regions of AtCk1/4 (determined by the U12857 transcript) and BoCk1a, corresponding to 340 bases upstream of the ATG codon of AtCk1/4, had 54% shared identity. AtCk1/4 displayed a (GA)₈ microsatellite sequence at 163 bases upstream of its ATG codon. This sequence was also present in BoCk1a but it consisted only of four GA copies.

BoN-myr: The sequences of BoN-myr and its two homologs, AtN-myr/2 and AtN-myr/5, lack introns interrupting their coding regions. Alignment of the three coding sequences shows some differences between the three corresponding genes. The two Arabidopsis coding sequences are not identical: AtN-myr/2 is larger than AtN-myr/5 in its 5' extremity. In their common part, the Arabidopsis homologs display 74% nucleotide identity. The BoN-myr gene differs from its Arabidopsis homologs by the presence of a 253-bp deletion in the central part of the gene, which results in a shorter predicted protein product (Fig 4). The conserved part of BoN-myr has amino acid sequence identities of 82.4 and 77.9% with the Arabidopsis N-myr/5 and N-myr/2 proteins, respectively. For the AtN-myr/5 gene, several expressed sequence tags (ESTs) are available in the databases. One of them, T76600, allowed us to identify an untranslated 5' exon of 144 bp followed by a 315-bp intron in the AtN-myr/5 gene, upstream of the ATG translation initiation signal. The 3' border of this intron is located 3 bp upstream of the ATG triplet. Such a structure can also be recognized in the BoN-myr gene by the following features: First, the 128 bp of the 5' end of the AtN-myr/5 exon can be aligned to the sequence upstream of the translation initiation signal in the BoN-myr gene with 71.8% identity. Second, the nucleotide sequences around the 5' and 3' borders of the AtN-myr/5 intron are conserved in the corresponding region of the BoN-myr gene. These are not found in the AtN-Myr/2 gene.

View larger version (60K): In this window In a new window Download PPT slide   Figure 4. Protein alignment for BoN-myr (upper sequence) and AtN-myr/5 (lower sequence) based on At EST T76600 and P1 clone MHM7. The missing portion in BoN-myr is indicated by dashes. In their common parts, both proteins are 90% homologous if only identical amino acids are considered (+). Identity is 96% taking into account conservative amino acids (.).

BoABI1: Only the 5' end of this gene was available for comparison between B. oleracea and A. thaliana. This included the promoter region, the first exon, and part of the first intron. Both A. thaliana homologs, AtABI1 and AtABI2, have four exons and three introns each, all of comparable sizes. The identity of the BoABI1 5' portion was higher with AtABI1 than with AtABI2. The first exon in BoABI1 has 507 bases and is shorter than its Arabidopsis homologs (549 bp for AtABI1 and 512 bp for AtABI2). At the amino acid level, homologies of the first exon of BoABI1 to those of AtABI1 and AtABI2 are 82 and 72%, respectively. The first intron in the A. thaliana homologs is small (70 bp for ABI1 and 82 bp for ABI2). In the B. oleracea homolog, this intron is much larger, with a partial sequence of 1044 bp. This intron lacks identity to its Arabidopsis counterparts.

Spacer BoRps2-BoCk1a:
The intergenic spacers for these two genes in A. thaliana and B. oleracea were structurally different. In Arabidopsis the spacer between these genes (559 bp) is formed by the overlapping ends of the 3' ends of the transcribed but nontranslated sequences (from stop to stop codons) of these two genes that are in opposite orientations (Fig 5A). There are only 12 nucleotides separating the end of the Rps2 transcript from the Ck1 stop signal. On the other hand, the 3' ends of the Rps2 and Ck1 genes in B. oleracea are 2212 bases apart, including the BoN-myr gene. The spacer sizes for these genes are shown in Fig 2A. The spacer between the 5' end of the BoN-myr gene and the 3' end of the BoCk1a gene has a complex structure consisting of partially overlapping sequences of both the N-myr gene and the 3' end of the Rps2 gene. These Rps2 interstitial sequences are homologous to the transcribed, nontranslated 3' end of both AtRps2 and cognate cDNA (U12860) and are in the expected orientation for Rps2 (Fig 3A and Fig 5B).

View larger version (19K): In this window In a new window Download PPT slide   Figure 5. Diagram depicting homologies in the spacers of two orthologous segments. Arrows represent direction of transcription for each gene. (A) AtRps2-AtCK1/4 spacer from stop to stop codon of both genes. The vertically hatched box represents Rps2 transcript U12860 and the solid bar represents the CK1 transcript U12857. The transcripts of both genes overlap at their ends, and the AtRps2 transcript ends 12 nucleotides before the AtCK1/4 stop codon. (B) BoN-myr-BoCK1 spacer. The diagonally hatched boxes depict homologous regions of AtN-myr sequences MHM17 (intron lacking homology to BoN-myr shown as open box) and EST T76600. The vertically hatched boxes show the region of homology to AtRps2 transcript U12860, which overlaps with AtCk1 sequences (F20B18 and U12857), shown as solid bars.

Spacer BoABI1-BoRps2:
These two genes are in opposite orientation in B. oleracea as well as in A. thaliana. The spacer between the translation initiation signals of both genes has 3446 bp in B. oleracea, whereas in the corresponding A. thaliana segment on chromosome 4 it has only 2767 bp (Fig 2A). This spacer includes the promoter region for both genes in both species. In the BoABI1 putative promoter region, the 666 bases upstream of the ATG initiation signal, which include a (GA)₁₆ microsatellite, align well with the 623 bases located upstream of the AtABI1 ATG triplet, with an overall 63% nucleotide identity. This microsatellite sequence was also present in AtABI1 and AtABI2 but in fewer copies. The corresponding region in AtABI2 is less conserved since only a few segments can be aligned with both BoABI1 and AtABI1 putative promoters. At the opposite side of the BoABI1-BoRps2 spacer, in the BoRps2 promoter region, a stretch of 207 nucleotides located upstream of the putative TATA domain already mentioned can be aligned with two putative reverse transcriptase sequences contained in the A. thaliana accessions AC006300 and AF058914. No similarity with known sequences was found for the middle part of the BoABI1-Rps2 spacer.

Other related segments:
A fifth clone (clone 5, Fig 1) in the Brassica cosmid library hybridized only to the Ck1 Arabidopsis probe and displayed a unique restriction profile different from those observed for clones 1 and 2. It was partially sequenced (GenBank accession no. AF180356), covering a total of 6639 bp of the HindIII fragment. This clone contains a Ck1 homolog (named BoCk1b) that maps on B. oleracea chromosome 7 (J. SADOWSKI, D. BABULA and M. KACZMAREK, unpublished results) as well as homologs for a hypothetical protein gene (named BohypothA) and transcription factor eiF4A gene (named BoeiF4A). The Brassica sequence AF180356 was found to have its counterpart in A. thaliana BAC F28P22 on chromosome 1 (AC010926). Although this Arabidopsis clone is nonannotated, its high sequence identity with its Brassica counterpart permitted identification of the homologous genes. The overall identity of the homologs in these two segments is shown in Fig 3B.

BoCk1b: Like BoCk1a, BoCk1b was truncated at the 5' end, but only its first two exons and the promoter were missing in this case. We completed the two missing exons and promoter by sequencing a BAC clone as explained above. The amino acid identity between the A. thaliana Ck1 homolog on chromosome 1 (named AtCk1/1 hereafter) and BoCk1b is 91% (97% amino acid conservation), which is the highest among other homologs in both species (Table 2). The exons compared, 1–14, were identical in size in both homologs, including exons 6, 13, and 14, which were variable among other homologs. The overall nucleotide identity for the exons of AtCk1/1 and BoCk1b was 91%. Intron nucleotide identity for these two genes was 70%, excluding introns 2 and 6, which are the largest exons and thus showed less similarity (Table 1). BoCk1b also displayed high identity to three A. thaliana ESTs that probably correspond to a single mRNA from AtCk1/1 (Z25497, R90041, and T13780).

The region upstream of the ATG codon of BoCk1b of 520 bases could be aligned to its corresponding 560-base region of AtCk1/1 with 48% identity. Several stretches of a few nucleotides shared the same sequence or one very similar, permitting alignment of these two regions corresponding to the promoters of the two genes.

BohypothA: This gene was found next to and in opposite orientation to BoCk1b. This is also the case for its A. thaliana homolog (hereafter named AthypothA) found in BAC F28P22 (AC010926), described as a gene coding for a "hypothetical protein," which is next to AtCk1/1 and in opposite orientation. The amino acid identity between AthypothA and BohypothA is 68.5% (85% conservation), whereas the nucleotide identity from the ATG translation initiation signal to the stop signal is 75%. Both genes are small and intronless, differing in size by only 6 bp (Fig 2C). The spacer between the Ck1 and hypothA genes in both species is also similar in size, 756 bases for Brassica and 724 bases for Arabidopsis. There are two stretches with higher sequence similarity: one of 230 bases downstream of the hypothA translation stop signal (59.6% identity) and the other 200 bases downstream of the Ck1 translation stop signal (78% identity). The rest of the spacer showed little similarity in the two species.

In Arabidopsis we found two other chromosomes carrying homologs for the gene coding for the hypothetical protein AthypothA. The first one is on chromosome 2 (hereafter named AthypothB, AC005917) and the second one is on chromosome 3 (hereafter named AthypothC, AB019229). The ORFs of these homologs are 350 and 309 bp, respectively. Similarly to AthypothA and BohypothA, none of these homologs contained introns. Interestingly, AthypothB was next to a Ck1 homolog (hereafter named AtCk1/2) on chromosome 2, mimicking the arrangement and orientation observed for these two genes on A. thaliana chromosome 1 and B. oleracea chromosome 7. Two more loci were observed, making a total of five genes coding a hypothetical protein of similar amino acid composition and size: a second locus on chromosome 2 (AC002535) and another one on chromosome 4 (AL049481). The identity correspondence among these proteins of the Arabidopsis hypothetical protein genes was lower in comparison to that observed between AthypothA and BohypothA.

BoeiF4A: BoeiF4A is next to the BohypothA gene; both are in the same orientation. This is also the case for the corresponding A. thaliana segment on chromosome 1 (BAC F28P22), which contains the homologs for these two genes (Fig 2C). The sequence of BoeiF4A and that of its Arabidopsis counterpart (hereafter named AteiF4A/1a) could only partially be compared because BoeiF4A was truncated at the 5' end starting in the second intron, making a total length of 1063 bases available for analysis. AteiF4A/1a has four exons; therefore, the promoter and first two exons of the Brassica gene could not be inspected. The two homologs displayed high amino acid identity, as well as the mRNA for AteiF4A/1a (GenBank accession no. AJ010472; 96.9% identity, 99.7% conserved). However, the sequence corresponding to the third intron of the Arabidopsis gene was completely absent in the Brassica homolog. The coding sequences corresponding to exons 3 and 4 have 89.5% identity between both homologs. These sequences have the same size in both species, indicating exon size conservation. The spacer between the eiF4A and hypothA genes was 53 bases longer in Arabidopsis than in Brassica (Fig 2C), displaying an overall nucleotide identity of 57.2%. The promoter of these genes seems to be 70 bases upstream of the ATG translation initiation signal, where TATA-like sequences could be observed. The sequences corresponding to the translated but nontranscribed sequences of both genes had higher sequence similarity than the rest of the spacer.

In A. thaliana there are at least two other homologs to the eiF4A gene. There is a second locus on chromosome 1 (hereafter named AteiF4A/1b; BAC clone AC005287) 20 kb upstream of the first one (BAC F28P22). An mRNA sequence is available in GenBank for this gene. The third eiF4A homolog was located on chromosome 3 (hereafter named AteiF4A/3, P1 clone AB019229). There is also an mRNA accession in GenBank (AJ010472), described as a DEAD box RNA helicase, another name given to this gene. The ORFs of these genes are 1524 and 1494 bases, respectively. All three A. thaliana eiF4A homologs have four exons and three introns. None of them lack the third intron as does BoeiF4A. These are large genes with total regions covered by transcription of 2373 bp for AteiF4A/3 and 2590 bp for AteiF4A/1b. The identity of the amino acid sequences between species and within Arabidopsis was very high, on the order of 95%. The highest identity, however, was observed between BoeiF4A and AteiF4A/1a. All three Arabidopsis chromosome segments carrying the eiF4A homologs also had homologs for a B-Ca⁺ trans ATPse gene nearby (Fig 2B).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sequencing of the B. oleracea segment spanning from BoABI1 to BoCk1a allowed us to disclose changes in gene content with respect to Arabidopsis in this part of the genome.

On the basis of sequence similarity and gene content we were able to detect two sets of orthologous chromosome segments in Arabidopsis and Brassica. The first one on chromosome 4, spanning genes ABI1 to Ck1, and the second one on chromosome 1, spanning genes eiF4A to Ck1. The orthology of the ABI1-Ck1 segments in both species is supported by the following facts:

1. Highest sequence similarity between the chromosome 4 segments from both species than to any other Arabidopsis segments carrying homologs for ABI1 and Ck1.

2. Single-copy status of Rps2 in both species and high sequence similarity of the two homologs (MINDRINOS et al. 1994 ; WROBLEWSKI et al. 2000 ). The single-copy status of Rps2 in A. thaliana is further supported by the absence of other ESTs or any other sequences displaying high similarity levels throughout the whole length of the gene.

3. The presence of three additional genes in common to both species downstream of their Ck1 respective homologs. These genes have been detected by genetic mapping and pulsed-field gel electrophoresis in B. nigra (SADOWSKI and QUIROS 1998 ) and B. oleracea (J. SADOWSKI, D. BABULA and M. KACZMAREK, unpublished results). They code for a nucleosome assembly protein (NAP1), for a NPR1-like protein, and for an "uncharacterized" protein.

The alternative possibility is that the ABI2-Ck1 segment on chromosome 5, which carries a N-myr gene next to Ck1, is orthologous to the B. oleracea ABI1-Ck1 segment. This is unlikely because of the following facts: (1) the absence of Rps2 in this segment, which is replaced by three other genes; (2) the absence of a NAP1 sequence contiguous to Ck1, although there is a NAP1 homolog 40 kb from Ck1 and in the same strand (in the other chromosomes these two genes are in opposite strands) on this chromosome; and (3) higher sequence identity for the ABI1 and Ck1 homologs in the chromosome 4 segments of both species.

The structural changes distinguishing the orthologous ABI-Ck1 segments in A. thaliana and B. oleracea most likely have occurred after the separation of the lineages leading to the formation of the Arabidae and Brassiceae tribes. The question is, which arrangement is ancestral? We can only speculate that perhaps the A. thaliana arrangement may be recent. In such case we assume that a copy of an ancestral N-myr gene was present in the ancestor of A. thaliana chromosome 4 between the AtRps2 and AtCK1/4 genes, in the same orientation as we found them today in the B. oleracea orthologous segment. This possibility is supported by the virtual fusion of the ends of the AtRps2 and AtCk1/4 genes, whose transcripts overlap at their 3' termini, which might have resulted from the excision of an N-myr gene. CONNER et al. 1998 reported that the lack of the self-incompatibility locus in A. thaliana might be due to a similar event. Furthermore, the contiguous arrangement of Ck1 and N-myr on A. thaliana chromosome 5 also supports the assumption that this is the ancestral arrangement in the Brassicaceae. An important consideration, however, is that the occurrence of the syntenic change resulting in the presence of BoN-myr between BoRps2 and BoCk1a can be tested in other species of this family, which will tell us more about the time of occurrence of this event and its implication on the origin and evolution of these species. The absence of the N-myr sequence on the A. thaliana chromosome 4 segment carrying Rps2 and the lack of analysis of all N-myr homologs in B. oleracea impede orthology assigment to BoN-myr. However, higher sequence identity of this gene to AtN-myr/5 rather than to AtN-myr/2 suggests a common origin for BoN-myr and AtN-myr/5. This is further supported by the similar structure of BoN-myr and AtN-myr/5. All N-myr proteins described to date present a structure comparable to that of the two Arabidopsis genes, AtN-myr/2 and AtN-myr/5. The absence of an interstitial segment in BoN-myr does not shift its ORF; however, it puts in question the functionality of this gene. Additional homologs of N-myr in B. oleracea will have to be characterized to know more about the origin and relationships of these genes.

Orthology assignment for the eiF4A-Ck1 segments from Arabidopsis chromosome 1 and B. oleracea chromosome 4 was straightforward on the basis of the high level of homology of the three pairs of genes compared. Furthermore, gene content is identical and even the spacers are not very different in size in the two segments.

Sequence identity conservation sheds light on the level of divergence among the genes compared. The Ck1 sequences are the most informative for this type of inference because of their multiple copies in A. thaliana and B. oleracea. Several conclusions can be reached from this analysis:

1. Greater similarity exists between homologs of different species than within the same species; e.g., BoCk1a and AtCk1/4 and BoCk1b and AtCk1/1 are two pairs of orthologs since they share higher identity than their respective homologs within the same species.

2. Divergence of these two pairs of orthologs from each other has taken place before tribal separation of the two species.

3. Intraspecific homologs share different levels of sequence identity. In Arabidopsis, the most divergent Ck1 homolog is AtCk1/2, which might represent an ancient paralog. On the other hand, the relatively higher sequence identity AtCk1/5 to AtCk1/4 indicates that this might represent a paralog of more recent origin.

4. Changes in exon size due to the loss of an intron do not reflect the level of sequence divergence among Ck1 or eiF4A homologs. For example, fusion of exons 11 and 12 due to the lack of intron 11 in AtCk1/5 was unique and might represent a recent structural change to this homolog, since it was absent in all other Ck1 homologs. On the other hand, the sizes of the last two exons located at the C-terminal part of the Ck1 genes (exons 13 and 14) were variable, with the notable exception of BoCk1b and AtCk1/1, which had the highest level of sequence identity.

A likely explanation for this variation is that the first 11–12 exons of these genes encode for the kinase portion of the protein product, which is generally conserved among all kinases. The situation observed for the 5' end portion of the ABI homologs available for analysis is apparently somewhat different. Although the information is quite limited, for these genes exon size might not be as conserved as it is for the Ck1 and eiF4A homologs, since the first exon of BoABI was smaller than that of AtABI1.

The presence of the three pairs of homologs, eiF4A and B-⁺Ca trans ATPse, close to each other on three different chromosome segments of Arabidopsis, is unlikely to occur by chance. These segments probably represent ancient duplications followed by rearrangements in the Arabidopsis genome. Six of the seven genes we compared have two to four copies, with the exception of Rps2, which was in single copy. The high level of gene replication observed is in agreement with the recent finding that the level of gene duplication in Arabidopsis is much higher than reported previously (LIN et al. 1999 ; BLANC et al. 2000 ; GRANT et al. 2000 ). This demonstrates that the Arabidopsis genome is not a simple and primitive genome. Although sequencing information is currently quite limited in Brassica, most likely a similar or higher level of complexity will be disclosed in these species. In B. oleracea we were able to compare only two Ck1 genes, BoCk1a and BoCk1b. In any case, it is clear that homologs for specific genes in both species can be readily distinguished by their sequences as well as their exonic/intronic structure. Now that the complete genome of Arabidopsis is sequenced (ARABIDOPSIS GENOME INITIATIVE 2000), a full reassessment of these duplications in this species as well as in Brassica species will be possible.

Spacer size difference between Arabidopsis and Brassica has been an issue that various laboratories have tried to address, using mostly genetic mapping data (LAGERCRANTZ and LYDIATE 1996 ; SADOWSKI et al. 1996 ; CONNER et al. 1998 ; SADOWSKI and QUIROS 1998 ). In general, there is the impression that the difference in genome size between Arabidopsis and other cruciferae species was due in part to a smaller spacer size in the former (SADOWSKI et al. 1996 ). However, comparative sequence analysis shows that the situation is much more complex than previously thought.

Inspection of spacer sequences for the Brassica genes studied failed to disclose an extensive retrotransposon sequence as reported in maize (SANMIGUEL et al. 1997). Thus, for the chromosome segments analyzed, it was not possible to explain rearrangements in synteny solely by the action of these elements in Brassica. The only evidence for the presence of retrotransposons was in the spacer between ABI1 and Rps2 in Brassica. This is not surprising, considering that these elements are located in high density in the pericentromeric regions of Arabidopsis (COPENHAVER et al. 1999 ). The spacers examined contained mostly the putative promoters and untranslated trailers of their respective genes. In general they disclosed poor sequence conservation among homologs, except for a few short nucleotide stretches.

Comparative sequencing allows detection of synteny breaks caused by chromosomal rearrangements that distinguish the genomes of Arabidopsis and Brassica. Genetic mapping based on DNA hybridization often fails to detect these changes since it affords only a rough approximation and must be followed by sequence analysis to gather precise information on the structure of complex genomes such as those of the crucifers. The latter approach will tell us more about the evolutionary paths followed by these species in the near future.

	ACKNOWLEDGMENTS

We are indebted to Vincent D'Antonio, Dinh Li, and Russell Wrobel for technical assistance and to Genyi Li, Sheila McCormick, and Roger Chetelat for reviewing the manuscript, and to Karen Olson for editing it. We are also indebted to Dean Lavelle at the UCD Plant Genetics Facility for sequencing our DNA samples. This work was supported in part by United States Department of Agriculture National Research Initiative grant no. 9600835 to C.F.Q. and by the Polish Committee for Scientific Research grant no. PO6A016 11 to J.S.

Manuscript received May 8, 2000; Accepted for publication July 1, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALTSCHUL, S. F., W. GISH, W. MILLER, E. MYIERS, and D. LIPMAN, 1990  Basic local alignment search tool. J. Mol. Biol. 215:403-410[Medline].

Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. (2000) Nature 408:796-815[Medline].

BLANC, G., A. BARAKAT, R. GUYOT, R. COOKE, and M. DELSENY, 2000  Extensive duplication and reshuffling in the Arabidopsis genome. Plant Cell 12:1093-1102[Abstract/Free Full Text].

BORODOVSKY, M. and J. MCININCH, 1993  Genemark: parallel gene recognition for both DNA strands. Comput. Chem. 17:123-133.

BURGE, C. B. and S. KARLIN, 1998  Finding the genes in genomic DNA. Curr. Opin. Struct. Biol. 8:346-354[Medline].

CONNER, J. A., P. CONNER, P. E. NASRALLAH, and J. B. NASRALLAH, 1998  Comparative mapping of the Brassica S locus region and its homeolog in Arabidopsis: implications for the evolution of mating systems in the Brassicaceae. Plant Cell 10:801-812[Abstract/Free Full Text].

COPENHAVER, G. P., K. NICKEL, T. KUROMORI, M. I. BENITO, and S. KAUL et al., 1999  Genetic definition and sequence analysis of Arabidopsis centromeres. Science 286:2468-2474[Abstract/Free Full Text].

GALE, M. D. and K. M. DEVOS, 1998  Plant comparative genetics after 10 years. Science 282:656-658[Abstract/Free Full Text].

GRANT, D., P. CREGAN, and R. C. SHOEMAKER, 2000  Genome organization in dicots: genome duplication in Arabidopsis and synteny between soybean and Arabidopsis. Proc. Natl. Acad. Sci. USA 97:4168-4173[Abstract/Free Full Text].

HERBSGAARD, S. M., P. G. KORNING, N. TOLSTRUP, J. ENGELBRECHT, and P. ROUZE et al., 1996  Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information. Nucleic Acids Res. 24:3439-3452[Abstract/Free Full Text].

HIGO, K., Y. UGAWA, M. IWAMOTO, and H. HIGO, 1998  PLACE: a database of plant cis-acting regulatory DNA elements. Nucleic Acids Res. 26:358-359[Abstract/Free Full Text].

KIANIAN, S. F. and C. F. QUIROS, 1992  Generation of a Brassica oleracea composite RFLP map: linkage arrangements among various populations and evolutionary implications. Theor. Appl. Genet. 84:544-554.

KOWALSKI, S. P., T-H. LAN, K. A. FELDMANN, and A. H. PATERSON, 1994  Comparative mapping of Arabidopsis thaliana and Brassica oleracea chromosomes reveals islands of conserved organization. Genetics 138:499-510[Abstract].

LAGERCRANTZ, U., 1998  Comparative mapping between Arabidopsis thaliana and Brassica nigra indicates that Brassica genomes have evolved through extensive genome replication accompanied by chromosome fusions and frequent rearrangements. Genetics 150:1217-1228[Abstract/Free Full Text].

LAGERCRANTZ, U. and D. LYDIATE, 1996  Comparative genome mapping in Brassica.. Genetics 144:1903-1910[Abstract].

LIN, X., S. KAUL, S. ROUNSLEY, T. SHEA, and M. I. BENITO et al., 1999  Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana.. Nature 402:761-767[Medline].

MARCK, C., 1988  "DNA Strider": a "C" program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 16:1829-1836[Abstract/Free Full Text].

MINDRINOS, M., F. KATAGIRI, G. YU, and F. M. AUSUBEL, 1994  The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78:1089-1099[Medline].

PATERSON, A., T-H. LAN, K. REISCHMANN, C. CHANG, and Y. R. LIN et al., 1996  Toward a unified genetic map of higher plants, transcending the monocot-dicot divergence. Nat. Genet. 14:380-382[Medline].

PRICE, R. A., J. D. PALMER and I. A. AL-SHEHBAZ, 1994 Systematic relationships of Arabidopsis: a molecular and morphological perspective, pp. 7–19 in Arabidopsis, edited by E. M. MEYEROWITZ and C. M. SOMERVILLE. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SADOWSKI, J. and C. F. QUIROS, 1998  Organization of an Arabidopsis thaliana gene cluster on chromosome 4 including the RPS2 gene, in the Brassica nigra genome. Theor. Appl. Genet. 41:226-235.

SADOWSKI, J., J. HU, M. DELSENY, and C. F. QUIROS, 1994  Genetical and physical mapping of an Arabidopsis gene complex in Brassica genomes. Cruciferae Newslett. 16:47-48.

SADOWSKI, J., P. GAUBIER, M. DELSENY, and C. F. QUIROS, 1996  Genetic and physical mapping in Brassica diploid species of a gene cluster defined in Arabidopsis thaliana.. Mol. Gen. Genet. 251:298-306[Medline].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SANMIGUEL, P., A. TIKHONOV, Y.-K. JIN, N. MOTCHOULSKAIA, and D. SAKHAROV et al., 1996  Nested retrotransposons in the intergenic regions of the maize genome. Science 274:765-768[Abstract/Free Full Text].

WROBLEWSKI, T., S. COULIBALY, J. SADOWSKI, and C. F. QUIROS, 2000  Variation and phylogenetic utility of the Arabidopsis thaliana Rps2 homolog in various species of the tribe Brassiceae. Mol. Phylogenet. Evol. 16:440-448[Medline].

YANG, Y.-W., K. N. LAI, P. Y. TAI, and W.-H. LI, 1999  Rates of nucleotide substitution in Angiosperm mitochondrial DNA sequences and dates of divergence between Brassica and other Angiosperm lineages. J. Mol. Evol. 48:597-604[Medline].

ZHANG, M. Q., 1997  Identification of protein coding regions in the human genome based on quadratic discriminant analysis. Proc. Natl. Acad. Sci. USA 94:565-568[Abstract/Free Full Text].

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