bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and expression of a mammalian peptide chain release factor with sequence similarity to tryptophanyl-tRNA synthetases.
Authors: Lee C C , Craigen W J , Muzny D M , Harlow E , Caskey C T
Published: May 2006

The termination of protein synthesis is encoded by in-frame nonsense (stop) codons. Most organisms use three nonsense codons: UGA, UAG, and UAA. In contrast to sense codons, which are decoded by specific tRNAs, nonsense codons are decoded by proteins called release factors (RFs). Here we report the cloning of a mammalian RF cDNA by the use of monoclonal antibodies specific for rabbit RF. Functional studies showed that, when expressed in Escherichia coli, the protein encoded by this cDNA has in vitro biochemical characteristics similar to those of previously characterized mammalian RFs. DNA sequencing of this eukaryotic RF cDNA revealed a remarkable sequence similarity to bacterial and mitochondrial tryptophanyl-tRNA synthetases, with the greatest similarity confined to the synthetase active site, and no obvious similarity to bacterial RFs.
Excitatory amino acid uptake and N-methyl-D-aspartate-mediated secretion in a neural cell line.
Authors: Morimoto B H , Koshland D E
Published: May 2006

A functional N-methyl-D-aspartate (NMDA) receptor has been identified on HT-4 cells, a clonal neural cell line, in which glutamate activates the receptor to elicit neurotransmitter secretion. Specific inhibitors of the NMDA receptor block glutamate-mediated secretion, and the characteristics of NMDA-mediated secretion parallel the reported properties of the NMDA receptor. Excitatory amino acid secretion can be elicited by potassium-evoked depolarization and is not the simple reversal of the uptake system. 2-Amino-4-phosphonobutyrate (APB) inhibits depolarization-induced secretion of excitatory amino acids but has no effect on excitatory amino acid uptake, suggesting that the APB binding protein in the brain represents a component involved in the secretion of excitatory amino acids.
Defective ATP-dependent bile canalicular transport of organic anions in mutant (TR-) rats with conjugated hyperbilirubinemia.
Authors: Kitamura T , Jansen P , Hardenbrook C , Kamimoto Y , Gatmaitan Z , Arias I M
Published: May 2006

TR- mutant Wistar rats secrete markedly fewer organic anions other than bile acids from the liver into the bile than do control rats. Fluorescence-image analysis of isolated normal and TR- hepatocyte "doublets", which retain a bile canaliculus between them, revealed that normal hepatocytes readily transport a fluorescent bile acid (fluorescein isothiocyanate glycocholate) and a nonbile acid organic anion (carboxydichlorofluorescein diacetate) into the canaliculus. Hepatocyte doublets from TR- rats also transported fluorescein isothiocyanate glycocholate normally, but transport of carboxydichlorofluorescein diacetate into the canaliculus was negligible. Vesicles derived from the canicular domain of the plasma membrane of hepatocytes (CMV) from control and TR- rats were used to characterize the transport process for 35S-labeled bromosulphthalein and 35S-labeled bromosulphthalein glutathione, which represent nonbile acid organic anions. CMV from normal rat hepatocytes had an ATP- and temperature-dependent, saturable transport process for these 35S-labeled compounds that was absent in CMV from TR- rats. CMV from TR- rats retained normal ATP-dependent transport of daunomycin, and immunologic blots with a monoclonal antibody against the multidrug resistance gene product, P-glycoprotein, revealed no difference between normal and TR-CMV. These studies reveal that the bile canaliculus in normal rats contains an ATP-dependent organic anion transport system that is functionally absent in TR- mutant rats. The defect in TR- mutant rats is phenotypically similar to that seen in mutant Corriedale sheep and in the Dubin-Johnson syndrome in man.
Evidence that retroviral transduction is mediated by DNA not by RNA.
Authors: Goodrich D W , Duesberg P H
Published: May 2006

Retroviral transduction of cellular nucleic acid sequences requires illegitimate RNA or DNA recombination. To test a model that postulates transduction via efficient illegitimate recombination during reverse transcription of viral and cellular RNAs, we have measured the ability of Harvey sarcoma viruses (HaSVs) with artificial 3' termini to recover a retroviral 3' terminus from helper Moloney virus (MoV) by illegitimate and homologous recombination. For this purpose, mouse NIH 3T3 cells were transformed with Harvey proviruses and then superinfected with MoV. The proviruses lacked the 3' long terminal repeat and an untranscribed region of the 5' long terminal repeat to prevent virus regeneration from input provirus. Only 0-11 focus-forming units of HaSV were generated upon MoV superinfection of 3 x 10(6) cells transformed by Harvey proviruses with MoV-unrelated termini. This low frequency is consistent with illegitimate DNA recombination via random Moloney provirus integration 3' of the transforming viral ras gene in the 10(6)-kilobase mouse genome. When portions of murine viral envelope (env) genes were attached 3' of ras, 10(2)-10(5) focus-forming units of HaSV were generated, depending on the extent of homology with env of MoV. These recombinants all contained HaSV-specific sequences 5' and MoV-specific sequences 3' of the common env homology. They were probably generated by recombination during reverse transcription rather than by recombination among either input or secondary proviruses, since (i) the yield of recombinants was reduced by a factor of 10 when the env sequence was flanked by splice signals and (ii) HaSV RNAs without retroviral 3' termini would be inadequate templates for provirus synthesis. We conclude that there is no efficient illegitimate recombination in retroviruses. In view of known precedents of illegitimate DNA recombination, the structure of known viral onc genes, and our evidence for illegitimate DNA recombination via provirus integration, we favor the DNA model of transduction over the RNA model.
Degradation of nuclear oncoproteins by the ubiquitin system in vitro.
Authors: Ciechanover A , DiGiuseppe J A , Bercovich B , Orian A , Richter J D , Schwartz A L , Brodeur G M
Published: May 2006

Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the N-myc, c-myc, c-fos, p53 and E1A genes. Each of these nuclear oncoproteins was synthesized in vitro by transcription of the appropriate cDNA and translation of the resulting mRNA in the presence of [35S]methionine. Degradation of labeled proteins was monitored in the ubiquitin cell-free system. ATP stimulated the degradation of all the proteins between 3- and 10-fold. The degradation was completely inhibited by neutralizing antibody directed against the ubiquitin-activating enzyme, E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, degradation in E1-depleted lysates could be restored in each case by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the degradation of these oncoproteins in vitro. Degradation of other proteins, such as superoxide dismutase, cytochrome c, enolase, RNase A, and ornithine decarboxylase, is not mediated by the ubiquitin cell-free system. This suggests that the nuclear oncoproteins studied here possess specific signals that target them for rapid turnover by this proteolytic pathway. Furthermore, the relative sensitivity to degradation of various E1A mutants in vivo is also maintained in the cell-free system, suggesting that the ubiquitin pathway may play a role in the cellular degradation of these proteins as well.
Melanosis and associated tumors in transgenic mice.
Authors: Klein-Szanto A , Bradl M , Porter S , Mintz B
Published: May 2006

Melanosis was found to various extents in a wide array of tissues of all 23 autopsied mice whose transgene consisted of the tyrosinase promoter fused to the simian virus 40 early-region oncogenic sequences. Pigmentation in a given animal was attributable to any or all of the following; an increase in numbers of some normally pigmented cells of neural crest origin (a result compatible with early stages of transformation); elicitation of melanin synthesis in some cells that normally have little melanin, or none at all (the latter possibly signaling metaplasia); unusual intercellular transfer of pigment granules from melanocytes into certain normally unpigmented epithelia and endothelia; and profusion of melanin-phagocytizing cells. Neoplasms, occasionally also containing melanin, arose in association with some of these melanotic tissues and included three choroid plexus tumors, three endocardial tumors, two peripheral nerve sheath tumors (schwannomas), two cochlear tumors, two pineal gland tumors, one salivary gland tumor, and one nasal mucosa tumor. These apparently originated independently of the ocular and cutaneous melanomas found in the same animals. The events involved in melanosis may thus contribute to neoplastic conversion.
Prothymosin alpha antisense oligomers inhibit myeloma cell division.
Authors: Sburlati A R , Manrow R E , Berger S L
Published: May 2006

The function of prothymosin alpha has been investigated by using four different antisense oligodeoxyribonucleotides directed at selected regions of its mRNA. In every case, when synchronized human myeloma cells were released from stationary phase by incubation in fresh medium containing antisense oligomers, cell division was prevented or inhibited; sense oligomers and random antisense oligomers had no effect. A detailed analysis of synchronized cell populations indicated that sense-treated and untreated cells divided approximately 17 hr after growth initiation, whereas cells incubated with antisense oligomer 183, a 16-mer targeted 5 bases downstream of the initiation codon, entered mitosis approximately one cell division late. The failure to divide correlated directly with a deficit in prothymosin alpha and with the continued presence of intact intracellular antisense oligomers over a period of at least 24 hr. Because antisense oligomers had no effect either on the timing of the induction of prothymosin alpha mRNA upon growth stimulation or on mRNA levels seen throughout the cell cycle, we concluded that antisense DNA caused specific hybrid arrest of translation. Our data suggest that prothymosin alpha is required for cell division. However, there is no evidence that prothymosin alpha directly regulates mitosis.
Somatic mosaicism and female-to-female transmission in a kindred with hemophilia B (factor IX deficiency).
Authors: Taylor S A , Deugau K V , Lillicrap D P
Published: May 2006

Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which greater than 95% are single nucleotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cytosine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. We used differential termination of primer extension to confirm and measure the degree of mosaicism. Our study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation of sequences including the mutant factor IX gene on the X chromosome inherited from the affected male.
Platelet-derived growth factor receptor alpha-subunit gene (Pdgfra) is deleted in the mouse patch (Ph) mutation.
Authors: Stephenson D A , Mercola M , Anderson E , Wang C Y , Stiles C D , Bowen-Pope D F , Chapman V M
Published: May 2006

Platelet-derived growth factor receptors are composed of two subunits (alpha and beta) that associate with one another to form three functionally active dimeric receptor species. The two subunits are encoded by separate loci in humans and other species. In this study, we used conventional interspecific backcross mapping and an analysis of a deletional mutation to establish close linkage between the alpha-subunit gene (Pdgfra) and the dominant spotting (W) locus on mouse chromosome 5. Further, by analyzing the restriction fragment length polymorphisms in interspecific F1 hybrids, we were able to demonstrate that the closely associated patch (Ph) locus carries a deletion in Pdgfra. This observation was confirmed by both DNA and RNA analysis of 10.5-day fetuses produced from crosses between Ph heterozygotes. Out of 16 fetuses analyzed, Pdgfra genomic sequences were absent and no mRNA for the receptor was detected in 6 fetuses that were developmentally abnormal (the presumptive Ph homozygotes). We also determined that the deletion associated with the Ph mutation does not extend into the coding sequences of the adjacent Kit gene, by analysis of the genomic DNA from both the interspecific F1 hybrids and the presumptive Ph homozygotes. The absence of Pdgfra genomic sequences and the lack of detectable message associated with the Ph mutation should make this mutant a valuable asset for understanding the role of the receptor alpha subunit during mammalian development.
iciA, an Escherichia coli gene encoding a specific inhibitor of chromosomal initiation of replication in vitro.
Authors: Thöny B , Hwang D S , Fradkin L , Kornberg A
Published: May 2006

The gene encoding the protein that binds the three 13-mers in the origin (oriC) of Escherichia coli to block initiation of replication in vitro has been cloned, sequenced, and overexpressed. The gene possesses an open reading frame for 297 amino acids (mass of 33,471 Da). The protein has a motif for DNA-binding (helix-turn-helix) and has homology to a diverse set of prokaryotic regulatory proteins, known as the LysR family. The protein, previously referred to as the 33-kDa protein, has been named IciA (for inhibitor of chromosome initiation). The iciA gene is at 62.8 min on the chromosomal map. Cells with enhanced levels of the protein grow at a normal rate but generally exhibit a pronounced lag upon transfer to a fresh medium.
Polymerase chain reaction-mediated gene synthesis: synthesis of a gene coding for isozyme c of horseradish peroxidase.
Authors: Jayaraman K , Fingar S A , Shah J , Fyles J
Published: May 2006

The synthesis of a gene coding for horseradish peroxidase (HRP, isozyme c; EC 1.11.1.7) is described using a polymerase chain reaction (PCR)-mediated gene synthesis approach developed in our laboratory. In this approach, all the oligonucleotides making up the gene are ligated in a single step by using the two outer oligonucleotides as PCR primers and the crude ligation mixture as the target. The PCR facilitates synthesis and purification of the gene simultaneously. The gene for HRP was synthesized by ligating all 40 oligonucleotides in a single step followed by PCR amplification. The gene was also synthesized from its fragments by using an overlap extension method similar to the procedure as described [Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. (1989) Gene 77, 61-68]. A method for combining different DNA fragments, in-frame, by using the PCR was also developed and used to synthesize the HRP gene from its gene fragments. This method is applicable to the synthesis of even larger genes and to combine any DNA fragments in-frame. After the synthesis, preliminary characterization of the HRP gene was also carried out by the PCR to confirm the arrangement of oligonucleotides in the gene. This was done by carrying out the PCR with several sets of primers along the gene and comparing the product sizes with the expected sizes. The gene and the fragments generated by PCR were cloned in Escherichia coli and the sequence was confirmed by manual and automated DNA sequencing.
Coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor.
Authors: Gubler U , Chua A O , Schoenhaut D S , Dwyer C M , McComas W , Motyka R , Nabavi N , Wolitzky A G , Quinn P M , Familletti P C
Published: May 2006

Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells. Coexpression of the two subunit cDNAs in COS cells is necessary for the secretion of biologically active CLMF; COS cells transfected with either subunit cDNA alone do not secrete bioactive CLMF. Recombinant CLMF expressed in mammalian cells displays biologic activities essentially identical to natural CLMF, and its activities can be neutralized by monoclonal antibodies prepared against natural CLMF. Since this heterodimeric protein displays the properties of an interleukin, we propose that CLMF be given the designation interleukin 12.
Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis.
Authors: Brunetto M R , Giarin M M , Oliveri F , Chiaberge E , Baldi M , Alfarano A , Serra A , Saracco G , Verme G , Will H
Published: May 2006

Using an oligonucleotide hybridization assay, we studied the clinical implication of wild-type hepatitis B virus (HBV) and a HBV mutant that is unable to secrete hepatitis B e antigen (HBeAg) because of a translational defect due to a stop codon in the pre-C region in 106 hepatitis B surface antigen-positive patients with chronic hepatitis B. Wild-type HBV was detected in 31 of 42 (73.8%) HBeAg-positive patients, whereas a mixed viral population was present in 10 (23.8%). Significant differences in the severity and outcome of liver disease were not observed in the two groups of patients. However, the emergence of HBeAg-minus HBV in wild-type HBV carriers was associated with an exacerbation of liver disease and was followed by the presence of antibodies against HBeAg (anti-HBe) in serum in 50% of the cases. In 61 of 64 (95.3%) anti-HBe-positive patients, HBeAg-minus HBV was the predominant virus: HBeAg-minus HBV was detected in 42 patients (65.6%), whereas both wild-type and HBeAg-minus HBV were present in 19 (29.7%). HBeAg-minus HBV was associated with a course of hepatitis characterized by flare-ups of liver cell necrosis interspersed with periods of asymptomatic HBV carriage (P less than 0.01). These data support the hypothesis that genetic heterogeneity of HBV significantly influences the course and outcome of chronic hepatitis B. Wild-type HBV secreting HBeAg induces immunologic tolerance and causes chronic infection. HBeAg-minus HBV might be unable to induce chronic infection without the helper function of wild-type HBV, but it appears to be more pathogenic. Once chronic infection is established, HBeAg-minus HBV variants may prevail and displace wild-type virus.
Footprinting analysis of mammalian RNA polymerase II along its transcript: an alternative view of transcription elongation.
Authors: Rice G A , Kane C M , Chamberlin M J
Published: May 2006

Ternary complexes of RNA polymerase II, bearing the nascent RNA transcript, are intermediates in the synthesis of all eukaryotic mRNAs and are implicated as regulatory targets of factors that control RNA chain elongation and termination. Information as to the structure of such complexes is essential in understanding the catalytic and regulatory properties of the RNA polymerase. We have prepared complexes of purified RNA polymerase II halted at defined positions along a DNA template and used RNase footprinting to map interactions of the polymerase with the nascent RNA. Unexpectedly, the transcript is sensitive to cleavage by RNases A and T1 at positions as close as 3 nucleotides from the 3'-terminal growing point. Ternary complexes in which the transcript has been cleaved to give a short fragment can retain that fragment and remain active and able to continue elongation. Since DNA.RNA hybrid structures are completely resistant to cleavage under our reaction conditions, the results suggest that any DNA.RNA hybrid intermediate can extend for no more than 3 base pairs, in dramatic contrast to recent models for transcription elongation. At lower RNase concentrations, the transcript is protected from cleavage out to about 24 nucleotides from the 3' terminus. We interpret this partial protection as due to the presence of an RNA binding site on the polymerase that binds the nascent transcript during elongation, a model proposed earlier by several workers in preference to the hybrid model. The properties of this RNA binding site are likely to play a central role in the process of transcription elongation and termination and in their regulation.
Expression of mammalian gamma-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes.
Authors: Polenzani L , Woodward R M , Miledi R
Published: May 2006

Gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is known to interact with two classes of GABA receptors denoted GABAA and GABAB. Using Xenopus oocytes, we compared the electrical and pharmacological properties of GABA receptors expressed by poly(A)+ RNA isolated from mammalian brain and retina. RNA from cerebral cortex expressed GABA responses with features characteristic of currents mediated by GABAA receptors. In contrast, RNA from retina expressed responses mediated by GABAA receptors and, in addition, GABA responses that were insensitive to the GABAA antagonist bicuculline and the GABAB agonist baclofen and showed no modulation by barbiturates or benzodiazepines. The bicuculline/baclofen-insensitive GABA response was a Cl- current that was blocked by picrotoxin but showed little desensitization or outward rectification. Our results suggest that mammalian retina contains RNAs encoding GABA receptors with distinct pharmacology.
Identification of a different-type homeobox gene, BarH1, possibly causing Bar (B) and Om(1D) mutations in Drosophila.
Authors: Kojima T , Ishimaru S , Higashijima S , Takayama E , Akimaru H , Sone M , Emori Y , Saigo K
Published: May 2006

The Bar mutation B of Drosophila melanogaster and optic morphology mutation Om(1D) of Drosophila ananassae result in suppression of ommatidium differentiation at the anterior portion of the eye. Examinations was made to determine the genes responsible for these mutations. Both loci were found to share in common a different type of homeobox gene, which we call "BarH1." Polyptides encoded by D. melanogaster and D. ananassae BarH1 genes consist of 543 and 604 amino acids, respectively, with homeodomains identical in sequence except for one amino acid substitution. A unique feature of these homeodomains is that the phenylalanine residue in helix 3, conserved in all metazoan homeodomains so far examined, is replaced by a tyrosine residue. By Northern blotting, considerably more BarH1 RNA was detected in the Bar mutant than in wild type. P element-mediated transformation showed Bar-like eye malformation to be induced by transient overexpression of the BarH1 gene in the late third-instar larvae. Somatic recombination analysis indicated normal gene functions of the Bar region, including the BarH1 gene, to be required for normal eye morphogenesis.
Isolation and expression of a cDNA encoding Renilla reniformis luciferase.
Authors: Lorenz W W , McCann R O , Longiaru M , Cormier M J
Published: May 2006

Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein. A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.
A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor.
Authors: Bass S H , Mulkerrin M G , Wells J A
Published: May 2006

A mutational strategy is presented that allowed us to identify hormone-binding determinants in the extracellular portion of the human growth hormone receptor (hGHbp), a 238-residue protein with sequence homology to a number of cytokine receptors. By systematically replacing side chains with alanine we probed the importance of charged residues (49 total, typically located on the surface), aromatic residues (9 total), and neighbors of these (26 total). The alanine substitutions that were most disruptive to hormone binding are located predominantly in four segments of a cysteine-rich domain in the hGHbp, and collectively they form a patch when mapped upon a structural model proposed for cytokine receptors. Control experiments with monoclonal antibodies confirmed that most of these alanine substitutions do not disrupt the overall antigenic structure of the hGHbp. This high-resolution functional analysis will complement structural studies and provides a powerful basis for evaluating and engineering the energetics of hormone-receptor interactions. Moreover, the hormone-binding determinants identified here may be similarly located in other, homologous, receptors.
Spo0A binds to a promoter used by sigma A RNA polymerase during sporulation in Bacillus subtilis.
Authors: Satola S , Kirchman P A , Moran C P
Published: May 2006

Examination of the effects of 56 single-base-pair substitutions in the spoIIG promoter and studies of the interaction of the spo0A product (Spo0A) with this promoter in vitro demonstrated that Spo0A acts directly to enable this promoter to be used by sigma A-associated RNA polymerase (EC 2.7.7.6). The spoIIG operon from Bacillus subtilis is transcribed during sporulation by a form o RNA polymerase containing sigma A, the primary sigma factor in vegetative cells. The spoIIG promoter is unusual in that it contains sequences that are similar to those found at the -10 and -35 regions of promoters that are used by sigma A-associated RNA polymerase, but these sigma A-like recognition sequences are separated by 22 base pairs rather than the typical 17 or 18 base pairs. We found that single-base-pair substitutions in the around the -35-like sequence, and substitutions in a region upstream from this position, around position -87, reduced promoter activity. DNase I protection and electrophoretic gel mobility shift assays were used to demonstrate that Spo0A binds specifically to these regions in vitro. Evidently, the -35-like sequence is part of a Spo0A binding site and therefore is possibly not a sigma A-recognition sequence. These results support a model in which Spo0A activates the spoIIG promoter after the onset of endospore formation.
The MCK enhancer contains a p53 responsive element.
Authors: Weintraub H , Hauschka S , Tapscott S J
Published: May 2006

p53 is an antioncogene that is defective or absent in a large number of human tumors. Its function in normal cells is not known. We show that co-transfection of mouse p53 with muscle-specific creatine kinase-chloramphenicol acetyltransferase reporter gene, containing 3.3 kilobase of upstream control sequence for the muscle-specific creatine kinase gene, results in a 10- to 80-fold activation. The p53 responsive element maps to a region distinguished from the known MyoD binding region. Identification of a p53 responsive element should allow a more focused analysis of the effects of p53 in controlling gene activity.
Immunization of simian immunodeficiency virus-infected rhesus monkeys with soluble human CD4 elicits an antiviral response.
Authors: Watanabe M , Levine C G , Shen L , Fisher R A , Letvin N L
Published: May 2006

Since the CD4 molecule is a high-affinity cell-surface receptor for the human immunodeficiency virus (HIV), it has been suggested that a soluble truncated form of CD4 may compete with cell-surface CD4 for HIV binding and thus be of use in the therapy of AIDS. We have utilized the simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys to explore another possible therapeutic application of CD4 in AIDS--the use of recombinant soluble CD4 (rsCD4) as an immunogen. SIVmac-infected rhesus monkeys immunized with human rsCD4 developed not only an anti-human CD4 but also an anti-rhesus monkey CD4 antibody response. Coincident with the generation of this antibody response, SIVmac could not be isolated easily from peripheral blood lymphocytes and bone marrow macrophages of these animals. Furthermore, the decreased number of both granulocyte/macrophage and erythrocyte colonies grown from the bone marrow of these immunized monkeys rose to normal levels. These findings suggest that a modified human CD4 molecule serving as an immunogen might elicit an antibody response in man that could induce a beneficial therapeutic response in HIV-infected individuals.
Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants.
Authors: Scharfmann R , Axelrod J H , Verma I M
Published: May 2006

Toward the goal of gene therapy, we have been attempting to establish model somatic cell systems with the potential of sustained expression of the foreign gene. We report here that long-term expression of foreign genes in mouse embryo fibroblast implants can be achieved if a housekeeping gene promoter is used to drive transcription. Specifically, we have shown that in implants containing a beta-galactosidase gene linked to either an immediate early promoter of cytomegalovirus or a dihydrofolate reductase (DHFR) gene promoter, only the DHFR promoter allows long-term expression. We propose that choice of the promoter manifests significant influence on the long-term expression of genes introduced in fibroblast implants by retroviral vectors.
Bispecific antibodies that mediate killing of cells infected with human immunodeficiency virus of any strain.
Authors: Berg J , Lötscher E , Steimer K S , Capon D J , Baenziger J , Jäck H M , Wabl M
Published: May 2006

Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.
Alternative translocation of protons and halide ions by bacteriorhodopsin.
Authors: Dér A , Száraz S , Tóth-Boconádi R , Tokaji Z , Keszthelyi L , Stoeckenius W
Published: May 2006

Bacteriorhodopsin (bR568) in purple membrane near pH 2 shifts its absorption maximum from 568 to 605 nm forming the blue protein bRacid605, which no longer transports protons and which shows no transient deprotonation of the Schiff base upon illumination. Continued acid titration with HCl or HBr but not H2SO4 restores the purple chromophore to yield bRHCl564 or bRHBr568. These acid purple forms also regain transmembrane charge transport, but no transient Schiff base deprotonation is observed. In contrast to bR568, no rate decrease of the bRacidpurple transport kinetics is detected in 2H2O; however, the transport rate decreases by a factor of approximately 2 in bRHBr568 compared with bRHCl564. The data indicate that in the acid purple form bR transports the halide anions instead of protons. We present a testable model for the transport mechanism, which should also be applicable to halorhodopsin.
Mutation in the gene encoding the alpha chain of platelet glycoprotein Ib in platelet-type von Willebrand disease.
Authors: Miller J L , Cunningham D , Lyle V A , Finch C N
Published: May 2006

Platelet-type von Willebrand disease (PT-vWD) is an autosomal dominant bleeding disorder characterized by abnormally enhanced binding of von Willebrand factor (vWF) by patient platelets. Although the platelet glycoprotein (GP) Ib/IX complex is known to constitute the platelet's ristocetin-dependent receptor for vWF, a unique structural abnormality within this complex has not previously been identified in PT-vWD. Using the polymerase chain reaction to amplify genomic DNA coding for the alpha chain of GP Ib (GP Ib alpha) and then sequencing the amplified DNA following cloning into M13mp18 and M13mp19 phage vectors, we have found a single point mutation in the GP Ib alpha coding region of PT-vWD DNA resulting in the substitution of valine for glycine at residue 233. This substitution within the vWF-binding region of GP Ib alpha is likely to exert a significant influence on the conformation of the resulting protein. Competitive oligonucleotide primer assay for this mutation showed a homozygous wild-type pattern in genomic DNA from the 161 normal volunteers studied and from 6 phenotypically normal members of a PT-vWD family. All 7 affected members of this family studied were heterozygous for the mutant allele. Platelet GP Ib alpha mRNA reverse-transcribed and studied by competitive oligonucleotide primer assay showed similar expression of the mutant and wild-type alleles in the affected PT-vWD patients. Absence in the normal population, tight linkage with phenotypic expression of disease, and absence of any additional abnormality of GP Ib alpha in these patients identify the glycine-to-valine substitution as a point mutation underlying functional abnormality of the vWF receptor in PT-vWD.
A molecular defect causing fish eye disease: an amino acid exchange in lecithin-cholesterol acyltransferase (LCAT) leads to the selective loss of alpha-LCAT activity.
Authors: Funke H , von Eckardstein A , Pritchard P H , Albers J J , Kastelein J J , Droste C , Assmann G
Published: May 2006

Epidemiological as well as biochemical evidence of recent years has established that a low plasma level of high density lipoprotein-cholesterol is a predictor for the risk of coronary artery disease. However, there is a heterogeneous group of rare familial disorders, characterized by severe high density lipoprotein deficiency, in which the predicted increased risk is not clearly apparent. One such disorder has been called fish eye disease to reflect the massive corneal opacification seen in these patients. In this report, we describe the biochemical and genetic presentation of two German fish eye disease homozygotes and their family members. Vertical transmission of a decrease in the specific activity of lecithin-cholesterol acyltransferase (EC 2.3.1.43) indicated that this enzyme was a candidate gene for harboring the defect responsible for this disorder. Direct sequencing of DNA segments amplified by the polymerase chain reaction (PCR) that encode the exons of the lecithin-cholesterol acyltransferase gene led to the identification of a homozygous mutation resulting in the substitution of threonine at codon 123 for an isoleucine residue in both individuals. Family analysis in an extended pedigree was used to establish a causal relationship between this mutation and the biochemical phenotype for fish eye disease. The homozygous presence of this mutation in two phenotypically homozygous members of an unrelated Dutch family with fish eye disease further supports this finding.
Kinesin is responsible for centrifugal movement of pigment granules in melanophores.
Authors: Rodionov V I , Gyoeva F K , Gelfand V I
Published: May 2006

Kinesin is a mechanochemical ATPase that induces translocation of latex beads along microtubules and microtubule gliding on a glass surface. This protein is thought to be a motor for the movement of membranous organelles in cells. Recently Hollenbeck and Swanson [Hollenbeck, P. J. & Swanson, J. A. (1990) Nature (London) 346, 864-866] showed that kinesin is involved in the positioning of tubular lysosomes in macrophages. However, the role of this protein in the movement of organelles was not yet clear. We used a polyclonal antibody against the kinesin heavy chain that inhibited kinesin-dependent microtubule gliding in vitro to study the role of kinesin in the movement of pigment granules in melanophores of the teleost black tetra (Gymnocorymbus ternetzi). Microinjection of the antibody into cultured melanophores did not produce any specific effect on the aggregation of pigment granules in melanophores, but it did result in a strong dose-dependent inhibition of the dispersion. Immunoblotting of melanophore extracts showed that the kinesin antibody reacted in these cells with a single protein component with a molecular mass of 135 kDa. Thus, kinesin is responsible for the movement of pigment granules from the center to the periphery of the melanophore.
An alternative spliced form of FosB is a negative regulator of transcriptional activation and transformation by Fos proteins.
Authors: Yen J , Wisdom R M , Tratner I , Verma I M
Published: May 2006

Two forms of FosB transcript and their products can be identified in mouse NIH 3T3 cells following serum induction. The larger RNA codes for a 338-amino acid protein, whereas the smaller RNA results from the removal of an additional 140 nucleotides from FosB mRNA by alternative splicing. This alternative splicing event places a stop codon following the "leucine zipper" region and results in a shorter protein (FosB2) of 237 amino acids that lacks 101 amino acids at the carboxyl terminus. FosB2 is able to form heterodimers with c-Jun and bind to an AP-1 site but is not able to activate the transcription of promoters containing AP-1 sites. Furthermore, FosB2 can not only suppress the transcriptional activation by c-Fos and c-Jun of promoters containing an AP-1 site but also interferes with the transforming potential of viral and cellular Fos proteins. We propose that FosB2 protein functions as a trans-negative regulator.
Are eukaryotic microorganisms clonal or sexual? A population genetics vantage.
Authors: Tibayrenc M , Kjellberg F , Arnaud J , Oury B , Brenière S F , Dardé M L , Ayala F J
Published: May 2006

We argue that the mode of reproduction of microorganisms in nature can only be decided by population genetic information. The evidence available indicates that many parasitic protozoa and unicellular fungi have clonal rather than sexual population structures, which has major consequences for medical research and practice. Plasmodium falciparum, the agent of malaria, is a special case: the scarce evidence available is contradictory, some suggesting that uniparental lineages may exist in nature. This is puzzling (because P. falciparum is known to have a sexual stage) and poses a challenge that can be readily settled by ascertaining the frequency distribution of genotypes in natural populations.
Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings.
Authors: Nagatani A , Kay S A , Deak M , Chua N H , Furuya M
Published: May 2006

We have examined the biological activity of rice type I phytochrome (PI) in transgenic tobacco seedlings. The progeny of four independent transformants that expressed the rice PI gene segregated 3:1 for shorter hypocotyl length under dim white light (0.04 W/m2). By contrast, this phenotype was not observed either in the dark or under white light at higher intensity (6.0 W/m2). This suggests that the phenotype is dependent not only on light but also on light intensity. The increased light sensitivity cosegregated with the kanamycin-resistance marker as well as with the rice PI polypeptides, indicating that this phenotype is directly related to the expression of the transgene. The transgenic plants showing short hypocotyls exhibited a reduced growth rate throughout the elongation period, and the resulting shorter hypocotyl length was attributable to shorter epidermal cell length but not to reduced cell number. Furthermore, successive pulse irradiations with red light elicited short hypocotyls similar to those obtained under dim white light, and the effect was reversed by immediate far-red light treatment, providing a direct indication that the phenotype is caused by biologically active rice PI. Therefore, the far-red-absorbing form of the introduced rice PI appears to regulate the hypocotyl length of the transgenic tobacco plants through endogenous signal-transduction pathways. This assay system will be a powerful tool for testing the biological activity of introduced phytochrome molecules.
The activity of the CIII regulator of lambdoid bacteriophages resides within a 24-amino acid protein domain.
Authors: Kornitzer D , Altuvia S , Oppenheim A B
Published: May 2006

The CIII protein of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes. We have isolated a set of missense mutations in the cIII gene of phage lambda and of phage HK022 that yield inactive CIII proteins. All the mutations are located in the relatively conserved central region of the protein. A comparative analysis of the CIII protein sequence in lambda, HK022, and the lambdoid bacteriophage P22 leads us to suggest that this central region assumes an amphipathic alpha-helical structure. This part of the lambda cIII gene was cloned within a fragment of the lacZ gene (the alpha-complementing fragment). The resulting fusion protein displays CIII activity. Mutations that yield a nonfunctional fusion protein map within its CIII moiety. These results indicate that the central portion of the CIII protein is both necessary and sufficient for CIII activity.
Retention of embryonic features by an adult neuronal system capable of plasticity: polysialylated neural cell adhesion molecule in the hypothalamo-neurohypophysial system.
Authors: Theodosis D T , Rougon G , Poulain D A
Published: May 2006

The neural cell adhesion molecule, N-CAM, changes at the cell surface during development, from a highly sialylated form [polysialic acid (PSA)-linked N-CAM, PSA-N-CAM] to several isoforms containing less sialic acid. N-CAM and its polysialic acid may serve to regulate cell apposition, thus affecting a variety of cell interactions. In the nervous system, PSA-N-CAM has until now been localized in developing tissues where it is thought to participate in the structuring of neuronal groups and tissue pattern formation. It has been proposed, however, that PSA-N-CAM may also be expressed in the adult, where it may take part in plasticity and cell reshaping. In the present study, the use of immunoblot and immunocytochemical procedures with a monoclonal antibody that specifically recognizes PSA-N-CAM revealed that the adult rat hypothalamo-neurohypophysial system, which undergoes important neuronal-glial and synaptic rearrangements in response to physiological stimuli, contains high levels of PSA-N-CAM immunoreactivity. The use of a polyclonal serum reacting with all N-CAM isoforms indicated that PSA-N-CAM is expressed together with "adult" forms of N-CAM. Light and electron microscopy demonstrated the presence of PSA-N-CAM immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus and in the neurohypophysis; the immunoreactivity was seen in dendrites, axons, and terminals and in associated astrocytes but not in neuronal somata. We propose that the continued expression of PSA-N-CAM confers to magnocellular neurons and their astrocytes the ability to reversibly change their morphology in adulthood. In addition, our observations suggest that evidence for polysialylation may serve to identify other neuronal systems capable of morphological plasticity in the adult central nervous system.
Interferon gamma abrogates the differentiation block in v-myc-expressing U-937 monoblasts.
Authors: Oberg F , Larsson L G , Anton R , Nilsson K
Published: May 2006

Extensive studies suggest a role for the myc protooncogene family in the control of cell proliferation and differentiation in vertebrates. Previously, deregulated expression of exogenous myc genes has been shown to inhibit induced differentiation in Friend erythroleukemia (MEL) cells and in human monoblastic U-937 cells. To examine the nature of the block of phorbol 12-myristate 13-acetate-induced differentiation in v-myc-expressing U-937 cells, we have studied the effect of other inducers utilizing signal pathways distinct from phorbol 12-myristate 13-acetate (i.e., 1 alpha, 25-dihydroxycholecalciferol and retinoic acid). We show that v-myc also inhibits differentiation associated with these inducers. However, the v-myc-associated block of phorbol 12-myristate 13-acetate-, 1 alpha,25-dihydroxycholecalciferol-, and retinoic acid-induced differentiation retinoic acid-induced differentiation can be overcome by adding interferon gamma as a costimulatory factor. Costimulation with interferon gamma restores terminal differentiation, as shown by acquisition of a macrophage phenotype and an irreversible growth arrest in the G0/G1 phase of the cell cycle, but induces only limited differentiation on its own. The differentiation is accomplished without altering the expression or nuclear localization of the v-myc protein. These results argue against the widely held view that down-regulation of myc expression is a general prerequisite for terminal differentiation of hematopoietic cells and suggests that interferon gamma induces a signal(s) that circumvents the v-myc activity.
Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide.
Authors: Takasugi M , Guendouz A , Chassignol M , Decout J L , Lhomme J , Thuong N T , Hélène C
Published: May 2006

On the basis of the structure of DNA-psoralen bis adducts (formed by psoralen with two thymines on opposite strands), a psoralen-oligonucleotide conjugate was designed to photoinduce a cross-link between the two DNA strands at a specific sequence. Psoralen was attached via its C-5 position to a 5'-thiophosphate group of an 11-mer homopyrimidine oligonucleotide. The 11-mer binds to an 11-base-pair homopurine.homopyrimidine sequence of a DNA fragment, where it forms a triple helix. Upon near-UV-irradiation, the two strands of DNA are crosslinked at the TpA step present at the triplex-duplex junction. The reaction is specific for the homopurine.homopyrimidine DNA sequence and requires both oligonucleotide recognition of the DNA major groove and intercalation of psoralen at the triplex-duplex junction. The yield of the photo-induced cross-linking reaction is quite high (greater than 80%). Such psoralen-oligonucleotide conjugates are probes of sequence-specific triple-helix formation and could be used to selectively control gene expression or to induce site-directed mutations.