Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.     

Effect of nucleosome distortion on the linking deficiency in relaxed minichromosomes

The wrapping of closed circular DNA on a protein surface, followed by relaxation with a topoisomerase and removal of proteins, produces a characteristic DNA linking deficiency, delta Lk. We show that the magnitude of delta Lk depends upon the surface shape, and we calculate changes in delta Lk caused by particular distortions of the protein wrapping surface. If the DNA remains attached to the surface during distortion, the DNA winding number, phi, is not altered. The change in delta Lk is then equal to the change in the surface linking number, SLk, which is a straightforward measure of the wrapping of the DNA around the surface. For left-handed wrapping, as in a nucleosome, SLk = -n, the number of times that the DNA axis winds around the axis of the protein complex. We calculate values of SLk for the helical wrapping of a constant length of DNA on protein surfaces having the shapes of cylinders and of ellipsoids and hyperboloids of revolution. If the equatorial radius of the protein is fixed, change in shape from a cylinder to a hyperboloid increases SLk, while the corresponding change to an ellipsoid reduces SLk. We apply the general results to the interpretation of experiments in which minichromosomes are relaxed with topoisomerase at various temperatures and delta Lk is determined. The result is that a distortion of the nucleosome core by at most 5% (the change in the radius at the axial extremity relative to the equator) is sufficient to explain the observed delta Lk changes.     

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.     

Chemotaxis,induced gene expression and competitiveness in the rhizosphere

Rhizobia are soil bacteria which symbiotically infect legume roots and generate nodules in which they fix atmospheric nitrogen for the plant in exchange for photosynthetically fixed carbon. A crucial aspect of signal exchange between these symbionts is the secretion of phenolic compounds by the host root which induce nodulation gene expression in the bacteria. Stimulation of nod gene expression by host phenolics is required for nodule formation, is biochemically specific at 10^-6 M, and is mediated by nodD. We and others have shown that rhizobia display chemotaxis to 10^-9 M of the same phenolic compounds. Chemotaxis to inducer phenolics is selectively reduced or abolished by mutations in certain nod genes governing nodulation efficiency or host specificity. Conversely, mutations in rhizobia that affect general motility or chemotaxis have substantial effects on nodulation efficiency and competitiveness. These findings suggest that microbes entering the rhizosphere environment may utilize minor, non-nutrient components in root exudates as signals to guide their movement towards the root surface and elicit changes in gene expression appropriate to this environment.     

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.     

PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

Conformational properties of the —35 region of the trp promoter in solution: comparison of the wild-type sequence with an AT transversion

The majority of the ¹H NMR resonances of the protons in a tetradecamer containing the —35 region of the trp promoter d(GCTGTTGACAATTA): d(TAATTGTCAACAGC) and in the TA transversion have been assigned. The conformational properties of the nucleotides have been determined and compared in the two duplexes. Analysis of spin-spin coupling and NOES shows that all sugar puckers are in the south domain (i.e. near C2 endo) and the glycosidic torsion angles are anti (110°). The NMR data are consistent with the duplex being in the B family of conformations. Significant differences in chemical shifts between the two molecules were observed only for nearest neighbours to the transversion site, suggesting the absence of long range conformational effects. This was confirmed by the similarity of coupling constants and NOEs. Other properties are also not greatly affected at positions more than two base pairs from the mutation site. These results are consistent with the hypothesis that unconstrained oligonucleotides are highly flexible, and can readily accommodate significant perturbations of the local structure, such as a transversion.     

Regulation of glycerol metabolism in Enterococcus faecalis by phosphoenolpyruvate-dependent phosphorylation of glycerol kinase catalyzed by enzyme I and HPr of the phosphotransferase system.

Using a polyclonal antibody against glycerol kinase from Enterococcus faecalis, we could demonstrate that glycerol kinase is inducible by growth on glycerol-containing medium and that during growth on glycerol the enzyme is mainly phosphorylated. Glucose and other sugars metabolized via the Embden-Meyerhof pathway strongly repressed the synthesis of glycerol kinase, while if glycerol was also present during growth, low activity, reflecting partial induction and the presence of mainly unphosphorylated, less active enzyme, was found. With gluconate, which is also a substrate of the phosphotransferase system, repression of glycerol kinase was less severe, but the enzyme was mainly present in the less active, unphosphorylated form. Effects of growth on different carbon sources on glycerol uptake are also reported.