bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat.
Authors: Grandchamp B , Romeo P H , Dubart A , Raich N , Rosa J , Nordmann Y , Goossens M
Published: May 2006

A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.
Crown gall oncogenesis: evidence that a T-DNA gene from the Agrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid.
Authors: Thomashow L S , Reeves S , Thomashow M F
Published: May 2006

Stable incorporation of tumor-inducing (Ti) plasmid sequences, the T-DNA, into the genomes of dicotyledonous plants results in the formation of crown gall tumors. Previous genetic studies have suggested that the products of the genes encoding transcripts 1 and 2, which are encoded by the TL-DNA region of pTiA6, are responsible for inducing the auxin-independent phenotype of crown gall tissues. Here we report the construction of a plasmid, pMTlacT2, which directs the synthesis of the Mr 49,800 polypeptide encoded by the transcript 2 gene. Cell-free extracts prepared from Escherichia coli harboring this plasmid converted indoleacetamide to indoleacetic acid, the natural auxin of plants; extracts prepared from plasmidless strains of E. coli or strains harboring the cloning vehicle pBR322 did not carry out this reaction. We conclude that the transcript 2 gene of pTiA6 codes for an enzyme that participates in auxin biosynthesis, probably an indoleacetamide hydrolase.
Squid rhodopsin and GTP-binding protein crossreact with vertebrate photoreceptor enzymes.
Authors: Saibil H R , Michel-Villaz M
Published: May 2006

The activation of photoreceptor GTP-binding protein by rhodopsin was studied in squid photoreceptors and in crossreactions between the squid and bovine proteins. Turbidity changes were observed in the far-red after photoexcitation of rhodopsin with brief flashes and were used to probe interactions between photoreceptor membrane suspensions and soluble protein extracts. Our findings are squid photoreceptors contain a GTP-binding protein detectable by light- and GTP-sensitive turbidity changes and by limited sequence homology of a 46-kilodalton polypeptide to the alpha-subunit of bovine GTP-binding protein; the squid membranes activate bovine GTP-binding protein qualitatively in the same way as bovine rhodopsin; the 46-kilodalton component is present in a membrane-bound fraction but is more abundant in a crude, soluble fraction of squid rhabdomes, and this soluble fraction can interact with either squid or bovine rhodopsin-containing membranes; light-activated GTPase activities in all of these preparations are consistent with the light-induced turbidity changes. These results show that rhodopsin activation of GTP-binding protein is highly conserved in vertebrate and cephalopod photoreceptors. Since squid rhodopsin is immobilized in precisely ordered microvilli, this suggests that activation of GTP-binding protein in cephalopod photoreceptors occurs in the absence of rhodopsin diffusion. The rhodopsin immobility may be compensated by higher mobility of the soluble GTP-binding protein.
Seasonal aggression independent of seasonal testosterone in wood rats.
Authors: Caldwell G S , Glickman S E , Smith E R
Published: May 2006

Levels of inter-male aggression, both in laboratory encounters and in the field, rise dramatically during the breeding season, closely paralleling the seasonal rise in testosterone. However, post-pubertally castrated males also show the dramatic seasonal rise in aggression in laboratory encounters with castrated opponents and show no decrement in fighting ability when paired with intact opponents, clearly demonstrating the independence of seasonal aggression from the proximate modulating effects of testosterone in wood rats.
Distinctive sequence organization and functional programming of an Alu repeat promoter.
Authors: Perez-Stable C , Ayres T M , Shen C K
Published: May 2006

Plasmid clones containing a human Alu family repeat can be transcribed efficiently by RNA polymerase III in HeLa cell extract. This generated three RNA species, all of which initiated from the first base (+1) of the repeat. By studying the transcriptional properties of deletion clones, subclones, and topologically different DNA templates, we demonstrated that: supercoiled DNA templates are transcribed 3- to 5-fold more efficiently than are linear or nicked circular DNA molecules; a contiguous DNA helix in the transcription complexes that extends into the 5' flanking region of positions -30 to -85 is absolutely required for initiation to occur (this interaction does not involve recognition of specific DNA sequences); and similar to the adenovirus VAI RNA and tRNA genes, the Alu repeat 3' to the alpha 1-globin gene (designated 3'-alpha 1 Alu) contains a split intragenic promoter: an anterior element (positions +4 to +37) and a posterior element (positions +70 to +82). However, the promoter of the Alu repeat functions in distinctive ways in comparison to those of other RNA polymerase III-dependent genes. The posterior promoter element alone is sufficient and necessary for an accurate initiation to occur. The presence of the anterior promoter element, which by itself does not initiate transcription, enhances the transcriptional efficiency by a factor of 10- to 20-fold. Furthermore, the distance between the initiation sites and the posterior promoter element, but not the anterior promoter element, remains constant. These results suggest that the promoter of this Alu family repeat consists of at least two functionally different domains: a "directing element" (the posterior promoter element) that determines the accuracy of initiation and an "enhancing element" (the anterior promoter element) that is mainly responsible for the transcriptional efficiency.
Characterization and purification of a phage phi 29-encoded DNA polymerase required for the initiation of replication.
Authors: Blanco L , Salas M
Published: May 2006

The phage phi 29 protein p2, required for the formation of the protein p3-dAMP initiation complex, has been purified from Escherichia coli cells harboring a gene 2-containing recombinant plasmid. The purified protein p2, of molecular weight 68,000, had a specific DNA polymerase activity that elongated the p3-dAMP initiation complex when phi 29 DNA-protein p3 was used as template. In addition, the purified protein p2 was active in catalyzing the initiation reaction when complemented with phi 29 mutant sus2-infected Bacillus subtilis or plasmid-containing E. coli extracts providing protein p3, in the presence of phi 29 DNA-protein p3 as template. However, when purified protein p3 was used in the complementation assay, a very low amount of initiation complex was formed; addition of extracts from uninfected B. subtilis or E. coli strongly stimulated the initiation reaction, indicating that, in addition to proteins p2 and p3 and the phi 29 DNA-protein p3 template, some host factor(s) is required for the formation of the p3-dAMP initiation complex. The results show that phage phi 29 encodes a DNA polymerase that is required at the initiation step of protein-primed DNA synthesis.
Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.
Authors: Hellerman J G , Cone R C , Potts J T , Rich A , Mulligan R C , Kronenberg H M
Published: May 2006

In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recombinant retrovirus encoding preproPTH, and this recombinant retrovirus was used to infect GH4 rat pituitary cells. Clonal lines of the infected GH4 cells contained copies of the recombinant provirus stably integrated via the long terminal repeats, and the expected RNA transcripts of proviral DNA accumulated in the cytoplasm, although no infectious virus was produced. The infected cells synthesized and processed preproPTH appropriately and secreted PTH in response to thyrotropin-releasing hormone, a secretagogue for GH4 cells. Use of recombinant retrovirus permits the introduction of DNA encoding normal and mutant secreted proteins into a number of cell types specialized for secretion. Analysis of the fate of the resultant proteins will help define the specific molecular interactions involved in transmembrane transport and processing of precursor proteins.
Synthesis and biological activity of human inhibin-like peptide-(1-31).
Authors: Yamashiro D , Li C H , Ramasharma K , Sairam M R
Published: May 2006

A peptide containing 31 amino acids with inhibin-like activity has been synthesized by the solid-phase method. The synthetic product was shown to be homogeneous in high-performance liquid chromatography and paper electrophoresis. In the mouse pituitary incubation system, the follitropin-suppressing activity of the synthetic peptide was comparable to that of the natural product. It was also active in suppressing lutropin-releasing hormone-induced follitropin release in vivo in immature male rats.
Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli.
Authors: Schoner B E , Hsiung H M , Belagaje R M , Mayne N G , Schoner R G
Published: May 2006

The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escherichia coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon and either the E. coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene. The expression of Met-bGH was low with either system. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation.
Magnetic susceptibility of oxy- and carbonmonoxyhemoglobins.
Authors: Savicki J P , Lang G , Ikeda-Saito M
Published: May 2006

The room temperature magnetic susceptibilities of human and carp oxy- and carbonmonoxyhemoglobin solutions were measured in a 30-gauss (1 G = 10(-4) T) field with a superconducting magnetometer. To within experimental uncertainty, the susceptibility was the same for both the oxy and carbonmonoxy forms, and salt concentration did not effect it. A variety of sample preparations were used; the iron chemical state was verified by Mössbauer spectroscopy. A value of -0.580 +/- 0.010 X 10(-6) centimeter-gram-second (cgs) system was obtained for the mass susceptibility of the protein. We attribute the paramagnetism sometimes observed in oxyhemoglobin solutions to the presence of a small amount of the deoxy form.
In vitro transcription of a silkworm 5S RNA gene requires an upstream signal.
Authors: Morton D G , Sprague K U
Published: May 2006

The RNA polymerase III transcription complex from Bombyx mori silkworms, in contrast to that from Xenopus frogs, requires a signal that is upstream from the transcription initiation site. We have found that part of the normal 5' flanking sequence is essential for transcription of Bombyx 5S RNA genes by homologous cell-free extracts. The critical region is structurally similar to the upstream signal that directs Bombyx tRNA gene transcription. The association of such a control element with two classes of polymerase III templates argues that it acts through a general component of the Bombyx transcription apparatus.
Calcium channel activation: a different type of drug action.
Authors: Freedman S B , Miller R J
Published: May 2006

Depolarization of NG108-15 (neuroblastoma-glioma) cells causes an increase in 45Ca2+ influx. This effect is blocked by low concentrations of dihydropyridines such as nitrendipine and by other blockers of voltage-sensitive calcium channels such as D-600, diltiazem, and Cd2+. Two other dihydropyridines, BAY K8644 and CGP 28392, have the opposite effect. Low concentrations of these compounds enhance depolarization-induced 45Ca2+ influxes. BAY K8644 is more effective than CGP 28392. Both agents have no effect on fluxes measured under nondepolarizing conditions. The effects of BAY K8644 and CGP 28392 can be inhibited by nitrendipine, D-600, diltiazem, or Cd2+. Whereas the interaction between nitrendipine and BAY K8644 is shown to be competitive in nature, that between BAY K8644 and D-600 is shown to be noncompetitive. These results indicate that dihydropyridines show a variety of effects on calcium channels, ranging from agonistic through partially agonistic to antagonistic. Moreover, the results also indicate that dihydropyridines and D-600 exert their effects on calcium channels at different sites.
Single-channel properties of the reconstituted voltage-regulated Na channel isolated from the electroplax of Electrophorus electricus.
Authors: Rosenberg R L , Tomiko S A , Agnew W S
Published: May 2006

The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (approximately equal to 11 pS in 95 mM Na+), mean open times of approximately equal to 1.9 msec, and 7-fold selectively for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of approximately equal to 28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel.
Elicitor rapidly induces chalcone synthase mRNA in
Phaseolus vulgaris
cells at the onset of the phytoalexin defense response
Authors: Ryder Thomas B. , Cramer Carole L. , Bell John N. , Robbins Mark P. , Dixon Richard A. , Lamb Chris J.
Published: May 2006

DNAs complementary to poly(A)
+
RNA present in elicitor-treated cells of
Phaseolus vulgaris
L. were inserted into pBR325 and used to transform
Escherichia coli
strain JA221. A clone was identified that contained sequences complementary to mRNA encoding chalcone synthase, a regulatory enzyme of phenylpropanoid biosynthesis, which catalyzes the first reaction of a branch pathway specific to flavonoid and isoflavonoid biosynthesis. Rapid, marked but transient increases in chalcone synthase mRNA in response to elicitor treatment were observed by RNA blot hybridization with
32
P-labeled chalcone synthase cDNA sequences. Induction of chalcone synthase mRNA governs the rate of enzyme synthesis throughout the phase of rapid increase in enzyme activity at the onset of accumulation of isoflavonoid-derived phytoalexins. The data are consistent with the hypothesis that elicitor causes a rapid transient stimulation of transcription of chalcone synthase gene(s) as an early event in the expression of the phytoalexin defense response.
Differential adhesion of tumor cells to capillary endothelial cells in vitro.
Authors: Alby L , Auerbach R
Published: May 2006

Adhesion studies were carried out to determine the relative ability of glioma cells and ovary-derived teratoma cells to adhere to endothelial cells obtained from mouse brain capillaries (designated MBE cell line) or mouse ovaries (designated MOE cell line). The teratoma cells showed preferential adhesion to MOE cells, whereas the glioma cells showed preferential adhesion to the MBE cell line. In contrast, the glioma and teratoma cells adhered equally to L929 and 3T3 fibroblasts. A testicular teratoma with ovary-seeking properties in vivo also adhered preferentially to MOE cells, while the preference for MBE cells was shared by glioma cells with an endothelioma and a bladder tumor line. The endothelioma, interestingly, showed a marked preferential adhesion to 3T3 cells, thus distinguishing it from the glioma. The experiments demonstrate that capillary endothelial cells derived from different sources are not alike and that differences expressed at the cell surface of these cells can be distinguished by tumor cells.
Injection of partially purified estrogen receptor protein from Xenopus liver nuclei into oocytes activates the silent vitellogenin locus.
Authors: Knowland J , Theulaz I , Wright C V , Wahli W
Published: May 2006

Injection of extracts from Xenopus liver nuclei that are enriched 2000 times in estradiol receptor into Xenopus oocytes induces transcription of the silent vitellogenin locus, which is activated in liver by estradiol, but not of the albumin locus, which is active in liver but suppressed by high levels of estradiol. Transcription initiates within the 5'-end region of the gene we have studied and probably continues into the 3' third. The activation seems to be very efficient, but most of the primary transcripts are probably rapidly and inaccurately processed. New proteins are also made and secreted by the oocytes.
Influence of early or late dietary restriction on life span and immunological parameters in MRL/Mp-lpr/lpr mice.
Authors: Kubo C , Day N K , Good R A
Published: May 2006

Reduced food intake doubles and even triples the life span of (NZB X NZW)F1 (B/W) mice and greatly influences of food intake while keeping vitamin and mineral intake constant in mice of the MRL/Mp-lpr/lpr (MRL/l) strain. Restriction of food intake greatly prolongs life. This influence also was seen when dietary restriction was imposed later in life. Dietary restriction inhibited development of lymphoproliferative disease and greatly decreased the numbers of cells in thymus, lymph nodes, and spleen. It also delayed development of glomerulonephritis and maintained certain immunological responses. Proliferative responses to phytohemagglutinin, pokeweed mitogen, or allogeneic spleen cells were maintained in the mice fed a low-calorie diet from 6 wk. Imposing diet at 12 wk had a lesser influence than earlier restriction. These dietary influences did not depress formation of anti-DNA antibodies or circulating immunocomplexes. MRL/l mice show an apparently extremely low production of interleukin 2, and dietary restriction increased the capacity of lymph node cells but not spleen cells to produce this immunomodulator.
Facilitation of Ca2+-channel currents in bovine adrenal chromaffin cells.
Authors: Hoshi T , Rothlein J , Smith S J
Published: May 2006

Ca2+-channel currents in primary cultures of bovine adrenal chromaffin cells were studied using the whole-cell patch-clamp method. Parameters of a double-pulse protocol were systematically varied to characterize facilitation by a prepulse (P1) of Ca2+-channel current during a test pulse (P2). The pulses were usually separated by 30 msec, an interval sufficient for decay of any measurable P1 tail currents. The Ca2+-channel current amplitude during P2 increased when P1 voltage was more positive than 0 mV. The effect became progressively greater with more positive P1 voltage. With a 60-msec P1 to +80 mV, the current amplitude typically increased by 25%-35% during a 60-msec P2. Comparison of facilitated and control inward Ca2+-channel current I(V) curves showed that facilitation was also strongly dependent on P2 test voltage. Facilitation of Ca2+-channel currents is a voltage-dependent phenomenon and is not dependent on Ca2+ entry. When short repetitive voltage-clamp pulses were applied, the Ca2+-channel current amplitude increased with each pulse. This suggests that Ca2+-channel facilitation could enhance release of catecholamines from chromaffin cells during a train of action potentials.
In vitro reprogramming of the photoperiodic clock in an insect brain-retrocerebral complex.
Authors: Bowen M F , Saunders D S , Bollenbacher W E , Gilbert L I
Published: May 2006

Pupal diapause in the tobacco hornworm Manduca sexta is a photoperiodically induced event expressed by the absence of release of the cerebral peptide prothoracicotropic hormone. A short-day photoperiod (light-dark, 12:12) during larval development programs the brain to forego the release of prothoracicotropic hormone in the pupal stage. Prothoracicotropic hormone release can occur in the pupae and diapause can be averted if developing larvae experience an intervening long-day photoperiod (light-dark, 16:8) for 3 days early in the last larval instar. Implanted brains from larvae that received such long-day stimulation can reverse the diapause commitment of short-day-reared diapause-destined larvae, thus overriding the diapause program of the recipient. A system was established that demonstrated that this photoperiodic reprogramming of the insect's brain occurs in vitro. Brain-retrocerebral complexes from day 1 instar larvae were cultured in vitro for 3 days under long-day conditions. The effect of this non-diapause photoperiod on the diapause program of these brain-retrocerebral complexes was assessed by assaying the effect of the brains from these cultures on the incidence of non-diapause development in diapause-destined larval hosts. Brains that received a long-day photoperiod reversed the pupal diapause program of the recipient larvae, whereas brains that received a short-day photoperiod were ineffective. Therefore, the larval brain-retrocerebral complex of this insect is capable of photoperiodic light reception in vitro, in that it can sense the number of light-dark cycles to which it is exposed, store this environmental input, and express the information 9 days later at another stage of development. These findings demonstrate that the photoperiodic clock of an animal can be reprogrammed by a light-dark cycle in vitro.
Specific absorption with monoclonal antibodies to muramyl dipeptide of the pyrogenic and somnogenic activities of rabbit monokine.
Authors: Chedid L , Bahr G M , Riveau G , Krueger J M
Published: May 2006

It is well established that muramyl dipeptide (MDP) can induce fever and enhance slow-wave sleep. Recently, crude or purified supernatants of activated macrophages containing endogenous pyrogen (EP) were also shown to enhance slow-wave sleep. These similarities and the recent finding that a mammalian factor that enhances slow-wave sleep is a muramyl peptide triggered us to study the possibility of the presence of this bacterial structure in the EP molecule. In the present study, EP was produced by stimulation of rabbit peritoneal cells with a nonpyrogenic, nonsomnogenic analog of MDP. The EP-containing supernatant lost its pyrogenicity and somnogenicity after passage over an immunoadsorbent column of monoclonal anti-MDP but not of another monoclonal antibody of different specificity. High percentage of the EP was recovered by elution of the anti-MDP columns with HCl/glycine buffer. Results suggest that bacterial muramyl peptides may be incorporated by mammalian cells into substances that act in picomole quantities to mediate immunological and physiological processes. In addition, the technique may be useful to extract interleukin 1 for structural studies.
In vitro regulation of phage lambda cII gene expression by Escherichia coli integration host factor.
Authors: Peacock S , Weissbach H , Nash H A
Published: May 2006

The effect of Escherichia coli integration host factor (IHF) on phage lambda gene expression has been examined in a simplified DNA-directed in vitro system that measures the formation of the first dipeptide of the gene product. Plasmid pKC30cII, which contains the phage lambda genes N, cII and O, under control of the PL promoter, was used as template to study the expression of the first dipeptide of the gene products--i.e., fMet-Asp for N protein, fMet-Val for cII, and fMet-Thr for O. Purified IHF stimulates the DNA-directed synthesis of fMet-Val (cII) and fMet-Thr (O) 2-3-fold but has no effect on the synthesis of fMet-Asp (N). In this in vitro system, the stimulation by IHF of cII and O gene expression is at the level of transcription. Phage lambda repressor completely inhibits dipeptide synthesis in the presence or absence of IHF. The results are consistent with a role of IHF as a transcription antiterminator, perhaps functioning at or near the tR1 site preceding the cII gene.
erythro-9-[3-(2-Hydroxynonyl)]adenine is an effective inhibitor of cell motility and actin assembly.
Authors: Schliwa M , Ezzell R M , Euteneuer U
Published: May 2006

erythro-9-[3-(2-Hydroxynonyl)]adenine (EHNA) has been reported previously to be an agent that arrests sperm motility by inhibiting the axonemal dynein ATPase activity and has been used to probe the involvement of putative cytoplasmic dyneins in mitosis and intracellular organelle transport. We report here that EHNA profoundly and reversibly affects several actin-dependent processes, both in vivo and in vitro. It induces dramatic changes in actin organization in cultured cells, inhibits cell translocation, blocks actin-dependent cytoplasmic streaming, interferes with actin-dependent gelation of cytoplasmic extracts, and inhibits actin assembly. Just as the cytochalasins, EHNA appears to be a highly effective inhibitor of actin-based motility, whose effects in complex biological systems should be interpreted with caution.
Group selection for a polygenic behavioral trait: estimating the degree of population subdivision.
Authors: Crow J F , Aoki K
Published: May 2006

For assessing the degree of population subdivision, and therefore the extent to which group selection might favor an altruistic trait, an appropriate measure is Nei's GST, defined by (F0-F)/(1-F). F0 is the probability that two alleles drawn from the same group are identical in state and F is the probability for two alleles drawn at random from the entire population. These probabilities can be assessed from molecular polymorphisms. GST has a number of properties that make it useful for empirical studies. When the mutation rate is small relative to the migration rate and the reciprocal of the group size, GST depends mainly on the absolute number of migrants per generation, moves rapidly to near equilibrium, and is independent of the number of alleles. The relative homogenizing effect of migration in the island and stepping-stone models is not as different as might be expected; one immigrant chosen randomly from the rest of the population is only one to two times as effective as one from a neighboring group, appreciably exceeding 2 only when there are 1000 or more groups. The use of molecular data to estimate the degree of population subdivision may permit testable predictions of the extent of altruistic behavior.
Mn
2+
and Co
2+
toxicity in chlorophyll biosynthesis
Authors: Csatorday K. , Gombos Z. , Szalontai B.
Published: May 2006

The metal ion-induced inhibition of tetrapyrrole biosynthesis was studied in the cyanobacterium
Anacystis nidulans
. The accumulation of protoporphyrin and Mg protoporphyrin due to the effect of Co
2+
and Mn
2+
treatment, respectively, pointed to two different sites of inhibition.
Correction of complex heteroduplexes made of mouse H-2 gene sequences in Escherichia coli K-12.
Authors: Cami B , Chambon P , Kourilsky P
Published: May 2006

We have prepared heteroduplexes between two plasmids that carry, in the same orientation, two H-2 cDNA inserts, 1.15 and 1.0 kilobase long, respectively. Their sequences encode two distinct class I transplantation antigens of the mouse and differ by 8% of their nucleotides. Molecules with a rearranged array of restriction sites were found after transformation and cloning in an Escherichia coli recA- host. Nucleotide sequences showed that the rearranged molecules derived their nucleotides from the two parental strands. Thus, correction of these complex heteroduplexes takes place in E. coli and probably involves repair mechanisms. It provides the basis for a mutational process in which several nucleotides (amino acids) can be altered in a single event. It also offers a practical means of making genetic variants. Several other implications are discussed.
Chromosome assignments of four mouse cellular homologs of sarcoma and leukemia virus oncogenes.
Authors: Sakaguchi A Y , Lalley P A , Zabel B U , Ellis R W , Scolnick E M , Naylor S L
Published: May 2006

Molecular probes for the oncogenes of Rous sarcoma virus (v-src), avian myeloblastosis virus (v-myb), Kirsten murine sarcoma virus (v-Ki-ras), and Harvey murine sarcoma virus (v-Ha-ras) were hybridized to the DNA from mouse-Chinese hamster somatic cell hybrids. The v-src, v-myb, v-Ki-ras, and v-Ha-ras genes each detected one or a few homologous mouse DNA fragments whose segregation was analyzed in cell hybrids. Mouse cellular homologs c-src, c-Ki-ras, c-Ha-ras, and c-myb segregated concordantly with chromosomes 2, 6, 7, and 10, respectively. Comparison with the known locations of human c-src (chromosome 20) and human c-Ha-ras1 (chromosome 11 short arm) suggests that the human and mouse homologs of these two viral oncogenes reside in conserved linkage groups. The c-Ki-ras gene on mouse chromosome 6 might reside also in a conserved linkage group, along with glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase. However, direct confirmation of this suggestion must await a demonstration that c-Ki-ras on mouse chromosome 6 is homologous to c-Ki-ras2 on the short arm of human chromosome 12.
Molecular cloning and expression of the mouse ornithine decarboxylase gene.
Authors: McConlogue L , Gupta M , Wu L , Coffino P
Published: May 2006

We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue.
Activation of Ca-dependent K channels by carbamoylcholine in rat lacrimal glands.
Authors: Trautmann A , Marty A
Published: May 2006

Electrical properties of the membranes of lacrimal gland cells were investigated using patch-clamp techniques [Hamill, O.P., Marty A., Neher, E., Sakmann, B. & Sigworth, F.J. (1981) Pflügers Arch. 391, 85-100]. The membranes were found to contain a specific kind of voltage- and Ca2+ -activated K+ channel ("BK channels"). These channels account for the strong rectification of the cell current-voltage curve as obtained in tight-seal whole-cell recordings. Application of low concentrations of carbamoylcholine (CbmCho, 0.5 microM) activated the BK channels. No effect was obtained in the presence of atropine (2 microM) or when dialyzing the cell with a strong CaEGTA buffer. The latter result, together with other findings, suggests that CbmCho exerts its action on BK channels by increasing the intracellular Ca2+ concentration. This Ca2+ concentration increase presumably occurred via liberation from a cytoplasmic Ca2+ store, because the response remained unaffected in the absence of extracellular Ca2+. At higher CbmCho concentration (2 microM), an inward current was observed, which was assumed to result from activation of another type of Ca2+ -regulated channel.
Remission of leukemia and loss of feline leukemia virus in cats injected with Staphylococcus protein A: association with increased circulating interferon and complement-dependent cytotoxic antibody.
Authors: Liu W T , Good R A , Trang L Q , Engelman R W , Day N K
Published: May 2006

We have injected purified Staphylococcus aureus protein A intraperitoneally into leukemic cats infected with feline leukemia virus, into cats persistently infected with feline leukemia virus but without hematologic or cytologic abnormalities, and into healthy cats without feline leukemia virus infection. Pre- and post-treatment serum samples were evaluated sequentially for interferon activity and for complement-dependent cytotoxic antibody. Our results indicate that serum interferon increased dramatically (less than 3 to 324 units/ml) during treatment only in cats that responded to staphylococcal protein A therapy. Increase of interferon preceded or was closely associated with increasing levels of cytotoxic antibody, loss of viremia, and correction of cytological and hematological abnormalities of three leukemic cats. The cytotoxic antibody was shown to be specific for envelope glycoprotein gp70 of the feline leukemia virus. One persistently feline leukemia virus-infected cat without leukemia that became nonviremic also developed high levels of interferon and specific cytotoxic antibody. By contrast, interferon levels of cats not responding to treatment had levels of less than 3 to 27 units/ml. Normal healthy cats injected with staphylococcal protein A showed moderate transient increases of interferon but no detectable cytotoxic antibodies to FL-74 cells. These data suggest that interferon and cytotoxic antibody may play important, possibly complementary roles in inducing remission of leukemia and loss of viremia in cats treated with staphylococcal protein A.
Heat shock regulatory gene (htpR) of Escherichia coli is required for growth at high temperature but is dispensable at low temperature.
Authors: Yura T , Tobe T , Ito K , Osawa T
Published: May 2006

Nonsense mutations affecting the positive regulatory gene (htpR) of heat shock response have been obtained in a strain of Escherichia coli carrying no suppressor. The mutants can grow only at temperatures below 34 degrees C-35 degrees C. Heat, ethanol, and coumermycin induce major heat shock proteins in the wild-type but not in the htpR mutants. In contrast, the level of heat shock proteins synthesized at low temperature is unaffected. The htpR gene product is thus required for induction of heat shock proteins by heat or other stresses but not for their "basal-level" synthesis. Nucleotide sequence has been determined for the wild-type and the mutant alleles of htpR. The coding region appears to consist of 852 nucleotide pairs that correspond to 284 amino acids. Sequences commonly considered as signals for transcriptional initiation and termination were found flanking the coding region. Within this region, six amber, one opal, and two missense mutations were identified; the nonsense mutations are scattered along the gene, some being very close to the presumed amino terminus. These results indicate that the absence of htpR gene product is directly responsible for the failure to respond to heat shock or other stresses and for the inability to grow at high temperature. We propose that htpR represents a new class of genes that are essential for growth only at high temperatures (greater than 35 degrees C). Implications of the sequence homologies found among htpR, rpoD, and nusA proteins are discussed.
The cromolyn binding protein constitutes the Ca2+ channel of basophils opening upon immunological stimulus.
Authors: Mazurek N , Schindler H , Schürholz T , Pecht I
Published: May 2006

Ca2+ channel opening has been proposed to be induced in the plasma membrane of mast cells and basophils upon crosslinking their Fc epsilon receptors. Here we report direct conductance measurements on planar lipid bilayers containing membrane components of rat basophils (RBL-2H3 line). These studies identify the Ca2+ channel-forming membrane component as the cromolyn binding protein [CBP, in which cromolyn is the anti-asthmatic drug 1,3-bis(2-carboxy-chromon-5-yloxy)-2-hydroxypropane]. Planar membranes were first formed from lipid vesicles containing unfractionated plasma membrane components prepared from RBL-2H3 cells. Conductance of these bilayers was induced by crosslinking IgE bound to the Fc epsilon receptors of this membrane by a specific polyvalent antigen. Channel conductance in the presence of only Ca2+ ions (2 mM) was 2 pS. When only sodium ions were present (150 mM), conductance was 10 pS. Upon addition of Ca2+ (2 mM) to the Na+ ion-containing solution, the conductance decreased from 10 pS to that of the Ca2+ ions--namely, 2pS. Open channel times were in the range of several hundred milliseconds. Conductance amplitudes and time characteristics were independent of the applied voltage. As our earlier studies revealed the essential role of the CBP in Ca2+ conductance of basophil membranes, we formed planar bilayers containing this isolated protein alone. Crosslinking of the CBP by a monoclonal antibody specific to it resulted in the appearance of channel conductances. All characteristics of these channels exhibited great similarity to those observed in planar membranes containing unfractionated RBL-2H3 membrane components. Moreover, in the latter membranes, the monoclonal anti-CBP antibody induced channel conductances that display an even closer similarity to those observed in membranes containing CBP alone. Conductances of both types of planar membranes, irrespective of the mode of activation used, were inhibited by cromolyn. Furthermore, the conductance induced in RBL membranes by polyvalent antigen was inhibited on dissociation of the crosslinked aggregates by a monovalent hapten. The detailed resemblance in channel behavior observed in experiments with the two types of planar bilayers provides compelling evidence that the CBP is the essential and sufficient component forming Ca2+ channels in basophil plasma membranes.
Is ATP a central synaptic mediator for certain primary afferent fibers from mammalian skin?
Authors: Fyffe R E , Perl E R
Published: May 2006

The possibility that ATP acts as a synaptic mediator at the central terminals of primary afferent fibers was examined by applying it iontophoretically to neurons of the outer layers of the cat spinal cord in vivo. ATP proved to be selectively excitatory for a limited subset of spinal neurons. Those units consistently excited by ATP iontophoresis with very small currents (2-15 nA) responded to gentle mechanical stimulation of the skin and usually evidenced excitatory input from unmyelinated primary afferent fibers. Most units excited by ATP were specifically mechanoreceptive; a few neurons receiving excitatory input from both low-threshold mechanoreceptors and nociceptors also responded to ATP. Selectively nocireceptive neurons were unresponsive. Generally, the mechanoreceptive neurons excited by ATP were located in the deeper substantia gelatinosa or in the immediately adjacent nucleus proprius of the dorsal horn. The results suggest the presence of a purinergic excitatory receptor on central neurons receiving excitatory projection from tactile mechanoreceptors with fine-diameter afferent fibers and are consistent with the possibility that an ATP-like agent may mediate central synaptic excitation for this set of sense organs.
Prevention of some electrophysiologic and biochemical abnormalities with oxygen supplementation in experimental diabetic neuropathy.
Authors: Low P A , Tuck R R , Dyck P J , Schmelzer J D , Yao J K
Published: May 2006

Endoneurial hypoxia has been suggested as a mechanism of human and experimental diabetic neuropathy (EDN). We found that rats rendered diabetic for 4 months had reduced nerve blood flow (NBF) and nerve oxygen tension (PnO2). The NBF was reduced by at least 33% in EDN and 60% of the oxygen tensions in the endoneurial O2 histogram were less than 25 mm Hg (3.3 kPa) in EDN compared with only 19% in the controls. To test the hypothesis that EDN may in part be due to hypoxia, we studied the effectiveness of oxygen supplementation in preventing some electrophysiologic and biochemical abnormalities. Rats with EDN had reduced caudal nerve conduction velocity and had a resistance to ischemic conduction block. When a matched groups of rats with EDN were O2 supplemented for 4 weeks, the time to 50% block of nerve conduction and nerve conduction velocity was no longer statistically different from controls. Endoneurial free sugars (glucose, fructose, sorbitol) were markedly increased in EDN. Oxygen supplementation resulted in no change in plasma glucose; by contrast, these increased endoneurial free sugars were significantly reduced (towards normal) by 60%, 33%, and 34%, respectively. myo-Inositol, however, was further decreased by oxygen supplementation. These findings of a partial prevention of electrophysiologic and biochemical abnormalities support a role of hypoxia in the pathogenesis of EDN.
Multicomponent RNA plant virus infection derived from cloned viral cDNA
Authors: Ahlquist Paul , French Roy , Janda Michael , Loesch-Fries L. Sue
Published: May 2006

In vitro
transcripts from mixtures of appropriate brome mosaic virus (BMV) cDNA clones are infectious when inoculated onto barley plants. Infectivity depends on
in vitro
transcription and on the presence of transcripts from clones of all three BMV genetic components. Infectivity is destroyed by RNase after transcription, but it is insensitive to RNase before or to DNase after transcription. Virion RNAs from plants infected with cDNA transcripts hybridize to BMV-specific probes and coelectrophorese with virion RNAs propagated from conventional inoculum. Direct RNA sequencing shows that a deletion in the noncoding region of one infectious BMV clone is preserved in viral RNA from plants systemically infected with transcript mixtures representing that clone.
Yeast mitochondrial genomes consisting of only A.T base pairs replicate and exhibit suppressiveness.
Authors: Fangman W L , Dujon B
Published: May 2006

Mutants, called p-, that result from extensive deletions of the 75-kilobase Saccharomyces cerevisiae mitochondrial genome arise at high frequency. The remaining mitochondrial DNA is amplified in the p- cells, often as head-to-tail multimers, producing a cell with the normal amount of mitochondrial DNA. In matings, some of these p- mutants exhibit zygotic hypersuppressiveness, excluding the wild-type mitochondrial genome (p+) from all the diploids that are produced. From a hypersuppressive p- strain, we isolated two mutants with reduced suppressiveness. These mutants, one moderately suppressive and one nonsuppressive, retain only 89 and 70 base pairs, respectively, of the wild-type mitochondrial genome. Their sequences consist of 100% A . T base pairs. Replication of DNA in the mitochondrion, formation and amplification of new deletion genomes, and exhibition of suppressiveness do not require a single G . C base pair.
Slow virus visna: reproduction in vitro of virus from extrachromosomal DNA.
Authors: Harris J D , Blum H , Scott J , Traynor B , Ventura P , Haase A
Published: May 2006

Under permissive conditions of growth in tissue culture, the retrovirus visna multiples over the course of a few days to high titer and kills the host cell. We show that in this lytic life cycle, viral DNA is tightly associated with, but not covalently linked to, chromosomal DNA. This finding provides explanations for a number of the unusual properties of the lentivirus subfamily of retroviruses, and suggests potential mechanisms for the block in virus gene expression in vivo responsible for the slow infection in nature.
Characterization of epidermal growth factor receptor gene expression in malignant and normal human cell lines.
Authors: Xu Y H , Richert N , Ito S , Merlino G T , Pastan I
Published: May 2006

To investigate the possibility that the epidermal growth factor (EGF) receptor functions as an oncogene product, we have determined the levels of EGF receptor protein and RNA in a variety of malignant and normal human cells, using a specific polyclonal antibody to the EGF receptor and a cDNA clone (plasmid pE7) that encodes the EGF receptor, respectively. Besides A431 epidermoid carcinoma cells, which are known to make large amounts of EGF receptor, cell lines from two ovarian cancers, two cervical cancers, and one kidney cancer were found to contain substantial amounts of receptor protein (11-22% of A431). Normal human fibroblasts (Detroit 551), a human lymphocyte line (IM-9), and a leukemic lymphocyte line (CEM) contained low or undetectable levels of EGF receptor. RNA blot analysis showed that among the human cell lines examined the levels of a 10- and a 5.6-kilobase species of pE7-specific RNA generally correlated with the amount of the EGF receptor protein. Genomic DNA blot analysis revealed that except for A431 none of these cell lines expressing high levels of EGF receptor protein possessed amplified receptor gene sequences. A431 cells are known to secrete a truncated form of the EGF receptor. An abundant 2.9-kilobase RNA is found only in A431 cells; it could encode the truncated form of the EGF receptor.