Functionally acceptable substitutions in two alpha-helical regions of lambda repressor

A method of targeted random mutagenesis has been used to investigate the informational content of 25 residue positions in two alpha-helical regions of the N-terminal domain of lambda repressor. Examination of the functionally allowed sequences indicates that there is a wide range in tolerance to amino acid substitution at these positions. At positions that are buried in the structure, there are severe limitations on the number and type of residues allowed. At most surface positions, many different residues and residue types are tolerated. However, at several surface positions there is a strong preference for hydrophilic amino acids, and at one surface position proline is absolutely conserved. The results reveal the high level of degeneracy in the information that specifies a particular protein fold.     

Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase

Cre is a 38-kD protein from bacteriophage P1 that catalyzes site-specific recombination between 34-bp loxP sequences. Our previous work has shown that Cre can perform site-specific excisive recombination not only in prokaryotes, but also in eukaryotes such as yeast and cultured mammalian cells. In this work we show that intermolecular Cre-mediated recombination can specifically direct the integration of a loxP-containing circular DNA into a chromosomal loxP site, both in yeast and in mammalian cells. The resulting integrants are predominantly simple single-copy insertions. Cre-mediated recombination thus provides a simple way to direct single-copy site-specific integration of exogenous DNA into the eukaryotic genome.     

Sequence-specific binding of arc repressor to DNA. Effects of operator mutations and modifications

A set of arc operators with transition and/or transversion mutations at each operator base pair has been constructed. By determining the ability of Arc to bind these variant operators, the importance of each base pair for Arc recognition has been assessed. Methylation protection experiments have also been used to probe points of close contact between Arc and most of the mutant operators. These data, together with phosphate interference results obtained previously for the wild type operator, provide information about the operator surface that is contacted when Arc binds.     

Identification of functionally important residues in the DNA binding region of the mnt repressor

Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator.     

Notes on the karyosystematics ofAnnonaceae

Information and illustrations on somatic chromosomes, structure of interphase nuclei and hair development are presented for 9 genera and 14 species ofAnnonaceae. A few suggestions tend to improve the unsatisfactory current classification. The spectrum of polyploids found in the family ranges from neo- to meso- and paleopolyploids.     

Helical structures in the cytoplasm of differentiating cells ofMicrasterias thomasiana

Summary Helical structures so far not known inMicrasterias are observed in the cytoplasm of differentiating cells ofMicrasterias thomasiana which were pretreated by a special fixation procedure. The structures measure about 45 nm in diameter and are situated in the area of the postmitotic nucleus and its surrounding microtubule system, sometimes close to different kinds of vesicle. Nature and function of the helical structures are still obscure; a relationship to microtubules is discussed.     

Non-invasive LC-PolScope imaging of biominerals and cell wall anisotropy changes

The formation of defined shapes by cells is one of the challenging questions in biology. Due to the anisotropy of cell walls and of certain biominerals, the LC-PolScope represents a promising tool for tracking dynamic structural changes in vivo non-invasively and, to some extent, quantitatively. A complex three-dimensional biogenic system, the in vitro precipitation of calcium oxalate induced by cellulose stalks produced by Dictyostelium discoideum, was analyzed. Although the retardance values and orientation of the crystals with respect to the stalk were quickly and easily detected, this study raised a number of issues that were addressed in this work. The effect of the refractive index of the embedding medium was examined by taking advantage of the homogeneous size and shape distribution of kiwifruit raphides, a biologically controlled calcium oxalate biomineral and of cotton (Gossypium) seed fibers. The retardance remained consistent when embedding these samples in media with increasing refractive indices from 1.33 to 1.42 or 1.47 for sucrose or glycerol gradients, respectively. The general applicability of LC-PolScope image processing for biominerals and cell wall formation during development in vivo was demonstrated in a particular group of green algae, the Desmidiaceae. Various organization levels of the cell wall were identified, thus confirming earlier findings based on electron microscopy and immunostaining investigations. It can be concluded that LC-PolScope microscopy is an attractive tool for studying dynamic ordering of biomolecules, such as plant cell walls, when additional parameters regarding the structure, composition, and refractive indices of the specimen are available.     

Stochastic versus deterministic variability in simple neuronal circuits: I. Monosynaptic spinal cord reflexes.

Long time series of monosynaptic Ia-afferent to alpha-motoneuron reflexes were recorded in the L7 or S1 ventral roots in the cat. Time series were collected before and after spinalization at T13 during constant amplitude stimulations of group Ia muscle afferents in the triceps surae muscle nerves. Using autocorrelation to analyze the linear correlation in the time series demonstrated oscillations in the decerebrate state (4/4) that were eliminated after spinalization (5/5). Three tests for determinism were applied to these series: 1) local flow, 2) local dispersion, and 3) nonlinear prediction. These algorithms were validated with time series generated from known deterministic equations. For each experimental and theoretical time series used, matched time-series of stochastic surrogate data were generated to serve as mathematical and statistical controls. Two of the time series collected in the decerebrate state (2/4) demonstrated evidence for deterministic structure. This structure could not be accounted for by the autocorrelation in the data, and was abolished following spinalization. None of the time series collected in the spinalized state (0/5) demonstrated evidence of determinism. Although monosynaptic reflex variability is generally stochastic in the spinalized state, this simple driven system may display deterministic behavior in the decerebrate state.