Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp. CHTI 55 and its effect on the thermal stability of DNA

In the deoxyribonucleoprotein complex of Methanosarcina sp. CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) (Mr 10,760) and MC2 (Mr 17,000). Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1. The effect of Methanosarcina sp. CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E. coli DNA-binding protein II. Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms. Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA.     

Fragments of LINE-1 retrotransposons flanked by inverted telomeric repeats are present in the bovine genome. Homology with human LINE-1 elements

In the bovine genome we found two intrachromosomal DNA fragments flanked by inverted telomeric repeats (GenBank Accession Nos. AF136741 and AF136742). The internal parts of the fragments are homologous exclusively to the human sequences and to the consensus sequence of the L1MC4 subfamily of LINE-1 retrotransposons which are widespread among mammalian genomes. We found that distribution of homologous human sequences within our fragments is not random, reflecting a complicated pattern of insertion mechanisms of and maintenance of retrotransposons in mammalian genomes. One of the possible explanations of the origin of LINE-1 truncated elements flanked by inverted telomeric repeats in the bovine genome is that extrachromosomal DNA fragments may be modified by telomerase and subsequently, transferred into chromosomal DNA.     

Differential activation of p53 by the various adducts of mitomycin C

Mitomycin C (MC) is a cytotoxic chemotherapeutic agent that causes DNA damage in the form of DNA cross-links as well as a variety of DNA monoadducts and is known to induce p53. The various DNA adducts formed upon treatment of mouse mammary tumor cells with MC as well as 10-decarbamoyl MC (DMC) and 2,7-diaminomitosene (2,7-DAM), the major MC metabolite, have been elucidated. The cytotoxicity of DMC parallels closely that of MC in a number of rodent cell lines tested, whereas 2,7-DAM is relatively noncytotoxic. In this study, we investigate the ability of MC, DMC, and 2,7-DAM to activate p53 at equidose concentrations by treating tissue culture cell lines with the three mitomycins. Whereas MC and DMC induced p53 protein levels and increased the levels of p21 and Gadd45 mRNA, 2,7-DAM did not. Furthermore, MC and DMC, but not 2,7-DAM, were able to induce apoptosis efficiently in ML-1 cells. Therefore the 2,7-DAM monoadducts were unable to activate the p53 pathway. Interestingly, DMC was able to initiate apoptosis via a p53-independent pathway whereas MC was not. This is the first finding that adducts of a multiadduct type DNA-damaging agent are differentially recognized by DNA damage sensor pathways.     

DNA bending versus DNA end joining activity of HMGB1 protein is modulated in vitro by acetylation

The ability of HMGB1 protein to recognize bent DNA and to induce bending in linear duplex DNA defines HMGB1 as an architectural factor. It has already been demonstrated that the binding affinity of the protein for various bent DNA structures is enhanced upon in vivo acetylation at Lys2. Here we investigate how this modification of HMGB1 affects its ability to bend DNA. We report that the modified protein cannot bend short DNA fragments but, instead, stimulates joining of the same fragments via their ends. The same properties are exhibited in vivo by acetylated HMGB1 lacking its acidic tail. Further, in vitro acetylation of the truncated protein at Lys81 (possible upon tail removal only) restores the protein's bending ability, while the level of stimulation of DNA end joining is strongly reduced. We conclude, therefore, that the ability of HMGB1 to bend DNA or to stimulate end joining is modulated in vitro by acetylation. In an attempt to explain the properties of in vivo-acetylated HMGB1, its complexes with DNA have been analyzed by both protein-DNA cross-linking and atomic force microscopy. Unlike the parental protein, bound mainly within the internal sequences, acetylated HMGB1 binds preferentially to DNA ends. We propose that the loading of acetylated protein on DNA ends accounts for both the failure to bend DNA and the stimulation of DNA end joining.     

Transformation in bacillus subtilis: fate of transforming DNA in transformation deficient mutants.

Summary Transformation deficient mutants were isolated by means of selection for sensitivity to methylmethane-sulfonate (MMS). The mutations were introduced into a multiple auxotrophic highly transformable recipient. The transformation deficient strains were characterized with respect to their sensitivity to UV-irradiation and treatment with MMS and mitomycin-C (MC) and with respect to both the physico-chemical and biological properties of reextracted donor DNA.As has been established previously (Davidoff-Abelson and Dubnau, 1973b) in the transformation proficient wild-type, double-stranded fragments (DSF), single-stranded fragments (SSF) and donorrecipient complex (DRC) are formed from donor DNA.The mutants we report on are of various types: Mutant 7G-73 (transformation frequency about 25 times lower than in the wild-type) is sensitive to UV-irradiation and treatment with MMS and MC, and is extremely deficient in the production of DRC.Mutant 7G-84 (transformation frequency about 12 times lower than in the wild-type) shows also sensitivity to UV-irradiation and treatment with MMS and MC. However, although it forms DSF, SSF, and DRC, the biological activity of DNA re-extracted from transforming cultures of 7G-84 is much reduced as compared to that of the wild-type.Mutant 7G-97 (transformation frequency about 500 times lower than in the wild-type) shows approximately wild-type resistance to UV-irradiation and treatment with MMS and MC, and forms DSF exclusively; the donor DNA is not processed further.Double mutants6G-103 and 6G-105, constructed by transformation of mutant 7G-97, with DNA from 7G-84 and 7G-73, are about 1250 and 5000 times less transformable than the wild-type respectively. They are sensitive to UV-irradiation and treatment with MMS and MC. Mutants 6G-103 and 6G-105 also produce DSF, which are not processed further.     

Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda

The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages.     

Hydrodynamic studies of a DNA-protein complex. Elongation of single stranded nucleic acids upon complexation with the gene 32 protein of phage T4 deduced from electric field-induced birefringence experiments

Short DNA and RNA fragments complexed with the helix destabilizing protein of bacteriophage T4, GP32, have been studied in solution by electric birefringence and circular dichroism. The birefringence of the complexes is positive and the magnitude indicates that the DNA and RNA fragments become linear and rigid upon protein binding. The field free decay is biphasic. On the basis of a rigid rod approximation the slow relaxation time leads to a base-base distance along the helix axis in the complex from 4.3 to 5.6 A, an elongation of at least 50% compared to single-stranded DNA.     

M protein of a Streptococcus dysgalactiae human wound isolate shows multiple binding to different plasma proteins and shares epitopes with keratin and human cartilage

Besides group A (GAS), Lancefield group C beta-haemolytic streptococci (GCS) have been implicated as a causative agent in outbreaks of purulent pharyngitis. In this study we have investigated a class CI M protein of a Streptococcus dysgalactiae1:256, revealed that 26% of these sera showed serological cross-reactivity between a 68-kDa cartilage protein and the N-terminal part of MC. Only 8% of the sera of healthy patients showed this property. In additional, MC also cross-reacted with antibodies recognising epidermal keratins. The cross-reacting 68-kDa protein from cartilage was different from human serum albumin, but was recognised with anti-vimentin immune serum. The MC was cloned and the gene sequenced. By using PCR, recombinant gene fragments encoding characteristic peptide fragments of MC were expressed in Escherichia coli. The peptides were used to map the binding sites for plasma proteins and to locate the cross-reacting epitopes on the MC molecule. In consequence, sequence alignments revealed that MC shared homologous regions with vimentin and different keratins. Our data, obtained with MC, suggest that not only infections with GAS but also infections with GCS and possibly GGS (the latter species can also produce class CI M-like proteins) may be responsible for the formation of streptococcal-associated sequel diseases.     

Atypical recognition of particular DNA sequences by the archaeal chromosomal MC1 protein

The MC1 protein is a chromosomal protein likely involved in the DNA compaction of some methanogenic archaea. This small and monomeric protein, structurally unrelated to other DNA binding proteins, bends DNA sharply. By studying the protein binding to various kinds of kinked DNA, we have previously shown that MC1 is able to discriminate between different deformations of the DNA helix. Here we investigate its capacity to recognize particular DNA sequences by using a SELEX procedure. We find that MC1 is able to preferentially bind to a 15 base pair motif [AAAAACACAC(A/C)CCCC]. The structural parameters of this sequence are characterized by molecular dynamics simulation experiments, and the binding mode of the protein to the DNA is studied by footprinting experiments. Our results strongly suggest that the protein realizes an indirect readout of the DNA sequence by binding to the DNA minor groove.     

Identification of the avian myeloblastosis virus genome. I. Identification of restriction endonuclease fragments associated with acute myeloblastic leukemia.

The proviral DNA of chicken peripheral blood leukemic myeloblasts was analyzed by restriction endonuclease digestion and Southern blotting. Two restriction endonuclease-generated fragments, an EcoRI 2.2-megadalton (Md) and a HindIII 2.6-Md fragment, were present upon enzyme cleavage of all leukemic myeloblast DNA preparations in addition to endogenous or helper-specific fragments. Neither of these fragments was derived from viral DNA of the two known myeloblastosis-associated viruses (MAV-1 and MAV-2). In contrast, DNA similarly treated from the erythrocytes of leukemic chickens showed only small amounts of the two avian myeloblastosis virus-specific fragments, whereas the helper virus-specific fragments were present in the amount seen in MAV-producing chicken embryo fibroblasts. The appearance of the EcoRI 2.2-Md and HindIII 2.6-Md specific fragments in all leukemic myeloblast DNA preparations indicates they are presumably part of the leukemogenic genome that must be present to induce acute myeloblastic leukemia.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Response of a DNA-binding protein to radiation-induced oxidative stress

The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHTI55. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis.Upon irradiation, the regular complex between MC1 and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.A model has been proposed to explain these experimental results. Two targets, R(1) and R(2), are concomitantly destroyed in the protein, with different kinetics. R(2) oxidation has no effect on the regular binding, whereas R(1) oxidation modifies the functioning of MC1: loss of preferential site and structure recognition, weaker bending. The destruction of both R(1) and R(2) targets leads to a total inactivation of the protein. This model accounts for the data obtained by titrations of DNA with irradiated proteins.When the protein is irradiated in the complex with DNA, bound DNA protects its binding site on the protein very efficiently.The highly oxidisable tryptophan and methionine could be the amino acid residues implicated in the inactivation process.     

Interaction of DNA with lysine-rich polypeptides and proteins. The influence of polypeptide composition and secondary structure

Using X-ray diffraction we have studied fibres obtained from complexes of DNA with lysine-rich polypeptides and with proteins that have different conformations, to ascertain whether the conformations of the polypeptides and the DNA are maintained upon interaction. Substances investigated include N-acetyl-Lys-Ala-Tyr-Ala-Lys-ethylamide, random poly(Leu50, Lys50), sequential poly(Leu-Lys), poly(Val-Lys), poly(Ala-Lys), poly(Lys-Ala-Ala-Lys), poly(Lys-Ala-Ala), poly(Lys-Leu-Ala), poly(Lys-Ala-Gly), protein phi 0 from sea cucumber spermatozoa, histone H1 and two fragments of this protein obtained by chemical cleavage. In general, the B form of DNA with ten base-pairs per helical turn is maintained upon interaction at high levels of humidity. The A form is never observed; it appears to be forbidden in a protein environment. No evidence for transition into any novel DNA conformation has been observed, although the B form is altered in some cases, in particular upon dehydration. Such alteration occurs always in the sense of tightening the double helix, so that the number of base-pairs per helical turn diminishes. The polypeptides may interact with DNA in both the alpha and beta conformations. We have found different types of complexes in which either a monolayer or a double layer of beta-pleated sheets is intercalated between layers of DNA molecules. Alternatively, the polypeptide chain may be wrapped around the DNA, following one of the grooves. The polypeptide conformation may be either maintained or changed upon interaction. The charge density of the polypeptide is an important parameter of the interaction. When it matches the charge density of the DNA, the polypeptide conformation is maintained in most cases; otherwise it is modified. The globular part of histone H1 gives a unique X-ray pattern upon interaction, indicative of a loss of order of DNA in the complex. On the other hand, the C-terminal part of histone H1 gives a very well-ordered complex, similar to a nucleoprotamine, in spite of its lower charge density.     

Binding of microtubule protein to DNA and chromatin: possibility of simultaneous linkage of microtubule to nucleic and assembly of the microtubule structure.

Microtubule protein binds to DNA through microtubule associated polypeptides (MAPs). Among MAPs there is one high molecular weight polypeptide (MAP2) which interacts with DNA fundamentally through certain polynucleotide sequences. This interaction is not affected by the presence of histones and other chromosomal proteins. DNA can associate to assembled microtubules and when a determinate DNA/protein ratio is reached the nucleic acid behaves as a microtubule associated molecule. The nucleic acid fragments which preferentially bind to microtubules have been isolated and characterized. These fragments contain DNA regions enriched in repetitive sequences that hybridizes preferentially to the pericentromeric zone of metaphase chromosomes. These results give further support to the model of interaction microtubule-chromosome based upon the mediator function of the microtubule associated proteins.     

Characterization of two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A 190L and their deoxyribonucleic acids

Two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A strain 190L and their deoxyribonucleic acids (DNAs) were purified and characterized. Phage alpha 1, which is unable to form plaques on any strain of C. botulinum, was produced in large quantities after treatment with mitomycin C (MC), whereas phage alpha 2, which was induced in much lower quantities than phage alpha 1, propagated in cultures of type A strain Hall. The phage DNAs were exclusively synthesized after induction with MC. Alpha 1 and alpha 2 DNAs had sedimentation coefficients of 34.0 and 30.6 S, corresponding to molecular weights of 31.9 x 10(6) and 23.5 x 10(6), respectively. The buoyant density in CsC1 was 1.682 g/cm3 for alpha 1 DNA and 1.680 g/cm3 for alpha 2 DNA. Based on thermal denaturation characteristics, the genomes of both phages were shown to be double-stranded DNAs. Agarose gel electrophoretic profiles of the phage DNAs digested with restriction endonuclease EcoRI revealed nine fragments for alpha 1 DNA and six fragments for alpha 2 DNA. The molecular weights of the phage DNAs as determined by restriction enzyme analysis were 30.55 x 10(6) for alpha 1 DNA and 25.83 x 10(6) for alpha 2 DNA. Nontoxigenic mutants obtained from strain 190L could, like the toxigenic parent strain, produce the two phages after treatment with MC. Lysogenic conversion to toxigenicity by phage alpha 2 was not observed with the nontoxigenic mutants. It seems likely that there is no relationship between either phage genome and the toxigenicity of C. botulinum type A.     

Lethal effect of mitomycin C on Haemophilus influenzae.

The sensitivity of ultraviolet-sensitive strains to inactivation by mitomycin C (MC) is at the most only a factor of two greater than that of the wild type. The presence of inducible prophage has very little effect on the sensitivity. Genes which control excision of ultraviolet-induced pyrimidine dimers also control repair of MC-induced cross-links, as measured by resistance of denatured deoxyribonucleic acid (DNA) from treated cells to S1 nuclease digestion. However, endonucleolytic breaks in MC-damaged DNA, as judged by decreased single-strand molecular weight upon incubation of treated cells, are independent of these genes and probably are caused by monoadducts. After long periods of incubation there is a return to the molecular weight of untreated DNA. DNA degradation after MC treatment of various strains is not correlated with sensitivity to inactivation. Stationary-phase cells of all strains are more than twice as sensitive to MC as exponentially growing cells, and the sensitivity difference agrees with the measured difference in the number of cross-links after MC treatment of cells in the two growth stages. Evidence has been obtained that these phenomena result from differences in uptake of MC, which can be influenced by cyclic adenosine monophosphate. Small deviations in MC sensitivity from that of the wild type observed in mutants lacking the adenosine 5'-triphosphate-dependent nuclease are postulated to result from differences in MC uptake. These mutants, although no more ultraviolet sensitive than the wild type, are more sensitive to streptomycin, which also must be taken up by the cell to be effective.     

Molecular cloning of DNA from a sorted human minichromosome

A human supernumerary minichromosome (MC), found in a newborn baby and sorted on a fluorescence-activated cell sorter (FACS-440) has been previously described [Ferretti et al., Cytotechnology 1 (1987) 7-12]. We report here on the construction of a library of EcoRI fragments in the phage lambda gtWES.lambda B', starting from 7.5 ng of MC DNA, and describe the isolation of single-copy DNA clones from the library in a two-step procedure. We employed in situ hybridization to unambiguously select the clones specific for the MC, and used three of these clones to demonstrate that it originated from chromosome 9.