A new method for automatic metaphase finding adaptable to different chromosome preparations

A FORTRAN computer program, running on a Digital PDP 11-34 minicomputer, has been developed for use in conjunction with a Cambridge Quantimet 720 image analyzer for the investigation of metaphase preparations in routine cytogenetics. During a short initiation phase the program is adapted to the type of metaphase being analyzed. The program is fast and its performance is good, even at low microscopic magnifications. It has other uses in biology for all investigations and characterizations of small distinct elements widely spread within a preparation (e.g., autoradiography, bacteriology).     

Role of nonhistone proteins in metaphase chromosome structure

In this paper, we show that HeLa metaphase chromosomes still possess a highly organized structure retaining the familiar metaphase morphology following removal of virtually all the histones and most of the nonhistone proteins. The structure is stabilized by a relatively small number of nonhistones, which we call scaffolding proteins.These results are based on a method which allows the removal of the histones, and most of the nonhistone proteins, by competition with polyanions such as dextran sulfate and heparin.The histone-depleted chromosomes sediment in sucrose gradients as a broad peak between 4000 to 7000S. These structures are dissociated by mild trypsin or chymotrypsin treatment, or by 4 M urea, but are stable in 2 M NaCl and insensitive to treatment with RNAase A. The histone-depleted chromosomes have a DNA to protein ratio of about 6:1; gel electrophoresis reveals the presence of about 30 nonhistone proteins and the virtual absence of histones. These experiments suggest that nonhistone proteins exist in metaphase chromosomes which maintain the DNA chain in a highly folded conformation.Structural studies support this conclusion. Analysis by fluorescence microscopy of histone-depleted chromosomes stained with ethidium bromide shows that each chromatid is still paired with its sister chromatid, and consists of a central structure surrounded by a halo of DNA. The length of the central structure in each chromatid is about 2–3 times longer than the chromatid length in the original chromosome.     

Cadmium toxicity affects photosynthesis and plant growth at different levels

In this article we discuss and update some of the effects of Cd toxicity on the photosynthetic apparatus in a model crop Lactuca sativa. Seeds of L. sativa were germinated in solutions with 0, 1, 10 and 50 μM of Cd(NO₃)₂ and then transferred to a hydroponic culture medium. After 28 days, the effects of Cd on the photosynthetic apparatus of lettuce were analysed. Exposure of lettuce to 1 μM Cd(NO₃)₂ affected already plant growth (dry biomass), but, did not induce serious damages in the photosynthetic apparatus. However, increasing concentrations of this metal to 10 and 50 μM promoted a strong reduction of the maximum photochemical efficiency of PSII and an impairment of net CO₂ assimilation rate, putatively due to Rubisco activity decrease. This ultimately results in a strong inhibition of plant growth. Nutrient uptake and carbohydrate assimilation were also severely affected by Cd.     

Mitochondrial structure and function are disrupted by standard isolation methods

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca(2+) handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca(2+) challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H(2)O(2) production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.     

Refined structure of dienelactone hydrolase at 1.8 A

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at 1.8 A resolution, is described. The final molecular model of DLH has a conventional R value of 0.150 and includes all but the carboxyl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014 A and the error in atomic co-ordinates is estimated to be 0.15 A. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the beta-ketoadipate pathway found in bacteria and fungi. DLH is an alpha/beta protein containing seven helices and eight strands of beta-pleated sheet. A single 4-turn 3(10)-helix is seen. The active-site Cys123 residues at the N-terminal end of an alpha-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The beta-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central beta-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases. As in papain and actinidin, the active thiol is partially oxidized during X-ray data collection. The positions of both the reduced and the oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the gamma-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.     

Improved methods for working with fish chromosomes with a review of metaphase chromosome banding

Improved methods for obtaining, preparing, and staining fish chromosomes are described. Included are procedures for resolving serial or G-type bands. A brief review of various metaphase banding procedures and their use in fishes is also presented.     

Refined structure of porcine pepsinogen at 1.8 A resolution

The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.     

Refined structure of glutathione reductase at 1.54 A resolution

The crystal structure of human glutathione reductase has been established at 1.54 A resolution using a restrained least-squares refinement method. Based on 77,690 independent reflections of better than 10 A resolution, a final R-factor of 18.6% was obtained with a model obeying standard geometry within 0.025 A in bond lengths and 2.4 degrees in bond angles. The final 2Fo-Fc electron density map allows for the distinction of carbon, nitrogen and oxygen atoms with temperature factors below about 25 A2. Apart from 461 amino acid residues and the prosthetic group FAD, the model contains 524 solvent molecules, about 118 of which can be considered an integral part of the enzyme. The largest solvent cluster is at the dimer interface and contains 104 interconnected solvent molecules, part of which are organized in a warped sheet-like structure. The main-chain dihedral angles are well-concentrated in the allowed regions of the Ramachandran plot. The spread of dihedral angles in beta-pleated sheets is much larger than in alpha-helices and especially in alpha-helix cores, indicating the higher plasticity of beta-structures. The analysis revealed a large amount of 3(10)-helix. The side-chain conformations cluster at the staggered positions, and show well-defined preferences. Also, a mobility gradient is observed for side-chains. Non-polar and polar side-chains show average temperature factor increases per bond of 10% and 25%, respectively. A number of alternative conformations of internal side-chains, in particular serines and methionines, have been detected. The extended FAD molecule also shows a mobility gradient between the very rigid flavin (mean value of B) = 8.7 A2) and the more mobile adenine (mean value of B = 16.2 A2). The entire active center is particularly well ordered, with temperature factors around 10 A2. The dimer interface consists of a rigid contact area, which is well conserved in the Escherichia coli enzyme, and a flexible area that is not. Altogether, the buried surfaces at the crystal contacts are half as large as at the dimer interface, but less specific. The refined structure shows clearly that there are no buried cations compensating the charge of the pyrophosphate moiety of FAD. The flavin deviates slightly from standard geometry, which is possibly caused by the polypeptide environment. In contrast to an earlier interpretation, atom N5 of the flavin can accommodate a proton, and it is conceivable that this proton proceeds to the redox-active disulfide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure

To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our method, the internal structure of nucleosomes or the pattern of nucleosome packing within chromatin fibers. In particular, phosphorylation of histones H1 and H3 at mitosis does not affect chromatin fiber structure, since the same x-ray results are obtained whether or not histone dephosphorylation is prevented by isolating metaphase chromosomes in the presence of 5,5'-dithiobis(2- nitrobenzoate) or low concentrations of p-chloromercuriphenylsulfonate (ClHgPhSO3). We also compared metaphase chromosomes isolated by several different published procedures, and found that the isolation procedure can significantly affect the x-ray diffraction pattern. High concentrations of ClHgPhSO3 can also profoundly affect the pattern.     

Induction of metaphase chromosome condensation in human sperm by Xenopus egg extracts

Cell-free extracts of Xenopus eggs cause permeabilized Xenopus sperm to form pronuclei, which condense into metaphase chromosomes when the cytosol from metaphase-arrested unfertilized eggs is added to the extracts. In this paper, the ability of these cell-free extracts to cause similar changes in permeabilized human sperm was examined. Sperm that had been treated with the disulfide reducing agent dithiothreitol formed pronuclei, whereas untreated sperm did not. The addition of metaphase cytosol to the extracts caused the pronuclei to form metaphase chromosomes but only after incubation times that were two to three times longer than those required for Xenopus sperm nuclei. These results indicate that despite species differences, the Xenopus egg extracts can be used to visualize the chromosomes of human sperm and possibly those of other species.     

Comparison of different myelin isolation methods by use of nonequilibriumpH gradient gel electrophoresis

Myelin was isolated from bovine white matter by five published procedures and several modifications of two of them. Comparison of the protein profiles of the preparations by nonequilibrium pH gradient gel electrophoresis, revealed clear differences in myelin protein content and composition between preparations obtained by different methods. In isolation methods where the medium contained salts, some of the myelin proteins were solubilized, the phenomenon being most pronounced in long-period isolations in buffered CsCl solution.     

Recent development of image analysis methods in plant chromosome research

Image analysis methods have provided effective tools in chromosome research along with the development both in computer software and hardware. A chromosome image analyzing system, CHIAS, for plant chromosomes was developed in 1985 and was subsequently revised so that with CHIAS3 one can take advantage of Internet use for downloading the program. In this review, the recent developments of imaging methods in plant chromosome research for automating chromosome identification, constructing a map of a pachytene chromosome, and patterning of interphase nuclei are described.     

Refined crystal structure of carboxypeptidase A at 1.54 A resolution

The crystal structure of bovine carboxypeptidase A (Cox) has been refined at 1.54 A resolution using the restrained least-squares algorithm of Hendrickson & Konnert (1981). The crystallographic R factor (formula; see text) for structure factors calculated from the final model is 0.190. Bond lengths and bond angles in the carboxypeptidase A model have root-mean-square deviations from ideal values of 0.025 A and 3.6 degrees, respectively. Four examples of a reverse turn like structure (the "Asx" turn) requiring an aspartic acid or asparagine residue are observed in this structure. The Asx turn has the same number of atoms as a reverse turn, but only one peptide bond, and the hydrogen bond that closes the turn is between the Asx side-chain CO group and a main-chain NH group. The distributions of CO-N and NH-O hydrogen bond angles in the alpha-helices and beta-sheet structures of carboxypeptidase A are centered about 156 degrees. A total of 192 water molecules per molecule of enzyme are included in the final model. Unlike the hydrogen bonding geometry observed in the secondary structure of the enzyme, the CO-O(wat) hydrogen bond angle is distributed about 131 degrees, indicating the role of the lone pair electrons of the carbonyl oxygen in the hydrogen bond interaction. Twenty four solvent molecules are observed buried within the protein. Several of these waters are organized into hydrogen-bonded chains containing up to five waters. The average temperature factor for atoms in carboxypeptidase A is 8 A2, and varies from 5 A2 in the center of the protein, to over 30 A2 at the surface.     

Refined structure of alpha beta-tubulin at 3.5 A resolution.

We present a refined model of the alpha beta-tubulin dimer to 3.5 A resolution. An improved experimental density for the zinc-induced tubulin sheets was obtained by adding 114 electron diffraction patterns at 40-60 degrees tilt and increasing the completeness of structure factor amplitudes to 84.7 %. The refined structure was obtained using maximum-likelihood including phase information from experimental images, and simulated annealing Cartesian refinement to an R-factor of 23.2 and free R-factor of 29.7. The current model includes residues alpha:2-34, alpha:61-439, beta:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion at the non-exchangeable nucleotide site, and one putative zinc ion near the M-loop in the alpha-tubulin subunit. The acidic C-terminal tails could not be traced accurately, neither could the N-terminal loop including residues 35-60 in the alpha-subunit. There are no major changes in the overall fold of tubulin with respect to the previous structure, testifying to the quality of the initial experimental phases. The overall geometry of the model is, however, greatly improved, and the position of side-chains, especially those of exposed polar/charged groups, is much better defined. Three short protein sequence frame shifts were detected with respect to the non-refined structure. In light of the new model we discuss details of the tubulin structure such as nucleotide and taxol binding sites, lateral contacts in zinc-sheets, and the significance of the location of highly conserved residues.     

Refined structure of cytochrome c3 at 1.8 A resolution

The structure of cytochrome c3 from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki has been successfully refined at 1.8 A resolution. The crystallographic R factor is 0.176 for 9907 significant reflections. The isotropic temperature factors of individual atoms were refined and a total of 47 water molecules located on the difference map were incorporated in the refinement. The four heme groups are closely packed, with adjacent pairs of heme planes being nearly perpendicular to each other. The fifth and the sixth ligands of the heme iron atoms are histidine residues with N epsilon 2-Fe distances ranging from 1.88 A to 2.12 A. The histidine co-ordination to the heme iron is different for each heme group. The heme groups are all highly exposed to solvent, although the actual regions exposed differ among the hemes. The four heme groups are located in different environments, and the heme planes are deformed from planarity. The differences in the heme structures and their environments indicate that the four heme groups are non-equivalent. The chemical as well as the physical properties of cytochrome c3 should be interpreted in terms of the structural non-equivalence of the heme groups. The characteristic secondary structural non-equivalence of the heme groups. The characteristic secondary structures of the polypeptide chain of this molecule are three short alpha-helices, two short beta-strands and ten reverse turns.