THE STRANDEDNESS OF MEIOTIC CHROMOSOMES FROM ONCOPELTUS

Meiotic chromosomes were isolated from male Oncopeltus fasciatus by dissecting the testes under insect Ringer's solution and spreading the living cells on the Langmuir trough. After being dried by the critical point method, preparations were examined under the electron microscope. Chromosomes at all stages of prophase prove to be multistranded. A significant increase in the number of parallel 250 A fibers in the chromosomes occurs between zygotene and diakinesis. Parallel folding, rather than true multistrandedness, is interpreted as the mechanism responsible for this observed increase in multistrandedness. It has not been possible to determine whether the multistrandedness observed at leptotene represents true multistrandedness or is the result of parallel folding. Apparent multistrandedness is lost at metaphase when the 250 A fibers of the chromosomes become coiled more tightly. In preparations isolated by these methods, no structures other than the 250 A chromosome fibers are visible in the chromomeres, which appear as regionally coiled or folded areas of the fibers along the arm of the chromosome.     

Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.     

Observations on the structure of mechanically stretched chromatin fibrils

During the spreading of human chromosomes prepared from cultured human lymphocytes, the peripheral fibers attaching the chromosome to the support film are mechanically stretched. The stretching reveals structural elements of the fibers; some of these features have been described earlier, for extracted chromatin. The molecule of DNA is bared through stretching and binds labeled actinomycin D in consequence. One of the prominent features of stretched fibers is the irregularity of resulting structures. This paper further demonstrates the excellent resolution obtainable by electron impact evaporation of tungsten.     

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.     

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.     

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.     

Properties of membrane-associated folded chromosomes of E. coli related to initiation and termination of DNA replication

Analysis of folded chromosomes prepared from amino acid-starved E. coli cells or from a dnaC initiation mutant indicates that a unique structure is associated with completion or near completion of rounds of chromosome replication in E. coli. Chromosomes remain associated with portions of the bacterial cell envelope throughout the DNA replication cycle, but become more rapidly sedimenting as replication proceeds in the absence of reinitiation. Before reinitiation of chromosome replication occurs after restoring required amino acids to amino acid-starved cells or after lowering the temperature in a thermosensitive dnaC mutant, sedimentation velocities of the membrane-associated folded chromosomes decrease substantially. The decrease in sedimentation velocity does not depend on renewed DNA synthesis, but does require the activity of at least the dnaC gene product.     

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake.     

DNA topology in chromosomes: a quantitative survey and its physiological implications

Using a simple geometric model, we propose a general method for computing the linking number of the DNA embedded in chromatin fibers. The relevance of the method is reviewed through the single molecule experiments that have been performed in vitro with magnetic tweezers. We compute the linking number of the DNA in the manifold conformational states of the nucleosome which have been evidenced in these experiments and discuss the functional dynamics of chromosomes in the light of these manifold states.     

Sequential folding of transfer RNA: A nuclear magnetic resonance study of successively longer tRNA fragments with a common 5′ end

Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time.The three-dimensional structure of yeast tRNA^Phe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5′ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5′ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.     

Novel structure of the conserved gram-negative lipopolysaccharide transport protein A and mutagenesis analysis

Lipopolysaccharide (LPS) transport protein A (LptA) is an essential periplasmic localized transport protein that has been implicated together with MsbA, LptB, and the Imp/RlpB complex in LPS transport from the inner membrane to the outer membrane, thereby contributing to building the cell envelope in Gram-negative bacteria and maintaining its integrity. Here we present the first crystal structures of processed Escherichia coli LptA in two crystal forms, one with two molecules in the asymmetric unit and the other with eight. In both crystal forms, severe anisotropic diffraction was corrected, which facilitated model building and structural refinement. The eight-molecule form of LptA is induced when LPS or Ra-LPS (a rough chemotype of LPS) is included during crystallization. The unique LptA structure represents a novel fold, consisting of 16 consecutive antiparallel β-strands, folded to resemble a slightly twisted β-jellyroll. Each LptA molecule interacts with an adjacent LptA molecule in a head-to-tail fashion to resemble long fibers. Site-directed mutagenesis of conserved residues located within a cluster that delineate the N-terminal β-strands of LptA does not impair the function of the protein, although their overexpression appears more detrimental to LPS transport compared with wild-type LptA. Moreover, altered expression of both wild-type and mutated proteins interfered with normal LPS transport as witnessed by the production of an anomalous form of LPS. Structural analysis suggests that head-to-tail stacking of LptA molecules could be destabilized by the mutation, thereby potentially contributing to impair LPS transport.     

Architecture of the Chinese hamster metaphase chromosome

The development of procedures for the isolation of unfixed metaphase chromosomes has made feasible a direct analysis of their morphology. Wholemount stereo electron microscopy was used to examine intact and partially disrupted chromosomes produced by physical shearing and extraction with salt and urea solutions. A model of chromosome architecture was developed to accommodate evidence from studies using both light and electron microscopy. In the proposed model the chromatid (anaphase chromosome) consists of two half-chromatids; each half-chromatid contains two deoxyribonucleoprotein ribbons wound into a single fiber (termed the core), with many loops of chromatin (termed epichromatin) attached along its length. The core ribbons are each about 50 Å thick by 4000 Å wide and are composed of many parallel deoxyribonucleoprotein strands. The epichromatin loops appear to be 250 Å supercoiled fibers containing about 75 per cent of the chromosomal DNA. The epichromatin can be selectively removed from the core fibers by extraction with 2.0 M NaCl or 6.0 M urea solutions.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage lambda carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.     

Electron microscopy of membrane-associated folded chromosomes of Escherichia coli

Membrane-associated folded chromosomes were purified from log-phase cultures of Escherichia coli 15 TAU-bar and prepared for electron microscopy by aqueous spreading techniques. A spectrum of structures was observed, ranging from condensed structures with no DNA fibers visible, to extended structures with DNA fibers. In the extended structures, loops of DNA radiated from residual envelope, the loops sometimes appeared super-coiled, and both their number and apparent contour length approximated previous estimates from physical and biochemical data. It is proposed that the structures with free DNA arose from the condensed structures.     

The fibrous structure of human chromosomes in relation to rearrangements and aberrations; a theoretical consideration.

Human chromosomes as a type-sample for mammalian chromosomes consist of 200-A fibers, folded to chromomeres, which are interconnected by about a dozen longitudinal fibers. The average fiber at both interphase and metaphase contains 28.3 lengths of one double helix of DNA per length of fiber. The orientation of DNA imparts polarity to the fiber and thus to the chromosome and is an important constraint in concepts of chromosomal aberrations and rearrangements, some of which are being interpreted on the basis of fiber-fiber exchanges. Chromosomal rearrangements discernible by light microscopy are not likely to be fully synonymous with change in gene sequence. Chromosomes are considered to possess a plane of symmetry originating from semiconservative replication. Implications for chromosomal structure, centromeric function, and chromatid cohesion are discussed. Fibers connecting one chromosome to others are discussed in light of the proposal that fiber regions of repeated nucleotide sequences exist that facilitate fiber-fiber exchanges. No free fiber or DNA ends are thought to occur at any time in the nucleus.     

Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure

We have used light microscopy and serial thin-section electron microscopy to visualize intermediates of chromosome decondensation during G1 progression in synchronized CHO cells. In early G1, tightly coiled 100-130-nm "chromonema" fibers are visualized within partially decondensed chromatin masses. Progression from early to middle G1 is accompanied by a progressive uncoiling and straightening of these chromonema fibers. Further decondensation in later G1 and early S phase results in predominantly 60-80-nm chromonema fibers that can be traced up to 2-3 microns in length as discrete fibers. Abrupt transitions in diameter from 100-130 to 60-80 nm along individual fibers are suggestive of coiling of the 60-80-nm chromonema fibers to form the thicker 100-130-nm chromonema fiber. Local unfolding of these chromonema fibers, corresponding to DNA regions tens to hundreds of kilobases in length, reveal more loosely folded and extended 30-nm chromatin fibers. Kinks and supercoils appear as prominent features at all observed levels of folding. These results are inconsistent with prevailing models of chromosome structure and, instead, suggest a folded chromonema model of chromosome structure.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.