A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-κB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685–839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-κB to the nucleus. N-CAM also activated NF-κB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-κB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-κB and may be important in other N-CAM-mediated signaling.

Changes of Mitochondrial Properties in Maize Seedlings Associated with Selection for Germination at Low Temperature. Fatty Acid Composition, Cytochrome c Oxidase, and Adenine Nucleotide Translocase Activities1

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5°C. When seedlings of the two populations were grown at 14°C (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome c oxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.

Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley1

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F1-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.

Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B.subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ∼0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (∼0.9 min–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (∼40 min–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.

Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.

Genetic code differences prevent expression of nuclear genes within Saccharomyces cerevisiae mitochondria. To bridge this gap a synthetic gene, ARG8m, designed to specify an arginine biosynthetic enzyme when expressed inside mitochondria, has been inserted into yeast mtDNA in place of the COX3 structural gene. This mitochondrial cox3::ARG8m gene fully complements a nuclear arg8 deletion at the level of cell growth, and it is dependent for expression upon nuclear genes that encode subunits of the COX3 mRNA-specific translational activator. Thus, cox3::ARG8m serves as a mitochondrial reporter gene. Measurement of cox3::ARG8m expression at the levels of steady-state protein and enzymatic activity reveals that glucose repression operates within mitochondria. The levels of this reporter vary among strains whose nuclear genotypes lead to under- and overexpression of translational activator subunits, in particular Pet494p, indicating that mRNA-specific translational activation is a rate-limiting step in this organellar system. Whereas the steady-state level of cox3::ARG8m mRNA was also glucose repressed in an otherwise wild-type strain, absence of translational activation led to essentially repressed mRNA levels even under derepressing growth conditions. Thus, the mRNA is stabilized by translational activation, and variation in its level may be largely due to modulation of translation.

The single Cys2-His2 zinc finger domain of the GAGA protein flanked by basic residues is sufficient for high-affinity specific DNA binding.

Specific DNA binding to the core consensus site GAGAGAG has been shown with an 82-residue peptide (residues 310-391) taken from the Drosophila transcription factor GAGA. Using a series of deletion mutants, it was demonstrated that the minimal domain required for specific binding (residues 310-372) includes a single zinc finger of the Cys2-His2 family and a stretch of basic amino acids located on the N-terminal end of the zinc finger. In gel retardation assays, the specific binding seen with either the peptide or the whole protein is zinc dependent and corresponds to a dissociation constant of approximately 5 x 10(-9) M for the purified peptide. It has previously been thought that a single zinc finger of the Cys2-His2 family is incapable of specific, high-affinity binding to DNA. The combination of an N-terminal basic region with a single Cys2-His2 zinc finger in the GAGA protein can thus be viewed as a novel DNA binding domain. This raises the possibility that other proteins carrying only one Cys2-His2 finger are also capable of high-affinity specific binding to DNA.

Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.

Both stem cells and mast cells express c-kit and proliferate after exposure to c-kit ligand. Mutations in c-kit may enhance or interfere with the ability of c-kit receptor to initiate the intracellular pathways resulting in cell proliferation. These observations suggested to us that mastocytosis might in some patients result from mutations in c-kit. cDNA synthesized from peripheral blood mononuclear cells of patients with indolent mastocytosis, mastocytosis with an associated hematologic disorder, aggressive mastocytosis, solitary mastocytoma, and chronic myelomonocytic leukemia unassociated with mastocytosis was thus screened for a mutation of c-kit. This analysis revealed that four of four mastocytosis patients with an associated hematologic disorder with predominantly myelodysplastic features had an A-->T substitution at nt 2468 of c-kit mRNA that causes an Asp-816-->Val substitution. One of one patient examined who had mastocytosis with an associated hematologic disorder had the corresponding mutation in genomic DNA. Identical or similar amino acid substitutions in mast cell lines result in ligand-independent autophosphorylation of the c-kit receptor. This mutation was not identified in the patients within the other disease categories or in 67 of 67 controls. The identification of the point mutation Asp816Val in c-kit in patients with mastocytosis with an associated hematologic disorder provides insight not only into the pathogenesis of this form of mastocytosis but also into how hematopoiesis may become dysregulated and may serve to provide a means of confirming the diagnosis, assessing prognosis, and developing intervention strategies.

A mitochondrial DNA clone is associated with increased risk for Alzheimer disease.

Severe mitochondrial genetic mutations lead to early degeneration of specific human tissues; milder mitochondrial mutations may cause degeneration at a later point in life. A mutation at position 4336 was reported to occur at increased frequency in individuals with Alzheimer disease (AD) and Parkinson disease [Shoffner, J. M., Brown, M. D., Torroni, A., Lott, M. T., Cabell, M. F., Mirra, S. S., Beal, M. F., Yang, C.-C., Gearing, M., Salvo, R., Watts, R. L., Juncos, J. L., Hansen, L. A., Crain, B. J., Fayad, M., Reckord, C. L. & Wallace, D. C. (1993) Genomics 17, 171-184]. We have investigated the notion that this mutation leads to excess risk of AD by using a case-control study design of 72 AD autopsies and 296 race- and age-matched controls. The 4336G mutation occurred at higher frequency in AD autopsies than age-matched controls, a statistically significant difference. Evolutionary analysis of mtDNAs bearing the 4336G mutation indicated they were more closely related to each other than to other mtDNAs, consistent with the model of a single origin for this mutation. The tight evolutionary relatedness and homoplasmy of mtDNAs that confer elevated risk for a late-onset disease contrast strikingly with the distant relatedness and heteroplasmy of mitochondrial genomes that cause early-onset disease. The dichotomy can be explained by a lack of selection against mutations that confer a phenotype at advanced age during most of the evolution of humans. We estimate that approximately 1.5 million Caucasians in the United States bear the 4336G mutation and are at significantly increased risk of developing mitochondrial AD in their lifetime. A mechanism for 4336G-mediated cell death is proposed.

The pre-S domain of the large viral envelope protein determines host range in avian hepatitis B viruses.

In addition to their well-recognized hepatotropism, all hepatitis B viruses (HBVs) display marked species specificity, growing poorly or not at all in species other than those closely related to their natural hosts. We have examined the molecular basis for this narrow host range, using duck HBV (DHBV) and heron HBV (HHBV) as a model system. HHBV virions will not infect ducks in vivo and infect cultured duck hepatocytes extremely inefficiently in vitro. Mutant HHBV genomes lacking all viral envelope proteins (HHBV env-) can be complemented in trans with DHBV envelope proteins; the resulting pseudotyped virions can efficiently infect duck hepatocytes. Further complementation analysis reveals that of the two viral surface proteins (L and S), it is the L protein that determines host range. Pseudotyping of HHBV env- with DHBV/HHBV chimeric envelope proteins reveals that replacement of as few as 69 amino acids of the pre-S domain of the HHBV L protein by their DHBV counterparts is sufficient to permit infection of duck hepatocytes. These studies indicate that the species-specificity of hepadnaviral infection is determined at the level of virus entry and is governed by the pre-S domain of the viral L protein.

Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues.

The integrase protein of human immunodeficiency virus type 1 is necessary for the stable integration of the viral genome into host DNA. Integrase catalyzes the 3' processing of the linear viral DNA and the subsequent DNA strand transfer reaction that inserts the viral DNA ends into host DNA. Although full-length integrase is required for 3' processing and DNA strand transfer activities in vitro, the central core domain of integrase is sufficient to catalyze an apparent reversal of the DNA strand transfer reaction, termed disintegration. This catalytic core domain, as well as the full-length integrase, has been refractory to structural studies by x-ray crystallography or NMR because of its low solubility and propensity to aggregate. In an attempt to improve protein solubility, we used site-directed mutagenesis to replace hydrophobic residues within the core domain with either alanine or lysine. The single substitution of lysine for phenylalanine at position 185 resulted in a core domain that was highly soluble, monodisperse in solution, and retained catalytic activity. This amino acid change has enabled the catalytic domain of integrase to be crystallized and the structure has been solved to 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266, 1981-1986]. Systematic replacement of hydrophobic residues may be a useful strategy to improve the solubility of other proteins to facilitate structural and biochemical studies.

Mitochondrial DNA replication but no nuclear DNA replication during development of Dictyostelium.

Dictyostelium discoideum cells initiate development when nutrients are depleted. DNA synthesis decreases rapidly thereafter but resumes during late aggregation, only in prespore cells. This observation has been previously interpreted as indicating progression of prespore cells through the cell cycle during development. We show that developmental DNA replication occurs only in mitochondria and not in nuclei. We also show that the prestalk morphogen known as differentiation-inducing factor 1 can inhibit mitochondrial respiration. A model is proposed for cell type divergence, based on competition to become prespores, that involves mitochondrial replication in prespore cells and reduction of mitochondrial activity in prestalk cells.