[Sequence-specific endonucleases involved in genetic recombination].
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Amino Acid Sequence↗
Biomedical subjects
N Morishima
Publications and source records attributed to N Morishima.
A subunit of yeast site-specific endonuclease SceI is a mitochondrial version of the 70-kDa heat shock protein.
Endo.SceI of Saccharomyces cerevisiae is a heterodimeric site-specific endonuclease, which is distinguishable from prokaryotic restriction endonucleases in the mode of recognition of its cleavage site. We have used monoclonal antibodies specific to the larger subunit (75 kDa) of Endo.SceI to isolate the gene for the subunit (ENS1) from S. cerevisiae. Unexpectedly, ENS1 was found to encode a 70-kDa heat shock protein-related polypeptide and to be identical to recently cloned SSC1. Subcellular fractionation experiments on yeast cells revealed that the primary target site of the larger subunit is mitochondria, where almost all the Endo.SceI activity is localized. Molecular genetic analysis of ENS1 demonstrated its indispensability for growth and the requirement of a high level of its expression at the sporulation and germination stages. The data suggest that ENS1 plays an important role, especially at these differentiation stages.
Amino Acid Sequence↗
Cytochrome P-450scc-catalyzed production of progesterone from 22R-hydroxycholest-4-en-3-one by way of 20,22-dihydroxycholest-4-en-3-one.
Transient accumulation of a dihydroxylated steroid was found when 22R-hydroxycholest-4-en-3-one was used as the substrate for a reconstituted cholesterol side-chain cleavage system derived from bovine adrenocortical mitochondria. The indications were that the accumulated steroid was an intermediate in the cytochrome P-450scc-catalyzed reaction. The retention time of the accumulated intermediate was identical with that of authentic 20,22-dihydroxycholest-4-en-3-one on HPLC. When 22R-hydroxycholesterol and 22R-hydroxycholest-4-en-3-one were incubated simultaneously, the total amount of reaction products was essentially the same as that observed with 22R-hydroxycholest-4-en-3-one alone. Under the conditions employed, the apparent turnover number of cytochrome P-450scc for 22R-hydroxycholesterol was calculated to be 77 nmol/min/nmol P-450 from the amount of pregnenolone formed, whereas the apparent turnover number for 22R-hydroxycholest-4-en-3-one was 64 nmol/min/nmol P-450 with respect to the intermediate formation and 77 nmol/min/nmol P-450 with respect to the progesterone formation. The apparent turnover number for 20,22-dihydroxycholest-4-en-3-one was about 125 nmol/min/nmol P-450, which was not significantly different from that of 20,22-dihydroxycholesterol. The apparent Km for 22R-hydroxycholesterol was about 20 microM and those for 22R-hydroxycholest-4-en-3-one and 20,22-dihydroxycholest-4-en-3-one were 50 and 40 microM, respectively. Thus, 22R-hydroxycholest-4-en-3-one was efficiently metabolized to progesterone by way of 20,22-dihydroxycholest-4-en-3-one by cytochrome P-450scc.
Adrenal Glands↗
Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells.
A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.
ADP-Ribosylation Factors↗
Sensitive assay of cytochrome P450scc activity by high-performance liquid chromatography.
We have developed a simple procedure for analyzing the reaction intermediates and product of the cholesterol side-chain cleavage system by high-performance liquid chromatography with uv absorption monitoring. After the cholesterol side-chain cleavage system had been incubated and the reaction then halted by heat treatment, the product was converted into 3-one-4-en steroid showing intense absorption at 240 nm upon reaction with cholesterol oxidase. The converted steroids were then analyzed by normal-phase HPLC. In consequence, the catalytic activity of the reconstituted adrenocortical cytochrome P450scc system was readily assayed with a sensitivity more than 10-fold higher by this conversion. Also, it was shown that 22R-hydroxy-cholest-4-en-3-one could serve as a good substrate for cytochrome P450scc and that the 20R,22R-dihydroxy derivative could be clearly detected as a reaction intermediate in the reconstituted system.
Animals↗
Concurrent poststreptococcal acute glomerulonephritis and Schönlein-Henoch purpura.
A 5-year-old Japanese boy developed concurrent poststreptococcal acute glomerulonephritis (PSAGN) and Schönlein-Henoch purpura (SHP). An elevated titer of ASK on admission confirmed the preceding streptococcal infection. Arthritis of the left knee and petechiae on admission were regarded as features of SHP. The presence of SHP was further confirmed by the pathological finding of leukocytoclastic vasculitis in the skin. PSAGN was strongly suspected due to the findings of microscopic hematuria and hypocomplementemia in the acute phase. The concurrence of SHP and PSAGN suggests similar underlying pathophysiological processes as poststreptococcal sequelae. At the height of the illness, peripheral blood lymphocyte subset analysis showed a marked increase in the suppressor inducer T subset and a reciprocal decrease in the helper T subset. This alteration in T lymphocyte subsets was regarded as indicative of the immunological derangement in this patient.
Child, Preschool↗
Gene structure of cytochrome P-450(M-1) specifically expressed in male rat liver.
Cytochrome P-450(M-1) [P-450(M-1)] is specifically expressed in adult male rat liver [Yoshioka, H., Morohashi, K., Sogawa, K., Miyata, T., Kawajiri, K., Hirose, T., Inayama, S., Fujii-Kuriyama, Y., & Omura, T. (1987) J. Biol. Chem. 262, 1706-1711]. Isolation and analysis of the gene for P-450(M-1) revealed that the coding region of the gene is interrupted by eight introns and is dispersed over a 35-kilobase pair region of chromosomal DNA. Intron insertion sites of the P-450(M-1) gene are located at equivalent positions to those of cytochrome P-450b and P-450e, which are phenobarbital-inducible. Sequence analysis of the 5'-upstream region of the P-450(M-1) gene shows that there is a homologous sequence to glucocorticoid regulatory elements (GRE) identified in glucocorticoid-responsive genes.
Amino Acid Sequence↗
Active site organization of bacterial type I fatty acid synthetase.
Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Mapping of acyl carrier domain within the subunit of type I bacterial fatty acid synthetase.
A fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, was used to label the acyl carrier site of the bacterial fatty acid synthetase from Brevibacterium ammoniagenes. The reagent bound preferentially to the 4'-phosphopantetheine thiol group of the acyl carrier domain and irreversively inactivated the enzyme. The modified enzyme was cleaved by proteinases for the mapping of the labeled site. The fluorescent fragment was readily detected on a polyacrylamide gel after electrophoresis. The region of 45 kDa containing the 4'-phosphopantetheine was located on the polypeptide at around two-thirds of the full length from the N-terminal.
Acyl Carrier Protein↗
Role of subunit interactions in the self-assembly of oligomeric proteins.
In oligomeric proteins, the native conformation and its functional properties depend on the interactions which exist between the different chains. The role of these subunit interactions can be studied using either the unfolded state or the native state as a starting point. During the folding process, the properties which appear following a bimolecular reaction are related to the formation of an association area. Similarly, the properties which are lost upon partial dissociation of the native state are related to the association area which is disrupted. Four examples are presented in this article: phosphofructokinase and aspartokinase-homoserine dehydrogenase from E. coli are studied through their folding process, and fatty acid synthetase from B. ammoniagenes and reptilian ovomacroglobulin are studied through their dissociated forms. In all cases, the function of the protein is a sensitive index of the formation of the subunit interactions, and can be more conveniently measured than other size/shape parameters. The extrapolation from the folding of small proteins to the assembly of large and complex structures can be reasonably achieved by admitting that subunit interactions are coupled to the subtle adjustments required by the protein to exert its biological function.
Animals↗
Structure of bacterial fatty acid synthetase from Brevibacterium ammoniagenes.
Hydrodynamic measurements and a cross-linking study with dimethyl suberimidate have shown that the native fatty acid synthetase from Brevibacterium ammoniagenes is a hexameric protein having a molecular weight of 1.56 . 10(6). The subunits of the enzyme are identical in size (Mr 2.6 . 10(5). The negatively stained fatty acid synthetase had an electron microscopic image of ellipsoidal structure with major and minor axes approximately equal to 270 A and 180 A, respectively. The electron microscopic image is similar to that of the yeast enzyme, which is quite distinct from the B. ammoniagenes enzyme with respect to the subunit composition. The inactivated enzyme prepared by dialysis against a lower ionic strength solution was partially reactivated by raising the ionic strength. Ellipsoidal images similar to those of the native enzyme were found in the electron micrograph of the reactivated enzyme. Sucrose density gradient centrifugation of the reactivated enzyme sample showed that the active component had almost the same sedimentation coefficient as the native hexamer. These results indicate that the enzyme is active only in its hexameric state.
Amino Acids↗
[Silver's syndrome; a case report and literature review].
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Body Constitution↗
A sequence-specific endonuclease, Endo.SceI, can efficiently induce gene conversion in yeast mitochondria lacking a major exonuclease.
Endo.SceI most likely initiates homologous recombination of yeast mitochondrial genome through sequence-specific double-strand scission of DNA. According to the double-strand break-repair model for the mechanism of homologous recombination, DNA ends created by sequence-specific endonucleases have to be processed by exonucleases. The major mitochondrial exonuclease (encoded by NUC1) has been shown to greatly affect the length of conversion tracts at the 21S rRNA locus when site-specific gene conversion is induced by omega endonuclease. In order to examine the role of the NUC1 nuclease in the Endo.SceI-induced recombination, recombination frequencies were measured after crossing of parental strains either in the presence or absence of NUC1 nuclease activity. The frequency of gene conversion in the oli2 locus induced by Endo.SceI was not reduced by disruption of the NUC1 gene. This result strongly implicates the presence of multiple exonucleases for the processing of the DNA ends created by sequence-specific endonucleases.
Base Sequence↗
Indocyanine green angiographic findings of lacquer cracks in pathologic myopia.
Lacquer cracks are thought to represent healed mechanical breaks in the retinal pigment epithelium, Bruch's membrane, and choriocapillaris complex. In this study, we analyzed the indocyanine green (ICG) angiographic features of lacquer cracks and compared them with findings using fluorescein angiography. Complete ophthalmologic examinations, fluorescein angiography, and ICG angiography were performed in 29 consecutive patients (37 eyes) with lacquer cracks. Fluorescein angiograms of the cracks revealed linear hyperfluorescence in all 37 eyes. Using ICG angiography, we observed linear hypofluorescence in all 37 eyes. In 15 of 37 eyes, the length of the hypofluorescent lesion detected by ICG angiography was longer than the hyperfluorescent lesion observed by fluorescein angiography. In 17 of 37 eyes, more lacquer cracks were observed by ICG angiography than by fluorescein angiography. These findings indicate that ICG angiography can detect the development of the lesion more precisely, and may provide useful information for diagnosing pathologic myopia.
Adolescent↗