bluuebunny/crossref_metadata_2025 huggingface dataset:
Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus.
Authors: Panicali D , Paoletti E
Published: May 2006

We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by cotransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.
Tissue-specific expression of an unusual H-2 (class I)-related gene.
Authors: Cosman D , Kress M , Khoury G , Jay G
Published: May 2006

Sequence analysis of mouse H-2 cDNA clones has suggested the existence of an unusual class of H-2 (class I)-related antigens that, unlike the classical membrane-associated molecules, retains only the extracellular portion and is likely to be secreted. The expression of this class of H-2-related mRNA is tissue restricted; it is detectable in liver but not in brain, kidney, testis, thymus, or spleen. In the liver, its accumulation represents about one-fourth of all the H-2 (class I)-specific transcripts. This class of transcripts is present in mice of different inbred strains, but the level of expression differs markedly among them. A model is presented in which such a soluble form of the H-2 antigen would play the role of a blocking factor in maintaining peripheral inhibition of H-2 recognition. This would ensure tolerance of the H-2 molecule as a self antigen while permitting it to act as a guidance molecule for the associative recognition of viral and tumor antigens by cytotoxic T cells.
Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts.
Authors: Bagley K , Dollinger G , Eisenstein L , Singh A K , Zimányi L
Published: May 2006

Fourier transform infrared difference spectroscopy has been used to obtain the vibrational modes in the chromophore and apoprotein that change in intensity or position between light-adapted bacteriorhodopsin and the K and M intermediates in its photocycle and between dark-adapted and light-adapted bacteriorhodopsin. Our infrared measurements provide independent verification of resonance Raman results that in light-adapted bacteriorhodopsin the protein-chromophore linkage is a protonated Schiff base and in the M state the Schiff base is unprotonated. Although we cannot unambiguously identify the Schiff base stretching frequency in the K state, the most likely interpretation of deuterium shifts of the chromophore hydrogen out-of-plane vibrations is that the Schiff base in K is protonated. The intensity of the hydrogen out-of-plane vibrations in the K state compared with the intensities of those in light-adapted and dark-adapted bacteriorhodopsin shows that the conformation of the chromophore in K is considerably distorted. In addition, we find evidence that the conformation of the protein changes during the photocycle.
In vitro replication of bacteriophage phi 29 DNA.
Authors: Watabe K , Shih M F , Sugino A , Ito J
Published: May 2006

We have been studying the mechanisms of linear DNA replication by using Bacillus bacteriophage phi 29 as a model system. To isolate and characterize the proteins required for phi 29 DNA replication, we have developed a cell-free replication system. A cell-free extract prepared from phi 29-infected Bacillus subtilis catalyzes the semiconservative replication of phi 29 DNA, but only if exogenous phi 29 DNA-protein complex is used as the template. This template consists of linear duplex DNA with a 30,000-dalton terminal protein attached covalently to both 5' ends. Replication starts nonsimultaneously at or near both ends of the template. The extract also catalyzes the specific binding between dATP and the phi 29 terminal protein. Thus, the in vitro system closely mimics the in vivo replication of phi 29 DNA. This system should allow characterization of the phi 29 DNA replication machinery.
Transcriptional control of the murine albumin/alpha-fetoprotein locus during development.
Authors: Tilghman S M , Belayew A
Published: May 2006

The ontogeny of expression of the alpha-fetoprotein (AFP) and albumin genes was examined in livers from late prenatal to 1-month-postpartum C3H/He mice. A parallel accumulation of both AFP and albumin mRNAs before birth, followed by a selective nonreciprocal decrease in AFP mRNA after birth, was observed. The decrease in AFP mRNA was the result of a decrease in transcription of the AFP gene, as measured by an in vitro nuclear transcription assay. We suggest a model for hepatic expression of the AFP and albumin gene cluster in which transcription of the two genes is activated simultaneously during differentiation and each gene is thereafter modulated independently in committed cells.
Post-tetanic potentiation at an identified synapse in Aplysia is correlated with a Ca2+-activated K+ current in the presynaptic neuron: evidence for Ca2+ accumulation.
Authors: Kretz R , Shapiro E , Kandel E R
Published: May 2006

We have examined the presynaptic changes underlying post-tetanic potentiation (PTP) in Aplysia by using voltage-clamp techniques combined with specific pharmacological blocking agents. The amplitude and time course of PTP parallel a slow outward clamp current that we have identified as a Ca2+-activated K+ current. Because this current is proportional to intracellular Ca2+ concentration our findings provide evidence for the "residual Ca2+ hypothesis," according to which PTP is caused by the accumulation of intracellular Ca2+ after tetanus. To obtain further evidence for this mechanism we injected EGTA intracellularly and found that it decreased the duration of both PTP and the Ca2+ -activated K+ current.
Reconstruction of the chloroplast noncyclic electron transport pathway from water to NADP with three integral protein complexes
Authors: Lam Eric , Malkin Richard
Published: May 2006

Reconstruction of photosynthetic noncyclic electron transport from water to NADP has been accomplished by using three integral protein complexes isolated from chloroplast thylakoid membranes: photosystems I and II and the cytochrome
b
6
-
f
complex. This system shows an absolute dependence on the presence of all three protein complexes for NADP reduction, in addition to plastocyanin, ferredoxin, and ferredoxin-NADP reductase. The reconstructed system was found to be sensitive to low concentrations of known inhibitors of noncyclic electron transport. Depletion of the Rieske iron-sulfur center and bound plastoquinone from the cytochrome
b
6
-
f
complex resulted in an inhibition of the photoreduction of NADP.
Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP.
Authors: Peñalva M A , Salas M
Published: May 2006

Incubation of extracts of phi 29-infected Bacillus subtilis with [alpha-32P]dATP produced a labeled protein having the electrophoretic mobility of p3, the 5'-terminal protein of phi 29 DNA. The reaction product was resistant to treatment with micrococcal nuclease, phosphatase, and RNases A and T1 and sensitive to proteinase K. Incubation of the 32P-labeled protein with piperidine under conditions in which the phi 29 DNA-protein p3 linkage is hydrolyzed released 5'-dAMP. The reaction with [alpha-32P]dATP was strongly inhibited by anti-p3 serum and required the preence of phi 29 DNA-protein p3 complex; no reaction took place with proteinase K-treated phi29 DNA. These results, together with those of acid hydrolysis and partial proteolysis, indicated that a covalent complex between protein p3 and 5'-dAMP is formed in vitro. The initiation complex (protein p3-dAMP) formed in the presence of 0.5 microM [alpha-32P]dATP can be elongated by addition of 40 microM dNTPs. Treatment with piperidine of the product elongated in the presence of 2',3'-dideoxycytidine 5'-triphosphate released the expected oligonucleotides, 9 and 12 bases long, taking into account the sequence at the left and right DNA ends, respectively.
Affinity labeling of the plasma membrane 3,3',5-triiodo-L-thyronine receptor in GH3 cells.
Authors: Horiuchi R , Johnson M L , Willingham M C , Pastan I , Cheng S
Published: May 2006

The binding of 3,3',5-triiodo-L-thyronine (T3) to GH3 rat pituitary tumor cells was studied at 15 degrees C and was shown to be saturable, reversible, and stereospecific. Least-squares analysis of the binding data showed two classes of binding sites with dissociation constants of 1.8 +/- 0.2 nM and 260 +/- 30 nM and binding capacities of (5.2 +/- 0.2) X 10(4) and (1.6 +/- 0.2) X 10(6) sites per cell, respectively. Affinity labeling of intact cells was carried out by incubation of cells with 0.3 nM N-bromoacetyl-[125I]T3 at 15 degrees C for 1 hr. Analysis of the cellular extracts by sodium dodecyl sulfate gel electrophoresis showed three labeled protein bands with apparent molecular masses of 55, 47, and 33 kilodaltons (kDal) in a ratio of 86:7:7. The labeling of only the 55-kDal protein band was selectively reduced to 50% by 20 microM unlabeled T3. Highly purified plasma membranes of GH3 cells were prepared and shown to be free of nuclei. Affinity labeling of the purified plasma membranes gave the same labeling pattern as with intact cells. Peptide mapping by Staphylococcus aureus V8 digestion of the 55-kDal protein from cells or plasma membranes gave the identical peptide fragments. Thus the 55-kDal protein labeled from intact cells is the same protein as that from purified plasma membranes. These results together with our earlier findings [Horiuchi, R., Cheng, S.-y., Willingham, M. & Pastan, I. (1982) J. Biol. Chem. 257, 3139-3144] suggest that the 55-kDal protein may be involved in mediating the uptake of T3 in GH3 cells.
Molecular mechanical studies of DNA flexibility: Coupled backbone torsion angles and base-pair openings
Authors: Keepers Joe W. , Kollman Peter A. , Weiner Paul K. , James Thomas L.
Published: May 2006

Molecular mechanics studies have been carried out on “B-DNA-like” structures of [d(C-G-C-G-A-A-T-T-C-G-C-G)]
2
and [d(A)]
12
·[d(T)]
12
. Each of the backbone torsion angles (ψ, φ, ω, ω′, φ′) has been “forced” to alternative values from the normal B-DNA values (g
+
, t, g
-
, g
-
, t conformations). Compensating torsion angle changes preserve most of the base stacking energy in the double helix. In a second part of the study, one purine N3-pyrimidine N1 distance at a time has been forced to a value of 6 Å in an attempt to simulate the base opening motions required to rationalize proton exchange data for DNA. When the 6-Å constraint is removed, many of the structures revert to the normal Watson-Crick hydrogen-bonded structure, but a number are trapped in structures ≈5 kcal/mol higher in energy than the starting B-DNA structure. The relative energy of these structures, some of which involve a non-Watson-Crick thymine C2(carbonyl)[unk]adenine 6NH
2
hydrogen bond, are qualitatively consistent with the ΔH for a “base pair-open state” suggested by Mandal
et al.
of 4-6 kcal/mol [Mandal, C., Kallenbach, N. R. & Englander, S. W. (1979)
J. Mol. Biol.
135, 391-411]. The picture of DNA flexibility emerging from this study depicts the backbone as undergoing rapid motion between local torsional minima on a nanosecond time scale. Backbone motion is mainly localized within a dinucleoside segment and generally not conformationally coupled along the chain or across the base pairs. Base motions are much smaller in magnitude than backbone motions. Base sliding allows imino N—H exchange, but it is localized, and only a small fraction of the N—H groups is exposed at any one time. Stacking and hydrogen bonding cause a rigid core of bases in the center of the molecule accounting for the hydrodynamic properties of DNA.
Human T-cell leukemia-lymphoma virus (HTLV) is in T but not B lymphocytes from a patient with cutaneous T-cell lymphoma.
Authors: Gallo R C , Mann D , Broder S , Ruscetti F W , Maeda M , Kalyanaraman V S , Robert-Guroff M , Reitz M S
Published: May 2006

A human type C retrovirus, designated HTLV, previously was isolated from or identified in some patients with leukemias and lymphomas of mature T lymphocytes. HTLV is genetically and serologically distinct from any known animal retroviruses. The absence of HTLV proviral sequences in DNA from normal humans showed that HTLV is not a ubiquitous endogenous (germ-line transmitted) virus of humans. Antibodies to HTLV core proteins have been identified in some people with T-cell neoplasias and are particularly prevalent in Japanese with adult T-cell leukemia, suggesting that HTLV is acquired horizontally. However, it was possible that HTLV is transmitted through the germ line of some (possibly rare) families and is then expressed in the HTLV- positive malignancies. An opportunity to study this question was provided by the development of several T-cell lines and a B-cell provided by the development of several T-cell lines and a B-cell line from one HTLV-positive patient with a cutaneous T-cell lymphoma. Here we report that HTLV proteins or nucleic acids (or both) are found in three independently derived T-cell lines, all shown by HLA typing to have originated from the patient. In contrast, the B-cell line, the identity of which was also ascertained by HLA typing, contained no detectable HTLV protein, RNA, or proviral DNA. Because the sensitivity of the latter assay is more than sufficient to detect one proviral equivalent per haploid genome, the results indicate that HTLV was not transmitted to this patient through the germ line but rather was acquired by infection.
Mapping of polypeptides encoded by the Epstein-Barr virus genome in productive infection.
Authors: Hummel M , Kieff E
Published: May 2006

Over 30 viral-specified polypeptides are translated in vitro from RNA of cells productively infected with Epstein-Barr virus (EBV). The polypeptides map to sites in EBV DNA by hybrid selection. Almost all of the polypeptides are reactive with EBV immune human serum. Several of the polypeptides are part of the early antigen complex. Two others are likely to be major structural components of the virus. Genes encoding persistent early and late polypeptides are intermixed through most of the EBV genome.
Two forms of murine epidermal growth factor: rapid separation by using reverse-phase HPLC.
Authors: Burgess A W , Knesel J , Sparrow L G , Nicola N A , Nice E C
Published: May 2006

Epidermal growth factor (EGF) has been isolated from acid extracts of C57BL6/J mouse submaxillary glands by using hydrophobic chromatography. High yields of EGF in large amounts (10 mg) can be isolated reliably from the acid extract of the glands in less than 4 hr. The reverse-phase HPLC techniques used to purify the EGF initially yielded what appeared to be a single homogeneous EGF molecule. However, ion pairing reagents (e.g., heptafluorobutyric acid) altered the chromatographic properties, revealing two distinct species: EGF-alpha and EGF-beta. The apparent molecular weights, isoelectric points, and antigenic properties of EGF-alpha and EGF-beta were identical, and both forms stimulated a mitogenic response in 3T3 cells. Analysis of different preparations of purified EGF (commercial and experimental) indicated the presence of EGF-alpha and EGF-beta in constant proportion. Previous EGF binding studies must have used mixtures of 125I-labeled EGF-alpha and 125I-labeled EGF-beta. The two molecules appear to compete for an identical receptor on the cell surface.
Modulation by calcium of the potassium permeability of dog heart sarcolemmal vesicles.
Authors: Caroni P , Carafoli E
Published: May 2006

The movement of K+ in heart sarcolemmal vesicles has been followed through the opposing movement of the tetraphenylphosphonium ion. Ca2+ (5-50 microM) stimulates the efflux of K+ from K+-loaded vesicles [Km(Ca2+) approximately equal to 10 microM]. and the activation requires that Ca2+ be present inside the vesicles together with K+. The efflux of 86Rb+ from K+-, Rb+-loaded vesicles is similarly stimulated by 5-50 microM Ca2+ [Km(Ca2+) approximately equal to 10 microM]. The Ca2+-induced increase of K+ permeability does not become spontaneously inactivated. The effects of some inhibitors have been tested under conditions in which Ca2+ promotes the entry of K+ into vesicles. In this system, direct interaction of the drug with the Ca2+ and K+ membrane binding site(s) was therefore studied. Tetraethylammonium ion, which inhibits the potential-dependent K+ channel, does not interfere with the effect of Ca2+ whereas quinidine (IC50 = 12 microM) and trifluoperazine (IC50 = 8 microM at 50 micrograms of sarcolemmal protein per ml) inhibit.
Conjugation of ubiquitin to denatured hemoglobin is proportional to the rate of hemoglobin degradation in HeLa cells.
Authors: Chin D T , Kuehl L , Rechsteiner M
Published: May 2006

Ubiquitin was radioiodinated and introduced into HeLa cells by the erythrocyte-mediated fusion procedure. Fractionation of injected HeLa cells and subsequent NaDodSO4/polyacrylamide gel electrophoresis showed that HeLa nuclei contained two major labeled proteins: ubiquitin and the histone H2A-ubiquitin conjugate, protein A24. HeLa cytosol contained ubiquitin and a series of ubiquitin-protein conjugates of diverse molecular weights. When injected HeLa cells were treated with phenylhydrazine to denature the cotransferred hemoglobin, a series of prominent ubiquitin-globin conjugates appeared. The identity of these conjugates was established by microinjection experiments in which both proteins were labeled. At low doses of phenylhydrazine, the intracellular concentration of globin-ubiquitin conjugates was proportional to the rate of hemoglobin degradation. This result, together with the observation that ubiquitin conjugation to globin is markedly enhanced by phenylhydrazine-induced denaturation of hemoglobin, provides support for the hypothesis that the covalent attachment of ubiquitin to proteins signals proteolysis.
Nonrandom distribution of repeated DNA sequences with respect to supercoiled loops and the nuclear matrix.
Authors: Small D , Nelkin B , Vogelstein B
Published: May 2006

The DNA in a eukaryotic nucleus is arranged into a series of supercoiled loops that are anchored at their bases to the nuclear matrix. We have analyzed the DNA sequences that are closest to the matrix attachment points for their relative content of specific repeated sequences. Sequences were enriched (mouse satellite, human Alu family) or depleted (mouse EcoRI repeat, monkey alpha component), depending on the specific sequence and species examined. These results can be understood in terms of a nonrandom arrangement of DNA sequences with respect to nuclear DNA loops.
Message-specific sequestration of maternal histone mRNA in the sea urchin egg.
Authors: Showman R M , Wells D E , Anstrom J , Hursh D A , Raff R A
Published: May 2006

Nucleate and anucleate fragments of sea urchin eggs were prepared by centrifugation on sucrose step gradients. The amount of total RNA, poly(A)+ RNA, histone mRNA, actin mRNA, alpha-tubulin mRNA, and mitochondrial rRNA was determined for each fragment. Total RNA, poly(A)+ RNA, actin mRNA, and alpha-tubulin mRNA all distributed in the same ratio as the volume of the fragments. In contrast, the mitochondrial rRNA was found preferentially distributed in the anucleate fragments, coinciding with the distribution of the mitochondria. Histone mRNAs did not follow the fragment volume ratios, but rather were always found associated with the fragment containing the nucleus. To distinguish between nuclear association and possible artifacts associated with centrifugation, eggs were manually cut into nucleate and anucleate fragments and the amount of histone mRNA was determined for each set. Again only the fragments containing the nucleus had detectable amounts of histone mRNA. Although histone mRNAs were always associated with the nucleate fragment, very little histone mRNA was found associated with isolated egg nuclei prepared under gentle isotonic isolation conditions. Furthermore, embryos that have had first nuclear breakdown blocked with 6-dimethylaminopurine still initiated the recruitment of histone mRNAs into polysomes at the same time as control embryos, thus indicating that nuclear breakdown is not necessary for normal histone message utilization. These results demonstrate a message-specific sequestration of maternal histone mRNA which is physically different from that of other maternal mRNAs and which may govern the timing of maternal histone synthesis in sea urchin embryos.
Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors.
Authors: Hartman J R , Nayak D P , Fareed G C
Published: May 2006

We have cloned and expressed the hemagglutinin (HA) gene of a human influenza virus (A/WSN/33) in monkey kidney cells by linking it to deleted simian virus 40 (SV40) genomes that contain the entire early gene region, the origin of replication, and late leader sequences. The HA gene (1775 base pairs long) was originally inserted by the dG . dC tailing technique into the multicopy plasmid of Escherichia coli, pBR322, using cDNA made from viral RNA. The cloned gene was further modified by treatment with nuclease Bal 31 to remove the dG . dC tails and some of the untranslated sequences and recloned in E. coli after addition of BamHI restriction endonuclease linkers. A number of SV40 and HA recombinants (SV--HA) were constructed by inserting recloned HA DNA into the late gene region of SV40. The SV--HA recombinants, when complemented in a lytic infection of monkey cells by the helper function of SV40 early deletion mutants expressed influenza HA as detected by immunofluorescence and immunoprecipitation of in vivo-labeled proteins using either heterogeneous anti-influenza rabbit antibodies or monoclonal antibodies against HA. Furthermore, the WSN HA expressed by the SV--HA recombinants was also glycosylated and possessed the same molecular weight (approximately 70,000) as the uncleaved HA of WSN virus in monkey cells.
Chemoelectronic mobilization of chemical species in low-conductivity fluids: new electrokinetic effect.
Authors: Haber N
Published: May 2006

An electrokinetic phenomenon is reported here which differs from its classical counterparts most distinctively by nonlinear conductivity and mobility. Neither purely electrolytic nor electrostatic in nature, this phenomenon is presumed to involve subtle charge transfer effects and association reactions permitting a controlled "chemoelectric" mobilization. In its electrokinetic manifestation, this phenomenon can be used to mobilize chemical species commonly with migration rates orders of magnitude greater than can be achieved electrophoretically and is shown to induce the movement of nonpolar molecules, such as aromatic hydrocarbons, at rates exceeding several centimeters per minute in easily achievable voltage gradients. The operational technique, developed as a separations method used for demonstrating the effect, is called "electromolecular propulsion".
Mechanisms of DNA uptake by mammalian cells: fate of exogenously added DNA monitored by the use of fluorescent dyes.
Authors: Loyter A , Scangos G A , Ruddle F H
Published: May 2006

Coprecipitation of DNA with calcium phosphate is a commonly used method of gene transfer in mammalian cells. We have found that DNA forms a tight complex with Ca Pi and that DNA in this complex is resistant to nucleases present in serum or added externally. Under optimal conditions, virtually all of the recipient mouse Ltk- Aprt- cells take up Ca Pi--DNA complexes, as determined by fluorescent dyes specific for DNA (4',6-diaminilo-2-phenylindol dihydrochloride) or for calcium salts (chlorotetracycline). However, only a small proportion of the cells have detectable CA Pi--DNA complexes in the nucleus. Uptake of the Ca Pi--DNA complexes was highly dependent upon the pH at which the Ca Pi--DNA complexes was formed and upon the concentration of DNA in the complex.
Inducer-mediated commitment of murine erythroleukemia cells to differentiation: a multistep process.
Authors: Chen Z , Banks J , Rifkind R A , Marks P A
Published: May 2006

There are a number of agents which, when added to cultures of murine erythroleukemia cells (MELC), markedly increase the probability of commitment to express the characteristics of terminal erythroid differentiation, including loss of proliferative capacity and increased accumulation of globin mRNA and hemoglobin. Some characteristics of inducer-mediated commitment of MELC to terminal erythroid differentiation were examined by determining the effects of dexamethasone (an inhibitor of inducer-mediated MELC differentiation) and of hemin (an inducer of globin mRNA accumulation). Previously, it was shown that exposure of MELC to hexamethylene-bisacetamide (HMBA) leads to commitment, detectable within 12 hr. MELC cultured with both HMBA and dexamethasone do not express commitment. MELC transferred from culture with HMBA and dexamethasone to cloning medium without these agents express commitment to terminal erythroid differentiation, indicating that MELC retain a "memory" for some early HMBA-mediated changes leading to commitment which occur even in the presence of the inhibitory steroid. The kinetics of commitment in experiments in which exposure to HMBA is interrupted, or dexamethasone is added to the culture in HMBA, suggest that there is a rate-limiting step early in the commitment process. The memory for this step persists for more than one cell cycle. Addition of hemin to cultures with HMBA and dexamethasone initiated accumulation of globin mRNA but does not reverse the steroid-mediated inhibition of terminal cell division (that is, the cells retain their proliferative capacity). Inducer-mediated MELC commitment is associated with accumulation of the chromatin protein IP25; dexamethasone does not inhibit this accumulation. Accumulation of IP25 may be inducer-related, but it is not sufficient to cause expression of terminal erythroid differentiation.
Inheritance and expression of lysine plus threonine resistance selected in maize tissue culture
Authors: Hibberd K. A. , Green C. E.
Published: May 2006

Selection for resistance to growth inhibition by lysine plus threonine in equimolar concentrations (LT) in tissue cultures of maize yielded a stable resistant line, LT19. Genetic analysis of progeny of plants regenerated from LT19 showed that LT resistance was inherited as a single dominant nuclear gene, temporarily designated
Ltr
*
-19. Tissue cultures initiated from resistant embryos required 5-10 times higher levels of LT to inhibit growth than did cultures from LT-sensitive embryos. LT resistance in
Ltr
*
-19 was expressed as much reduced inhibition of root and shoot growth in the presence of LT. The free pool of threonine was increased 6 times in cultures initiated from immature embryos of LT-resistant plants, and 75-100 times in kernels homozygous for
Ltr
*
-19, as compared to cultures and kernels from LT-sensitive embryos and plants, respectively. Overproduction of free threonine increased the total threonine content in homozygous
Ltr
*
-19 kernels by 33-59%. The results demonstrate that LT resistance selected with tissue culture methods is heritable and is expressed in cultures, seedlings, and kernels. Furthermore, they demonstrate a method to obtain amino acid-overproducer mutants in maize, which have the potential to increase substantially specific amino acids in kernels. The capability to increase specifically the nutritionally limiting amino acid(s) could have important nutritional implications for the grain of cereals and other crops.
Developmentally regulated interconversions between end product-inhibitable and noninhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by protein dephosphorylation-phosphorylation reactions.
Authors: Frisa P S , Sonneborn D R
Published: May 2006

During the life cycle of Blastocladiella emersonii, dramatic shifts occur in the sensitivity of the first hexosamine biosynthetic pathway-specific enzyme [amidotransferase; 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring), EC 5.3.1.19] to end product inhibition. These shifts are developmentally correlated with changes in the utilization of the end product (uridine-5'-diphospho-N-acetylglucosamine) for chitin synthesis [Selitrennikoff, C. P., Dalley, N. E. & Sonneborn, D. R. (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. Alterations in amidotransferase sensitivity to end product inhibition can be mimicked by in vitro protein dephosphorylation-phosphorylation reactions, as follows: (i) Zoospore end product-inhibitable amidotransferase activity can be converted to a noninhibitable form by an endogenous (zoospore) protein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) reaction; this noninhibitable form can be converted back to an inhibitable form either by an endogenous cAMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) reaction or with an added cAMP-dependent protein kinase. (ii) Noninhibitable amidotransferase activity from growing cells can also be converted to the inhibitable form with added protein kinase.
Microinjection of monoclonal antibody to protein p53 inhibits serum-induced DNA synthesis in 3T3 cells.
Authors: Mercer W E , Nelson D , DeLeo A B , Old L J , Baserga R
Published: May 2006

Monoclonal antibody directed against the transformation-related protein p53 was microinjected manually into the nuclei of quiescent Swiss 3T3 mouse cells. The cells were subsequently stimulated with 10% fetal calf serum. Microinjection of p53 antibody at or around the time of serum stimulation clearly inhibited the subsequent entry of Swiss 3T3 cells into the S phase of the cell cycle. p53 antibody had no effect on serum-stimulated DNA synthesis when it was microinjected 4 hr or later after serum stimulation. Monoclonal antibody to an unrelated antigen, Lyt-2.2, had no effect on serum-stimulated DNA synthesis regardless of the time it was microinjected. Under similar experimental conditions, p53 antibody had no effect on simian virus 40- or adenovirus 2-induced DNA synthesis. These experiments add strength to the suggestion that p53 is involved in the regulation of cell proliferation.
Internal structure of a mitochondrial intron of Aspergillus nidulans.
Authors: Waring R B , Davies R W , Scazzocchio C , Brown T A
Published: May 2006

The intron of the mitochondrial apocytochrome b gene, cobA, of Aspergillus nidulans has been subjected to sequence analysis. It contains an open reading frame of 957 base pairs contiguous with the preceding exon. Regions of the translated open reading frames of cobA and the third intron of the cob gene in yeast show high amino acid homology. Comparison of the cobA intron with this and other yeast introns indicates that cobA codes for a maturase protein that splices out the intron encoding it and possibly other mitochondrial introns. Two very similar decamer peptides are found in the protein sequences of the cobA intron, four mitochondrial yeast introns, and the yeast mitochondrial sequence reading frame 1 (RF-1) and may be diagnostic of one class of maturase-coding introns. Four short DNA sequences, two of which are in the region defined by box9 and box2 mutations in the cob gene of yeast, are conserved in cobA and certain yeast introns. Comparison with three yeast introns strongly suggests that the first 200 base pairs of the open reading frame of the cobA intron do not code for any amino acids present in the putative maturase protein but are required for splicing or the control of splicing, or both.
Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms.
Authors: Watanabe S M , Goodman M F
Published: May 2006

Enzyme kinetic measurements are presented showing that Km rather than maximum velocity (Vmax) discrimination governs the frequency of forming 2-aminopurine X cytosine base mispairs by DNA polymerase alpha. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. We now report that the ratio of Km values KCm/KTm = 25 +/- 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. We also report that VCmax is indistinguishable from VTmax. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides.
Antagonism between steady light and phosphodiesterase inhibitors on the kinetics of rod photoresponses.
Authors: Capovilla M , Cervetto L , Torre V
Published: May 2006

Treatment of toad rods with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, caffeine, theophylline, papaverine, and RO 20-1724) modifies the properties of the intracellular voltage responses to dim flashes of light. 3-Isobutyl-1-methylxanthine at 1-20 microM causes an increase in flash sensitivity and a slowing down of the kinetics of the photoresponses. When the drug concentration is greater than 20 microM, rods also show supralinear behavior, whereby doubling the intensity of a dim flash may increase the response by greater than 2-fold. Sensitivity, kinetics, and supralinear behavior can be restored to normal by steady background illumination while still in the presence of 3-isobutyl-1-methylxanthine. However, the intensity of the steady light needed to restore the sensitivity to control levels is not sufficient to accelerate the kinetics back to control values. The antagonism between the effects of 3-isobutyl-1-methylxanthine and the effects of background illumination is explained by assuming that: (i) the length of time to peak voltage responses to dim flashes of light is inversely proportional to the rate of a chemical reaction; (ii) the rate of this reaction is controlled by an enzyme that is inhibited competitively by 3-isobutyl-1-methylxanthine with a Ki of 3 x 10(-6) M; and (iii) the concentration of a cofactor of this reaction increases proportionally with the intensity of the background illumination.
The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.
Authors: Gorman C M , Merlino G T , Willingham M C , Pastan I , Howard B H
Published: May 2006

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.
Isolation and characterization of cDNA clones for human apolipoprotein A-I.
Authors: Breslow J L , Ross D , McPherson J , Williams H , Kurnit D , Nussbaum A L , Karathanasis S K , Zannis V I
Published: May 2006

We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states.
Unifying concept for the coupling between ion pumping and ATP hydrolysis or synthesis.
Authors: Hammes G G
Published: May 2006

A mechanism is proposed for the coupling between ion transport and enzyme catalysis. The basic concept is that enzymes associated with transport exist in two possible conformations. Each conformation has the potential of catalyzing the enzymatic reaction, and pumping is associated with the conversion of one conformational form to the other. The conformational transition is triggered by the kinetic blockage of specific mechanistic steps for each conformation. Such blockages can cause a cycling between the two conformations concomitant with catalysis. This mechanistic concept is consistent with a variety of results obtained with the Na+,K+-ATPase, the Ca2+,Mg2+-ATPase, and ATP synthesizing enzymes (coupling factors).
Affinity chromatography of human platelet α
2
-adrenergic receptors
Authors: Regan John W. , Barden Nicholas , Lefkowitz Robert J. , Caron Marc G. , DeMarinis Robert M. , Krog Arnold J. , Holden Kenneth G. , Matthews William D. , Hieble J. Paul
Published: May 2006

Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving α
2
-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with α
2
-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing α
2
-adrenergic receptors, can be specifically labeled with [
3
H]yohimbine and can be solubilized with digitonin without loss of their α
2
-adrenergic binding characteristics. Chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in the adsorption of 70-80% of the initial [
3
H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both α-adrenergic antagonists (phentolamine ≥ yohimbine > prazosin) and by α-adrenergic agonists [
p
-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [
3
H]yohimbine binding activity from the affinity gel is effected with the aforementioned agonists and antagonists in the same order of potency. Other drugs that do not interact appreciably with α-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [
3
H]yohimbine binding activity from the affinity gel. In addition to the specificity of the interaction, chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in a 40-50% overall yield and an approximately 200-fold increase in the specific binding activity for [
3
H]yohimbine. The results indicate that the SKF 101253-Sepharose CL-4B affinity adsorbent should provide a powerful tool for the purification of the adenylate cyclase-inhibitory α
2
-adrenergic receptor of human platelets.
Identification of the origins of T4 DNA replication.
Authors: King G J , Huang W M
Published: May 2006

Two physical origins of T4 DNA replication were determined by hybridization of viral DNA prepared 2.5 min after infection to a display of total T4 DNA. This is the earliest time after T4 infection of Escherichia coli at 37 degrees C that labeled and hybridizable DNA can be detected. The two origins, separated by about 25 kilobases, were identified and localized in the early region of the T4 map. One of them is located in a 5.6-kilobase EcoRI fragment containing genes 62-46. The other is located between genes rI and e in a 1.9-kilobase EcoRI fragment. Both of these T4 fragments have been cloned and their interactions with the host cell are discussed.
Radical pair state in photosystem II
Authors: Rutherford A. W. , Thurnauer M. C.
Published: May 2006

A stable light-induced EPR signal is reported in photosystem II particles and in chloroplasts at 5 K. Characteristic spectral features indicate that the signal arises from dipole-dipole interactions of a radical pair triplet state. From its dependence on potential, its relationship to the spin-polarized triplet state, and the redox state of the pheophytin acceptor (Ph) and because it is present in Tris-washed chloroplasts but not in untreated chloroplasts, we conclude that the signal is formed when the reaction center is in the state D
+
P
680
Ph
-
(P
680
is the primary chlorophyll donor and D
+
is an oxidized donor to P
680
). The low-temperature photochemical sequence is thought to occur as follows. (
i
) Donation from D to the P
680
+
Ph
-
state occurs at liquid helium temperature in low quantum yield; this reaction is reversible at temperatures above 5 K. (
ii
) In normal chloroplasts, donation occurs to the D
+
P
680
Ph
-
state, but this does not occur in Tris-washed chloroplasts or in the photosystem II particles at 77 K or lower. (
iii
) Illumination, at 200 K, of photosystem particles or Tris-washed chloroplasts results in donation to the D
+
P
680
Ph
-
state from another donor. From experiments in the absence of redox mediators and the temperatures dependence of the splitting of the signal, it is suggested that the state D
+
P
680
Ph
-
itself may be the origin of the radical pair triplet signal. The signal has been simulated by assuming the presence of at least two distinct radical pairs that differ slightly in the distance separating the radicals of the pairs. The distance between the radicals of the pair is calculated to be 6-7 Å.
Structural genes on the Y chromosome of Drosophila melanogaster.
Authors: Goldstein L S , Hardy R W , Lindsley D L
Published: May 2006

Testis proteins of Drosophila melanogaster deficient for six different Y-chromosome regions were fractionated by means of a sodium dodecyl sulfate/polyacrylamide gel system designed to separate high molecular weight polypeptides (Mr, greater than 200,000). Analysis of the banding patterns indicates that the three regions containing fertility genes kl-2, kl-3, and kl-5 are responsible for three different high molecular weight polypeptides. Several observations indicate that these polypeptides are structural components of the sperm axoneme. They are present in seminal vesicles, which are highly enriched for mature sperm. They are first detected during development at a time when the first spermatids are elongating. Finally, deletion of either kl-5 or kl-3 leads to the absence of the outer dynein arm of the peripheral doublets of the axoneme. Although absence of the kl-2 region eliminates the third polypeptide, an associated structural defect in the axoneme has yet to be identified. The three polypeptides are in the Mr 300,000-350,000 range, and their mobilities are similar to those of dynein polypeptides from Chlamydomonas axonemes. Experiments using dosage variation and a temperature-sensitive sterile mutation in kl-5 suggest that the Y-chromosome regions contain the coding sequences for the polypeptides.
A new Salmonella tester strain (TA102) with A X T base pairs at the site of mutation detects oxidative mutagens.
Authors: Levin D E , Hollstein M , Christman M F , Schwiers E A , Ames B N
Published: May 2006

A new tester strain, TA102, is described as an addition to the set of strains for the Salmonella/microsome mutagenicity test. This strain contains A X T base pairs at the site of the mutation (determined by DNA sequence analysis) in contrast to the other Salmonella tester strains that detect mutagens damaging G X C base pairs. This strain differs from previous tester strains in that the mutation has been introduced into a multicopy plasmid, so that approximately equal to 30 copies of the mutant gene are available for back mutation. The new strain detects a variety of oxidative mutagens, including x-rays, bleomycin, hydrogen peroxide and other hydroperoxides, streptonigrin and other quinones, and phenylhydrazine; a variety of aldehydes, including formaldehyde, glyoxal, kethoxal, glutaraldehyde, and malondialdehyde; a number of psoralens (in the presence of near-UV light), mitomycin C, neocarzinostatin, and UV light. Some of these mutagens have been previously shown to damage thymine in DNA. Several auxiliary tester strains also are described, including TA96, a frameshift tester strain with a hot spot for mutation at a run of five A X T base pairs with a specificity similar to that of TA102. The importance of oxidative mutagens is discussed.
Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3.
Authors: Sanchez-Madrid F , Krensky A M , Ware C F , Robbins E , Strominger J L , Burakoff S J , Springer T A
Published: May 2006

Monoclonal antibodies were prepared to anti-HLA-DR cytolytic T lymphocytes (CTLs) and screened for inhibition of CTL-mediated killing. Binding of monoclonal antibodies to four types of molecules, LFA-1, LFA-2, LFA-3, and HLA-DR, inhibited killing, suggesting that these molecules participate in the CTL-target cell interaction. The antigens were characterized by immunoprecipitation, crosslinking, NaDodSO4/polyacrylamide gel electrophoresis, and immunofluorescence flow cytometry. The LFA-1 antigen contains alpha and beta polypeptide chains of Mr 177,000 and 95,000 that are noncovalently associated in an alpha 1 beta 1 structure. It is present on both B and T lymphocytes and marks subpopulations that differ in quantitative expression. Human LFA-1 appears to be the homologue of mouse LFA-1. Human LFA-2 is of Mr 49,000 with a minor component of Mr 36,000. It is expressed on CTL lines but not on a B-cell line and in peripheral blood preferentially on T lymphocytes. Human LFA-3 is of Mr 60,000 and is expressed on both B and T lymphocytes.