Dual regulation of open-complex formation and promoter clearance by Arc explains a novel repressor to activator switch.

In studies of variants of the P(ant) promoter of bacteriophage P22, the Arc protein was found not only to slow the rate at which RNA polymerase forms open complexes but also to accelerate the rate at which the enzyme clears the promoter. These dual activities permit Arc, bound at a single operator subsite, to act as an activator or as a repressor of different promoter variants. For example, Arc activates a P(ant) variant for which promoter clearance is rate limiting in the presence and absence of Arc but represses a closely related variant for which open-complex formation becomes rate limiting in the presence of Arc. The acceleration of promoter clearance by Arc requires occupancy of the operator subsite proximal to the -35 region and is diminished when Arc bears a mutation in Arg-23, a residue that makes a DNA-backbone contact in the operator complex.

Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor.

The interaction of the hormone erythropoietin and its receptor (EpoR) is though to be required for normal hematopoiesis. To define the role of EpoR in this process, the murine EpoR was disrupted by homologous recombination. Mice lacking the EpoR died in utero at embryonic day 11-12.5 with severe anemia. Embryonic erythropoiesis was markedly diminished, while fetal liver hematopoiesis was blocked at the proerythroblast stage. Other cell types known to express EpoR, including megakaryocytes, mast, and neural cells were morphologically normal. Reverse transcription-coupled PCR analysis of RNA from embryonic yolk sac, peripheral blood, and fetal liver demonstrated near normal transcripts levels for EKLF, thrombopoietin (Tpo), c-MPL, GATA-1, GATA-2, and alpha- and embryonic beta H1-globin but non for adult beta maj-globin. While colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) colonies were not present in cultures derived from EpoR-/- liver or yolk sac cells, hemoglobin-containing BFU-E colonies were detected in cultures treated with recombinant Tpo and Kit ligand or with Tpo and interleukin 3 and 11. Rescued BFU-E colonies expressed adult beta-globin and c-MPL and appeared morphologically normal. Thus, erythroid progenitors are formed in vivo in mice lacking the EpoR, and our studies demonstrate that a signal transmitted through the Tpo receptor c-MPL stimulates proliferation and terminal differentiation of these progenitors in vitro.

Auxin as a positional signal in pattern formation in plants.

By using a novel, extremely sensitive and specific gas chromatography-mass spectrometry technique we demonstrate in Pinus sylvestris (L.) trees the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid, over the lateral meristem responsible for the bulk of plant secondary growth, the vascular cambium. This is the first evidence that plant morphogens, such as indole-3-acetic acid, occur in concentration gradients over developing tissues. This finding gives evidence for a regulatory system in plants based on positional signaling, similar to animal systems.

Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

The hippocampal formation participates in novel picture encoding: evidence from functional magnetic resonance imaging.

Considerable evidence exists to support the hypothesis that the hippocampus and related medial temporal lobe structures are crucial for the encoding and storage of information in long-term memory. Few human imaging studies, however, have successfully shown signal intensity changes in these areas during encoding or retrieval. Using functional magnetic resonance imaging (fMRI), we studied normal human subjects while they performed a novel picture encoding task. High-speed echo-planar imaging techniques evaluated fMRI signal changes throughout the brain. During the encoding of novel pictures, statistically significant increases in fMRI signal were observed bilaterally in the posterior hippocampal formation and parahippocampal gyrus and in the lingual and fusiform gyri. To our knowledge, this experiment is the first fMRI study to show robust signal changes in the human hippocampal region. It also provides evidence that the encoding of novel, complex pictures depends upon an interaction between ventral cortical regions, specialized for object vision, and the hippocampal formation and parahippocampal gyrus, specialized for long-term memory.

Formation of junctions involved in excitation-contraction coupling in skeletal and cardiac muscle.

During excitation-contraction (e-c) coupling of striated muscle, depolarization of the surface membrane is converted into Ca2+ release from internal stores. This process occurs at intracellular junctions characterized by a specialized composition and structural organization of membrane proteins. The coordinated arrangement of the two key junctional components--the dihydropyridine receptor (DHPR) in the surface membrane and the ryanodine receptor (RyR) in the sarcoplasmic reticulum--is essential for their normal, tissue-specific function in e-c coupling. The mechanisms involved in the formation of the junctions and a potential participation of DHPRs and RyRs in this process have been subject of intensive studies over the past 5 years. In this review we discuss recent advances in understanding the organization of these molecules in skeletal and cardiac muscle, as well as their concurrent and independent assembly during development of normal and mutant muscle. From this information we derive a model for the assembly of the junctions and the establishment of the precise structural relationship between DHPRs and RyRs that underlies their interaction in e-c coupling.

Stabilization of diverged tandem repeats by mismatch repair: evidence for deletion formation via a misaligned replication intermediate.

A functional methyl-directed mismatch repair pathway in Escherichia coli prevents the formation of deletions between 101-bp tandem repeats with 4% sequence divergence. Deletions between perfectly homologous repeats are unaffected. Deletion in both cases occurs independently of the homologous recombination gene, recA. Because the methyl-directed mismatch repair pathway detects and excises one strand of a mispaired duplex, an intermediate for RecA-independent deletion of tandem repeats must therefore be a heteroduplex formed between strands of each repeat. We find that MutH endonuclease, which in vivo incises specifically the newly replicated strand of DNA, and the Dam methylase, the source of this strand-discrimination, are required absolutely for the exclusion of "homeologous" (imperfectly homologous) tandem deletion. This supports the idea that the heteroduplex intermediate for deletion occurs during or shortly after DNA replication in the context of hemi-methylation. Our findings confirm a "replication slippage" model for deletion formation whereby the displacement and misalignment of the nascent strand relative to the repeated sequence in the template strand accomplishes the deletion.

Heterodimer formation and activity in the human enzyme galactose-1-phosphate uridylyltransferase.

One of the fundamental questions concerning expression and function of dimeric enzymes involves the impact of naturally occurring mutations on subunit assembly and heterodimer activity. This question is of particular interest for the human enzyme galactose-l-phosphate uridylyl-transferase (GALT), impairment of which results in the inherited metabolic disorder galactosemia, because many if not most patients studied to date are compound heterozygotes rather than true molecular homozygotes. Furthermore, the broad range of phenotypic severity observed in these patients raises the possibility that allelic combination, not just allelic constitution, may play some role in determining outcome. In the work described herein, we have selected two distinct naturally occurring null mutations of GALT, Q188R and R333W, and asked the questions (i) what are the impacts of these mutations on subunit assembly, and (ii) if heterodimers do form, are they active? To answer these questions, we have established a yeast system for the coexpression of epitope-tagged alleles of human GALT and investigated both the extent of specific GALT subunit interactions and the activity of defined heterodimer pools. We have found that both homodimers and heterodimers do form involving each of the mutant subunits tested and that both heterodimer pools retain substantial enzymatic activity. These results are significant not only in terms of their implications for furthering our understanding of galactosemia and GALT holoenzyme structure-function relationships but also because the system described may serve as a model for similar studies of other complexes composed of multiple subunits.

Cellular mechanisms for the formation of a soluble form derivative of T-cell receptor alpha chain by suppressor T cells.

Upon stimulation with anti-CD3, suppressor T-cell (Ts) hybridomas and homologous transfectants of T-cell receptor a (TCRalpha) cDNA in the T-cell hybridoma formed a 55-kDa TCRalpha chain derivative that bound both the monoclonal anti-TCRalpha chain and polyclonal antibodies against glycosylation inhibiting factor (GIF). The peptide is a subunit of antigen-specific suppressor T-cell factor (TsF), and is considered to be a posttranslationally-formed conjugate of TCRalpha chain with GIF peptide. The TCRalpha derivative is synthesized by the transfectant after stimulation with anti-CD3, and not derived from TCR present on the cell surface. Stimulation of the stable homologous transfectants with anti-CD3 induced translocation of the 13-kDa GIF peptide into endoplasmic reticulum (ER). When a helper Ts hybridoma or a stable transfectant of the same TCRalpha cDNA in a helper cell-derived TCRalpha- clone was stimulated with anti-CD3, translocation of GIF peptide was not detected, and these cells failed to secrete a TCRalpha derivative. However, further transfection of a chimeric cDNA encoding a procalcitonin-GIF fusion protein into the helper cell-derived stable transfectant of TCRalpha cDNA resulted in translocation of the GIF protein and formation of bioactive 55-kDa GIF. The results indicated that translocation of GIF peptide through ER is unique for Ts cells, and that this process is essential for the formation/secretion of the soluble form derivative of TCRalpha chain by T cells.

Formation of stable cationic lipid/DNA complexes for gene transfer.

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

Positive genetic feedback governs cAMP spiral wave formation in Dictyostelium.

The aggregation stage of the life cycle of Dictyostelium discoideum is governed by the chemotactic response of individual amoebae to excitable waves of cAMP. We modeled this process through a recently introduced hybrid automata-continuum scheme and used computer simulation to unravel the role of specific components of this complex developmental process. Our results indicated an essential role for positive feedback between the cAMP signaling and the expression of the genes encoding the signal transduction and response machinery.

A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Gly-93--> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the FALS mutant, G93A, and wild-type cDNA of human Cu,Zn-SOD, overexpressed them in Sf9 insect cells, purified the proteins, and studied their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as substrate. Our results showed that both enzymes contain one copper ion per subunit and have identical dismutation activity. However, the free radical-generating function of the G93A mutant, as measured by the spin trapping method, is enhanced relative to that of the wild-type enzyme, particularly at lower H2O2 concentrations. This is due to a small, but reproducible, decrease in the value of Km for H2O2 for the G93A mutant, while the kcat is identical for both enzymes. Thus, the ALS symptoms observed in G93A transgenic mice are not caused by the reduction of Cu,Zn-SOD activity with the mutant enzyme; rather, it is induced by a gain-of-function, an enhancement of the free radical-generating function. This is consistent with the x-ray crystallographic studies showing the active channel of the FALS mutant is slightly larger than that of the wild-type enzyme; thus, it is more accessible to H2O2. This gain-of-function, in part, may provide an explanation for the association between ALS and Cu,Zn-SOD mutants.

Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.

A JAK-STAT pathway regulates wing vein formation in Drosophila.

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.

Cellular stress inhibits transposition of the yeast retrovirus-like element Ty3 by a ubiquitin-dependent block of virus-like particle formation.

Many stress proteins and their cognates function as molecular chaperones or as components of proteolytic systems. Viral infection can stimulate synthesis of stress proteins and particular associations of viral and stress proteins have been documented. However, demonstrations of functions for stress proteins in viral life cycles are few. We have initiated an investigation of the roles of stress proteins in eukaryotic viral life cycles using as a model the Ty3 retrovirus-like element of Saccharomyces cerevisiae. During stress, Ty3 transposition is inhibited; Ty3 DNA is not synthesized and, although precursor proteins are detected, mature Ty3 proteins and virus-like particles (VLPs) do not accumulate. The same phenotype is observed in the constitutively stressed ssa1 ssa2 mutant, which lacks two cytoplasmic members of the hsp70 family of chaperones. Ty3 VLPs preformed under nonstress conditions are degraded more rapidly if cells are shifted from 30 degrees C to 37 degrees C. These results suggest that Ty3 VLPs are destroyed by cellular stress proteins. Elevated expression of the yeast UBP3 gene, which encodes a protease that removes ubiquitin from proteins, allows mature Ty3 proteins and VLPs to accumulate in the ssa1 ssa2 mutant, suggesting that, at least under stress conditions, ubiquitination plays a role in regulating Ty3 transposition.