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The genomic DNA in cells of Escherichia coli is localized in one or two compact, phase-like regions with characteristic shapes. Nucleoids undergo progressive changes in shape and compaction in the presence of drugs such as chloramphenicol or puromycin. Forces which influence nucleoid shape and compaction are reviewed, with particular emphasis on crowding effects of the cytoplasm and confinement effects of the cell envelope. Based in part on the theory of Kornyshev and Leikin for interaction between DNA duplexes, the folding of DNA caused by binding of DNA-associated proteins is suggested to antagonize DNA condensation and, thereby, increase access to DNA sequences. These views are incorporated into a working model for nucleoid organization.

The genomic DNA of Escherichia coli occurs in compact bodies known as nucleoids. Organization and structure of nucleoids are poorly understood. Compact, characteristically shaped, nucleoids isolated by the polylysine-spermidine procedure were visualized by DNA fluorescence microscopy. Treatment with urea or trypsin converted compact nucleoids to partially expanded forms. The transition in urea solutions was accompanied by release of most DNA-associated proteins; the transition point between compact and partially expanded forms was not changed by the loss of the proteins nor was it changed in nucleoids isolated from cells after exposure to chloramphenicol or from cells in which Dps, Fis, or H-NS and StpA had been deleted. Partially expanded forms became dispersed upon RNase exposure, indicating a role of RNA in maintaining the partial expansion. Partially expanded forms that had been stripped of most DNA-associated proteins were recompacted by polyethylene glycol 8,000, a macromolecular crowding agent, in a cooperative transition. DNA-associated proteins are suggested to have relatively little effect on the phase-like behavior of the cellular nucleoid. Changes in the urea transition indicate that a previously described procedure for compaction of polylysine-spermidine nucleoids may have an artifactual basis, and raise questions about reports of repetitive local structures involving the DNA of lysed cells.

This study revisits the classical x-ray diffraction patterns from hydrated, noncrystalline fibers originally used to establish the helical structure of DNA. We argue that changes in these diffraction patterns with DNA packing density reveal strong azimuthally dependent interactions between adjacent molecules up to approximately 40 A interaxial or approximately 20 A surface-to-surface separations. These interactions appear to force significant torsional "straightening" of DNA and strong azimuthal alignment of nearest neighbor molecules. The results are in good agreement with the predictions of recent theoretical models relating DNA-DNA interactions to the helical symmetry of their surface charge patterns.

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.

The genomic DNA of Escherichia coli is localized in one or a few compact nucleoids. Nucleoids in rapidly grown cells appear in complex shapes; the relationship of these shapes to underlying arrangements of the DNA is of structural interest and of potential importance in gene localization and nucleoid partition studies. To help assess this variation in shape, limited three-dimensional information on individual nucleoids was obtained by DNA fluorescence microscopy of cells as they reoriented in solution or by optical sectioning. These techniques were also applied to enlarged nucleoids within swollen cells or spheroplasts. The resulting images indicated that much of the apparent variation was due to imaging from different directions and at different focal planes of more regular underlying nucleoid shapes. Nucleoid images could be transformed into compact doublet shapes by exposure of cells to chloramphenicol or puromycin, consistent with a preexisting bipartite nucleoid structure. Isolated nucleoids and nucleoids in stationary-phase cells also assumed a doublet shape, supporting such a structure. The underlying structure is suggested to be two subunits joined by a linker. Both the subunits and the linker appear to deform to accommodate the space available within cells or spheroplasts ("flexible doublet" model).

The genomic DNA of bacteria is highly localized in one or a few bodies known as nucleoids. A number of restraints to the unfolding of the DNA of spermidine nucleoids from Escherichia coli were previously associated with characteristic urea concentrations (U(m) values). The dominant restraint to unfolding was sensitive to pancreatic RNase and underwent a cooperative transition at U(m) = 3.2 M urea. The losses of the RNase-sensitive restraint caused by urea or pancreatic RNase appear to result from breakage of cotranslational insertion linkages which joined the nucleoid to the cell envelope in growing cells. This conclusion is based upon effects from exposures of cells to antibiotics (chloramphenicol, rifampicin, streptomycin), treatment of nucleoid preparations with formaldehyde or concentrated NaCl solutions, and effects of urea on purified ribosomes. The specific RNase-sensitive and urea-sensitive components of the spermidine nucleoids are suggested to be the mRNA and ribosomes, respectively, of cotranslational insertion linkages., (Copyright 2002 Elsevier Science B.V.)

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.

The genomic DNA of bacteria is contained in one or a few compact bodies known as nucleoids. We describe a simple procedure that retains the general shape and compaction of nucleoids from Escherichia coli upon cell lysis and nucleoid release from the cell envelope. The procedure is a modification of that used for the preparation of spermidine nucleoids (nucleoids released in the presence of spermidine) (T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. USA 71:3189--3193, 1974). Polylysine is added to prevent the normal decompaction of nucleoids which occurs upon cell lysis. Nucleoids retained their characteristic shapes in lysates of exponential-phase cells or in lysates of cells treated with chloramphenicol or nalidixate to alter nucleoid morphology. The notably unstable nucleoids of rifampin-treated cells were obtained in compact, stable form in such lysates. Nucleoids released in the presence of polylysine were easily processed and provided well-defined DNA fluorescence and phase-contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde fixation and heating with sodium dodecyl sulfate.

The DNA of bacteria is compacted into nucleoids. We have lysed cells of Escherichia coli under conditions in which the cell envelope is retained. The extent of DNA compaction was determined by light microscopy, comparing DAPI fluorescence and phase contrast images. The release of cytoplasm upon lysis allowed the nucleoidal DNA to expand to fill the residual cell boundaries, supporting the role of cytoplasmic crowding in nucleoid compaction. The addition of polylysine allowed lysis with retention of DNA compaction. Furthermore, chloramphenicol treatment of cells resulted in nucleoids which were more resistant to decompaction upon lysis., (Copyright 2001 Academic Press.)

Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged., (Copyright 2000 Academic Press.)

The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC and yejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.

The ability of low centrifugal forces to convert denatured spermidine nucleoids from Escherichia coli into nonsedimentable macroscopic clumps is the basis of a rapid and simple, nonisotopic assay for nucleoid denaturation.

Nucleoids from Escherichia coli were isolated in the presence of spermidine at low salt concentrations. The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedimenting zones and/or macroscopic aggregates upon sucrose gradient centrifugation. Denaturation is accompanied by a loss of a characteristically compact shape as visualized by light and electron microscopy. Addition of polyethylene glycol or dextran prevents these changes, extending the range of stability of the isolated nucleoids to temperatures and ionic conditions like those which commonly occur in vivo. The effects of the polymers are consistent with stabilization by macromolecular crowding. Enzymatic digestion of the nucleoid DNA primarily releases three small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as residual lysozyme from the cell lysis procedure. If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabilized conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained. These extracted nucleoids maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E. coli, is not a necessary component for maintaining compaction in these preparations.

Nucleoids isolated from Escherichia coli at low salt concentrations in the presence of spermidine (Kornberg et al., Proc. Natl. Acad. Sci. USA, 71, 3189-3193 (1974)) retain large amounts of protein and RNA and are, thus, potentially useful in structural and other studies. However, these preparations have neither been visualized nor extensively characterized with regard to their protein and other components. We have investigated this type of nucleoid preparation and here supply both light and electron microscope appearances and a description of the DNA-associated proteins. Light microscopy is used to follow the stages of nucleoid release and to demonstrate characteristically rounded nucleoids after chloramphenicol treatment of the cells from which the nucleoids were isolated. The nucleoids are "envelope-associated" particles. Electron microscopy shows an irregular central core that is partially covered with small, membranous vesicles. A significant fraction of the nucleoids have a characteristic doublet/dumbbell-shaped appearance by light microscopy. The nucleoids contain large amounts of protein and RNA in addition to DNA. The DNA and RNA are rendered acid-soluble by very low levels of nucleases, indicating an open structure. A small group of proteins, including H-NS, FIS, HU, and RNA polymerase, is released from the particles upon enzymatic digestion of the DNA.

Cellular DNA in bacteria is localized into nucleoids enclosed by cytoplasm. The forces which cause condensation of the DNA into nucleoids are poorly understood. We suggest that direct and indirect macromolecular crowding forces from the surrounding cytoplasm are critical factors for nucleoid condensation, and that within a bacterial cell these crowding forces are always present at such high levels that the DNA is maintained in a condensed state. The DNA affected includes not only the preexisting genomic DNA but also DNA that is newly introduced by viral infection, replication or other means.

Polyethylene glycol (PEG) and polyethylene glycol derivatives are analyzed by a modification of the sodium dodecyl sulfate-polyacrylamide stacking gel electrophoresis procedure of Kurfürst. Gels using a discontinuous buffer system but which do not have a separate stacking gel are used without loss of resolution and with less tendency to form artifactual multiple or distorted bands. Examination of several commercial preparations of PEGs and PEG derivatives on such gels indicates heterogeneity other than the expected unimodal size distributions. SDS-gel electrophoresis of proteins or other materials in samples containing PEGs may yield gels with zones of contamination from the PEGs. Methods of reducing such contamination are suggested.

DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable ('condensed') form. The transition occurs over a relatively small increment in extract concentration. The extract appears to play two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations. The two roles of the extract are suggested by properties of fractions prepared by absorption of extracts with DNA-cellulose. The DNA-binding fraction and the DNA-nonbinding fractions from these columns are separately poorer condensing agents than the original extract, but when rejoined are similar to the original extract in the amount required for condensation. The dual role for the extract is supported by model studies of condensation with combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or polyethylene glycol 8000); in each case, crowding agents and DNA-binding materials jointly reduce the amounts of each other required for condensation. The condensation reaction as studied in extracts or in the purified systems may be a useful approach to the forces which stabilize the compact form of DNA within the bacterial nucleoid. The effect of condensation on the reactivity of the DNA was measured by changes in the rate of cohesion between duplex DNA molecules bearing the complementary single-strand termini of lambda DNA. Condensation caused large increases in the rates of cohesion of both lambda DNA and of restriction fragments of lambda DNA bearing the cohesive termini. Cohesion products of lambda DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers. We suggest a simple mechanism based upon condensation at the site of viral injection which may explain this discrepancy.

DNA-binding protein fractions from exponential and stationary phase cell extracts of E. coli were isolated by affinity chromatography on native DNA-cellulose. The ability of these fractions to convert DNA into a readily-sedimented form was compared in the absence or presence of added polymers. In the absence of polymers, large amounts of the proteins were required. In the presence of polyethylene glycol or polyvinylpyrrolidone, much smaller amounts of the DNA-binding proteins were required, indicating a macromolecular crowding effect from these polymers. The enhanced binding under crowded conditions appears to resolve a paradox between the cellular abundance of the DNA-binding proteins and the amounts required in earlier in vitro studies. The 'histone-like' protein HU from the DNA-binding protein fraction was preferentially incorporated into the pelleted DNA in the presence of polymers. Purified HU at roughly similar amounts caused a similar conversion of DNA to a readily-sedimentable ('condensed') form. Crowding-enhancement of DNA condensation by promoting the binding of proteins to the DNA provides a model for the stabilization of systems such as the bacterial nucleoid or kinetoplast DNA.

The distribution coefficients of single- and double-stranded oligodeoxynucleotides in a PEG 8000/phosphate two-phase system are a function of their chain length. Values of the distribution coefficients are in general agreement with a simple extension of a model for excluded volume effects (the "available volume model") which was applied previously to the distribution of proteins in this system. The current results therefore provide a second set of examples for molecules of very different geometry where the distribution added molecules is controlled by excluded volume interactions between those molecules and the PEG 8000 of the two-phase system.

The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects. To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm of Escherichia coli. Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions. These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts. Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration. Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction. That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intact E. coli. Macromolecule concentrations in the cytoplasm of E. coli in either exponential or stationary growth phase are estimated to be approximately 0.3 to 0.4 g/ml. Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol. Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in vivo. Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies. Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects. The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed.

A procedure for the quantitation of reactions between specific members of a set of DNA restriction fragments is presented. Quantitation of the cohesive fragments in NruI nuclease digests of lambda DNA is used as an example. Restriction fragments are resolved on agarose gels and their amounts are estimated from densitometer scans of photographic negatives of ethidium bromide-stained gels. A linear relationship is found between the peak height of given fragment on the scan and the logarithm of the molecular weight of the fragment, arising in part from the stoichiometry of the digest; this relationship allows simple interpolation between the peak heights of the nonreacting fragments in each gel lane to determine the theoretical maximal amount of each reactive fragment in that gel lane. Similar procedures should be applicable to enzymatic ligation or to site-specific cleavage of specific restriction fragments or to autoradiographic detection of the fragments. Since each lane of the gel is analyzed independently, the method is largely self-correcting for variations in amounts applied to the gel.

Partition parameters of several proteins and other macromolecules are measured in an aqueous two-phase liquid system containing polyethylene glycol and phosphate buffer. Distribution of macromolecules is a function of the relative volume excluded to the macromolecules in the two phases. A simple model with no adjustable parameters yields covolumes of the macromolecules with the polyethylene glycol. Covolumes are used to estimate effective molecular volumes and the magnitudes of excluded volume effects. The same approach is applied to mixtures of macromolecules.

Because of an increase in hospital marketing efforts, medical illustrators may be asked to assist in developing and implementing hospital identity manuals, which specify the graphic standards for visual communications media. A checklist survey of existing identity manuals and a literature review were conducted by one of the authors, a medical illustrator, to help her to develop a manual for her hospital employer. This article documents the literature review, the study, and the identity manual development, and presents recommendations to help other medical illustrators who might become involved in similar assignments.

The rates of blunt-end and cohesive-end ligation of DNA by T4 DNA ligase are increased by orders of magnitude in the presence of high concentrations of a variety of nonspecific polymers such as polyethylene glycol, Ficoll, bovine plasma albumin, or glycogen. Blunt-end ligation of small self-complementary oligodeoxyribonucleotides is also stimulated. The use of polyethylene glycol 6000 in such systems is characterized in some detail. Conditions are suggested which either greatly increase the rate of formation of and size of linear ligation products or which allow smaller but significant improvements in the amounts of circular ligation products.

A new unsaturated glutamic acid analog, 4-amino-3-chloro-2-pentenedioic acid ( ACPA ) was isolated from a fermentation broth produced by a strain of Streptomyces. ACPA has a very narrow antibacterial spectrum, which is virtually limited to Micrococcus luteus.

T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations.

A set of complexes of calf thymus DNA and n-butylamine, covalently cross-linked to the DNA bases with CH2O, has been examined by X-ray diffraction. The attachment of this amine to DNA has been previously shown to result in a profound reduction of the rotational strength of the positive band above 260 nm in the circular dichroism (CD) spectrum in 20 mM NaCl, pH 7, without changing any of the properties which are characteristic of a native base-stacked duplex [Chen, C., Kilkuskie, R., & Hanlon, S. (1981) Biochemistry 20, 4987]. In 20 mM NaCl, pH 7, substitution levels of 0-0.15 mol of amine/mol of nucleotide are sufficient to produce a family of CD spectra which range from the conservative one normally seen for protein-free DNA in this solvent to the nonconservative one ascribed to the C form of DNA [Hanlon, S., Brudno, S., Wu, T. T., & Wolf, B. (1975) Biochemistry 14, 1648-1660]. Fibers of the DNA X amine complexes at 79% relative humidity (rh) and 33% rh do indeed show C-type X-ray patterns whereas the controls exhibit A forms under the same conditions. The same preparations, however, exhibit only B patterns when the fibers are examined at 98% rh and in the wet fiber form. Although we cannot rule out the possibility that molecular interactions in the fiber have imposed conformational restraints on the DNA structure that are not present in solution, these present results suggest that the conformation of DNA giving rise to the "C" CD spectrum is a variant of the B form.

Macromolecular crowding increases the rate of nonenzymatic cohesion of the complementary ends of lambda DNA. Both lambda DNA and DNA fragments bearing the cohesive ends of lambda DNA are similarly affected. High concentrations of plasma albumin or Ficoll 70 increase the rate of cohesion by ca. 100-fold whereas high concentrations of polyethylene glycol 8000 cause greater than 2000-fold stimulation in this rate. These results have implications for the mechanism of polymer-stimulated enzymatic ligation of DNA or RNA. In addition, these crowding effects may help to explain the rapid cohesion of lambda DNA observed in vivo. An improved procedure for the recovery of DNA fragments separated by agarose gel electrophoresis is also described.

L-681,217 is a new broad spectrum antibiotic isolated from fermentation broth. The compound is a structurally unique member of the efrotomycin family of growth permittant antibiotics.

Cefoxitin is a new, cephalosporin-like antibiotic which is highly resistant to hydrolysis by beta-lactamase. Ninety-one cultures were selected either for their general resistance to cephalosporin antibiotics or for their ability to produce beta-lactamase. Some of these cultures were resistant to cefoxitin. The capacity of each of the 91 strains to hydrolyze cefoxitin with beta-lactamase was determined. Only seven of the cultures degraded the antibiotic as determined by a general assay for beta-lactamase. Several others were able to hydrolyze cefoxitin after enzyme was induced by low concentrations of the antibiotic. The role of the constitutive and inducible enzyme in bacterial resistance to the antibiotic was investigated. Enzymatic destruction of cefoxitin was found to be an important factor contributing to bacterial resistance. However, the complete and rapid degradation of cefoxitin is not essential to resistance since one strain, Enterobacter cloacae 1316, hydrolyzed the antibiotic very slowly but was able to grow unaffected in the presence of cefoxitin. The presence of the enzyme is not necessarily sufficient to confer resistance since another culture, Klebsiella D535, readily hydrolyzed the antibiotic but was susceptible to it.

The amount of reaction catalyzed by T4 polynucleotide kinase on a variety of its substrates is greatly increased in the presence of polyethylene glycol 8000 (PEG 8000). Both the forward and reverse reactions as well as the exchange reaction can be stimulated. The stimulation is a general effect on T4 polynucleotide kinase reactions involving high molecular weight DNA substrates. The use of PEG 8000 is particularly advantageous for labeling or removing terminal 5'-phosphate groups which are only slowly or incompletely labeled or removed under ordinary conditions, such as those at recessed termini or at "nicks" in duplex DNA, although the reaction on blunt-ended or protruding termini is also increased. It is further advantageous for labeling very low concentrations of substrates.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.

Recent survey research has often been used to study trends and developments in biomedical communication. Guidelines for conducting effective surveys are presented and followed by content analysis of 14 survey research reports published in the Journal of Biocommunication from 1980 through 1984 which revealed strengths and weaknesses. In general, information about survey purpose, population identification, analysis and conclusions was reported well, and information about sampling techniques, instrument design and data collection methods was inadequate. Recommendations are presented for future survey research.

In the presence of high concentrations of any of several types of macromolecules, DNA ligase preparations from rat liver nuclei or from Escherichia coli actively catalyze the blunt-end ligation of DNA. This is in contrast to the lack of activity on such substrates by these enzymes under conventional assay conditions. In addition, the previously established activity of T4 DNA ligase on blunt-ended molecules is greatly increased in the presence of high concentrations of macromolecules. Because such crowded solutions may well be a more adequate model for intracellular conditions than assays in dilute solutions, we suggest that blunt-end ligation may be a widely occurring reaction in vivo.

Intermediate filaments (IF) were prepared under identical conditions from Chinese hamster ovary (CHO), baby hamster kidney (BHK), and primary mouse epidermal cells. CHO and BHK cells each have a major IF protein (Mr = 55,000); these proteins comigrate on two-dimensional gels. BHK cells have an additional protein constituent (Mr = 54,000) which is absent from CHO. The IF preparation from the epidermal cells was considerably more complex, containing at least five proteins with molecular weights between 50,000 and 70,000, none of which has a mobility similar to the CHO protein. If assembled in vitro from crude CHO IF preparations were morphologically indistinguishable from filaments assembled from other cell types. Analysis of these in vitro polymerized filaments from CHO cells using circular dichroism and wide angle x-ray diffraction revealed that the CHO filaments were structurally similar to filaments prepared from BHK or epidermal cells. The Mr = 55,000 proteins from CHO and BHK cells were found to have identical one- and two-dimensional peptide maps, demonstrating that their amino acid sequences must be very similar. These studies indicate that CHO cells possess the simplest system thus far described for studying the assembly of intermediate filaments.

31P nuclear magnetic resonance of highly oriented DNA fibers has been observed for three different conformations, namely, the A, B, and C forms of DNA. At a parallel orientation of the fiber axis with respect to the magnetic field, DNA fibers in both the A and B forms exhibit a single, abnormally broad resonance; in contrast, fibers in the C form show almost the full span of the chemical shift anisotropy (170 ppm). The spectra of the fibers oriented perpendicular indicate that the DNA molecules undergo a considerable rotational motion about the helical axis, with a rate of greater than 2 x 10(3) s-1 for the B-form DNA. Theoretical considerations indicate that the 31P chemical shift data for the B-form DNA fibers are consistent with the atomic coordinates of the phosphodiester group proposed by Langridge et al. [Langridge, R., Wilson, H. R. Hooper, C. W., Wilkins, M. H. F., & Hamilton, L. D. (1960) J. Mol. Biol. 2, 19--37] but not with the corresponding coordinates proposed by Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Coomun. 47, 1504--1509], and also lead to the conclusion that the phosphodiester orientation must vary significantly along the DNA molecule. The latter result suggests that DNA has significant variations in its backbone conformation along the molecule.

Difficidin and oxydifficidin, two novel macrocyclic polyene lactone phosphate esters were discovered in fermentation broths of each of two strains of Bacillus subtilis: ATCC 39320 and ATCC 39374. Difficidin and oxydifficidin each showed a broad spectrum of activity against aerobic and anaerobic bacteria. Many of the susceptible aerobes and anaerobes were human pathogens resistant to one or more antibiotics. Difficidin and oxydifficidin when administered intraperitoneally protected mice against an otherwise lethal bacteremia caused by Klebsiella pneumoniae (ED50 in mg/kg of 1.31 and 15.6 respectively). Neither difficidin nor oxydifficidin were effective when administered via the subcutaneous route.

The antiprotozoal drug 3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,2-benzisoxazole (I), which exhibits activity against trypanosomiasis, is also antibacterial in vivo. Since the urine from a dog dosed with I showed a broader spectrum of antibacterial activity than I itself, metabolites from this urine were isolated and partially characterized. The metabolites were mono- and dihydroxy-substituted species with the hydroxyl groups on carbons 4--7 of the hexahydrobenzisoxazole ring. These observations led to the synthesis of several such hydroxy derivatives of I, and their properties fully supported the proposed positions of metabolic hydroxylation. One synthetic compound, the 6,7-cis-dihydroxy compound, exhibited higher antibacterial activity against Salmonella schottmuelleri in mice and greater trypanocidal activity in vivo against Trypanosoma cruzi (Brazil strain) than I.