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Fish & Shellfish Immunology (2000) 10, 61–85Article No. fsim.1999·0230Available online at http://www.idealibrary.com on

Cloning and sequencing hybrid striped bass (Moronesaxatilis x M. chrysops) transforming growth factor-�(TGF-�), and development of a reverse transcriptionquantitative competitive polymerase chain reaction

(RT-qcPCR) assay to measure TGF-� mRNA of teleost fish

C. A. HARMS1*, S. KENNEDY-STOSKOPF

1, W. A. HORNE2, F. J. FULLER

1AND

W. A. F. TOMPKINS1

1Department of Microbiology, Pathology and Parasitology, and 2Departmentof Anatomy, Physiological Sciences and Radiology, College of Veterinary

Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh,NC 27606, U.S.A.

(Received 21 April 1999, accepted 7 June 1999)

A transforming growth factor (TGF)-� was isolated and cloned from hybridstriped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclearcells. This isolate (Genbank accession number AF140363) contains an openreading frame of 1146 bases coding for a 382 amino acid protein most similar torainbow trout TGF-� (57·3 and 78·6% identity with precursor and activeprotein, respectively) and rat TGF-�1 (41·1 and 68·8% identity with precursorand active protein, respectively). Consensus primers were demonstrated toamplify specifically by polymerase chain reaction (PCR), a TGF-� segmentfrom 14 species of teleost fish comprising 10 taxonomic families in 7 orders. Areverse transcription quantitative competitive polymerase chain reaction(RT-qcPCR) assay was devised to measure TGF-� mRNA expression in teleostfish. Higher levels of TGF-� mRNA expression were detected in mononuclearcells of peripheral blood than from spleen or anterior kidney.

� 2000 Academic Press

Key words: transforming growth factor-�, TGF-�, sequence, hybrid stripedbass, Morone saxatilis X M. chrysops, quantitative PCR.

I. Introduction

Fish immunology is receiving increasing attention as fish assume greaterimportance as models in environmental toxicology, and as alternative modelsin biomedical research (Stolen & Fletcher, 1994). Yet the molecular mecha-nisms of immune cell interactions that are becoming well characterised inmammalian systems are poorly known for fish. Study of fish immunology ishampered by a paucity of specific reagents, including those for detectingcytokines. The quantity and balance of cytokines produced in the course of animmune response can determine the type (e.g. TH1 v. TH2) and e$cacy of that

*Corresponding author.

611050–4648/00/010061+25 $35.00/0 � 2000 Academic Press

62 C. A. HARMS ET AL.

response (Janeway & Travers, 1997). The ability to detect and measure fishcytokines would provide valuable insight into the immunologic status of fishin response to infectious agents and toxins.

Based on biological activity, biological cross-reactivity and antigenic cross-reactivity, there is evidence in fish for interleukin (IL)-1, IL-2, type 1 and 2interferons (IFN), macrophage activating factor and macrophage migrationinhibition factor, tumor necrosis factor (TNF)-� and transforming growthfactor (TGF)-� (Secombes, 1994; Secombes et al., 1996). However, fish cytokinesequence data are limited. Sequences from the Japanese flatfish have beenpublished for purported homologues of IL-2 and an IFN (Tamai et al., 1993,1994) though the homology to known sequences is marginal when only regionsderived independently of the original primers are considered (Secombes et al.,1996).

A partial sequence for TGF-�2 from carp (Cyprinus carpio, Genbank acces-sion number U66874, Sumathy et al., 1997), and a complete sequence for arainbow trout (Oncorhynchus mykiss) TGF-� (Genbank AJ007836) groupingwith Xenopus laevis TGF-�5, chicken TGF-�4 and mammalian TGF-�1 (Hardieet al., 1998), were recently published. Evidence for the conserved nature ofTGF-� activity in fish is provided by both biological and antigenic cross-reactivity. Bovine TGF-�1 enhanced respiratory burst activity of restingrainbow trout macrophages at low doses. However, at higher doses bovineTGF-�1 inhibited activated rainbow trout macrophages and countered thee#ects of activating signals on resting macrophages (Jang et al., 1994).Chicken antiporcine TGF-�1 enhanced the ability of rainbow trout leucocyte-derived supernatants to stimulate respiratory burst activity of anterior kidneymacrophages (Jang et al., 1995).

TGF-� immunoregulatory properties are primarily suppressive. Immunefunctions downregulated by TGF-� include the following: MHC and FcRexpression, some cytokine production, thymocyte proliferation, T and B cellproliferation, IgG and IgM production, IL-2 receptor expression, cytotoxic Tcell generation and function, LAK and NK cell activation and function,macrophage activation, macrophage respiratory burst activity, neutrophiladhesion to endothelium and haematopoiesis (Ruscetti & Palladino,1991). TGF-� also has some pro-inflammatory e#ects: promotion ofmacrophage and neutrophil chemotaxis, IgA production, granulopoiesis andproduction of some cytokines. Whether TGF-� is pro- or anti-inflammatorydepends on its concentration, the state of di#erentiation of target cells and theconcentration of other pro-inflammatory compounds (McCartney-Francis &Wahl, 1994).

The ability to measure di#erences in TGF-� production could provide avaluable tool for evaluating the impact of environmental contaminants,aquaculture management practices, vaccines and infectious agents onfish immunity. This paper reports first, nucleotide and deduced aminoacid sequences for TGF-� from the hybrid striped bass (Morone saxatilis�M. chrysops) and second, the development of a reverse transcriptionquantitative–competitive polymerase chain reaction (RT-qcPCR) techniquefor measuring TGF-� transcription by lymphoid cells of multipleteleost species.

TGF-� IN HYBRID STRIPED BASS 63

II. Materials and Methods

FISH

Hybrid striped bass purchased from a commercial aquaculture facility wereheld in a 1000 l freshwater tank at the NCSU College of Veterinary Medicineunder Institutional Animal Care and Use Committee approval. Waterconditions were as follows: 18� C, pH 6·8–7·2, ammonia<0·1 mg l�1, nitrite<0·1 mg l�1. Other fish species [Southern stingray Dasyatis americana,rainbow trout Oncorhynchus mykiss, brook trout Salvelinus fontinalis, chan-nel catfish Icalurus punctatus, fathead minnow Pimephales promelas, giantzebrafish Danio malabaricus, red pacu Piaractus brachypomus (=Colossomabrachypomum), black crappie Pomoxis nigromaculatus, bluegill Lepomismacrochirus, black sea bass Centropristis striata, white perch Moroneamericana, tilapia Oreochromis mossambicus, southern flounder Paralichthyslethostigma, planehead filefish Monacanthus hispidus] were obtained singlyfrom other researchers and clinical cases opportunistically.

COLLECTION OF LYMPHOID ORGAN AND PERIPHERAL BLOOD MONONUCLEAR CELLS

Fish were anaesthetised with 100 mg l�1 tricaine methanesulfonate (MS-222), bled from the caudal vein into heparinised syringes, then killed with anoverdose of 250 mg l�1 tricaine (Harms, 1998). As much blood as possible wascollected in order to deplete lymphoid organs of peripheral blood. Spleen andanterior kidney were removed aseptically and placed in sterile cell culturemedium (RPMI 1640 medium plus 10% heat-inactivated foetal bovine serum,100 U ml�1 penicillin, 100 �g ml�1 streptomycin, 2 mM EDTA; subsequentlyreferred to as complete RPMI). Blood and organs were kept on wet ice untilprocessed, in all cases in less than 24 h. Organs were minced finely, forcedthrough a fine wire mesh and resuspended in complete RPMI. Blood wascentrifuged at 400�g for 5 min at 4� C and the leucocyte layer was harvestedand resuspended in complete RPMI. Organ homogenates and leucocytes werecentrifuged on two-step Percoll gradients (40% and 51% stock solution in0·15 M saline; specific gravity 1·053 and 1·066 g ml�1, respectively) at 400�gfor 5 min then 800�g for 20 min at 4� C, and mononuclear cells harvested fromthe 40/51% interface. Cells were washed twice in complete RPMI. Viable cellcounts were performed with cells suspended in 0·2% trypan blue, and di#er-ential counts were performed on cytospin preparations stained with Leuko-StatTM (Fisher Scientific, Pittsburgh, PA, U.S.A.). Cell viability typicallyexceeded 95%.

ISOLATION OF RNA

Total RNA was isolated by the guanidine thiocyanate method (Tri Reagent,Molecular Research Center, Cincinnati, OH, U.S.A.). One ml of Tri Reagentfor each 5–10�106 cells was added to the cell pellet. The cells were lysed bypipetting multiple times, and RNA isolated according to kit instructions. The


RNA pellet was washed with 75% ethanol and resuspended in sterile diethylpyrocarbonate (DEPC)-treated water at a concentration of 5�104 cellequivalents �l�1.

REVERSE TRANSCRIPTION OF MRNA

mRNA was reverse transcribed to cDNA with oligo dT15 priming. TotalRNA (3�106 cell equivalents) and 300 pmol oligo dT15 were mixed in a 72 �lvolume and heated to 72� C for 5 min and chilled on ice. Samples were adjustedwith additional reagents in a final volume of 150 �l to the following con-ditions: 1�RT bu#er (50 mM Tris–HCI, pH 8·3; 75 mM KCI, 3 mM MgCl2),0·4 mM each dNTP, 10 mM DTT, 0·8 U �l�1 RNAsin and 2 U �l�1 reversetranscriptase (Superscript II RT, Gibco-BRL, Gaithersburg, MD, U.S.A.).Reactions were incubated at 45� C for 60 min, heat inactivated at 94� C for10 min, and chilled on ice. Samples of cDNA were diluted 1:2 in DEPC-treatedwater to 300 �l and stored at �20� C until used. Negative RT controls wererun in parallel.

POLYMERASE CHAIN REACTION

PCR conditions were as follows: 1�PCR bu#er [10 mM Tris–HCI (pH 8·3),1·5 mM MgCI2, 50 mM KCI] 250 �M each dNTP, 0·375 �M each primer,30 �U �l�1 Taq DNA polymerase (Boehringer Mannheim, Indianapolis, IN,U.S.A.), and 5 �l of the cDNA template mixed in a total volume of 40 �l.Oligonucleotide primers are listed in Table 1. The reaction mix was overlaidwith mineral oil and thermal cycled (Hybaid Omnigene thermal cycler,Vangard International, Neptune, NJ, U.S.A.) at 94� C 1 min hot start, (94� C30 s denature, 50 or 55� C 1·5 min anneal, 72� C 2 min extend) �25–39 cycles,72� C 7 min final extension.

ISOLATION AND SEQUENCING OF HYBRID STRIPED BASS TGF-�

A battery of consensus mammalian cytokine primers (Rottman et al., 1996)were employed on cDNA from hybrid striped bass. PCR products near theexpected size were cloned into the pGEM-T Easy vector (Promega) andsequenced for verification.

PCR products were column purified by Wizard PCR Preps DNA PurificationSystem (Promega, Madison, WI, U.S.A.) and ligated into pGEM-T Easy vectorby T-A cloning according to kit directions. Plasmids were isolated with theQIAprep spin miniprep kit (QIAGEN, Chatsworth, CA, U.S.A.). Plasmids weredigested with EcoR I to verify appropriate insert size. Alternatively, positiveclones were identified by colony hybridisation using an internal oligonucle-otide probe (Table 1) (Sambrook et al., 1989). Inserts were cycle sequencedfrom the 5� and 3� ends, starting with universal forward and reverse primersanchored in the vector, followed by primer walking to the interior of theinsert, using the ABI PRISM Dye Terminator Cycle Sequencing ReadyReaction Kit with AmpliTaq DNA Polymerase, FS (Perkin-Elmer, Foster City,CA, U.S.A.). Reaction conditions were set according to manufacturer’s

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instructions, with 500 ng plasmid DNA, and cycled on a Perkin ElmerGeneAmp PCR System 9600 at (96� C 10 s, 50� C 5 s, 60� C 4 min)�25 cycles.Extension products were spin purified with Centri-Sep columns (PrincetonSeparations, Adelphia, NJ, U.S.A.) or precipitated with 70% ethanol with0·5 mM MgCl2, resuspended in loading bu#er (for ABI 377) or TemplateSuppression Reagent (for ABI 310), denatured and loaded into an ABI 310 or377 automated fluorescent sequencer. Sequences obtained were screened andanalysed using ABI Prism Sequencing Analysis 2·1·2 (Perkin Elmer) andLasergene (DNASTAR, Inc., Madison, WI, U.S.A.) software.

A PCR product with strong homology to the precursor segment of knownTGFs-� was selected for rapid amplification of cDNA ends (RACE) to derivethe entire coding sequence. The RACE reactions were performed with the 5�/3�RACE Kit (Boehringer Mannheim), according to directions, using specificprimers derived from the partial TGF-� sequence, and oligo-dT anchor andPCR anchor primers supplied with the kit (Table 1). Briefly, for 3� RACE,cDNA was synthesised using the oligo-dT anchor primer and AMV RT. PCRwas performed using a gene-specific primer (TGF 3� RACE #2) and the PCRanchor primer. A hemi-nested PCR with a second specific primer (TGF 3�RACE #1) was also necessary for TGF-� 3� cDNA end recovery. For 5� RACE,cDNA synthesis was primed with a gene-specific primer (TGF con B2), andcDNA was purified with High Pure PCR Purification Kit (BoehringerMannheim). Purified cDNA was poly-A tailed with dATP and terminaldeoxytransferase, and then treated as for the 3� RACE, using gene specificprimers TGF B and TGF 5� RACE #3. RACE products were sequenced asabove. TGF 5� RACE #4 was used as a probe for colony screening of the 5�RACE clones.

After the entire coding sequence was determined, a full-length clone of thecoding sequence was constructed from hybrid striped bass cDNA using PCRproducts of primers from the 5� and 3� untranslated regions (TGF 5�UTR #3and TGF 3�UTR #1) inserted in the pGEM T-Easy vector. This full lengthclone was resequenced from both ends to verify the derived sequence(Genbank accession number AF140363). For comparison with other TGFs-�and TGF-� superfamily members, sequence alignments and phylogenetic treeswere constructed using the Clustal method in MEGALIGN (DNASTAR)with Percent Accepted Mutations (PAM) 250 residue weighting. Genbankaccession numbers for sequences used in comparisons were as follows: activinbeta B (goldfish, AF 004669; zebrafish, X76051), TGF-�1 (bovine, M36271; ovine,X76916; equine, X99438; porcine, X12373; human, P01137; vervet, M16658;mouse, M13177; rat, X52498), TGF-�2 (carp, U66874; Xenopus, P17247; chicken,P30371; human, P08112; mouse, P27090), TGF-�3 (chicken, P16047; porcine,X14150; human, P10600; mouse, P17125; rat, Q07258), TGF-�4 (chicken, M31160X08012 S41706), TGF-� (Xenopus, J05180), and rainbow trout TGF-� (X52498).
5
NORTHERN BLOT

Total RNA was isolated from hybrid striped bass anterior kidney mono-nuclear cells, as above. Poly-A+RNA (mRNA) was isolated using oligo(dT)25-coated magnetic beads [Dynabeads� Oligo(dT)25� Dynal Inc., Lake Success,


NY, U.S.A.] according to the manufacturer’s directions. Total RNA (75 �g) in100 �l of DEPC-treated water was heated to 65� C for 2 min. Magnetic beads(200 �l suspension) were washed and resuspended in 100 �l of 2� bindingbu#er (20 mM Tris pH 7·5, 1 M LiCl, 2 mM EDTA). Total RNA was mixed withthe magnetic beads at room temperature for 5 min, and the beads weremagnetically separated from the supernatant. Supernatant (total RNA minusmRNA) was saved. Beads were washed twice with 200 �l wash bu#er (10 mM

Tris pH 8·0, 0·15 M LiCl, 1 mM EDTA) and resuspended in 10 �l elution bu#er(10 mM Tris pH 7·5) at 65� C for 2 min. Beads were separated magnetically fromthe mRNA-containing supernatant.

The resulting mixtures [5·4 �l each of total RNA minus mRNA, and mRNA,in a final concentration of 8 mM sodium phosphate pH 7·4, 1·7 M formaldehyde,18 M formamide, 1� RNA loading bu#er (final concentration 0·1% SDS, 5%glycerol, 5 mM EDTA, 0·05% bromophenol blue)] were electrophoresed in asodium phosphate-bu#ered, formaldehyde, 0·8% agarose gel (10 mM sodiumphosphate pH 7·4, 1·1 M formaldehyde), and stained with ethidium bromide forvisualisation on a 300 nm UV transilluminator. The gel contents were trans-ferred overnight by capillary action to a nylon membrane with 20� SSC.The membrane was rinsed with 2� SSC, air-dried and UV cross-linked at144 mJ cm2�1.

A random prime labelled probe was prepared from a 732 bp hybrid stripedbass PCR product generated by the TGF A and TGF con B2 primers (Table 1)using the RTS RadPrime DNA Labeling System (Gibco BRL, Gaithersburg,MD, U.S.A.) and �32P-dCTP according to kit instructions (25 ng template in45 �l of 10 mM Tris pH 7·5 heated to 95� C for 5 min and iced; template added tothe RTS RadPrime reaction mix, along with 5 �l of 10 �Ci �l�1 �32P-dCTP, andincubated 10 min at 37� C; reaction stopped with 5 �l of 0·2 M EDTA). Follow-ing prehybridisation of the nylon membrane (0·25 M sodium phosphate pH 7·4,7% SDS) at 65� C for 20 min, the membrane was hybridised overnight at 65� Cwith the random prime labelled probe (same conditions) and washed twice in0·1� SSC/0·1%SDS at room temperature for 30 min each, then once in 20 mM

sodium phosphate pH 7·4/1% SDS at 55� C for 30 min. After air-drying, themembrane was exposed to radiographic film at �80� C for 3 days.

SOUTHERN BLOT

Internal oligonucleotide probes (Table 1) were end-labelled with �32P-ATPand T4 polynucleotide kinase (Promega) and spin purified with Chromospin-10columns (Clontech, Palo Alto, CA, U.S.A.). PCR products were separated in2% agarose gels in TAE. Gels were soaked in 0·5 M NaOH/1·5 M NaCl for30 min at room temperature with gentle agitation, then in 0·5 M Tris/1·5 M

NaCl twice for 15 min, and reaction products were transfered to nylonmembranes with 20� SSC. Nylon membranes were rinsed in 2� SSC and UVcross-linked at 144 mJ cm2�1. Membranes were prehybridised (2� SSC, 5�Denhardt’s solution, 2·5 mM sodium phosphate pH 7·5, and 0·1 mg ml�1

herring sperm DNA) at 42� C 6 h. Membranes were transferred to freshprehybridisation solution plus labelled probe (105–106 cpm ml�1 in 5–10 ml) forhybridisation at 42� C overnight. Transfers were rinsed twice in 1� SSC/0·5%


SDS for 5 min at room temperature, then three times at 37� C, placed in plasticwrap and exposed to radiographic film 1–21 h at �80� C.

CONSENSUS PRIMERS AND PROBES FOR TELEOST TGF-� AND BETA-ACTIN

Consensus primers and probes for multiple species of teleost fish weredesigned for TGF-� and beta-actin (Table 1). Primers and probes for TGF-�were based on alignments of rat TGF-�1 (Genbank X52498), rainbow troutTGF-� (Genbank X99303), and hybrid striped bass TGF-�, using Primer Select3·01 software (DNASTAR). The primers were designed to span one knownintron of mammalian TGF-�1, in order to permit detection of genomic contami-nation, if present. Subsequently, it transpired that the primers also span aknown intron of rainbow trout TGF-� (Genbank AJ007836, Daniels &Secombes, 1999). The 3� end of the downstream primer (TGF con B2) wasdesigned with intentional mismatches with rat TGF-�1, carp TGF-�2, porcineTGF-�3, chicken TGF-�4, and Xenopus TGF-�5 sequences to reduce the chanceof amplifying isoforms other than the specific target. Consensus TGF-�primers (TGF con A1 and TGF con B2) amplified a 225 bp fragment. Primerspecificity was verified by product size and by Southern blot with a consensusoligonucleotide internal probe (TGF con probe).

Primers for beta-actin were developed to allow use of beta-actin as ahousekeeping gene in the quantitative PCR assay (see below). Beta-actinprimers were selected based on alignments of beta-actin sequences fromhuman (Genbank X00341, J00074, M10278) and five teleost fish species (stripedbass, carp, grass carp, fugu and medaka; Genbank L36342, M24113, M25013,U38849, S74868, U39357 and M10278, respectively). Beta-actin primers (beta-actin con A1 and beta-actin con B1) were designed to span two known intronsbased on the fugu genomic sequence, and amplified a 439 bp fragment. Primerspecificity was verified by PCR product size and Southern blot using aconsensus oligonucleotide internal probe (beta-actin con probe).

PRODUCTION OF HOMOLOGOUS COMPETITIVE FRAGMENTS FOR RT-QCPCR

Homologous competitive fragments for TGF-� and beta-actin reverse tran-scription quantitative–competitive polymerase chain reaction (RT-qcPCR)were constructed by creating deletion mutations by PCR (Köhler et al., 1995).Competitive fragment construction primers were designed with a 5� tagcomprised of the downstream consensus primer and a 3� end complementary toa region located approximately 85% of the normal PCR product length fromthe upstream primer. PCR was then performed with the consensus upstreamprimer (TGF con A1 or beta-actin con A1) and the competitive fragmentconstruction primer (TGF CF construction or beta-actin CF construction)(Table 1). PCR products were column purified and cloned into a pGEM-T Easyvector and transfected into JM-109 cells as described above. Minipreps(QIAprep Spin Miniprep Kit, QIAGEN Inc., Valencia, CA, U.S.A.) of theplasmids containing competitive fragment inserts were used as templates forPCR with the consensus primers, and a clone which produced the expectedproduct size (192 bp for TGF-� and 365 bp for beta-actin) was selected for each


target. Plasmids were quantified by OD260 readings, and stock solutions ofeach competitive fragment were prepared and stored at �20� C. Dilutionseries were made in DEPC-treated water, from 50 amol (3·01�107 copies) �l�1

down to 1·02�10�6 amol (0·6 copies) �l�1 in 12 five-fold steps.

REVERSE TRANSCRIPTION QUANTITATIVE–COMPETITIVE POLYMERASE CHAIN REACTION

RT-qcPCR was performed based on a previously described procedure(Rottman et al., 1996). Reverse transcription and dilution of cDNA wasperformed as above, and 2·5 �l of this template was included in each competi-tive PCR. Final reaction volume of the competitive PCR was 25 �l, reached byadding 20 �l of a PCR master mix to 5 �l of each competitive fragment dilution(1·5�108 copies down to 6 copies) in a Thermowell� 96 well thin-wallpolycarbonate plate (Costar, Acton, MA, U.S.A.) on ice. Final reactionconcentrations were as described above {1� PCR bu#er [10 mM Tris–HCl (pH8·3), 1·5 mM MgCl2, 50 mM KCl], 250 �M each dNTP, 0·375 �M each primer,30 �U �l�1 Taq DNA polymerase}. Reactions cycled on a PTC-100� thermo-cycler (MJ Research, Inc., Watertown, MA, U.S.A.) at 94� C 1 min, (94� C 30 sdenature, 55� C 1·5 min anneal, 72� C 2 min extend) �35 cycles, 72� C 7 minfinal extension.

ANALYSIS OF RT-QCPCR PRODUCTS

PCR products were separated in 2% agarose gels in TAE, stained withethidium bromide and photographed on a 300 nm UV transilluminator. Theimages were digitised and analysed with the Alphaimager 2000 Documen-tation and Analysis System (Alpha Innotech, Co., San Leandro, CA, U.S.A.).Fluorescence of target and competitor bands in each lane were measured andexpressed as area under the curve. Fluorescence di#erences due to molecularweight di#erences were compensated by the formula:

corrected fluorescence ratio=[target fluorescence (area)/competitor fluorescence (area)]�[competitor size (bp)/target size (bp)]

The log of the corrected fluorescence ratio was plotted against the log of thenumber of copies of competitor in the sample, and the point of molecularequivalence (the point at which the copy number of target cDNA equals thecopy number of competitor DNA) is the X intercept. Finally, to control forsample-to-sample variation in RNA isolation, reverse transcription, amplifi-cation and gel loading during quantification, TGF-� results were normalisedto those of the house-keeping gene, beta-actin.

RT-QCPCR ASSAY VALIDATION

Coe$cients of variation (CV) were calculated for TGF-�: beta-actin ratiosdetermined from RT-qcPCR assays of a hybrid striped bass anterior kidneymononuclear cell cDNA sample processed in triplicate on the same day (inparallel) and di#erent days (in series). The CV was also calculated starting


with a mononuclear cell sample in triplicate and processed in parallel.Dilutional parallelism for TGF-� and beta-actin RT-qcPCR, and maintenanceof constant TGF-�: beta-actin ratios independent of starting cDNA concen-tration, was demonstrated on three-fold dilutions (to 1:81) of hybrid stripedbass anterior kidney mononuclear cell cDNA.

MONONUCLEAR CELL SOURCE DIFFERENCES IN TGF-�: BETA-ACTIN RATIOS

Mononuclear cells from spleen, peripheral blood and anterior kidney wereharvested from 17 hybrid striped bass and used to compare constitutive TGF-�:beta-actin ratios derived by RT-qcPCR.

STATISTICAL ANALYSIS

Statistical analyses were performed with a commercial statistical package(JMP 3, SAS Institute Inc., Cary, NC, U.S.A.). TGF-�: beta-actin ratios fromspleen, anterior kidney and peripheral blood mononuclear cells were com-pared by Welch Anova for detecting di#erences in group means allowing forunequal variances, and Tukey-Kramer HSD to determine which pairs di#eredsignificantly. Di#erential cell counts in the mononuclear cell preparationswere compared according to organ source also by Welch Anova and Tukey-Kramer HSD. Analysis of covariance was used to examine the e#ects of organsource and di#erential count cell percentage on measured TGF-� mRNAexpression. One-way Anova was used to determine correlation of TGF-�mRNA expression between organs of the same fish (i.e. do high levels ofexpression from splenic mononuclear cells correlate with high levels fromanterior kidney or peripheral blood in a given fish?).

III. Results

HYBRID STRIPED BASS TGF-�

No PCR reactions using mammalian consensus cytokine primers yielded theexpected products, but the consensus IL4 primers yielded a product withstrong homology to the precursor segment of several TGFs-� on a BLASThomology search (Altschul et al., 1990; Gish et al 1993). The entire codingsequence plus portions of the 5� and 3� UTRs were derived by RACE (Fig. 1).The compiled sequence was used to design primers for amplifying a new 1268nucleotide clone containing the coding sequence, which was resequenced fromthe 5� and 3� ends to verify the compiled sequence. Primer and probe locationsare shown in Fig. 2. An open reading frame of 1146 bases was identified whichtranslates to a 382 amino acid protein (Figs 1 and 3).

The coding sequence has a high degree of homology with known TGF-�sequences and the deduced protein contains characteristic motifs of the TGF-�superfamily (Table 2, Figs 1 and 3). A hydrophobic 18 amino acid leadersequence is followed by a 252 amino acid region corresponding to the LatencyAssociated Peptide (LAP), and a 112 amino acid mature protein monomer.Within the LAP there are five potential N-linked glycosylation sites (N-X-T/S),three of which are shared with rainbow trout TGF-� and two with mammalian


Fig. 1. Nucleotide sequence and translated amino acid sequence of hybrid striped bassTGF-� (Genbank accession number AF140363). Amino acid sequence and all dec-orations lie below the corresponding nucleotide sequence. Predicted hydrophobicleader, latency-associated peptide (LAP) and active TGF-� segments indicated bysingle-headed arrows between nucleotide and amino acid sequences. PotentialN-linked glycosylation sites (N-X-T/S) in the LAP denoted by double-T-bars belowamino acid sequence. RGD integrin binding site and RKKR tetrabasic cleavage sitemarked by double-headed arrows below amino acid sequence. Potential poly-Atailing site (AATAAA) shown by box below nucleotide sequence.


Fig. 2. Oligonucleotide primer and probe locations on the hybrid striped bass TGF-�sense strand nucleotide sequence. Primer and probe 3� ends shown by<or >. Actualprimer and probe sequences are in Table 1. Start codon indicated by single-headedarrow; stop codon indicated by double T-bar.

TGFs-�1. Additional conserved features present in the hybrid striped bassTGF-� LAP segment are an RGD integrin binding site, a C-X-C site and anRKKR tetrabasic cleavage site, beyond which lies the mature protein. Themature protein segment contains nine conserved cysteines, although both thehybrid striped bass TGF-� and the rainbow trout TGF-� di#er from other


Fig. 3. Predicted amino acid sequence of hybrid striped bass (sbx) TGF-� aligned withrainbow trout (rbt) TGF-� and rat TGF-�1. Identical amino acids are boxed. Rulerabove alignment corresponds to hybrid striped bass sequence; shaded areas indicategaps inserted into the hybrid striped bass sequence.


Table 2. Percent nucleotide and amino acid identities of hybrid striped bass TGF-�sequences with other TGFs-�

Mature sequence Full-length sequenceNucleotide Amino acid Nucleotide Amino acid

rbtTGF-� 75·2 78·6 59·2 57·3ratTGF-�1 66·4 68·8 44·4 41·1humTGF-�1 67·8 68·8 45·3 40·6humTGF-�2 55·2 58·0 38·0 35·3humTGF-�3 59·9 62·5 41·3 36·1chknTGF-�4 59·6 64·3 44·4 39·1xenTGF-�5 53·1 60·7 41·2 40·3

139.9100120 80

TGFs-β

060

bov tgf beta 1

40 20

1

2

3

ovis tgf beta 1porcine tgf beta 1

hum tgf beta 1vervet tgf betaequine tgf beta 1mus tgf beta 1rat tgf beta 1

chickent tgf beta 4rbt tgf beta

SBX TGF betaxen tgf beta 5

hum tgf beta 2mus tgf beta 2chicken tgf beta 2

xen tgf beta 2carp tgf beta 2

mus tgf beta 3rat tgf beta 3hum tgf beta 3porcine tgf beta 3

chicken tgf beta 3goldfish activin beta Bdanio activin beta B

Fig. 4. Rooted cladogram of selected TGF-� superfamily members using Clustal methodin MEGALIGN (DNASTAR) with Percent Accepted Mutations (PAM) 250 residueweighting. Scale denotes substitution distance. The two fish activins are TGF-�superfamily members used as outgroups for the TGFs-�. TGF-� isoform groupings1–3 are indicated. (bov=bovine, hum= human, mus=mouse, rbt=rainbow trout,SBX=hybrid striped bass, xen=Xenopus, danio=zebrafish).

known TGF-� isotypes in having the first cysteine of the mature protein atposition 8 rather than position 7. The degree of homology with other TGFs-� isgreatest in the mature protein segment (Table 2).

No regulatory regions were identified in the 88 nucleotide segment of the 5�UTR that was sequenced. In the 277 nucleotides sequenced in the 3� UTR, onepotential polyadenylation signal (AATAAA) was detected (Fig. 1).

A rooted cladogram was constructed from Genbank protein sequences ofTGFs-� and TGF-� superfamily members (whole length) using the Clustalmethod and the PAM 250 residue weighting table (Fig. 4). The hybrid stripedbass sequence clearly groups within the TGFs-�, and is most closely allied withthe rainbow trout sequence. The fish TGFs-� are somewhat more loosely alliedwith Xenopus TGF-�5, chicken TGF-�4 and mammalian TGFs-�1. Alternateresidue weighting (PAM 100, Identity) results in making the fish sequencesoutgroups within the TGF-� cluster (not shown). However, considered in the


context of an unrooted cladogram the branching patterns do not di#er and thesubstitution distance from hybrid striped bass TGF-� is least to rat TGF-�1

(excepting rainbow trout TGF-�).Northern blot of hybrid striped bass mRNA was performed to determine the

TGF-� transcript size. A major band was detected at 4·2 Kb, and a lesserproduct at 2·9 Kb (Fig. 5). No transcripts were observed in the total RNAminus mRNA control lane.

� �

��

��

Fig. 5. Northern blot of hybrid striped bass anterior kidney mononuclear cell mRNA(lane 1) and total RNA minus mRNA (lane 2), probed with random prime labelledhybrid striped bass PCR product generated by TGF A and TGF con B2 primers.

CONSENSUS TELEOST TGF-� AND BETA-ACTIN PRIMERS

In order to validate consensus primer use in multiple species, RT-PCR andSouthern blot with an internal consensus oligonucleotide probe were per-formed. Specificity of consensus primers for TGF-� (TGF con A1, TGF con B2,Table 1) was verified by appropriate RT-PCR product size and Southern blotwith a TGF-� consensus probe (TGF con probe, Table 1) in 14 species of teleost


fish (Fig. 6, Table 3). Consensus TGF-� primers failed to amplify cDNA from adomestic cat and an elasmobranch fish (southern stingray). Consensus primerspecificity for beta-actin (beta-actin con A1, beta-actin con B2, Table 1) wasverified in the same 14 teleost fish species by appropriate RT-PCR product sizeand Southern blot with a beta-actin consensus probe (beta-actin con probe,Table 1; data not shown).

��

�

��

��

� � � � �

� � � � � � �

��

Fig. 6. Ethidium bromide stained gels (first and third rows) and Southern blots (secondand fourth rows) of RT-PCR products from multiple species using TGF con A1 andTGF con B2 primers. Row 2 exposed for 1 h, row 4 exposed for 21 h. Top two rows,lane 1) brook trout, 2) rainbow trout, 3) black crappie, 4) bluegill, 5) black sea bass,6) hybrid striped bass, 7) white perch, 8) tilapia, 9) southern flounder, 10) planeheadfilefish. Bottom two rows, lane 1) channel catfish, 2) red pacu, 3) fathead minnow, 4)giant zebrafish, 5) domestic cat, 6) negative RT control.

QUANTITATIVE PCR FOR TGF-�

A representative RT-qcPCR gel is shown in Fig. 7. Coe$cients of variationfor TGF-�: beta-actin ratios from hybrid striped bass anterior kidney mono-nuclear cell triplicate samples processed in parallel, and cDNA triplicatesamples processed in parallel and in series are 68, 21 and 40%, respectively.Assay independence from starting cDNA concentration is demonstrated bythe close correspondence to the expected copy numbers for both TGF-� andbeta-actin, and maintenance of a nearly constant TGF-�: beta-actin ratio,throughout a three-fold dilution series extending to 1:81 (Fig. 8).


TGF-� expression varied by organ source of mononuclear cells (Fig. 9;Welch Anova, P<0·0001), with expression being greater in mononuclear cellsfrom peripheral blood (TGF-�: beta-actin ratio mean�standard deviation:0·034�0·016) than from spleen (0·014�0·0070), and greater from spleen thanfrom anterior kidney (0·0043�0·0019) (Tukey-Kramer HSD, P<0·05). Cellularcomponents of the mononuclear cell preparations, though derived by the samePercoll density gradient protocol, also di#ered by source based on di#erentialcounts of cytospin slides (Table 4). In particular, lymphocytes were lessabundant and blast cells were more abundant in the anterior kidney prepara-tions than for spleen or peripheral blood. Analysis of covariance with organsource and cell percentages as main e#ects revealed that only lymphocytepercentage had a significant e#ect on TGF-� expression, and that organ sourcewas the primary significant e#ect when considered along with all cell typepercentages, including lymphocytes (e#ect test: organ source P<0·0001, lym-phocyte percentage P=0·0038; for all other cell types, organ source P< 0·0001,cell type percentages P>>0·05). TGF-� expression did not correlate betweenorgans from the same fish (one-way ANOVA, P>0·05).

Table 3. Teleost fish species for which TGF-� and beta-actin primers have been verifiedby appropriate RT-PCR product size and Southern blot with an internal consensus

oligonucleotide probe

Species Order

Fathead minnow (Pimephales promelas) CypriniformesGiant zebrafish (Danio malabaricus) CypriniformesRed pacu (Piaractus brachypomus) CharaciformesChannel catfish (Ictalurus punctatus) SiluriformesRainbow trout (Oncorhynchus mykiss) SalmoniformesBrook trout (Salvelinus fontinalis) SalmoniformesBlack Crappie (Pomoxis nigromaculatus) PerciformesBluegill (Lepomis macrochirus) PerciformesBlack Sea Bass (Centropristis striata) PerciformesHybrid striped bass (Morone saxatilis�M. chrysops) PerciformesWhite perch (Morone americana) PerciformesTilapia (Oreochromis mossambicus) PerciformesSouthern flounder (Paralichthys lethostigma) PleuronectiformesPlanehead filefish (Monacanthus hispidus) Tetraodontiformes

IV. Discussion

The nucleotide sequence for the entire coding section and a portion of the 5�and 3� UTRs of hybrid striped bass TGF-� is reported and used to designconsensus primers and probes for measuring TGF-� mRNA from multiplespecies of teleost fish by RT-qcPCR. The initial fortuitous priming of a hybridstriped bass TGF-� fragment from consensus mammalian IL-4 primers resultedfrom very close matches in the 3� half of both primers. The deduced precursorsequence of 382 amino acids corresponds in length to that for rainbow troutTGF-� and Xenopus TGF-�5, compared with 390 for TGF-�1, 412 for TGFs-�2&3


� � � � � �

��

��

��

Fig. 7. Representative reverse transcription quantitative–competitive polymerasechain reaction (RT-qcPCR) gel for TGF-� and beta-actin from hybrid striped bassperipheral blood mononuclear cell cDNA. Lane numbers indicate the competitivefragment dilutions. In this example, the top row for TGF-� utilises the second(3�107 copies) through the ninth (3·8�102 copies) of five-fold TGF-� competitivefragment dilutions; bottom row for beta-actin utilises the first (1·5�108 copies)through the eighth (1·9�103 copies) of five-fold beta-actin competitive fragmentdilutions. In both cases the competitive fragment is the lower molecular weightband (TGF-� competitive fragment 192 bp, TGF-� target 225 bp, beta-actin competi-tive fragment 365 bp, beta-actin target 439 bp). See text for details on quantification.

and 304 for TGF-�4, while the 112 amino acid processed active segment is thesame length as all other TGF-� isoforms except TGF-�4 which is 114 aminoacids long (Hardie et al., 1998; Roberts & Sporn, 1990). The presumed leadersequence of 18 amino acids is shorter than the predicted rainbow trout leadersequence (20 amino acids) but still indicates that it is a secreted protein(Hardie et al., 1998). The hybrid striped bass LAP segment contains morepotential N-linked glycosylation sites (five) than rainbow trout TGF-� andmammalian TGF-�1 (three sites). One site in the hybrid striped bass sequence(NMT108–110) that is not shared by rainbow trout corresponds to NMK110–112 inthe trout sequence, and is in close proximity to NNT113–115 (NTS116–118 trout),such that only one of the two adjacent sites might actually be glycosylated.The mature protein segment contains nine conserved cysteines involved informing intra- and inter-chain disulfide bonds and the cysteine knot typical ofthe TGF-� superfamily (Kingsley, 1994). In both fish sequences, the firstcysteine is in position 8 of the processed protein rather than position 7 in otherknown TGFs-�.


25 000 000

01 to 3

Cop

y n

um

ber

5 000 000

15 000 000

10 000 000

undiluted 1 to 9Dilution

20 000 000

1 to 811 to 27

TGF-b (× 100)TGF-b expected (× 100)beta-actinbeta-actin expectedTGF-b:beta-actin (× 107)

Fig. 8. RT-qcPCR results are independent of starting cDNA concentration. Fourthree-fold dilutions of hybrid striped bass anterior kidney mononuclear cell cDNAhad the expected e#ect on starting copy number for both TGF-� and beta-actin, andminimal e#ect on the TGF-�: beta-actin ratio. Scales are adjusted as indicated to aidvisualisation.

The potential polyadenylation signal detected in the hybrid striped bassTGF-� sequence is considerably closer to the stop codon (94 nucleotidesdownstream) than that for rainbow trout (503 nucleotides, Hardie et al., 1998),and may not play a major role. The 3� UTR was determined 277 nucleotidesbeyond the stop codon for the hybrid striped bass TGF-�. Regulatorysequences such as ATTTA stability motifs in the 3� UTR were not identifiedwithin that span.

The rooted cladogram of TGF-� superfamily members, using the Clustalmethod with PAM 250 residue weighting, groups rainbow trout and hybridstriped bass TGFs-�, Xenopus TGF-�5 and chicken TGF-�4 into a loosely alliedcluster with the mammalian TGFs-�1 (Fig. 4). This result is in accord with aproposal that TGFs-�4&5 be considered homologs of TGF-�1 (Burt & Law, 1994).Previous analyses of the rainbow trout TGF-� sequence (Hardie et al., 1998)and genomic organisation (Daniels & Secombes, 1999) suggest that it also is ahomolog of TGF-�1. Percent identity of the hybrid striped bass TGF-� fullamino acid sequence is greatest with rat TGF-�1 than to any other TGF-�except the rainbow trout sequence (Table 2). TGF-�1 is the isoform with thegreatest immunologic relevance in mammals (Derynck, 1994), implying thatthe hybrid striped bass and rainbow trout isolates are also important inregulating immune responses in these fish species. In contrast with theproximal branching pattern for the TGF-�1 clade (including �4, �5 and thehybrid striped bass and rainbow trout isolates, Fig. 4), TGF-� types 2 and 3cluster in more clearly defined groups, even with avian, amphibian and fish


0.06

0.00pbmc

tgf-

b:b-

acti

n

0.01

0.05

0.04

0.03

0.02

kidney spleen

Source

Fig. 9. TGF-�: beta-actin ratios vary by organ source of mononuclear cells (WelchAnova P<0·0001). TGF-� transcription is greater in mononuclear cells of peripheralblood than from spleen, and greater from spleen than from anterior kidney(Tukey-Kramer HSD P<0·05). Means dots with standard error bars and standarddeviation lines are shown. Horizontal line indicates total sample mean.

Table 4. Di#erential counts of hybrid striped bass monouclear cells derived fromspleen, anterior kidney and peripheral blood (mean and standard deviation)

Cell type Spleen Anterior kidney Peripheral blood

Lymphocyte 49·9 (11·0)* 33·0 (17·8)* 65·4 (16·3)*Monocyte 15·6 (10·7) 14·4 (13·1) 10·4 (7·1)Melanomacrophage 0·4 (1·0) 0·06 (0·2) 0 (0)Blast 7·0 (7·9) 27·9 (18·8)* 0·8 (1·4)Granulocyte 8·2 (5·9) 14·6 (13·6)# 3·4 (3·2)†Thrombocyte 17·6 (14·2) 7·8 (13·5) 18·4 (13·5)Erythrocyte 4·7 (4·2)* 2·1 (1·6) 1·6 (1·3)

* Values di#er significantly from other values in row; #, † granulocyte component from anteriorkidney is significantly greater than from peripheral blood, neither di#ers significantly fromspleen; Welch Anova followed by Tukey-Kramer HSD, P<0·05.

TGFs-�2 considered. It is possible that as TGF-� sequences are determinedfrom more non-mammalian species, the distinctions between the fiveoriginally designated isoforms will become less evident.

Northern blot revealed two transcripts: a major product at 4·2 Kb and alesser product at 2·9 Kb. Both fall within the wide range of known TGF-�transcripts (1·7–6·5 Kb; Roberts & Sporn, 1990). The 2·9 Kb transcript could bethe result of alternate poly-A tailing sites (two are present in some TGFs-�,Scotto et al., 1990), alternate splicing, or presence of another isoform with lessspecific binding by the random prime labelled probe. Other isoforms are likelypresent, as a TGF-�2 partial sequence has been reported from carp, andDaniels & Secombes (1999) state that all three tetrapod TGF-� isoforms are


present in bony fish. For PCR, the 3� end of primer TGF con B2 was designedwith intentional mismatches with rat TGF-�1, carp TGF-�2, porcine TGF-�3,chicken TGF-�4, and Xenopus TGF-�5 sequences to reduce the chance ofamplifying other TGF-� isoforms.

TGF-� consensus primers were specific in 14 species of teleost fish tested(Table 3), based on product sequencing (hybrid striped bass) and Southern blotwith a consensus oligonucleotide internal probe (13 additional species). Thisassemblage represents fish of 10 taxonomic families (Salmonidae, Cyprinidae,Characidae, Ictaluridae, Percicthyidae, Serranidae, Centrarchidae, Cichlidae,Bothidae, Balistidae) of 7 orders (Salmoniformes, Cypriniformes, Characi-formes, Siluriformes, Perciformes, Pleuronectiformes and Tetraodontiformes),inhabiting a wide range of habitats (cold, temperate and warm freshwater, andtemperate estuarine and marine waters). Probe binding was least intense forPCR products from channel catfish, red pacu, giant zebrafish and fatheadminnow, as demonstrated by the 21 h exposure time in Fig. 6 for these fourspecies, v. 1 h for all others. These four species are members of the superorderOstariophysi, indicating TGF-� sequence divergence within the probe’s 17nucleotide span for that taxonomic group. The primers did not amplify cDNAsuccessfully from one cartilaginous fish.

Perfectly-matching consensus primers could not be designed from thealignment of hybrid striped bass and rainbow trout TGF-� sequences. There-fore, a single base pair mismatch near the extreme 5� end of the primer existsbetween primer TGF con A1 and both the hybrid striped bass and rainbowtrout sequences, and between primer TGF con B2 and the rainbow troutsequence. Thus, while the primers amplify specifically from a wide range offish species, they may not bind and amplify with equal e$ciency. Forquantitative applications (RT-qcPCR) then, cross-species comparisons maynot be valid, and baseline comparative values should be established in eachspecies examined.

For RT-qcPCR, beta-actin was selected as the internal standard housekeep-ing gene to control for sample-to-sample variation in RNA isolation, reversetranscription, amplification and gel loading during quantification. Primers forother housekeeping genes [consensus primers for glyceraldehyde-3-phosphatedehydrogenase (G3PD, Rottman et al., 1996) and commercial consensusprimers for cyclophillin] were attempted but failed to amplify specificallyfrom teleost fish samples, based on Southern blot and on cloning andsequencing the PCR products. The beta-actin consensus primers weredesigned based on alignments of human and five fish sequences, and containno mismatches with the fish sequences and a single mismatch in the5� half of the human sequence. Primer specificity was confirmed bySouthern blot and appropriate product size for the 14 teleost species(Table 3). Although expression of beta-actin mRNA is more variable thanG3PD under some conditions (Spanakis, 1993), beta-actin expression isactually somewhat less variable in response to glucocorticoids than isG3PD mRNA in chick embryo tendon and heart (Oikarinen et al., 1991). Areduced glucocorticoid response may make beta-actin more appropriateas an internal standard for investigations of constitutive cytokineexpression in immunosuppressed fish. In addition, TGF-� itself has minimal


e#ects on beta-actin transcription in human colon carcinoma cells (Dodgeet al., 1990).

Homologous competitive fragments for TGF-� and beta-actin RT-qcPCRwere employed rather than a multi-standard competitive fragment with anon-homologous internal spacer (e.g. Rottman et al., 1996; Levy et al., 1998).Homologous competitive fragments amplify with nearly the same e$ciency astheir associated target over the entire cycle range, allowing quantificationeven when reactions reach plateau (Köhler et al., 1995).

The CVs for the TGF-� RT-qcPCR assay were similar to those reportedpreviously in other quantitative PCR applications, though CVs were calcu-lated from di#erent starting points (Rottman et al., 1996; Morrison et al., 1998).Continuous fluorescence observation of amplifying DNA for a single target,starting with aliquots of cDNA, and not subsequently normalised to a secondset of reactions for a housekeeping gene, averaged a CV of 34% (Morrisonet al., 1998). Another RT-qcPCR assay (Rottman et al., 1996) yielded CVs of 17and 22% under the same parameters. Using data provided in Table 3 of thelatter paper, CVs for cytokine: housekeeping gene ratios, starting with cDNAaliquots processed in parallel ranged from 5–26%, and starting from mRNAaliquots processed in duplicate, CVs ranged from 0–51%. In the present case,variability increased with number of manipulations from the starting sample,being least when starting from cDNA aliquots processing in parallel (21%)and greatest when starting from aliquots of cells and processed through RNAisolation and subsequent RT-qcPCR (68%), analyses not performed in theother two studies cited. Power analysis (JMP) based on sample sizes of 10, anda CV of 68% reveals that the assay can reliably detect true di#erences of meanTGF-�: beta-actin ratios separated by as little as 40% between two popu-lations, and di#erences of only 14% for a CV of 21%. For comparison,constitutive cytokine: housekeeping gene mRNA ratio di#erences of three-foldand substantially greater have been reported in an infectious disease modelutilising RT-qcPCR methodology (Levy et al., 1998). Therefore, precision of theRT-qcPCR assay reported here more than su$ces to detect di#erences ofthe magnitude of interest.

Mononuclear cell preparations from spleen, anterior kidney and peripheralblood di#ered in cellular composition, even though separated on identicalPercoll gradients. A 40/51% Percoll stock two-step gradient was selectedbased in part on previous reports in rainbow trout (Graham et al., 1988; Hardieet al., 1994; Sharp et al., 1991) and catfish (Waterstrat et al., 1988). Preliminaryfindings in this laboratory with hybrid striped bass showed this methodmaximised lymphocyte and macrophage content at the 40/51% interface andreduced thrombocytes and cellular debris with the lower density 40% Percollstock layer. The greater blast cell composition in the anterior kidney prep-arations is consistent with the role of that organ in haematopoiesis (Kennedy-Stoskopf, 1993). In analysis of covariance, lymphocyte percentage was the onlycellular component that had a significant e#ect, along with mononuclear cellsource, on constitutive TGF-�: beta-actin ratios. The finding that TGF-�:beta-actin ratios depended in part on lymphocyte percentage in mononuclearcell preparations supports the working hypothesis that this TGF-� isolate is ofimmunologic relevance. Organ source of mononuclear cells had the primary


e#ect on TGF-�: beta-actin ratios, as would be expected if cells are in di#erentstates of activation based on location.

In summary, a TGF-� from hybrid striped bass that has close a$nities torainbow trout TGF-� and TGF-�1 (inclusive of TGFs-�4&5) has been cloned andsequenced. Consensus PCR primers were designed which amplify a specificproduct from a wide range of teleost species occupying multiple habitats. Useof RT-qcPCR demonstrated tissue-specific di#erences in TGF-� transcriptionlevels. The RT-qcPCR technique described herein may prove useful as an aidin assessing teleost fish immune responses to environmental contaminants,aquaculture management practices and infectious diseases.

We thank Tedd Childers, Joe Bucci, Ed Noga and Stephanie Perry for advice andassistance. Funding sources include a US EPA Science to Achieve Results Fellowship(U-915209–01–0) and a North Carolina State University College of Veterinary MedicineState Research Grant.

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