bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of an 86-kilodalton glycoprotein in an idiopathic mammary adenocarcinoma of a BALB/c mouse.
Authors: Sarkar N H , Etkind P R , Lasfargues E Y , Whittington E S
Published: May 2006

An epithelial cell line (BALB/c-ST) was established from a mammary adenocarcinoma that developed spontaneously in a 20-month-old BALB/c mouse. Like uninfected normal tissues, the cell line was found to contain three endogeneous murine mammary tumor virus (MuMTV) proviruses, but MuMTV particles and antigens were not detected. The cultured cells, after being inoculated subcutaneously, produced tumors in syngeneic mice, and sera from a high percentage of the tumor-bearing mice specifically immunoprecipitated an 86-kilodalton antigen from the extracts of BALB/c-ST cells. This antigen was found to be glycosylated, but whether or not it was exposed to the cell surface could not be demonstrated by cell-surface iodination and immunoprecipitation studies. The 86-kilodalton antigen, a glycoprotein designated gp86, was not detected in immunoprecipitates from extracts of normal mammary cells or from the extracts of GR, C3H, and BALB/cfC3H mammary tumor cells. After being infected in vitro with RIII-derived MuMTV but not with C3H-derived MuMTV, the BALB/c-ST cells appeared to undergo a phenotypic change in that they did not produce tumors in syngeneic mice, and the expression of gp86 was inhibited. Our results indicate that the expression of gp86 in the mammary cells of BALB/c mice is a consequence of neoplastic transformation and that MuMTV infection modulates its expression.
Avian myeloblastosis virus and E26 virus oncogene products are nuclear proteins.
Authors: Boyle W J , Lampert M A , Lipsick J S , Baluda M A
Published: May 2006

The defective acute leukemia viruses avian myeloblastosis virus (AMV) and E26 virus each contain an inserted cellular sequence related to the same highly conserved cellular gene, proto-amv. The oncogenes of these two retroviruses differ from this cellular proto-oncogene in gene structure, transcript structure, and gene product. The product of the AMV oncogene (myb) is a 48,000 Mr protein, p48myb, encoded by a transduced segment (amv) of proto-amv flanked by short helper-virus-derived terminal sequences. The E26 virus oncogene product is a 135,000 Mr protein, p135gag-amve-ets, encoded by significant portions of a viral structural gene (gag), sequences related to proto-amv (amve), and additional E26-specific sequences (ets) transduced from cellular proto-ets. Both p48myb and p135gag-amve-ets transforming proteins are located in the nucleus of cells transformed by these viruses. A protein of 110,000 Mr which is specifically immunoprecipitated by antisera to amv peptides and may be the product of the normal cellular gene (proto-amv) has been located in the cytoplasm of cells that express proto-amv mRNA.
An
Escherichia coli
mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme
Authors: Chang Ying-Ying , Cronan John E.
Published: May 2006

The pyruvate oxidase (pyruvate:ferricytochrome
b
1
oxidoreductase, EC 1.2.2.2) of
Escherichia coli
is markedly activated by phospholipids
in vitro
. To test the physiological relevance of this activation, we isolated an
E. coli
mutant producing an oxidase that is deficient in activation by (and binding to) phospholipids. The mutant oxidase could be fully activated by a specific proteolytic cleavage, indicating that the catalytic site is normal. The mutant enzyme functions poorly
in vivo
, indicating that activation of the oxidase by phospholipids plays an important physiological role.
Soliton-assisted unidirectional circulation in a biochemical cycle
Authors: Careri Giorgio , Wyman Jeffries
Published: May 2006

The unidirectional circulation of an enzyme round a working cycle can be understood in terms of the formation and decay of a soliton that acts as a ligand for the protein and for which the surrounding heat bath serves as a sink. An experimental confirmation of this interpretation is suggested in which the soliton is of the Davidov type and is created by infrared photon absorption as in recent experiments on acetanilide.
Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts.
Authors: Iba H , Takeya T , Cross F R , Hanafusa T , Hanafusa H
Published: May 2006

The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.
Long terminal repeat nucleotide sequence and specific insertion of the gypsy transposon.
Authors: Freund R , Meselson M
Published: May 2006

We have determined the nucleotide sequences of the long terminal repeats of the transposable element gypsy from the cloned mutant alleles sc1, bx3, and bx34e. These mutations are suppressible by the suppressor of Hairy-wing, su(Hw). The long terminal repeats are 482 base pairs long and are highly conserved. In each case, gypsy is inserted into the sequence T-A-C-A-T-A and generates a duplication of the sequence T-A-C-A. This was verified by sequencing an empty site in the wild-type bx gene. Consideration of the sequence of the long terminal repeats and their surroundings limits the possible explanations for the mechanism of mutation by these gypsy insertions and for their suppression by su(Hw).
Nuclease S1 mapping of a homozygous mutation in the carboxyl-propeptide-coding region of the pro alpha 2(I) collagen gene in a patient with osteogenesis imperfecta.
Authors: Dickson L A , Pihlajaniemi T , Deak S , Pope F M , Nicholls A , Prockop D J , Myers J C
Published: May 2006

The molecular defect in a patient with a moderately severe form of osteogenesis imperfecta was characterized by nuclease S1 mapping. Single-stranded 5' and 3' end-labeled DNA probes coding for 80% of the carboxyl-propeptide of the pro alpha 2(I) collagen gene were hybridized to mRNA isolated from cultured fibroblasts of the patient and his parents. Nuclease S1 digestion revealed a homozygous mutation in the patient and a heterozygous pattern in the consanguineous parents. As a result of the defect in the gene, none of the pro alpha 2(I) chains synthesized by the patient's fibroblasts were incorporated into a type I procollagen heterotrimer consisting of two pro alpha 1(I) chains and one pro alpha 2(I) chain. Cultured skin fibroblasts from the patient have previously been shown to secrete only pro alpha 1(I) trimers. As shown here, fibroblasts from both parents, who do not have osteogenesis imperfecta, secrete both pro alpha 1(I) trimers and normal type I procollagen. A further observation was that synthesis of pro alpha 2(I) chains was decreased in fibroblasts from the patient and his parents. The decrease in the synthesis of pro alpha 2(I) chains is not caused by decreased transcription of the pro alpha 2(I) collagen alleles, since the pro alpha 1(I)/pro alpha 2(I) mRNA ratios were normal in the patient and his parents.
Specific binding of leukotriene C4 to ileal segments and subcellular fractions of ileal smooth muscle cells.
Authors: Krilis S , Lewis R A , Corey E J , Austen K F
Published: May 2006

A specific high-affinity receptor for leukotriene C4 (LTC4) has been identified on segments of longitudinal smooth muscle from guinea pig ileum, in disrupted cells obtained from the ileal segments, and in subcellular fractions enriched for mitochondrial membranes and plasma membranes, respectively. Specific [3H]LTC4 binding at a fixed input at 4 degrees C reached a plateau at 60 min with each of the four preparations and was greater than 80% reversible after binding reached equilibrium by the introduction of excess unlabeled homoligand. LIGAND analysis demonstrated a single high-affinity receptor on the smooth muscle segments, the disrupted cells, and the subcellular fractions enriched for mitochondrial membranes and for plasma membranes with respective Kd values of 7.6 nM, 1.3 nM, 13 nM, and 8.5 nM. These Kd values overlap with the concentration of LTC4 known to elicit a spasmogenic response in the ileal muscle, indicating that the radioligand recognizes a receptor that mediates the biological response. Competition analysis with disrupted ileal cells and subcellular fractions with a fixed input of LTC4 radioligand and incremental concentrations of the natural sulfidopeptide leukotrienes demonstrated leukotriene D4 (LTD4) to be 1-3 logarithms less active than LTC4 and leukotriene E4 (LTE4) to be essentially inactive. Thus, ileal longitudinal smooth muscle cells possess a high-affinity receptor that is selective for LTC4 and that receptor exhibits both a plasma membrane and subcellular distribution.
Modulation of human lymphocyte responses by low density lipoproteins (LDL): enhancement but not immunosuppression is mediated by LDL receptors.
Authors: Cuthbert J A , Lipsky P E
Published: May 2006

The role of low density lipoprotein (LDL) receptors in mediating the immunomodulatory effects of LDL was examined by comparing responses of normal lymphocytes with those obtained from a patient with familial hypercholesterolemia (FH) lacking receptors for LDL. The function of LDL receptors in supporting lymphocyte growth was demonstrated by blocking endogenous sterol synthesis with mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and culturing cells in lipoprotein-deficient medium with supplemental LDL as the only source of cholesterol. Mevinolin inhibited mitogen-induced proliferation of normal and FH lymphocytes. Whereas inhibition was overcome by mevalonate, the product of the inhibited enzyme, low concentrations of LDL (less than 10 micrograms of protein/ml) restored the responses of normal but not FH lymphocytes. When normal and FH lymphocytes were cultured in the absence of mevinolin, high concentrations of LDL (greater than 100 micrograms of protein/ml) inhibited mitogen-induced lymphocyte proliferation. The inhibitory effects on normal and FH lymphocytes were similar in that both required comparably large concentrations of LDL and could be completely reversed by transferrin. When normal lymphocytes were cultured in serum-free medium supplemented with transferrin, low concentrations of LDL (less than 10 micrograms of protein/ml) caused marked augmentation of proliferation. By contrast, no enhancement of FH lymphocyte growth was observed. These results indicate that LDL-mediated enhancement of lymphocyte growth in the presence or absence of endogenous sterol biosynthesis involves specific receptors for LDL whereas the immunosuppression caused by LDL is independent of these receptors. Moreover, the results suggest that peripheral lymphocytes can be used to evaluate the functional integrity of the receptor-mediated uptake of LDL.
Radioprotection by glutathione ester: transport of glutathione ester into human lymphoid cells and fibroblasts.
Authors: Wellner V P , Anderson M E , Puri R N , Jensen G L , Meister A
Published: May 2006

Glutathione is not effectively transported into human lymphoid cells, normal human skin fibroblasts, and fibroblasts from patients with genetic deficiencies of gamma-glutamylcysteine synthetase or glutathione synthetase. On the other hand, the monoethyl ester of glutathione, in which the carboxyl group of the glycine residue is esterified, is readily transported into these cells and is hydrolyzed intracellularly. This leads to greatly increased cellular levels of glutathione, which often exceed those found normally. Glutathione ester was found to protect human lymphoid cells of the CEM line against the lethal effects of irradiation. Under the conditions employed, complete protection was found when the ester was added prior to irradiation. Addition of the ester after irradiation was partially effective, suggesting that GSH may also function in repair processes.
Characterization of a cDNA coding for human protein C.
Authors: Foster D , Davie E W
Published: May 2006

Protein C is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it readily inactivates factor Va and factor VIIIa, two proteins that participate as cofactors in the blood coagulation cascade. In the present studies, a lambda gt11 library containing cDNA inserts prepared from human liver mRNA has been screened with an antibody to human protein C. Seven positive clones were isolated from 2 X 10(6) phage and were plaque-purified. The cDNA inserts of two of these phage were sequenced and shown to code for human protein C. Each cDNA insert coded for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a 3'-noncoding region, and a poly(A) tail. The length of the noncoding sequence on the 3' end differed in the two clones, but each contained a processing or polyadenylylation signal followed by a poly(A) tail. The amino acid sequence as determined from the cDNA indicates that protein C is synthesized as a single-chain polypeptide containing the light chain and the heavy chain connected by a dipeptide of Lys-Arg. The single-chain molecule is then converted to the light and heavy chains by cleavage of two or more internal peptide bonds. In plasma, the heavy and light chains of protein C are linked together by a disulfide bond. The amino acid sequence of human protein C shows a high degree of homology with that of the bovine molecule. The DNA sequence coding for the catalytic region near the active site serine in human protein C also showed a high degree of DNA and amino acid sequence identity with prothrombin, factor IX, and factor X, three of the other vitamin K-dependent serine proteases that are present in plasma.
Detection of cis and trans X-Pro peptide bonds in proteins by 13C NMR: application to collagen.
Authors: Sarkar S K , Young P E , Sullivan C E , Torchia D A
Published: May 2006

Heretofore the complexity of natural abundance spectra has precluded the use of 13C NMR to detect cis peptide bonds in proteins. We have incorporated [4-13C]proline into chicken calvaria collagen and report here well-resolved C gamma signals, arising from cis and trans X-Pro and X-Hyp peptide bonds (where X is any amino acid residue) in the 13C NMR spectrum of the thermally unfolded protein. Measurement of 13C signal areas shows that 16% of the X-Pro and 8% of X-Hyp bonds are cis in the unfolded collagen. These results strongly support the conclusion drawn from kinetic studies that cis-trans isomerization of peptide bonds is the rate-limiting step in helix propagation after nucleation. Our method can be applied to other proteins as well and should aid in testing the generality of the hypothesis of Brandts, Halvorson, and Brennan that cis-trans isomerization is the rate-limiting step in protein folding when proline is present.
Isoelectric focusing of plant cell membranes
Authors: Griffing Lawrence R. , Quatrano Ralph S.
Published: May 2006

An isoelectric focusing method is described that discriminates plant cell organelle populations on the basis of surface charge. The isoelectric points (pI values) of the Golgi apparatus, the mitochondria, and putative plasma membrane from etiolated pea stem cells are reported. The pI of the pea Golgi apparatus is similar to that of the
35
SO
4
-labeled membranes of developing
Fucus
embryos. The pI of
Fucus
35
SO
4
-labeled membranes depends on whether the membranes are being polarly transported to the growing tip or randomly transported to the entire periphery of the zygote. Those subject to polar tip transport have a significantly greater negative surface charge than those being randomly transported. The implication of this result toward an understanding of the localization mechanism is discussed. The method is also capable of subfractionating glucan synthetase II-containing membranes (putative plasma membrane) from peas. The pI of putative plasma membrane from a pea stem homogenate is similar to the pI of the plasma membrane of whole protoplasts of
Catharanthus
leaf cells. Isoelectric focusing appears to be a useful technique to discriminate membranes and, hence, to provide new information and approaches to study cellular and developmental phenomena.
Eukaryotic chromosome transfer: linkage of the murine major histocompatibility complex to an inserted dominant selectable marker.
Authors: Weis J H , Nelson D L , Przyborski M J , Chaplin D D , Mulligan R C , Housman D E , Seidman J G
Published: May 2006

We have developed an approach for genetic analysis of the murine H-2 complex that has broad general applicability to the study of eukaryotic genome organization. We have used a retroviral vector to introduce a selectable marker into the mouse genome close to the major histocompatibility complex (MHC). Chromosomal segments containing large portions of the MHC from these donor cells have been transferred both to hamster and monkey cell recipients. The procedure involved the following steps. First, a murine cell line was multiply infected with a defective recombinant murine leukemia virus that contains the neomycin-resistance gene (a gene that confers resistance to G418). In this way, the neomycin-resistance gene was introduced at multiple sites in the mouse genome. Second, metaphase chromosomes, prepared from this infected cell population, were transferred to hamster cell recipients. Third, two G418-resistant transferents were identified that expressed murine H-2 antigens on their cell surface. These transferents were shown to contain a large segment of the murine MHC (H-2K and I regions) by DNA hybridization. The neomycin-resistance gene and the mouse MHC genes must be physically linked in these cells since they could be cotransferred from the hamster cells to monkey cells. Fourth, the murine cell carrying the neomycin-resistance gene near the MHC was identified from the original donor cell population. This cell will serve as a useful source of chromosome fragments for analysis of larger portions of the MHC. This series of steps can serve as a paradigm for the first steps in a detailed genetic analysis of any specific region of a mammalian genome to which one or more genes have already been mapped.
ADP-dependent common receptor mechanism for binding of von Willebrand factor and fibrinogen to human platelets.
Authors: Timmons S , Kloczewiak M , Hawiger J
Published: May 2006

Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by prostaglandin I2, a known activator of platelet adenylate cyclase. A purine nucleotide affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies the ADP binding sites on the human platelet membrane, prevented binding of vWF induced with ADP, as well as with human thrombin and with ionophore A23187, agents known to cause platelet ADP secretion. By comparison, FSBA did not inhibit binding of vWF induced by ristocetin, indicating that the ristocetin mechanism is not dependent on ADP. Human fibrinogen inhibited in a competitive manner the ADP-induced binding of 125I-labeled vWF (9 micrograms/ml) with an IC50 of 25 micrograms/ml. Conversely, unlabeled vWF inhibited ADP-induced binding of 125I-labeled fibrinogen (60 micrograms/ml) with an IC50 of 16 micrograms/ml. A synthetic dodecapeptide (Mr, 1188), analogous with the specific platelet receptor recognition site of human fibrinogen gamma chain (gamma 400-411), inhibited binding of both 125I-labeled vWF and 125I-labeled fibrinogen to ADP-treated platelets, whereas it was without effect on binding of 125I-labeled vWF to ristocetin-treated platelets. These data indicate that vWF and fibrinogen have a common receptor mechanism for their interaction with human platelets that is dependent on ADP occupancy of its binding sites and is recognized by the sequence of 12 amino acid residues at the carboxyl terminus of the human fibrinogen gamma chain.
Purification of the muscarinic acetylcholine receptor from porcine atria.
Authors: Peterson G L , Herron G S , Yamaki M , Fullerton D S , Schimerlik M I
Published: May 2006

The muscarinic acetylcholine receptor from porcine atria has been purified 100,000-fold to homogeneity by solubilization in digitonin/cholate and sequential chromatography on wheat germ agglutinin-agarose, diethylaminoethylagarose, hydroxylapatite, and 3-(2'-aminobenzhydryloxy)tropane-agarose. The yield of purified receptor was 4.3% of that found in the membrane fraction, and the purified receptor bound 11.1-12.8 nmol of L-[3H]quinuclidinyl benzilate per mg of protein, corresponding to a binding component Mr of 78,400-90,000. The purified receptor preparation consisted of two polypeptides in approximately equimolar amounts when examined on silver-stained sodium dodecyl sulfate/polyacrylamide gels. The larger polypeptide (Mr 78,000 on 8% polyacrylamide gels) was specifically alkylated with [3H]propylbenzilylcholine mustard, whereas the smaller polypeptide (Mr 14,800) was not labeled. The possibility that the small polypeptide is a contaminant fortuitously appearing in equimolar amounts with the large polypeptide cannot be ruled out at this time. The purified preparation was highly stable, with no measurable change in the number of ligand binding sites or the gel pattern after 1 month's storage on ice. Scatchard analysis showed a single class of binding sites for the antagonist L-[3H]quinuclidinyl benzilate with a dissociation constant of 61 +/- 4 pM. Equilibrium titration experiments demonstrated that the antagonist L-hyoscyamine displaced L-[3H]quinuclidinyl benzilate from a single class of sites (Kd = 475 +/- 30 pM), whereas the agonist carbamoylcholine interacted at two populations of sites (53% +/- 3% high affinity, Kd = 1.1 +/- 0.3 microM; 47% +/- 3% low affinity, Kd = 67 +/- 14 microM). The ligand binding data were very similar to that for the membrane-bound receptor, suggesting that the receptor has not been altered radically during purification.
Rat apolipoprotein A-IV contains 13 tandem repetitions of a 22-amino acid segment with amphipathic helical potential.
Authors: Boguski M S , Elshourbagy N , Taylor J M , Gordon J I
Published: May 2006

Apolipoprotein (apo) A-IV is a 46,000 Mr protein that is a major component of rat high density lipoproteins and chylomicrons. We have isolated, from a rat intestinal cDNA library, a full-length apo A-IV clone of 1423 base pairs and determined its nucleotide sequence. The 1173-nucleotide coding region specifies a protein of 391 amino acids, which includes a 20-amino acid signal peptide. The portion of the cDNA sequence representing the mature plasma protein contains a 66-nucleotide subsequence that is repeated at least 13 times. Although each repeated unit shows some sequence variation, base changes in comparably positioned codons generally conserve the chemical type, if not the identity, of the corresponding amino acids. Nine of the thirteen 22-amino acid repeat units in the derived protein sequence begin with proline and most of these docosapeptides are predicted to have a high content of alpha-helix according to Chou-Fasman rules. When hydration potentials of individual residues are considered, many of the helices are shown to be amphipathic and may thus constitute lipid-binding domains with the ability to activate lecithin:cholesterol acyltransferase. The structure and organization of the repeat units of rat apo A-IV bear a striking similarity to a corresponding repeated sequence block in human apo A-I. This finding raises the possibility of close tandem linkage of the apo A-I and A-IV genes and suggests unequal crossing-over as a mechanism for the evolution of these genes.
Iron-carbon bond lengths in carbonmonoxy and cyanomet complexes of the monomeric hemoglobin III from Chironomus thummi thummi: a critical comparison between resonance Raman and x-ray diffraction studies.
Authors: Yu N T , Benko B , Kerr E A , Gersonde K
Published: May 2006

Soret-excited resonance Raman spectroscopy yields direct information regarding the iron-carbon bonding interactions in the cyanomet and carbonmonoxy complexes of hemoglobin III from Chironomus thummi thummi (CTT III) in solution. By isotope exchange in cyanide (13CN-, C15N-, and 13C15N-) and carbon monoxide (13CO, C18O, and 13C18O), we have assigned the Fe(III)-CN- stretching at 453 cm-1, the Fe(III)-C-N- bending at 412 cm-1, the Fe(II)-CO stretching at 500 cm-1, the Fe(II)-C-O bending at 574 cm-1, and the C-O stretching at 1960 cm-1. The resonance Raman data, in conjunction with those obtained from heme model complexes with well-known Fe-C bond distances, strongly suggest that the Fe(III)-CN- bond (approximately 1.91 A) is longer (hence weaker) than the Fe(II)-CO bond (approximately 1.80 A). This result disagrees with those of x-ray crystallographic studies [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338] in which the Fe-C bond lengths were reported as 2.2 A in cyanomet and 2.4 A in carbonmonoxy CTT III. Based on Badger's rule and normal mode calculations, the x-ray data would lead to the prediction of 279 cm-1 for the Fe(II)-CO stretching frequency in CTT III . CO, which was not observed. On the other hand, we estimate the Fe-CO bond as approximately equal to 1.82 A, which is very similar to the 1.80-A value in human Hb . CO crystals. Furthermore, we have used isotope shift data to estimate the Fe-C-O angle as 169 +/- 5 degrees, somewhat larger than the 161 degrees value found by Steigemann and Weber. We therefore conclude that there must be errors in the x-ray crystallographic refinement for the ligand geometry in carbonmonoxy and cyanomet CTT III.
Estimation of mating system parameters when outcrossing events are correlated
Authors: Schoen Daniel J. , Clegg Michael T.
Published: May 2006

Many plant species have mating systems characterized by a mixture of self-fertilization and outcrossing. Statistical estimation of the outcrossing rate has relied on a model of the mating process that assumes that successive outcross events within a family arise from independent draws of pollen from the total population of male plants. Although this assumption is likely to be most appropriate for wind-pollinated plants, it is not appropriate in certain insect-pollinated plants. An alternative model is developed that assumes that successive outcross events within a family involve pollen drawn from a single male parent. The estimation of the parameters that index this model is outlined and a procedure for calculating the variances of the parameter estimates is presented. Monte Carlo simulations of the sampling processes assumed by each model are also presented. The simulations show that application of the incorrect estimation model to data can lead to a large bias in parameter estimates.
Inhibition of DNA synthesis by an electrophilic metabolite of benzo[a]pyrene.
Authors: Busbee D L , Joe C O , Norman J O , Rankin P W
Published: May 2006

Mitogen-stimulated scheduled DNA synthesis and DNA excision repair in human lymphocytes, as well as DNA polymerase a activity in a cell-free system, were inhibited by an electrophilic metabolite of benzo[a]pyrene. This metabolite, (+/-)-anti-(7r,8t)-dihydroxy-(9,10t)-epoxy-7,8,9,10-tetrahyd robenzo[a]pyrene (BPDE), covalently binds to cellular macromolecules and is mutagenic, carcinogenic, and cytotoxic. Human lymphocytes treated with BPDE at concentrations greater than 500-800 ng/ml showed decreases in both mitogen-stimulated DNA synthesis and excision repair of damaged DNA but did not exhibit overt cytotoxicity (excluded trypan blue and maintained an adenylate charge of greater than 0.7). Formation of, and total concentration of, BPDE-DNA adducts was not correlated with inhibition of DNA synthesis. DNA polymerase alpha studies using a cell-free system showed that enzymatic activity was not diminished when purified polymerase was treated with BPDE prior to the addition of template DNA. When the template DNA concentration was varied, BPDE inhibition of enzyme activity was uncompetitive. BPDE inhibition of enzyme activity was found to be noncompetitive when concentrations of dATP, dCTP, or dTTP were varied and competitive when the concentration of dGTP was varied. The data indicate that BPDE competitively inhibits interaction of dGTP with the template-DNA polymerase alpha complex.
A position 12-activated H-ras oncogene in all HS578T mammary carcinosarcoma cells but not normal mammary cells of the same patient.
Authors: Kraus M H , Yuasa Y , Aaronson S A
Published: May 2006

Among 21 human mammary tumors analyzed for transforming genes by transfection of NIH/3T3 cells, only DNA of a carcinosarcoma cell line, HS578T, registered as positive. A Harvey (H)-ras oncogene identified in this line was cloned in biologically active form and the activating lesion was identified as a single nucleotide substitution of adenine for guanine in the 12th codon. This results in substitution of aspartic acid for glycine at this position of the p21 coding sequence. Knowledge that this alteration creates a restriction site polymorphism for Msp I/Hpa II in the H-ras protooncogene made it possible to survey for the presence of the activated H-ras allele in normal cells as well as in clonally derived tumor cell lines of the same patient. We demonstrated the presence of unaltered H-ras alleles in normal HS578 cells. In contrast, every clonally derived HS578T tumor cell line analyzed contained the H-ras oncogene possessing the genetic alteration at position 12. These findings establish that activation of this oncogene was the result of a somatic event selected within all HS578T tumor cells.
Isolation of immortal cell lines from the first stage of murine leukemia virus-induced leukemia.
Authors: Oliff A , Oliff I , Schmidt B , Famulari N
Published: May 2006

Friend murine leukemia virus (F-MuLV) is a replication-competent retrovirus that induces a rapidly fetal leukemia in susceptible mice (stage I disease). Leukemia cells obtained from these animals do not grow in cell culture using standard tissue culture conditions. However, in the presence of WEHI-3 cell-conditioned medium (CM), 100% of spleen or bone marrow explants from diseased mice yield immortal cell lines. These cell lines exhibit the same growth properties, produce the same viruses, and express the same oncogenes as the leukemia cells found in mice with stage I disease. No cell lines were obtained from leukemic mice in the absence of CM. No cell lines were obtained from uninfected adult, newborn, or phenylhydrazine-treated animals with or without CM. We conclude that some of the hematopoietic cells in F-MuLV-diseased mice will proliferate indefinitely in the presence of CM. The development of this abnormal response to CM is one of the early changes associated with F-MuLV-induced leukemia.
Repeatability of taxon longevity in successive foraminifera radiations and a theory of random appearance and extinction
Authors: Levinton Jeffrey S. , Ginzburg Lev
Published: May 2006

An analysis of taxonomic longevity for species of the two Cenozoic radiations of Caribbean planktonic foraminifera shows strong similarity in longevity for extinct taxa, though extant species of the latter radiation are biased towards longer-lived forms. In this case, the present is not the key to the past. A simple one-parametric stochastic model predicts generally the shape of the distributions, though there is an excess of species with model longevity and a deficiency of longer-lived forms, relative to the prediction of the model. Although the one parameter may relate to the biology of the group under consideration, the distribution about the mode may reflect random appearance-extinction processes.
Expression of the Thy-1 glycoprotein gene by DNA-mediated gene transfer.
Authors: Evans G A , Ingraham H A , Lewis K , Cunningham K , Seki T , Moriuchi T , Chang H C , Silver J , Hyman R
Published: May 2006

We isolated a gene encoding the Thy-1.2 glycoprotein from a recombinant library constructed from BALB/c mouse DNA. To evaluate the expression of this cloned gene in different genomic environments, we introduced it into cell lines derived from fibroblast, lymphoid, and neuronal tissues by DNA-mediated gene transfer. When integrated into the genome of mouse L cells, cell-surface Thy-1 can be detected with anti-Thy-1 monoclonal antibodies. These L-cell lines contain between two and four copies of the cloned Thy-1 gene stably integrated in the host genome. After subcloning into a plasmid vector containing the bacterial Eco-gpt gene as a selectable marker, the Thy-1 gene was introduced into the Thy-1-deficient mouse lymphoma AKR1 (Thy-1-d), and the rat neuronal cell line, B50. The resulting transformants also contain two to four copies of the cloned Thy-1 gene but express up to 50-fold more cell-surface Thy-1.2 than the L cell transformants. The expression of vastly differing amounts of cell-surface Thy-1 from similar numbers of genes suggests that the gene encoding this differentiation antigen is under tissue-specific regulation.
Spider venoms inhibit L-glutamate binding to brain synaptic membrane receptors.
Authors: Michaelis E K , Galton N , Early S L
Published: May 2006

The venoms from three spider species, Araneus gemma, Neoscona arabesca, and Argiope aurantia, were shown to inhibit the high-affinity, sodium-independent L-glutamate-binding sites in rat brain synaptic membranes. The same three venoms caused concentration-dependent inhibition of the activity of the glutamate-binding glycoprotein purified from rat brain synaptic membranes. The venom milked from the glands of Araneus gemma was the most active inhibitor of L-glutamate binding, causing 60-80% inhibition of both synaptic membrane and purified protein binding activity at 0.01 unit. The inhibitory activity of this venom was associated with a single protein peak obtained from gel permeation chromatography of the venom. Finally, the effect of the venom from Araneus gemma on the synaptic membrane glutamate-binding sites was slowly reversible. These observations indicate that the spider venoms have a direct effect on the recognition sites for L-glutamic acid in brain synaptic membranes and that these sites are related to the physiologic glutamate receptors.
Transient complementation of xeroderma pigmentosum cells by microinjection of poly(A)+ RNA.
Authors: Legerski R J , Brown D B , Peterson C A , Robberson D L
Published: May 2006

An assay has been developed in which excision repair deficiency of xeroderma pigmentosum cells is transiently complemented, as measured by unscheduled DNA synthesis, by microinjection of cytoplasmic poly(A)+ RNA derived from HeLa cells. Four different complementation groups of xeroderma pigmentosum have been assayed. Groups A and G showed complementation, whereas groups D and F did not. Survival for cells in each of the groups subsequent to microinjection was approximately equal to 75%. Approximately 10-25% of surviving cells from groups A and G were complemented, as judged by near-normal unscheduled DNA synthesis. Fractionation of cytoplasmic poly(A)+ RNA on a 15-30% nondenaturing sucrose gradient and subsequent microinjection of the individual fractions indicate that repair mRNAs that complement xeroderma pigmentosum groups A and G sediment at approximately 11 S and 12 S, respectively. This assay should be of great utility in the cloning and biochemical analysis of DNA repair genes.
Theory of Mössbauer spectra of proteins fluctuating between conformational substates.
Authors: Nadler W , Schulten K
Published: May 2006

Mössbauer spectra of 57Fe in proteins fluctuating between different conformational substates are evaluated by means of a two-sided Padée approximation, which can reproduce the low and high frequency dependence of the spectral line shape I(omega) to any desired accuracy. The dynamics of the atom is modeled as Brownian motion in a multiminimum potential and described by a Fokker-Planck equation. The Mössbauer spectrum is expanded in terms of Lorentzian contributions, which can be attributed separately to fluctuations between conformational substates (potential minima) and to relaxation within the substates. In the limit of closely spaced substates, the Mössbauer spectra can be accounted for by an effective diffusion coefficient with Arrhenius-type temperature dependence. We demonstrate that the observed temperature dependence of Mössbauer spectra of proteins [Parak, F., Knapp, E.W. & Kucheida, D. (1982) J. Mol. Biol. 161, 177-194] can be accounted for by stochastic motion in a multiminimum potential.
Localization of senescent cell antigen on band 3.
Authors: Kay M M
Published: May 2006

Senescent cell antigen is a glycosylated polypeptide, migrating in the band 4.5 region of NaDodSO4/polyacrylamide gels, that appears on the surface of senescent and damaged cells. Appearance of the senescent cell antigen initiates specific binding of IgG autoantibodies to it and the removal of erythrocytes (RBCs). Previous experiments suggested that the senescent cell antigen may be immunologically related to an integral membrane protein designated band 3 that is involved in anion transport across the RBC membrane. In the present studies, senescent cell antigen was mapped along the band 3 molecule by using topographically defined fragments of band 3. Both binding of IgG eluted from senescent RBCs ("senescent cell IgG") to defined proteolytic fragments of band 3 in immunoblots and two-dimensional peptide mapping of senescent cell antigen, band 3, and defined proteolytic fragments of band 3 were used to localize senescent cell antigen along the band 3 molecule. The data suggest that the antigenic determinants of the senescent cell antigen that are recognized by physiologic IgG autoantibodies reside on an external portion of a naturally occurring transmembrane fragment of band 3 that has lost a Mr approximately equal to 40,000 cytoplasmic (NH2-terminal) segment and part of the anion-transport region. A critical cell-age-specific cleavage of band 3 appears to occur in the transmembrane, anion-transport region of band 3.
Natural interspecies transfer of mitochondrial DNA in amphibians.
Authors: Spolsky C , Uzzell T
Published: May 2006

mtDNAs of two Central European water frog species, Rana ridibunda and Rana lessonae, were examined by electrophoresis of restriction enzyme fragments. Two types of mtDNA occur in R. ridibunda. One shares with mtDNA of R. lessonae 25.8% of 132 fragments generated by 19 enzymes, corresponding to a nucleotide sequence divergence of 8.1%; the other has diverged from R. lessonae mtDNA by only 0.3%. This latter type is a variant R. lessonae mtDNA that has been transferred into R. ridibunda; the introgression may have occurred via the hybridogenetic hybrid lineages collectively known as Rana esculenta. Of 37 R. ridibunda from Poland, 59% had the typical R. ridibunda mtDNA; 41% had the modified R. lessonae mtDNA as did a single individual from Switzerland (introduced). A single R. ridibunda from Turkey, outside the present range of R. lessonae, had the typical R. ridibunda mtDNA phenotype. Discordancies between inheritance of mitochondrial and nuclear genomes point up the danger of relying on a single molecular feature in reconstructing phylogeny. In addition, studies of mtDNA provide otherwise inaccessible information on complex evolutionary histories of closely related species. A knowledge of these complexities is important to an understanding of phylogenetic relationships and of the genetic processes that underlie the evolution of clonal taxa.
Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK.
Authors: Sclafani R A , Fangman W L
Published: May 2006

The herpes simplex virus type 1 thymidine kinase gene TK complements the defect in five temperature-sensitive mutants and in vitro constructed insertion and deletion mutants of the CDC8 gene of Saccharomyces cerevisiae. The herpes thymidine kinase enzyme acts as both a thymidine kinase and a thymidylate kinase (dTMP kinase). The latter activity is responsible for the cdc8 complementation since all thermosensitive cdc8 mutants are deficient in dTMP kinase activity at all temperatures. However, an intragenic revertant, cdc8-320, which was selected by demanding mitotic growth at the restrictive temperature, exhibits thermolabile dTMP kinase activity. We conclude that CDC8 is the structural gene for dTMP kinase, which catalyzes an essential step in DNA precursor biosynthesis. Previously, it has been shown that the DNA replication defect of cdc8 mutants could not be bypassed by the addition of deoxyribonucleoside triphosphates to permeabilized cells. This apparent discrepancy can be explained by hypothesizing a multiprotein yeast DNA replication complex containing the CDC8 protein.
Structure of the human renin gene.
Authors: Miyazaki H , Fukamizu A , Hirose S , Hayashi T , Hori H , Ohkubo H , Nakanishi S , Murakami K
Published: May 2006

The human renin gene was isolated from a Charon 4A human genomic library and characterized. The gene spans about 11.7 kilobases and consists of 10 exons and 9 introns that map at points that could be variable surface loops of the enzyme. The complete coding regions, the 5'- and 3'-flanking regions, and the exon-intron boundaries were sequenced. The active site aspartyl residues Asp-38 and Asp-226 are encoded by the third and eighth exons, respectively. The extra three amino acids (Asp-165, Ser-166, Glu-167) that are not present in mouse renin are encoded by the separate sixth exon, an exon as small as 9 nucleotides. The positions of the introns are in remarkable agreement with those in the human pepsin gene, supporting the view that the genes coding for aspartyl proteinases have arisen as the result of duplication of a common ancestral gene. As in most eukaryotic genes, the putative T-A-T-A and C-A-A-T sequences, which may play a role in the initiation of gene transcription, are found in the vicinity of -29 and -51 nucleotides of the cap site. Further upstream, at nucleotides -456 to -451, is located the hexanucleotide T-G-T-T-C-T, which has recently been suggested as a binding site for the glucocorticoid receptor. In the 3'-flanking region, there is the conserved hexanucleotide sequence A-A-T-A-A-A, thought to be necessary for polyadenylylation. Blot-hybridization analyses of the isolated gene clone and the total cellular DNA after digestion with restriction enzymes revealed that human renin is encoded by a single gene.
Oxygen regulation of anaerobic and aerobic genes mediated by a common factor in yeast.
Authors: Lowry C V , Zitomer R S
Published: May 2006

The expression of a number of yeast genes is regulated by oxygen levels. While many of these are known to be induced in the presence of oxygen, we have described a gene, ANB1, that responds in the opposite fashion, being expressed only under anaerobic conditions. To identify genes involved in regulation of ANB1 and other oxygen-regulated genes, we selected mutations causing constitutive expression of ANB1, using a fusion of the ANB1 modulator segment to the CYC1 gene. A number of trans-acting mutations affecting a gene designated ROX1 caused constitutive expression of both the fused and wild-type genes, indicating that the ROX1 gene product operates through the ANB1 modulator sequence at the level of transcription. The mutant alleles of ROX1 fall into two phenotypic classes. The rox1-a class is semi-dominant, and the rox1-b class is recessive. One mutant, rox1-a1, is pleiotropic and causes constitutive expression of three oxygen-induced genes--CYC1, SOD (superoxide dismutase), and tr-1 (an oxygen-induced gene with homology to ANB1)--as well as constitutive expression of the oxygen-repressed ANB1 gene. Alleles of the rox1-b class cause constitutive expression of ANB1 but do not affect expression of the oxygen-induced genes tested. The pleiotropy of the rox1-a1 mutant indicates that the ROX1 gene product is involved in coordinate expression of both oxygen-induced and oxygen-repressed genes.
Diversity at the variable-joining region boundary of lambda light chains has a pronounced effect on immunoglobulin ligand-binding activity.
Authors: Azuma T , Igras V , Reilly E B , Eisen H N
Published: May 2006

By recombining lambda light (L) chains having known variable (V) region amino acid or nucleotide sequences with a heavy (H) chain from a myeloma protein or a monoclonal antibody, we obtained reconstituted Igs that differed from each other in sequence by only one or a few amino acid substitutions at known L chain positions. Differences in affinity of the reconstituted Igs for 2,4-dinitrophenyl (DNP) ligands revealed a pronounced effect on Ig binding activity of amino acids at the V-J boundary of the lambda chains. In one instance, two reconstituted Igs that differed about 1000-fold in affinity for epsilon-DNP-aminocaproate differed in primary structure by only a single tyrosine-phenylalanine substitution at the V-J junction (position 98) of their lambda 2 chains--i.e., by only one out of approximately 660 amino acid residues (L + H chains). By focusing on affinity changes, chains with unusual V lambda-J lambda junctional residues were identified. It is possible that because of a critical effect on tertiary structure junctional amino acid variations arising from gene segment assembly (V/J and perhaps V/D/J) constitute an important source of ligand-binding diversity of antibodies.
Increased expression of Ia antigens on resting B cells: an additional role for B-cell growth factor.
Authors: Noelle R , Krammer P H , Ohara J , Uhr J W , Vitetta E S
Published: May 2006

The present studies demonstrate that both T-cell-derived supernatants containing B-cell growth factor (BCGF or BSF) and a partially purified preparation of the B-cell growth factor (BSF-p1) induce an increase in the expression of IA and IE-encoded antigens on small resting B cells. This increase is detectable by 6-8 hr after initiation of culture and is relatively selective, since levels of surface immunoglobulin and H-2 antigens do not increase to the same extent. Although interferon-gamma induces increased expression of Ia antigens on macrophages and dividing neoplastic B cells, it does not induce an increase in the expression of Ia antigens on resting B cells. These results demonstrate that BSF-p1 may play two roles: (i) it acts on resting B cells to increase the levels of Ia antigen expression; and (ii) it sustains the growth of B cells that have been previously activated with mitogens, antigens, or anti-Ig.