Preliminary analysis of length and GC content variation in the ribosomal first internal transcribed spacer (ITS1) of marine animals.

Length and guanine-cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species.

Retinoic acid given at late embryonic stage depresses sonic hedgehog and Hoxd-4 expression in the pharyngeal area and induces skeletal malformation in flounder (Paralichthys olivaceus) embryos.

During the development of pharyngeal cartilages, signal molecules, including sonic hedgehog (shh) and various growth factors, as well as Hox genes are expressed in the pharyngeal area. To elucidate whether shh and Hoxd-4 function in pharyngeal cartilage formation in teleost jaw and gill primordia, spatial and temporal patterns of shh expression in flounder (Paralichthys olivaceus) embryonic pharynx were examined. The effects of retinoic acid (RA) on shh and Hoxd-4 expression and the patterning of pharyngeal cartilages were analyzed. At the prim-5 stage, when cartilage precursor cells aggregate in the pharyngeal primordia, pharyngeal endoderm expressed shh in two domains, in portions of the mandibular and hyoid primordia and in the gill primordia. After a further 40 h, shh domains expanded at the posterior edge of the endoderm of each mandibular, hyoid and gill primordium, concurrent with the growth of the primordia. A new shh expression domain appeared at the endodermal border of the mouth. Retinoic acid treatment depressed shh and Hoxd-4 expression, and also reduced the amount of expansion of the shh expression domains. Pharyngeal cartilages that formed in these embryos were malformed; their growth direction was shifted posteriorly and size was reduced. This provides the possibility that shh and Hoxd-4 regulate the growth and direction of pharyngeal cartilage precursor cells and that RA disturbs their expression, causing skeletal malformation.

Hoxd-4 expression during pharyngeal arch development in flounder (Paralichthys olivaceus) embryos and effects of retinoic acid on expression.

Teleost fish develop seven pharyngeal arches (mandible, hyoid and five gill arches) which give rise to the jaw and gills, and skeletal cell populations which originate from the cranial neural crest. The anterior border of expression of the Deformed (Dfd) group is located in the hindbrain and pharyngeal region. To investigate pharyngeal skeletal formation in the teleost fish, we cloned the cDNA coding Hoxd-4 from a cDNA library for flounder (Paralichthys olivaceus) embryos, and analyzed gene expression pattern during embryogenesis and the effects of retinoic acid (RA) on this gene expression. Between the 21-somite and prim-5 stages, Hoxd-4 was expressed in the central nervous system from rhombomere 7 to the spinal cord, and in the pharyngeal region posterior from gill arch 2. Its expression then became restricted to cartilage precursor cells of gill arches 2-5. When embryos in the early shield stage were exposed to RA at concentrations above 10(-7) M, the anterior border of Hoxd-4 expression shifted anteriorly in a dose-dependent manner, both in the central nervous system and pharyngeal region. We propose that, during gill skeleton formation, Hoxd-4 functions in the specification of regional identity between gill arches 1 and 2, and that their identity is affected by treatment with RA.

Mitochondrial DNA sequence analysis of the masu salmon--phylogeny in the genus Oncorhynchus.

We determined 2162-bp sequences from the mitochondrial genome of the masu salmon Oncorhynchus masou masou, chum salmon Oncorhynchus keta, and Atlantic salmon Salmo salar by using polymerase chain reactions. These DNA sequences span from the 3' region of the gene for ATPase subunit 6 to the 5' region of the gene for NADH dehydrogenase subunit 4L. With the aid of the sequence data from other Pacific salmonid species (Thomas and Beckenbach, 1989, J. Mol. Evol. 29: 233-245), the evolutionary distances among the masu salmon and other species in the genus Oncorhynchus were calculated. These evolutionary distances were then used to construct a neighbor-joining tree. Further, a maximum parsimony tree was constructed. The evolutionary trees that were obtained suggest that masu salmon first diverged from the common ancestor of the genus Oncorhynchus.

Genetic relationship among three subspecies of Oncorhynchus masou determined by mitochondrial DNA sequence analysis.

It is generally accepted that there are 3 subspecies of Oncorhynchus masou in Japan, namely, Masu salmon (Oncorhynchus masou masou (Brevoort)), Amago salmon (O. masou ishikawae Jordan & McGregor), and Biwa salmon (O. masou rhodurus Jordan & McGregor or O. masou subsp. Kimura). Since the genetic relationship of these three taxa is not well known, there has been considerable confusion over their nomenclature. We have clarified the genetic relationship among these three taxa by partially sequencing their mitochondrial DNA. Sequences of 948 base pairs from the 3' region of the ATPase subunit 6 gene to the 5' region of the cytochrome oxidase subunit 3 gene were obtained for 20 individuals including wild Biwa salmon, wild and farmed Amago and Masu salmon. Furthermore, 2,162 base pairs from the 3' region of ATPase subunit 6 gene to the 5' region of NADH dehydrogenase subunit 4L gene were determined in 4 individuals. In total, there were 26 sites of base substitutions. The haplotypes of Masu salmon and Amago salmon were similar. On the other hand, 17 of the 26 sites had substitutions characteristic of Biwa salmon. A matrix of genetic distances and maximum parsimony analysis among the haplotypes indicated that Biwa salmon is genetically more distant from Masu and Amago salmon, than Masu salmon is from Amago salmon. This means that Biwa salmon diverged from the common ancestor of the Oncorhynchus masou complex before the divergence between Masu salmon and Amago salmon.

Spectroscopic studies on histone-DNA interactions. I. The interaction of histone (H2A, H2B) dimer with DNA: DNA sequence dependence.

Binding of the histone (H2A, H2B) dimer with chicken erythrocyte DNA has been studied by salt-titration spectroscopy in equilibrium conditions. The circular dichroism of DNA near 275 nm is depressed by the interaction with (H2A, H2B) at low concentrations of salt. The depression increases with increasing amounts of (H2A, H2B), and reaches a plateau at an (H2A, H2B) to DNA ratio of 1.5 (w/w), at which one (H2A, H2B) dimer occupies 28 base-pairs of DNA. The fluorescence emission intensity of the tyrosine residues in (H2A, H2B) is depressed by the H2A, H2B)-DNA interaction. When the DNA-(H2A, H2B) complex is titrated with NaCl, these two signals show transitions with increasing ionic strength of the buffer, whose normalized transition curves agree well. The midpoint of the transition is about 0.42 M-NaCl for a sample with a DNA concentration of 0.05 mg/ml and an (H2A, H2B) to DNA ratio of 0.4 (w/w). The fluorescence titration curves have been analyzed to obtain the binding constant for the (H2A, H2B) dimer with DNA. The sample concentration dependence of the titration profiles is consistent with the model of non-cooperative binding of (H2A, H2B) dimer to DNA. The titration profiles are reversible. The obtained binding constant for the (H2A, H2B) dimer with chicken erythrocyte DNA at 20 degrees C (pH 7.6), as a function of the ionic strength, I, is as follows: log10K = -14.9 log10(I)-1.2. The change of enthalpy delta H accompanied by the binding of the (H2A, H2B) dimer is nearly equal to zero, within an error of +/- 1.4 kcal/mol (1 cal = 4.184 J). DNA sequence dependence of the stability of DNA-(H2A, H2B) interactions is observed using reconstituted materials of synthetic DNAs. A decreasing stability of the interaction is observed following the order: the duplex of poly[(dA)-(dT)] greater than chicken erythrocyte DNA or the copolymer duplex of poly(dA).poly(dT) greater than the duplex of poly[(dG)-(dC)]. The difference in free energy of the association of the (H2A,H2B) dimer between the two copolymers is 0.8 kcal/mol.

Spectroscopic studies on histone-DNA interactions. II. Three transitions in nucleosomes resolved by salt-titration.

The multiple-step transitions in DNA-histone interactions in chicken erythrocyte nucleosomes with increasing ionic strength are resolved by salt-titration spectroscopy. Both the circular dichroism of the DNA and the fluorescence of the histones in nucleosomes change during the titration process with concentrations of NaCl from 0.1 M to 2.5 M. By differentiating the titration curves, three distinct peaks corresponding to three structural transitions are observed. The two peaks near 0.95 M and 1.45 M-NaCl are common to the circular dichroism and fluorescence curves. The circular dichroism curve has another peak near 0.55 M-NaCl. Because the derivative of the fluorescence titration curve for the DNA-(H3, H4) complex has only one peak near 1.45 M-NaCl, that peak is attributed to the dissociation of the histone dimer (H3, H4). The peak near 0.95 M-NaCl corresponds to the dissociation of the dimer (H2A, H2B) from the DNA-(H3, H4) complex, as shown by binding experiments of (H2A, H2B) to the DNA-(H3, H4) complex at the salt concentration near this peak. The peak near 0.55 M-NaCl reflects some inner-core structural change. As the change of the circular dichroism signal is reversible, salt-titration spectroscopy is applicable to equilibrium studies of the physical chemical properties of DNA-histone interactions. By the assumption of a non-co-operative model, the binding constant for the chicken erythrocyte (H2A, H2B) dimer to the DNA-(H3, H4) complex is calculated as 2.8 X 10(6) M-1 at 1.0 M-NaCl (20 degrees C, pH 7.6). The DNA sequence dependence of the stability of the DNA-(H3, H4) interaction is observed in the salt-titration profiles of reconstituted material. Decreasing stability of the interaction of (H3, H4) is observed following the order: poly[(dG)-(dC)] much greater than chicken erythrocyte DNA greater than poly[(dA)-(dT)]. It is concluded that histones (H3, H4) have a different DNA sequence dependence from histones (H2A, H2B).

Reconstitution mechanism of nucleosome core particles mediated by poly(L-glutamic acid).

Poly(L-glutamic acid) has been reported to mediate in vitro nucleosome assembly (Stein, A., Whitlock, J.P., Jr. and Bina, M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76,5000-5004). To study the reaction mechanism, we have reconstituted nucleosome core particles from chicken erythrocyte core DNA and core histones in the presence of poly(L-glutamic acid) and analyzed the assembly products by polyacrylamide gel electrophoresis. Poly(L-glutamic acid), which binds and forms a large complex with core histones, is replaced with core DNA in the reconstitution process. When histone-poly(L-glutamic acid) complex and core DNA are mixed with a histone:DNA ratio of 1.0, the yield of core particles increases by prolonged reconstitution time. Two phases with a distinct time range appear in the process. In the fast phase within 30 min, 60% of the DNA is involved in products containing histones: reconstituted core particles, a larger nucleoprotein complex and aggregation. In the second phase, the remaining DNA and the DNA in the aggregation decrease, and the core particles increase slowly. The yield of core particles is approx. 60% after 24 h. The slow phase is not observed by reconstitution with a histone:DNA ratio of 2.0 in the initial mixture. The reaction scheme of the assembly process derived from these data is given. Based on the in vitro reaction scheme, the possible role of in vivo 'nucleosome assembly factors' is also discussed.