Plant Molecular Biology 11:641-649 (1988) © KluwerAcademic Publishers, Dordrecht - Printed in the Netherlands
Isolation of an alfalfa histone H3 gene: structure and expression Sheng-Cheng Wu, I_Aszl6 B/Sgre, l~va Vincze, Gy6rgy B. Kiss and D6nes Dudits*
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary (*author for correspondence) Received 7 June 1988; accepted 16 August 1988
Key words: codon usage, gene expression, histone H3 gene, Medicago sativa, somatic embryos Abstract A histone H3 gene was isolated from a dicotyledonous plant, alfalfa (Medicago sativa). The sequence analysis o f this gene revealed no obvious GC preference in its codon usage. Apart from containing most o f the typical consensus sequences found in both animal and plant histone genes, the alfalfa H3 gene exhibits distinct structural features such as (1) the unusual location o f two GATCC motifs in its 5' flanking sequence, (2) the existence o f a CGCGGATC on the nonsense strand at position - 2 3 2 , (3) the existence of a long palindromic structure, and (4) several polyadenylation signal-like sequences in the 3' flanking region. There are about 160 copies of histone H3 gene in alfalfa tetraploid genome. Using the alfalfa H3 gene as a probe to study the pattern o f histone H3 transcripts in the alfalfa, we found that the H3 RNAs are undetectable in leaves, more in stems than in roots, and highest in somatic embryos. Moreover, the RNA products of H3 genes in all alfalfa tissues tested show unusually long nontranslated region compared to those o f animal histone genes. An additional high molecular weight species o f H3 transcript was detected only in somatic embryos.
Introduction In the past twenty years, significant progress has been made in the analysis of histone genes and their expression (see reviews [10, 16]). The available data indicate that the structure, organization and expression o f histone genes is quite variable. Recently, emphasis has been on the evolutionary diversification o f histone genes and the specific regulation o f their expression during cell cycle and developmental changes [12, 13, 14, 21]. Our recent knowledge about the organization and structure o f plant histone genes is mostly based on histone H3 and H4 genes from three monocotyledonous species [5, 20, 26, 28, 29, 36] and one dicotyledonous plant, Arabidopsis thaliana . Several studies have revealed specific features of
plant histone genes, such as the consensus sequences, ACGTCA and CGCGGATC, found in the 5' flanking region [3, 5, 17]. Interestingly, the corn histone H3 and H4 mRNAs have been recently demonstrated to be polyadenylated . However, in contrast to the abundant knowledge about the expression o f histone genes in animals, little is known in plants. Here we describe the structural analysis o f a histone H3 gene isolated from an alfalfa genomic library and its comparison to the nucleotide sequences o f histone H3 genes from different sources. Furthermore, we present data about the amount of histone H3 transcripts in various parts of the alfalfa plant, undifferentiated callus tissues and somatic embryos.
642 Materials and methods
Southern hybridization and estimation of copy number
Plant materials Medicago sativa L. cv. Nagyszenfisi plants, commercially available in Hungary, were used for the construction of the genomic library. In vitro grown plants of M. sativa L. cv. Regen S, genotype RA3, kindly provided by D. A. Stuart , were used for tissue culture and gene expression experiments.
Construction of genomic library Total DNA isolated according to Wu  was partially digested with Mbo I and fractioned in 1 0 - 40°7o sucrose gradient by the method o f Maniatis et al. . The 10-15 kb alfalfa DNA fragments were then inserted into pGY97, a vector which can be used either as a plasmid or as a bacteriophage lambda (E. Vincze et al., in preparation). The library was stored as bacteria at - 7 0 ° C in small aliquots.
Genomic DNA or cloned DNAs were digested with appropriate restriction enzymes, separated on an agarose gel and blotted according to the standard procedure . The copy number of histone H3 genes was estimated according to Gullis . Cloned DNA and genomic DNA was dot blotted onto a nitrocellulose filter according to White . After hybridization, the radioactivity of each dot was measured and used to calculate the copy number. Hybridization conditions were the same as used for RNA hybridization.
DNA sequencing DNA fragments were subcloned into M13 mp18/19 vectors and sequenced by the dideoxy chain termination method according to the standard procedure provided by Amersham Inc.
R N A preparation
Plant tissue culture
RNAs were prepared by the method of Cathala  and quantified by spectrophotometric assay as well as by agarose gel electrophoresis. As a source of plants, we used two-week-old alfalfa plants developed from nodal cuttings on agar-solidified UM medium  without plant hormones.
General procedure for somatic embryo induction was used as described by Stuart  with a few modifications. We initiated soft and rootless calli from roots on SH medium  containing 1/~M 6benzyl aminopurine (BAP) and 5/~M 2,4dichlorophenoxyacetic acid (2,4-D).
Northern blot hybridization
RNAs were separated on a formaldehyde agarose gel and blotted onto a nitrocellulose filter . Hybridization was carried out in a solution containing 50°70 formamide, 3 × SSC, 0.1070 SDS, 0.25070 milk powder (Gloria, Belgium) and 2×107 c p m / m l universal primer-labeled probe DNA at 42 °C. After hybridizing for 2 4 - 30 h, the filters were washed in 0.1 x SSC and 0.1070 SDS at 67 °C for 5 h.
The isolation and structural analysis of an alfalfa histone H3 gene To isolate histone H3 genes, about 105 clones from an alfalfa genomic library (E. Vincze et al., in preparation) were screened by in situ plaque hybridization . A rice histone H3 gene, H3RI1 (S.-C. Wu, in preparation), served as a probe. One positive clone out of four, designated XALH3-1, was further analyzed. The cloned 10.7 kb genomic DNA fragment bears only one histone H3 gene (Fig. 1). Other his-
643 EE H ,, I
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B IKb ,
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Fig. 1. The restriction m a p of XALH3-I (a), ALH3-I.1 (b) and the sequencing strategy (c) for the alfalfa histone H3 gene. The dark box indicates the coding sequence of ALH3-1.1. B = B a m HI, C = Cla I, E = Eco RI, Ev = Ego RV, H = Hind III, S = Sac I.
tone genes (H2a, H2b and H4) have not been detected by DNA hybridizations (data not shown). A 1.1 kb fragment carrying this H3 gene, ALH3-1.1, was then subcloned and sequenced. The complete nucleotide sequence o f ALH3-1.1 is presented in Fig. 2. In comparison with the histone genes from other species, we observed that both the coding region and the flanking sequences of the alfalfa gene exhibited several characteristic features. The amino acid sequence of alfalfa histone protein deduced from the nucleotide sequence of ALH3-1.1 shows very high homology (over 95070) with those determined in other species (Table 1). It has the same amino acid sequence as those in wheat and pea, and only one amino acid replacement at position 90 compared to those in corn and Arabidopsis, but more than five replacements can be found in comparison to rice and animal H3 proteins. However, despite the high homology at the protein level, the coding sequence o f ALH3-1.1 has only about 80°70 homology with all the genes compared (Table 1). According to the comparison described above, it can be concluded that the isolated alfalfa gene is a histone H3 gene, though its activity has yet to be tested. Based on the available data on biased codon usage in plant histone genes we have analyzed the codon usage in ALH3-1.1. By comparing the codons for the abundant amino acids (ala, arg, leu, lys and thr), three codons were revealed to be preferentially used. GCT among four possible codons, encoding ala, is used 60070 of the time; CGT among six possible codons, encoding arg, is used 6007o o f the time and CTC among six possible codons, encoding leu, is used 83°7o o f the time. Additionally, we compared
the nucleotides present at the third position o f codons in various histone genes from different organisms (Table 2). In contrast to the exclusively preferential use o f the G/C-ended codons in all of the monocotyledonous histone genes compared, the three histone genes from three dicotyledonous plants show no such G / C preference. The 284 bp o f the 5' flanking region exhibits most o f the typical consensus sequences found in both animal and plant histone genes (Fig. 2). The TATA box and CAAT box are located at position -111 and - 188, respectively. A "cap site"-like motif TCAATTC which is probably the transcription initiation point  is located about 30 bp downstream from TATA box. Instead of their usual locations , two GATCC motifs are found outside the CAAT box, at positions - 2 3 5 and - 2 7 9 . A hexamer, ACGTCA, found in wheat and other plant histone H3 and H4 genes  is also observed at position - 2 4 2 . Strikingly, a highly conserved octamer,
Table 1. H o m o l o g y between alfalfa H3 gene and deduced H3 protein with those determined in other species (%). C o m p a r e d species Clone Corn Rice Wheat Arabidopsis Pea Human Sea urchin
Protein DNA* Source
H3C2 99.3 pRH3-2 96.3 TH012 100 H3 A713 99.3 100 HUH3-149 94.8 SpL22 95.6
80.8 81.5 81.5 79.8 76.4 82
C h a u b e t  Xie  Tabata  Chaboute  Patthy  Wells  Kaumeyer 
*Comparison including only the 405 bp coding sequences.
-241 -231 -221 -211 -271 -261 -251 CCTCATCACA CAAACAAAACACATCCACAC GCCACGTCAT CG,A.TC,CGCGT GTCGCAAATACTCCAAATAA -171 -161 -151 -141 -201 -191 -181 ACGACACCCG TCGATTA~-ff'~AATCAAC GGCCACAATT ACACCCCATT CACCCACTTC TCAAATTTCA -131
AAACCCGCAC AAAATCATAT C A C ~ - ~
-101 -91 -81 -71 CTCACCCCTT TCATCTCTTC TTCCTCATCACTCTCAATTC
-61 -51 -41 -31 -21 -11 -1 TTCAAAGCAC AAAAACAACC TTCAAGTTTC TCTGTTTGAT ACTGTTCTTT TCAGTTATTT TTCATAATCA 30 60 ATG GCA CGT ACC AAG CAA ACC GCT CGC AAA TCC ACC GGT GGC AAA GCT CCA AGG AAG CAA MET AIa Arg Thr Lys Gin Thr AIa Arg Lys Ser Thr Gly Gly Lys Ala Pro Arg Lys Gin 90 120 CTC GCC ACA AAA GCC GCT CGC AAA TCT GCT CCG GCC ACC GGC GGA GTG AAG AAA CCT CAC Leu Ala Thr Lys Ala Ala Arg Lys Set Ala Pro Ala Thr Gly Gly Val Lys Lys Pro His 150 180 CGT TTC AGG CCA GGA ACC GTC GCT CTC CGT GAG ATC CGC AAG TAC CAG AAG AGC ACT GAG Arg Phe Arg Pro Gly Thr Val Ala Leu Arg Glu l i e Arg Lys Tyr Gin Lys Ser Thr Glu 210 240 CTC CTC ATC CGC AAA CTC CCC TTC CAG CGT CTT GTC CGT GAG ATC GCT CAG GAT TTC AAG Leu Leu l i e Arg Lys Leu Pro Phe Gin Arg Leu Val Arg Glu l i e Ala Gin Asp Phe Lys 270 300 ACT GAT CTC CGT TTC CAG AGC TCC GTC GTG TCG GCT TTG CAA GAA GCG GCC GAG GCT TAT Thr Asp Leu Arg Phe Gin Set Ser Val Val Ser Ala Leu Gin Glu Ala Ala Glu Ala Tyr 330 360 CTC GTC GGT CTC TTT GAG GAT ACT AAC CTC TGC GCC ATT CAT GCT AAG CGT GTC ACT ATC Leu V81 Gly Leu Phe Glu Asp Thr Asn Leu Cys Ala l l e His AIa Lye Arg Vsl Thr l i e 390 ATG CCT AAG GAT ATC CAG CTC GCT AGG CGT ATC CGT GGC GAG CGT GCT TGA MET Pro Lys Asp l i e Gin Leu AIa Arg Arg l i e Arg Gly Glu Arg Ala 450 460 470 480 420 430 440 TCTTGTTGA TTCGCTTTGT TAGGGTTTGT GTAGATAGGT TCATGATGTAGTTAAATCACAAACCGTTGC 520 530 540 550 490 500 510 TATAAGTTTC TCTATGGATT TTGTTATATT GTAATGTGCT TAACGCTTAA TCAATGAAATCGATCATCTT 600 610 620 560 570 580 590 TTGTTAAACT CTTTGTTCAA TTACTTATGC TTTTTTTTTA TCTTTTCTTA ACCCTAATTT TCTGTCATTT 670 680 690 630 640 650 660 TATTACACTT TCCGAACTTT TGTTATCCCTAATTGGATT~ GAAATCAAAA TTAGGGTTGATA~GGCATA 740 750 760 700 710 720" 730 TTGTATAATG TTGAAATTCT TGTTAAATAT ATT~rA'T-~'AT ACAATTATTT CAAAGTAAAA ATTAAATGCT 770 780 790 800 GTTGATTACA TCTTGAAATG TAAAAATAAT TGCATGCATA AGCTT Fig. 2. The nucleotide sequence of an alfalfa histone H3 gene, ALH3-1.1. The coding sequence starting from the initiation codon is arranged in triplets, and deduced a m i n o acids are given below the corresponding codons. In the 5' flanking region, the TATA box and CAAT box are boxed, the "cap" site-like sequence is underlined, the GATCC motifs are dotted, the CGCGGATC octamer is indicated with asterisk a n d the A C G T C A consensus is marked with interrupted lines. In the 3' flanking region, the palindromic sequence is indicated by inverted arrows and the polyadenylation signal-like sequences are overlined.
Table 2. Comparison of the nucleotides present at the third position of the codons (%). Species
Corn Rice Wheat Alfalfa
H3 H3 H3 H3 H3 HI H3.3 H3
41.6 42.4 37.2 21.9 28.5 32.5 22.6 28.5
51.8 54.7 60.6 37.2 22.2 18.1 21.2 46.0
1.5 0.7 0.7 12.4 25.9 24.9 22.6 5.8
5.1 2.2 1.5 28.5 22.4 24.5 33.6 19.7
Arabidopsis Pea* Human Sea urchin
*Histone HI cDNA of pea from Gantt and Key ; alfalfa histone H3 gene from Fig. 2 in this paper; other data are from the same sources as Table 1.
C G C G G A T C , a m o n g all p l a n t histone genes  was n o t seen o n the sense strand b u t o n the opposite s t r a n d at p o s i t i o n - 2 3 2 (Fig. 2). T h e 3 ' f l a n k i n g region o f the alfalfa histone H3 gene is characterized by a n A / T - r i c h n u c l e o t i d e comp o s i t i o n (72°7o AT). A long p a l i n d r o m e believed to f o r m stable h a i r p i n structure is observed 235 b p d o w n s t r e a m from the stop c o d o n . Moreover, several sequences similar to the classical p o l y a d e n y l a t i o n signals are also observed a d j a c e n t to the 3' e n d o f this p a l i n d r o m i c structure. Interestingly, a putative p o l y a d e n y l a t i o n signal, 5 ' - A T G ( G ) A A A T G - 3 ' , f o u n d in c o r n histone genes  was also seen in the alfalfa H3 gene at p o s i t i o n +533, 182 b p d o w n stream from the stop c o d o n .
The repetition and genomic organization o f histone H3 genes in alfalfa A b o u t 160 reiterated copies o f histone H3 gene were estimated per tetraploid g e n o m e according to the m e a s u r e m e n t s h o w n i n Fig. 3A, a s s u m i n g the size o f the tetraploid g e n o m e o f Medicago sativa to be 1.7 × 109 b p . To s t u d y the g e n o m i c o r g a n i z a t i o n o f the 160 copies o f H3 genes, we a n a l y z e d the alfalfa g e n o m i c D N A by S o u t h e r n hybridization. As s h o w n i n Fig. 3B, the 160 copies o f h i s t o n e H3 genes are located o n m o r e t h a n 20 b a n d s in the double-digested alfalfa g e n o m i c D N A . It seems that a regular repeat
Fig. 3. The copy number estimation (A) and genomic organization (B) of histone H3 genes in alfalfa. (A) 83.25, 166.5, 333.0, 832.5 and 1665 pg ofALH3-1.1 DNA subcloned in M13mpl8 vector and 1, 2 and 5 pg of alfalfa Nagyszen~isiDNA weredot-blotted (1-8), respectively. After hybridization with the 32p-labeled 195 bpEco RI/Sac I fragment of ALH3-1.1 (Fig. 1), each dot was cut out and the radioactivity measured. The data were used for calculating the copy number. (B) Bam HI and Hind III-digested DNAs were separated on a 1.0% agarose gel, Southern blotted and hybridized with 32p-labeled 1.1 kb fragment of ALH3-1.1. Lane 1, 10 #g of Nagyszen~isi DNA and lane 2-4, ALH3-1.1 DNA equal to 5, 10and 20 copies of histone H3 genes corresponding to 10/xg of alfalfa genomic DNA, assuming the genomic size of alfalfa to be 1.7x109 bp .
u n i t o f H3 genes c a n be excluded in alfalfa. However, it is possible that some H3 genes are closely linked in certain fragments as is the case for rice , since some fragments c o n t a i n obviously m o r e t h a n o n e copy o f the H3 gene (Fig. 3B).
The expression of histone H3 genes in alfalfa To study the expression o f histone H3 genes, we analyzed the total amount of m R N A homologous to ALH3-1.1 during the development o f RA3 plants through somatic embryogenesis. Total RNAs were isolated from different parts of in vitro grown alfalfa plants, calli and somatic embryos. The RNAs were analyzed by Northern hybirdization with the alfalfa histone H3 gene ALH3-1.1 as a probe. As shown in Fig. 4, the quantitative variation o f H3 transcripts between tissues is significant. Although H3 transcripts are almost undetectable in leaves and roots, H3 RNA is accumulated at a relatively high level in stems, similar to that in calli cultured in vitro. However, the level o f H3 mRNA in the different plant tissues and in the callus is considerably lower than those in somatic embryos differentiated from callus cells. Since the development of somatic embryos in RA3 culture system is unsynchronized, we artificial-
Fig. 4. The pattern of histone H3 RNAs in alfalfa. 10/zg of RNA was isolated from various sources, separated on a 1.5 °70 formaldehyde agarose gel and hybridized with 32P-labeled I.I kb fragment of ALH3-1.1. L = leaf, S = stem, R = root, C = calli, E1 = early stage including round and ellipsoid embryos, E2 = bottle-like embryos called torpedo stage in the text and E3 = cotyledonary embryos . Arrows indicate the different species of H3 transcript detected. Northern hybridization with the chicken/3-tubulin gene [311, indicating constant/3-tubulin RNA in leaves, stems and roots, was used as internal control (data not shown). Molecular markers were the Cla I and Bst NI-digested M13mpl8 RF DNA.
ly divided them into three stages according to Stuart . As shown in Fig. 4, the histone H3 transcripts remain at high levels at all the three stages of embryonic development, and little quantitative changes can be seen. Interestingly, we observed two species of RNAs that showed hybridization to the alfalfa H3 gene. The predominant band is about 900 bp and exists in all of the tissues tested. Another species is over 2 500 bp found only in embryos (Fig. 4). The level of this RNA species increases until the torpedo stage (E2 in Fig. 4) and then starts to decline.
The unique structure of ALH3-L1 Alfalfa is the second dicotyledonous plant of which the histone H3 gene has been characterized. Despite the fact that this gene shares several c o m m o n features with other histone genes of animals and plants, its special characteristics are described as follows. 1) A GATCC-Iike pentamer has been found 10 bp upstream from the TATA box for most histone genes and has been described as a histone gene-specific motif . In the case of the alfalfa histone H3 gene, however, such motifs can only be observed far upstream from the TATA box, at positions - 2 3 5 and - 2 7 9 . The unusual location of the GATCC motifs raises the question whether their position could have an influence on their presumed role in the regulation of gene expression. 2) A highly conserved octamer, CGCGGATC, found ubiquitously in plant histone genes , was found to overlap one o f the GATCC motifs in the 5' flanking region on the nonsense strand, that is, 5'GATCCGCG-3' at position - 232. Similar findings have been noticed in corn histone gene H3C3 , wheat histone H3 gene TH081  and rice histone H3 genes (S.-C. Wu, in preparation). It is unknown whether the orientation of the octamer motif has any significance in the expression of plant histone genes. 3) Another striking feature o f the alfalfa histone H3 gene is its unique codon usage. Comparing the use o f individual codons for abundant amino acids in
647 the H3 protein of alfalfa, we have found three preferentially used codons (CGT, G C T and CTC) different from those in monocot or dicot plants [3, 5]. In contrast to monocot plants , the alfalfa H3 gene shows a preferential use o f G C T and CGT, whereas no significant codon preference is observed in a histone H3 gene (H3A713) o f A. thaliana . GC preference in codon usage has been shown to be a c o m m o n feature of all histone H3 and H4 genes in monocot plants studied [5, 20, 26, 28, 35]. This seems not to be the case in dicot plants, according to the comparison shown in Table 2. In fact, the nonrandom codon usage is a c o m m o n feature among eukaryotic genes and its has been suggested that the difference in codon usage between genes reflects the modulation of gene expression rather than different mechanisms of gene evolution [8, 32]. According to this suggestion, the activity of histone gene may be somewhat different between monocot and dicot plants. To confirm the difference in codon usage between monocot and dicot histone genes, a further systematic survey and comparison of histone genes is necessary. 4) The 3' flanking sequence of the alfalfa H3 gene is characterized by three structural features: the ATrich nucleotide composition, the long inverted repeat sequence found far downstream from the stop codon, and the several classical polyadenylation signal-like motifs, a motif frequently missing from the majority of histone genes in animals and in plants [3, 10]. In animals, most o f the histone genes contain a typical T-hyphenated palindrome structure about 40 bp downstream from the stop codon. This T-hyphenated palindrome has been demonstrated to be necessary for the 3' termination o f transcription . In alfalfa, the long palindrome has no structural similarity to the classical T-hyphenated palindrome and lies far away from stop codon. However, since the H3 m R N A in alfalfa has an unusually long 3' nontranslated region (see below), the palindrome might be involved in termination o f transcription. 5) Several polyadenylation signal-like sequences are found in the 3' flanking region of ALH3-1.1 and the size o f the H3 transcript in all tissues tested is unusually long (about 900 bp) compared to the histone mRNAs in animals . This means that the nontranslated region of H3 m R N A in alfalfa is about 500 bp long. Based on the locations o f the TATA box
and the "cap site" (Fig. 2), the 3' nontranslated sequence can be suggested to the 400 bp long. In comparison with the usual termination point of transcription in wheat  and corn  histone genes, it seems reasonable to postulate that most (if not all) of the H3 transcripts in alfalfa may be polyadenylated. Our suggestion is consistent with the recent finding that the pea H1 cDNA contains a polyadenylated tail  and most o f maize H3 and H3 mRNAs are polyadenylated . To confirm this suggestion, further experiments are in progress.
The expression of histone H3 genes in alfalfa In RNA hybridization experiments, we have found a significant variation in the level of H3 mRNA in different parts and developmental stages of alfalfa plant. The differences in H3 gene activity between the tissues studied cannot be simply explained by using an analogy with differentiated animal cells, where most of histone RNA synthesis is cell cycledependent . Nevertheless, the high amount of H3 mRNAs in somatic embryos may suggest a similarity with the DNA replication-uncoupled histone synthesis that is seen in animal embryos . However, differences have to be considered as, for example, the source of histone mRNA from which the uncoupled histones are translated. In zygotic embryogenesis, the high demand for histone RNAs is provided by an RNA pool at the early stage of embryogenesis, while the expression o f histone genes remains at a low level . Probably, in contrast, the histone genes are highly active in somatic embryogenesis because of the lack of oogenesis which provides the histone mRNA pool. Consistent with this hypothesis, V. Raghavan (see Abstracts o f 14th Botanical Congress, 1987, Berlin) found that the H4 gene is highly expressed in newly formed cells in rice anther cultures during embryogenic division. In addition to the increased level o f H3 transcripts in somatic embryos, a high molecular weight RNA species (over 2 500 bp) homologous to H3 gene has been detected only in the embryos (Fig. 4). We think that this RNA species might be an embryo-specific H3 transcript, though we have no explanation for its origin or possible function. The pattern of histone H3 transcripts in alfalfa
648 s u g g e s t s t h a t t h e h i s t o n e H 3 g e n e s c a n s e r v e as a molecular marker for studies on plant development including embryonic differentiation from somatic cells.
Acknowledgement W e t h a n k Dr. Z o l t f i n V 6 g h f o r c o m p u t e r a n a l y s i s .
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