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Endo-LysC cleavage of VERL excised from SDS gels was performed by the PAN Facility (Stanford University)
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note
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PCR primers were TAYTGDATNARCATCATRTT and GTNCCNATHACNCARGA (Y = C or T; D = A, G, or T; N = A, G, C, or T; R = A or G; H = A, C, or T), template was ovary cDNA primed with oligo(dT). PCR products were cloned and sequenced by using ABI prism FS chemistry on an Applied Biosystems model ABI 370.
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20
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3543027074
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note
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A pink abalone ovary, random-primed cDNA library was made in pSport (BRL), and 92,160 independent clones were arrayed (Q-Bot) on five high-density filters. Filters were hybridized with a random-primed probe of the VERL repeat. Nested deletions (Promega) were generated and sequenced.
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21
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note
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PCR was performed with genomic DNA or with cDNA from the other abalone species. PCR primers are as in (8). The products were cloned and individual colonies were sequenced. To avoid biased amplification, two or three PCRs were done for each species.
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note
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Ten micrograms of genomic DNA were digested, separated on a 0.8% agarose gel, denatured, neutralized, transferred to nylon, and probed as in (9).
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23
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3543016972
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note
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One hundred micrograms of total RNA were separated on a 0.8% agarose formaldehyde gel, transferred to nylon, and probed as in (9). A pink VERL repeat is 459 bp. Assuming 500 bp is untranslated, 13 kb codes for 28.3 repeats. The average molecular mass of the pink repeats is 16,560 daltons, which gives a total molecular mass of ≈470 kD. Recalculating the binding stoichiometry (6) with this VERL molecular mass yields 58 to 66 lysins per VERL.
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0030054305
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Maximum-likelihood estimates of Dn and Ds were calculated [S. V. Muse, Mol. Biol. Evol. 13, 105 (1996)]. Identical values were obtained by the Nei and Gojobori method [M. Nei and T. Gojobori, Mol. Biol. Evol. 3, 418 (1986)].
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3543008567
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note
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A neighbor-joining tree was constructed with MEGA (S. Kumar, K. Tamura, M. Nei, Pennsylvania State University) using Jukes-Cantor corrected nucleotide distances. Positions with alignment gaps were omitted only in pairwise comparisons.
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note
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Abbreviations for amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
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We thank E. H. Davidson, Y.-H. Lee, T. Walkup, E. C. Metz, M. E. Hellberg, J. R. Schulz, R. S. Burton, R. R. Hudson, J. D. Calkins, J. M. Swanson, C. F. Aquadro, M. Schug, K. Schmid, J. B. Walsh, and R. R. McConnaughey for their research support and editorial help. Supported by NIH grant HD12986 to V.D.V.
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