Application of Physical Methods to the Study of Serum Lipoprotein

Abstract

One of the primary characteristics distinguishing prokaryotic from eukaryotic cells is the absence of a nucleus with a clearly defined nuclear membrane. In prokaryotic cells the DNA is condensed into a structure called the nucleoid. This structure has also been referred to attimes as the nuclear body, prokaryotic nucleus, bacterial chromosome, folded genome, or folded bacterial chromosome. The nomenclature sometimes becomes confusing because unfolded bacterial DNA free of other components of the nucleoid has also been referred to as the bacterial chromosome. To avoid such confusion, it would be preferable to reserve the terms nucleoid or bacterial chromosome to describe the condensed prokaryotic DNA structures which have some features analogous to the eukaryotic metaphase chromosome and condensed interphase chromatin. If this convention is followed, the terms “folded chromosome” or “folded genome” become ambiguous because they could equally mean “folded nucleoid.” These latter terms will, therefore, be avoided throughout this article.     

Membrane-bound mitochondrial DNA: isolation, transcription and protein composition.

Mitochondrial membrane-bound DNA complex from bovine heart mitochondria lysed in the presence of Triton X-100 was isolated by differential centrifugation. The yield of "nucleoid" is about 30 microgram protein/mg mitochondrial protein. It contains about 3-5 microgram DNA/mg protein and varying amounts of RNA. The heart mitochondrial nucleoid actively synthesizes RNA. The nucleoid fraction contains about sixteen different proteins as evidenced by urea-SDS gel electrophoresis and about twenty-one proteins as evidenced by acid-urea gel electrophoresis. It appears that the nucleoid is attached to the inner membrane since it does contain cytochromes.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

Properties of a membrane-attached form of the folded chromosome of Escherichia coli

Two types of DNA-containing particles are released from lysozyme-produced Escherichia coli spheroplasts after gentle lysis with non-ionic detergents in 1.-0 m-NaCl. Lysis at 25 °C releases the folded chromosomes (1300 S to 2200 S particles). Lysis at 10 °C results in faster sedimenting structures (3000 S to 4000 S). Both types of particles coexist in extracts of cells lysed at intermediate temperatures, i.e. 15 °C.The 3000 S to 4000 S particles are folded chromosomes attached to membrane fragments; they contain membrane proteins and phospholipids in addition to the folded DNA and nascent RNA chains. Incubation of the membrane-attached chromosomes with 1% Sarkosyl releases the folded chromosomes; this Sarkosyl treatment removes the membrane proteins and phospholipids, and halves the sedimentation velocity of the particles, but has no effect on the folded DNA and nascent RNA chains.Membrane-attached chromosomes cannot be isolated from amino acid-starved cells which have completed their rounds of DNA replication; all of the DNA then appears as released folded chromosomes. After resumption of protein synthesis, chromosome attachment to the membrane precedes the initiation of DNA replication. Controls strongly suggest that the changes observed, i.e. the attachment and release from the membrane of the folded chromosome, are related to the act of DNA replication itself.     

Association of the Folded Chromosome with the Cell Envelope of Escherichia coli: Nature of the Membrane-Associated DNA

Membrane-associated folded chromosomes isolated from Escherichia coli in the presence of spermidine sedimented at about 5,800S. The folded chromosome and the membrane fragment were each stable in the absence of the other; a 1,700S folded chromosome was obtained after removal of the membrane by a Sarkosyl treatment, and a 4,000S membrane fragment remained after digestion of the chromosomal DNA with deoxyribonuclease I. The interaction between the folded chromosome and the membrane fragment was stable, and, even when the DNA was unfolded, both components remained associated and cosedimented. The large frictional effect of the unfolded DNA reduced the sedimentation rate of the complex to about 2,000S. Partial removal of this unfolded DNA with restriction endonucleases caused the membrane fragments and the remaining associated DNA to sediment faster, at about 3,500S. The DNA remaining associated with the membrane fragments after restriction endonuclease treatment, about 4.5% of the total DNA when EcoRI was used, was indistinguishable from the DNA released from the membranes by three criteria: (i) DNA size distribution in agarose gels after electrophoresis, (ii) reassociation kinetics, and (iii) thermal elution from hydroxylapatite. This finding, that random DNA sequences rather than specific ones were responsible for the majority of the DNA-membrane interactions, argues against the folded chromosome's being a static structure with specific DNA sequences interacting with the cell envelope.     

Folded chromosome structure and DNA-binding protein of Streptomyces hygroscopicus

The isolation of the folded chromosomal structure from a Streptomyces strain is described. It is shown that the compact DNA structure of Streptomyces hygroscopicus is stabilized by DNA-RNA interactions. Nucleolytic cleavage decreases the sedimentation rate of the originally isolated (membrane-free) folded Streptomyces chromosome from 1500 S to 800 S or 300 S and finally to completely unfolded DNA. Data on ethidium bromide intercalation suggest a dye-induced relaxation and reintroduction of DNA supertwisting. Like naturally occurring covalently closed circular DNA and like the Escherichia coli chromosome, the folded chromosomal DNA of S. hygroscopicus has about one negative superhelix turn per 200 bp. The results further demonstrate that the isolated Streptomyces nucleoid contains a characteristic S protein which exhibits gel electrophoretic properties of a histone-like component similar to that found in E. coli or Thermoplasma acidophilum. Digestion of the nucleoid with proteinase K at 37°C causes elimination of the S protein and unfolding of the compact structure. The S protein is also present in other species of the genus Streptomyces.     

Some physico-chemical properties of the compact chromosome of Ananbaena variabilis]

It was shown that total DNA of the blue-green algae A. variabilis can be isolated as a compact particle with the sedimentation constant of 2700--2900S by lysis of the cells with the TritonX-100--sodium deoxycholate mixture in 1.0 M NaCl. The structure isolated is sensitive to ribonuclease and the hydrodynamic shift. It was found that within a compact chromosome A. variabilis DNA is circular and negatively superhelicized. The density of superturns of the DNA in 0,2 M NaCl at 20 degrees C is 0,061, which corresponds to one negative turn per 165 pairs of DNA bases.     

Cellular location of Mu DNA replicas.

To ascertain the form and cellular location of the copies of bacteriophage Mu DNA synthesized during lytic development, DNA from an Escherichia coli lysogen was isolated at intervals after induction of the Mu prophage. Host chromosomes were isolated as intact, folded nucleoids, which could be digested with ribonuclease or heated in the presence of sodium dodecyl sulfate to yield intact, unfolded nucleoid DNA. Almost all of the Mu DNA in induced cells was associated with the nucleoids until shortly before cell lysis, even after unfolding of the nucleoid structure. We suggest that the replicas of Mu DNA are integrated into the host chromosomes, possibly by concerted replication-integration events, and are accumulated there until packaged shortly before cell lysis. Nucleoids also were isolated from induced lambda lysogens and from cells containing plasmid DNA. Most of the plasmid DNA sedimented independently of the unfolded nucleoid DNA, whereas 50% or more of the lambda DNA from induced lysogens cosedimented with unfolded nucleoid DNA. Possible explanations for the association of extrachromosomal DNA with nucleoid DNA are discussed.     

Proteins firmly bound to DNA in the E. coli nucleoid

The nucleoid isolated from E. coli cells was subjected to further deletion by treatment with 2 M NaCl. After disintegration of this nucleoid by ultrasonication, two fractions were obtained, i. e., a rapidly (RS) and slowly sedimenting (SS) ones. The protein, RNA and DNA patterns in the RS fraction are similar to that of the eukaryotic cell nuclear matrix. Electrophoretic analysis of total non-dissociating by 2 M NaCl proteins revealed that the RS and SS fractions predominantly contain proteins with Mr 31,27 and 23 kD. The protein with Mr = 31 kD is firmly bound to DNA, does not dissociate in the guanidine hydrochloride (4 M)-urea (5 M) mixture as well as in solution of 1% sodium-dodecyl sulphate and may be responsible for the chromosome binding to the E. coli membrane.     

Biochemical properties of nucleic acids of chloroplasts in Spirogyra1

In chloroplasts and nuclei of Spirogyra separated by discontinuoussucrose dnesity gradient centrifugation, five types of nucleicacids were found by MAK column chromatography; tRNA, low molecularweight rRNA, DNA, light and heavy rRNAs. Chloroplast rRNA was found to differ from cytoplasmic rRNA,by comparative studies on its base composition, its elutionpattern by MAK column chromatography and its sedimentation profilewith sucrose density gradient centrifugation. Molecular species of chloroplast DNA differed from those ofnuclear DNA in their thermal denaturation points (Tm), theirbehaviour on an IRC-50 column, their buoyant density in CsCland their base analyses. (Received September 13, 1972; )     

Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore protein.

The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native-like, heat- and SDS-stable form requires incubations with isolated cell envelopes and Triton X-100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat- and SDS-stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat-stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat-stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half-life that will form heat-stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process.     

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.     

Characterization of rapidly labelled RNA associated with DNA in Chlorella

Summary After pulse-labelling with ³²P-orthophosphate and fractionation of the nucleic acids from synchronously cultured cells of the green alga Chlorella pyrenoidosa on methylated serum albumin and kieselgur (MAK) the DNA contained a species of ³²P-RNA. About 3% of the total ³²P radioactivity incorporated in the cells' RNA were confined to this DNA-associated component. Its base ratio differed significantly from that of soluble and ribosomal RNA but varied only slightly during the life-cycle of the cells. About 4% of the DNA-associated ³²P-RNA resisted ribonuclease digestion suggesting a stable binding of RNA to DNA in the form of a complex. Gel filtration and sucrose gradient centrifugation of the nucleic acids isolated in the DNA region during previous MAK column chromatography resulted in a separation of most of the ³²P-RNA from the DNA. The remaining ribonuclease-resistent but alkali labile ³²P radioactivity bound to the latter was in the order of 4%. No evidence has been obtained so far that it represents rapidly synthesized RNA associated with DNA in stable and functional complex.     

The LHIα and LHIIα Complexes in Association with Phospholipids Are Able to Be Inserted in Heavy Membranes of Rhodobacter capsulatus B10

We show in this paper that a complex constituted by phospholipids and LHI and LHII α polypeptides was inserted in a heavy membrane fraction in a nonextractable form, indicating a transmembrane localization. The best accepting membranes originated from aerobically grown cells. Addition of ATP during the insertion inhibited this reaction 25 to 30% in heavy membranes isolated from aerobically grown cells (HM_aer) and a higher inhibition (60 to 65%) was detected when using heavy membranes isolated from photosynthetically grown cells (HM_pho). Purification by gel filtration of a crude Na₂CO₃ extract yielded three phosphate-labeled fractions. Two of them contained protein and phospholipids in a stable association. However, only fractions containing phosphatidylethanolamine were shown to be reconstituted. The third radioactive fraction contained labeled ATP and protein, but no phospholipids and could not be reassociated to the heavy membranes of any origin. A model for the insertion of the LH polypeptides is presented in which the recently synthesized polypeptides are phosphorylated and become associated to anionic phospholipids. The interaction of this complex to the membrane spontaneously leads to stable insertion. Received: 16 February 1999 / Accepted: 22 March 1999     

Intrastrand self-complementary sequences in Bacillus subtilis DNA.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.     

Preparative isolation and study of the reassociation kinetics of a T2 phage DNA fraction containing readily melting regions]

A DNA fraction comprising 6% of total DNA and containing readily-melting regions is isolated from phage T2 DNA using preparative chromatography on MAK columns at T congruent to T m--3 degrees C. Two denaturation regions, differing in the stability for 7 degrees, were observed on this DNA melting curve. A sharp increase of the reassociation rate at initial moments under reassociation temperatures T r approximately less than m --25 degrees C was observed. Thermodynamic characteristics obtained under the repeated melting of DNA fragments after reassociation confirm the fact, that under these reassociation temperatures the incorporation of readily-melting regions into spiral duplexes takes place.     

Association of the folded chromosome with the cell envelope of E. coli: Characterization of the proteins at the DNA-membrane attachment site

Gentle lysis of E. coli cells in the presence of a DNA counterion (either 1.0 M NaCl or 5 mM spermidine) permits the isolation of the folded intact bacterial chromosome associated with membrane fragments. Most of the proteins in these chromosomes are also found in purified membrane preparations, and they can be identified as belonging to either the inner or the outer bacterial membrane.Ultraviolet irradiation of the membrane-attached chromosomes causes the formation of a stable complex between two inner membrane proteins (molecular weight 80,000 and 56,000 daltons) and 5-bromodeoxyuridine (BrdU)-substituted DNA. The photochemical attachment of BrdU-substituted DNA to specific membrane proteins suggests that these proteins may be bound to the DNA in vivo. Such DNA-membrane-binding proteins may have a role in the attachment of the folded chromosome to the bacterial envelope.     

Bacterial chromosome segregation: evidence for DNA gyrase involvement in decatenation

Nucleoids isolated from a temperature-sensitive gyrB mutant of E. coli, incubated at restrictive temperatures, exhibit increased sedimentation rates and an abnormal doublet or dumbbell-shaped morphology. Shifting cells from restrictive to permissive temperature prior to nucleoid isolation leads to decreases in the percentage of doublet nucleoids and in nucleoid sedimentation rates. When nucleoids isolated from mutant cells exposed to restrictive temperature are incubated with purified gyrase, the percentage of doublet nucleoids decreases as the total number of nucleoids increases. These results, together with the demonstrated ability of gyrase to decatenate small circular DNA molecules in vitro, suggest that gyrase participates in bacterial chromosome segregation through its decatenating activity.