Identification of a soybean chloroplast DNA replication origin-binding protein

Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS-PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.     

Isolation, characterization and restriction endonuclease mapping of the Petunia hybrida chloroplast DNA

A procedure is developed for the isolation of intact chloroplast DNA (ctDNA) from Petunia hybrida. The molecular weight, calculated from contour length measurements, is 96.0 +/- 4.5 x 10(6) daltons. This value is in good agreement with the value of 101.2 x 10(6) daltons that was estimated from the electrophoretic mobilities of restriction endonuclease fragments of ctDNA. Analysis of petunia ctDNA in neutral CsCl gradients revealed the presence of only one type of DNA at a buoyant density of 1.6987 +/- 0.0005 gcm-3. This corresponds with a GC-content of 39.3 +/- 0.5%. A physical map of petunia ctDNA was constructed by using the restriction endonucleases Sal I, Bgl I, Hpa I and Kpn I. The map indicates that petunia ctDNA contains two copies of a sequence in an inverted orientation. The inverted repeat regions have a minimum length of 10 x 10(6) daltons. Hybridization data indicate that part of the inverted repeat regions contain the genes for chloroplast ribosomal RNAs.     

Ribonucleic acid synthesis in chloroplasts

Chloroplasts isolated from young spinach leaves incorporate [(3)H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7x10(6) and two minor products of molecular weight 1.2x10(6) and 0.47x10(6). These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of (32)P(i) into chloroplast rRNA species (mol.wt. 1.05x10(6) and 0.56x10(6)) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34x10(6) and 0.7x10(6)). Incorporation of (32)P(i) into chloroplast RNA species of molecular weight 2.7x10(6), 1.2x10(6), 0.65x10(6) and 0.47x10(6) proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2x10(6) and 0.65x10(6) being precursors to the more slowly labelled rRNA species of molecular weight 1.05x10(6) and 0.56x10(6) respectively.     

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.     

RNA-dependent RNA polymerase activity in coronavirus- infected cells

An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.     

Purification and characterization of the corticosteroid-binding globulin of pregnant guinea pig serum.

The corticosteroid-binding globulin from guinea pig pregnancy serum was purified by the sequential use of affinity chromatography, hydroxylapatite chromatography, and gel filtration chromatography at a cumulative yield of 80%. The protein was found to be homogeneous by analytical gel electrophoresis, equilibrium sedimentation ultracentrifugation, immunoelectrophoresis, and stoichiometry (1:1) of steroid binding. Guinea pig corticosteroid-binding globulin has a molecular weight of 43 300 and contains 29% carbohydrate. The intrinsic fluorescence of the corticosteroid-binding globulin is quenched by about 73% when 1 mol of cortisol is bound. The association constants (pH 7.4) at 4 and 37 degrees C are 2.5 X 10(7) and 1.5 X 10(6) M-1 for cortisol and 1.4 X 10(6) and 0.2 X 10(6) M-1 for progesterone, respectively.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship.

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58-69% recovered in the 100 000 X g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 +/- 13.6% (S.D.) and triacylglycerol lipase 11.9 +/- 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, The hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent Km for substrate and molecular weight 3-10(5)-6-10(5) by gel filtration and a minor component of lower apparent Km and heterogeneous molecular weight above 1 million, which was found mostly in complex and lipid.     

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.     

Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage.

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.     

Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves.