Indian Journal of Biotechnology Vo l I, April 2002, pp 164- 169

Cloning and Characterization of a Gene Encoding Ubiquitin Conjugating Enzyme from the Mangrove Species, Avicennia marina (Forsk.) Vierh.

M Parani, M N Jithesh, M Lakshmi and A Parida*

M S Swaminathan Research Foundation, III Cross Street, Taramani Institutional Area, Chennai 600 113, India

Received 20 April 2001 ; revised 7 February 2002

Covalent attachment of ubiquitin has been implicated in mediating proteolysis of the cellular proteins by Ubiq­uitin-proteasome pathway. Ubiquitin activating enzyme (EI), ubiquitin conjugating enzyme (E2), and ubiquitin pro­tein ligase (E3) are the three enzymes involved in this process. This paper reports the isolation of a gene that codes for the ubiquitin conjugating enzyme in Avicennia marilla (AmUBC2), and regulation of its expression at RNA level under salt stress. Deduced amino acid sequence of AmUBC2 showed 96% identity with UBC2 of Arabidopsis thalialla and also 73-78 % identity with RAD6 DNA repair protein of Homo sapiens, Rattus llorvegicus, Caenorhabditis elegalls, Drosophila melallogaster, Arabidopsis thalialla and Saccharomyces cerevisiae. Multiple alignment analysis showed that the amino acid residues in the core region of UBC2 were highly conserved across different taxa in the evolution­ary hierarchy. While some ubiquitin conjugating enzymes were induced under salt, heat and heavy metal stress in different tissues in plants, Northern analysis in the present study has clearly shown that the expression of UBC2 is not induced by salt stress either in root or in leaf tissues in A. marilla. Southern hybridization of genomic DNA with gene-specific probe showed that AmUBC2 is a single copy gene.

Keywords: ubiquitin conjugating enzyme, Avicenllia marina, proteolysis, mangroves

Introduction The state of proteins in living organisms is dy­

namic being continuously synthesized and degraded. Degradation of proteins could be non-specific like hydrolysis in lysosomes (Knop et ai, 1993) or selec­tive like degradation by ubiquitin-proteasome path­way (Hershko & Ciechanover, 1992). Three enzymes are involved in conjugating a particular protein to ubiquitin before being presented to 26S proteasomes for selective degradation . The C-terminus of ubiquitin is adenylated by the ubiquitin activating enzyme (ubi or El). The resultant ubiquitin-AMP intermediate of the ternary complex is then attacked by a sulfhydryl group of the enzyme, yielding an E l-ubiquitin thiolester. Then, El is replaced by ubiquitin conju­gating enzyme (ubc or E2) by transthiolation yielding E2-ubiquitin thiolester. Mostly, E2-ubiquitin binds to the target protein in conjunction with ubiquitin­protein ligase (ubr or E3) or rarely on its own. Subse­quently, consuming ATP, the 26S proteasomes de­grade the protein and release the ubiquitin (Finley & Chau, 1991; Ciechanover, 1994). Apart from protein

*Author for correspondence: Tel: +91-44-2541 229/2541698; Fax: + 91-44-2541319 Ema il: pmb@mssrf.res. in

degradation , UBC is also involved in DNA repair, cell cycle control and removal of incorrectly folded pro­teins under normal and stress conditions (Hochstrasser, 1996). In plants, some UBC's are also induced under stress conditions such as heat shock, heavy metal treatment (Feussner et aI, 1997) and high salt (Emilie et ai, 2000), and have been implicated in selective degradation of incorrectly folded proteins caused by stress (Seufert & Jentsch, 1990). Several ubiquitin genes are present in plants for the protea­some-ubiquitin pathway and hence the diversity in functional characteristics, expression and regulation of plant UBC's (Ingvardsen & Veierskov, 2001). A multi gene family encodes the ubiquitin-conjugating enzyme. For example, there are 18 structurally related genes belonging to the UBC super family in Arabi­dopsis thaliana. Super family can be divided into five gene families based on the protein structure and the physiological functions . AtUBC 1 gene family in­cludes AtUBC1, 2 and 3; AtUBC4 family with AtUBC4, 5 and 6; AtUBC7 family includes AtUBC7, 13 and 14; AtUBC8 family with AtUBC8, 9, 10, 11 and 12; AtUBC15 family with AtUBCI5, 16, 17, 18 (Sullivan et ai, 1994; Nacker et ai, 1996). There are two types of ubiquitin genes, polyubiquitin genes and ubiquitin extension genes. Polyubiquitin genes are

PARANI el ai: UBIQUITIN FROM A VICENNIA MARINA 165

arranged tandem head-to-tail repeats, and post­translational processing results in monomeric units of the protein. Ubiquitin extension genes are single monoubiquitins linked via its C-terminus to an unre­lated protein of either 52 or 76 to 81 amino acids (Ni­shi et aZ, 1993).

This paper reports the isolation and characterisation of a full-length cDNA clone from a mangrove species, Avicennia marina that codes for the ubiquitin­conjugating enzyme. Mangroves inhabit the estuaries and backwaters of the tropical and sub-tropical coasts, and A. marina is one of the true mangrove tree species capable of withstanding salinity that is even higher than that of normal sea-water.

Materials and Methods

c-DNA Library ConstruCtion

One-year-old wild plant of Avicennia marina (Forsk.) Vierh. was collected and treated with 0.5 M NaCI for 48 hrs. Total RNA from the leaf tissue was isolated following the GITC method (Chomzynski & Sacchi, 1987) with minor modifications (Parani et al, 1999). Poly (At RNA was purified over oligo-(dT) cellulose column and used as template for cDNA synthesis. The SuperScript™ Lambda System for cDNA Synthesis and A Cloning (Life Technologies, USA) was used for cDNA synthesis. First strand cDNA synthesis was primed with Notl-primer adapter, and the double stranded cDNA was direc­tionally cloned in plasmid vector (pSPORT 1) using the SalI adapter ligated at the 5' end. The SaLI adapter ligated cDNAs were size fractionated over SizeSepTM-400 Sepharose CL-4B spun column (Pharmacia Bio­tech, USA) before cloning in the plasmid vector. The ligated cDNAs were transformed in to the DH5a strain of Escherichia coli.

Sequencing of Expressed Sequence Tags (ESTs)

Several clones from A. marina cDNA library were randomly selected, and the insert size in each of the clones was determined by PCR using the universal M13 forward and reverse primers. The Plasmid DNA from the cDNA clones having cDNA of above 600 bp size were isolated by alkaline lysis method (Birnboim & Doly, 1979). The 5' end of the cDNAs were sub­jected to single-read sequencing using M 13 reverse primer and Big-Dye™ Terminators in an automated sequencing machine (ABI3lO, Applied Biosystems, USA). The DNA sequences were clipped for remov-

ing vector and adapter sequences and manually edited for sequencing errors. The edited DNA sequences were used for searching nucleotide and protein ho­mology to the existing genes in the databases at www.ncbi.nlm.nih.gov using BLASTN and BLASTX algorithms, respectively.

While the clones identified to be partial or having homology with unknown proteins were reserved for future studies, the clones having potentially full­length genes were completely sequenced from both the strands and further characterized. This study re­ports the characterization of a full -length cDNA that codes for ubiquitin conjugating enzyme in A. marina (Genbank Accession No. AF262934). Multiple align­ment analysis of A. marina gene coding for the ubiq­uitin conjugation enzyme with other homologues was performed using a CLUSTALW Program (Thompson etaZ, 1994).

Northern Analysis

The seeds of A.marina were collected from the mangrove forests in Pichavaram, Tamil Nadu, and seedlings were raised in pots, and irrigated with half­strength MS solution. Full-grown plants (two months after planting) were kept either in half-strength MS solution (control) or half-strength MS solution supplemented with 500 mM NaCl (treated). Total RN A from leaf and root tissues from control and treated plants were isolated at 12 hrs interval up to 48 hrs after imposing the stress, and up to 24 hI'S after withdrawing the stress according to the procedure described previously. Total RNA (15 flg) was separated in 1.2% formaldehyde gel and transferred to nylon membranes (Sambrook et ai, 1989). A 200 bp long 3' untranslated region (UTR) from AmUBC2 was amplified by PCR using forward (5'­TCCCTTACT AGACGGTTGG) and reverse (5'­AGTGACGCGTTCCTT ACA) primers, and eluted from agarose gel. This fragment was radioactively labeled with 32p and used as gene specific probe for Northern analysis. Following hybridizations (Sambrook et al, 1989), the blot was finally washed at IX SSC and 0.1 % SDS stringency conditions and exposed to X-ray films for 2 days at 70°C. Estimation of RNA sizes was based on the mobility of RNA Ladder 0.24-9.5 Kb of GIBCO BRL and computing the relative size of AmUBC2 using a Fragment Sizer Program of SEQAID II (tm) version 2.01.

166 INDIAN J SIOTECHNOL, APRIL 2002

Southern Anaiysis Total genomic DNA from A. marina leaves was

isolated using the CT AB method with minor modifi­cations (Pat'ani et aI, 1997). 10 ~g of genomic DNA was digested with four different restriction endonu­cleases, EcoRI, EcoRV, HindIII and BamHI, fraction­ated in a 0.8% agarose gel and then transferred on to a nylon membrane (Hybond N+, Amersham) by South­ern blotting (Southern, 1975). The blot was hybrid­ized with a radioactive labeled 200 bp fragment in the 3' untranslated region (UTR) of AmUBC2 as ex­plained in the previous section. After hybridizations (Sambrook et aI, 1989), the blot was finally washed at high stringency (0.1 X SSC and 0.1 % SDS) and ex­posed for 3 days at 70°C.

Results and Discussion The mangrove ecosystem harbours several inter­

esting plants, animals and microbes (Rao, 1987). The plant species of the mangrove forest that grow luxuri­ously under saline seawater is a rich source for genes conferring salinity tolerance. Therefore, authors have constructed a cDNA library from one of the highly salt tolerant ·mangrove species, A. marina, in order to isolate the genes involved in salt stress or osmotic stress. Amino acid residues in the N-terminal region of the proteins are conserved across the species, and therefore, by sequencing a few hundred nucleotides in the 5' end of the cDNA, it is possible to predict the gene function without complete sequencing of the corresponding gene. That is, the 5' nucleotide se­quence can serve as a tag fol' the intact expressed gene, and hence, called as Ex'pressed Sequence Tags (ESTs). Large numbers of ESTs are sequenced in sev­eral species for rapid gene discovery (Rounsley et ai, 1996; Covitz et ai, 1998). The approach was used for gene isolation from the A. marina cDNA library. One of the ESTs (Am494) showed significant homology with ubiquitin conjugating enzyme. This clone was approximately 900bp as determined by PCR and the open reading frame (ORF) was intact at the 5' end from 60 bp. As the ORF of the reported UBCs were about 500 bp, this could be a full-length clone. Since some of the ubiquitin conjugating enzymes were re­ported to be upregulated under salt stress, Am494 was completely sequenced and further characterized.

Complete sequencing of Am494 revealed that it is 850bp long with a longest ORF of 459bp (60bp-518bp) coding for 152 amino acids. The 5' untrans­lated region (UTR) is 59bp and the 3'UTR is 332bp

with a 22-residue Poly (At tail from 829bp. The 3'UTR did not contain a typical poly-adenylation sig­nal but a putative signal A TT AAA is located at posi­tion 763-768bp.

Homology search for the deduced amino acid se­quence of the longest ORF of Am494 was carried out with the global database using BLASLX algorithm. It showed strong homology with UBC2 of A. thaliana (AtUBC2). AtUBC2 is a member of a UBC gene family AtUBCl that includes AtUBCI, 2 and 3. Am494 clone showed 95, 96 and 87% identical amino acids with AtUBC 1 (Accession No. L19351), AtUBC2 (Accession No. L19353) and AtUBC3 (Ac­cession No. Ll9352), respectively. It also showed 96% identical amino acids with UBC2 from Medi­cago sativa and Triticum aestivum. However, it showed only 35-45% identical amino acids with UBCs outside this family like UBC4, 7, 8, 15 of A.

thaliana (Accession No. Ll9354, U33757, Z14989, AF028338) (Table 1). Percentage of identical amino acids was only 45-46% with UBC7 of maize (Acces­sion No. AF034946), and UBC4 of tomato and pea (Accession No. L23762, L29077). Similarity percent­age between the nucleotide sequences of AmUBC and AtUBC2 was 85%. But it showed no significant similarity with any of the UBC outside the AtUBCl gene family (Table 1). This clearly showed Am494 to be a homologue of AtUBC2 of the AtUBCl gene family, and hence named AmUBC2. In addition, AmUBC2 also showed 73-78% identical amino acids with RAD6 DNA repair protein of Homo sapiens, Rattus norvegicus, Caenorhabditis elegans, Droso­phila melanogaster, Arabidopsis thaliana and Sac­charomyces cerevisiae (Accession Nos P49459, M62388, U08139, M63729, Ll9353, P06104 respec­tively). Multiple alignment analysis of AmUBC2 and AtUBC2 from plants and the RAD6 DNA repair pro­tein homologues in yeast and animals presented in Fig. 1, reveals that the UBC enzymes across different taxa carries a highly conserved cysteine residue. It is hypothesized that ubiquitinylation takes place via a thiol-ester at this cysteine residue (Sullivan & Vier­stra, 1991). Mutation of the highly conserved cys­teine-88 residue in the yeast Saccharomyces cere­visiae RAD6 protein has been experimentally shown to destroy the protein's vital ubiquitin-conjugating activity (Sung et ai, 1990).

The Ubiquitin dependent proteolysis pathway plays a vital role in the selective protein degradation in cel­lular organisms. UBC2-proteasome c0I!lplex in yeast

HOIro Rattus Caenorhabdi tis Drosophila Avicennia Arab idops is Saccharomyces

HonD Rattus CaenorhalJeiitis Drosophila Avicennia Arabidopsis Saccharorl1yces

Horro Rattus CaenorhalJdi tis Drosophila Avicennia Arabiciopsis S acc haromyc es

HOlro Rattus CaenorhalJeiitis Drosophila Avicennia Arab idops is Saccharomyces

PARANI el af: UBIQUITIN FROM A VICENNIA MARINA

HSTP ARRRL HRDFKRLQEDPP AGV5GAPSENN nrV'lJN AIlIFGPEGTPFGDGTFKL TIEFT It3TPARRRLHRDFKRLQEDPPVGIl5GAPSEI%JIHQlJNAVIFGPEGTPFEDGTFKLVIEFS HTTPSRRRLHRDFKKLQEDPP AGVSGAPTEDNIL nJEAIIFGPQETPFEDGTFKLSLE FT It3TP ARRRL HRDFKRLQEDPPTGVSGAPTDNNIHIlJN AVIFGPHDTPFEDGTFKL TIE FT It3TP ARKRL HRDFKRLQQDPP AG ISGAPQDNN IHL lJNAVIFGPDDTPlJDGGTFKL TLQFT It3TP ARKRL HRDFKRLQQD PP AG ISGAPQDNN IHL lJNAVIFGPDDTPlJDGGTFKLSLQFS 1l'3TP ARRRL HRDF KRl'IKED APPGIlSAS PLPDNVHIJ~JNAHI IGPADTPYEDGTFRLLLEFD

EEYPNKPPTIlRFVSK1IFHPN"lTl ADG5 ICLD ILQNRlJSPTYDVS5 IL TS IQ5LLDEPNPNS EEYPNKPPTVRFLSK1IFHPNVYADGS ICLD ILQNRlJSPTYDVS:3 IL TS IQ::~LLDEPNPNS EEYPNKPPTVl'::F' ISK1IFHPN"lTl ADGS ICLD ILQNRlJSPTYDV.o.AIL TS IQSLLDEPNPNS EE\'PNKPPTVRFV:3KVFHPNITl .u.DGG I CLD ILQNRlJSP RYDV:3A IL TS IQSLLSDPNPNS EDYPNKPPTVRFVSRlIFHPNIYADGS ICLD ILQNQVJSP!YD I.o..o.IL TS IQSLLCDPNPN:3 EDYPNKPPTVRFVSRl'IFHPNIYADGS ICLD ILQNQlJSP IYDVAAIL TS IQSLLCDPNPNS EEYPNKPPHVKFLSEHFHPNVY .uJIJGE ICLD ILQNRlJTPTYDV.o.S IL TS IQSLFNDPNP ."_3

PPJ'JSQ.u_u.QL YQENKREYEKRV:3AIVEQSlJRDC---------------------------­PPJ··JSQ.u_u.QL YQENKREYEKRIlS.o.IVEQS~JNDS---------------------------­P .U·JSL.u_U.QL YQENRREYEKRVQQII,IEQSlJLNFGENEGDAVLKDDVE lEE I AAPG.uJIJD.o.DD P.uJ'J5T.u_u.QLYKENRREYEKRVK.o.CVEQSFID----------------------------­P."J'JSE."_u.RHFSENKREYNRRVREIVEQSlJTAD---------------------------­P.uJ'JSE.u_u.RHFSESKREYNRRVREVVEQSlJTAD---------------------------­P.uJ'JVEAATLFKDHKSQYVKRVKETVEKSlJEDDHDDHDDDDDDDDDDDDDEAD--------

DRHDEGASGSNA

167

Fig. I - Comparison of the deduced amino-acid sequences from different organisms using the ClustalW algorithm. The sequence identi­ties shown here include GenBank accessions from the ncbi.nlm.nih.gov database. The accession numbers of the organisms listed here include Homo sapiens (P49459) , Ra/lus norvegiclis (M62388), Caenorhabdilis elegans (U08139), Drosophila melanogaster (M 63729), Avicennia marina (AF262934), Arabidopsis thaliana (L19353) and Saccharomyces cerevisiae (P06104). The amino-acid residues marked in bold indicates the cysteine and hi stidine residues, active sites which are conserved in all the UBC enzymes across various taxa and amino acids which are marked with aSlerix represent the conserved sequences.

could efficiently target H2B for ubiquitylation (Prasad et ai, 2000). It is also hypothesized that the interaction between UBC and the proteasome may be regulated by DNA repair and could be induced upon physio­logical stress. One report shows that mcUBCl coding for ubiqutin conjugating enzyme in the halophytic ice plant, Mesembryanthemum crystallinum, is upregu­lated under salt stress. In ice plant, mRNA level of UBC4 (but named as mcUBCl) was induced under 200 mM and 400 mM NaCI stress only in root but not in leaf tissues. However, UBC9 (but named as mcUBC2) was not induced either in root or leaf tissue (Yen et ai, 1999; Emilie et ai, 2000). A UBC gene

was induced under heat as well as heavy metal stress (Feussner et ai, 1997). However, it could not be prop­erly annotated and assigned to a particular UBC gene family, because its sequence homology with other plant UBCs covered only smaller sequence parts. Therefore, it would be interesting to study the spatial and temporal regulation of AmUBC2 in A. marina. Northern hybridization of total RNA from A. marina roots and leaves with gene specific probe for AmUBC2 was used to study regulation of this gene at RNA level. A single mRNA species (approx 830 bp) was observed with this probe indicating the expres­sion of RNA transcripts for AmUBC2. However, the

168 INDIAN 1 BIOTECBNOL, APRIL 2002

Table I-Percentage of identical amino acids and nuc leotides (given in parenthesis) between the UBC from Avice/1IIia marilla (annotated as UBC22 and other UBCs from Arabidopsis thaliana. One gene from each gene family of A. thalialla UBC super family was taken for

comparison

AmUBC2 AtUBC2 AtUBC4 AtUBC7 AtUBC8 AtUBCI5 (AF262934) (Ll9353) (Ll9354) (U33757) (ZI4989) (AF028338)

Amubc2 100 (100)

Atubc2 96 (85) 100 (100)

Atubc4 32 (*) 32 (*) 100 (100)

Atubc7 37 (*) 39 (*) 26 (*)

Atubc8 45 (*) 46 (*) 34 (*)

AtUBCI5 35 (*) 35 (*) 27 (*)

(*) No significant similarity between the nuc leotide sequences

1.35 Kb - .

0.83 Kb --+

O.24Kb -

1.35 Kb

O.83Kb -+

0.24 Kb

2.37 Kb

1.90Kb --+ 1.35 Kb -

1 2 3 5 6

a

b

c

Fig. 2 - Northern blot analysis of AmUBC2 RNA transcripts in Leaf tissues (a) and Root tissues (b) of Avicennia marina under salt stress. Total RNA was isolated from leaves (a) and roots (b)

. of plants that had not received NaCI treatment, Control (Lane I) and plants that were sal t stressed (0.5M) for 12 hours (Lane 2); 24 hours (Lane 3); 48 hours (Lane 4); 12 hours after withdrawal from the salt stress (Lane 5); 24 hours after withdrawal from the salt stress (Lane6). 15 Ilg of total RNA was loaded and probed with 32p labeled 3' UTR AmUBC2 cDNA' probe. To confirm equal loading, blot (a) was stripped and reprobed for 18S r-RNA (c). The same was done for root also (data not shown). For size esti­mation, GIBCO BRL 0.24-9.5 Kb size ladder was used.

100 (100)

34 (*) 100(100)

31 (*) 37 (*) 100 (100)

;;:.. 1-4 ~

s:! - :I: ~ ~

~ ... ~ ~ ~ .... ~ ~ ..

5.5Kb --. ... ~

4.0Kb --. 3.1Kb =; . 2.8 Kb

+ •

/

Fig. 3 - Southern blot analysis of Avicennia marina genomic DNA with the AmUBC2 3' UTR specific cDNA probe. 10 Ilg of genomic DNA was digested with restriction enzymes EcoRI, EcoRV, HindIII and BamBI. The blot was hybridized with 32p

labeled AmUBC2 3' UTR cDNA probe.

expression of UBC2 is not induced under salt stress either in the root or in the leaves even after 48 hours of salt treatment (Fig. 2). This result establishes that up regulation of UBC mRNA is specific to only cer­tain members of the gene family. However, the steady

PARANI el al: UBIQUITIN FROM A VICENNIA MARINA 169

state level of different USCs in different tissues and reason for up regulation of only specific UBCs under stress remains to be studied. In this context, it has to be noted that UBC2 is not only involved in proteoly­sis but also in DNA repair, which is an essential con­stitutive function for any organism.

Southern hybridization studies using AmUBC2 cDNA was done to find out the gene copy number. In hybridizations of 3' UTR AmUBC2 specific probes to the Avicennia marina total genomic DNA, at high stringency washing conditions, all the four enzymes (EcoRI, EcoRV, HindIII and BamHI) used to digest total genomic DNA, showed one major hybridizing fragment (Fig. 3). Thus, it is concluded that AmUBC2 gene is present as a single copy gene in the genome of A vicennia marina.

Thus, the present communication would help in understanding and elucidating the functions of this crucial enzyme of protein turnover and DNA repair in different organisms.

Acknowledgement The authors gratefully acknowledge the Depart­

ment of Biotechnology and the Department of Atomic Energy, Government of India, New Delhi, for finan­cial support.

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