We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase ␤. At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C 18 -linoleic acid (LA) or C 24 -nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48 -51, and 79 -87) and residues adjacent to an ⍀-type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K D ) of 1.02 or 2.64 mM, respectively.We reported previously that long-chain fatty acids strongly inhibited the activities of mammalian DNA polymerase ␣ (pol ␣) 1 and DNA polymerase ␤ (pol ␤) in vitro and plant DNA polymerases, albeit less potently, but that at the concentrations used, the fatty acids hardly influenced the activities of prokaryotic DNA polymerases or other DNA metabolic enzymes such as DNase I (1, 2). The most potent inhibitors were fatty acids, which have the following characteristics: hydrocarbon chain containing 18 or more carbons, a free carboxyl end, and the cis-configuration is preferred to the trans-configuration. Fatty acids in the trans-configuration have a much weaker inhibitory effect on pol ␤, and those in which the carboxyl end is chemically modified can lose the inhibitory effect on both pol ␣ and pol ␤. The mode of inhibition by longer chain fatty acids showed the same characteristics, except that the minimum inhibitory doses of these longer chain fatty acids were much lower (2, 3). Lineweaver-Burk plots of the fatty acids indicated that both the substrate (i.e. deoxynucleotide)-binding and the template DNA-binding sites of pol ␣ were nonantagonistically inhibited by the fatty acids, but they were effective as antagonists against the sites of pol ␤. For pol ␤, fatty acids acted by competing with not only the substrate but also the template-primer DNA. In screening inhibitors of eukaryotic DNA polymerase, we also found several natural compounds which inhibited pol ␤ in the same manner as fatty acids (4 -10).Pol ␤ is the smallest known DNA polymerase in animal cells with a molecular mass of 39 kDa, and its structure is highly conserved among mammals (11). This protein has a modular two-domain structure, with apparent flexibility within a protease-sensitive region between residues 82 and 86, which separates the two domains. Treatment with trypsin yields an N-terminal domain fragment (8 kDa), which retains binding affinity for single-stranded DNA (ssDNA), and a C-terminal domain fragment (31 kDa) ...

Three families of tRNA-derived repeated retroposons in the genomes of salmonid species have been isolated and characterized. These three families differ in sequence, but all are derived from a tRNALYS or from a tRNA species structurally related to tRNALyS. The salmon Sma I family is present in the genomes of two species of the genus Oncorhynchus but not in other species, including five other species of the same genus. The charr Fok I family is present only in four species and subspecies of the genus Salvelinus. The third family, the salmonid Hpa I family, appears to be present in all salmonid species but is not present in species that are not members of the Salmonidae. Thus, the genome of protoSalmonidae was originally shaped by amplification and dispersion of the salmonid Hpa I family and then reshaped by amplification of the Sma I and Fok I families in the more recently evolved species of salmon and charr, respectively. We speculate that amplification and dispersion of retroposons may have played a role in salmonid speciation.Gene duplication is believed to be of major importance in creating genetic diversity (1). The genes for immunoglobulins, histocompatibility complexes, and globins are examples of this gene duplication. This mechanism operates at the DNA level and probably has as old a history as DNA genomes themselves. Another mechanism for maintaining the fluidity of eukaryotic genomes is that recently characterized retroposition, in which information in nonviral cellular RNA can flow back into the genome via cDNA intermediates (2,3). Retroposition creates additional sequence combinations through dispersal of genetic information and can shape and reshape eukaryotic genomes in many different ways (3,4). The precise mechanism of retroposition is at present speculative. Recently, Weiner and Meizels (5) presented an interesting hypothesis concerning the mechanism of generation of duplex DNA at the beginning of the DNA world, proposing that duplex DNA genomes may have been derived from earlier DNA genomes that replicated like retroviruses through an RNA intermediate. This suggests that the mechanism of retroposition might be closely linked to that of replication of retroviruses (6).The highly repetitive sequences that are interspersed throughout eukaryotic genomes have been classified into two categories based on size: long interspersed repetitive elements (LINEs), which include Li sequences, and short interspersed repetitive elements (SINEs), such as the primate Alu and rodent type 1 or 2 Alu families (7). Previously, highly repetitive and transcribable sequences have been found in the genome of the chum salmon (Oncorhynchus keta) (8,9). Like all SINE families examined so far (10-14) other than Alu (15, 16), this Sma I family [formerly the salmon polymerase (Pol) III/SINE family] has been shown to be derived from a tRNA; moreover the Sma I family has several of the characteristic features of retroposons and appears to be the youngest SINE family characterized to date.The genus Oncorhynchus has many s...

Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 g/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was L-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or Staphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) of D-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and ␤-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (K a ) of STL2 to lipovitellin and ␤ component were 3.44 ؋ 10 6 and 4.99 ؋ 10 6 , respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor superfamily.

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Various 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA synthesis in vitro. RNA polymerase I utilized the 5-methyl-, chloro, bromo, and iodo derivatives of UTP more efficiently than unmodified UTP, but RNA polymerase II utilized UTP most efficiently. 5-Methyl-4-thiouridine 5'-triphosphate (4-thio TTP) was utilized more efficiently than UTP by RNA polymerase I. On the other hand, it was found that 4-thio TTP was a selective substrate for RNA polymerase I and that its incorporation by RNA polymerase II was very slow. Thus recognition of UTP analogues as substrates by RNA polymerase I and II was different. These observation were attributed from kinetic analyses to differences in catalytic activity (Vmax).