Classification and expression of a family of cyclin gene homologues in Brassica napus
Steven Szarka, Melanie Fitch, Santiago Schaerer and Maurice Moloney* Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada, T2N 1N4 (* author for correspondence)
Received 26 July 1994; accepted in revised form 5 October 1994
In order to investigate the role of cell division in plant development, we isolated several plant genes which encode homologues of animal and yeast cell cycle regulators known as cyclins.
Through the use of degenerate primers and the polymerase chain reaction (PCR) we isolated a Brassica sequence which showed homology to the 'cyclin box' functional domain found within cyclin proteins. Southern blot analysis indicated that Brassica napus has a large number of genes containing cyclin box-related sequences. This was further supported by the isolation of cyclin box sequences from six different genomic clones. In addition, we have isolated two different cyclin cDNA clones, BnCYC1 and BnCYC2, from a Brassica napus shoot apical cDNA library. Both of the cDNA clones contain a 'de- struction box' regulatory domain similar to animal mitotic cyclins.
Northern blot analysis using BnCYC2 shows mRNA levels which correlate well with the level of cell division in various tissues. Messenger RNA abundance was highest in 1-3 mm leaves, root tips and shoot apices. The m R N A detected using BnCYC1 was restricted to young leaves and the shoot apex, suggesting divergent, organ-specific roles for cyclin family members. The results demonstrate that the plant cyclin gene family is more extensive than previously demonstrated and consists of genes expressed in all di- viding tissues as well as a subset of developmentally specific members.
Introduction
Cell division is a fundamental process affecting plant morphogenesis and development. Yet very little is known about the mechanism of cell divi- sion regulation in plants. Over the past decade significant advances have been made in our un-
derstanding of eukaryotic cell division regulation using animal and yeast systems (reviewed by [21]). Central to this progress was the discovery of a 34 kDa serine/threonine protein kinase (en- coded by the fission yeast cdc2 gene) which has been shown to be a major regulator controlling the transitions from G1 to S phase in yeast [35]
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers L25398 (BrCYC), L25399 (BnCYCI-4), L25400 (BnCYCI-6), L25401 (BnCYC2-1), L25402 (BnCYC2- 3), L25403 (BnCYC2-4), L25404 (BnCYC2-9), L25405 (BnCYC1) and L25406 (BnCYC2)
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and G2 to M in both yeast and animal cell cycles [7, 35, 43]. Homologues of p34 °a°2 have been isolated from a broad range of eukaryotic organ- isms [24, 25, 27, 36] including plants [3, 8, 10, 14, 17, 19, 20, 31 ]. The cdc2 gene represents the most well known prototype of the cyclin-dependent kinase (CDK) family of cell cycle regulatory genes.
CDK proteins function in association with in- dividual members of the cyclin class of cell cycle control factors [34]. The phase-specific function of individual CDK/cyclin complexes is due to their differential activity during the cell division cycle [5, 40, 44]. Cyclins are classified according to their timing of activity within the cell cycle. The G1 cyclins are regulated to produce a peak in CDK/cyclin complexes prior to the initiation of DNA synthesis [46]. Conversely, the peak of G2 cyclin/CDK complexes occurs during G2 prior to M phase [42]. Since the function of CDKs is dependent on the association with specific cyclin subunits, the accumulation of cyclins has an im- portant impact on CDK activity. Regulation of cyclin abundance during the cell cycle has been shown to occur by both transcriptional [46] and post-translational [6] mechanisms. The post- translational fluctuation in G2 cyclin proteins during the cell division cycle is the result of con- tinual synthesis during interphase followed by targeted proteolysis during mitosis. The signal for proteolysis is localized to the N-terminal domain of G2 cyclins and includes the 'destruction box' amino acid motif and adjacent lysine-rich se- quences [12, 33].
Cyclin proteins, like CDKs, are conserved in all eukaryotes studied thus far and have been isolated from the following plants: Daucus carom and G lycine max [ 15 ], A rabidopsis thaliana [4,16 ], Medicago sativa [ 18 ], and Antirrhinum majus [ 11 ]. Although cyclin clones have been isolated from several different plant species, the size of the plant gene family and the extent of functional special- ization of its various members have not yet been determined. We are currently studying cyclins and their effect on plant development as a result of their role in cell cycle regulation. In order to ad- dress the questions of diversity and differential
function, we have investigated the range of cyclin- like genes present in the plant species Brassica napus.
Materials and methods
DNA probes
A plant cyclin box probe was isolated using PCR and degenerate oligonucleotides designed to en- code two highly conserved amino acid domains found in animal cyclins. The forward (5'- ATGCGIGGIATHYTIRYIGAYTGG-3 ') and reverse (5' -GGRTAIATYTCYTCRTAYTT-3 ' ) primers encode the amino acids MRGILI/VDW and KYEEIYP, respectively. PCR amplification was carried out using 600 pmol of each primer and 2 #g of Brassica rapa genomic DNA. The reaction also contained 200 #M dNTPs, 10 mM Tris-HC1 pH 8.3, 50 mM KC1, 1.5 mM MgCI2, 0.001 ~o (w/v) gelatin, and 2.5 U of Taq DNA polymerase (Pharmacia) in a final 100 #1 volume. Temperature conditions for amplification were 94 °C for 4 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The PCR product was end-repaired with T4 DNA polymerase (Pharmacia) and cloned as a blunt fragment into the Sma I site of pUC19 (BrCYC). A DNA fragment of the B. napus protein phos- phatase 2A sequence [ 30 ] (EMBL accession num- ber X57439) was also isolated by PCR ampli- fication. The forward (5 ' -CGCGGATCCCCG AGCAACAGGTTAGAGC-3 ' ) and reverse (5 ' -CGCGGATCCCTCACTCAGAAGACAA GGCG-3') primers coincide to sequences span- ning nucleotides 71 to 89 and 1007 to 1026 of Fig. 2 in MacKintosh et al. [30] and include Bam HI restriction enzyme sites (underlined). Lambda DNA isolated from a B. napus apical cDNA library [2] was used as a template for PCR. Amplification was performed for 30 cycles of 94 °C for 1 rain, 55 °C for 45 s, and 72 °C for 2 min. The 957 bp fragment was digested with Barn HI and cloned into the equivalent site of pUC19. The clone Bnpp2A was confirmed by sequence analysis. The B. napus CAB cDNA (labelled as
BnCAB in this report) was kindly provided by Jas Singh (Agriculture Canada, Ottawa) and encodes a type I P/S II chlorophyll a/b-binding-protein.
Probes used for library screening and northern blot analysis were labelled using the random hex- amer priming method [9]. Cyclin box probes used for Southern blot analysis were labelled using the cyclin box-redundant primers and PCR essen- tially as described [ 1 ]. Changes to the PCR la- belling protocol included using 5 ng of plasmid template DNA, Taq polymerase at 1.25 U per reaction, and reaction buffer from Pharmacia (Baie D'Urf6, Quebec). The labelled nucleotide was e-[32P]dCTP (3000 Ci/mmol; Amersham). PCR conditions were: 94 °C for 2 min followed by 30 cycles of 1 rain at 94 ° C, 30 s at 48 ° C, 30 s a t72 °C, and a final 72 °C incubation for 10 rain.
Screening of cDNA and genomic libraries
A B. napus cv. Westar apical cDNA library [2] was screened with the B. rapa cyclin box PCR fragment. The 2ZAP II library was plated ac- cording to Stratagene and transferred to Hybond N membrane (Amersham). Membranes were fixed by baking for 1 h at 80 °C. Both the pre- hybridization and hybridization were carried out at 55 °C in 6 x SSC, 5 x Denhardt's solution, 0.5~o SDS, and 5 mg of denatured yeast tRNA. Hybridized library filters were washed to 2 x S SC at 55 °C. Kodak X-OMAT AR X-ray film was exposed with the library membranes using inten- sifying screens for ca. 48 h at -80 °C. Two of the primary clones, BnCYC1 and BnCYC2, were pu- rified in a secondary screen. A B. napus cv. Bridger genomic library from Clonetech was screened using the Xba I to Hind III partial insert from BnCYC1. Conditions were the same as those indicated for the cDNA library screen.
Sequence determination and analysis
All sequencing was performed using Sequenase (United States Biochemical) and the dideoxy ter-
265
mination method [41]. Subclones for sequencing each cDNA were generated by either ExoIII/ mung bean nuclease deletions (according to Stratagene) or selected restriction enzymes.
PCR amplification of genomic cyclin box sequences from lambda clones
Primary isolates from the genomic screen were analysed by PCR amplification and sequence de- termination of the cyclin box. An aliquot (5-20 #1) of each primary phage suspension in SM buffer (100 mM NaC1, 100 mM MgSO4"7H20, 50 mM Tris-HC1 pH 7.5, 0.01~o gelatin) was pre- treated at 100 °C for 5 min prior to PCR. PCR conditions and primers were the same as outlined above for the original B. rapa cyclin box fragment. Amplified fragments were end-repaired with Es- cherichia coli DNA polymerase Klenow fragment or T4 DNA polymerase and cloned into the Hind II or Sma I site of pUC19 for sequencing.
Southern blots
Genomic DNA was extracted from B. rapa, B. napus and B. oleracea [45]. DNA (5 #g) was di- gested with Hind III and separated on a 0.8~o agarose gel in 1 x TBE buffer. The DNA was transferred by capillary blot to Hybond N as per the manufacturer's instructions (Amersham). Prehybridization and hybridization was per- formed at 65 ° C with constant agitation. Both the prehybridization and hybridization steps were carried out using RAPID-HYB buffer (Amer- sham). PCR probes were generated from each cyclin box clone in pUC19. Labelled fragments were purified on a Sephadex G-50 column and added to a final activity of 1.0 x 10 6 cpm per ml of RAPID-HYB buffer. After hybridization, the membranes were washed twice with 2x SSC 0.1~o SDS at 25 °C for 15 min and then once with l x SSC0.1~o SDS at 6 5 ° C f o r 15rain. Autoradiography was performed at -80 °C with Kodak X-OMAT AR X-ray film and an intensi- fying screen.
266
Northern blots
Total RNA was isolated from B. napus tissues using the guanidine HC1 procedure of Logemann et aI. [29]. Four-day old hypocotyls and shoot apices, as well as 24 h post-germination root tips, were isolated from B. napus cv. Westar seeds germinated on 0.5x Murashige-Skoog (MS) minimal organic media with 3~o sucrose and 0.7~o phytagar (Gibco) (24 °C with 60-80 #E m - 2 s - 1 light intensity for a 16 h light/8 h dark photoperiod). Young (1-3 mm) and intermediate (10-30 mm) leaves were taken from 35 day old B. napus cv. Westar plants grown under green- house conditions. Various stages of late flower buds, whole roots, and mature leaves (> 150 mm) were harvested from growth chamber-grown B. napus cv. Topaz plants (15 °C 16 h day/10 °C 8 h night with 400 /~E m -2 S-i light intensity). Poly(A) + RNA was purified from total RNA samples using the PolyATtract mRNA isolation system (Promega). Based on OD26 o measure- ments, five micrograms of Poly(A) + RNA was estimated for each tissue sample and separated in a 1.2~o agarose gel containing 2.05 M formalde- hyde. Relative loading was confirmed subsequent to running the samples using UV fluorescence of ethidium bromide stain. The RNA was trans- ferred to GeneScreen Plus membrane according to NEN Dupont and hybridized to the DNA probes indicated in Fig. 6. Hybridization was performed at 43 °C in 5 x SSC, 50~o deionized formamide, 5 x Denhardt's solution, 1~o SDS, and 10 mg denatured yeast tRNA. BnCYC1 was washed down to 2 x SSC 0.2~o SDS at 43 °C, while BnCYC2, Bnpp2a and BnCAB were each washed to 0.2 x SSC 0.5~o SDS at 65 °C. Ex- posures were carried out with intensifying screens at -80 ° C.
Phylogenetic analysis
The cyclin box sequences for each of the cDNA and genomic clones were analysed with PAUP (Phylogenetic Analysis Using Parsimony; version 3.0s, D.L. Swofford, Illinois Natural History Sur-
vey). For each of the genomic templates only the exon sequence was used. Since the primer se- quence is incorporated into each PCR-generated cyclin box, and may not represent the template sequence, these sequences were not included in the analysis. Transitions were weighted as 1 and transversions were weighted as 10. An exhaustive search was performed and one representative minimal trees is presented.
Results
Isolation of Brassica cyclin sequences
A genomic DNA fragment from Brassica rapa cv. Tobin (BrCYC) was isolated using redundant primers and PCR amplification (Fig. 1A). Trans- lation of the putative exons yielded an amino acid sequence with 21 of the 29 (72.4 ~o) animal cyclin A and 16 of the 23 (69.6~o) animal cyclin B con- sensus amino acids (Fig. 1B). The mixed identity with animal cyclin A or cyclin B consensus amino acids did not allow the assignment of this plant cyclin to one of the animal mitotic classes, as has been noted for other plant cyclins [11, 15, 16]. Based on the observed homology, the B. rapa fragment was presumed to encode a plant cyclin functional domain and was used to probe a shoot apical cDNA library in 2ZAPII from B. napus cv. Westar. Two cDNA clones, designated BnCYC1 and BnCYC2, were isolated and sequenced.
The BnCYC1 cDNA is 1838 nucleotides in length and contains a poly(A) tail (Fig. 2). The largest open reading frame (ORF) is 1302 nucle- otides. This ORF is predicted to encode a 434 amino acid protein with an estimated size of 49004 Dal. The BnCYC2 cDNA contains 1573 nucleotides (Fig. 3). The largest B n C Y C 2 0 R F is 1275 nucleotides. This ORF begins at the first A1TG following the in frame stop codon (T _96AA). TWO additional methionine codons are present within the first 43 amino acids of this same ORF (A61TG and Ai27TG); although the first ATG codon shows the most similarity to the plant start codon consensus TAAACAAUG- GCT [22]. The largest ORF is predicted to en-
267
A 1 ATGCGGGGGATTCTGGTGGATTGGCTTGTGGAGgt t agt gatt t agtt caacct ctgatg 60
M R G I L V D W L V E 61 ctaccgtccaaagtactcacaaaagtctcatctcactcacagGTCGCTGAGGAATACACG 120
..L..V...V..HLR.H ..... LFL..QL...FLAEHS.SKGK ....... AMFI.S ...... P
..A..I...V..QMK.R ..... MFM..GI...FLQEHP.PKNQ ....... AMFL.A ...... P
..A..I...V..QMK.R ..... MYM..SI...FMQNNC.PKKM ....... AMFI.S ...... P
Fig. 1. Cyclin box probe sequence from Brassica rapa. A. DNA and predicted amino acid sequence of the B. rapa cyclin box PCR product. Redundant primer sites are double-underlined. Predicted intron sequences are indicated in lower case. Amino acids are indicated in single-letter code below the predicted exon codons shown in upper case. B. Predicted amino acid sequence of B. rapa
cyclin box aligned with animal cyclin A and cyclin B consensus sequences. Identical amino acids in the animal cyclin sequences are indicated by ('). Matches between B. rapa and animal cyclin A and B conserved amino acids are indicated by (1). The amino acids encoded by the PCR primers are double underlined and are not included in the comparison.-
code a protein of 47988 Dal. The two smaller ORFs would encode 45761 and 43483 Dal pro- teins respectively.
Both BnCYC1 and BnCYC2 exhibit the over- all structure of mitotic cyclins. This includes 'de- struction box' motifs (RXALG(V/D/E/N)IXN, [12]) located in the N-terminal region (Figs. 2 and 3) of both proteins. A PEST domain has also been localized to amino acids 11-21 in BnCYC2 (Fig. 3) using the PESTFIND program from PC- GENE (Intelligenetics, Mountain View, CA). PEST sequences have been correlated with pro- teins which have short half lives [39] and have been found in cyclins from other species [13]. The PEST domain is N-terminal to the second
in-frame methionine residue and would not be encoded in the CYC2 protein if either of the in- ternal methionine codons were used for the start of translation.
A subclone from the BnCYC1 cDNA was used to screen a B. napus cv. Bridger genomic library. Thirty seven putative positives were isolated from the primary screen. Of the 37 clones, 6 were analysed by PCR amplification and sequence analysis of the cyclin box domains. The six clones generated from the PCR cyclin box fragments are: BnCYC1.4, BnCYC1.6, BnCYC2.1, Bn- CYC2.3, BnCYC2.4 and BnCYC2.9. Each of the six clones analysed was found to encode a dif- ferent cyclin box sequence.
aagacactctctttccccaaggg cggagcggattcaccatctcgatctgcccccaccctccaagctcctttcttttcctgaagggttttctttctggagcttc gcagatcggaggaagatga•gcagtcctgaaacagcaagactttcaaggctctgtgtcttcttctagtgggattttaaac ATG GGGAAG GAAAAT GTT GTC TCT CGT CCT CTC ACT CGT GCC TTT GCG TCT GCT TTG CC-C M G K E N V V S R P L GCT TCA ACT ACA GAG AAT CAA CAG AGA GCA AAC A S T T E N Q Q R A N AAC GTC ACT GCG CCA CCC AAT AAG AAG AAG AAG N V T A P P N K K K K GCT AGC TTC AGT GCA GCT AAA CTT GAG GCT AGA A S F S A A K L E A R GGG TTG GCG AGT C-CA TCT TGT GTT ACT TCA GAA G L A S A S C V T S E GCA AAA GCT GAA GTT GTA TCA GTG ACA GCA GGA A K A E V V S V T A G ATC GAG AAA CAC AAA TTG CCT CCT AGA CCT CTT I E K H K L P P R P L AAA AGT GGT GTT ATT CGT AGT TCA ACA GCA CTG K S G V I R S S T A L TCT GAT GAC AAG GAT CCT TTA TTG TGC TGC CTC S D D K D P L L C C L CGT GTC TCA GAG CTT AAA CGC AGA CCG GTT CCT R V S E L K R R P V P
ACA TT GTG CCT GAC ACT TT TAC CAA ACA GTG T L V P D T L Y Q T V
~ C TAC CTG GAA AGA CAG AGA CTT CAA CTC CTC Y L E R Q R L Q L L
T R A F A S A L R ACA AAA AGA CCA GCC TCG GAG GAT GTT T K R P A S E D V
D I K Q V K K S Q GTC ACA GAT CTT CAG TCC GGG ACC GAG V T D L Q S G T E AAC ACAAAT GAC ACA GCT GAT AAC TGT N T N D T A D N C GGG AGA TCA TCA GCT TCT ATA GTT GAG G R S S A S I V E GAT CTC CCAAAA TTC ACA GAC ATT GAT D L P K F T D I D TAC GCC CCT GAAATC TAC TAC AAT TTG Y A P E I Y Y N L AAC TTT ATG GAG AGG ATA CAG AAG GAC N F M E R I Q K D TGG CTT GTG GAG GTC TCT GAG GAA TAC W L V E V TAT CTC ATC GAC TGG Y L I D W GGC ATC ACT TGC ATG G I T C M GAG TTC TGC TTC ATC E F C F I
s • ~ y TTC CTC AT ~GA F L H CTA ATT GCC ~CG L I A
ACGT DGAT NAAC ACCT
TAC ACA AGA GAT CAG GTC CTG GAG ATG GAG AAC CAA GTA CTT GCG CAT TTT AGC TTT CAG Y T R D Q V L E M E N Q V L A H F S F Q ATA TAC ACT CCC ACT CCAAAA ACG TTC CTA AGG AGA TTT CTC AGA GCA GCT CAA GCC TCT I Y T P T P K T F L R R F L R A A Q A S TAC CTG ATC CCT CGC CGT GAA CTC GAA TGT CTA GCC AGC TAT CTA ACG GAG GTG ACG TTG Y L I P R R E L E C L A S Y L T E V T L ATA GAC TAT CAC TTC TTGAAG TTT CTT CCT TCT GTC ATC GCT GCT TCA GCG GTT TTT CTT I D Y H F L K F L P S V I A A S A V F L GCC AAG TGG ACA TTG GAC CAA TCA AAC CAC CCA TGG AAT CCA ACA CTT GAG CAC TAC ACA A K W T L D Q S N H P W N P T L E H Y T ACG TAC AAG GCG TCA GAT CTC AAA GCA TCT GTT CAT GCC TTA CAA GAT CTG CAG CTT AAC T Y K A S D L K A S V H A L Q D L Q L N ACC AAA GGT TGC CCT TTG AGC GCT ATA CGC ATG AAG TAT AAG CAA GAG AAG TTC AAA TCT T K G C P L S A I R M K Y K Q E K F K S GTG GCG GTT CTC ATG TCT CCT AAA CTA CTT GAC ACG CTA TTC TGA agggctaaaccgaacggttc V A V L M S P K L L D T L F *** aactcctaaccgataatagatctctgtgacattgatggtccggttcgtctttactctttttgttcataaaacaggctccc a•agt•c•a•t•atctaatacctgattagaattcggtttag•ttggtttagtttgat•tggaggtttggtt•tt••aaa• accaga•ctggtttaatctaatcggtag•aagaggttattttagttgtatcgagaatgta•attttct•••atcttgagg cg•aatgtaaaaa•agttt•gggaattt•ata•ttactatttatgtatttt••gatga•gtagtttaattaaaaaaaaaa aaaaaaaaaa
Fig. 2. DNA and amino acid sequence of the Brassica napus BnCYC1 cDNA. The region of the cyclin box dundant primers used in this study is outlined. The destruction box motif is highlighted in black.
The amino acid sequences from the two B. napus cDNA clones and the six genomic clones were aligned with other published plant cyclin se- quences (Fig. 4). The aligned plant cyclin box se- quences were divided into four subgroups based on sequence similarity. The first group consists of five B. napus cyclins (BnCYC1, BnCYC1.6, BnCYC2.1, BnCYC2.4, and BnCYC2.9). BnCYC2.3 does not appear to have homologues from other plant species yet isolated and repre- sents a separate group. BnCYC2 and the previ- ously published D. carom cyclin appear to belong to the same subgroup. The final group has
BnCYC1.4 aligned with cyclins from A. thaliana, G. max, A. majus and M. sativa, all of which have been grouped together previously by others [11, 18]. The majority of plant cyclins isolated from other plant species fall within this final subgroup, and suggests that until now studies of plant cy- clins have only focused on a single subgroup within the superfamily. Alignment of all cyclin box sequences indicates conservative amino acids unique to each of the subgroups (Fig. 4). As well, a general clustering of amino acid differences ap- pears in certain regions of the cyclin box. Al- though functional amino acids have been pro- posed for some of the conserved animal positions [48], the structure and function of the cyclin box
1 ATG TCG AAG ACT ACT AAT CAG AAC CGG CGG CCT TCT TTC ACC TCG TCG ACG GAA TCG TCG 60 1 M S K T T N Q N R R P S F T S S T E S S 20
61 ATG AGG AAA CGT CAC GGA CCA TCT TCT TCT TCT TCG GCA GTG AAA CCA ATC TCT AAC ACG 120 21M__.. R K R H G P S S S S S A V K P I S N T 40
121 GCG GTT ATG GTG GCC AAG AAA CGA GCC CCA CTG GGT AAC ATC ACT AAT CAA AGG AAG GAT 180 41 A V M V A K K - R K D 60
181 TCA CGAATA TTC CCAAAT TCATCT TCT GCT GAT TCC GCA CAT TGT CCGAACAAG TCT GCG 240 61 S R I F P N S S S A D S A H C P N K S A 80
241 AAA TTG AAG TTG GCG GCA CCA ACA CAA CCT GTT TGC GTT AAT GCC TGT GAG ACAAAG TCT 300 81 K L K L A A P T Q P V C V N A C E T K S i00
301 ACA TGT GAAGAAGAAGTG GTT CCC ATCGAG AGGAAGGCA TTT AGCAAT CTA TGC ATA ACC 360 I01 T C E E E V V P I E R K A F S N L C I T 120 361 CCG AGT TCA GAC ACAACT ACT AAT GTC ATG TCT GAAACGGAGAATAAG GAG GAG AAG TTT 420 121 P S S D T T T N V M S E T E N K E E K F 140 421 ATGAAC ATC GAC AAT AAG GAT GAT GCG GAT CCG CAG CTC TAT GCA ACG TTT GCT TGT GAT 480 141 M N I D N K D D A D P Q L Y A T F A C D 160 481 ATT TAC AAT CAT CTC CGT GCC GCC GAG GCAAAG AAG CAG CCT GCC GTT GAC TAC ATG GAG 540 1611 Y N ~ ~ R A a E A K K e P A v D Y ~ E 180 541 ACT GTT CAG AAA GAT GTC AAC TCT ACT ATG CGC GGA ATC CTT GTT GAT TGG CTT GTT GAG 600 181~ v Q K D v N s T Ix R G I L V D W L V Z I 200
l TAT 6Ol cco GAG 66O N fT
~TG pc I 201 IV S E E Y R L V P E T L Y T D 220
AAT 7 720 AGT AGA CAGAAG CTT CAG CTT ~TG GGT ~ TG ATT 221,RYLSGcGCAAAAN ~ A A S R Q K I L Q G c P E~GG~TTC-T-~TAT 240 TAT TGT A CCA CAA GTG
• v o v c 260 221,RYLSGcGCAAAAN AG GTA 780 241 ~M M I 781 ATC ACT GAT AAT ACA TAT CTC AAG GAT GAG GTT CTT GAC ATG GAA TCT GCT GTT TTG AAC 840 261 I T D N T Y L K D E V L D M E S A V L N 280 841 TAC TTG AAG TTT GAA ATG TCA GCT CCA ACA GTC AAG TGC TTT CTA AGA CGA CTT TTC TCG 900 281 Y L K F E M S A P T V K C F L R R L F S 300 901 GGC TGC CCA AGG GTT CAT GAG GCT CCG TGT ATG CAG TTA GAA TGT ATG GCC AGC TAC ATT 960 301 G C P R V H E A P C M Q L E C M A S Y I 320 961 GCG GAA CTG TCG CTT TTG GAG TAT ACA ATG CTC TCT CAT CCT CCT TCA CTT GTT GCT GCT 1020 321 A E L S L L E Y T M L S H P P S L V A A 340
1021 TCT GCC ATT TTC TTG GCT AAG TAT ACT CTA GAC CCA ACA AGA AGA CCG TGG AAT TCG ACG 1080 341 S A I F L A K Y T L D P T R R P W N S T 360
1081 TTG CGACAC TAT ACGCAG TAC GAAGCG ATG GAATTG AGA GGG TGT GTG ATG GAT CTT CAA 1140 361 L R H Y T Q Y E A M E L R G C V M D L Q 380
1141 CGG TTG TGT AGT AAC GCT CAT GTC TCT ACT CTT CCT GCT GTA AGG GAC AAG TAT AGT CAA 1200 381 R L C S N A H V S T L P A V R D K Y S Q 400
1201 CAC AAA TAC AAG TTT GTG GCA AAG AAG TTT TGT CCA TCA ATT ATC CCA CCA GAT TTC TTC 1260 401 H K Y K F V A K K F C P S I I P P D F F 420
1261 AAG AAC AGC TTG TAT TGA ttgatcaattttcaaattaactttgttccaatcttgogttggattgtcctttgttc 1334 421 K N S L Y *** 426
Fig. 3. DNA and amino acid sequence of BnCYC2 cDNA. Both the cyclin box and the destruction box are indicated as in Fig. 2. The putative PEST domain is underlined.
domain is not understood well enough to classify the differences in plant cyclin box sequences as functional or silent evolutionary changes.
Since the cyclin box has been shown, by dele- tion analysis in animals, to be the functional do- main of cyclin proteins [23, 26] we performed a phylogenetic analysis of the B. napus cyclin box coding sequences to determine the similarity and possible functional relationships between the dif- ferent clones. The result of this analysis is con- sistent with the amino acid alignments and is shown in Fig. 5A. Distinct branches made up of BnCYC1.4, BnCYC2.3, and the BnCYC1 cluster are seen on the phylogram. The only discrepancy between the phylogram and the amino acid align- ment was that BnCYC2 was not placed as an individual branch but instead shown to be related
to the BnCYC1 cluster. However, when consen- sus phylograms were generated BnCYC2 was placed on a fourth and separate branch (data not shown). As well, pattern differences from both S outhern and northern analysis support BnCYC2 as a group separate from the BnCYC1 cluster (see below).
Southern blot analysis was performed on three Brassica species; the amphidiploid B. napus and its two diploid parental species B. rapa and B. oleracea. By probing genomic D N A with each cyclin box sequence we hoped to estimate the number of cyclin genes in each branch of the phylogenetic tree. The sequence relatedness of the clones in the BnCYC1 cluster was substantiated by the repetitive hybridization pattern seen with the BnCYC1, BnCYC1.6, BnCYC2.1,
Conserved AA MR ILVDWL V L TLYLTV LQLLG LIAS KYEEI I F I V V
Fig. 4. Amino acid comparison of plant cyclin box sequences. Aligned amino acid cyclin box sequences from Brassica napus (this paper), Arabidopsis thaliana [4, 16], Daucus carom [15], Glyeine max [15], Medieago sativa [18], and Antirrhinum majus [11]. Se- quences determined by the redundant primers in PCR products were not included. The predicted intron position and size (in base pairs) within genomic sequences are indicated in square brackets; cDNA sequences are artificially spaced at introns to allow proper alignment of amino acids. Gaps are indicated by spaces. Conserved plant amino acids in the cyclin box region are indicated below the sequence alignments. Black highlights identify conserved amino acids within each subgroup. Non-conservative amino acid differences within each subgroup are indicated in bold. EMBL databank accession numbers for each of the sequences are shown in the right column.
BnCYC2.4, and BnCYC2.9 probes (Fig. 5B). Hybridization with either BnCYC2.3 or Bn- CYC1.4 gave distinct patterns from the BnCYC1 cluster and each other; supporting their assign- ment as distinct subgroups. Four A. thaliana clones [4] have been isolated which fall into the same subgroup as BnCYC1.4 (Fig. 4). The A. thaliana cycl clone isolated from Day and Reddy [4] shows 93.5~o amino acid identity and 83~o nucleotide identity with BnCYC1.4 in the cyclin box region suggesting that BnCYC1.4 and the A. thaliana cycl clone may be equivalent genes from each species. The results in Arabidopsis showing that four genes are encoded in this branch of the cyclin family is supported by the BnCYC1.4 hy- bridization pattern in Brassica. Each branch of the cyclin family appears to have multiple mem- bers, except for BnCYC2.3, which appears to be a single gene subgroup. BnCYC2 also gives a distinct hybridization pattern on the Southern
blot analysis, supporting its assignment as a sep- arate subgroup. Some of the hybridizing bands align with the BnCYC1 cluster pattern. Although it is unknown if the similarity of restriction frag- ment length polymorphism bands is meaningful.
Expression of cyclins
To determine the expression of both cyclin cDNAs in B. napus, poly(A) + RNA was ex- tracted from various tissues for northern analysis (Fig. 6). The BnCYC1 m R N A signal was stron- gest in the young leaf sample and lower in the shoot apex. The absence of significant BnCYC1 m R N A in tissues which are known to contain a large population of dividing cells (e.g. root tip) suggests that expression of this clone does not correlate with the amount of cell division but is restricted to young leaves and the shoot apex,
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Fig. 5. Phylogenetic and Southern blot analysis of Brassica napus cyclin box sequences. A. Phylogenetic relationship between B. napus cyclin box coding sequences was performed using PAUP v3.0s, cDNA (CYC1 and CYC2) and genomic clone (1.4, 1.6, 2.1, 2.3, 2.4, 2.9) labels are indicated below each phylogram branch. Numbers indicate the absolute nucleotide changes for each branch. B. Southern blot analysis of cyclin genes in three Brassica species. Genomic DNA (5 #g) from B. rapa (R), B. napus (N), and B. oleracea (O) was digested with Hind III and hybridized with each cyclin box probe. Southern blots for each cyclin box are shown below their respective phylogenic branch label.
thereby displaying tissue or organ specificity. Faint bands were also visible in other tissues. Although we have not determined if these faint signals are due to reduced BnCYC1 mRNA abundance or a low level of cross hybridization between the BnCYC1 probe and the related cy- clins (i.e. BnCYC1.6, BnCYC2.1, BnCYC2.4 and BnCYC2.9). The levels of BnCYC2 mRNA more accurately correlated with the level of cell division in the various tissue samples. The signal was highest in tissues known to have a large percent- age of dividing cells: i.e. young leaves, shoot apex, and root tip. Reduced signals were also seen in other tissues tested. The same northern blot was reprobed with a protein phosphatase 2A sequence (Bnpp2a), and a chlorophyll a/b-binding protein sequence (BnCAB) to further judge loading and support the integrity of mRNA in each of the samples.
Discussion
Many of the cell division cycle regulators identi- fied in animal and yeast systems are presumed to be ubiquitous in all eukaryotes, including plants. Although others have demonstrated that plant cy- clins exist and are encoded by a multi-gene family [4, 11, 15, 18], as expected from the results of other eukaryotic systems, until now the size of the gene family has not been accurately represented. The isolation of many different cyclin genes from a single plant species, B. napus, has provided us with the first insight into the size and variation of the plant cyclin gene family. We have chosen to use the cyclin box domain as the region for com- parison since deletion analysis on animal cyclins have shown that the cyclin box is the minimum functional domain required for CDK binding and activation [23, 26]. Therefore, functional differ-
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Fig. 6. Tissue expression of B. napus cyclins. Northern blot hybridization with the BnCYC1 cDNA, the BnCYC2 cDNA, the B. napus protein phosphatase 2A DNA fragment, and the B. napus CAB (chlorophyll a/b-binding protein) cDNA. Lanes correspond to 5/~g Poly(A) + RNA from mature seeds (MS), whole roots (WR), root tips (RT), shoot apices (SA), hypo- cotyls (HY), 1-3 mm leaves (YL), 10-30 mm leaves (IL), mature leaves (ML), and late flower buds (FB).
ences in the various plant cyclins are likely to manifest themselves as differences in the amino acid sequence, especially within the cyclin box domain. A limitation to the approach taken in this report is that by selecting the animal G2 cyclin A and B classes for the design of the initial redun- dant primers we have undoubtedly favored the isolation of mitotic plant cyclins. Since sequence divergence between other eukaryotic G1 and G2 subclasses is greater than that seen within each subclass [ 13, 28], we may have failed to detect plant G1 cyclins through the use of G2-specific primers. For this reason our results are likely, if anything, to underrepresent the diversity of the plant cyclin family. Although it might be argued that the use of an amphidiploid species likely overestimates the minimal required gene family found in diploid species the Southern blot analy- sis (Fig. 5) clearly shows that the diversity indi- cated by the BnCYC1, BnCYC2, BnCYC2.3, and BnCYC1.4 types from B. napus also appears to hold for the progenitor diploid species B. rapa
and B. oleracea. Although, on average, a slightly higher degree of complexity (not necessarily two- fold) exists in the amphidiploid species.
The multiplicity of members in three of the four phylogenetic subgroups may imply a functional redundancy, as seen with the budding yeast CLN cyclins [38], or differentially regulated members, as seen with the animal cyclin D class [32, 47]. In contrast, since the BnCYC2.3 clone shows a single hybridizing band in diploid Brassica spe- cies it is likely to have a unique function from the other cyclin subclasses.
Sequence comparisons between plant and ani- mal cyclins has not allowed unambiguous assign- ment of function to individual plant cyclins (Fig. 1 and [11, 15, 16]). Although the first three sub- groups in Fig. 4 show more similarity with animal cyclin A sequences and the last subgroup shows more similarity with animal cyclin B sequences enough differences exist to question assignments based on sequence alone. Therefore, comparative assignments between animal and plant cyclins will likely require a functional basis. Certain features, such as the presence and position of a destruction box motif (involved in animal G2 cyclin proteoly- sis during mitosis) are also found in plant cyclins (Figs. 2 and 3 and [11, 15, 16]) and suggest that cyclins with this motif may be post-translationally regulated consistent with a G2 function. Although the presence of such a motif is suggestive, direct correlation of post-translational regulation and phase-specific function are still required for indi- vidual plant cyclins.
A more historic relatedness of the cyclin genes may be inferred from the intron structure within the cyclin box domains. Of the six genomic clones only BnCYC1.4 lacks the two introns which are conserved in their position in the remaining five genes (Fig. 4). The presence of these conserved introns suggest that the five cyclin genes shared a common ancestor after the divergence of Bn- CYC1.4; and have subsequently evolved sepa- rately into the three subgroups. This is consistent with the cyclin A and B similarity stated above.
Northern blot analysis using the two cyclin cDNAs, BnCYC1 and BnCYC2, clearly shows a difference in the tissue-specific expression of these two cyclins. Transcription of both animal [ 37, 46 ] and plant [11] cyclins has been shown to be dif- ferentially regulated during the phases of the cell
cycle. Therefore, it appears that transcriptional control of individual plant cyclins can occur on two levels, either during specific cell cycle phases [11] or in certain tissues and organs during de- fined developmental stages (BnCYC1). Since plant cell divisions involved in organogenesis are predominantly postembryonic and restricted to distinct meristematic centers, developmental and/or organ specific regulation of cyclins may be more prominent in plants. An important implica- tion from this result is that dividing cells within the young leaves and shoot apex which express the BnCYC1 cyclin protein may be some how qualitatively different from the remaining dividing cell population of the shoot apex. For example, the terminal divisions in a leaf may be inherently different from earlier, indeterminate divisions in the shoot apical meristem (e.g. endoreplication). The BnCYC1 gene will provide a tool to test these types of questions and potentially provide some understanding of how cell division is con- trolled to affect plant morphogenesis and devel- opment.
Analysis of the size and variation within the B. napus cyclin gene family has allowed a prelimi- nary classification of cyclins into four subgroups based on sequence similarity. If the cyclin group- ings presented in this report also represent a func- tional classification then we propose the Bn- CYC2, BnCYC1.4, BnCYC2.3 and BnCYC1 branch groups be referred to as subclass I through IV respectively. Confirmation of this classifica- tion will require further analysis in Brassica and other species. We have also performed expression analysis using the BnCYC1 and BnCYC2 cDNA clones. Although the role of BnCYC1 and Bn- CYC2 in cell division cycle regulation has not yet been demonstrated, expression data from north- ern blot analysis suggests that while BnCYC2 may be involved in the cell divisions of all tissues Bn- CYC1 is strongly expressed only in young leaves and the shoot apex. Similar analysis on the re- maining cyclin genes is necessary to begin defin- ing the role(s) of the various cyclin subclasses. Further characterization of cell division regula- tors in plants is clearly needed, for although some of the basic mechanisms of cell division are highly
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conserved among eukaryotes the unique organi- zation of morphogenesis and development in higher plants may be accompanied by unique spe- cialization of some of these ubiquitous cell cycle genes.
Acknowledgements
Santiago Juan Schaerer is supported by a Post- doctoral Fellowship from the Swiss National Sci- ence Foundation and by grants from the Soci6t6 Acad6mique Vaudoise and from the Fondation du 450e Anniversaire of the University of Lau- sanne. The authors would also like to thank Dr J. Silverman for the PEST analysis of cyclin se- quences.
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