176
M.R. Coscia and U. Oreste
disclosed a major peak, (190 kDa) corresponding to the monomer [6]. Both
plasma and bile FPLC purified peaks were then resolved, by SDS-PAGE
under reducing conditions, into two separated bands, corresponding to H
and L chains (Fig. 1 C). In addition, bile Ig gave at least two distinct Lchain bands, of very similar molecular mass (about 25-27 kDa), in
agreement with data reported by Lobb et aI. for Ichtalurus punctatus serum
Ig [8]; it is not clear if these L chain variants represent distinct L chain
classes. We did not find any difference in relative molecular mass between
plasma and bile Ig H chain, as seen in Archosargus probatocephalus [9].
The pI spectrum of purified plasma Ig, ranging between 4.0 and 6.0, was
found lower than that of higher vertebrates (Fig. ID). This result suggests
the prevalence of acidic residues in the carbohydrate andlor amino acid
moieties. In fact, the sialic acid linked a(2-3) to galactose was selectively
recognized as terminal residue by Maackia amurensis agglutinin (Coscia et
aI., in preparation).
Antisera specific for either H or L chains were obtained by immunizing
rabbits with the H-chain or L-chain band, electroeluted from an SDSPAGE slab gel of purified T bernacchii Ig, run under reducing conditions.
The anti-L chain antiserum reacted with human IgG, whereas T bernacchii
Ig was recognized, in double immunodiffusion, by commercial antihuman
L chain (Iv) antiserum and antimouse H chain (y) antiserum; nonimmune
rabbit sera were used as controls [7]. The antigenic crossreactivity
observed between T bernacchii Ig and mammalian Ig suggests the
conservation of structurally important epitopes during evolution.
To better understand how the Antarctic fish Ig can work at very low
temperatures we looked for the structural characteristics accounting for this
property. For this purpose, and to overcome the difficulties related to the
amino acid sequencing, we constructed a cDNA library in ZAP Express
vector.
Total RNA was extracted from the spleen of a T bernacchii specimen by
the guanidinium thiocyanate method. Poly(A)+ mRNA was purified from
total RNA through an oligo( dT)-cellulose column and was used for cDNA
synthesis. Rabbit anti-T bernacchii Ig H chain antibodies, purified by
MabTrap G affinity chromatography, were used to immunoscreen the
cDNA library. Fourteen clones were found positive; five of them were
partially sequenced in both directions by the Sanger dideoxynucleotide
chain termination method with T7 DNA polymerase, using T3 or universal
primers. Due to its length (1.9 kb), the clone 2C2.3 was expected to
represent the most length of the Ig H chain transcript.
When the partial nucleotide sequences were used to search the databases,
significant homologies were obtained with the VH region of a primitive
teleost (Elops saurus), rainbow trout (Oncorhynchus mykiss), Xenopus
laevis, human, mouse, and rabbit. On the basis of this homology, the clone
M.R. Coscia and U. Oreste
disclosed a major peak, (190 kDa) corresponding to the monomer [6]. Both
plasma and bile FPLC purified peaks were then resolved, by SDS-PAGE
under reducing conditions, into two separated bands, corresponding to H
and L chains (Fig. 1 C). In addition, bile Ig gave at least two distinct Lchain bands, of very similar molecular mass (about 25-27 kDa), in
agreement with data reported by Lobb et aI. for Ichtalurus punctatus serum
Ig [8]; it is not clear if these L chain variants represent distinct L chain
classes. We did not find any difference in relative molecular mass between
plasma and bile Ig H chain, as seen in Archosargus probatocephalus [9].
The pI spectrum of purified plasma Ig, ranging between 4.0 and 6.0, was
found lower than that of higher vertebrates (Fig. ID). This result suggests
the prevalence of acidic residues in the carbohydrate andlor amino acid
moieties. In fact, the sialic acid linked a(2-3) to galactose was selectively
recognized as terminal residue by Maackia amurensis agglutinin (Coscia et
aI., in preparation).
Antisera specific for either H or L chains were obtained by immunizing
rabbits with the H-chain or L-chain band, electroeluted from an SDSPAGE slab gel of purified T bernacchii Ig, run under reducing conditions.
The anti-L chain antiserum reacted with human IgG, whereas T bernacchii
Ig was recognized, in double immunodiffusion, by commercial antihuman
L chain (Iv) antiserum and antimouse H chain (y) antiserum; nonimmune
rabbit sera were used as controls [7]. The antigenic crossreactivity
observed between T bernacchii Ig and mammalian Ig suggests the
conservation of structurally important epitopes during evolution.
To better understand how the Antarctic fish Ig can work at very low
temperatures we looked for the structural characteristics accounting for this
property. For this purpose, and to overcome the difficulties related to the
amino acid sequencing, we constructed a cDNA library in ZAP Express
vector.
Total RNA was extracted from the spleen of a T bernacchii specimen by
the guanidinium thiocyanate method. Poly(A)+ mRNA was purified from
total RNA through an oligo( dT)-cellulose column and was used for cDNA
synthesis. Rabbit anti-T bernacchii Ig H chain antibodies, purified by
MabTrap G affinity chromatography, were used to immunoscreen the
cDNA library. Fourteen clones were found positive; five of them were
partially sequenced in both directions by the Sanger dideoxynucleotide
chain termination method with T7 DNA polymerase, using T3 or universal
primers. Due to its length (1.9 kb), the clone 2C2.3 was expected to
represent the most length of the Ig H chain transcript.
When the partial nucleotide sequences were used to search the databases,
significant homologies were obtained with the VH region of a primitive
teleost (Elops saurus), rainbow trout (Oncorhynchus mykiss), Xenopus
laevis, human, mouse, and rabbit. On the basis of this homology, the clone