Search

HIC1(hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified bothin vivoandin vitrowith O-linkedN-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of[3H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites forO-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown thatO-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that theO-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399–714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised againstO-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins areO-GlcNAc modified in their C-terminal tail (residues 670–711) and not in the DNA-binding domain, as for the full-length proteins. Thus, theO-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein, p53, selectively binds to supercoiled (sc) DNA lacking the specific p53 consensus binding sequence (p53CON). Using p53 deletion mutants, we have previously shown that the p53 C-terminal DNA-binding site (CTDBS) is critical for this binding. Here we studied supercoil-selective binding of bacterially expressed full-length p53 using modulation of activity of the p53 DNA-binding domains by oxidation of cysteine residues (to preclude binding within the p53 core domain) and/or by antibodies mapping to epitopes at the protein C-terminus (to block binding within the CTDBS). In the absence of antibody, reduced p53 preferentially bound scDNA lacking p53CON in the presence of 3 kb linear plasmid DNAs or 20 mer oligonucleotides, both containing and lacking the p53CON. Blocking the CTDBS with antibody caused reduced p53 to bind equally to sc and linear or relaxed circular DNA lacking p53CON, but with a high preference for the p53CON. The same immune complex of oxidized p53 failed to bind DNA, while oxidized p53 in the absence of antibody restored selective scDNA binding. Antibodies mapping outside the CTDBS did not prevent p53 supercoil-selective (SCS) binding. These data indicate that the CTDBS is primarily responsible for p53 SCS binding. In the absence of the SCS binding, p53 binds sc or linear (relaxed) DNA via the p53 core domain and exhibits strong sequence-specific binding. Our results support a hypothesis that alterations to DNA topology may be a component of the complex cellular regulatory mechanisms that control the switch between latent and active p53 following cellular stress. [ABSTRACT FROM AUTHOR]

The NMR structure of 2′,5′ d(GGGGCCCC) was determined to gain insights into the structural differences between 2′,5′- and 3′,5′-linked DNA duplexes that may be relevant in elucidating nature's choice of sugar-phosphate links to encode genetic information. The oligomer assumes a duplex with extended nucleotide repeats formed out of mostly N-type sugar puckers. With the exception of the 5′-terminal guanine that assumes the syn glycosyl conformation, all other bases prefer the anti glycosyl conformation. Base pairs in the duplex exhibit slide (−1.96 Å) and intermediate values for X-displacement (−3.23 Å), as in ADNA, while their inclination to the helical axis is not prominent. Major and minor grooves display features intermediate to A and BDNA. The duplex structure of iso d(GGGGCCCC) may therefore be best characterized as a hybrid of A and BDNA. Importantly, the results confirm that even 3′ deoxy 2′,5′ DNA supports duplex formation only in the presence of distinct slide (≥−1.6 Å) and X-displacement (≥−2.5 Å) for base pairs, and hence does not favor an ideal BDNA topology characterized by their near-zero values. Such restrictions on base pair movements in 2′,5′ DNA, which are clearly absent in 3′,5′ DNA, are expected to impose constraints on its ability for deformability of the kind observed in DNA during its compaction and interaction with proteins. It is therefore conceivable that selection pressure relating to the optimization of topological features might have been a factor in the rejection of 2′,5′ links in preference to 3′,5′ links. [ABSTRACT FROM AUTHOR]

The NMR structure of 2′,5′ d(GGGGCCCC) was determined to gain insights into the structural differences between 2′,5′- and 3′,5′-linked DNA duplexes that may be relevant in elucidating nature's choice of sugar-phosphate links to encode genetic information. The oligomer assumes a duplex with extended nucleotide repeats formed out of mostly N-type sugar puckers. With the exception of the 5′-terminal guanine that assumes the syn glycosyl conformation, all other bases prefer the anti glycosyl conformation. Base pairs in the duplex exhibit slide (−1.96 Å) and intermediate values for X-displacement (−3.23 Å), as in ADNA, while their inclination to the helical axis is not prominent. Major and minor grooves display features intermediate to A and BDNA. The duplex structure of iso d(GGGGCCCC) may therefore be best characterized as a hybrid of A and BDNA. Importantly, the results confirm that even 3′ deoxy 2′,5′ DNA supports duplex formation only in the presence of distinct slide (≥−1.6 Å) and X-displacement (≥−2.5 Å) for base pairs, and hence does not favor an ideal BDNA topology characterized by their near-zero values. Such restrictions on base pair movements in 2′,5′ DNA, which are clearly absent in 3′,5′ DNA, are expected to impose constraints on its ability for deformability of the kind observed in DNA during its compaction and interaction with proteins. It is therefore conceivable that selection pressure relating to the optimization of topological features might have been a factor in the rejection of 2′,5′ links in preference to 3′,5′ links. [ABSTRACT FROM AUTHOR]

During DNA replication, transcription and DNA repair in eukaryotes, the cellular machineries performing these tasks need to gain access to the DNA that is packaged into chromatin in the nucleus. Chromatin is a dynamic structure that modulates the access of regulatory factors to the genetic material. A precise coordination and organization of events in opening and closing of the chromatin is crucial to ensure that the correct spatial and temporal epigenetic code is maintained within the eukaryotic genome. This review will summarize the current knowledge of how chromatin remodeling and histone modifying complexes cooperate to break and remake chromatin during nuclear processes on the DNA template. [ABSTRACT FROM AUTHOR]

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5′...CC*T/AGG...3′ DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl- l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH2 group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5′...CCT/AGG...3′ sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA–M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl- l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl- l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs. [ABSTRACT FROM AUTHOR]

Reports on the isolation of a cDNa encoding a DNA-binding protein which binds specifically to the regulatory element. Deduction of amino-acid sequence of the insert identical to that corresponding to the DNA-binding domain; Disruption of the NF-IL6-binding site within the regulatory element that resulted in the disappearance of the transcriptional enhancing activity.

Analyzes the properties that cause the overexpression of DNA methyltransferase in myoblast cells. Acceleration of myotube formation; Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA; Methyl states of the HpaII sites in exon 1 through exon 2.

Demonstrates the properties of the strand-exchange protein 1 in Saccharomyces cerevisiae to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single-stranded DNA in vitro. Delineation of the mechanism by which the gene acts in the strand-exchange reaction; Analysis of mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1.

Eel natriuretic peptide receptor C (NPR-C) was cloned, characterized and found to have a unique interchain disulfide linkage when compared to that of mammalian NPR-C. The NPR-C cDNA was obtained from an eel gill cDNA library; the open reading frame codes for a polypeptide of 502 amino acids exhibiting the known features of NPR-C, including a weak ligand specificity and a disulfide-linked homodimeric structure. The deduced amino acid sequence shares approximately 60% similarity with the mammalian NPR-C but it lacks the Gly-rich prosequence present in the mammalian counterparts. Site-directed mutagenesis revealed that eel and mammalian NPR-C are quite different in their interchain disulfide-bonding pattern; eel uses the second Cys residue and mammals the fifth Cys residue for the covalent dimerization. The ligand-binding activity of the extracellular domain is not independent of the short cytoplasmic tail. RNase protection analysis revealed that the eel receptor is highly expressed in the gill and heart and, to a much lesser extent, in other tissues including the brain and intestine. The NPR-C mRNA levels were found to be down-regulated in most tissues when eels were transferred from fresh water to seawater; however, in the anterior intestine, the levels were up-regulated, suggesting that NPR-C plays a role in the adaptation to salinity changes in the euryhaline eel. [ABSTRACT FROM AUTHOR]

cDNA libraries constructed from poly(A)-rich RNA isolated from Allium sativum (garlic), Allium cepa (onion) and Allium ascalonicum (shallot) were screened for cDNA clones encoding the alliinase using colony hybridization. Sequence analysis of the alliinase cDNA clones from different Alliaceae species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level. Apparently, the alliinases are translated from mRNA species of approximately 2200 nucleotides. The primary translation products are preproproteins which are converted into the mature alliinases following post-translational modifications. In the case of the garlic alliinase, the mRNA encodes a 486-amino-acid polypeptide with a molecular mass of 55623 Da. Cleavage of the signal peptide (28 amino acids) results in a preprotein which extends 10 amino acids before the first amino acid of the mature protein of 51451 Da. Southern-blot analysis of genomic DNA has shown that the alliinases are most probably encoded by a family of closely related genes, which is in good agreement with the sequence heterogeneity found between different alliinase cDNA clones of one species. [ABSTRACT FROM AUTHOR]

A confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL-2H3). After stimulation with antigen (2,4-dinitrophenol-conjugated bovine serum albumin), fluo-3-fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Time-dependent profiles of the fluo-3-fluorescence intensities resembled closely the patterns of the sequential fluorescence-ratio images of fura-2, which were used to measure the intracellular free-calcium concentration ([Ca2+]i) in individual RBL-2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo-3-fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL-2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (bisbenzimide H 33342, a DNA- specific probe) which were produced by excitation with a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDA1 gene. To this end, the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDA1 gene abolished production of the E1α subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh-mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rhoo], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDA1 gene. Explanations for these phenotypes are discussed. [ABSTRACT FROM AUTHOR]

NAD+-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220–215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499–8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (> 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60–66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. α2 dimeric subunit structure, isoelectric pH, Km and kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity. [ABSTRACT FROM AUTHOR]

The promoter of the chicken α1(VI) collagen gene resembles the 5′-flanking regions of many housekeeping genes. It lacks a canonical TATAA box but contains potential binding sites for transcription factors AP1 and SP1. The promoter region has a relatively high GC content and forms a typical CpG island. In accordance with the absence of a TATAA element, the gene contains multiple transcription-initiation sites distributed over 80 bp genomic DNA. A 621-bp fragment derived from the 5′ end of the αl (VI) collagen gene is able to direct transcription of a heterologous reporter gene in transient-expression assays. Other DNA fragments that are either shorter or longer than the 621- bp fragment show markedly reduced promoter activity. Thus, the basic promoter element of the α1(VI) collagen gene must reside within this 621-bp fragment. [ABSTRACT FROM AUTHOR]

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11–14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiences or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate. [ABSTRACT FROM AUTHOR]

Ring-substituted diaqua(1,2-diphenylethylenediamine)platinum(II) sulfate shows unusual kinetics in its reaction with salmon testis DNA. The mechanism for diaqua[meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]platinum(II) sulfate, [Pt(H2O)2(meso-6)]2+ SO42-, a representative of this series, has been investigated and compared with that for cis-[Pt(NH3)2(H2O)2]2+. Reactions were followed by atomic absorption, analytical HPLC of Pt-DNA digests, arrest of enzymatic DNA synthesis/degradation, ultraviolet and fluorescence spectrophotometry. Except for the formation of monofunctional DNA adducts, the kinetics of the platinum(II) complexes are comparable. The pseudo-first-order rate constant for the attack of DNA by [Pt(H2O)2(meso-6)]2+ follows the concentration of DNA in a hyperbolic fashion, which is in contrast to the linear dependence for cis- [Pt(NH3)2(H2O)2]2+. The hyperbolic dependence is typical for a dissociable DNA/drug complex preceding the coordination reaction. By studying the binding of free ligand to DNA, and by correlating ligand structures and electrostatic charges with effects on adduct formation, both the phenyl residues and the positive charge of the platinum(II) complex are shown to be crucial for the stability of the dissociable complex. A non-intercalative mode of binding to the DNA backbone is suggested. At the high concentrations of DNA found in cell nuclei, the reaction of the dissociable complex can, principally, become rate-limiting in the attack of DNA and thus reduce the cytotoxic efficiency of a drug. [ABSTRACT FROM AUTHOR]

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. The first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N-terminal sequencing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence.In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12-kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASAII cDNA clones is almost identical to that of the second domain of the ASAI eDNA clones. [ABSTRACT FROM AUTHOR]

The origin of the anomalous H8 chemical shifts observed in ¹H-NMR spectra of oligonucleotides cross-linked at a GpG sequence with cis-(Pt(NH3)2]2+ has been investigated and clarified. The main contributions that distinguish the H8 resonances of the two platinum-ligating guanines from other GH8 signals and from each other are: (a) the inductive effect of platinum binding which we have recently quantified as a downfield shift of 0.48 ± 0.07 ppm (M. H. Fouchet. D. Lemaire, J. Kozelka and J.-C. Chottard, unpublished results); (b) the ring-current effect of one GpG guanine on the H8 resonance of the other guanine, which is negative (shielding) for the 5′-H8 and positive (deshielding) for the 3′-H8 in single-stranded adducts, but has the opposite sign in double-stranded adducts; (c) a deshielding polarization effect of the phosphate 5′ to the GpG unit. The different signs of the ring-current effects in single-stranded and double-stranded oligonucleotides originate from the orientation of the guanines in the cis-[Pt(NH3)2(Gua)2]2+ moiety (Gua, guanine), which is left-handed helicoidal in single strands and right-handed helicoidal in double strands. In the platinated dinucleotides (cis-[Pt(NH3)2(GpG)]4 m-[Pt(NH3)2{d(GpG)}]+ and cis-[Pt(NH3)2{d(pGpG)}]), the guanines assume either the left-handed or the right-handed arrangement, depending on the sugar moiety (ribose or deoxyribose), protonation state at N1 and, in the solid state, on crystal forces. This work shows that chemical shifts contain valuable structural information which is complementary to that extracted from correlated spectroscopy and nuclear Overhauser spectroscopy data. [ABSTRACT FROM AUTHOR]

The structural gene of the lanthionine-containing peptide antibiotic epidermin is located on a 54kb plasmid of Staphylococcus epidermidis [Schnell et al. (1988) Nature 333, 276-278]. A 13.5-kb DNA region neighbouring the epidermin structural gene (epiA) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, epic and epiD shared no homology with previously described proteins stored in data bases. They were located 3' adjacent to epiA. Using epiB as a probe, a 5-kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3-kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3' to epiA, epiB, epic and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis. [ABSTRACT FROM AUTHOR]

A DNA fragment encompassing the first exon and about 750 bp of the 5'-flanking sequence has been isolated and sequenced. The gene has multiple start sites of transcription which are dispersed over about 200 bp. The promoter lacks TATA and CAAT boxes and is very G+C-rich, with putative binding sites for the transcriptional factors Sp1 and AP2. Similar features are shared with two other brain-specific genes encoding thy-1 antigen and γ-enolase. The existence of a conserved block of similarity upstream of the human and rat aldolase C genes suggests that this region could be involved in tissue-specific expression whose mechanism seem to be, at least in part, transcriptional. [ABSTRACT FROM AUTHOR]

The remodeling of nucleoproteins during dog-fish spermiogenesis involves two successive nuclear protein transitions: the first from somatic-type histones to transition proteins during the nuclear elongation of spermatids and the second leading to protamine-DNA association in mature spermatozoa. The chromatin of elongating spermatids contains two transition proteins called S1 and S2. The amino acid sequence of protein S1, a polypeptide of 87 residues was determined previously [Chauvière, M., Martinage, A., Briand, G., Sautière, P. & Chevaillier, Ph. (1987) Eur. J. Biochem. 169, 105-111]. In the present paper, we report the elucidation of the primary structure of the minor transition protein S2 established by automated Edman degradation of the protein and of its fragments generated by cleavage at methionine and aspartate residues. S2 contains 80 residues and has a molecular mass of 9726 Da. S2 is mainly characterized by a high content of basic amino acids mostly represented by lysine, a relatively high level of hydrophobic residues, the presence of six phosphorylatable residues and the lack of cysteine. Its amino acid sequence shows that the N-terminal half is highly basic, while the acidic residues are located in the C-terminal part of the protein where more diversity in amino acids is noticed. The two transition proteins S1 and S2 share striking structural similarities. Few but significative similarities have been detected with the mammalian transition protein TP1 [Kistler, W. S., Noyes, C., Hsu, R. & Heinrikson, R. L. (1975) J. Biol. Chem. 250, 1847-1853], suggesting similar functions for all these proteins in chromatin remodeling during sperm differentiation. By contrast, the two dog-fish spermatid-specific proteins are structurally unrelated to sperm protamines and cannot be considered as their precursors. [ABSTRACT FROM AUTHOR]

Cultures of Escherichia coli (strains ML30 and K12 ABl157), synchronized by repeated phosphate starvation, were submitted to nutritional shifts-up at various cell ages. The progression of the replication forks was assessed by DNA-DNA hybridization of pulse-labelled chromosomal DNA with plasmid DNA probes containing specific chromosomal sequences. The rate of phospholipid synthesis and its cyclic discontinuities were measured by continuous and pulse labelling with palmitate. The DNA-DNA hybridization experiments showed that a shift-up induces a burst of initiation from the oriC region. These hybridization results, taken together with older data from the literature, suggest that most DNA initiations belonging to this burst are not followed by complete replication. Following a shift-up, the rate of phospholipid synthesis is maintained for 13–20 min, depending on cell age at shift-up, then doubles. The new steady-state rate of phospholipid synthesis is reached through a series of three doublings, while the cell mass doubles approximately twice. This discrepancy brings the rate of phospholipid synthesis per mass unit to its steady-state postshift value. [ABSTRACT FROM AUTHOR]

We have previously identified a patient with familial hypercholesterolaemia (FH), where the defect appears to be caused by a deletion in the 3′ region of the low-density lipoprotein (LDL)-receptor gene. We have now isolated the LDL-receptor gene from the patient and have studied the defect at the DNA level. Restriction mapping and sequence analysis demonstrate that a 4-kb DNA deletion has occurred between two alu-repetitive sequences that are in the same orientation, one in intron 12 and the other in intron 14. This deletion eliminates exons 13 and 14, and changes the reading frame of the resulting spliced mRNA such that a stop codon is created in the following exon. Immuno- and ligand-blot analysis using cultured fibroblasts from this patient revealed the normal gene product, but failed to detect any smaller receptor protein. This implies that the truncated receptor protein that is synthesised is rapidly degraded. We suggest that in this patient the deletion is caused by an unequal crossing-over event that occurred between two homologous chromosomes at meiosis. [ABSTRACT FROM AUTHOR]

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) Of the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp. Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors. [ABSTRACT FROM AUTHOR]

Protein extracts were prepared at various times after serum stimulation of growth-arrested mouse 3T3 fibroblasts. The extracts were fractionated by sucrose gradient centrifugation and used to determine the activities of DNA polymerase α and DNA primase. We found that polymerase and primase appeared in close association in one homogeneous 8.2-S peak. Neither polymerase, free of associated primase, nor primase, free of polymerase, could be detected at any time after serum stimulation. The activities of both enzymes started to increase concomitantly at the beginning of the DNA replication phase of the cell cycle. We found five to six times more DNA primase activity in replicating than in resting 3T3 cells. Besides DNA primase, a second additional priming activity could be detected. This activity sedimented at 12.5 S and corresponded most probably to RNA polymerase I. [ABSTRACT FROM AUTHOR]

A recombinant plasmid, pHW1, directing the overproduction of the enzyme deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) from Escherichia coli has been constructed. A 1900-base DNA fragment carrying the structural gene for the enzyme (dut) has been recloned into a runaway replication vector that also carries the strong leftward promoter (PL) of bacteriophage λ. Upon temperature shift, an E. coli strain carrying the new plasmid gives an increase in dUTPase activity of about 600-fold in rich medium compared to wild-type bacteria. The 64-kDa protein corresponding to the mature form of the enzyme reaches 20% of the total protein content of the bacterial cell. Using this strain, a simplified procedure has been developed for the purification of dUTPase. The purification steps consist of extraction of the cytoplasmic proteins, ammonium sulfate precipitation, anion-exchange chromatography and gel filtration on FPLC. The new overproducing plasmid and the simplified purification procedure developed will make it possible to purify dUTPase in sufficient amounts for detailed characterization studies. [ABSTRACT FROM AUTHOR]

Since the nucleotide sequence of bovine adrenodoxin cDNA is at variance with protein sequencing data in that it encodes an additional 14 amino acids at the COOH terminus, we used a specific antibody raised against this 14-amino-acid segment to examine its presence in: (a) nascent precursor polypeptide chains, (b) the processed mature adrenodoxin and (c) mitochondria of both steroidogenic and nonsteroidogenic tissues. These studies reveal the presence of the extra peptide in the precursor form derived from in vitro translation and in the newly synthesized mature form as shown by [35S]methionine labeling of proteins in adrenocortical cells. Both the purified COOH-terminal synthetic peptide and purified mature adrenodoxin competed with radiolabeled adrenodoxin for immunoprecipitation by the anti-peptide antibody. Immunoblots revealed the presence of the extra peptide in purified adrenodoxin and in bovine adrenocortical, corpus luteal, kidney and liver mitochondria while it was not detectable in heart mitochondria. Thus, we conclude that mature adrenodoxin and its homologs in non-steroidogenic tissues contain the C-terminal extension following uptake into mitochondria. These results indicate structural homology between adrenodoxin and the iron-sulfur proteins of the kidney and liver and also suggest the presence of a second iron-sulfur protein in kidney and liver. [ABSTRACT FROM AUTHOR]

Presents the results of the proton nuclear magnetic resonance (NMR) imaging studies in water carried out on the self-complementary DNA octamer and compared with similar studies on dimethylated analogue. Assignment of resonances by means of two-dimensional nuclear Overhauser spectroscopy; Analysis of proton-proton couplings to extract thermodynamic parameters for duplex-to-coil tarnsition.

Inverted repeats of pBR 322 and ColE1 DNAs have been analyzed for the presence of cruciform structures upon formation of nucleosomes, using S1, P1 and restriction enzyme analysis. In both cases the fraction of molecules showing nuclease-sensitive sites is unaffected by the DNA relaxation, owing to the formation of nucleosomes, A kinetic mechanism, based on the freezing of cruciform structures on the nucleosome surface or nearby, is proposed. This hypothesis is supported by a preferential location of nucleosomes at the DNA sequences containing the nuclease-sensitive sites, as indicated by restriction enzyme analysis and electron microscopy visualization after psoralen cross-linking. [ABSTRACT FROM AUTHOR]

The enzyme GTP: α-D-mannose-1-phosphate guanylyltransferase from porcine thyroid tissue has been purified 69900-fold on columns of blue-Sepharose, DEAE-Sepharose, phenyl-Sepharose and agarose-GTP affinity materials. Although it exhibits a tendency to aggregate, the enzyme travelled, upon sucrose velocity sedimentation, as a single oligomer with a molecular mass of 412 kDa. Michaelis constants were determined to be 1.0μM, 1.0 mM, 3.5 mM and 0.4 mM for GDP-α-D-mannose, pyrophosphate, GTP and mannose-1-phosphate, respectively. The enzyme appears to be specific for the mannose moiety but will accept an inosine replacement for guanine and a deoxyribose replacement for ribose in GTP. [ABSTRACT FROM AUTHOR]

Restriction endonucleases are enzymes which recognize short DNA sequences anti cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonuclease are homodimers which recognize short palindromic sequences 4-8 bp in length and, in the presence of Mg2+ cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10-12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriction endonucleases are not only functionally but also evolutionarily related. [ABSTRACT FROM AUTHOR]

We have investigated the influence of the nucleotide sequence adjacent to the recognition site on the rate of cleavage of DNA by the restriction endonuclease EcoRI. For this purpose two decadeoxynucleotides, d(G-G-G-AA-T-T-C-T-T) (Ia) and d(A-A-G-A-A-T-T-C-C-C) (Ib) were synthesized. The duplex Ia -Ib is cleaved by EcoRI preferentially in the dA-rich strand (approximately 10 times over the dG-rich strand). The individual nucleotides Ia and Ib are also cleaved by EcoRI, Ib at a higher rate than Ia and both at a lower rate than Ia · Ib. The temperature dependence of the reaction rate shows that only double-stranded oligodeoxynucleotides are substrates for the EcoRI endonuclease. We have, furthermore, synthesized oligomers of d(G-G-A-A-T-T-C-C), which contain two, three and four EcoRI sites, respectively. These oligodeoxynucleotides are preferentially cleaved at the sites next to the 5′ end, where the recognition site is only flanked by one dG · dC base pair, in contrast to the other sites which are flanked by three such pairs. These data indicate that sequences adjacent to the recognition site influence the rate of cleavage: dA · dT base pairs enhance and dG · dC base pairs slow down the hydrolytic activity of the EcoRI endonuclease. [ABSTRACT FROM AUTHOR]

We have investigated the sequence organization of the very small genome (DNA content/haploid cell c = 0.4 - 0.5 pg) of a tetraodontid fish, Arothron diadematus, by using two main experimental approaches. The first one, renaturation kinetics, showed that slowly reassociating, intermediate, fast and foldback sequences represented 87 %, 7 %, 5 % and 1 %, respectively, of A. diadematus DNA, which is, so far, the vertebrate DNA lowest in repeated sequences. The second approach, centrifugation in CS2SO4/BAMD density gradients [BAMD -- bis(acetate-mercurimethyl) dioxane], showed that A. diadematus DNA can be resolved into several components, characterized by buoyant densities of 1.700, 1.7045, 1.708, 1.712 and 1.723 g/cm³ and representing 15%, 73%, 4%, 4% and 2.5 %, respectively, of total DNA. The last component comprised a satellite DNA and ribosomal DNA. A family of interspersed repeats, possibly related to the AluI family of warm-blooded vertebrates, showed an extremely specific genomic distribution, being present in only the 1.708 g/cm³ component, which it matched in base composition. [ABSTRACT FROM AUTHOR]

Transcription of a cloned cauliflower mosaic (CaMV) DNA fragment (plasmid pCa8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination. ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3′-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment. [ABSTRACT FROM AUTHOR]

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG · dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S l protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 × 103 bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals. [ABSTRACT FROM AUTHOR]

The toxicity of ascorbate towards phage lambda and the phages T2-T7 has been investigated. At room temperature the T-odd and lambda bacteriophages arc highly susceptible to ascorbate-induced damage, whereas the T-even phages are practically resistant. The toxicity of ascorbate is dependent on the presence of copper (or iron) and oxygen, although oxygen is not required in the presence of H2O2. Hydrogen peroxide is essential for the ascorbate- induced phage inactivation and the damage is prevented by catalase. At the concentrations used, most of the copper ions are bound to the phage particles. Chelating agents such as EDTA or histidine fully protect the phages, whereas sallicylate only reduces the rate of phage inactivation. OH scavengers such as sucrose, formate, mannitol, tert-butyl alcohol or poly(ethylene glycol) have no protective effect. Experiments with DNA labeled phages indicate that both phage adsorption and DNA injection are impaired as a result of the exposure to ascorbate and copper. The failure to express the viral genetic information as a result of single and double-strand breaks in the DNA, probably also contribute to the loss of the plaque-forming ability of the phages. The results are interpreted in terms of a ‘site-specific’ Fenton mechanism according to which the binding of the transition metal ions to the biological target is a prerequisite for the production of damage. The bound metal ion is reduced either by O[This symbol cannot be presented in ASCII format], ascorbate or other reductants and is subsequently reoxidized by H2O2 yielding OH' radicals. This cyclic redox rcaction of the metal gcnerates OH' radicals which react with vital macromolecules with a high probability of causing ‘multi-hit’ damage. This ‘site-specific’ formation of OH' radicals, which lakes place near the target molecules, accounts both for the high damaging efficiency and for the failure of OH' scavengers to protect against it. [ABSTRACT FROM AUTHOR]

Proton NMR studies at 500 MHz in aqueous solution were carried out on the G-G chelated deoxytrinucleosidediphosphate platinum complex cis-Pt(NH3)2{d(GpCpG)}, on the uncoordinated trinucleotide d(GpCpG) and on the constituent monomers cis-Pt(NH3)2{d(Gp)}2, cis-Pt(NH3)2{d(pG)}2, d(Gp), d(pCp) and d(pG). Complete NMR spectral assignments are given and chemical shifts and coupling constants are analysed to obtain an impression of the detailed structure of d(GpCpG) and the distortion of the structure due to chelation with [cis-Pt(NH3)2]2+. Platination of the guanosine monophosphates affects the sugar conformational equilibrium to favour the N conformation of the deoxyribose ring. This feature is also apparent in ribose mononucleotides and is possibly caused by an increased anomeric effect. In cis-Pt(NH3)2{d(pG)}2 the phase angle of pseudorotation of the S-type sugar ring is 20° higher than in 'free' d(pG) which might be an indication for an ionic interaction between the positive platinum and the negatively charged phosphate. It appears that d(GpCpG) reverts from a predominantly random coil to a normal right-handed B-DNA-like single-helical structure at lower temperatures, whereas the conformational features of cis-Pt(NH3)2{d(GpCpG)} are largely temperature-independent. In the latter compound much conformational freedom along the backbone angles is seen. The cytosine protons and deoxyribose protons exhibit almost no shielding effect as should normally be exerted by the guanine bases in stacking positions. This is interpreted in terms of a 'turning away' of the cytosine residue from both chelating guanines. Conformational features of cis-Pt(NH3)2{d(GpCpG)} are compared with the 'bulge-out' of the ribose-trinucleotide m62ApUpm4... [ABSTRACT FROM AUTHOR]

The sequence of the deoC gene of Escherichia coli K12 and the amino acid sequence of the corresponding protein, deoxyriboaldolase, has been established. The protein consists of 259 amino acids with a molecular weight of 27 737. The purified enzyme may exist both as a monomer and as a dimer. On the basis of amino acid composition, molecular weight and catalytic properties, the enzymes from E. coli and Salmonella typhimurium seem to be almost similar. They belong to the class I aldolases, which form Schiff base intermediates. Using data for the S. typhimurium enzyme, the lysine residue involved in the active site in the E. coli enzyme was tentatively identified. [ABSTRACT FROM AUTHOR]

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work was to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength. [ABSTRACT FROM AUTHOR]

The DNA-dependent RNA polymerase II or B from the lower eukaryote Physarum polycephalum has been purified to apparent homogeneity by a new method employing poly(ethylene imine) precipitation and elution, and heparin-Sepharose affinity chromatography. The method is readily scaled up or down and affords a purification of over 5000-fold with a yield of 35-45%. The procedure is easy to perform and can be carried out in less than three days even on a large scale. Furthermore, it gives enzyme of higher purity and in at least 10-fold greater yield than previously published procedures for its purification from this organism. These improvements have allowed the detection of a series of subforms of the enzyme. The combination of precipitation using poly(ethylene imine) with chromatography on heparinSepharose may prove useful in the preparation of other proteins which interact with nucleic acids. [ABSTRACT FROM AUTHOR]

The c2 repressor of phage P22 has been purified to homogeneity. It specifically binds to λimm21 and P22 DNA. Its affinity for the presumed operator mutant P22 virB is reduced. The initial dissociation rates of the complex between c2 repressor and λimm21 DNA are 0.02 min-1 at 0 °C, 0.08 min-1 at 20 °C and 0.17 min-1 at 32 °C. The dissociation rates of complexes formed between the c2 repressor and the λimm21 operators OR, OL and OR vira were measured and compared to the corresponding rates obtained with 21 cl repressor. [ABSTRACT FROM AUTHOR]

A deoxyribonuclease has been purified more than 2000-fold from the green algae. Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is at pH 8.5, in 80 mM Tris-HCl buffer and 2 mM CaCl2. Other divalent cations can replace Ca2+ with varying lower efficiency. EDTA and inorganic phosphate are strongly inhibitory, while ATP and high concentrations of 2-mercaptoethanol are slightly inhibitory. The molecular weight is approximately 35000, the Stokes radius is 2.7 nm, and the sedimentation coefficient 2.8 S. It is a single polypeptide chain, and the frictional ratio of 1.27 suggests it is only slightly asymetrical. The isoelectric point is 9.5. This enzyme has been termed exonuclease. [ABSTRACT FROM AUTHOR]

An F+ derivative of Escherichia coli E 508 thermosensitive in dnaA function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E 508 (ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M 13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal. phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also, but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates.: (a) parental replicative form DNA synthesis is not dependent on DNA function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dnaA mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dnaA function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M 13 DNA synthesis is discussed. [ABSTRACT FROM AUTHOR]

Purified T5+ DNA in nicked form and after repair of the specific single-strand interruptions with DNA ligase was used in transcription studies using Escherichia coli RNA polymerase (holoenzyme) in the presence and absence of E. coli termination protein rho. The transcriptional products were analyzed with respect to their size distribution and the sequences transcribed from the different templates. The results indicate that the single-strand breaks in the DNA of bacteriophage T5, though in genetically defined positions, do not have any specific effect on transcription in vitro. Furthermore, the E. coli rho protein, although it depresses net RNA synthesis and reduces the average molecular weight of the transcripts, seems to act in a non-specific way in this system. [ABSTRACT FROM AUTHOR]

1. The α and β anomers of 5-ethyl-2'-deoyuridine, the latter of which is a thymidine analogue with antiviral activity, have been subjected to a conformational analysis in aqueous medium by proton magnetic resonance (PMR) spectroscopy with the aid of the iteration program LAOCOON-3. 2. The results demonstrated a marked preference for the conformation C-2'-endo of the deoxyribose rings of both anomers; and this appears to be an inherent property of the deoxyribose ring inself. As regards the exocyclic 5'-CH2OH group, the β anomer exhibited a preference for the gauchegauche conformation; in the case of the α anomer, all three classical conformers were equally favoured. 3. A particularly extensive analysis of the conformation about the glycosidic bond, based on the chemical shifts of several sugar-protons, pointed to the conformation anti for both anomers. 4. The data for the protons of the 5-ethyl side chain are in accord with a symmetrical distribution of the rotamers of this substituent relative to the plane of the pyrimidine ring. 5. The overall findings are compared with those earlier reported for the α and β anomers of thyroidine, as well as a number of other nucleosides nucleotides and polynucleofides. 6. PMR spectral data were similarly utilised to perform a conformafional analysis of the α and β anomers of 5-ethyl-2'-deoxycrytidine, an analogue of 5-methyldeoxycytidine; the results did not differ appreciably from those for the 5-ethyldeoxyuridine anomers. [ABSTRACT FROM AUTHOR]

Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine. Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1-phosphate and/or the level of thymidine phosphorylase. Thymine incorporation in wild-type cells could be observed if the cells possessed an increased deoxyribose 1-phosphate pool. The kinetics of thymine uptake in wild-type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration. The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that th3Tnidine is synthesized from deoxyribose 1-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside. [ABSTRACT FROM AUTHOR]

It is shown that preinitiator or storage stretches on TS-DNA are not a consequence of the existence of natural nicks in this template. Nicks do not contribute to the formation of heparin-labile and stable starters. Analysis of the number of pppA and pppG starters and their products on T7-DNA in enzyme saturation in the presence of heparin yielded 8 stable pppA starters, all synthesizing one product from the r-strand sedimenting with 32 S and with a molecular weight of 1.9×106 (from sedimentation rate) or 2.2×l06 (from chain length). Six labile pppG starters, yielding products from the 1-strand and, in addition, two very labile pppG starters seen only in the absence of heparin, also on the 1-strand, were demonstrated. These results give further evidence for the existence of pre-initiator (or storage) stretches, width another template for which no assumption about the number of reading units have to be made. The polyanion heparin is well suited for the elimination of artificial starts in vitro. [ABSTRACT FROM AUTHOR]

The distribution of the mitochondrial DNA among the allomorphic forms, supercoiled circle, open circle and linear rod is a characteristic feature of each cell line. After pulse-chase labeling with [Me-3H]thymidine the bulk of the radioactivity can be located either in the supercoiled circular mitochondrial DNA (monkey Vero, ATCC; CCL 81) or in the open circular mitochondrial DNA (mouse bTCTC 929, ATCC; CCL 1). The open circular mitochondrial DNA from Vero cells contains one single nick, which is located in the heavy strand (alkaline CsC1 gradient). In contrast, the open circular mitochondrial DNA from mouse cells contains one nick in one strand and at least two nicks in the opposite strand. The bulk of the supercoiled Vero mitochondrial DNA has a heat-labile site in the heavy strand and one site in both strands at which the phosphodiester backbone can be hydrolyzed by pancreatic RNAase. [ABSTRACT FROM AUTHOR]

A purification procedure to prepare highly purified DNA-dependent RNA polymerase from Bacillus subtilis vegetative cells is described. The enzyme consists of four different subunits, β′, β, α (molecular weights 154000 for β′ and β, 56000 for α and 43000 for α) in a molar ratio 1:1:1:2. By phosphocellulose chromatography RNA polymerase has been disseociated in α subunit and core enzyme, containing β′, β and α subunits in a molar ratio 1:1:2 the α subunit stimulates the activity of core polyrnerase with ϕ 29 DNA but not with poly[d(A--T)]. The general properties of the enzyme are also described. [ABSTRACT FROM AUTHOR]