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MALDITOF mass spectrometry of preCol-P, preCol-D, and their proteolytically derived fragments indicates that apparent masses are overestimated by 20% on SDS-polyacrylamide gel electrophoresis.
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11
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9844238352
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4 dilution of the RNA probe. The location of the probe on the membrane was detected with alkaline phosphatase-conjugated anti-digoxigenin (DIG) (Boehringer Mannheim, Indianapolis, IN) and visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate toluidinium (NBT/BCIP). The full-length sequence for α-preCol-P is 2800 bases with an open reading frame of 2706 bases and a translated amino acid sequence of 902 residues. We verified the size of the cDNA transcript by Northern blot analysis. The absence of preCol-P mRNA in non-foot tissue confirmed its tissue-specific expression in adult mussels. Double-stranded DNA probes for Northern blots were synthesized by incorporation of DIG-11-deoxyuridine triphosphate into products during polymerase chain reaction (PCR) amplification (32). We synthesized a probe specific for preCol-P by PCR amplification of deletion plasmid P14-7aF and with T3 and T7 primers. This deletion plasmid includes the last 900 bases of the sequence for preCol-P. We synthesized a second probe for actin in the same manner from a cloned actin cDNA from the sea scallop Placopecten magellanicus (a generous gift from M. Patwary, National Research Council of Canada). Probes constructed from the actin clone of this sea scallop have been shown to cross-react with actin RNAs from other bivalve species, including M. edulis (33). We fractionated mRNA from foot and non-foot tissue on a 0.44 M formaldehyde gel in duplicate and transferred them to positively charged nylon membranes (Boehringer Mannheim) by standard techniques. We incubated the blots overnight at 42°C in separate chambers: one with the preCol-P probe and the other with the actin probe. The probe-mRNA hybrid on the membranes was localized by incubation with alkaline phosphatase-conjugated anti-DIG and visualized with NBT/BCIP.
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33
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Bell, E.C.1
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42
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9844260730
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note
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Abbreviations for the amino acids are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, He; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.
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43
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9844264524
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note
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We thank A. Hunt and J. MacDonald for the use of their sequencer. M. L. Tanzer, E. C. Bell, D. Urry, and three anonymous reviewers provided useful comments for improvement of the manuscript. Supported by grants from the National Institute of Dental Research (Biomaterials) and the Office of Naval Research (MIMI Program) to J.H.W.
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