Distribution of 59- and 55-kDa Catalase in Dark- and Light-grown Pumpkin and Various Other Plant Tissues

The catalase molecule in germinating pumpkin cotyledons is synthesizedas a precursor (59-kDa) form, whose relative molecular massis larger than the mature enzyme (55-kDa). Although both typesof molecules are localized in the microbodies, the 59-kDa specieshas been shown to be present predominantly in the leaf peroxisomesisolated from green cotyledons, while the 55-kDa species ispredominantly in the glyoxysomes from etiolated cotyledons [Yamaguchiet al. (1984) Proc. Natl. Acad. Sci. USA, 81: 4809]. We examinedthe distribution of the 59- and 55-kDa catalase molecules indark- and light-grown tissues of pumpkin seedlings as well asin other plant species, using the immunoblotting technique.The ratios of the 59- and 55-kDa catalase species differed inthe pumpkin tissues examined. Light interferes with the conversionof the 59-kDa precursor to the 55-kDa form, especially in thecotyledons. The effect of light was less pronounced in the rootsand hypocotyls, indicating that the light regulation of theconversion is tissue-specific. Dark- and light-grown cotyledonsfrom cucumber and watermelon seedlings showed a similar lightregulation, suggesting that cucurbitaceous plants possess similarlight-regulatory mechanism. From the analysis of catalase proteinfrom various plant tissues, a limited correlation between molecularforms of catalase and different microbody populations was observed. (Received September 6, 1986; Accepted December 4, 1986)     

Healing modes correlate with visuotectal pattern formation in regenerating embryonic Xenopus retina

After removal of the nasal or the temporal two-thirds of the embryonic (stage 32) eye, the remaining one-third sized fragment undergoes wound healing and then, in most cases, regenerates to form a new eye. Using gross anatomy and histology techniques, we categorized eye fragments into three healing mode categories over the first 24 hr after surgery (stage 37-38). Representative animals were reared through metamorphosis and their visuotectal projections were assayed using standard electrophysiology techniques. In the "rounded-up" healing mode, the cut edges of the fragment pinch to close the wound; retinal cell type layers (pigmented retinal epithelium (pre), photoreceptors, interneurons, ganglion cells) and a lens are present by 24 hr postsurgery. No extraneous or disorganized cells are present either internal or external to the fragments. These fragments regenerated to form normal projections 83% of the time and pattern duplicated projections only 17% of the time. In the "intermediate" healing mode, wound closure is not complete by 24 hr post surgery and groups of disorganized cells are present in the fragment and amassed between the healing cut edges. These fragments formed pattern duplicated projections 72% of the time. In the tongue healing mode, an ectopic mass of cells, contiguous with the main body of the fragment, forms a supernumerary retina in the region of the ablation. At 24 hr post surgery, the cells of the main body fragment form retinal layers; the cells of the tongue, excluding the presence of differentiated pre cells, remain undifferentiated, resembling ciliary margin. The cut edges of the main body fragment eventually fuse with the tongue to form a single eyeball. Tongue fragments formed pattern duplicated projections 100% of the time. In addition, pattern duplicated points derived from nasal fragments appeared most often in the posterior region of the tectum, the normal site of innervation of the nasal retina. This differed significantly from temporal fragment derived duplicated points which appeared more often in the front of the tectum, the normal site of innervation by temporal retina. Thus, the specificity of pattern duplicated innervation is related to the positional values remaining in the fragment after partial retinal ablation. The data indicate that cell movements during healing, whether overt as in the tongue healing mode, or remaining internal to the fragment as in the intermediate healing mode, are intimately correlated with pattern forming mechanisms which underlie pathological visuotectal duplication.     

Positional heterogeneity of interaction between fragments of avian limb bud in organ culture

We have previously succeeded in culturing whole leg bud from stage 21-23 chick embryos and observed a leg structure with typical cartilage pattern in vitro. In the present study, we have attempted the organ culture of the fragmented leg bud and investigated its capacity to form cartilage. Leg buds from stages 17-21 chick embryos were dissected into four pieces in the anteroposterior sequence (named 1, 2, 3, and 4, respectively) and cultured on a membrane filter in a medium consisting of Ham's F-12, chick serum, and chick embryo extract. After 6 days in culture, two central fragments (2 and 3) developed into large cartilaginous masses, while anterior (1) and posterior (4) fragments formed few or small cartilaginous masses. In addition, when these less chondrogenic fragments were combined, pinned together, and cultured, large cartilaginous masses were formed from 1 + 4 combinations but not from 1 + 1 or 4 + 4 combinations. These observations were analyzed quantitatively by measurement of 35SO4 incorporation into the sulfated glycosaminoglycan (S-GAG) and of final DNA content per explant, and by histological reconstruction of the chick-quail chimera explant. The results showed that (a) the 1 + 4 combination resulted in higher S-GAG synthesis and final DNA content than the 1 + 1 or 4 + 4 combinations in stage 18 and 21 leg buds (P less than 5%); (b) removal of ectoderm from the leg bud inhibited the increase observed for the 1 + 4 combination; c) in chick-quail chimera explants the cartilage formed from the 1 + 4 combination was largely of fragment 1 origin. These results demonstrate, first, the presence of a difference in chondrogenic capacity along the anteroposterior axis in the leg bud and, second, the occurrence of an interaction between anterior and posterior fragments which mimics the effects of grafting a zone of polarizing activity (ZPA). The mechanism of ZPA function is still unknown but the ectoderm may play some role. Some roles for ectoderm in ZPA function and differences in mesodermal responsiveness to ZPA factor(s) are suggested.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Taxonomic studies ofAsarum sensu lato

The floral vascular anatomy of 12 species representing each ofAsarum s. str.,Asiasarum, Geotaenium, Heterotropa andHexastylis are compared to clarify intergeneric relationships. The five genera have basically similar structures in floral morphology and vasculature, and consistently have a six-carpelled compound ovary and the associated similar placental vasculature. They show, however, a significant difference in the position and the constituent of the “ventral” carpellary bundles in the placental axis betweenAsiasarum-Heterotropa-Hexastylis andAsarum-Geotaenium. InAsiasarum, Heterotropa andHexastylis the ventral bundles of each carpel are basically free and antilocular as expected in the least specialized compound ovary of angiosperms; in contrast, inAsarum andGeotaenium the ventral carpellary bundles are antiseptal and heterogenous (i.e., formed by the lateral fusion of ventral bundles of adjacent carpels). Shared probable apomorphic floral vasculature, as well as shared single style-column, suggests the closest mutual relationships betweenAsarum andGeotaenium. In terms of floral morphology and anatomy,Asiasarum, Heterotropa andHexastylis retain plesiomorphies. Possible chromosomal evolution in the related genera is also discussed.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Mutations in {beta}-Tubulin Block Transhyphal Migration of Nuclei in Dikaryosis in the Homobasidiomycete Coprinus cinereus

ß-Tubulins from fourteen benomyl-resistant strainsof the homobasidiomycete Coprinus cinereus, which carry thebenA, benB, benC or benD mutations, were analyzed by urea SDS-PAGEor isoelectric focusing and subsequent immunoblot analysis.Electrophoretic aberrations in a major ß-tubulin isotype,denoted ß1 were found in two strains, BEN154 and BEN215,both of which carry benomyl resistance mutations in benA ⁺ Theaberrations of ß1 in BEN154 and BEN215 cosegregatedwith benomyl resistance among the progeny of outcrosses of BEN154 and BEN215 to wild type, indicating that the ß1aberrations were caused by the benA mutations. Both the mutantand wild-type ß1 tubulins were present in the heterozygousdikaryons, BEN 154/wild-type and BEN215/wild-type, ruling outpost-translational modification as a possible cause for theaberrations in ß1. Thus, we conclude that benA isa structural gene for ß1. Transhyphal migration ofnuclei in dikaryosis was blocked in the mycelia of BEN 154 andin its progeny that carried benA (ß1 mutation), demonstratingthat microtubules are involved in the migration process. Nuclearmigration in dikaryosis seems to differ in terms of mechanism,at least in part, from the migration of tetrad nuclei from basidiainto prespores during formation of basidiospores and from themigration of nuclei from basidiospores into hyphae during germination,because a benA mutation blocked the former without affectingthe latter two processes. (Received May 19, 1989; Accepted August 30, 1989)     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.