bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site.
Authors: Collins C J , Underdahl J P , Levene R B , Ravera C P , Morin M J , Dombalagian M J , Ricca G , Livingston D M , Lynch D C
Published: May 2006

A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for von Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span approximately 175 kilobases of human DNA sequence, and hybridization analysis suggests that the vWf coding unit is approximately 150 kilobases in length. Within one of these clones, the vWf transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical "TATA box," a downstream "CCAAT box," and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWf translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species.
Sequence homologies between proteins of bacterial phosphoenolpyruvate-dependent sugar phosphotransferase systems: identification of possible phosphate-carrying histidine residues.
Authors: Bramley H F , Kornberg H L
Published: May 2006

The DNA sequences for some of the genes involved in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) of Escherichia coli and Salmonella typhimurium have been reported. Comparison of the deduced amino acid sequences of enzyme IIBgi, enzyme IIMtl, and enzyme IIGlc/enzyme IIIGlc, which catalyze the uptake and concomitant phosphorylation of beta-glucosides, mannitol, and glucose, respectively, reveals considerable sequence homology. In particular, the carboxyl-terminal region of enzyme IIBgl is so homologous to the whole of enzyme IIIGlc as to suggest a common function. We postulate that His-547 of enzyme IIBgl receives a phosphate group directly from the cytoplasmic protein HPr and transfers this phosphate to His-306 located in the amino-terminal half of enzyme IIBgl. This latter histidine is conserved in enzyme IIBgl and enzyme IIGlc and, in both proteins, occurs in a region that shows homology with the His-15 region of HPr, which is known to act as the phosphate carrier. An equivalent histidine residue, His-195, is also present in enzyme IIMtl, although here the flanking sequence is different. None of these specified histidine residues is likely to be buried within the membrane.
Exonucleolytic proofreading by calf thymus DNA polymerase delta.
Authors: Kunkel T A , Sabatino R D , Bambara R A
Published: May 2006

The fidelity of DNA synthesis by calf thymus DNA polymerase delta (pol delta) in vitro has been determined using an M13lacZ alpha nonsense codon reversion assay. Pol delta is highly accurate, producing on average less than 1 single-base substitution error for each 10(6) nucleotides polymerized. This accuracy is 10- and 500-fold greater than that of DNA polymerases alpha and beta, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol delta. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by pol delta are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of pol delta. These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.
Molecular cloning and sequence analysis of the catalytic subunit of bovine type 2A protein phosphatase.
Authors: Green D D , Yang S I , Mumby M C
Published: May 2006

We have isolated a cDNA clone corresponding to the Mr 38,000 catalytic subunit of bovine type 2A protein phosphatase. The cDNA was isolated from a bovine adrenal gland cDNA library through the use of oligonucleotide probes whose sequences were based on partial amino acid sequence obtained from cyanogen bromide fragments of the purified cardiac enzyme. The entire 1724-base-pair cDNA has been sequenced and found to contain an open reading frame coding for a protein of 325 amino acids having a calculated molecular weight of 37,320. The deduced amino acid sequence contains the experimentally determined sequences of five different cyanogen bromide peptides. Transfection of COS-m6 cells with the cloned cDNA resulted in transient expression of a protein that could be detected by immunoblot analysis with a monoclonal antibody directed against the purified cardiac protein phosphatase. The expressed protein had the same apparent molecular weight as the purified enzyme when analyzed by NaDodSO4/polyacrylamide gel electrophoresis, suggesting that this clone contains the entire coding region of the phosphatase mRNA. The cloned cDNA hybridizes to a mRNA of 2.0 kilobases in bovine heart and adrenal gland. Under conditions of reduced stringency, the cDNA also hybridizes to a mRNA species of 1.2 kilobases in cardiac tissue.
Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells.
Authors: Luby-Phelps K , Castle P E , Taylor D L , Lanni F
Published: May 2006

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size-fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.
Depolarizing influences regulate preprotachykinin mRNA in sympathetic neurons.
Authors: Roach A , Adler J E , Black I B
Published: May 2006

We have been studying mechanisms regulating neurotransmitter plasticity in sympathetic neurons. Neurons of the rat superior cervical ganglion (SCG) synthesize multiple putative transmitters, including the peptide substance P (SP). We have now examined steady-state levels of the mRNA encoding preprotachykinin (PPT), the SP precursor. A cloned cDNA probe was used to examine regulation mRNA levels in culture and in vivo. In RNA gel blot experiments, a single band (1.1 kilobases long) was observed in all cases in which an RNA was detected. A low level of PPT mRNA was detected by RNase protection assay in uncultured ganglia, suggesting that the low levels of SP previously observed in the normal ganglion in vivo are synthesized locally. When ganglia were maintained in culture, with consequent denervation, the steady-state level of PPT mRNA increased by 25-fold over the first 24 hr, and the high level was maintained for at least 7 days. RNase protection experiments indicated that the major message in the SCG is the beta-PPT mRNA, encoding both SP and neurokinin A peptide regions. Accumulation of the PPT mRNA in cultured ganglia was sharply inhibited by the depolarizing agent veratridine, and this effect was blocked by tetrodotoxin. Therefore, one form of neuronal plasticity, change in neurotransmitter metabolism, is regulated at least in part by altering steady-state levels of specific mRNA. More generally, extracellular signals may contribute to neuronal plasticity through changes in gene expression.
Amino-terminal extension generated from an upstream AUG codon is not required for mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferase.
Authors: Ellis S R , Hopper A K , Martin N C
Published: May 2006

The TRM1 gene of Saccharomyces cerevisiae is necessary for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNAs. The DNA sequence of the TRM1 locus and the 5' ends of mRNAs expressed from this gene have been determined. The majority of the 5' ends map within a large open reading frame between two in-frame ATGs at positions +1 and +49. A small fraction of the 5' ends are located upstream of the first ATG. Both AUGs of the TRM1 mRNAs are used to initiate translation, and two forms of N2,N2-dimethylguanosine-specific tRNA methyltransferase, which differ by an amino-terminal extension of 16 amino acids, are made. Mitochondrial tRNAs are modified when the initiation of translation is limited to one or the other of the AUGs, suggesting that the amino-terminal extension is not necessary for import of the protein into mitochondria. Mitochondrial targeting information must, therefore, be located in a region of N2,N2-dimethylguanosine-specific tRNA methyltransferase that is found in both forms of the enzyme.
Rapid and reversible changes in nucleosome structure accompany the activation, repression, and superinduction of murine fibroblast protooncogenes c-fos and c-myc.
Authors: Chen T A , Allfrey V G
Published: May 2006

A procedure for the isolation of transcriptionally active nucleosomes was used to monitor changes in chromatin structure during the activation, repression, and superinduction of the protooncogenes c-fos and c-myc. Nuclei were isolated from murine fibroblasts at successive times after stimulation of quiescent cell cultures with serum or platelet-derived growth factor. The nucleosomes released by a brief micrococcal nuclease digestion were fractionated by HgII-affinity chromatography to separate the unfolded nucleosomes of transcriptionally active genes (in which the sulfhydryl groups of histone H3 are accessible for binding to HgII) from the compactly beaded nucleosomes of transcriptionally inert DNA sequences (in which the H3 sulfhydryl groups are not accessible). The DNA sequence contents of the HgII-bound and unbound nucleosome fractions were compared by slot-blot hybridizations to 32P-labeled cloned probes for c-fos and c-myc. The binding of the c-fos and c-myc nucleosomes to the HgII column accurately reflected both the timing and the degree of their expression, as determined by run-off transcription assays with the isolated nuclei. The superinduction of c-fos and c-myc expression by an inhibitor of protein synthesis (cycloheximide) was reflected in the persistence of the unfolded, transcriptionally active state of their component nucleosomes. These results provide direct evidence that rapid and reversible changes in nucleosome topography accompany the program of oncogene expression, and they suggest a way to monitor aberrant gene activity during malignant transformation.
Phylogenetic relationships between chlorophytes, chrysophytes, and oomycetes.
Authors: Gunderson J H , Elwood H , Ingold A , Kindle K , Sogin M L
Published: May 2006

The phylogenetic relationships among the chlorophyte Chlamydomonas reinhardtii, the chrysophyte Ochromonas danica, and the oomycete Achyla bisexualis were explored by comparing the sequences of their small-subunit ribosomal RNA coding regions. Comparisons of similarity values or inspection of phylogenetic trees constructed by distance matrix methods reveal a very close relationship between oomycetes and chrysophytes. The separation of chrysophytes from chlorophytes is comparable to that of plants from animals, and both separations are far antedated by the divergence of a number of other protist groups.
The PapG protein is the alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose-binding adhesin of uropathogenic Escherichia coli.
Authors: Lund B , Lindberg F , Marklund B I , Normark S
Published: May 2006

Uropathogenic Escherichia coli adhere to uroepithelial cells by their digalactoside alpha-D-galactopyranosyl-(1----4)-beta-D-galactopyranose [alpha-D-Galp-(1----4)-beta-D-Galp or Gal alpha (1----4)Gal]-binding pili, which are composed of repeating identical subunits. The major subunit (PapA) of these pili is not required for binding, but the papF and papG gene products are essential for adhesion. Transcomplementation analysis between the pap gene cluster and a related gene cluster encoding a different binding specificity showed that PapG and not PapF is the Gal alpha (1----4)Gal-specific adhesin. Antibodies against PapG were obtained upon immunizing with whole Pap pili, showing that the adhesin is a pilus component. Antisera specific for different Pap proteins were used to demonstrate that a pilin protein, either PapA or PapE, together with both PapG and PapF, must be exposed on the cell surface to allow E. coli to bind. The DNA sequence of the papG gene is presented, and the deduced primary structure showed similarities both to the B-chain sequence of the digalactoside-binding Shigella toxin and to established amino acid sequences of pilins.
Evolution of P transposable elements: sequences of Drosophila nebulosa P elements.
Authors: Lansman R A , Shade R O , Grigliatti T A , Brock H W
Published: May 2006

P elements have been cloned and sequenced from Drosophila nebulosa. Their sequences have diverged less than 6% from P elements of Drosophila melanogaster. However D. nebulosa P elements have nucleotide changes that close all four open reading frames found in the D. melanogaster P element. Microinjection experiments show that D. nebulosa P elements cannot provide transposase function for D. melanogaster P elements, nor are D. nebulosa P elements mobilized by the transposase provided by a D. melanogaster P factor. Three D. nebulosa P elements appear to have integrated into the same position of a complex, centromeric repeated sequence. Comparison of nucleotide sequences suggests that D. nebulosa P elements have diverged upon different pathways from a common ancestor that was 99% homologous to the P elements of D. melanogaster.
Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli.
Authors: Eisenstein B I , Sweet D S , Vaughn V , Friedman D I
Published: May 2006

The on-and-off expression (phase variation) of type 1 fimbriae, encoded by fimA, in Escherichia coli is controlled by the inversion of a promoter-containing 314-base-pair DNA element. This element is flanked on each side by a 9-base-pair inverted, repeat sequence and requires closely linked genes for inversion. Homology analysis of the products of these genes, fimB and fimE, reveals a strong similarity with the proposed DNA binding domain of lambda integrase, which mediates site-specific recombination in the presence of integration host factor. Integration host factor, encoded by himA and hip/himD, binds to the sequence 5' TNYAANNNRTTGAT 3', where Y = pyrimidine and R = purine, in mediating integration-excision. In analyzing the DNA flanking the fim 314-base-pair inversion sequence, we found the adjacent sequence 5' TTTAACTTATTGAT 3', which corresponds perfectly with the consensus integration host factor binding site. To characterize the role of himA in phase variation, we transduced either a deletion of himA or an insertionally inactivated hip/himD gene into an E. coli strain with a fimA-lacZ operon fusion. We found the rate of phase variation decreases sharply from 10(-3) to less than 10(-5) per cell per generation. Southern hybridization analysis demonstrates that the himA mutation results in a failure of the switch-generated genetic rearrangement. When the transductant was transformed with a himA+ plasmid, normal switching returned. Thus integration host factor is required for normal type 1 fimbriae phase variation in E. coli.
Monte Carlo-minimization approach to the multiple-minima problem in protein folding.
Authors: Li Z , Scheraga H A
Published: May 2006

A Monte Carlo-minimization method has been developed to overcome the multiple-minima problem. The Metropolis Monte Carlo sampling, assisted by energy minimization, surmounts intervening barriers in moving through successive discrete local minima in the multidimensional energy surface. The method has located the lowest-energy minimum thus far reported for the brain pentapeptide [Met5]enkephalin in the absence of water. Presumably it is the global minimum-energy structure. This supports the concept that protein folding may be a Markov process. In the presence of water, the molecules appear to exist as an ensemble of different conformations.
Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells.
Authors: Wilhelm S M , Collier I E , Kronberger A , Eisen A Z , Marmer B L , Grant G A , Bauer E A , Goldberg G I
Published: May 2006

We have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO4/PAGE of 60 and 57 kDa. The minor 60-kDa polypeptide is a glycosylated form of the major 57-kDa protein containing N-linked complex oligosaccharides. Zymogen activation by trypsin results in the removal of 84 amino acids from the amino terminus of the enzyme generating a 45-kDa active enzyme species. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively (1-2 micrograms per 10(6) cells per 24 hr). Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. Our data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.
A cis-acting element is necessary and sufficient for translational regulation of human ferritin expression in response to iron.
Authors: Hentze M W , Rouault T A , Caughman S W , Dancis A , Harford J B , Klausner R D
Published: May 2006

Ferritin plays a key role in determining the intracellular fate of iron and is highly regulated by the iron status of the cell. We have identified a cis-acting element in the transcribed but nontranslated 5' leader sequence of human ferritin heavy-chain mRNA. In transiently transfected murine fibroblasts, the presence of a 157-nucleotide region of the 5' leader sequence was found to be necessary for iron-dependent regulation of ferritin biosynthesis. Further, this 5' leader region is sufficient to transfer iron-mediated translational control to the expression of a heterologous gene product, chloramphenicol acetyltransferase.
East African cheetahs: evidence for two population bottlenecks?
Authors: O'Brien S J , Wildt D E , Bush M , Caro T M , FitzGibbon C , Aggundey I , Leakey R E
Published: May 2006

A combined population genetic and reproductive analysis was undertaken to compare free-ranging cheetahs from east Africa (Acinonyx jubatus raineyi) with the genetically impoverished and reproductively impaired south African subspecies (Acinonyx jubatus jubatus). Like that of their south African counterparts, the quality of semen specimens from east African cheetahs was poor, with a low concentration of spermatozoa (25.3 X 10(6) per ejaculate) and a high incidence of morphological abnormalities (79%). From an electrophoretic survey of the products of 49 genetic loci in A. jubatus raineyi, two allozyme polymorphisms were detected; one of these, for a nonspecific esterase, shows an allele that is rare (less than 1% incidence) in south African specimens. Estimates of polymorphism (2-4%) and average heterozygosity (0.0004-0.014) affirm the cheetah as the least genetically variable felid species. The genetic distance between south and east African cheetahs was low (0.004), suggesting that the development of genetic uniformity preceded the recent geographic isolation of the subspecies. We propose that at least two population bottlenecks followed by inbreeding produced the modern cheetah species. The first and most extreme was ancient, possibly late Pleistocene (circa 10,000 years ago); the second was more recent (within the last century) and led to the south African populations.
Purification and characterization of a nicotinic acetylcholine receptor from rat brain.
Authors: Whiting P , Lindstrom J
Published: May 2006

We previously reported the immunoaffinity purification of an acetylcholine receptor from chicken brain that did not bind alpha-bungarotoxin but did bind nicotine and other cholinergic agonists. Antisera and monoclonal antibodies raised against this receptor crossreacted with a receptor from rat brain that had similar pharmacological properties, and also bound to functional acetylcholine receptors in chicken ciliary ganglion cells and rat PC12 cells. Here we report purification of the receptor from rat brain using monoclonal antibody (mAb) 270 raised against receptor from chicken brain. This receptor, similar in size to monomers of receptor from Torpedo electric organ, contained two subunits--apparent Mr, 51,000 and 79,000. The Mr 51,000 subunit was bound by antisera to alpha subunits of receptor from Torpedo electric organ and by mAb 270, which is specific for the Mr 49,000 subunit analogue of receptor from chicken brain. Both subunits were bound by mAb 286, which also binds both subunits of receptors from chicken brain. The alpha-bungarotoxin binding component was purified from the same extracts. It consisted of four subunits of apparent Mr 44,700, 52,300, 56,600, and 65,200. The basic structure of receptors from muscle had evolved to an (alpha)2 beta gamma delta subunit stoichiometry by the time of primitive elasmobranches and is now little changed in mammals. The apparent (alpha)2(beta)2 or (alpha)3(beta)2 structure of the neuronal acetylcholine receptors that we have purified may derive from an early gene duplication event in the evolution of the extended gene family, which now also includes receptors from ganglia and muscle as well as neuronal alpha-bungarotoxin binding sites.
Complete tracking of transient proton flow through active chloroplast ATP synthase
Authors: Junge Wolfgang
Published: May 2006

Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CF0-CF1 (where CF0 is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of P
i
and ADP, the electric potential transients elicited transients of proton flow via CF0-CF1, typically 1.3 H
+
per CF1 and flash group. Proton flow was blocked by CF0-CF1 inhibitors:
N,N
′-dicyclohexylcarbodiimide, acting on the channel component CF0, and tentoxin, acting on the catalytic component CF1. The half-rise time was 40 ms in
1
H
2
O and 78 ms in
2
H
2
O. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a
K
m
(P
i
) of only 14 μM, contrasting with a
K
m
of several hundred micromolar for continuous ATP synthesis at high rate. This might reflect a resistance to P
i
diffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CF0-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CF0-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.
Comparison of the effects of microtubule-associated protein 2 and tau on the packing density of in vitro assembled microtubules.
Authors: Black M M
Published: May 2006

I have compared the effects of microtubule-associated protein 2 (MAP-2) and tau on the packing density of sedimented microtubules. Microtubules assembled in vitro in taxol were pelleted by centrifugation. The volumes of the resulting pellets were calculated from their weights assuming a specific gravity of 1 and then were normalized to the amount of protein in the pellet, yielding a value for pellet specific volume in microliter/mg of protein. The specific volume of the pellets reflects the intermicrotubule spacing within the pellet. Microtubules were assembled from tubulin alone or tubulin plus various amounts of MAP-2 or tau and collected by centrifugation, and the pellet specific volume was measured. The specific volume of microtubules composed of pure tubulin ranged from 6.4 to 7.7 microliter/mg of protein. Tau had no detectable effect on this value even at saturating levels on the microtubules. In contrast, MAP-2 increased pellet specific volume as the MAP-2/tubulin weight ratio increased; at the highest ratio examined, 0.43, the pellet specific volume was approximately 33. Even at the relatively low MAP-2/tubulin ratio of 0.09, pellet specific volume was approximately 2-fold greater than that of microtubules containing tubulin alone or tubulin plus tau. Electron microscopy confirmed that the observations on pellet specific volume reflected differences in the effects of MAP-2 and tau on the packing density of sedimented microtubules. These results are discussed in the context of observations showing that neighboring microtubules are more widely spaced in dendrites than in axons and that MAP-2 is enriched on microtubules in dendrites compared to microtubules in axons, whereas the converse is true for tau.
Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide.
Authors: Hayes G R , Lockwood D H
Published: May 2006

The oxidant H2O2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H2O2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the beta subunit of the insulin receptor was increased approximately 2-fold by treatment of intact cells with 3 mM H2O2, a concentration that maximally stimulates 2-deoxyglucose uptake. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H2O2-treated adipocytes showed that 32P incorporation into phosphotyrosine and phosphoserine residues of the beta subunit was enhanced. Furthermore, partially purified receptors from H2O2-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu80Tyr20. In contrast, the direct addition of H2O2 to partially purified insulin receptors did not stimulate tyrosine kinase activity or insulin receptor autophosphorylation. This was not due to breakdown of H2O2 or oxidation of ATP or the required divalent cations. To define the factors involved in H2O2's effect, we have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H2O2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H2O2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H2O2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H2O2 on the insulin receptor and requires nonplasma membrane cellular constituents.
Cloning vectors for expression of cDNA libraries in mammalian cells.
Authors: Murphy A J , Efstratiadis A
Published: May 2006

We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient expression of the released library in a cell line containing a packaging mutant of Moloney murine leukemia virus, followed by cocultivation of these producers with recipient cells. Transcription of cDNAs in the recipient cells is driven by the strong long terminal repeat promoter, and the transcripts, even from truncated cDNAs, can be expressed because translational start sites have been provided in all three reading frames (tri-initiator). Sequences conferring a recognizable phenotype can be rescued by cell fusion. The functionality of the tri-initiator and the rescue of a rare cDNA have been successfully tested using model systems.
Bacteriophage PRD1 DNA polymerase: evolution of DNA polymerases.
Authors: Jung G H , Leavitt M C , Hsieh J C , Ito J
Published: May 2006

A small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase that utilizes terminal protein as a primer for DNA synthesis. The PRD1 DNA polymerase gene has been sequenced, and its amino acid sequence has been deduced. This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300. Thus, it appears to be the smallest DNA polymerase ever isolated from prokaryotic cells. Comparison of the PRD1 DNA polymerase sequence with other DNA polymerase sequences that have been published yielded segmental but significant homologies. These results strongly suggest that many prokaryotic and eukaryotic DNA polymerase genes, regardless of size, have evolved from a common ancestral gene. The results further indicate that those DNA polymerases that use either an RNA or protein primer are related. We propose to classify DNA polymerases on the basis of their evolutionary relatedness.
Structure of the chromosomal chicken progesterone receptor gene.
Authors: Huckaby C S , Conneely O M , Beattie W G , Dobson A D , Tsai M J , O'Malley B W
Published: May 2006

We have isolated cosmid clones containing the chromosomal chicken progesterone receptor gene. The gene consists of eight exons and is approximately 38 kilobases long. Individual exons correlate well with conserved functional domains of the receptor molecule. Alternative polyadenylylation in the second intron results in a putative non-hormone-binding protein. The cap site of the gene is heterogeneous over at least 14 base pairs and lies in a very G + C-rich region. The promoter lacks "TATA" and "CAAT" boxes, but CCGCCC motifs exist in the surrounding region.
A bifurcated hydrogen-bonded conformation in the d(A.T) base pairs of the DNA dodecamer d(CGCAAATTTGCG) and its complex with distamycin.
Authors: Coll M , Frederick C A , Wang A H , Rich A
Published: May 2006

The crystal structures of d(CGCA3T3GCG) complex to the antitumor drug distamycin and the DNA fragment alone were solved by x-ray diffraction at 2.2 and 2.5 A resolution, respectively. The drug lies in the narrow minor groove near the center of the B-DNA fragment covering 5 of the 6 A.T base pairs. It is bound to the DNA by hydrogen bonding, van der Waals, and electrostatic interactions. In addition, the DNA was found to have an unusual conformation in the (dA)3.(dT)3 regions. These base pairs have a high positive propeller twist so that in the major groove the adenine amino group is located intermediate between the carbonyl O-4 groups of two adjacent thymines of the opposite strand, making bifurcated hydrogen bonds to the two thymine residues. This suggests a model to explain the unusual properties of poly-(dA).poly(dT) in which a modified B conformation is associated with a large propeller twist of the bases and a set of continuous bifurcating hydrogen bonds along the major groove, which may provide incremental stability to these segments. In addition, shorter segments of (dA)3-6.(dT)3-6 may have this conformation in the midst of B-DNA and stabilize bends in the DNA that may be associated with stacking on one of the high propeller-twisted bases at the ends of these segments.
Bacterial heme synthesis is required for expression of the leghemoglobin holoprotein but not the apoprotein in soybean root nodules.
Authors: O'Brian M R , Kirshbom P M , Maier R J
Published: May 2006

In Bradyrhizobium japonicum/soybean symbiosis, the leghemoglobin (legume hemoglobin) apoprotein is a plant product, but the origin of the heme prosthetic group is not known. B. japonicum strain LO505 is a transposon Tn5-induced cytochrome-deficient mutant; it excreted the oxidized heme precursor coproporphyrin III into the growth medium. Mutant strain LO505 was specifically deficient in protoporphyrinogen oxidase (protoporphyrinogen-IX:oxygen oxidoreductase, EC 1.3.3.4) activity, and thus it could not catalyze the penultimate step in heme biosynthesis. Soybean root nodules formed from this mutant did not contain leghemoglobin, but the apoprotein was synthesized nevertheless. Data show that bacterial heme synthesis is required for leghemoglobin expression, but the heme moiety is not essential for apoleghemoglobin synthesis by the plant. Soybean leghemoglobin, therefore, is a product of both the plant and bacterial symbionts.
Transfer of resistance traits from carrot into tobacco by asymmetric somatic hybridization: Regeneration of fertile plants
Authors: Dudits Denes , Maroy Eszter , Praznovszky Tunde , Olah Zoltan , Gyorgyey Janos , Cella Rino
Published: May 2006

Transfer of methotrexate and 5-methyltryptophan resistance from carrot (
Daucus carota
) to tobacco (
Nicotiana tabacum
) was achieved by fusion between leaf mesophyll protoplasts of tobacco and irradiated cell culture protoplasts of carrot. Some of the regenerated somatic hybrids exhibited normal tobacco morphology with coexpression and independent segregation of the transferred resistance markers. Chromosomal instability resulted in aneuploid somatic hybrids with significantly lower chromosome number than predicted by simple addition of parental chromosome number. The methotrexate resistance phenotype was correlated with the expression of carrot-specific dihydrofolate reductase as judged by isozyme and immunological characteristics of the enzyme. The genomic construct of these somatic hybrids made the transmission of the resistance character into the next sexual generation possible.
Role of diaxial versus diequatorial hydroxyl groups in the tumorigenic activity of a benzo[a]pyrene bay-region diol epoxide.
Authors: Chang R L , Wood A W , Conney A H , Yagi H , Sayer J M , Thakker D R , Jerina D M , Levin W
Published: May 2006

Tumorigenic activities of the (7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro derivatives of benzo[a]pyrene [(+)-B[a]P diol epoxide-2] and 6-fluorobenzo[a]pyrene (6-FB[a]P diol epoxide-2) were evaluated in newborn CD-1 mice. A total dose of 14 nmol of either diol epoxide was administered to preweanling mice, and tumorigenic activity was determined when the mice were 32 to 36 weeks old. At the termination of the study, 13% of solvent-treated control mice had developed lung tumors with an average of 0.19 tumor per mouse. No other tumors were observed in control animals. (+)-B[a]P diol epoxide-2 induced pulmonary tumors in 60% of the mice with an average of 1.9 tumors per mouse, and 14% of the male mice developed hepatic tumors with an average of 0.18 tumor per mouse. In contrast, 6-FB[a]P diol epoxide-2 had no significant tumorigenic activity at the 14-nmol dose. Although both bay-region diol epoxides have the same absolute configuration, (7R,8S,9S,10R), the hydroxyl groups of (+)-B[a]P diol epoxide-2 prefer the pseudoequatorial conformation whereas the hydroxyl groups of 6-FB[a]P diol epoxide-2 prefer the pseudoaxial conformation. The tumorigenicity results reported here are the first direct demonstration that conformation of the hydroxyl groups in a bay-region diol epoxide, in addition to the documented effect of absolute configuration, is an important determinant in the tumorigenic activity of these ultimate carcinogens.
Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium.
Authors: Storz G , Christman M F , Sies H , Ames B N
Published: May 2006

Salmonella typhimurium strains containing deletions of oxyR, a positive regulator of defenses against oxidative stress, show 10- to 55-fold higher frequencies of spontaneous mutagenesis compared to otherwise isogenic oxyR+ control strains. The high spontaneous-mutation frequency in oxyR deletion strains is decreased by a factor of 3 when the strains are grown anaerobically. oxyR deletion strains show an increase in small deletion mutations and at least three of the six possible base-substitution mutations (T.A to A.T, C.G to T.A, and C.G to A.T). However, the largest increase in mutation frequency is observed for T.A to A.T transversions (40- to 146-fold), the base-substitution mutation most frequently caused by chemical oxidants. The introduction into oxyR deletion strains of multicopy plasmids carrying the oxyR-regulated genes for catalase (katG) or alkyl hydroperoxide reductase (ahp) results in overexpression of the respective enzyme activities and decreases the number of spontaneous mutants to wild-type levels. The introduction into oxyR deletions of a plasmid carrying the gene for superoxide dismutase (sodA) decreases the mutation frequency by a factor of 5 in some strain backgrounds. Strains that contain a dominant oxyR mutation and overexpress proteins regulated by oxyR show lower spontaneous-mutation frequencies by a factor of 2. These results indicate that oxyR and oxyR-regulated genes play a significant role in defense against spontaneous oxidative DNA damage. The role of oxidative damage to DNA in "spontaneous" mutagenesis is discussed.
Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast, and nuclear DNAs.
Authors: Wolfe K H , Li W H , Sharp P M
Published: May 2006

Comparison of plant mitochondrial (mt), chloroplast (cp) and nuclear (n) DNA sequences shows that the silent substitution rate in mtDNA is less than one-third that in cpDNA, which in turn evolves only half as fast as plant nDNA. The slower rate in mtDNA than in cpDNA is probably due to a lower mutation rate. Silent substitution rates in plant and mammalian mtDNAs differ by one or two orders of magnitude, whereas the rates in nDNAs may be similar. In cpDNA, the rate of substitution both at synonymous sites and in noncoding sequences in the inverted repeat is greatly reduced in comparison to single-copy sequences. The rate of cpDNA evolution appears to have slowed in some dicot lineages following the monocot/dicot split, and the slowdown is more conspicuous at nonsynonymous sites than at synonymous sites.
Reactive free radical generation in vivo in heart and liver of ethanol-fed rats: correlation with radical formation in vitro.
Authors: Reinke L A , Lai E K , DuBose C M , McCay P B
Published: May 2006

Rats fed a high-fat ethanol-containing diet for 2 weeks were found to generate free radicals in liver and heart in vivo. The radicals are believed to be carbon-centered radicals, were detected by administering spin-trapping agents to the rats, and were characterized by electron paramagnetic resonance spectroscopy. The radicals in the liver were demonstrated to be localized in the endoplasmic reticulum. Rats fed ethanol in a low-fat diet showed significantly less free radical generation. Control animals given isocaloric diets without ethanol showed no evidence of free radicals in liver and heart. When liver microsomes prepared from rats fed the high-fat ethanol diet were incubated in a system containing ethanol, NADPH, and a spin-trapping agent, the generation of 1-hydroxyethyl radicals was observed. The latter was verified by using 13C-substituted ethanol. Microsomes from animals fed the high-fat ethanol-containing diet had higher levels of cytochrome P-450 than microsomes from rats fed the low-fat ethanol-containing diet. The results suggest that the consumption of ethanol results in the production of free radicals in rat liver and heart in vivo that appear to initiate lipid peroxidation.
B protein of bacteriophage mu is an ATPase that preferentially stimulates intermolecular DNA strand transfer.
Authors: Maxwell A , Craigie R , Mizuuchi K
Published: May 2006

A DNA strand-transfer reaction is an early step in the transposition of phage Mu. It has been shown that an efficient reaction in vitro requires, in addition to buffer and salt, only the Mu A protein, Mu B protein, host protein HU, ATP, and Mg2+. We have determined that, of the three protein factors involved, only the Mu B protein has an ATPase activity. The Mu B ATPase is stimulated by Mu A protein and DNA but not by either of these factors alone. Double-stranded DNA is a much better cofactor than single-stranded DNA, but there is no apparent sequence specificity. In the absence of the Mu B protein and/or ATP, the intermolecular Mu DNA strand-transfer reaction is extremely inefficient, and the strand-transfer products are predominantly the result of an intramolecular reaction. This contrasts with the efficient intermolecular reaction that occurs if Mu B protein and ATP are provided. The Mu B protein, in the presence of Mu A protein and protein HU, therefore, seems to facilitate interactions between potential DNA target sites and pairs of Mu DNA ends.
Introduction of the human pro alpha 1(I) collagen gene into pro alpha 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen.
Authors: Schnieke A , Dziadek M , Bateman J , Mascara T , Harbers K , Gelinas R , Jaenisch R
Published: May 2006

The Mov-13 mouse strain carries a retroviral insertion in the pro alpha 1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro alpha 2 mRNA are synthesized. We have introduced genomic clones of either the human or mouse pro alpha 1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro alpha 1(I) chains can associate with the endogenous mouse pro alpha 2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human alpha 1 chains and one mouse alpha 2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both alpha 1(I) and alpha 2(I) chains in the human-mouse hybrid molecules were retarded, compared to the alpha (I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse alpha 1 and alpha 2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human alpha chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow us to study the effect specific mutations introduced in transfected pro alpha 1(I) genes have on the synthesis, assembly, and function of collagen I.
Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae.
Authors: Madaule P , Axel R , Myers A M
Published: May 2006

The rho genes comprise an evolutionarily conserved family with significant homology to the ras oncogene family. Two members of the rho family were isolated from the yeast Saccharomyces cerevisiae and characterized by DNA sequence analysis. The yeast genes RHO1 and RHO2 are 70% and 57% identical, respectively, to the rho gene of the marine snail Aplysia, and they are 53% identical to each other. Inactivation of these genes showed that RHO1 is required for cell viability, while RHO2 is not an essential gene. A mutant allele of RHO1 (RHO1-His68) was constructed with a mutation analogous to one that activates the transforming potential of the human HRAS gene. Diploid strains containing RHO1-His68 in either low or high copy number are unable to sporulate, and the mutant allele is dominant over wild-type RHO1. The requirement for RHO1 cannot be circumvented by introduction of high copy number plasmids containing either the gene encoding the catalytic subunit of cAMP-dependent protein kinase or the mutant allele RAS2-Val19. Despite the conservation between the rho and ras gene families, the finding that RHO1 functions independently of the adenylate cyclase cAMP-dependent protein kinase cascade suggests that rho and ras are involved in distinct biochemical pathways.