bluuebunny/crossref_metadata_2025 huggingface dataset:
Reciprocal and nonreciprocal recombination in diploid clones from
Bacillus subtilis
protoplast fusion: Association with the replication origin and terminus
Authors: Gabor Magda H. , Hotchkiss Rollin D.
Published: May 2006

The primary heterodiploid bacteria regenerated after
Bacillus subtilis
fusion, although generally noncomplementing diploids, behave in pedigree analysis as multipotential systems. Individual diploid colonies yielding complete reciprocal recombinant (RR) progeny—often accompanied by one or both parents—constitute 10-30% of the total recombinant-forming units. The RR (reciprocal for 8-11 genes) usually occur in equivalent numbers both among and within individual colonies. Novel for bacteria, they demonstrate that entire parental genomes brought together within a diploid protoplast are retained as two independent replicons able to undergo classical recombination characteristic of eukaryotic gametogenesis. Parental or recombinant genomes are also subject to multiple rounds of recombination without obligate segregation and often not reciprocal. Diploid recombinant clones, sharing streptomycin resistance but reciprocal for auxotrophic markers, have displayed a partial ability to make a facultative shift in chromosome expression. They have also produced two types of prototrophs: a stable one (presumably haploid and recombinant) and an unstable one, (diploid and temporarily complementing at low frequency). It follows that chromosome extinction may affect both parental and recombinant chromosomes and does not interfere with recombination. Analysis of the number and chromosomal distribution of crossovers in all recombinants and those from single diploid clones shows increased frequency of exchange in the regions of the replication origin and terminus, possibly a result of the association of these sites with the cell wall or membrane.
Identification of a cDNA clone for mouse apoprotein A-1 (apo A-1) and its use in characterization of apo A-1 mRNA expression in liver and small intestine.
Authors: Miller J C , Barth R K , Shaw P H , Elliott R W , Hastie N D
Published: May 2006

A cDNA clone for mouse apoprotein A-1 (apo A-1), the major apoprotein of plasma high density lipoproteins, has been identified. In addition to structural and physiological evidence, a genetic polymorphism for mouse plasma apo A-1 has been used to confirm that this DNA sequence corresponds to the apo A-1 gene. Use of this clone in molecular hybridization studies has shown that the concentration of apo A-1 mRNA is similar in liver and small intestine and is constant along the entire length of the small intestine. We provide evidence that the same apo A-1 gene is expressed in both liver and small intestine. Apo A-1 mRNA is also present in the stomach and esophagus at 10-15% the concentration found in small intestine but is undetectable in other tissues (such as large intestine, pancreas, heart, kidney, spleen, and brain). Finally, we show that there is a differential effect of a diet high in saturated fat and cholesterol on apo A-1 mRNA levels in liver and small intestine.
Reversal of Rous sarcoma-specific immunoglobulin phosphorylation on tyrosine (ADP as phosphate acceptor) catalyzed by the src gene kinase.
Authors: Fukami Y , Lipmann F
Published: May 2006

To determine the equilibrium constant of the reaction between ATP and protein-bound tyrosine we used as catalyst the highly purified Rous sarcoma src gene transcript. J. M. Sturtevant had earlier found (personal communication) that free tyrosine O-phosphate, upon hydrolysis with alkaline phosphatase in a calorimeter (37 degrees C, pH 9), yielded a delta H degrees of -2.8 kcal/mol (1 kcal = 4.18 kJ), less than half of that found in ATP hydrolysis. Experience with protein-bound serine phosphate (in phosvitin) had shown it to be energy rich [Rabinowitz, M. & Lipmann, F. (1960) J. Biol. Chem. 235, 1043-1050]. We wondered if the same is true for tyrosine phosphate when it is protein bound. From the equilibrium constant of 2.62 (at pH 6.5 and 5 mM Mg2+), we calculate a delta G degrees' of -9.48 kcal/mol for hydrolysis of protein-bound tyrosine phosphate, assuming an approximate delta G degrees' of -10 kcal/mol for hydrolysis of ATP. The experiments show that protein-bound tyrosine phosphate is energy rich, like serine phosphate in phosvitin.
Extrachromosomal DNA of pea (
Pisum sativum
) root-tip cells replicates by strand displacement
Authors: Krimer D. B. , Hof J. Van't
Published: May 2006

In cultured pea roots there is extrachromosomal DNA associated with cells that differentiate from the G
2
phase of the cell cycle that is absent from those that differentiate from the G
1
phase. We examined this extrachromosomal DNA by electron microscopy and found that it consisted of three types: (
i
) double-stranded linear molecules with single-stranded branches (74%), (
ii
) double-stranded molecules without branches (26%), and (
iii
) free single-stranded molecules. The double-stranded molecules with or without branches were similar in length, having a modal length of 10-15 μm. The free single-stranded molecules were shorter and had a mean length of 3.8 μm. The length of the branches attached to the duplex molecules was only slightly less than that of the free form. The duplex molecules with branches were interpreted as configurations reflecting an ongoing strand-displacement process that results in free single-stranded molecules. Finally, measurements on duplex molecules with multiple branches suggested that the extrachromosomal DNA may exist in the form of tandemly repeated sequences.
Cloning and sequence analysis of cDNA for rat angiotensinogen.
Authors: Ohkubo H , Kageyama R , Ujihara M , Hirose T , Inayama S , Nakanishi S
Published: May 2006

A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxyl-terminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRag16, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecular consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.
Nuclear ribonucleoprotein release and nucleoside triphosphatase activity are inhibited by antibodies directed against one nuclear matrix glycoprotein.
Authors: Baglia F A , Maul G G
Published: May 2006

Circumstantial evidence suggests that nucleocytoplasmic exchange or transport is an active process involving the nuclear pore complex of the nuclear envelope. To test this hypothesis, antibodies were generated against nuclear envelope components from a highly enriched pore complex fraction from Spisula solidissima oocytes. Some of these antibodies inhibited ATP-dependent ribonucleoprotein release from prelabeled, isolated rat nuclei and inhibited nucleoside triphosphatase activity essential in nucleocytoplasmic transport. Inhibition of both functions by lectins indicated that the antigen was a glycoprotein. It was identified as lamin B, a major component of the nuclear envelope and nuclear matrix. This glycoprotein may not only be a structural nuclear protein but also may have nucleoside triphosphatase activity. We speculate that lamin B represents the solid support for ribonucleoprotein transport. This protein is expected to be highly conserved if active transport in and out of the nucleus is essential in the eukaryotic system.
Dihydropyridine receptor in rat brain labeled with [3H]nimodipine.
Authors: Bellemann P , Schade A , Towart R
Published: May 2006

Receptor binding sites for 1,4-dihydropyridine (DHP) calcium antagonists have been characterized by using [3H]-nimodipine, a potent analogue of nifedipine with cerebrovascular and neuro- and psychopharmacological properties. [3H]Nimodipine exhibited reversible and saturable binding to partially purified brain membranes. The equilibrium dissociation constant, Kd, was 1.11 nM and the maximal binding capacity, Bmax, was 0.50 pmol/mg of protein. The DHP receptor proved to be highly specific for various potently displacing DHP derivatives and discriminated between their optical isomers (stereoselectivity) with inhibition constants (Ki) in the nanomolar or even subnanomolar range. Structurally different calcium antagonists such as gallopamil (D-600), on the other hand, displayed much lower affinities, further substantiating the specificity of the receptor for DHP structures. Furthermore, the displacement potency of a series of DHP derivatives correlated well with that determined for inhibition of mechanical response in the intact smooth muscle over 5 orders of magnitude. [3H]Nimodipine binding thus may provide a molecular probe to elucidate the nature of the interaction of calcium entry blockers with specific membrane-located receptor sites that may be associated with the putative calcium channel. These receptor sites might well represent the loci of signaling events where the potent DHPs exert their pharmacological action.
Time course of the increase in the myocardial slow inward current after a photochemically generated concentration jump of intracellular cAMP.
Authors: Nargeot J , Nerbonne J M , Engels J , Lester H A
Published: May 2006

Voltage-clamped atrial trabeculae from bullfrog hearts were exposed to membrane-permeant photolyzable o-nitrobenzyl esters of cAMP and cGMP. UV flashes produced intracellular concentration jumps of cAMP or cGMP. With the cAMP derivative, flashes resulted in an increased slow inward current (Isi), producing a broadened action potential. The Isi reached a maximum 10-30 sec after the flash and decreased over the next 60-300 sec. The first increases were observable within 150 msec; this value is an upper limit imposed by the instrumentation. Responses to flashes lasted longer at higher drug concentrations and in the presence of the phosphodiesterase inhibitor papaverine; effects of flashes developed and decreased faster at higher temperature. Although the amplitude of the Isi was increased, its waveform and voltage sensitivity were not affected. Intracellular concentration jumps of cAMP failed to affect the muscarinic K+ conductance. There were no observable effects of cGMP concentration jumps. The data confirm (i) that cAMP regulates the Isi and (ii) that the 5- to 10-sec delay between application of beta-agonists and the onset of positive inotropic effects, observed in previous studies, has been correctly ascribed to events prior to the interaction between cAMP and protein kinase.
Transduction of a cellular oncogene: the genesis of Rous sarcoma virus.
Authors: Swanstrom R , Parker R C , Varmus H E , Bishop J M
Published: May 2006

The oncogene of Rous sarcoma virus (v-src) arose by transduction of a cellular gene (c-src). In an effort to explore the mechanism of transduction, we have identified the splice acceptor site used in the genesis of mRNA for v-src, shown that an equivalent site is used in the splicing of mRNA for c-src, and determined the nucleotide sequence from the boundaries of homology between v-src and c-src. Our data indicate that (i) only a portion of c-src is represented within v-src, (ii) the leftward recombination between the genome of the transducing virus and c-src occurred in an intron of the cellular gene, (iii) v-src is in part a spliced version of the corresponding portion of c-src, and (iv) nucleotide sequences represented once in the genome of the transducing virus become duplicated to flank v-src. These findings indicate that the first step in transduction is probably recombination between DNA forms of the transducing viral genome and c-src and otherwise support the prevailing model for transduction by retroviruses. The carboxyl termini of the proteins encoded by v-src and c-src differ appreciably. An unidentified domain of 127 or 128 nucleotides is located at different positions in the genomes of two strains of RSV and gives evidence of being a foreign element that entered the viral genomes by genetic transposition independent of the transduction of src.
Theory of measurement of Förster-type energy transfer in macromolecules
Authors: Epe Bernd , Steinhäuser Klaus G. , Woolley Paul
Published: May 2006

We describe the theoretical basis of an unconventional method for the determination of the amount of energy transferred between two fluorophores by the Förster mechanism. The method involves an internal comparison made by separation of the fluorophores
in situ
(i.e., in the optical cell), for example, by means of enzymic digestion; it eliminates several important sources of error and it simplifies calculation while making maximal use of the information contained in the fluorescence spectra. The validity of the method is demonstrated by determination of the known distance between two modifiable sites on the transfer RNA molecule, and its usefulness is exemplified by its application to triangulation of the ribosome of
Escherichia coli
.
p53, a transformation-related cellular-encoded protein, can be used as a biochemical marker for the detection of primary mouse tumor cells.
Authors: Rotter V
Published: May 2006

p53, a transformation-related cellular-encoded protein, was found to accumulate at high concentration in transformed cell lines. The results presented here show that p53 biosynthesis is also increased in most induced and spontaneous mouse tumors. Judged by the identity in antigenic determinants (estimated by binding to monoclonal antibodies), size, and partial peptide mapping, I conclude that the p53 molecule found in primary tumors is indistinguishable from that in established cell lines. The fact that p53 is found in heterogeneous populations of primary tumors makes it a convenient biochemical diagnostic marker for the detection of primary tumors in mice. It is found in primary tumors as a phosphoprotein, just as it was found previously in established cell lines. On the other hand, the p53 found at low concentration in normal thymocytes is labeled with [35S]methionine but cannot be found in its phosphorylated form.
Endogenous murine leukemia proviral long terminal repeats contain a unique 190-base-pair insert.
Authors: Khan A S , Martin M A
Published: May 2006

We have determined the nucleotide sequence in the U3-R regions of the long terminal repeat (LTR) associated with NFS-Th-1 xenotropic murine leukemia virus (MuLV) DNA and the LTR components of five endogenous proviruses cloned from BALB/c mouse chromosomal DNA. The five endogenous MuLV LTRs contained the regulatory signals thought to be important in viral transcription, such as "TATAA" and CCAAT-like boxes. A unique feature in four of the endogenous LTRs was the presence of a highly conserved 190-base-pair (bp) insert bounded by 6-bp direct repeats located 48 bp upstream from the C-C-A-A-T sequence. This segment was absent from LTRs associated with ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. All five endogenous LTR segments also contained a 14-bp duplication of a sequence located near the 5' end of the first component of the long (greater than 72-bp) direct repeat of ecotropic and MCF MuLV LTRs. An evolutionary scheme relating LTRs associated with endogenous MuLV proviral DNAs to those found in ecotropic or xenotropic proviruses is presented. Nucleotide sequence analysis also suggested that the U3 region of the MCF247 MuLV LTR is derived from an NFS xenotropic related MuLV.
Acquired immunodeficiency syndrome in a colony of macaque monkeys.
Authors: Letvin N L , Eaton K A , Aldrich W R , Sehgal P K , Blake B J , Schlossman S F , King N W , Hunt R D
Published: May 2006

A naturally occurring immunodeficiency syndrome has been seen in a captive colony of macaque monkeys. This syndrome is seen primarily in the species Macaca cyclopis. Affected animals died with lymphomas (a rare disease in macaques) or such opportunistic infections as Pneumocystis carinii and noma (necrotizing gingivitis). These M. cyclopis exhibited anemia, neutropenia, and a circulating bizarre immature monocyte. In addition, liver function tests suggested hepatitis. Pokeweed mitogen-, concanavalin A-, and xenogeneic cell-stimulated proliferative responses by lymphocytes of animals with the syndrome were dramatically diminished. The T4 (helper, inducer)/T8 (suppressor, cytotoxic) ratio in the peripheral blood mononuclear T-cell populations of M. cyclopis in this colony are decreased when compared with those from either Macaca mulatta in the same colony or normal humans. Epidemiologic evidence implicates a common source agent in this syndrome. The similarity of this syndrome in macaques to human acquired immunodeficiency syndrome suggests that it may provide an important model for studying the human syndrome.
Electrical activity and cytosolic calcium regulate levels of tetrodotoxin-sensitive sodium channels in cultured rat muscle cells.
Authors: Sherman S J , Catterall W A
Published: May 2006

Pharmacological blockade of the spontaneous electrical activity present in primary cultures of rat myotubes by growth in bupivacaine, tetrodotoxin, or KCl was found to increase the number of voltage-sensitive Na+ channels 38-83% as measured by the specific binding of [3H]saxitoxin. The inhibition of spontaneous electrical activity and increase in channel density by bupivacaine displayed an identical dose response, with a half-maximal effect at 3.0 microM. Growth of myotubes in the presence of 1 microM A23187, a Ca2+-specific ionophore, resulted in a 30-60% decrease in the number of tetrodotoxin-sensitive channels with no change in affinity for [3H]saxitoxin. A23187 was able to overcome the increase in channel density produced by bupivacaine. These results suggest the presence of a Ca2+-mediated negative feedback system in which electrical excitability may be regulated by altering the number of tetrodotoxin-sensitive Na+ channels.
Isolation and sequence of a human apolipoprotein CII cDNA clone and its use to isolate and map to human chromosome 19 the gene for apolipoprotein CII.
Authors: Jackson C L , Bruns G A , Breslow J L
Published: May 2006

cDNA clones encoding human apolipoprotein CII (apo CII) were identified by screening an adult human liver cDNA library with a mixed oligonucleotide probe corresponding to all possible codons for apo CII amino acid 6-10. One clone with an approximately equal to 500-base-pair (bp) insert, designated pCII -711, was selected for DNA sequence analysis. This clone contained a DNA sequence that corresponded with the previously reported amino acid sequence of apo CII with only minor differences. The DNA sequence specified a polypeptide of 79 amino acids, compared to the 78 amino acids previously reported. The pCII -711 clone contains a 36-bp DNA sequence upstream from that specifying the NH2-terminal threonine which, when read in frame, specifies the amino acid sequence Leu-Val-Leu-Leu-Val-Leu-Gly-Phe-Glu-Val-Gln-Gly and may be part of an apo CII signal peptide. The pCII -711 clone also contains a 144-bp region that corresponds to the 3' untranslated region of apo CII mRNA as well as a portion of the poly(A) tail. Clone pCII -711 was used to isolate and characterize by restriction endonuclease digestion the gene for apo CII from a human genomic library. In addition, through Southern blot analysis of DNA from human-rodent somatic cell hybrids, clone pCII -711 also was used to provisionally map the gene for apo CII to human chromosome 19.
Plasmid-linked
nif
and “
nod
” genes in fast-growing rhizobia that nodulate
Glycine max, Psophocarpus tetragonolobus
, and
Vigna unguiculata
Authors: Broughton William J. , Heycke Nina , Z.A. Heiner Meyer , Pankhurst Clive E.
Published: May 2006

Forty-nine fast-growing
Rhizobium
strains from the nodules of 26 different tropical legume genera were screened to find isolates that would (
i
) nodulate, e.g., winged beans, so producing large nodules for RNA and protein isolation; (
ii
) also nodulate various small-seeded legumes, thus allowing screening of large numbers of mutants; and (
iii
) harbor plasmids containing
nif
structural genes as well as other functions involved in nodulation. On the basis of six different criteria, this rhizobial group appeared intermediate between classical fast- and slow-growing organisms, yet all contained plasmids. Plasmid numbers varied from one to five. Hybridizations between DNA prepared from
nifDH
and the putatative “
nod
” region of
R. meliloti
and these plasmids bound to nitrocellulose filters suggested that
nif-nod
genes are linked on a single
sym
plasmid. A broad-host-range strain containing a single
sym
plasmid was chosen for further study. Its plasmid, pMPIK3030a, was isolated on cesium chloride gradients and cloned in the cosmid pJB8, and the overlapping fragments were mapped by homology with the
nif
and
nod
regions of
R. meliloti
. As the wild-type plasmid pMPIK3030a was not self-transmissible, confirmation that the
nod
genes detected by homology were responsible for nodulation was obtained by introducing the mobilization functions of RP
4
(together with Tn
5
) and selecting transconjugants resistant to kanamycin and neomycin. Transconjugants (obtained at a frequency of about 10
-6
per recipient) in
Agrobacterium tumefaciens
cured of the Ti plasmid produced ineffective nodules on
Vigna unguiculata
, those in nonnodulating (Nod
-
)
R. meliloti
were partially effective, while those in Nod
-
R. leguminosarum
were often fully effective.
DNA methylation pattern is determined by the intracellular level of the methylase.
Authors: Szyf M , Avraham-Haetzni K , Reifman A , Shlomai J , Kaplan F , Oppenheim A , Razin A
Published: May 2006

Extrachromosomal plasmid DNA is transiently undermethylated in Escherichia coli during amplification in the presence of chloramphenicol. In addition, undermethylation of phage lambda DNA was observed after thermal induction of a lambda c1857 lysogen while the integrated lambda phage DNA was found to be fully methylated. These methylation pattern changes occur under conditions (extensive replication) in which the intracellular methylase level becomes limiting. In an E. coli strain that harbors a plasmid that carries the dam methylase gene and therefore overproduces dam methylase, there is no undermethylation of dam sites in either of the extrachromosomal DNAs. The sites that are methylated by the mec methylase in both plasmid and lambda phage DNAs were undermethylated in the dam overproducer as well. These results indicate that the intracellular level of the E. coli methylase determines the DNA methylation pattern.
Insulin receptor is an insulin-dependent tyrosine protein kinase: copurification of insulin-binding activity and protein kinase activity to homogeneity from human placenta.
Authors: Petruzzelli L , Herrera R , Rosen O M
Published: May 2006

The insulin-binding activity and insulin-dependent tyrosine protein kinase activity of the insulin receptor have been purified 2000-fold to homogeneity from human placental membranes. The purified receptor has one high-affinity binding site for insulin per mol of receptor. Its Vmax for phosphorylating angiotensin is 80 nmol of phosphate per min per mg of protein at 23 degrees C. The procedure used to purify the receptor includes chromatography on wheat germ agglutinin-agarose and on insulin-Sepharose. The purified receptor was eluted from insulin-Sepharose with 0.5 M NaCl and 1 mM dithiothreitol at pH 5.5. The addition of dithiothreitol was essential for recovery of the protein kinase. A silver-stained gel of the reduced purified receptor showed two major bands, Mr 95,000 (beta subunit) and Mr 135,000 (alpha subunit). The component of Mr 95,000 comigrated with the autophosphorylated beta subunit of the receptor. The latter was phosphorylated exclusively on tyrosine residues by an intramolecular process. In the presence of insulin, approximately 2 mol of phosphate was incorporated per mol of beta subunit. Two major beta subunit tryptic phosphopeptides were resolved by high-pressure liquid chromatography after autophosphorylation of the purified receptor in the presence or absence of insulin. It is concluded that the insulin binding and the insulin-dependent protein kinase are intrinsic components of the same oligomer since (i) they copurify to homogeneity, (ii) the purified receptor protein kinase is immunoprecipitated by polyclonal and monoclonal antibodies to the human insulin receptor, and (iii) phosphorylation of the beta subunit of the receptor occurs by an intramolecular reaction.
Signal recognition particle is required for co-translational insertion of cytochrome P-450 into microsomal membranes.
Authors: Sakaguchi M , Mihara K , Sato R
Published: May 2006

Insertion of newly synthesized P-450(1), the major phenobarbital-inducible form of rabbit liver microsomal cytochrome P-450, into microsomal membranes was studied in a wheat germ cell-free translation system programed with total RNA from the liver of a phenobarbital-treated rabbit. P-450(1) synthesized in vitro had the same molecular weight as the mature molecule and was co-translationally inserted into dog pancreas rough microsomal membranes. In the presence of salt-washed microsomes, instead of unwashed ones, the insertion was greatly diminished. It could, however, be restored by supplementation of the system with purified signal recognition particle (SRP), a known component of the membrane translocation machinery for secretory proteins. In the absence of microsomes, SRP inhibited the translation of mRNA encoding P-450(1) and this translation arrest was released by the addition of salt-washed microsomes. On the other hand, SRP did not affect the translation of mRNAs encoding yeast porin and reticulocyte globin, which are mitochondrial and cytosolic proteins, respectively. We conclude that co-translational insertion of P-450(1) into microsomal membranes requires SRP and postulate that P-450(1) possesses an uncleavable signal sequence that can be recognized by SRP.
Differential induction of chalcone synthase mRNA activity at the onset of phytoalexin accumulation in compatible and incompatible plant-pathogen interactions
Authors: Bell John N. , Dixon Richard A. , Bailey John A. , Rowell Pat M. , Lamb Chris J.
Published: May 2006

Changes in the mRNA activity of chalcone synthase, the first enzyme of phenylpropanoid metabolism specific to flavonoid/isoflavonoid biosynthesis, have been investigated in relation to expression of the phytoalexin defense response in race-cultivar specific interactions between hypocotyls of
Phaseolus vulgaris
and the partially biotrophic fungus
Colletotrichum lindemuthianum
, causal agent of anthracnose. In an incompatible interaction (host resistant) there is an early but localized increase in chalcone synthase mRNA activity prior to the onset of accumulation of the phenylpropanoid-derived phytoalexin phaseoflin and expression of hypersensitive resistance. In contrast, in a compatible interaction (host susceptible) there is no induction of mRNA activity in the early stages of infection but rather a delayed, widespread increase during attempted lesion limitation at the onset of symptom development. The data indicate that control of phytoalexin gene expression is a key early component in the defense responses of biologically stressed cells during a race-cultivar specific host—pathogen interaction.
Human T-lymphocyte subset specificity of the regulatory effects of leukotriene B4.
Authors: Payan D G , Missirian-Bastian A , Goetzl E J
Published: May 2006

Leukotriene B4, but not (12S)-leukotriene B4, coupled to fluorescein-labeled human serum albumin interacts specifically with human T lymphocytes, as assessed by fluorescence-activated flow cytometry. Approximately 11% of blood T lymphocytes bind the fluorescent conjugate of leukotriene B4. The leukotriene B4-reactive T lymphocytes are distributed between the suppressor-cytotoxic (14%) and helper-inducer (8%) subsets, which were identified concurrently by phycoerythrin-labeled monoclonal antibodies to subset-specific antigens. Leukotriene B4, but not (12S)-leukotriene B4 or leukotriene C4, enhances significantly the proliferation of mitogen-stimulated purified suppressor-cytotoxic T lymphocytes, at concentrations of 10(-12) M to 10(-6) M, and inhibits significantly the proliferation of mitogen-stimulated purified helper-inducer T lymphocytes, at concentrations of 10(-8) M to 10(-6) M, as assessed by the uptake of [3H]thymidine. The net effect of leukotriene B4 on blood T lymphocytes is immunosuppression, which may be expressed in some human hypersensitivity and inflammatory diseases.
Tumors undergoing rejection induced by monoclonal antibodies of the IgG2a isotype contain increased numbers of macrophages activated for a distinctive form of antibody-dependent cytolysis.
Authors: Adams D O , Hall T , Steplewski Z , Koprowski H
Published: May 2006

Monoclonal antibodies of the IgG2a isotype specifically inhibit the growth of human tumors in nude mice; tumors in mice given no antibody, an antibody of the IgG2a isotype that does bind to the tumor cells, or an antibody of the IgG2b isotype grow progressively. In the present work it is demonstrated that tumors of mice given the IgG2a antibody are essentially masses of necrosis, while tumors from the control mice are sheets of healthy tumor cells. Tumors of the treated mice, in comparison to controls, contain an increased number of macrophages, and these macrophages are activated for a distinct form of cytolysis dependent on antibodies of the IgG2a isotype. The data suggest that changes in the number and function of intratumoral macrophages are a major component of tumor destruction mediated by IgG2a antibodies.
Decreased physiological sensitivity mediated by newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart.
Authors: Hunter D D , Nathanson N M
Published: May 2006

Treatment of chicken embryos in ovo for 8 hr with the muscarinic agonist carbachol results in an 85% reduction in the number of muscarinic acetylcholine receptors ( mAcChoR ) present in atrial membrane homogenates. Subsequent treatment of embryos with the muscarinic antagonist atropine results in a gradual increase in mAcChoR number, which returns to control levels after 14 hr. This recovery of receptor number is blocked by administration of the protein synthesis inhibitor cycloheximide, consistent with previous results in cell culture, which suggested that de novo protein synthesis is required for the recovery of mAcChoR after agonist-induced decreases. Measurements of the negative chronotropic response to applied carbachol with isolated atria show that even after recovery of receptor number to control levels the response to agonist is diminished. The IC50 for carbachol is shifted approximately equal to 10-fold from controls at 20 hr after atropine treatment, but less than 3-fold at 28 hr, with no further change in receptor number occurring over this time. This increase in physiological sensitivity is not blocked by cycloheximide. Receptors at 20 hr have binding constants for agonists and antagonists that are indistinguishable from controls. This implies that there is a defect in coupling of mAcChoR binding to the physiological response when mAcChoR reappear following agonist-mediated decreases in receptor number. Recovery of the ability of mAcChoR to inhibit adenylate cyclase parallels recovery of the beating response--that is, the IC50 is shifted approximately equal to 11-fold from controls at 20 hr after atropine treatment, yet only 3-fold at 28 hr. Thus, newly synthesized mAcChoR exhibit decreased physiological and biochemical responses to muscarinic agonists, suggesting that mAcChoR are initially synthesized in a less active form.
Potentiation of alpha 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.
Authors: Morgan N G , Charest R , Blackmore P F , Exton J H
Published: May 2006

Treatment of isolated hepatocytes with the alpha 1-adrenergic agonist norepinephrine induced a dose-dependent increase in free cytosolic Ca2+, as judged by fluorescence increases, in cells loaded with the Ca2+ indicator (2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8 -bis [carboxymethyl]aminoquinoline (quin-2). Pretreatment with either glucagon or dibutyryl cAMP increased the rate and magnitude of the quin-2 fluorescence response in hepatocytes treated with submaximal doses of norepinephrine and increased the cell sensitivity such that a physiological concentration of norepinephrine (7.5 nM) was able to provoke a quin-2 fluorescence response. Similar enhancement of norepinephrine-induced phosphorylase activation and pyridine nucleotide reduction in isolated hepatocytes and Ca2+ efflux from the perfused liver was also observed in the presence of glucagon. These potentiated responses correlated with a cAMP-dependent increase (mediated by glucagon, dibutyryl cAMP, or forskolin) in the binding of [3H]norepinephrine or [3H]epinephrine to sites present on isolated hepatocytes bearing the characteristics of alpha 1-adrenergic receptors. The data suggest that a cAMP-dependent mechanism is involved in the regulation of alpha 1-agonist binding to liver cells and, thereby, in the control of hepatic carbohydrate metabolism in response to catecholamines.
Structural gene for beta-nerve growth factor not defective in familial dysautonomia.
Authors: Breakefield X O , Orloff G , Castiglione C , Coussens L , Axelrod F B , Ullrich A
Published: May 2006

The developmental loss of neurons in sympathetic, sensory, and some parasympathetic ganglia in familial dysautonomia suggests an inherited defect in the action of beta-nerve growth factor (beta-NGF). The role of this growth factor in dysautonomia has been difficult to resolve as there is no known source of authentic human beta-NGF. The availability of a cloned DNA probe for the human beta-NGF gene has allowed identification of some copies of the gene (alleles) in six affected families. Alleles differ in the length of restriction endonuclease fragments that hybridize to DNA probes for the gene. In two families, affected children did not inherit the same two alleles at the beta-NGF locus. Since this disease is transmitted in an autosomal recessive manner, affected children must share the same alleles at the locus causing the disease. This analysis excludes the beta-NGF gene region as the cause of this neurologic disease but does not eliminate other genes involved in beta-NGF action, such as those coding for processing enzymes, receptors, or other subunits of the NGF complex.
Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.
Authors: Gilmour D S , Lis J T
Published: May 2006

We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.
Cleavage of single-stranded DNA by the ϕX174 A
*
protein: The A
*
-single-stranded DNA covalent linkage
Authors: Sanhueza Sonia , Eisenberg Shlomo
Published: May 2006

Phage ϕX174 A
*
protein cleaves single-stranded DNA and then binds to the 5′-phosphorylated terminus of the cleaved DNA fragment, forming a covalent protein-DNA complex. The bound A
*
protein can religate the termini to form covalently closed single-stranded circles. To determine the nature of the covalent linkage and the amino acid involved, we used A
*
protein to cleave DNA synthesized
in vivo
with [α-
32
P]dATP to form the A
*
-single-stranded DNA complex. The complex was then digested with DNase I and the
32
P-labeled A
*
protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was digested with either trypsin or Pronase. Incubation of the tryptic digest with snake venom phosphodiesterase gave
32
P-labeled products that migrated on electrophoresis on cellulose plates to the cathode, indicating covalent linkage of
32
P-labeled dAMP residues to a tryptic peptide. High concentrations of snake venom phosphodiesterase released all of the
32
P label as free dAMP. Formic acid/diphenylamine depurination (Burton reaction) of the [α-
32
P]dATP-labeled peptide-oligonucleotide complexes caused a transfer of the labeled phosphate from dAMP to the peptide. The phosphorylated peptides were isolated on cellulose plates and shown to be sensitive to bacterial alkaline phosphatase, indicating that a phosphodiester bond linked the peptides to the dAMP. The phosphorylated product of the Pronase digest was identified as free phosphotyrosine by its mobility in three different chromatography systems. Likewise, acid hydrolysis (5.6 M HCl, 110°C, 2 hr) of the phosphorylated tryptic peptides revealed linkage of the phosphate to a tyrosine. Thus, A
*
protein cleaves single-stranded DNA and binds covalently to the 5′-phosphorylated terminus via a tyrosyl-dAMP phosphodiester bond.
Monoclonal antibodies as probes for unique antigens in secretory cells of mixed exocrine organs.
Authors: Basbaum C B , Mann J K , Chow A W , Finkbeiner W E
Published: May 2006

In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.
Cloning, characterization, and DNA sequence of a human cDNA encoding neuropeptide tyrosine.
Authors: Minth C D , Bloom S R , Polak J M , Dixon J E
Published: May 2006

In vitro translation of the RNA isolated from a human pheochromocytoma demonstrated that this tumor contained a mRNA encoding a 10.5-kDa protein, which was immunoprecipitated with antiserum raised against porcine neuropeptide Y. Double-stranded cDNA was synthesized from total RNA and inserted into the Pst I site of pUC8. Transformants containing the neuropeptide Y cDNA were identified using the mixed hybridization probe d[A-(A,G)-(A,G)-T-T-(A,G,T)-A-T-(A,G)-T-A-(A,G)-T-G]. The probe sequences were based on the known amino acid sequence, His-Tyr-Ile-Asn-Leu, found in porcine neuropeptide Y. The nucleotide sequence of the cDNA was determined and contained 86 and 174 bases in the 5'- and 3'-untranslated regions, respectively. The coding sequence consisted of 291 bases, suggesting a precursor to neuropeptide Y that was 97 amino acids long (10,839 Da). The deduced amino acid sequence of the precursor suggested that there were at least two sites of proteolytic processing, which would generate three peptides having 28 (signal peptide), 36 (human neuropeptide Y), and 30 (COOH-terminal peptide) amino acid residues. A partial NH2-terminal sequence obtained by Edman degradation of the immunoprecipitated in vitro translation product identified the positions of methionine and leucine in the first 30 residues of the prepropeptide. A highly sensitive single-stranded complementary mRNA hybridization probe specific for neuropeptide Y mRNA was prepared using the bacteriophage SP6 promoter. This probe was used to identify a mRNA corresponding to neuropeptide Y of approximately 800 bases.
Separation of glutamine synthetase species with different states of adenylylation by chromatography on monoclonal anti-AMP antibody affinity columns.
Authors: Chung H K , Rhee S G
Published: May 2006

Glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] from Escherichia coli is composed of 12 identical subunits arranged in two superimposed hexagonal arrays. Therefore, partially adenylylated GS is a mixture of hybrid molecules containing different numbers (from 0 to 12) and distributions of adenylylated subunits. In an effort to separate these GS species into uniquely adenylylated molecular species, immunoadsorbants were prepared by covalent attachment of monoclonal antibodies, 72-76-1 (IgG2a and 37-2-1 (IgM), to Affi-Gel 10. The bound GS was eluted with AMP gradients. The results indicate that the IgG column and IgM column yield significantly different elution profiles; the column prepared with the IgG monoclonal antibody better resolves GS containing fewer than 3 adenylylated subunits per dodecamer, whereas the column prepared from the IgM monoclonal antibody better resolves the GS containing higher numbers (from 4 to 12) of adenylylated subunits. In addition, the results indicate that both the distribution and the number of adenylylated subunits are important factors in this separation.
Nucleotide sequence 5' of the chicken c-myc coding region: localization of a noncoding exon that is absent from myc transcripts in most avian leukosis virus-induced lymphomas.
Authors: Shih C K , Linial M , Goodenow M M , Hayward W S
Published: May 2006

We have determined the nucleotide sequence of the 2.2-kilobase-pair region upstream of the chicken c-myc coding exons. Using RNA blot analysis, we have localized a noncoding exon to a region that is separated from the c-myc coding sequences by an intron of 700-800 base pairs. In most avian leukosis virus-induced lymphomas proviral integration has occurred within, or downstream of, the first exon, thus presumably displacing the regulatory sequences that normally control c-myc expression. More than 70% of the integration sites were clustered in a 250-base-pair region in the first intron, immediately preceding the coding sequences. Sequences from the upstream noncoding exon were absent from the myc transcripts in these lymphomas; RNA transcripts from the normal c-myc allele were not expressed at detectable levels.
Outline of a theory of cellular heterogeneity.
Authors: Elsasser W M
Published: May 2006

In the effort to advance toward a theory of cellular function that is not completely mechanistic, I have encountered a variable whose significance is often overlooked. This is the degree of heterogeneity of organic tissue, which may vary from highly homogeneous conditions to quite large degrees of heterogeneity, primarily with respect ot chemical bonding. For several decades, I have dealt with the theoretical analysis of this type of problem, and here I give a condensed outline of the conceptual changes to which such an analysis is likely to lead. I believe the time is ripe to compare these theoretical arguments with observations. The data that occasion this note are those of Rubin [Rubin, H. (1984) Proc. Natl. Acad. Sci. USA 81, 5121-5125]; they and numerous similar observations suggest the possibility of an advance toward a nonreductionist model of cellular function along lines that are here indicated; the theoretical model is thought of as operating entirely within the framework of quantum mechanics.
Catalytic consequences of oligomeric organization: kinetic evidence for "tethered" acto-heavy meromyosin at low ATP concentrations.
Authors: Hackney D D , Clark P K
Published: May 2006

The influence of the supramolecular organization of myosin on its ATPase activity was investigated at a range of ATP concentrations, using as a model system subfragment 1 (S1) and heavy meromyosin (HMM), which are respectively monomeric and dimeric proteolytic fragments of myosin. At low ATP levels in the presence of a molar excess of actin, dimeric HMM showed an increased rate of ATP hydrolysis relative to that for monomeric S1. This increased ATPase for HMM was inhibited by high concentrations of ATP, which reduced the acto-HMM ATPase rate to the lower level of acto-S1. This observation is consistent with the rapid ATP hydrolysis of acto-HMM at low ATP being due to rapid product release from a "tethered" acto-HMM species, which has product bound to one head group while the other head group remains bound to actin. At high concentrations of ATP, ATP binds to both head groups, resulting in net dissociation of HMM from actin. This model is supported by 18O exchange data. Acto-HMM hydrolyzed ATP with extensive exchange of water oxygens into Pi at high ATP levels, but not at low ATP levels. Acto-S1 exhibited extensive exchange at both high and low ATP levels. This result is consistent with rapid product release from a tethered acto-HMM intermediate at low ATP.
Mouse kidney and submaxillary gland renin genes differ in their 5' putative regulatory sequences.
Authors: Panthier J J , Dreyfus M , Roux T L , Rougeon F
Published: May 2006

The genomic DNAs that specify the renin mRNAs found in the submaxillary gland and in the kidney of Swiss mice have been isolated by molecular cloning in phage lambda. The Ren1 and Ren2 genes, encoding the kidney and submaxillary renins, respectively, span about 9.5 kilobases each. Restriction maps of both renin genes and their flanking sequences are presented. To correlate the differential expression of the renin genes with their structures, a sequence analysis of the putative 5' regulatory sequences of both genes was conducted. The results show that the leader sequences and promoter regions of both genes are highly homologous to a point located 179 nucleotides upstream from the transcription start point; the sequences diverge beyond this position.