Striking effects of coupling mutations in the acceptor stem on recognition of tRNAs by Escherichia coli Met-tRNA synthetase and Met-tRNA transformylase.

We measured kinetic parameters in vitro and directly analyzed aminoacylation and formylation levels in vivo to study recognition of Escherichia coli initiator tRNA mutants by E. coli Met-tRNA synthetase and Met-tRNA transformylase. We show that, in addition to the anticodon sequence, mutations in the "discriminator" base A73 also affect aminoacylation. An A73----U change has a small effect, but a change to G73 or C73 significantly lowers Vmax/Kappm for in vitro aminoacylation and leads to appreciable accumulation of uncharged tRNA in vivo. Significantly, coupling of the G73 mutation with G72, a neighboring-base mutation, results in a tRNA essentially uncharged in vivo. Coupling of C73 and U73 mutations with G72 does not have such an effect. Elements crucial for Met-tRNA transformylase recognition of tRNAs are located at the end of the acceptor stem. These elements include a weak base pair or a mismatch between nucleotides (nt) 1 and 72 and base pairs 2.71 and 3.70. The natures of nt 1 and 72 are less important than the fact that they do not form a strong Watson-Crick base pair. Interestingly, the negative effect of a C.G base pair between nt 1 and 72 is suppressed by mutation of the neighboring nucleotide A73 to either C73 or U73. The presence of C73 or U73 could destabilize the C1.G72 base pair at the end of an RNA helix. Thus, in some tRNAs, the discriminator base could affect stability of the base pair between nt 1 and 72 and thereby the structure of tRNA at the end of the acceptor stem.


AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.

We isolated cDNA clones containing the entire coding region of the putative oncogene AKT2. Sequence analysis and in vitro translation demonstrated that AKT2 encodes a 56-kDa protein with homology to serine/threonine kinases; moreover, this protein contains a Src homology 2-like domain. AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. AKT2 was mapped to chromosome region 19q13.1-q13.2 by fluorescence in situ hybridization. In the two ovarian carcinoma cell lines exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. We conclude that AKT2 belongs to a distinct subfamily of protein-serine/threonine kinases containing Src homology 2-like domains and that alterations of AKT2 may contribute to the pathogenesis of ovarian carcinomas.


Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro.


Regulation of thyroid hormone receptor-mediated transcription by a cytosol protein.

Thyroid hormone receptors (TRs) are members of the steroid hormone/retinoic acid receptor superfamily, which regulate homeostasis, development, and differentiation. Their transcriptional activity is modulated by the thyroid hormone 3,3',5-triiodo-L-thyronine (T3). The present study evaluated the effect of the availability of cytoplasmic T3 on the modulation of transcriptional responses of the TRs. In human choriocarcinoma JEG-3 and monkey COS-1 cells, the cytosolic thyroid hormone binding protein is a monomer of the tetrameric pyruvate kinase, subtype M2, which does not bind T3. The in vivo monomer-tetramer interconversion is regulated by glucose via fructose 1,6-bisphosphate. At the physiological T3 concentration, lowering the glucose concentration led to an increase in the cellular concentration of the cytosolic thyroid hormone binding protein. By using a transient transfection system, a concomitant reduction in the transcriptional activity of the human beta 1 thyroid hormone receptor was detected in both cell lines. In the absence of glucose, the transcriptional activity of the human beta 1 thyroid hormone receptor in JEG-3 and COS-1 cells was reduced by 65-75% and 90-95%, respectively. However, glucose had no effect on the basal transcriptional activity. These findings demonstrate an important prenuclear step in the modulation of the gene regulating activity of the TRs.


A sarcomeric alpha-actinin truncated at the carboxyl end induces the breakdown of stress fibers in PtK2 cells and the formation of nemaline-like bodies and breakdown of myofibrils in myotubes.

In many nonmuscle cells, nonsarcomeric alpha-actinin is distributed in the dense bodies of stress fibers, adhesion plaques, and adherens junctions. In striated muscle, a sarcomeric isoform of alpha-actinin (s-alpha-actinin) is found in the Z-bands of myofibrils and subsarcolemmal adhesion plaques. To understand the role(s) of the alpha-actinin isoforms in the assembly and maintenance of such cytoskeletal structures, full-length or truncated s-alpha-actinin cDNAs were expressed in PtK2 cells and in primary skeletal myogenic cells. We found the following. (i) In transfected PtK2 cells the truncated s-alpha-actinin was rapidly incorporated into preexisting dense bodies, adhesion plaques, and adherens junctions. With time these structures collapsed, and the affected cells detached from the substrate. (ii) In myotubes the truncated s-alpha-actinin was incorporated into nascent Z-bands. Many of these progressively hypertrophied, forming nemaline-like bodies. With time the affected myofibrils fragmented, and the myotubes detached from the substrate. (iii) In both cell types the truncated s-alpha-actinin was significantly more disruptive of the cytoskeletal structures than the full-length molecule. (iv) Pools of "over-expressed" full-length or truncated protein did not self-aggregate into homogeneous, amorphous complexes; rather the exogenous proteins selectively colocalized with the same cohort of cytoskeletal proteins with which the endogenous alpha-actinin normally associates. The similarity among the hypertrophied Z-bands in transfected myotubes, the nemaline bodies in patients with nemaline myopathies, and the streaming Z-bands seen in various muscle pathologies raises the possibility that the genetically determined nemaline bodies and the pathologically induced Z-band alterations may reflect primary and/or post-translational modifications of s-alpha-actinin.


Emergence of the keratinocyte growth factor multigene family during the great ape radiation.

The structural gene for human keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, consists of three coding exons and two introns typical of other fibroblast growth factor loci. A portion of the KGF gene, located on chromosome 15, is amplified to approximately 16 copies in the human genome, and these highly related copies (which consist of exon 2, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.


Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant.


Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and a proposed uridine diphosphate binding site.

Erwinia herbicola, a nonphotosynthetic bacterium, is yellow colored due to the accumulation of unusually polar carotenoids, primarily mono- and diglucosides of zeaxanthin. We have cloned and expressed the gene for the enzyme that catalyzes the glucosylation of zeaxanthin. The enzyme has an apparent molecular mass of 45 kDa on an SDS/polyacrylamide gel, which is consistent with its calculated molecular mass. In vitro enzymatic activity was demonstrated using UDP-[14C]glucose and zeaxanthin as substrates. The product zeaxanthin diglucoside and its intermediate monoglucoside were identified by thin layer chromatography. The optimum pH and temperature ranges of the enzyme are 7.0-7.5 and 32-37 degrees C, respectively. A hydropathy plot indicates no apparent membrane-spanning regions, and biochemical experiments suggest that the enzyme is weakly membrane-associated. The amino acid sequence derived from the zeaxanthin glucosyltransferase gene shows a small region of high similarity with other glucuronosyl- and glucosyltransferases that use either UDP-activated glucuronic acid or a sugar as one of their substrates. Based on these similarities, we propose that this conserved sequence is part of the UDP binding site.


Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14C]guanine-containing DNA substrate by nick-translating DNA with [14C]dGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cell might protect them from exposure to metabolites of vinyl chloride.


Peptoids: a modular approach to drug discovery.

Peptoids, oligomers of N-substituted glycines, are described as a motif for the generation of chemically diverse libraries of novel molecules. Ramachandran-type plots were calculated and indicate a greater diversity of conformational states available for peptoids than for peptides. The monomers incorporate t-butyl-based side-chain and 9-fluorenylmethoxy-carbonyl alpha-amine protection. The controlled oligomerization of the peptoid monomers was performed manually and robotically with in situ activation by either benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate or bromotris(pyrrolidino)phosphonium hexaflurophosphate. Other steps were identical to peptide synthesis using alpha-(9-fluorenylmethoxycarbonyl)amino acids. A total of 15 monomers and 10 oligomers (peptoids) are described. Preliminary data are presented on the stability of a representative oligopeptoid to enzymatic hydrolysis. Peptoid versions of peptide ligands of three biological systems (bovine pancreatic alpha-amylase, hepatitis A virus 3C proteinase, and human immunodeficiency virus transactivator-responsive element RNA) were found with affinities comparable to those of the corresponding peptides. The potential use of libraries of these compounds in receptor- or enzyme-based assays is discussed.


Cloning of an apparent splice variant of the rat N-methyl-D-aspartate receptor NMDAR1 with altered sensitivity to polyamines and activators of protein kinase C.

Molecular cloning identified complementary DNA species, from a rat ventral midbrain library, encoding apparent splice variants of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 (which we now term NR1a). Sequencing revealed that one variant, NR1b, differs from NR1a by the presence of a 21-amino acid insert near the amino end of the N-terminal domain and by an alternate C-terminal domain in which the last 75 amino acids are replaced by an unrelated sequence of 22 amino acids. NR1b is virtually identical to NR1a in the remainder of the N- and C-terminal domains, at the 5' and 3' noncoding ends, and within the predicted transmembrane domains and extracellular and cytoplasmic loops. These findings suggest that the two forms of the receptor arise by differential splicing of a transcript from the same gene. Sequencing of other clones indicates the existence of a third variant, NR1c, identical to NR1b in its C terminus but lacking the N-terminal insert. NR1b RNA injected into Xenopus oocytes generated functional homomeric NMDA channels with electrophysiological properties distinct from those of NR1a homomeric channels. NR1b channels exhibited a lower apparent affinity for NMDA and for glutamate. NR1b channels exhibited a lower affinity for D-2-amino-5-phosphonovaleric acid and a higher affinity for Zn2+. The two receptor variants showed nearly identical affinities for glycine, Mg2+, and phencyclidine. Spermine potentiation of NMDA responses, prominent in oocytes injected with rat forebrain message, was also prominent for NR1a receptors, but was greatly reduced or absent for NR1b receptors. Treatment with the protein kinase C activator phorbol 12-myristate 13-acetate potentiated NMDA responses in NR1b-injected oocytes by about 20-fold; potentiation of NMDA responses in NR1a-injected oocytes was much less, about 4-fold. These findings support a role for alternate splicing in generating NMDA channels with different functional properties.


Cloning and chromosomal mapping of a human immunodeficiency virus 1 "TATA" element modulatory factor.

A critical regulatory element in many promoters transcribed by RNA polymerase II is the "TATA" box, which is located 25-30 nucleotides upstream of the transcription initiation site. TFIID is a biochemically defined HeLa cell nuclear fraction containing a transcription factor activity that binds specifically to the TATA box and is critical in determining both basal and regulated promoter activity. Recently, the gene for a TATA-binding protein was cloned and found to bind to various TATA elements and to substitute for TFIID in stimulating basal gene expression in in vitro transcription systems. However, it is possible that additional cellular factors can bind to the TATA element and influence the level of gene expression. By using lambda gt11 expression cloning with oligonucleotides corresponding to the human immunodeficiency virus 1 TATA element, we report the identification of a cellular protein with a calculated molecular mass of 123 kDa that we designate TATA element modulatory factor (TMF). TMF binds to the human immunodeficiency virus 1 TATA element in gel-retardation assays and inhibits activation of the viral long terminal repeat by the TATA-binding protein in in vitro transcription assays. TMF contains leucine-zipper amino acid motifs and exhibits homology in its DNA binding domain with the phage-encoded DNA binding protein Ner. Chromosomal mapping localizes the TMF gene to human chromosome 3p12-p21, which is a site of frequent rearrangements in lung and renal carcinomas. Thus, TMF is a transcription factor that likely regulates the expression of both viral and cellular genes.


Nitric oxide stimulates auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase.

Nitric oxide generation in brain cytosolic fractions markedly enhances ADP-ribosylation of a single 37-kDa protein. By utilizing a biotinylated NAD and avidin affinity chromatography, we purified this protein. Partial amino acid sequencing establishes its identity as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This is further confirmed by detection of GAPDH enzymatic activity in the purified 37-kDa protein. GAPDH is ADP-ribosylated in the absence of brain extract. This auto-ADP-ribosylation is enhanced by nitric oxide generation. ADP-ribosylation appears to involve the cysteine where NAD interacts with GAPDH so that ADP-ribosylation likely inhibits enzymatic activity. Such inhibition may play a role in nitric oxide-mediated neurotoxicity.


Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine.

We previously described a protein, isolated from human tissues and cells, that bound to a defined double-stranded oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine. It was further demonstrated that this protein was a glycosylase and released 1,N6-ethenoadenine. We now find that this enzyme also releases 3-methyladenine from methylated DNA and that 3-methyladenine-DNA glycosylase behaves in the same manner, binding to the ethenoadenine-containing oligonucleotide and cleaving both ethenoadenine and 3-methyladenine from DNA containing these adducts. The rate and extent of glycosylase activities toward the two adducts are similar.


Spontaneous insertion of polypeptide chains into membranes: a Monte Carlo model.

The Monte Carlo dynamics method was used to examine the process of protein insertion into model cell membranes. The water and lipid environments were taken into account via an effective medium approximation based on coordinate-dependent hydrophobic and hydrogen bond potentials. The polypeptide chain was represented in a full-backbone atom representation as a chain of diamond lattice vectors. The simulations support the idea that to a good approximation insertion may be depicted as a spontaneous thermodynamic process. The mechanism of membrane insertion of a simple lattice protein chain exhibits many features of theoretical predictions and is in good accord with experimental data. In the model, insertion begins with adsorption of the chain onto the interface, followed by the formation of helical fragments. These fragments, having partially saturated internal hydrogen bonds, can be transported into the lipid phase and then form transbilayer structures.


Activation of potassium channels: relationship to the heat shock response.

We examined the possibility that whole cell currents are involved and possibly trigger the response of mammalian cells to heat shock. Heat-sensitive cells from a radiation-induced fibrosarcoma (RIF-1) and heat-resistant variants (TR-4, TR-5) were heated at 45 degrees C for 3-30 min. We observed induction of voltage-dependent currents after heating in the heat-resistant cells. These currents decayed to nonmeasurable levels over a period of 6 h. In RIF-1 cells, however, voltage-dependent currents were detectable during heating only; these currents then decayed rapidly. Tetraethylammonium (TEA) cations blocked the currents; changing the concentration of extracellular K+ modified the current-voltage (I-V) relationship. These currents, therefore, resulted from the activation of voltage-dependent K+ channels. Addition of TEA during heating sensitized TR-4 cells to heat but had no effect on the heat response of the RIF-1 cells. Continuous exposure of the RIF-1 cells to 2% (vol/vol) dimethyl sulfoxide (DMSO) for 7 days induced the expression of additional functional, voltage-dependent K+ channels; these gave rise to currents that were measurable after heating. In parallel, these cells became heat resistant. Addition of TEA to DMSO-treated cells blocked channels and returned the heat response almost to the pre-DMSO levels. Our data show a correlation between heat resistance and expression of K+ channels. Because resistance to heat very likely relates to the heat shock response, our data suggest that activation of channels may be a very early event in initiation of the heat shock response.


Grammatical model of the regulation of gene expression.

Based on a formal proof that justifies the search for generative grammars in the study of gene regulation, a linguistic formalization of an exhaustive data base of Escherichia coli sigma 70 promoters and their regulatory binding sites has been initiated. The grammar presented here generates all the arrays of the collection plus those that are predicted as consistent with the principles of regulation of sigma 70 promoters. "Systems of regulation," sets of regulatory sites that collaborate in a mechanism of regulation, are represented by means of syntactic categories. A small set of phrase structure rules restricted by an X-bar principle and by a hierarchical, c-command relation generates a representation of arrays of sites of regulation where the selection of the protein(s) identifying the system(s) of regulation occurs. Based on the features of the proteins, optional duplicated proximal and remote sites are generated by means of transformational rules. Consistency with the data, the predictions that the grammar generates, and important similarities and differences with some aspects of the generative theory of natural language are discussed.


Dominant negative selection of heterologous genes: isolation of Candida albicans genes that interfere with Saccharomyces cerevisiae mating factor-induced cell cycle arrest.

We have used a genomic library of Candida albicans to transform Saccharomyces cerevisiae and screened for genes that act similarly to dominant negative mutations by interfering with pheromone-mediated cell cycle arrest. Six different plasmids were identified from 2000 transformants; four have been sequenced. One gene (CZF1) encodes a protein with structural motifs characteristic of a transcription factor. A second gene (CCN1) encodes a cyclin homologue, a third (CRL1) encodes a protein with sequence similarity to GTP-binding proteins of the RHO family, and a fourth (CEK1) encodes a putative kinase of the ERK family. Since CEK1 confers a phenotype similar to that of the structurally related S. cerevisiae gene KSS1 but cannot complement a KSS1 defect, it is evident that dominant negative selection can identify proteins that complementation screens would miss. Because dominant negative mutations exert their influence even in wild-type strain backgrounds, this approach should be a general method for the analysis of complex cellular processes in organisms not amenable to direct genetic analysis.


Complementation of a red-light-indifferent cyanobacterial mutant.

Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation.


Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] initiates repair of damaged DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonuclease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in parallel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutamine completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glutamine or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in Km and decreases in kcat of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamine was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.


Three-dimensional structure of two crystal forms of FabR19.9 from a monoclonal anti-arsonate antibody.

The three-dimensional structure of FabR19.9 from a well-characterized anti-p-azobenzenearsonate monoclonal antibody has been determined by x-ray diffraction techniques in two crystalline forms (I and II) to a resolution of 2.8 and 2.7 A, respectively. Essentially the same tertiary and quaternary structure of the Fab is observed in the two forms. The major difference resides in the intermolecular contacts, which are interpreted to favor an irreversible transition from the metastable form I to the more stable form II. The third complementarity-determining region of the heavy chain (H3) folds back over the combining site and requires rearrangement for hapten binding. This dynamic requirement on H3 is consistent with its mobility in the structure and can explain hapten binding to an otherwise inaccessible antibody combining site.


Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) strains display a high degree of heterogeneity in their biological properties that correlate with in vivo pathogenesis of the virus. We previously demonstrated that overlapping regions encompassing the third hypervariable domain (V3), within the envelope glycoprotein gp120 determine the tropisms of HIV-1 for T-cell lines and primary macrophages. Studies with mutant viruses carrying one or more amino acid substitutions in the V3 loop have now identified this hypervariable domain as a major determinant for these cellular host range properties. Three to five amino acid changes in this domain, but rarely a single amino acid substitution, can confer macrophage tropism and alter T-cell-line tropism. These findings emphasize the effect on cell tropism of small amino acid differences in the viral envelope and suggest that the overall conformation of the V3 loop plays the major role in determining the ability of HIV-1 to infect T-cell lines and primary macrophages.


Increased expression of beta-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage.

Despite increasing evidence for a pathogenetic role for the beta-amyloid precursor protein (beta APP) in Alzheimer disease, the physiological function of the protein remains unclear. The expression of the neural-specific isoform containing 695 amino acids, beta APP695, is consistent with a role for the protein in neuronal development. In this study, we analyzed the expression of beta APP during the retinoic acid-induced neuronal differentiation of P19 murine embryonal carcinoma cells. Northern blot and RNase protection analyses show a selective increase in beta APP695 expression, concomitant with the morphologic differentiation of P19-derived neurons. Moreover, the time course of increase observed for the beta APP695 mRNA is paralleled by other neuronal-specific transcripts. A similar increase in beta APP695 is observed at the protein level. Furthermore, we show that levels of beta APP695 protein progressively increase during the in vitro differentiation of primary hippocampal neurons. The finding that beta APP695 increases selectively and progressively during neuronal differentiation in two different cell culture systems suggests that this isoform has an important cellular function during this process in the brain. Unlike beta APP in most peripheral cell types, the increased levels of beta APP found in terminally differentiated neuronal cells are not processed in significant amounts by secretory cleavage. Thus, differentiation of neurons is accompanied by increased beta APP695 expression and membrane retention of the protein as intact, full-length molecules that could serve as potential substrates for amyloidogenesis.


Light regulates expression of brain-derived neurotrophic factor mRNA in rat visual cortex.

Specific sensory input has profound transient and long-lasting effects on the function of corresponding sensory cortical areas both during development and in adulthood. To study whether neurotrophic factors might play a role in such processes, we investigated the effects of light on the nerve growth factor and brain-derived neurotrophic factor (BDNF) mRNA levels in rat visual cortex. Keeping adult rats in the dark or preventing normal activity of retinal ganglion cells by intraocular injection of tetrodotoxin significantly decreased the levels of BDNF mRNA in the visual cortex but not in other cortical areas. Exposure to light after a period in darkness rapidly restored the mRNA to control levels. These alterations in visual input had no effect on nerve growth factor mRNA. The mRNA of trkB, the putative signal-transducing receptor unit for BDNF, was also decreased in darkness, although less than BDNF mRNA. BDNF mRNA levels increased in the visual cortex of newborn rats after eye-opening. This increase is retarded, although not completely abolished, by rearing the pups in darkness. Thus, the levels of BDNF mRNA are rapidly regulated by sensory input during development and in adulthood. BDNF may therefore play an important role in formation and in activity-dependent modulation of specific connections in the visual cortex.


Cell cycle-dependent localization of casein kinase I to mitotic spindles.

Casein kinase I (CKI) is a class of protein kinases ubiquitous to all eukaryotic cells. Recently, cDNA clones encoding several bovine CKI isoforms have been sequenced that show high sequence identity to the HRR25 gene product of the budding yeast Saccharomyces cerevisiae; HRR25 is required for normal cellular growth, nuclear segregation, DNA repair, and meiosis. We have raised polyclonal antibodies to a human erythroid 34-kDa CKI and have sequenced a portion of this kinase. The amino acid sequence identifies the CKI as the alpha-CKI isoform, which is 62% identical to the HRR25 protein kinase. By use of immunofluorescence, the alpha-CKI has been localized to vesicular cytosolic structures and to the centrosome in interphase cells. As cells progress into mitosis, centrospheric staining increases and, in mitosis, alpha-CKI associates with kinetochore fibers. This localization suggests that alpha-CKI, like HRR25, plays a role in the segregation of chromosomes during mitosis and may be cell cycle-regulated both in humans and in yeast.


Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258.

The arsenic resistance operon of Staphylococcus aureus plasmid pI258 consists of three genes, arsR (encoding the repressor regulatory protein), arsB (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsC (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). ArsC has now been shown to be an arsenate reductase, converting intracellular arsenate [As(V)] to arsenite [As(III)], which is then exported from the cells by an energy-dependent efflux process. The arsenate reductase activity was found in the soluble cytoplasmic fraction in Escherichia coli (and not associated with the periplasmic fraction or the sedimentable cell envelope). Purified ArsC protein coupled in vitro with thioredoxin plus dithiothreitol (but not 2-mercaptoethanol or reduced glutathione) to reduce arsenate to arsenite.


Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6.

In Saccharomyces cerevisiae, at least 17 DNA replication genes are coordinately expressed at the G1/S boundary during the cell cycle. All of these genes have the DNA sequence element ACGCGT in their 5' upstream regulatory regions. This sequence has been shown to be essential for periodic expression of the POL1, CDC9, and TMP1 genes. The cyclin (CLN1 and CLN2) and HO genes are another subset of genes that are expressed with the same timing as the DNA replication genes. Their periodic expression requires the participation of two well-characterized transcriptional activators: the SWI4 and SWI6 gene products. In this study, we present evidence that SWI6 contributes to the regulation of DNA replication genes as well. Surprisingly, a preferential requirement for SWI6 over SWI4 is observed in our studies of ACGCGT-dependent reporter gene expression in vivo. This selectivity has not been observed for the other G1/S genes. Correlating with the in vivo results, protein-DNA complexes formed in vitro on multimeric ACGCGT elements are either abolished or reduced in swi6 delta deletion mutants.


The repressor MDBP-2 is a member of the histone H1 family that binds preferentially in vitro and in vivo to methylated nonspecific DNA sequences.

MDBP-2 is a repressor that binds preferentially to methylated DNA. Peptides derived from MDBP-2 were sequenced. The sequences of the two peptides, KPAGPS-VTELITK and ALAAGGYDVEK, are identical to those found in the chicken histone H1 core protein. In SDS/polyacrylamide gels MDBP-2 has an apparent molecular mass of 21 kDa, and antibodies directed against calf thymus total histone H1 cross-react with MDBP-2. The preferential binding of affinity-purified MDBP-2 to methylated DNA is not sequence-specific but requires a minimum length of 30 base pairs and one pair of symmetrically methylated (i.e., methylated on both strands) CpG dinucleotides. As previously shown, there is a decrease in the binding activity of MDBP-2 to methylated DNA upon estradiol treatment. Immunoblots show that upon estradiol treatment the amount of immunocrossreacting MDBP-2 protein remains unchanged. MDBP-2 enables another protein to bind DNA which by itself does not bind methylated DNA. Ultraviolet crosslinking and selective immunoadsorption assays with anti-histone H1 antibodies show that in vivo MDBP-2 preferentially binds to the methylated repressed vitellogenin gene. It is concluded that MDBP-2 may participate in the long-term silencing of genes (formation of heterochromatin) through selective binding to methylated DNA.


Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.

Cellular progenitors of the coronary vasculature are believed to enter the chicken heart during epicardial morphogenesis between stages 17 and 27 (days 3-5) of egg incubation. To trace cells which give rise to the coronary arteries in vivo, we applied retroviral cell tagging procedures and analyzed clonal populations of vascular smooth muscle, endothelium, and connective tissue in the hearts of post-hatch chickens. Our data provide direct proof that (i) vascular smooth muscle progenitors begin to enter the heart at stage 17, substantially after the heart begins propulsive contractions; (ii) cardiac myocytes, vascular smooth muscle, perivascular fibroblasts, and coronary endothelial cells all derive from independent precursors when these cells migrate into the heart; (iii) endothelial cells of the coronary vessels have a different clonal origin than endothelial cells of the endocardium; (iv) coronary arteries form by the coalescence of discontinuous colonies (i.e., in situ vasculogenesis), each derived from a founder cell tagged at the time of retroviral injection (stages 17-18); and (v) coronary arteries contain discrete segments composed of "polyclones." These studies indicate the feasibility of gene targeting to coronary progenitors through the use of recombinant retroviruses.


Primary electron transfer kinetics in bacterial reaction centers with modified bacteriochlorophylls at the monomeric sites BA,B.

The primary electron transfer has been investigated by femtosecond time-resolved absorption spectroscopy in two chemically modified reaction centers (RC) of Rhodobacter sphaeroides, in which the monomeric bacteriochlorophylls BA and BB have both been exchanged by 13(2)-hydroxybacteriochlorophyll a or [3-vinyl]-13(2)-hydroxybacteriochlorophyll a. The kinetics of the primary electron transfer are not influenced by the 13(2)-hydroxy modification. In RCs containing [3-vinyl]-13(2)-hydroxybacteriochlorophyll a the primary rate is reduced by a factor of 10.


Prevention of autoimmune insulitis in nonobese diabetic mice by expression of major histocompatibility complex class I Ld molecules.

Nonobese diabetic (NOD) mice spontaneously develop a T-cell-mediated autoimmune disease that is similar in many respects to insulin-dependent diabetes mellitus in humans. NOD mice were shown to express major histocompatibility complex class I Kd and Db antigens. To examine the possible involvement of major histocompatibility complex class I molecules in the development of autoimmune insulitis, we attempted to express a different type of class I molecule in NOD mice by crossing C57BL/6 mice transgenic for the class I Ld gene with NOD mice. The backcross progeny expressed the Ld antigen on the peripheral blood lymphocytes at a level comparable with that of the BALB/c mice. The cell surface expression of endogenous class I and class II antigens on the peripheral blood lymphocytes was not affected. Analysis of these mice revealed that the expression of the class I Ld antigen significantly reduced the incidence of insulitis at 20 weeks of age. In situ hybridization of a biotinylated probe on mouse chromosomes showed that the Ld transgene was located in the E area of chromosome 6 with which no genetic linkage to insulin-dependent diabetes mellitus was demonstrated. These results suggest that the NOD-type class I molecules are involved in the development of insulitis in NOD mice.


Birth and migration of neurons in the central posterior/prepacemaker nucleus during adulthood in weakly electric knifefish (Eigenmannia sp.).

In contrast to mammals, fish maintain their capacity to generate neurons in the central nervous system even during adulthood for prolonged periods of life. By employing immunohistochemical, autoradiographic, and electron microscopic techniques, we studied such a postnatal neurogenesis within the complex of the central posterior/prepacemaker nucleus (CP/PPn) in knifefish (Eigenmannia sp.), a weakly electric teleost. The CP/PPn is a bilateral cluster of neurons in the thalamus. It controls frequency modulations of the electric organ discharge as they are used during social interactions. In the CP/PPn region adjacent to the wall of the third ventricle ("ventricular zone"), cells are born continuously and at high rates. They undergo multiple cell divisions before differentiating into neurons. Concomitant with this development, the newborn neurons migrate toward lateral regions of the CP/PPn. In the course of this lateral migration, they appear to acquire immunological and morphological characteristics that are typical for mature CP/PPn neurons. We hypothesize that at least some of the newly generated cells develop finally into functional CP/PPn neurons.


Decreased choline acetyltransferase mRNA expression in the nucleus basalis of Meynert in Alzheimer disease: an in situ hybridization study.

The subnormal choline acetyltransferase (ChoAcTase) activity in the cerebral cortex of patients with Alzheimer disease (AD) is thought to originate from the loss of cholinergic neurons in the nucleus basalis of Meynert (nbM). To examine possible changes in the functional activity of the remaining cholinergic neurons in the nbM of patients with AD, the level of expression of ChoAcTase mRNA was evaluated. A procedure for double-labeling cholinergic neurons to detect ChoAcTase mRNA and the corresponding protein in the same cell was developed, taking advantage of an anti-ChoAcTase antibody and the recently isolated cDNA complementary to a sequence of the human ChoAcTase mRNA. In the study of three controls and four patients with AD, the presence of both ChoAcTase mRNA and protein was observed in the same large neurons in both nbM and putamen. Specificity of in situ hybridization was further supported by the absence of neuronal staining with a sense probe. In AD patients a subnormal level of expression of ChoAcTase mRNA per cholinergic cell was detected in the nbM but not in the putamen. Our data support the hypothesis that expression of ChoAcTase mRNA might be down-regulated in the surviving cholinergic neurons in the nbM of patients with AD, raising the possibility of functional restoration by stimulating ChoAcTase synthesis.


Farnesylcysteine, a constituent of the alpha and beta subunits of rabbit skeletal muscle phosphorylase kinase: localization by conversion to S-ethylcysteine and by tandem mass spectrometry.

The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed.


Characterization of the genes encoding phycoerythrin in the red alga Rhodella violacea: evidence for a splitting of the rpeB gene by an intron.

The phycobilisome of the eukaryotic unicellular red alga Rhodella violacea presents in some respects an organization that is intermediate between those of the homologous counterparts found in cyanobacteria (the putative chloroplast progenitor) and more advanced, pluricellular red algae. This suggests evolutionary relationships that we investigated at the genome level. The present work describes the sequences of two rhodophytan phycobilisome genes, rpeA and rpeB. These chloroplast genes encode the alpha and beta subunits of phycoerythrin, the major component of the light-harvesting antennae and one of the most abundant cellular proteins in these algae. The amino acid sequences deduced from both rpeA and rpeB present strong homologies with those previously reported for phycoerythrin subunits of cyanobacteria, rhodophyta, and cryptomonads. The main difference with the corresponding cyanobacterial genes was the unexpected occurrence of an intervening sequence that split rpeB into two exons. This intervening sequence presents characteristics of group II introns but lacks several structural domains. Transcriptional analyses showed that the two rpe genes are cotranscribed and that the major RNA species detected corresponds to a mature mRNA lacking the intron. As the phycobiliproteins form a group of closely related polypeptides in cyanobacteria and rhodophyta, the molecular events affecting the corresponding genes, such as the rpeB intron, may be a clue to elucidate some aspects of the molecular processes involved in the evolution of plastid genes.


Structure of the host-specific toxins produced by the fungal pathogen Periconia circinata.

Four metabolites named peritoxins A and B and periconins A and B have been isolated together with the known metabolite circinatin from culture filtrates of the fungal pathogen Periconia circinata. Peritoxins A and B, which correspond to the P. circinata toxins Ia and IIa partially characterized in previous work, are selectively toxic to genotypes of Sorghum bicolor susceptible to the pathogen, whereas periconins A and B are biologically inactive. Combination of instrumental analysis and chemical degradation has led to structural assignments for each of the four compounds; only the configuration at some of the chiral centers remains undefined. Structural comparison suggests a precursor role for circinatin in the formation of the peritoxins and the periconins.


Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.


Imaging of luciferase secretion from transformed Chinese hamster ovary cells.

The blue luminescence characteristic of the marine ostracod crustacean Vargula hilgendorfii is from a simple, but highly specific, enzyme-substrate reaction. Light is emitted by the oxidation of Vargula luciferin (substrate) by molecular oxygen, a reaction catalyzed by luciferase. Stable transformants of Chinese hamster ovary cells carrying the Vargula luciferase gene secreted luciferase from discrete sites on the cell surface, and this secretion could be monitored in real time by the bioluminescence produced by the secreted luciferase in the presence of Vargula luciferin by using an image-intensifying technique. Addition of anti-Vargula luciferase IgG to the luminescing cells almost completely extinguished the luminescence, confirming that Vargula luciferase caused the luminescence.


Reductive detoxification as a mechanism of fungal resistance to singlet oxygen-generating photosensitizers.

Fungi that are resistant or sensitive to the singlet oxygen-generating toxin cercosporin were assayed for their ability to detoxify it by reduction. Cercosporin reduction was assayed microscopically by using bandpass filters to differentiate between fluorescence emission from cercosporin and reduced cercosporin. Hyphae of the resistant Cercospora and Alternaria species emitted a green fluorescence, indicative of reduced cercosporin. Hyphae of nonviable cultures and of cercosporin-sensitive fungi did not reduce cercosporin. Sensitive fungi occasionally reduced cercosporin when incubated with reducing agents that protect against cercosporin toxicity. Cercosporin could not be efficiently photoreduced in the absence of the fungus. Cercospora species were also resistant to eosin Y but were sensitive to rose bengal. Microscopic observation demonstrated that Cercospora species were not capable of reducing rose bengal but were capable of reducing eosin Y. These observations were supported by in vitro electrochemical measurements that revealed the following order with respect to ease of reduction: cercosporin >> eosin Y > rose bengal. The formal redox potential (E 0') of cercosporin at pH 7.5 was found to be -0.14 V vs. the normal hydrogen electrode. We conclude that Cercospora species protect themselves against cercosporin by the reduction and detoxification of the toxin molecule.


Mutational analysis of the integrase protein of human immunodeficiency virus type 2.

Purified integrase protein (IN) can nick linear viral DNA at a specific site near the ends and integrate nicked viral DNA into target DNA. We have made a series of 43 site-directed point mutants of human immunodeficiency virus type 2 IN and assayed purified mutant proteins for the following activities: site-specific cleavage of viral DNA (donor cut), integration (strand transfer), and disintegration. In general, the different activities were similarly affected by the mutations. We found three mutations that (almost) totally abolished IN function: Asp-64-->Val, Asp-116-->Ile, and Glu-152-->Leu, whereas 25 mutations did not affect IN function. A few mutations affected the different activities differentially. Near the amino terminus a zinc finger-like sequence motif His-Xaa3-His-Xaa20-30-Cys-Xaa2-Cys is present in all retroviral IN proteins. Two mutations in this region (His-12-->Leu and Cys-40-->Ser) strongly inhibited donor cut but had less effect on strand transfer. The central region of IN is most highly conserved between retroviral INs. Three mutants in this region (Asn-117-->Ile, Asn-120-->Leu, and Lys-159-->Val) were inhibited in strand transfer but were inhibited less strongly in donor cut. Mutation of Asn-120 (to glycine, leucine, or glutamate) resulted in changes in integration-site preference, suggesting that Asn-120 is involved in interactions with target DNA. We did not find a mutant in which one activity was lost and the others were unaffected, supporting the notion that IN has only one active site for the catalysis of donor cut and strand transfer.


1H-[13C] NMR measurements of [4-13C]glutamate turnover in human brain.

A limitation of previous methods for studying human brain glucose metabolism, such as positron emission tomography, is that metabolic steps beyond glucose uptake cannot be studied. Nuclear magnetic resonance (NMR) has the advantage of allowing the nondestructive measurement of 13C distribution in specific carbon positions of metabolites. In this study 1H-[13C] NMR spectroscopy in conjunction with volume localization was used to measure the rate of incorporation of 13C isotope from infused enriched [1-13C]glucose to human brain [4-13C]glutamate. In three studies C4 glutamate turnover time constants of 25, 20, and 17 min were measured in a 21-cm3 volume centered in the region of the visual cortex. Based on an analysis of spectrometer sensitivity the spatial resolution of the method can be improved to < 4 cm3. In conjunction with metabolic modeling and other NMR measurements this method can provide a measure of regional rates of the brain tricarboxylic acid cycle and other metabolic pathways.


Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages.

The legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular parasite. Its interaction with phagocytes has characteristics in common with several other intracellular parasites. Critical aspects of L. pneumophila intracellular multiplication are evasion of lysosomal host cell defenses and the presence of a nutritionally appropriate environment. Following phagocytosis, wild-type L. pneumophila multiply within a specialized phagosome which does not fuse with secondary lysosomes. Mutants which have lost the ability to grow within phagocytes no longer cause disease in animals, indicating that the capacity to multiply intracellularly is important for pathogenesis. One such mutant, 25D, has been shown to be defective in inhibiting phagosome-lysosome fusion. This phagolysosomal environment is not conducive to Legionella growth. We report the isolation of a region of the L. pneumophila genome (icm, intracellular multiplication) which restores the capacity of 25D to multiply in human macrophages. The complemented mutants also regain the capacity to interfere with phagosome-lysosome fusion and to cause lethal pneumonia in guinea pigs.


Activation of expression of genes coding for extracellular matrix proteins in Tat-producing glioblastoma cells.

The Tat protein of human immunodeficiency virus type 1 has been increasingly implicated in directly contributing to the disease AIDS by altering the expression of strategic cellular genes. In this study we demonstrate that the presence of the human immunodeficiency virus type 1 regulatory protein Tat is associated with a significant induction in the expression of certain protein components of the extracellular matrix in glial-derived cells. Northern blot analysis reveals that in cells expressing Tat there is a marked elevation in the steady-state RNA levels for fibronectin and types I and III collagen. Metabolic labeling of the Tat-producing cells demonstrates that this induction is also reflected at the level of protein synthesis. Transient transfection experiments indicate that the presence of Tat results in increased transcription of fibronectin and alpha I type I collagen promoters. Possible mechanisms for this phenomenon and their significance with regard to AIDS are discussed.


Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor.

The synaptic surface of the acetylcholine receptor in membranes from Torpedo californica electric organ has been imaged by scanning tunneling microscopy. The molecule appears pentameric, with one major and four minor protrusions rising above the surface, and these protrusions encompass a large central cavity. The outer diameter of the molecule is 69 +/- 10 A, while the diameter of the cavity, measured at the widest complete contour line delimiting the opening, is 26 +/- 7 A. The images and dimensions obtained are consistent with the structure determined from hybrid density maps obtained by x-ray diffraction and electron microscopy. Thus, scanning tunneling microscopy can be used to obtain overall dimensions and low-resolution structural features of the surface of a membrane-embedded protein.


Detection of transcripts for the receptor for macrophage colony-stimulating factor, c-fms, in murine osteoclasts.

Macrophage colony-stimulating factor (M-CSF), whose action is restricted to the cell populations of the mononuclear phagocyte system, has recently been found to be required for osteoclastogenesis and bone resorption. To investigate the cells involved in the action of M-CSF in these processes, expression of c-fms mRNA, encoding the M-CSF receptor, was studied by in situ hybridization. Paws from murine embryos and newborn mice, tibiae from 2-day-old animals, as well as isolated osteoclasts, were hybridized with a c-fms-specific RNA probe. In bone, c-fms mRNA was detected only in cells at the late stages of osteoclastogenesis and in mature osteoclasts. The findings strengthen the relation between osteoclasts and the mononuclear phagocyte system. Furthermore, they suggest that M-CSF acts directly on osteoclast precursors and on mature osteoclasts during osteoclastogenesis.


Symmetry in the mechanism of bacteriophage lambda integrative recombination.

During the strand-exchange events of bacteriophage lambda integration, pairs of phosphodiester bonds are broken and then rejoined to form novel DNA linkages. The reaction proceeds in vitro in the absence of an external energy source; the bond energy needed to rejoin broken strands of DNA must therefore be conserved during cleavage. Although some of this conservation involves a covalent intermediate between DNA and the recombinase Int, it is possible that such an intermediate is formed with only one of the two phosphodiesters. In such an asymmetric mechanism, the second phosphodiester would be attacked by a nucleophile that is exposed by cleavage of the first DNA strand. In contrast, a symmetric mechanism hypothesizes nucleophilic attack by Int on both phosphodiesters. We have distinguished these two mechanisms by removing potential nucleophiles from the integrative recombination reaction. Our data are inconsistent with an asymmetric mechanism. We conclude that during strand exchange both phosphodiesters proceed through a covalent protein-DNA intermediate.


Plasmodium chabaudi p68 serine protease activity required for merozoite entry into mouse erythrocytes.

To define the role of malaria parasite enzymes during the process of erythrocyte invasion, we have developed an in vitro serum-free invasion assay of mouse erythrocytes by purified Plasmodium chabaudi merozoites. The sensitivity of a merozoite-specific serine protease (p68) to various inhibitors and the effect of these inhibitors on invasion indicate a crucial role for p68. The substrate specificity of the purified enzyme has been partially defined using fluorogenic peptides. Consistent with this, in vitro incubation of mouse erythrocytes with the merozoite enzyme led to the cleavage of band 3 protein. The possible implication of erythrocyte band 3 truncation for the successful entry of the merozoite into the erythrocyte is discussed.


Distinct binding sites on HLA-DR for invariant chain and staphylococcal enterotoxins.

During biosynthesis, class II molecules of the major histocompatibility complex exist as complexes of the polymorphic alpha and beta chains in association with trimers of the invariant chain (Ii). The nonpolymorphic Ii contains sequences necessary for proper targeting of class II to endosomal compartments, where Ii is degraded. Ii also prevents the premature association of antigenic peptides with class II molecules. It is not known whether the effect of Ii on peptide binding extends to other ligands of class II, specifically exogenous superantigens. Cells expressing a mutant Ii molecule stably associated with HLA-DR at the cell surface were tested for their ability to interact with staphylococcal toxins. Most toxins (staphylococcal enterotoxins A-E and exfoliative toxin) were found to bind to cells expressing this alpha beta Ii complex with levels comparable to cells expressing only alpha beta chains at the cell surface. Cells expressing surface alpha beta Ii complexes stimulated polyclonal populations of peripheral blood T cells in association with these toxins. Binding of toxic shock syndrome toxin (TSST) and subsequent stimulation of T cells were reduced by the presence of the Ii. This reduction was not due to an alteration in the repertoire of T cells responding to TSST in the presence of Ii. Data from experiments with a T-cell clone suggest that interactions between class II molecules and T-cell antigen receptors occur during staphylococcal enterotoxin-mediated stimulation and that surface Ii does not interfere with such interactions.


Dendritic field size and morphology of midget and parasol ganglion cells of the human retina.

The visual system of the macaque monkey has provided a useful model for understanding the neural basis of human vision, yet, there are few detailed comparisons of neural populations other than photoreceptors for the two species. Using intracellular staining in an in vitro preparation of the isolated and intact human retina, we have characterized the relationship of dendritic field size to retinal eccentricity for the two major ganglion cell classes, the midget and the parasol cells. We report three findings. (i) The difference in dendritic field diameter between the parasol and midget cells increases from a ratio of approximately 3:1 in the retinal periphery to approximately 10:1 at 3 degrees eccentricity, suggesting that human midget cells may outnumber parasol cells by as much as 30:1 in the central retina. (ii) The dendritic fields of human ON-center parasol and midget cells are 30-50% larger in diameter than their OFF-center counterparts, suggesting a distinct asymmetry in the human ON-OFF visual pathways. (iii) The dendritic fields of parasol cells, but not midget cells, are larger in humans than in macaques. The difference increases from approximately 20% in the retinal periphery to approximately 90% at 5 degrees eccentricity. This result predicts that the human parasol cells should show a lower resolving ability and an increased sensitivity to luminance contrast than their equivalents in the macaque.


Quinolone binding to DNA is mediated by magnesium ions.

The binding of plasmid DNA to norfloxacin, a quinolone antibacterial agent, was investigated by fluorescence, electrophoretic DNA unwinding, and affinity chromatography techniques. The amount of quinolone bound to DNA was modulated by the concentration of Mg2+. No interaction was evident in the absence of Mg2+ or in the presence of an excess of Mg2+, whereas maximum binding was observed at a Mg2+ concentration of 1-2 mM. The experimental data can be fitted to the formation of three types of Mg adducts: a binary adduct with norfloxacin and Mg2+, a binary adduct with DNA and Mg2+, and a ternary adduct with quinolone, plasmid, and Mg2+. We propose a model for the ternary complex, in which Mg acts as a bridge between the phosphate groups of the nucleic acid and the carbonyl and carboxyl moieties of norfloxacin. Additional stabilization may arise from stacking interactions between the condensed rings of the drug and DNA bases (especially guanine and adenine), which may account for the preference exhibited by quinolones for single-stranded and purine-rich regions of nucleic acids. Other possible biochemical pathways of drug action are suggested by the observation that norfloxacin binds Mg2+ under conditions that are close to physiological.


Conformational substates in azurin.

Azurin is a small blue copper protein in the electron transfer chain of denitrifying bacteria. It forms a photolabile complex with nitric oxide (NO) at low temperatures. We studied the temperature dependence of the ligand binding equilibrium and the kinetics of the association reaction after photodissociation over a wide range of temperature (80-280 K) and time (10(-6)-10(2) s). The nonexponential rebinding below 200 K is independent of the NO concentration and is interpreted as internal recombination. The rebinding can be modeled with the Arrhenius law by using a single preexponential factor of 6.3 x 10(8) s-1 and a Gaussian distribution of enthalpy barriers centered at 23 kJ/mol with a width of 11 kJ/mol. Above 200 K, a slower, exponential rebinding process appears. The dependence of the kinetics on the NO concentration characterizes this reaction as bimolecular rebinding. The binding kinetics of NO to azurin show impressive analogies to the binding of carbon monoxide to myoglobin. We conclude that conformational substates occur not only in heme proteins but also in proteins with different active sites and secondary structures.


Sphingosine 1-phosphate, a specific endogenous signaling molecule controlling cell motility and tumor cell invasiveness.

Sphingosine 1-phosphate (Sph-1-P), the initial product of Sph degradation by Sph kinase, was shown to be a strong inhibitor of cell motility and phagokinesis of B16 melanoma and other types of cells at 10-100 nM concentration. It also inhibited "chemoinvasion" of tumor cells through a thick layer of Matrigel on a filter membrane. Such inhibitory effects were produced minimally or not at all by Sph, N-methyl derivatives of Sph, or other related sphingolipids and phospholipids. Sph-1-P did not inhibit cell proliferation or protein kinase C (PKC) activity, in contrast to Sph and N-methyl-Sph, which inhibit PKC activity and cell growth in general. Radiolabeled [3H]Sph and [14C]N-methyl-Sph were rapidly incorporated into B16 melanoma cells. However, [14C]N-methyl-Sph was not metabolically converted into other compounds, whereas [3H]Sph was efficiently converted within 10 min to Sph-1-P, followed by conversion to other sphingolipids and phospholipids. The inhibitory effect of Sph-1-P on cell motility and tumor cell invasiveness could be a specific phenomenon independent of PKC and other known transmembrane signaling mechanisms, based on an unknown mechanism. It may directly affect organizational assembly of actin filaments. Since exogenous Sph is rapidly converted into Sph-1-P, some reported effects of Sph may be ascribable to such conversion.


Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluid-phase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on beta 2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes.


Myristoylation of the G alpha i2 polypeptide, a G protein alpha subunit, is required for its signaling and transformation functions.

GTPase-inhibiting mutations of the alpha subunit (alpha i2) of the G protein, Gi2, result in constitutive activation of alpha i2 signal transduction functions. GTPase-inhibited alpha i2 mutant polypeptides, referred to as gip2 oncoproteins, have glutamine-205 mutated to leucine (alpha i2Q205L). Expression of the alpha i2Q205L polypeptide inhibits adenylyl cyclase stimulation, constitutively activates p42 mitogen-activated protein kinase, and transforms Rat 1a fibroblasts. The alpha i2 polypeptides are N-terminal-myristoylated, but the function of myristoylation is unclear in alpha i2 signal transduction. We have tested the requirement for myristoylation on the ability of the alpha i2Q205L mutant polypeptide to constitutively regulate signal pathways and cell transformation. When expressed in Rat 1a cells, the nonmyristoylated alpha i2Q205L polypeptide is membrane associated but is unable to regulate adenylyl cyclase or p42 mitogen-activated protein kinase and does not induce cellular transformation. We conclude that myristoylation is absolutely necessary for alpha i2Q205L signal transduction and regulation of effector enzymes in the cell.


Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation.

The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.


Isolation of DNA sequences deleted in lung cancer by genomic difference cloning.

To identify DNA sequences that are deleted in human lung cancer, genomic subtraction hybridization was used to construct plasmid libraries that are enriched for DNA sequences deleted in the small cell lung carcinoma cell line SK-LC-17. The clones of the libraries contained predominantly single copy sequences, allowing direct screening of normal and tumor DNA by genomic Southern blotting. Of 150 clones tested, three independent clones (del-27, del-118, and del-109) were identified that specifically hybridized with normal human DNA but not with tumor DNA from the cell line SK-LC-17. The corresponding DNA sequences are localized on human chromosomes 5, 8, and X/Y. The DNA regions identified by del-109 and del-118 were also found to be deleted in several other lung carcinoma cell lines. Moreover, del-118 was deleted in a freshly isolated lymph node metastasis of a human lung adenocarcinoma. It is therefore reasonable to speculate that the identified clones are derived from independent genetic loci encoding potential tumor suppressor genes.


Selective expression of human X chromosome-linked green opsin genes.

The human red and green photopigments are specified by genes on the long arm of the X chromosome (Xq28). In individuals with normal color vision, the locus was proposed to consist of a single red pigment gene upstream of one or more copies of green pigment genes. The presence of a single red pigment gene in the array was confirmed by demonstration of only one retinal mRNA transcript coding for the red opsin. In individuals with multiple green pigment genes, it is unknown whether all genes are expressed. We analyzed the sequence of red- and green-specific mRNA from retinas of individuals with multiple green pigment genes in comparison with the corresponding genomic DNA sequences. The data showed that only a single green pigment gene is expressed. We therefore suggest that a locus control-like element, already known to be located 3.8 kilobases upstream of the transcription initiation site of the red pigment gene, allows transcription of only a single copy of the green pigment genes, probably the most proximal copy. This finding provides an explanation for the not-infrequent presence of 5' green-red hybrid genes in individuals with normal color vision. Such hybrid genes are usually associated with defective color vision. We suggest that 5' green-red hybrid genes produce defective color vision only when their position in the gene array allows expression in the retinal cone cells.


Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity.

The substrate preference of recombinant Escherichia coli thymidylate synthase (TS) has been altered from 2'-deoxyuridylate (dUMP) to 2'-deoxycytidylate (dCMP) by site-directed mutagenesis of the codon for Asn177, which was changed to aspartic acid. The side-chain amide of Asn177 forms hydrogen bonds with O4 and N3 of dUMP bound to the crystalline enzyme [Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, J. S., Maley, G. F., Hardy, L., Maley, F. & Stroud, R. M. (1990) Biochemistry 29, 6964-6977]. This Asn is invariant in all natural sequences for TS known. The values of kcat for the mutant enzyme, TS(N177D), with dCMP and dUMP are, respectively, 0.09 and 0.002 times the value of kcat of wild-type TS with dUMP as substrate. TS(N177D) turns over dCMP at 35 times its rate of dUMP turnover, whereas wild-type TS turns over dCMP at < 10(-5) of its rate of dUMP turnover. Thus Asn177 is a major determinant of the pyrimidine nucleotide specificity of TS. The mutant enzyme, like wild-type TS, forms a covalent complex with 5-fluoro-dUMP in the presence of 5,10-methylenetetrahydrofolate. TS(N177D) also has a newly acquired ability to be transiently inactivated by dUMP. This time-dependent inactivation requires the presence of methylenetetrahydrofolate and may be due to the accumulation of the enzyme in the form of a catalytic intermediate. The likely mechanistic basis for discrimination by TS between dUMP and dCMP is their differing requirements for charge stabilization during covalent catalysis.


Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

We describe an autoantibody specificity present in a subgroup of patients with a severe form of autoimmune chronic active hepatitis. These antibodies precipitate a 90-nucleotide RNA from human whole cell extracts and recognize a 48-kDa polypeptide in immunoblotting assays. The RNA is a UGA suppressor serine tRNA that carries selenocysteine (tRNA[Ser]Sec)), as shown by sequence analysis. The protein does not appear to be seryl-tRNA synthetase; rather, it is an excellent candidate for a factor involved in cotranslational selenocysteine incorporation in human cells.


Circular dichroism and molecular modeling yield a structure for the complex of human immunodeficiency virus type 1 trans-activation response RNA and the binding region of Tat, the trans-acting transcriptional activator.

Transcription in the human immunodeficiency virus type 1 (HIV-1) retrovirus is regulated by binding the viral Tat protein (trans-acting transcriptional activator) to the trans-activation response (TAR) RNA sequence. Here, vacuum UV circular dichroism (VUV-CD) is used to study the structure of TAR and its complex with two peptide fragments that are important for Tat binding to TAR. The VUV-CD spectrum of TAR is typical of A-form RNA and is minimally perturbed when bound to either the short or the long Tat peptide. The CD spectra of the complexes indicate an extended structure in the arginine-rich region of Tat from amino acid residue 47 through residue 58 and a short alpha-helix within the adjacent 59-72 region. Models of TAR and its peptide complexes are constructed to integrate these spectroscopic results with current biochemical data. The model suggests that (i) the arginine-rich 49-58 region is primarily responsible for electrostatic interactions with the phosphates of the RNA, (ii) the arginine side chains can additionally interact with substituent groups of the nucleotide bases to confer base recognition in the complex, (iii) the recognition of uracil-23 in TAR is facilitated by the peptide backbone, and (iv) the glutamine-rich face of an alpha-helix within the 59-72 region pairs to bases UGG at nucleotide positions 31-33 in the TAR loop and thus provides an additional motif in the Tat trans-activating protein to recognize TAR RNA.


Trisomy 7 and trisomy 10 characterize subpopulations of tumor-infiltrating lymphocytes in kidney tumors and in the surrounding kidney tissue.

We performed conventional cytogenetic analysis and fluorescence in situ hybridization in short-term cultures of normal and neoplastic kidney tissues. Cell populations carrying an extra chromosome 7 or an extra chromosome 10 as the only chromosome change could be identified in kidney tumors, mostly renal cell carcinomas, and in the surrounding kidney tissue, but not in nonneoplastic kidneys. To identify the type of cells displaying these aneuploidies, we performed in situ hybridization (ISH) with probes specific for the centromeric region of chromosomes 7 and 10 on frozen kidney tissue sections. Trisomy 7 and trisomy 10 were restricted to infiltrating inflammatory cells in the tumor as well as in the surrounding tissue. Trisomy 7 and trisomy 10 were also found in subpopulations of peripheral blood T cells of cancer patients and of normal individuals, as well as in the thymus of five normal fetuses (21-29 weeks), but not in noninvaded reactive lymph node sections of patients without malignancy. When lymphocytes were enriched from kidney tumors and surrounding tissue by either Ficoll/Hypaque density gradient or immunomagnetic selection with anti-CD3, anti-CD4, or anti-CD8 monoclonal antibodies, it was confirmed that they contained a high percentage of trisomy 7 and trisomy 10 cells. Further proof for T-lymphocyte origin of the trisomy 7 and trisomy 10 cells was obtained by simultaneous staining of lymphocytes isolated from tumor tissue with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies and ISH. We conclude that trisomy 7 and trisomy 10, found in renal carcinomas and surrounding kidney tissue, characterize subpopulations of tumor-infiltrating lymphocytes. The biologic significance of this phenomenon is unknown and requires further investigation.


Recombinant antibody-metallothionein: design and evaluation for radioimmunoimaging.

We have produced a chimeric antibody (Ab) in which metallothionein, a well-characterized biological chelator of metals, was genetically fused to the F(ab') domain of the S107 Ab heavy chain. Coexpression with the Ab light chain that conveys specificity for the synthetic antigen phosphocholine was achieved in plasmacytoma cells. Metal- and antigen-binding domains of the Ab-metallothionein hybrid function with normal avidity and specificity. Ab-metallothionein can be efficiently loaded with 99mTc and used to specifically bind phosphocholine-haptenated cells in vitro or to localize plasma-cell ascites tumors in mice. The approach offers potential advantages for producing radiolabeled Ab for targeted radiotherapy and diagnostic imaging.


Metal-binding chimeric antibodies expressed in Escherichia coli.

Metallothionein, a well-characterized biological chelator of metals, has been genetically fused to the binding domain of an antibody and expressed in the periplasm of Escherichia coli. Specific delivery of 109Cd to immobilized hapten or to haptenated cells was demonstrated directly in periplasmic extracts. This approach is potentially useful for targeted radiotherapy and diagnostic imaging. We find six to seven atoms of metal per active antigen-combining site. Absence of the Fc portion of the immunoglobulin along with low immunogenicity of metallothionein-metal complexes should reduce immunologic reactions.


A broad molecular phylogeny of ciliates: identification of major evolutionary trends and radiations within the phylum.

The cellular architecture of ciliates is one of the most complex known within eukaryotes. Detailed systematic schemes have thus been constructed through extensive comparative morphological and ultrastructural analysis of the ciliature and of its internal cytoskeletal derivatives (the infraciliature), as well as of the architecture of the oral apparatus. In recent years, a consensus was reached in which the phylum was divided in eight classes as defined by Lynn and Corliss [Lynn, D. H. & Corliss, J. O. (1991) in Microscopic Anatomy of Invertebrates: Protozoa (Wiley-Liss, New York), Vol. 1, pp. 333-467]. By comparing partial sequences of the large subunit rRNA molecule, and by using both distance-matrix and maximum-parsimony-tree construction methods (checked by boot-strapping), we examine the phylogenetic relationships of 22 species belonging to seven of these eight classes. At low taxonomic levels, the traditional grouping of the species is generally confirmed. At higher taxonomic levels, the branching pattern of these seven classes is resolved in several deeply separated major branches. Surprisingly, the first emerging one contains the heterotrichs and is strongly associated with a karyorelictid but deeply separated from hypotrichs. The litostomes, the oligohymenophorans, and the hypotrichs separate later in a bush-like topology hindering the resolution of their order of diversification. These results show a much more ancient origin of heterotrichs than was classically assumed, indicating that asymmetric, abundantly ciliated oral apparatuses do not correspond to "highly evolved" traits as previously thought. They also suggest the occurrence of a major radiative explosion in the evolutionary history of the ciliates, yielding five of the eight classes of the phylum. These classes appear to differ essentially according to the cytoskeletal architecture used to shape and sustain the cellular cortex (a process of essential adaptative and morphogenetic importance in ciliates).


Molecular phylogenetic inference from saber-toothed cat fossils of Rancho La Brea.

A method for the successful extraction of sequestered cellular DNA from 14,000-year-old fossil bones was developed and applied to asphalt-preserved specimens of the extinct saber-toothed cat, Smilodon fatalis. Two distinct gene segments, the mitochondrial gene for 12S rRNA and nuclear FLA-I (the feline class I major histocompatibility complex gene), from three different individual fossil specimens were cloned and sequenced after PCR amplification. Comparison of fossil-derived DNA sequences to homologous regions in 15 living carnivorous species, including 9 species of Felidae and 6 nonfelids, affirmed the phylogenetic placement of Smilodon within the modern radiation of Felidae distinct from the Miocene paleofelid (Nimravidae) saber-toothed "cat" species. These results raise the prospect of obtaining genetically informative DNA from preserved bones of extinct fossil species, particularly among the 2 million specimens excavated from the asphaltic sediments at Rancho La Brea in metropolitan Los Angeles.


Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.

Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase. During infection of Escherichia coli, T7 annexes the host protein thioredoxin for use as a processivity factor for T7 DNA polymerase. We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E. coli thioredoxin. The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin. A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7. This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene 5 protein. Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain. Each suppressor mutation alone is necessary and sufficient to confer the revertant phenotype.


Establishment of a highly sensitive and specific exon-trapping system.

We have established a highly sensitive and specific exon-trapping system (SETS) with a specific plasmid vector in which an exon in a given DNA segment is identified by its ability to remain as a mature mRNA after splicing. The SETS provides us with the isolation of possible exons rapidly and easily from DNA fragments in chromosomal regions of more than 300 kilobase pairs. Genomic DNA fragments were partially digested and subsequently cloned into plasmid pMHC2, an exon-trapping vector we have constructed. These constructs were transfected into COS-7 cells, and consequent RNA transcripts were spliced in the cells. The resulting mature mRNA was harvested and amplified by using reverse transcription-PCR. Possible exons can be recognized by the sizes of PCR products and cloned into a plasmid vector. The SETS provides a direct means of cloning exons from genomic DNA of more than 300 kilobase pairs within a short period of time. Using this system, we have screened 300-kilobase-pair genomic DNA segments derived from human chromosome 11q13. Human chromosome 11q13 may contain genes responsible for human cancers, because DNA amplification is observed in several malignant tumors. We have successfully identified exon 2 of the HST1 gene and additional transcribed sequences.


Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis.

There is considerable evidence implicating tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of rheumatoid arthritis. This evidence is based not only on the universal presence of TNF-alpha in arthritic joints accompanied by the upregulation of TNF-alpha receptors but also on the effects of neutralizing TNF-alpha in joint cell cultures. Thus, neutralization of TNF-alpha in vitro results in inhibition of the production of interleukin 1, which like TNF-alpha, is believed to contribute to joint inflammation and erosion. To determine the validity of this concept in vivo, the effect of administering TNF-neutralizing antibodies to mice with collagen-induced arthritis has been studied. This disease model was chosen because of its many immunological and pathological similarities to human rheumatoid arthritis. TN3-19.12, a hamster IgG1 monoclonal antibody to murine TNF-alpha/beta, was injected i.p. into mice either before the onset of arthritis or after the establishment of clinical disease. Anti-TNF administered prior to disease onset significantly reduced paw swelling and histological severity of arthritis without reducing the incidence of arthritis or the level of circulating anti-type II collagen IgG. More relevant to human disease was the capacity of the antibody to reduce the clinical score, paw swelling, and the histological severity of disease even when injected after the onset of clinical arthritis. These results have implications for possible modes of therapy of human arthritis.


Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg.min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme displayed an absolute requirement for Fe2+ and ascorbate. The stoichiometry of the enzymatic reaction was determined: ACC + ascorbate + O2-->C2H4 + HCN + CO2 + dehydroascorbate + 2 H2O. A polyclonal antibody was raised against a synthetic tridecapeptide (PDLEEEYRKTMKE) whose sequence was deduced from the apple pAE12 cDNA [Dong, J. G., Olson, D., Silverstone, A. & Yang, S. F. (1992) Plant Physiol. 98, 1530-1531], which is homologous to tomato cDNAs encoding ACC oxidase. On a Western blot, this antibody specifically recognized the purified ACC oxidase protein. The amino acid composition of the purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KEFAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.


The path of carbon in photosynthesis: improved crop yields with methanol.

Foliar sprays of aqueous 10-50% methanol increased growth and development of C3 crop plants in arid environments. The effects of low levels (< 1 ml per plant) of methanol were observed for weeks after the brief time necessary for its rapid metabolism. Within several hours, foliar treatment with methanol resulted in increased turgidity. Plants treated with nutrient-supplemented methanol showed up to 100% increases in yields when maintained under direct sunlight in desert agriculture. In the shade and when winter crops were treated with methanol, plants showed no improvement of growth. When repeatedly treated with nutrient-supplemented methanol, shaded plants showed symptoms of toxicity. Repeated methanol treatments with glycine caused increased turgidity and stimulated plant growth without injury under indirect sunlight, but indoors with artificial illumination, foliar damage developed after 48 hr. Addition of glycerophosphate to glycine/methanol solutions allowed treatment of artificially illuminated plants indoors without injury. Plants with C4 metabolism showed no increase in productivity by methanol treatment. Plants given many applications of aqueous methanol showed symptoms of nutrient deficiency. Supplementation with a source of nitrogen sustained growth, eliminating symptoms of deficiency. Adjustment of carbon/nitrogen ratios was undertaken in the field by decreasing the source of nitrogen in the final application, resulting in early maturation; concomitantly, irrigation requirements were reduced.


Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure.

Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore. A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons. A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium. Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations. One of these had a deletion in the a subunit. The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211. The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity. To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated. Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix. These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase. The implications of these modifications for the mechanism of proton translocation are discussed.


Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission.


Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal alpha-actin transcription in embryonic myoblasts.

Muscle-restricted transcription of the skeletal alpha-actin gene is controlled in part by a positive regulator, serum response factor (SRF), and a negative regulator, F-ACT1, which bind competitively to the most proximal serum response element (SRE1). We show here that F-ACT1 is identical to a transcription factor recently cloned and described as YY1, NF-E1, delta, or UCRBP. We found that although the DNA-binding activity of SRF accumulates during myogenesis, that of YY1 diminishes simultaneously. Myoblasts rendered incapable of differentiation by BrdUrd treatment exhibited the highest level of YY1 and the lowest level of SRF activities. Transfected SRF could directly transactivate the skeletal alpha-actin promoter by overcoming the inhibitory effect of BrdUrd-induced YY1. The transactivation depends on intact SRE DNA elements and requires the DNA-binding/dimerization domain of SRF as well as its C-terminal half rich in serines and threonines. Since the functions of YY1 and SRF appear to be developmentally regulated, the convergence of their binding sites upon the SRE constitutes an integrated mechanism whereby temporal and spatial muscle gene expression may be accomplished.


Isolation and sequence of a cDNA encoding the precursor of a bombesinlike peptide from brain and early embryos of Xenopus laevis.

A cDNA encoding the precursor of a bombesinlike peptide was isolated from brain of Xenopus laevis. The predicted end product resembles neuromedin B, which was originally isolated from mammalian spinal cord. The mRNA for this precursor was also present in gastrointestinal tract and in ovaries. Moreover, it could be detected in early embryos (stage 2 and stage 10) of X. laevis. These findings suggest novel roles for peptides of the bombesin family in oocyte maturation and early amphibian development.


Ligation-anchored PCR: a simple amplification technique with single-sided specificity.

A simple, efficient, and sensitive technique has been developed for amplification of cDNAs encoding molecules with 5' regions of unknown sequence. In this ligation-anchored PCR, T4 RNA ligase is used to covalently link an "anchor" oligonucleotide to first-strand cDNAs. These anchored cDNAs are then amplified by using one PCR primer specific for the anchor and another specific for a sequence within the molecule of interest. The anchor oligonucleotide has been especially designed to facilitate subsequent analysis and cloning of the resultant PCR products. This three-stage procedure does not require purification of product between steps and avoids many of the technical difficulties associated with established anchored PCR protocols. The efficacy of ligation-anchored PCR was demonstrated by amplification of a specific IgG1 cDNA; total RNA equivalent to as few as 100 cells yielded the expected PCR product.