Developmental and Comparative Im by wuyunyi


									                                                 ARTICLE IN PRESS
                                                                                                            & Comparative
                             Developmental and Comparative Immunology 31 (2007) 889–902

   Identification of immune-relevant genes in histoincompatible
       rejecting colonies of the tunicate Botryllus schlosseri
            Matan Orena,b,Ã, Jacob Doueka, Zvi Fishelsonb, Baruch Rinkevicha
       Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 8030, Haifa 31080, Israel
        Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
                Received 22 November 2006; received in revised form 16 December 2006; accepted 18 December 2006
                                                Available online 24 January 2007


  The colonial ascidian Botryllus schlosseri manifests a unique allorecognition system that is controlled by a single
histocompatibility haplotype, the Fu/HC locus. When two allogeneic incompatible colonies come into direct contact, they
develop inflammatory-like rejection lesions, called points of rejection (POR). While screening for differentially expressed
genes during POR formation, we developed and analyzed a cDNA library of expressed sequence tags (ESTs) with 1693
unique ESTs that were clustered and assembled into 217 contigs and 1476 singlets. About 51% of these ESTs showed high
similarity (E-value p0.005) to known database sequences, of which 123 matches were identified as immune-relevant genes
encoding for stress proteins, pattern recognition receptors and complement proteins, proteases and protease inhibitors, cell
adhesion and coagulation proteins, cytokine-related proteins, programmed cell death and proteasome-associated proteins.
This first EST wide-screening analysis of the Botryllus allorecognition effector arm reveals a complex innate immune
system, hallmarked by a whole genome response to allorecognition challenge.
r 2007 Published by Elsevier Ltd.

Keywords: Allorecognition; Botryllus; Expressed sequence tags; Immunity; Subtraction library; Urochordata

1. Introduction                                                      nerve tube and segmented musculature in their
                                                                     tadpole larva [2]), and therefore have been con-
  Ascidians are sessile marine invertebrates of the                  sidered as a legitimate subject for evolution studies
subphylum Urochordata, occupying a key phyloge-                      [3]. This is further supported by the availability of
netic position in the origin of the vertebrates [1]. As              improved research tools, such as the publication of
chordates, this group of organisms features basic                    the draft genome from the solitary ascidian Ciona
morphological characteristics, similar to those ob-                  intestinalis [4].
served in the vertebrates (tail, notochord, dorsal                      One colonial urochordate group, the botryllid
                                                                     ascidians (family Styelidae, subfamily Botryllinae),
  ÃCorresponding author. Israel Oceanographic and Limnologi-
                                                                     exhibits a unique and well-defined allorecognition
cal Research, National Institute of Oceanography, P.O. Box           phenomenon, which has become a model system in
8030, Haifa 31080, Israel. Tel.: +972 4 8565273;
fax: +972 4 8511911.
                                                                     the study of the evolution of immunity [5]. A typical
    E-mail addresses:,          botryllid colony consists of few to several thousands
(M. Oren).                                                           morphologically and genetically identical modular

0145-305X/$ - see front matter r 2007 Published by Elsevier Ltd.
                                             ARTICLE IN PRESS
890                    M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

units, each called a zooid, which are typically                tag (EST) screening analysis that aims to reveal
arranged in oval, serpentine or star-shaped struc-             transcriptionally modulated genes during Botryllus
tures, termed systems, embedded within a translu-              rejection process.
cent and gelatinous matrix, the tunic. All zooids
within a colony are connected to each other via a              2. Methods
ramified vascular system that projects along the
peripheral colonial zones through numerous blind               2.1. Allorecognition assay
termini, referred to as ampullae. Ampullae are the
sites for self–nonself-recognition and allorecogni-               Four Fu/HC incompatible colonies of B. schlos-
tion effector-arm expression in botryllid ascidians            seri, originating from specimens collected some
[6,7]. In the model species, Botryllus schlosseri              years ago at Monterey Marina, California, were
allorecognition processes begin when the tunic edges           maintained under constant conditions, in the flow
of two allogeneic disparate colonies make contacts.            through facility at the National Institute of Ocea-
These contacts lead further to either fusion or                nography. Two similar size subclones were prepared
rejection between the colonies, a phenomenon that              from each genotype, as described [24]. Four
is controlled by a single highly polymorphic                   subclones, one from each genotype, were used for
histocompatibility haplotype termed the Fu/HC                  the allorecognition assays by positioning them in
locus, which contains multiple co-dominantly, ex-              two allorecognition incompatible pairs, each pair on
pressed alleles [8,9]. Fusion may occur between two            a different glass slide. The subclones were juxta-
different genotypes sharing one or both Fu/HC                  posed in a distance of less than 1 mm. This
alleles, whereas rejecting colonies share no Fu/HC             permitted the development of a full repertoire of
alleles. Fused colonies form a chimeric entity,                interactions between growing edges. When the first
leading to morphological resorption of one of the              point of rejection (POR) had been identified
partners [10] followed by development of somatic               (24–36 h after initiation; Fig. 1), the allogeneic
and germ cell parasitism [11]. Allogeneic incompa-             challenged subclones were harvested for RNA
tible encounters develop dark-golden to brown                  extraction. The four allogeneic naive subclones of
cytotoxic lesions that are usually followed by                 the same genotypes were harvested at the same time
formation of necrotic areas (called points of                  (illustrated schematically in Fig. 2).
rejection or PORs; Fig. 1) and in many cases,
amputation of interacting ampullae. Interactions               2.2. RNA extraction
terminate by disconnections between contacting
colonies, in many cases in concert with a retreat                 Total RNA was extracted separately from the
growth phenomenon [12]. Although scientists have               interacting pairs and from their naive homologues
recently claimed to have isolated a candidate Fu/              by EPICENTER MasterPureTM RNA Purification
HC locus [13], the genes participating in fusion or            kit. The integrity of the total RNA was verified by
rejection processes have remained unknown. It                  agarose gel electrophoresis.
should be noted, that although characteristics of
botryllid ascidians allorecognition resemble some              2.3. Subtraction library
characteristics of the vertebrates immunity [14], it is
regarded as innate and distinct from the adaptive                 First strand cDNA synthesis was performed on
graft rejection of the vertebrates. Observations               500 ng from each, experiment and naive total RNA,
suggest that allorecognition in botryllid ascidians            by Super SMARTTM PCR cDNA synthesis kit
is a blood-borne phenomenon initiated by humoral               (Clontech) according to manufacturer’s instruc-
factors [15]. To date, attempts to identify these              tions. cDNA subtraction was done in one direction
proteins have failed. Several Botryllus genes, which           (naive transcriptosome was subtracted from the
could be related to its innate immunity (which are             challenged transcriptosome) by Clontech PCR-
prevalent or upregulated at sites of interactions),            SelectTM cDNA Subtraction Kit according to
were identified in botryllid ascidians [16–23], but no          manufacturer’s instructions. All PCR reactions of
extensive screening of the allorecognition machinery           the process were done on a Perkin-Elmer GeneAmp
has been made. Based on a specific allorecognition              PCR System 5700 using Clontech original primers.
assay, developed previously [24] we present here, for          The amplified fragments were cloned in pTZ57R/T
the first time, a comprehensive expressed sequence              vector (Fermentas; the proportion of white to blue
                                                    ARTICLE IN PRESS
                           M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902                                 891

Fig. 1. B. schlosseri colonies during rejection: (A) general picture am. ampulla z. zooid and (B) enlarged picture of the marked area. por.
point of rejection. ia. Interacting ampulla. Scale bar indicates 50 mm.

clones was about 80%). A total of 2304 individual                        processing was done by SequencherTM program
positive clones were selected and re-cultured in 96                      (Gene Codes Corporation). It included trimming
wells LB plates with 100 mg/ml ampicillin. Sequen-                       of low quality sequence ends, pTZ57R vector
cing of the PCR products was performed in the Max                        sequences and of the Clontech primers
Planck Institute for Molecular Genetics, Berlin,                         (50 -CCCGGGCAGGT-30 , 50 -GCGGCCGAGGT-30 ).
Germany.                                                                 Further cleaning was done by a dedicated Perl
                                                                         script, which included removal of CDS5 (AC-
2.4. Sequences processing and assembly                                   GCGGG) and AC from the 50 ends and their
                                                                         complementary sequence from the 30 ends; trimming
  Sequences analysis was performed at the Bioin-                         polyA ends from the 30 ends and polyT from the 50
formatics Support Unit at the Ben-Gurion Uni-                            ends; and rejecting cleaned sequences shorter than
versity of the Negev, Israel. Initial sequence                           50 bp. Finally, manual check was done to ensure
                                             ARTICLE IN PRESS
892                   M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

                                 Colony A                                  Colony B

                                Subcloning                                Subcloning

                       Naive                      Rejection assay                         Naive

                                           Total RNA

                                         Tester cDNA

                                            Total RNA

                                         Driver cDNA

                      Fig. 2. The cDNA source of B. schlosseri subtraction library—experiment scheme.

that all remaining sequences were of high quality.              matches if their Blastx E-value cut-off was lower
The cleaned sequences were assembled into singlets              than 0.005 (only the top match for each sequence is
or contigs using CAP3 software [25].                            revealed). All sequences with E-value above this
                                                                threshold were considered sequences with no
2.5. Blast analysis                                             database match.

   Processed sequences (contigs and singlets) longer            2.6. RT PCR
than 100 base pairs were merged into one Fasta file
and submitted to database search using Blastx                      RNA was extracted from interacting and naive
algorithm. The blast search was done separately                 colonies (N ¼ 4) as described in Section 2.2. First
against both SwissProt and GenBank databases on                 strand cDNA was synthesized by first strand DNA
all unique sequences. Sequences were considered as              synthesis kit (Fermentas). The PCR amplification
                                                  ARTICLE IN PRESS
                            M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902               893

Table 1
Primers used for RT PCR

Gene match name             50 -primer                                 30 -primer                             Annealing

AsMASPb (seq. 39)           50 -GGCCTTGGTCAGTCTTCCTC-30                50 -TGACTTTCATCAACAGTTCGG-30           53
Cortical granule lectin     50 -AACCCTGGAAATCGTGAGC-30                 50 -CGTTCGGTATTCCTTTCGG-30             53.3
(seq. 33)
C-type lectin 2 (seq. 32)   50 -ACCGGTATTAGTGGCTGGAAG-30               50 -TCGTCATAACCAACCCACAAG-30           55
Humoral lectin              50 -TACCGCTGTGACCAACTTTG-30                50 -CCTGGTGCTAAGACGTTTCC-30            55.4
(seq. 31)
Rhamnospondin 2             50 -GGCACGATCACCTACACCAG-30                50 -ACGTAAGTTTGAATGCCAGGTC-30          55.5
(seq. 37)
Selectin P (seq. 73)        50 -GCCTAAATGCACCCTGATCAC-30               50 -GCGCGGGACATACAATCAG-30             56.2
BCAM (seq. 70)              50 -GATCCTTTGGCCGTGCATG-30                 50 -CACAAGGCAGCACCAAGGTC-30            57
Rhamnospondin 1             50 -ATGTATGTGCACCGTCCTGTC-30               50 -ACGGAGGAGAGTGTTGGCAC-30            57.2
(seq. 36)
40S ribosomal protein       50 -TTGTGACGGGTAACCAGATTC-30               50 -CTTGTAGTAGCGAGCGAGACG-30           57.2
S13 (Ctrl)
BS cytoplasmic actin        50 -GTAGGTAGTCTCGTGAATTC-30                50 -CACGCCATCTTGCGTCTGGA-30            57.2
(ref gene)

was performed using designed sets of primers (IDT                   sequences]/Total number of sequences) was calcu-
Inc.) as depicted in Table 1. In addition to the                    lated to be 21.6%.
specific sets of primers, actin primers were added to
all samples in order to amplify fragments of a                      3.2. Functional categories of EST matches
known database Botryllus actin (accession number:
AY159281), which served as a reference gene. The                       The BLASTX analysis revealed that 51% (861) of
PCR reaction was carried out for 30 cycles (94 1C,                  the ESTs had high similarity to known genes (E-
1 min; 53–57.2 1C, 1 min; 72 1C, 1 min) following by                value p0.005) whereas the remaining 49% (832)
additional 5 min at 72 1C. The RNA samples were                     had low or no similarity (E-value 40.005). The 861
normalized by a separate RT PCR reaction (ctrl)                     sequences with significant SwissPort matches were
using two sets of primers to amplify fragments of                   classified into functional categories according to
both housekeeping genes actin and a library 40S                     their blast results (Fig. 4): 336 ESTs (39%) were
ribosomal protein S13 (accession number:                            classified as genes with unknown function; 123
EH017358). This reaction was carried out for 27                     (14%) matched cell/organism defense genes; 112
cycles with same parameters as noted above. Four                    (13%) with gene expression and protein biosynth-
microliters of each PCR product was analyzed in                     esis genes; 57 (7%) with cell structure and motility
1.2% agarose/EtBr gel alongside DNA marker.                         genes; 57 (7%) with cell communication and
                                                                    signaling genes; 149 (17%) with metabolism genes;
3. Results and discussion                                           20 (2%) with transport-related genes; and 7 (1%)
                                                                    with cell cycle genes. Since urochordate genes are
3.1. General library statistics                                     under-represented in protein databases, the distri-
                                                                    bution pattern revealed here might not be fully
   The quality of 2161 ESTs (93.8% of total ESTs)                   representative of the tunicate transcriptome. The
was found adequate for assembly. The ESTs                           relatively high expression ratio of genes related to
length before assembly ranged between 50 and                        known cell and organism defense mechanisms
1000 bp (Fig. 3) with an average length of 350 bp                   indicates the high efficiency of the allorecognition
and standard deviation of 171. These sequences                      assay performed. It is also possible that part of the
were assembled into 1693 ESTs from which 217                        39% ESTs characterized as genes with unknown
are contigs and 1476 are singlets. Redundancy                       functions may be linked to the allorecognition
([Total number of sequencesÀnumber of unique                        effector arm. In addition, genes of the metabolism
                                                         ARTICLE IN PRESS
894                             M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

                                                               Distribution of Sequences Length






                                      0-100   101-200 201-300 301-400 401-500 501-600 601-700 701-800 801-900 901-1000
                                                                    Sequence Length (bp)

                                        Fig. 3. Distribution of Botryllus subtraction library EST lengths.

                                              Gene expression
                                                and protein          Cell cycle
                                                biosynthesis             1%
                                                                                                   Unknown function

                         Cell structure and                                                     Cell
                               motility                                                    communication
                                  7%                                                        and signaling

              Fig. 4. Distribution of 874 SwissPort EST matches (E-value p0.005) by major functional categories.

category include many of the mitochondrial elec-                              and actin (structure and motility category) and
tron transport chain such as ATP synthases,                                   transport proteins (transport category). It is, there-
cytochromes B and C, and NADH dehydrogenase,                                  fore, proposed that whole genome response to
possibly indicating high energy consuming processes                           allorecognition challenge is a hallmark of this
during the allorecognition assay. Other genes                                 phenomenon. The category of cell/organism de-
prominent to the subject of allorecognition are the                           fense-related genes includes 123 ESTs, matching
transcription and translation regulators (in the gene                         to 110 database entries, among them only six
expression and protein biosynthesis category),                                B. schlosseri known sequences (Table 2). All 123
kinases (cell communication and signaling cate-                               ESTs were deposited in the GeneBank (accession
gory), cytoskeleton genes such as myosin, tubulin                             numbers: EE743534 to EE743656).
                                                       ARTICLE IN PRESS
                                 M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902                                     895

Table 2
B. schlosseri cell/organism defense EST matches

Functional            Seq. no.      GeneBank           Gene match SwissPort ID   Gene match name                  Organism            E-value
classification                       accession number

Response to stress    1             EE743591           Q90Z20_ANAPL              Heat-shock protein 90 alpha      A. platyrhynchos    0
                      2             EE743602           Q6K045_PAROL              Heat-shock protein 90 beta       P. olivaceus        3.00EÀ32
                      3             EE743613           Q7YZJ0_9METZ              Heat-shock protein 90            S. fuscus           2.00EÀ23
                      4             EE743624           Q6USB9_9BIVA              Heat-shock protein 90            C. farreri          3.00EÀ16
                      5             EE743635           Q9NAX9_PARTI              Heat-shock protein 70            P. trichosuri       3.00EÀ47
                      6             EE743646           Q95V47_ARTSF              Heat-shock protein 70            A. franciscana      1.00EÀ45
                      7             EE743534           Q2MJK5_HALDI              Heat-shock protein 70            H. discus hannai    4.00EÀ43
                      8             EE743545           HSP70_PYRSA               Heat-shock protein 70            P. salina           5.00EÀ04
                      9             EE743556           GRP75_RAT                 Heat-shock protein 70            R. norvegicus       1.00EÀ37
                      10            EE743564           CH10_RAT                  Heat-shock protein 10            R. norvegicus       2.00EÀ09
                      11            EE743565           Q5RLR3_PIG                Heat-shock protein 10            S. scrofa           2.00EÀ07
                      12            EE743566           Q3UDS0_MOUSE              Heat-shock protein 8             M. musculus         5.00EÀ25
                      13            EE743567           Q27569_DROHY              Cu, Zn superoxide dismutase      D. hydei            2.00EÀ13
                      14            EE743568           Q7PXV9_ANOGA              Cu, Zn superoxide dismutase      A. gambiae          2.00EÀ10
                                                                                 like (ENSANGP00000018136)
                      15            EE743569           Q210I6_RHOPB              Glutathione peroxidase           R. palustris        2.00EÀ13
                      16            EE743570           OXR1_RAT                  Oxidation resistance protein 1   R. norvegicus       5.00EÀ09
                      17            EE743571           Q5DWF4_9CNID              Putative 8-lipoxygenase-allene   C. viridis          9.00EÀ35
                                                                                 oxide synthase fusion protein
                      18            EE743572           Q53GB9_HUMAN              Microsomal glutathione S-        H. sapiens          3.00EÀ25
                                                                                 transferase 3 variant
                      19            EE743573           DPYS_HUMAN                Dihydropyrimidinase              H. sapiens          4.00EÀ07
                      20            EE743574           Q6I666_CITLA              Cytochrome P450 like_TBP         C. lanatus          6.00EÀ11
                      21            EE743575           CP3AE_CAVPO               Cytochrome P450 3A14             C. porcellus        3.00EÀ05
                      22            EE743576           O04892_TOBAC              Cytochrome P450 like_TBP         N. tabacum          2.00EÀ04
                      23            EE743577           Q8CD72_MOUSE              NIMA (Nek1)                      M. musculus         6.00EÀ74

Pattern recognition   24            EE743578           C1QBP_BOVIN               Complement component 1 Q         B. taurus           3.00EÀ04
receptors/                                                                       subcomponent-binding protein
                      25            EE743579           Q9BP40_HALRO              Complement factor B              H. roretzi          5.00EÀ03
                      26            EE743580           Q966W1_HALRO              Ficolin 4                        H. roretzi          9.00EÀ43
                      27            EE743581           Q95P98_HALRO              Ficolin 3 precursor              H. roretzi          8.00EÀ37
                      28            EE743582           Q95P99_HALRO              Ficolin 2 precursor              H. roretzi          2.00EÀ45
                      29            EE743583           *                         *                                *                   1.00EÀ24
                      30            EE743584           Q29041_PIG                Ficolin                          S. scrofa           3.00EÀ11
                      31            EE743585           HMCT_BOMMO                Hemocytin precursor (humoral     B. mori             2.00EÀ07
                      32            EE743586           Q8AXR8_ANGJA              C-type lectin 2                  A. japonica         1.00EÀ11
                      33            EE743587           Q69HM9_CIOIN              Cortical granule lectin-like     C. intestinalis     7.00EÀ18
                      34            EE743588           Q5IWS5_HUMAN              Intelectin 1                     H. sapiens          1.00EÀ30
                      35            EE743589           Q5PPM0_XENLA              XEEL protein                     X. laevis           2.00EÀ10
                      36            EE743590           Q1PG16_HYDSY              Rhamnospondin 1                  H.                  2.00EÀ03
                      37            EE743592           Q1PG02_HYDSY              Rhamnospondin 2                  H.                  4.00EÀ10
                      38            EE743593           Q6NVT6_XENTR              Calreticulin                     X. tropicalis       9.00EÀ23
                      39            EE743594           O01655_HALRO              AsMASPb                          H. roretzi          3.00EÀ33

Proteases             40            EE743595           Q27458_BOTSH              Serine protease                  B. schlosseri       4.00EÀ27
                      41            EE743596           *                         *                                *                   6.00EÀ22
                      42            EE743597           *                         *                                *                   2.00EÀ09
                      43            EE743598           *                         *                                *                   6.00EÀ20
                      44            EE743599           Q8T3A0_CIOIN              Putative coagulation serine      C. intestinalis     1.00EÀ10
                      45            EE743600           DPP4_PIG                  Dipeptidyl peptidase 4 (T-cell   S. scrofa           4.00EÀ20
                                                                                 activation antigen CD26)
                      46            EE743601           Q3V5L6_POLMI              Trypsin                          P. misakiensis      2.00EÀ73
                      47            EE743603           O01310_BOTSH              Trypsinogen                      B. schlosseri       3.00EÀ43
                      48            EE743604           *                         *                                *                   9.00EÀ19
                      49            EE743605           *                         *                                *                   1.00EÀ33
                      50            EE743606           O01309_BOTSH              Trypsinogen                      B. schlosseri       8.00EÀ37
                      51            EE743607           O17439_9ASCI              Chymotrypsinogen                 B. villosa          2.00EÀ16
                      52            EE743608           *                         *                                *                   2.00EÀ20
                      53            EE743609           Q63ZG7_XENLA              Cathepsin A like (LOC494810      X. laevis           2.00EÀ07
                                                       ARTICLE IN PRESS
896                              M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

Table 2 (continued)

Functional            Seq. no.      GeneBank           Gene match SwissPort ID   Gene match name                    Organism          E-value
classification                       accession number

                      54            EE743610           Q864S1_BOVIN              Cathepsin   C                      B. taurus         2.00EÀ35
                      55            EE743611           Q45HJ6_9TREM              Cathepsin   D-like aspartic        O. viverrini      2.00EÀ38
                      56            EE743612           Q6A1I0_SUBDO              Cathepsin   L                      S. domuncula      2.00EÀ07
                      57            EE743614           Q6WNW6_BRABE              Cathepsin   L                      B. belcheri       4.00EÀ03
                      58            EE743615           Q2V9 Â 2_9METZ            Cathepsin L                        H. perlevis       6.00EÀ23
                      59            EE743616           Q5U4I5_XENLA              Aspartyl aminopeptidase            X. laevis         1.00EÀ11
                                                                                 (LOC495491 protein)
                      60            EE743617           Q5U2N2_RAT                Predicted ubiquitin-specific        R. norvegicus     2.00EÀ16
                                                                                 protease 14
                      61            EE743618           Q6PGT1_XENLA              Legumain like (MGC64351            X. laevis         2.00EÀ11
                      62            EE743619           Q504N0_MOUSE              Carboxypeptidase A2,               M. musculus       8.00EÀ31
                      63            EE743620           Q364L6_9GAMM              Peptidyl-dipeptidase A             Shewanella sp.    7.00EÀ27
                                                                                 precursor                          ANA-3

Protease inhibitors   64            EE743621           ALST_TAMSI                Alpha-1-antitrypsin-like           P. misakiensis    7.00EÀ06
                                                                                 protein CM55ST precursor
                      65            EE743622           Q25B53_BRALA              Serpin 1 precursor                 B. lanceolatum    2.00EÀ20
                      66            EE743623           Q27HF2_BOVIN              Squamous cell carcinoma            B. taurus         4.00EÀ04
                                                                                 antigen recognized by T-cells 1-
                                                                                 like protein
                      67            EE743625           ILEU_HORSE                Serpin B1                          E. caballus       1.00EÀ29
                      68            EE743626           Q2ES50_DABRR              Kunitz protease inhibitor-I        D. russelli       9.00EÀ03
                      69            EE743627           Q1LXL1_BRARE              Kunitz protease inhibitor-I        B. rerio          3.00EÀ18

Cell adhesion/        70            EE743628           Q99K86_MOUSE              Bcam protein                       M. musculus       5.00EÀ05
                      71            EE743629           SCRB2_MOUSE               Scavenger receptor class B         M. musculus       4.00EÀ21
                                                                                 member 2 (LIMP II)
                      72            EE743630           Q3U5F6_MOUSE              Selectin, endothelial cell         M. musculus       7.00EÀ09
                      73            EE743631           Q5TI49_HUMAN              Selectin P                         H. sapiens        7.00EÀ19
                      74            EE743632           Q2XNL5_CHICK              Tumor necrosis factor-             G. gallus         1.00EÀ10
                                                                                 inducible protein 6
                      75            EE743633           Q6WB00_TRYCR              Mucin-like protein                 T. cruzi          2.00EÀ03
                      76            EE743634           Q26878_TRYCR              Mucin-like protein                 T. cruzi          1.00EÀ03
                      77            EE743636           Q4CUU5_TRYCR              Mucin TcMUCII, putative            T. cruzi          4.00EÀ03
                      78            EE743637           MUA1_XENLA                Integumentary mucin A.1            X. laevis         5.00EÀ10
                      79            EE743638           CLD19_MOUSE               Claudin-19                         M. musculus       1.00EÀ18
                      80            EE743639           Q1ZXQ3_DICDI              Annexin VII                        D. discoideum     2.00EÀ11
                      81            EE743640           Q6NVI8_XENTR              Annexin A7                         X. tropicalis     9.00EÀ09
                      82            EE743641           Q2F5T8_BOMMO              Annexin isoform 1                  B. mori           4.00EÀ13
                      83            EE743642           Q7T391_BRARE              Annexin 11a, isoform 2             B. rerio          6.00EÀ25
                      84            EE743643           Q6PVV9_CIOIN              SPARC                              C. intestinalis   1.00EÀ28
                      85            EE743644           O76470_LYTVA              Echinonectin                       L. variegatus     6.00EÀ07
                      86            EE743645           *                         *                                  *                 3.00EÀ10
                      87            EE743647           *                         *                                  *                 6.00EÀ07
                      88            EE743648           Q5S3N1_SALSA              Zonadhesin-like                    S. salar          2.00EÀ20
                      89            EE743649           Q2PC93_CHICK              SCO-spondin                        G. gallus         5.00EÀ41
                      90            EE743650           Q7QCP4_ANOGA              Notch like                         A. gambiae        5.00EÀ03
                      91            EE743651           Q69HL6_CIOIN              Polydomain protein-like            C. intestinalis   2.00EÀ08
                      92            EE743652           O18977_BOVIN              Tenascin-X                         B. taurus         4.00EÀ06
                      93            EE743653           Q769I3_CIOIN              Von Willebrand Factor like 1       C. intestinalis   2.00EÀ40
                      94            EE743654           *                         *                                  *                 1.00EÀ39
                      95            EE743655           Q769I2_CIOIN              Von Willebrand Factor like 2       C. intestinalis   1.00EÀ20
                      96            EE743656           Q9BLJ1_CIOIN              Ci-META1                           C. intestinalis   2.00EÀ03
                      97            EE743535           *                         *                                  *                 5.00EÀ16
                      98            EE743536           Q9BLJ2_CIOIN              Ci-META2                           C. intestinalis   9.00EÀ11
                      99            EE743537           *                         *                                  *                 6.00EÀ10
                      100           EE743538           Q8MVQ1_9ASCI              Vwa1 protein                       B. villosa        3.00EÀ06
                      101           EE743539           *                         *                                  *                 1.00EÀ10
                      102           EE743540           APLP_LOCMI                Apolipophorins precursor           L. migratoria     9.00EÀ12
                      103           EE743541           Q5YD83_BIOGL              Fibrinogen related domain          B. glabrata       4.00EÀ17
                                                                                 variant 3
                                                       ARTICLE IN PRESS
                                 M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902                                    897

Table 2 (continued)

Functional            Seq. no.      GeneBank           Gene match SwissPort ID   Gene match name                   Organism          E-value
classification                       accession number

Cytokins and          104           EE743542           Q9R175_RAT                Interferon-inducible protein 16   R. norvegicus     7.00EÀ04
cytokine regulation
                      105           EE743543           Q4H3B3_CIOIN              Interferon regulatory factor      C. intestinalis   2.00EÀ17
                                                                                 like protein
                      106           EE743544           Q6GL57_XENTR              Interleukin enhancer binding      X. tropicalis     1.00EÀ36
                                                                                 factor 3
                      107           EE743546           Q49CF6_BOVIN              Myeloid differentiation           B. taurus         3.00EÀ10
                                                                                 primary response protein

Programmed cell       108           EE743547           Q7T166_BRARE              Death-associated protein 1a       B. rerio          2.00EÀ07
death                                                                            (Fragment)
                      109           EE743548           Q3HL92_AIPPA              Caspase-like protein              A. pallida        9.00EÀ06
                      110           EE743549           PARP1_CRIGR               Poly [ADP-ribose] polymerase      C. griseus        1.00EÀ34
                                                                                 1 (PARP-1)

Tumor proteins        111           EE743550           Q6P3J1_BRARE              Tumor protein D52-like 2,                           2.00EÀ28
                                                                                 isoform 2
                      112           EE743551           Q3TWS8_MOUSE              Tumor protein, translationally-   M. musculus       2.00EÀ15
                                                                                 controlled 1
                      113           EE743552           ZG14_HUMAN                Gastric cancer antigen Zg14       H. sapiens        2.00EÀ27
                      114           EE743553           BCL7B_HUMAN               B-cell CLL/lymphoma 7             H. sapiens        5.00EÀ20
                                                                                 protein family member B

Proteasome-           115           EE743554           P90725_BOTSH              LMP7-like protein                 B. schlosseri     1.00EÀ53
associated proteins
                      116           EE743555           *                         *                                 *                 1.00EÀ38
                      117           EE743557           LAMP3_HUMAN               LAMP-3                            H. sapiens        2.00EÀ05

Other                 118           EE743558           Q17280_BOTSH              FK506-binding protein             B. schlosseri     1.00EÀ34
                      119           EE743559           P90724_BOTSH              Soluble immunoglobulin            B. schlosseri     9.00EÀ53
                                                                                 molecule homologue,
                      120           EE743560           Q9CYV7_MOUSE              Helicase, lymphoid specific        M. musculus       3.00EÀ34
                      121           EE743561           Q5TBV0_HUMAN              Esterase D                        H. sapiens        1.00EÀ18
                      122           EE743562           Q6T4Q2_PYRCO              Putative senescence-associated    P. communis       2.00EÀ31
                      123           EE743563           Q1VTK4_9FLAO              Cold-shock protein                P. torquis        1.00EÀ10

*Different singlet correlated with the same SwissPort match as the one above it.

3.3. Differential expression of lectins                                      shock proteins (seq. 1–12), including HSPs 90, HSPs
                                                                             70, and two small HSPs (10 and 8 kDa). HSPs are
   Lectins were chosen for the RT PCR amplification                           members of the chaperone family that reach high
since this protein family is highly represented in our                       expression levels under diverse environmental stress
library and its members have been shown to                                   conditions, such as heat [26]. In vertebrates, HSPs
participate in innate immune response as described                           play a role in inflammation [27,28] and cell death
below. Eight lectins from the library were differentially                    [29], immune response to pathogens and detection
amplified by RT PCR using independent RNA from                                of stressed or damaged cells [30], all of which can be
naive and rejecting Botryllus colonies. The results                          relevant to Botryllus allogeneic rejection responses.
(Fig. 5) revealed specific amplification of lectins in                         It is interesting to note, that although two HSP70
allogeneic challenged colonies, further confirming the                        genes had been sequenced from B. schlosseri [17],
specificity of this EST library to the rejection process.                     neither of them matched the HSPs expressed here,
                                                                             reflecting a wider repertoire of HSP70 genes in this
3.4. Stress proteins                                                         species.
                                                                                Sequences 13–23 match proteins that are involved
   Sequences 1–23 in Table 2 show high similarity to                         in the protection of cells against oxidative stress and
stress-related proteins. Among them are 12 heat-                             other cytotoxic agents. Oxidative stress is known to
                                                                                ARTICLE IN PRESS
898                                 M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

                      N             R     N                   R   N                     R   N                    R   N                     R   N                 R   N          R   N                     R   N               R

Actin (ref.)


                                                                                                                         Rhamnospondin 2

                                                                                                                                                                                        Rhamnospondin 1
                                           Cortical granule

                                                                      C-type lectin 2

                                                                                                Humoral lectin

                                                                                                                                                                                                              40S ribosomal
                                                                                                                                                   Seclectin P


                                                                                                                                                                                                              protein S13

Fig. 5. RT PCR differential expression of lectins in rejecting (R) and naive (N) Botryllus colonies. An actin fragment (321 bp) served as the
reference gene and was expressed in all samples, whereas the approx. 200 bp lectin EST fragments were expressed only in the rejecting
colonies. RNA samples were normalized by two housekeeping genes actin and 40S ribosomal protein S13 (Ctrl).

be a major cause for cell damage in Botryllus [31].                                                                  acids, lipoproteins, peptidoglycans (PGN) and
Reactive oxygen metabolites (ROMs) such as                                                                           (1-3) b-D-glucans [35], known as pathogen-asso-
hydrogen peroxide, hydroxyl radicals and singlet                                                                     ciated molecular patterns (PAMPs). The two
oxygen can be products of innate immunity reac-                                                                      collagenous lectins, mannose-binding lectins
tions, such as respiratory burst and prophenolox-                                                                    (MBLs) and ficolins, have been proven to be pattern
idase activating system expressed in the immune                                                                      recognition receptors (PRRs) that recognize
circulating cells of Botryllus, the morula cells [32].                                                               PAMPs [36] and initiate the lectin complement
We have identified two new putative Cu and Zn                                                                         pathway through MBL-associated serine proteases;
superoxide dismutases (SOD; seq. 13, 14) and a                                                                       MASPs [37]. In this study, we have identified eight
glutathione peroxidase (seq. 15), known antioxi-                                                                     lectins (seq. 31–38), an ascidian MASP (AsMASPb;
dants that are capable of reducing ROMs such as                                                                      seq. 39) and four different ficolins (seq. 26–30), of
free hydrogen peroxide to water. SOD was found to                                                                    which three matched ascidian ficolins (ficolins 2–4).
inhibit the proPO cytotoxic activity in Botryllus                                                                    C1q binding protein (seq. 24) and Factor B (seq. 25)
[31,32] and to inactivate superoxide anion products                                                                  are the only representatives found of the classical
in H. roretzi hemocytes [33]. Other genes that may                                                                   and alternative complement pathways, respectively.
participate in protection from oxidative damage are                                                                  The high number of different PRRs: lectin and
the oxidation resistance protein 1,8-lipoxygenase-                                                                   ficolin matches together with several serine pro-
allene oxide synthase fusion proteins and the                                                                        teases (including MASP) implies a possible existence
glutathione S-transferase 3 variant (matching to                                                                     of a lectin-based opsonization system as suggested
seq. 16–18). Sequences 19–22 show similarity to                                                                      for other ascidians [38]. In this alloreactivity
detoxification proteins including three different                                                                     context, it is possible that the C1q, ficolins and
cytochrome P450 proteins. Sequence 23 matches                                                                        lectins play a role in opsonization of dead cells at
NIMA (Nek1), a protein involved in the response to                                                                   the POR and in the inflammatory process [39].
DNA damage and ionized radiation stress.                                                                             Although we have identified some of the comple-
                                                                                                                     ment lectin pathway components, as well as C1q
3.5. Pattern recognition receptors and complement-                                                                   binding protein and Factor B matches from the
associated genes                                                                                                     classical and alternative pathways, there is still no
                                                                                                                     evidence for the existence of a full complement
   Since, so far there has not been any evidence to                                                                  system as in vertebrates. This is due to the absence
the existence of specific adaptive immunity in                                                                        of downstream complement components (C3–C9) in
invertebrates [34], it is assumed that these organisms                                                               our library. Matches for most of the complement
are equipped with innate immune responses that                                                                       proteins were predicted based on C. intestinalis draft
rely on detection of conserved surface epitopes,                                                                     genome sequence [37], but classical complement
including lipopolysacharides (LPS), lipoteichoic                                                                     functionality has not been proven yet. Two putative
                                             ARTICLE IN PRESS
                       M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902                 899

complement proteins have been identified pre-                   coagulation-related protein matches in our library
viously in Botryllus: a complement control protein             must not be ignored and should be examined in the
containing a SCR domain [40] and a Botryllus                   context of Botryllus immune system.
C-type lectin [21]. Both are not presented in our
EST list. It should be noted that our subtraction              3.8. Cytokine-related proteins
approach only permits identification of genes that
undergo up-regulation in their level of transcription.            Sequences 104–107 share similarity with cytokine
It is possible that products of other genes, such as           interacting proteins. Two are interferon-related
complement C3, play a role in the alloreactivity               (interferon-inducible protein 16 and interferon
process but their level of synthesis is not modified.           regulatory factor like protein) and two are inter-
Function of complement C3 of H. roretzi in                     leukin-related (interleukin enhancer binding factor 3,
opsonization has been demonstrated [41].                       Myeloid differentiation primary response protein;
                                                               MyD88). The identification of these four proteins
3.6. Proteases and protease inhibitors                         implies a possible existence of the two major cytokines
                                                               in Botryllus. To date, none of the above proteins or
   Sequences 40–63 match 19 different proteases,               any other interferon or interleukin-related genes have
including eight serine proteases (seq. 40–53) from             been identified in Botryllus.
which five are new and three have been identified
before [16,19]. Sequences 64–69 show similarity to six         3.9. Programmed cell death and tumor proteins
new serine protease inhibitors (Serpins). Interestingly,
all serine proteases identified in the EST library                 Sequences 108–110 share similarity with pro-
match sequences from ascidian origin. Another                  grammed cell death proteins and sequences
protease group represented here is the cathepsin               111–114 with tumor proteins, some of which posses
group (seq. 54–58) including representative of cathe-          anti-apoptotic properties (such as tumor protein,
psins A, C, D and L. The cathepsins are lysosomal              translationally controlled 1). Programmed cell death
cysteine proteases that can degrade and digest                 events are important and integral parts of Botryllus
products of phagocytosis. However, they are also               cyclic blastogenesis, the weekly developmental
known to be involved in programmed cell death [42].            process characteristic to this group of organisms
                                                               [45], but apoptosis may be also involved in Botryllus
3.7. Cell adhesion and coagulation                             immune system.

   Sequences 70–103 show similarity to cell adhesion           3.10. Proteasome-associated proteins
and coagulation proteins. Among them are several
cell adhesion proteins that may play a major role in              Analysis reveals expression of LMP proteins in
inflammation, such as members of the selectin (seq.             the allorecognition EST library. LMPs are subunits
72, 73) and mucin (seq. 75–77) protein families. In            of the immunoproteasome that are regulated by
mammals these proteins help in leukocytes recruit-             interferon g and tumor necrosis factor [46]. Two
ment by promoting their adherence to blood vessel              putative LMP matches have been identified here: a
walls, enabling their penetration into the inflamed             Botryllus LMP 7 [20] (seq. 115–116) and a new LMP
tissue. Similarly, during Botryllus rejection re-              3 candidate (seq. 117). Proteasomes are essential
sponse, blood cells infiltrate through the vasculature          protein degradation machinery found in all eukar-
endothelium into the tunic, where they participate             yotic cells, including ascidians [47] and involved in
in the formation of PORs [43]. This process                    apoptosis [48]. The role of LMPs and, in general,
resembles the inflammatory response in higher                   proteasomes in alloresponsiveness still awaits clar-
vertebrates. To date there is no evidence for the              ification. Recently, upregulation of LMP7 expres-
existence of coagulation system in ascidians as                sion was described in epithelial and endothelial cells
known for vertebrates. Nevertheless, ascidians                 of ischemia kidney [49].
paralogs and/or constituent domains seem to be
present for all vertebrates key coagulation factors            3.11. Other
[44]. We have identified 19 coagulation-related
protein matches (seq. 85–103), of which 9 are from               A previously identified Botryllus soluble immu-
C. intestinalis genome. The presence of so many                noglobulin [18] was expressed in our EST library
                                             ARTICLE IN PRESS
900                    M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

(seq. 119). This protein could reveal primitive roles          hallmarked by a whole genome response to allor-
of immunoglobulin molecules in botryllid ascidians             ecognition challenge. In order to analyze further
alloresponses. The recently sequenced soluble im-              gene expression patterns during the rejection
munoglobulin that was defined as the Fu/HC                      process, we will apply microarray technology based
candidate of B. schlosseri [13] (though its function           on our ESTs library.
has yet to be revealed) is not represented in the EST
library. Another interesting EST match is the
Botryllus FKBP (seq. 118), which was identified                 Acknowledgments
previously [23]. Vertebrate FKBP is a target for
immunosuppressive agents FK 506 and its function                  This study was supported by a grant from the
in ascidians have yet to be clarified. Botryllus                Marine Genomics Europe Network of Excellence
putative CD94/NKR-P1-related receptor, recently                (EDD Node), from the United States–Israel Bi-
isolated from rejecting Botryllus colonies by non-             National Science Foundation (2003-010) and partly
biased screening strategy of differential display              from the Israel Academy of Science (550-06). We
library [22], was not found in our EST list.                   thank V. Caspi from the Bioinformatics Support
                                                               Unit at the Ben-Gurion University of the Negev for
4. Conclusion                                                  dedicated computer work and G. Paz for assistance
                                                               in manuscript preparation.
   While allorecognition and the complex effector
mechanisms involved are well described in the
vertebrates, invertebrate’s allorecognition is char-           References
acterized only on the morphological level. More-
over, no adult vertebrates undergo (as in the case of           [1] Berrill NJ. The origin of vertebrates. London: Oxford
Botryllus) natural transplantation in the wild (ex-                 University Press; 1955.
                                                                [2] Kowalevsky A. In Entwicklungsgeschichte der einfachen
cept of pregnancy in mammals). The remarkable                       Ascidien. Mem Acad St Petersburg 1866;10:1–19.
morphologically defined and predicted allorejection              [3] Satoh N, Jeffery WR. Chasing tails in ascidians: develop-
process in botryllid ascidians makes the colony                     mental insights into the origin and evolution of chordates.
allorecognition assay highly suitable for elucidating               Trends Genet 1995;11:354–9.
various aspects of ascidians’ immunity. Developing              [4] Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De
                                                                    Tomaso A, et al. The draft genome of Ciona intestinalis:
ESTs for the analysis of whole-genome expression                    insights into chordate and vertebrate origins. Science
towards a specific biological challenge has been                     2002;298:2157–67.
proven to be an efficient tool in identification of new           [5] Rinkevich B. Primitive immune systems: are your ways my
immune genes [50], including immune-related asci-                   ways? Immunol Rev 2004;198:25–35.
                                                                [6] Taneda Y, Watanabe H. Studies on colony specificity in the
dian genes [51]. The cDNA subtraction technology
                                                                    compound ascidian, Botryllus primigenus Oka, I: initiation
permitted us to screen large quantities of ESTs with                of ‘‘nonfusion’’ reaction with special reference to blood cells
high ratio of relevance to the biological process of                infiltration. Dev Comp Immunol 1982;6:43–52.
ascidian allo-rejection. In our functionality analysis,         [7] Taneda Y, Saito Y, Watanabe H. Self or non-self
we have focused on currently known immune/                          discrimination in ascidians. Zool Sci 1985;2:433–42.
defense-related database gene matches, although                 [8] Sabbadin A. Le basi genetiche della capacita di fusione fra
                                                                    colonie in Botryllus schlosseri (Ascidiacea). Atti Accad Naz
matches of other categories can be equally impor-                   Lincei Rend 1962;32:1031–5.
tant to the process. Other potentially relevant ESTs            [9] Rinkevich B, Porat R, Goren M. Allorecognition elements
may prove to be hidden in the 819 ESTs (48%) that                   on a urochordate histocompatibility locus indicate unprece-
show low or no similarity, or within the 343 ESTs                   dented extensive polymorphism. Proc R Soc London B
(20%) that match with genes with unknown                            1995;259:319–24.
                                                               [10] Rinkevich B, Weissman IL. A long-term study of fused
function. The last category can be of particular                    subclones of a compound ascidian: the resorption phenom-
interest for gene functionality studies in ascidians,               enon. J Zool (London) 1987;213:717–33.
because it can help to reveal the functions of these           [11] Stoner DS, Rinkevich B, Weissman IL. Heritable germ and
genes in higher vertebrates. We found 123 ESTs                      somatic cell lineage competitions in chimeric colonial
                                                                    protochordates. Proc Natl Acad Sci USA 1999;96:9148.
(14%) that show similarity to cell/organism defense
                                                               [12] Rinkevich B, Weissman IL. Retreat growth in the ascidian
genes. As expected, no classical elements of acquired               Botryllus schlosseri: the consequences of non-self recogni-
immunity has been found. Nevertheless, the overall                  tion. In: Grosberg RK, editor. Invertebrate historecognition.
figure drawn is of complex innate immune system,                     New York: Plenum Press; 1988. p. 93–109.
                                                      ARTICLE IN PRESS
                             M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902                                   901

[13] De Tomaso AW, Nyholm SV, Palmeril KJ, Ishizukal KJ,                [30] Stewart GR, Young DB. Heat-shock proteins and the
     Ludington WB, Mitchel K, et al. Isolation and characteriza-             host–pathogen interaction during bacterial infection. Curr
     tion of a protochordate histocompatibility locus. Nature                Opin Immunol 2004;16:506–10.
     2005;38:454–9.                                                     [31] Ballarin L, Cima F, Floreani M, Sabbadin A. Oxidative
[14] Scofield VL, Schlumpberger JM, West LA, Weissman IL.                     stress induces cytotoxicity during rejection reaction in the
     Protochordate allorecognition is controlled by an MHC-like              compound ascidian Botryllus schlosseri. Comp Biochem
     gene system. Nature 1982;295:499–502.                                   Physiol C 2002;133:411–8.
[15] Watanabe H, Taneda Y. Self and non-self recognition in             [32] Ballarin L, Cima F. Phenoloxidase and cytotoxicity in the
     compound ascidians. Am Zool 1982;22:775–82.                             compound ascidian Botryllus schlosseri. Dev Comp Immu-
[16] Muller WZ, Pancer Z, Rinkevich B. Molecular cloning and                 nol 1998;22:479–92.
     localization of a novel serine protease from the colonial          [33] Abe Y, Ishikawa G, Satoh H, Azumi K, Yokosawa H.
     tunicate Botryllus schlosseri. Mol Mar Biol Biotechnol                  Primary structure and function of superoxide dismutases
     1994;3:70–7.                                                            from the ascidian Halocynthia rortezi. Comp Biochem
[17] Fagan M, Weissman I. Sequence and characterization of                   Physiol B 1999;122:321–6.
     two HSP70 genes in the colonial protochordate Botryllus            [34] Rinkevich B. Invertebrates versus vertebrates innate im-
     schlosseri. Immunogenetics 1996;44:134–42.                              munity: in the light of evolution. Scand J Immunol
[18] Pancer Z, Cooper EL, Muller WE. A tunicate (Botryllus                   1999;50:456–60.
     schlosseri) cDNA reveals similarity to vertebrate antigen          [35] Iwanaga S, Lee BL. Recent advances in the innate immunity
     receptors. Immunogenetics 1996;45:69–72.                                of invertebrate animals. J Biochem Mol Biol 2005;38:128–50.
[19] Pancer Z, Leuck J, Rinkevich B, Steffen R, Muller I, Muller        [36] Holmskov U. Collections and ficolins: humoral lectins of the
     WE. Molecular cloning and sequence analysis of two                      innate immune defense. Annu Rev Immunol 2003;21:
     cDNAs coding for putative anionic trypsinogens from the                 547–78.
     colonial Urochordate Botryllus schlosseri (Ascidiacea). Mol        [37] Fujita T, Endo Y, Nonaka M. Primitive complement
     Mar Biol Biotechnol 1996;5:326–33.                                      system-recognition and activation. Mol Immunol 2004;41:
[20] Pancer Z, Leuck J, Rinkevich B, Steffen R, Muller I, Muller             103–11.
     WE. Cloning of sponge (Geodia cydonium) and tunicate               [38] Endo Y, Takahashi M, Fujita T. Lectin complement system
     (Botryllus schlosseri) proteasome subunit epsilon (PRCE):               and pattern recognition. Immunobiology 2006;211:283–93.
     implications about the vertebrate MHC-encoded homologue            [39] Kuraya M, Ming Z, Liu X, Matsushita M, Fujita T. Specific
     LMP7 (PRCC). Biochem Biophys Res Commun 1996;228:                       binding of L-ficolin and H-ficolin to apoptotic cells leads to
     406–10.                                                                 complement activation. Immunobiology 2005;209:689–97.
[21] Pancer Z, Diehl-Seifert B, Rinkevich B, Muller WE. A novel         [40] Pancer Z, Gershon H, Rinkevich B. Cloning of a urochor-
     tunicate (Botryllus schlosseri) putative C-type lectin                  date cDNA featuring mammalian short consensus repeats
     features an immunoglobulin domain. DNA Cell Biol                        (SCR) of complement-control protein superfamily. Comp
     1997;16:801–6.                                                          Biochem Physiol B 1995;111(4):625–32.
[22] Khalturin K, Becker M, Baruch R, Bosch TCG. Urochor-               [41] Nonaka M, Azumi K, Ji X, Namikawa-Yamada C, Sasaki
     dates and the origin of natural killer cells: identification of a        M, Saiga H, et al. Opsonic complement component C3 in the
     CD94/NKR-P1-related receptor in blood cells of Botryllus.               solitary ascidian, Holocynthia roretzi. J Immunol 1999;162:
     Proc Natl Acad Sci 2003;100:622–7.                                      387–91.
[23] Pancer Z, Gershon H, Rinkevich B. cDNA cloning of a                [42] Chwieralski CE, WElte T, Buhling F. Cathepsin-regulated
     putative protochordate FK506-binding protein. Biochem                   apoptosis. Apoptosis 2006;11:143–9.
     Biophys Res Commun 1993;197:973–7.                                 [43] Rinkevich B, Taratakover S, Gershon H. Contribution of
[24] Rinkevich B. Morphologically related allorecognition assays             morula cells to allogenic responses in the colonial urochor-
     in botryllid ascidians. In: Stolen JS, Fletcher TC, Smith SA,           date Botryllus schlosseri. Mar Biol 1998;131:227–36.
     Zelikoff JT, Kaattari SL, Anderson RS, Soderhall K, Weeks
                                                   ¨     ¨              [44] Jiang Y, Doolittle R. The evolution of vertebrate blood
     Perkins BA, editors. Techniques in fish immunology, 4:                   coagulation as viewed from a comparison of puffer fish and
     immunology and pathology of aquatic invertebrates. Fair                 sea squirt genomes. Proc Natl Acad Sci 2003;24(100):
     Haven, NJ: SOS Publishers; 1995. p. 17–21.                              7527–32.
[25] Huang X, Madan A. CAP3: a DNA sequence assembly                    [45] Cima F, Basso G, Ballarin L. Apoptosis and phosphatidyl-
     program. Genome Res 1999;9:868–77.                                      serine-mediated recognition during the take-over phase of
[26] Ellen A, Nollen A, Morimoto RI. Chaperoning signaling                   the colonial life-cycle in the ascidian Botryllus schlosseri. Cell
     pathways: molecular chaperons as stress-sensing ‘‘heat-                 Tissue Res 2003;312:369–76.
     shock’’ proteins. J Cell Sci 2002;115:2809–16.                     [46] Loukissa A, Cardozo C, Altschuller-Felberg C, Nelson JE.
[27] Quintana FJ, Cohen IR. Heat shock proteins as endogenous                Control of LMP7 expression in human endothelial cells by
     adjuvants in sterile and septic inflammation. J Immunol                  cytokines regulating cellular and humoral immunity. Cyto-
     2005;175:2777–82.                                                       kine 2000;12:1326–30.
[28] van Eden W, van der Zee R, Prakken B. Heat-shock proteins          [47] Kawahara H, Sawada H, Yokosawa H. The 26 S protea-
     induce T-cell regulation of chronic inflammation. Immunol-               some is activated at two points in the ascidian cell cycle.
     ogy 2005;5:318–30.                                                      FEBS Lett 1992;310:119–22.
[29] Yanari MA, Liu J, Zheng Z, Vexler ZS, Lee JE, Fiffard RG.          [48] Orlowski RZ. The role of the ubiquitin-proteasome pathway
     Antipoptotic and anti-inflammatory mechanisms of heat                    in apoptosis. Cell Death Differ 1999;6:303–13.
     shock protein protection. Ann N Y Acad Sci 2005;1053:              [49] Ostrowska H, Kruszewski K, Kasacka I. Immuno-protea-
     74–83.                                                                  some subunit LMP7 is up-regulated in the ischemic kidney in
                                                 ARTICLE IN PRESS
902                       M. Oren et al. / Developmental and Comparative Immunology 31 (2007) 889–902

     an experimental model of renovascular hypertension. Int           glabrata hemocytes. Dev Comp Immunol 2005;29:
     J Biochem Cell Biol 2006;38(10):1778–85.                          393–407.
[50] Mitta G, Galinier R, Tisseyre P, Allienne JF, Girerd-        [51] Davidson B, Swalla BJ. A molecular analysis of ascidian
     Chambaz Y, Guillou F, et al. Gene discovery and expression        metamorphosis reveals activation of an innate immune
     analysis of immune-relevant genes from Biomphalaria               response. Development 2002;129:4739–51.

To top