Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments.

One of the hallmarks of Alzheimer disease is the pathological aggregation of tau protein into paired helical filaments (PHFs) and neurofibrillary tangles. Here we describe the in vitro assembly of recombinant tau protein and constructs derived from it into PHFs. Though whole tau assembled poorly, constructs containing three internal repeats (corresponding to the fetal tau isoform) formed PHFs reproducibly. This ability depended on intermolecular disulfide bridges formed by the single Cys-322. Blocking the SH group, mutating Cys for Ala, or keeping tau in a reducing environment all inhibited assembly. With constructs derived from four-repeat tau (having the additional repeat no. 2 and a second Cys-291), PHF assembly was blocked because Cys-291 and Cys-322 interact within the molecule. PHF assembly was enabled again by mutating Cys-291 for Ala. The synthetic PHFs bound the dye thioflavin S used in Alzheimer disease diagnostics. The data imply that the redox potential in the neuron is crucial for PHF assembly, independently or in addition to pathological phosphorylation reactions.

Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines.

Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA)n.(dT)n mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

Nucleotide sequence of the cDNA encoding the proenzyme of phenol oxidase A1 of Drosophila melanogaster.

Clones encoding pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] A1 were isolated from a lambda gt10 library that originated from Drosophila melanogaster strain Oregon-R male adults. The 2294 bp of the cDNA included a 13-bp 5'-noncoding region, a 2070-bp encoding open reading frame of 690 amino acids, and a 211-bp 3'-noncoding region. A hydrophobic NH2-terminal sequence for a signal peptide is absent in the protein. Furthermore, there are six potential N-glycosylation sites in the sequence, but no amino sugar was detected in the purified protein by amino acid analysis, indicating the lack of an N-linked sugar chain. The potential copper-binding sites, amino acids 200-248 and 359-414, are highly homologous to the corresponding sites of hemocyanin of the tarantula Eurypelma californicum, the horseshoe crab Limulus polyphemus, and the spiny lobster Panulirus interruptus. On the basis of the phylogenetic tree constructed by the neighbor-joining method, vertebrate tyrosinases and molluscan hemocyanins constitute one family, whereas pro-POs and arthropod hemocyanins group with another family. It seems, therefore, likely that pro-PO originates from a common ancestor with arthropod hemocyanins, independently to the vertebrate and microbial tyrosinases.

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.

Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure.

Myotonic dystrophy is caused by an expansion of a CTG triplet repeat sequence in the 3' noncoding region of a protein kinase gene, yet the mechanism by which the triplet repeat expansion causes disease remains unknown. This report demonstrates that a DNase I hypersensitive site is positioned 3' of the triplet repeat in the wild-type allele in both fibroblasts and skeletal muscle cells. In three unrelated individuals with myotonic dystrophy that have large expansions of the triplet repeat, the allele with the triplet repeat expansion exhibited both overall DNase I resistance and inaccessibility of nucleases to the adjacent hypersensitive site. These results indicate that the triplet repeat expansion alters the adjacent chromatin structure, establishing a region of condensed chromatin, and suggests a molecular mechanism for myotonic dystrophy.

Operational RNA code for amino acids: species-specific aminoacylation of minihelices switched by a single nucleotide.

The genetic code is based on aminoacylation reactions where specific amino acids are attached to tRNAs bearing anticodon trinucleotides. However, the anticodon-independent specific aminoacylation of RNA minihelix substrates by bacterial and yeast tRNA synthetases suggested an operational RNA code for amino acids whereby specific RNA sequences/structures in tRNA acceptor stems correspond to specific amino acids. Because of the possible significance of the operational RNA code for the development of the genetic code, we investigated aminoacylation of synthetic RNA minihelices with a human enzyme to understand the sequences needed for that aminoacylation compared with those needed for a microbial system. We show here that the species-specific aminoacylation of glycine tRNAs is recapitulated by a species-specific aminoacylation of minihelices. Although the mammalian and Escherichia coli minihelices differ at 6 of 12 base pairs, two of the three nucleotides essential for aminoacylation by the E. coli enzyme are conserved in the mammalian minihelix. The two conserved nucleotides were shown to be also important for aminoacylation of the mammalian minihelix by the human enzyme. A simple interchange of the differing nucleotide enabled the human enzyme to now charge the bacterial substrate and not the mammalian minihelix. Conversely, this interchange made the bacterial enzyme specific for the mammalian substrate. Thus, the positional locations (if not the actual nucleotides) for the operational RNA code for glycine appear conserved from bacteria to mammals.

Differential subcellular mRNA targeting: deletion of a single nucleotide prevents the transport to axons but not to dendrites of rat hypothalamic magnocellular neurons.

It has previously been shown that mRNA encoding the arginine vasopressin (AVP) precursor is targeted to axons of rat magnocellular neurons of the hypothalamo-neurohypophyseal tract. In the homozygous Brattle-boro rat, which has a G nucleotide deletion in the coding region of the AVP gene, no such targeting is observed although the gene is transcribed. RNase protection and heteroduplex analyses demonstrate that, in heterozygous animals, which express both alleles of the AVP gene, the wild-type but not the mutant transcript is subject to axonal compartmentation. In contrast, wild-type and mutant AVP mRNAs are present in dendrites. These data suggest the existence of different mechanisms for mRNA targeting to the two subcellular compartments. Axonal mRNA localization appears to take place after protein synthesis; the mutant transcript is not available for axonal targeting because it lacks a stop codon preventing its release from ribosomes. Dendritic compartmentation, on the other hand, is likely to precede translation and, thus, would be unable to discriminate between the two mRNAs.

Catalytically critical nucleotide in domain 5 of a group II intron.

Domain 5 (D5) is a small hairpin structure within group II introns. A bimolecular assay system depends on binding by D5 to an intron substrate for self-splicing activity. In this study, mutations in D5 identify two among six nearly invariant nucleotides as being critical for 5' splice junction hydrolysis but unimportant for binding. A mutation at another site in D5 blocks binding. Thus, mutations can distinguish two D5 functions: substrate binding and catalysis. The secondary structure of D5 may resemble helix I formed by the U2 and U6 small nuclear RNAs in the eukaryotic spliceosome. Our results support a revision of the previously proposed correspondence between D5 and helix I on the basis of the critical trinucleotide 5'-AGC-3' present in both. We suggest that this trinucleotide plays a similar role in promoting the chemical reactions for both splicing systems.

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

Identification of mutations in the Wiskott-Aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at DXS6940, adjacent to the disease gene.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The disease gene has been localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The function of the encoded protein remains undetermined. In this study we have characterized mutations in 12 unrelated patients to confirm the identity of the disease gene. We have also revised the coding sequence and genomic structure for the WAS gene. To analyze further the transmittance of the disease gene, we have characterized a polymorphic microsatellite at the DXS6940 locus within 30 kb of the gene and demonstrate the inheritance of the affected alleles in families with a history of WAS.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

Isolation and molecular characterization of a human T-cell lymphotropic virus type II (HTLV-II), subtype B, from a healthy Pygmy living in a remote area of Cameroon: an ancient origin for HTLV-II in Africa.

We report characterization of a human T-cell lymphotropic virus type II (HTLV-II) isolated from an interleukin 2-dependent CD8 T-cell line derived from peripheral blood mononuclear cells of a healthy, HTLV-II-seropositive female Bakola Pygmy, aged 59, living in a remote equatorial forest area in south Cameroon. This HTLLV-II isolate, designated PYGCAM-1, reacted in an indirect immunofluorescence assay with HTLV-II and HTLV-I polyclonal antibodies and with an HTLV-I/II gp46 monoclonal antibody but not with HTLV-I gag p19 or p24 monoclonal antibodies. The cell line produced HTLV-I/II p24 core antigen and retroviral particles. The entire env gene (1462 bp) and most of the long terminal repeat (715 bp) of the PYGCAM-1 provirus were amplified by the polymerase chain reaction using HTLV-II-specific primers. Comparison with the long terminal repeat and envelope sequences of prototype HTLV-II strains indicated that PYGCAM-1 belongs to the subtype B group, as it has only 0.5-2% nucleotide divergence from HTLV-II B strains. The finding of antibodies to HTLV-II in sera taken from the father of the woman in 1984 and from three unrelated members of the same population strongly suggests that PYGCAM-1 is a genuine HTLV-II that has been present in this isolated population for a long time. The low genetic divergence of this African isolate from American isolates raises questions about the genetic variability over time and the origin and dissemination of HTLV-II, hitherto considered to be predominantly a New World virus.