bluuebunny/crossref_metadata_2025 huggingface dataset:
Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.
Authors: Lu R C , Moo L , Wong A G
Published: May 2006

The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (S1), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared in the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis was carried out in the presence of Mg2+ and ATP or Mg2+ and adenosine 5-[beta, gamma-imido]triphosphate (AdoPP[NH]P), a 70-kDa band, attributable to a crosslinked (50 kDa + 20 kDa) species, was also observed. This suggests that the conformational change induced by nucleotide binding reduces the distance between the 50-kDa region and the label on SH-1. Similar results were obtained when labeling and photolysis were carried out on trypsin-nicked S1, in which the 25-kDa, 50-kDa, and 20-kDa fragments are held together noncovalently. Further, when labeling with benzophenone-4-iodoacetamide was carried out in the presence of Mg-ATP, which increases the reactivity of another thiol, presumably SH-2, both 45-kDa and 70-kDa species were formed upon photolysis in the absence of ATP, suggesting that SH-2 is close to the 50-kDa region. More of the 70-kDa species was formed, at the expense of the 45-kDa species, when photolysis was carried out in the presence of Mg-ATP. Partial heat denaturation preferentially reduced the crosslinking between the reactive thiols and the 50-kDa region.
DNase I hypersensitive sites within the inducible qa gene cluster of Neurospora crassa.
Authors: Baum J A , Giles N H
Published: May 2006

DNase I hypersensitive regions were mapped within the 17.3-kilobase qa (quinic acid) gene cluster of Neurospora crassa. The 5'-flanking regions of the five qa structural genes and the two qa regulatory genes each contain DNase I hypersensitive sites under noninducing conditions and generally exhibit increases in DNase I cleavage upon induction of transcription with quinic acid. The two large intergenic regions of the qa gene cluster appear to be similarly organized with respect to the positions of constitutive and inducible DNase I hypersensitive sites. Inducible hypersensitive sites on the 5' side of one qa gene, qa-x, appear to be differentially regulated. Employing these and previously published data, we have identified a conserved sequence element that may mediate the activator function of the qa-1F regulatory gene. Variants of the 16-base-pair consensus sequence are consistently found within DNase I-protected regions adjacent to inducible DNase I hypersensitive sites within the gene cluster.
Inhibitory and stimulatory G proteins of adenylate cyclase: cDNA and amino acid sequences of the alpha chains.
Authors: Sullivan K A , Liao Y C , Alborzi A , Beiderman B , Chang F H , Masters S B , Levinson A D , Bourne H R
Published: May 2006

The G protein family of signal transducers includes five heterotrimers, which are most clearly distinguished by their different alpha chains. The family includes Gs and Gi, the stimulatory and inhibitory GTP-binding regulators of adenylate cyclase; Go, a protein of unknown function abundant in brain; and transducin 1 and transducin 2, proteins involved in retinal phototransduction. Using a bovine alpha t1 cDNA as a hybridization probe, we have isolated mouse cDNAs that encode alpha chains of two G proteins. One encodes a polypeptide of 377 amino acids (Mr 43,856), identified as alpha s because it specifically fails to hybridize with any transcript in an alpha s-deficient S49 mouse lymphoma mutant, cyc-; the other encodes a polypeptide of 355 amino acids (Mr 40,482), presumed to be alpha i. These alpha chains and those of the retinal transducins exhibit impressive sequence homology. Of the four, alpha t1 and alpha t2 are most alike (81% identical amino acid residues), whereas the presumptive alpha i is more similar than alpha s to alpha t1 (63% vs. 38% identical residues). Sequence homologies with p21ras and elongation factor Tu identify regions of the alpha chains that form the site for GTP binding and hydrolysis. Further comparison of the alpha-chain sequences suggests additional regions that may contribute to interactions with beta gamma subunits and the receptor and effector components of different signal transduction systems.
Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase.
Authors: Thompson R C , Ohlsson K
Published: May 2006

A potent inhibitor of human leukocyte elastase (EC 3.4.21.37) and cathepsin G (EC 3.4.21.20) and of human trypsin (EC 3.4.21.4) has been purified from human parotid secretions. The complete amino acid sequence of this protein has been determined. The sequence suggests that the protein has two domains of about 54 amino acids, each of which contains four disulfide bonds. On the basis of a limited homology to other protease inhibitors, the antielastase and antitrypsin activities are thought to be properties of the C-terminal and N-terminal domains, respectively. The affinity of the inhibitor for leukocyte elastase is very high, suggesting a functional role for the protein in preventing elastase-mediated damage to oral and possibly other mucosal tissues.
Loss of intercalated membrane particles by treatment with phorbols.
Authors: Zucker-Franklin D , Nabi Z F
Published: May 2006

Because brief exposure to phorbol esters renders normal cells vulnerable to deformation and cytolysis by lymphocytes, it was postulated that these tumor promoters might cause a hitherto unrecognized physical alteration in membrane architecture. To investigate this possibility, four tissue culture cell lines (K-562 erythroleukemia cells, melanoma cells, N1121 adult fibroblasts, and normal fetal fibroblasts) and three blood cell types (lymphocytes, monocytes, and platelets) were subjected to freeze-fracture analysis before and after brief treatment with phorbol myristate acetate. Phorbol myristate acetate caused a 50% reduction of intramembranous particles associated with the external leaflet (E face) of the plasma membrane of every cell except platelets. In contrast, no change in size or number of intramembranous particles associated with the protoplasmic membrane leaflet (P face) was evident. Since the platelet membrane is known to be turned "inside out," as regards the partition coefficient of the intramembranous particles, the disparity between the results obtained with platelets and other cells may serve to determine the nature of intramembranous particles affected by phorbols. Also, since phorbols affect primarily glycolipids and/or glycoproteins anchored in the external membrane leaflet, these findings may provide a useful tool for future exploration of membrane structure.
Promoters selected from random DNA sequences.
Authors: Horwitz M S , Loeb L A
Published: May 2006

We have selected a group of Escherichia coli promoters from random DNA sequences by replacing 19 base pairs at the -35 promoter region of the tetracycline resistance gene tetr of the plasmid pBR322. Substitution of 19 base pairs with chemically synthesized random sequences results in a maximum of 4(19) (about 3 X 10(11)) possible replacement sequences. From a population of about 1000 bacteria harboring plasmids with these random substitutions, tetracycline selection has revealed several functional -35 promoter sequences. These promoters have retained only partial homology to the -35 promoter consensus sequence. In three of these promoters, the consensus alignment shifts 10 nucleotides downstream, allowing the RNA polymerase to recognize another Pribnow box from within the original pBR322 sequence. Two of the sequences promote transcription more strongly than the native promoter. This technique may have application for the selection of additional DNA sequences with varied biological activity.
A potent, heat-stable protein inhibitor of [branched-chain alpha-keto acid dehydrogenase]-phosphatase from bovine kidney mitochondria.
Authors: Damuni Z , Humphreys J S , Reed L J
Published: May 2006

A heat- and acid-stable protein inhibitor of the [branched-chain alpha-keto acid dehydrogenase]-phosphatase was purified over 100,000-fold from extracts of bovine kidney mitochondria. The nearly homogeneous protein was recovered with a yield of 4-8%. The apparent molecular weight of the inhibitor is about 36,000. This protein is a noncompetitive inhibitor of the phosphatase, and the inhibitor constant (Ki) is about 0.13 nM. The inhibition was reversed 50% by about 1.3 mM Mg2+ and about 0.1 mM spermine. This protein inhibitor is different from the cytosolic protein phosphatase inhibitors 1 and 2.
Sequence evolution within populations under multiple types of mutation.
Authors: Golding G B , Aquadro C F , Langley C H
Published: May 2006

DNA sequence and restriction map data from natural populations can be used to estimate the phylogenetic history of observed sequences. Studies of this sort usually examine large regions of DNA where many evolutionary events have taken place. From such data, detailed phylogenies can be constructed and qualitatively different kinds of mutational and substitutional processes can be studied. The value of investigating more than one mutational type is in the power of comparing relative rates of these distinct substitutional processes. In this paper, we construct a neutral model to describe the frequencies of sequence haplotypes according to the haplotypes from which they arose. This theory of the frequency of haplotypes (incorporating their historical past) is applied to data from the alcohol dehydrogenase gene region of Drosophila melanogaster. The observed patterns of change associated with transposable elements around the Adh locus are not in accord with a neutral model. Values for the mutation rates cannot be found that will bring the observed data into agreement with a simple neutral model, but with the addition of mildly deleterious selection, the model can explain these patterns of change. These results suggest that transposable elements are deleterious to the organisms carrying them, but at levels only several times their rate of transpositional insertion. Similar analyses of small deletions in the Adh region suggest that they may experience mildly deleterious selection.
Evidence that a human soluble beta-galactoside-binding lectin is encoded by a family of genes.
Authors: Gitt M A , Barondes S H
Published: May 2006

Two cDNA clones were isolated by immunoscreening a human hepatoma cDNA library with an antiserum that bound specifically to a human soluble beta-galactoside-binding lectin with Mr of approximately 14,000. The deduced amino acid sequences of the inserts of these two clones show considerable homology with each other, the sequence of chicken skin beta-galactoside-binding lectin, and eight peptides derived from purified human lung lectin of Mr approximately 14,000. However, the sequence differences between the two hepatoma clones as well as among each clone and the lung peptides suggest that at least three variants of the gene encoding this lectin are expressed in human tissue.
Isolation and characterization of the cDNA for murine granulocyte colony-stimulating factor.
Authors: Tsuchiya M , Asano S , Kaziro Y , Nagata S
Published: May 2006

A cDNA sequence coding for murine granulocyte colony-stimulating factor (G-CSF) has been isolated from a cDNA library prepared with mRNA derived from murine fibrosarcoma NFSA cells, which produce G-CSF constitutively. Identification of murine G-CSF cDNA was based on the cross-hybridization with human G-CSF cDNA under a low-stringency condition. The cDNA can encode a polypeptide consisting of a 30-amino acid signal sequence, followed by a mature G-CSF sequence of 178 amino acids with a calculated Mr of 19,061. The nucleotide sequence and the deduced amino acid sequence of murine G-CSF cDNA were 69.3% and 72.6% homologous, respectively, to the corresponding sequences of human G-CSF cDNA. The murine G-CSF cDNA, when introduced into monkey COS cells under the simian virus 40 promoter, could direct the synthesis of a protein that can stimulate the granulocyte colony formation from mouse bone marrow cells and support the proliferation of murine NFS-60 myeloid leukemia cells.
Hemorrhage in lesions caused by cowpox virus is induced by a viral protein that is related to plasma protein inhibitors of serine proteases.
Authors: Pickup D J , Ink B S , Hu W , Ray C A , Joklik W K
Published: May 2006

Several recombinant cowpox viruses were constructed and used to identify a viral gene that controls the production of hemorrhage in lesions caused by the Brighton Red strain of cowpox virus (CPV-BR). This gene is located in the KpnD fragment of CPV-BR DNA, between 31 and 32 kilobases from the end of the genome. This position corresponds well with that predicted from analyses of the DNA structures of spontaneously generated deletion mutants. The gene responsible for hemorrhage encodes a 38-kDa protein that is one of the most abundant early gene products. The 11-base-pair sequence GAAAATATATT present 84 base pairs upstream of its coding region is also present upstream of three other early genes of vaccinia virus; therefore, this sequence may be involved in the regulation of transcription. There is extensive similarity between the predicted amino acid sequence of the 38-kDa protein and the amino acid sequences of several plasma proteins that are inhibitors of various serine proteases involved in blood coagulation pathways. This suggests that the viral protein may possess a similar biological activity, which may enable it to effect hemorrhage by inhibiting one or more of the serine proteases involved in the host's normal processes of blood coagulation and wound containment.
Introduction and expression of the bacterial PaeR7 methylase gene in mammalian cells.
Authors: Kwoh T J , Kwoh D Y , McCue A W , Davis G R , Patrick D , Gingeras T R
Published: May 2006

An approach is devised for studying the role of DNA methylation in eukaryotic gene expression. The approach is based on the expression of site-specific bacterial methylase genes in animal cells. A model system using the cloned PaeR7 (an isoschizomer of Xho I) methylase gene was constructed to test the feasibility of this approach. Expression plasmids for the PaeR7 methylase gene were introduced into mouse Ltk- cells by cotransfection with the cloned chicken thymidine kinase (tk) gene. Several of the cell strains derived from Tk+ colonies were found to express the PaeR7 gene as judged by four criteria: the cellular DNA of these strains showed increased resistance to cleavage by Xho I; these strains contained cellular proteins that comigrated with pure PaeR7 methylase protein, as visualized by immunoblotting; PaeR7 methylase activity was found in vitro in crude extracts of total cellular protein from these strains; and murine adenovirus genomes grown on cells expressing PaeR7 methylase showed resistance to cleavage to PaeR7 endonuclease. The potential applications of this approach for the study of cellular and viral gene regulation, DNA repair, and restriction modification are discussed.
Importance of a hydrophobic residue in binding and catalysis by dihydrofolate reductase.
Authors: Mayer R J , Chen J T , Taira K , Fierke C A , Benkovic S J
Published: May 2006

A conserved residue at the dihydrofolate binding site of dihydrofolate reductase (EC 1.5.1.3), leucine-54, was replaced with glycine to ascertain the role of this hydrophobic amino acid. The effect of the mutation is both to increase the dissociation rate of dihydrofolate and decrease the rate of hydride transfer thus changing the rate-limiting step in catalysis from product loss (leucine-54) to hydride transfer (glycine-54). The total stabilization by leucine-54 of the transition state for hydride transfer is ca. 10(4)-fold (delta delta G approximately 5.4 kcal/mol) at subsaturating dihydrofolate levels relative to free enzyme despite its location some 10 A from the site of chemical reaction.
Enhancement of galactose/N-acetylgalactosamine receptor activity on the surface of freshly isolated rat hepatocytes: evidence for masking of receptor sites by inhibitors derived from collagenase preparations.
Authors: Stults N L , Lee Y C
Published: May 2006

Rat hepatocytes prepared by collagenase perfusion of the liver are known to exhibit increased binding of asialoorosomucoid (ASOR) after prior treatment with EDTA or after warming at 37 degrees C. The cause of the apparent increase in the surface binding activity of the galactose/N-acetylgalactosamine (Gal/GalNAc) receptor on freshly isolated rat hepatocytes was investigated. Binding experiments using three different galactose-terminated ligands revealed up to a 2- to 6-fold increase in the level of surface receptor sites on rat hepatocytes upon prior incubation at 4 degrees C with 10 mM GalNAc or 10 mM EDTA or at 37 degrees C compared to untreated cells. With digitonin-permeabilized cells, it was shown that the newly exposed receptor sites most likely originated from masked surface receptor sites, as no alteration in the size of the internal pool of receptor was observed. Collagenase preparations were found to inhibit the binding of 125I-labeled ASOR to the Gal/GalNAc receptor. Exposure of hepatocytes to collagenase resulted in a significant decrease in 125I-labeled ASOR binding, which was reversible upon treatment with GalNAc or EDTA at 4 degrees C or upon warming at 37 degrees C. Perfusion of EDTA through the isolated whole liver at 0-2 degrees C to remove any possible bound endogenous ligands did not result in a significant increase in the level of 125I-labeled ASOR binding, while perfusion of collagenase caused a marked decrease in the binding activity of the liver. We conclude that the enhancement of Gal/GalNAc receptor activity on the surface of freshly isolated hepatocytes by temperature and EDTA is potentially an artifact of the collagenase perfusion method.
A phosphorylated, disulfide-linked membrane protein in murine cytotoxic T lymphocytes.
Authors: Lieberman J , Verret C R , Kranz D M , Hubbard S C , Saito H , Raulet D H , Tonegawa S , Eisen H N
Published: May 2006

The previously determined sequence of the murine T-cell gamma gene and its transcription in cloned T lymphocytes suggests that the polypeptide encoded by this gene is generally present in cytotoxic T cells as a 33-kDa monomer in a disulfide-bonded dimer. The gamma chain is also expected to be phosphorylated because a sequence in its cytoplasmic domain is homologous to an active site for serine phosphorylation in the regulatory subunit of cAMP-dependent protein kinase. We describe here a cytotoxic-T-cell-associated phosphorylated protein, many of whose properties suggest that it may be the product of the T-cell gamma gene. Its phosphorylation is greatly enhanced by interleukin 2 stimulation.
Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity.
Authors: Riedel N , Wise J A , Swerdlow H , Mak A , Guthrie C
Published: May 2006

Previous work showed that the simple eukaryote Saccharomyces cerevisiae contains a group of RNAs with the general structural properties predicted for small nuclear RNAs (snRNAs), including possession of the characteristic trimethylguanosine 5'-terminal cap. It was also demonstrated that, unlike their metazoan counterparts, the yeast snRNAs are present in low abundance (200-500 molecules per haploid cell). We have now used antibody directed against the 5' cap to investigate the total set size of snRNAs in this organism. We present evidence that the number of distinct yeast snRNAs is on the order of several dozen, that the length of the capped RNAs can exceed 1000 nucleotides, and that the relative abundance of a subset of these RNAs is 1/5th to 1/20th that of the class of snRNAs described previously. These findings suggest that the six highly abundant species of snRNAs (U1-U6) typically reported in metazoans may represent a serious underestimation of the total diversity of snRNAs in eukaryotes.
Mutagenic potential of O4-methylthymine in vivo determined by an enzymatic approach to site-specific mutagenesis.
Authors: Preston B D , Singer B , Loeb L A
Published: May 2006

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.
Identification of two anti-parallel beta-sheet conformations in the solution structure of murine epidermal growth factor by proton magnetic resonance.
Authors: Montelione G T , Wüthrich K , Nice E C , Burgess A W , Scheraga H A
Published: May 2006

Epidermal growth factor (EGF) is a small mitogenic protein. Proteins with sequence homology with EGF or with its membrane-bound protein receptor have been proposed to play a role in oncogenesis. This report describes solution NMR data that provide evidence that the solution conformation of murine EGF includes an anti-parallel beta-sheet structure involving residues S2-P4, V19-I23, and S28-N32; a small anti-parallel beta-sheet involving residues Y37-S38 and T44-R45; and a multiple-bend (or short irregular helix) structure for residues C6-C14 that is disulfide bonded to the V19-I23/S28-N32 beta-sheet. Implications of these results for structure and function studies of EGF and for molecular design of EGF and homologous alpha-type transforming growth factors are discussed.
A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins.
Authors: Ahnn J , March P E , Takiff H E , Inouye M
Published: May 2006

The DNA sequence of a gene (era) located immediately downstream of the gene (rnc) encoding ribonuclease III of Escherichia coli was determined and found to encode a protein of 316 amino acid residues. The amino acid sequence of this protein, Era, has significant similarity to the yeast RAS proteins. Overexpression of the Era protein was achieved and GTP cross-linking experiments demonstrated that the protein was indeed capable of binding GTP, as are the yeast and mammalian ras gene products. These data indicate that ras-related sequences occur not only in eukaryotes but also in prokaryotes.
Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system.
Authors: Williams N , Fox D K , Shea C , Roseman S
Published: May 2006

Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated IIMan, comprises lipid and two membrane proteins, II-AMan and II-BMan. The proteins are encoded by ptsM, located at approximately equal to 40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for lambda DNA penetration of the cytoplasmic membrane. Earlier studies suggested that both pel function and II-BMan are encoded by the same gene, while a different gene (also in ptsM) encodes II-AMan. In the present studies, a ptsM clone, pCS13, was isolated from an E. coli HindIII gene bank in pBR322 and restored both mannose termentation and pel+ function to ptsM mutants defective in II-BMan. Subclones of pCS13 show that two distinct genes, manY and manZ, encode the pel+ function and the II-BMan protein, respectively; each gene may have its own promoter; whereas the protein encoded by manY (Pel) alone seems sufficient for lambda sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the IIMan complex. The efficiency of the complex may depend on the ratio of Pel to IIMan.
Isolation of yeast mutants defective in protein targeting to the vacuole.
Authors: Bankaitis V A , Johnson L M , Emr S D
Published: May 2006

We have constructed a PRC1-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that "mislocalize" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent defects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic.
Stimulation of human neuroblastoma DNA polymerase alpha and primase activities by a protein factor isolated from rat liver chromatin.
Authors: Takada S , Torres-Rosado A , Ray S , Basu S
Published: May 2006

Nuclear protein factor type 1 (NPF-1) that simulates IMR-32 primase-associated DNA polymerase alpha 1 and alpha 2 activities has been purified from a high-salt extract of liver chromatin from 6-month-old rats. The final purified factor lacks DNA polymerase alpha, RNA polymerase, and DNA-unwinding or topoisomerase type I activities. The stimulatory activity is destroyed by trypsin (60 min at 37 degrees C), DNase II (60 min at 37 degrees C), and heat treatment (2 min at 68 degrees C). The 125I-labeled NPF-1 does not bind to activated calf thymus DNA or poly(dC). However, it forms a ternary complex with DNA in the presence of DNA polymerase alpha-primase complex (alpha 1 and alpha 2). The ternary complex sediments on sucrose density gradient as a heavier band (11S). The NPF-1 also stimulates (2.5-fold) primase-catalyzed incorporation of GMP and dGMP from the corresponding triphosphates on poly(dC) template even in the presence of a high concentration of alpha-amanitin (400 micrograms/ml). The labeled duplex containing the poly(dC) template, [32P]-GTP, and [3H]dGTP loses 80% of the 32P label and 70% of the 3H label after treatment with 0.3 M KOH and DNase I, respectively. The products were isolated from reaction mixtures incubated with and without NPF-1 and subjected to alkaline sucrose-density-gradient sedimentation analysis. The results suggest that the rate of synthesis of DNA short chains is increased in the presence of NPF-1 without a concomitant increase in the chain length of the newly synthesized products.
Structural analysis of a rat liver glutathione S-transferase Ya gene.
Authors: Telakowski-Hopkins C A , Rothkopf G S , Pickett C B
Published: May 2006

We have isolated and characterized a complete structural gene encoding a rat liver glutathione S-transferase (glutathione transferase; EC 2.5.1.18) Ya subunit. The gene spans approximately 11 kilobases and is comprised of seven exons separated by six introns. A sequence similar to the Goldberg-Hogness promoter ("TATA" box), TATTA, is located 32 base pairs upstream from the transcription initiation site. Exons 2 and 4 of the glutathione S-transferase gene encode amino acid sequences of the Ya subunit that are highly conserved in the Yc subunit, whereas exons 3 and 5 encode amino acids that are divergent in the Yc subunit. These data suggest that exons 2 and 4 may encode domains of the Ya subunits that have similar structural or functional properties to the corresponding domains in the Yc subunit (e.g., glutathione binding site), whereas exons 3 and 5 may encode domains of the Ya subunit that have unique structural or functional properties to the corresponding domains in the Yc subunit (e.g., substrate binding site).
Direct introduction of genes into rats and expression of the genes.
Authors: Benvenisty N , Reshef L
Published: May 2006

A method of introducing actively expressed genes into intact mammals is described. DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats. The injected genes have been taken up and expressed by the animal tissues. To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen. In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.
Tumorigenicity of hamster and mouse cells transformed by adenovirus types 2 and 5 is not influenced by the level of class I major histocompatibility antigens expressed on the cells.
Authors: Haddada H , Lewis A M , Sogn J A , Coligan J E , Cook J L , Walker T A , Levine A S
Published: May 2006

Inbred hamster and mouse cells transformed by the nononcogenic adenovirus (Ad) serotypes, Ad2 and Ad5, are nontumorigenic in syngeneic adult animals, while cells from these species transformed by the highly oncogenic Ad12 are tumorigenic in such rodents. By immunoprecipitation and flow cytometry, cells from four of six Ad2- and Ad5-transformed hamster and mouse lines expressed high levels of cell-surface class I major histocompatibility complex (MHC) antigens, while cells from two of these six lines expressed low levels of cell-surface class I MHC antigens. The levels of class I MHC proteins expressed by cells from these latter two lines were comparable to the levels of cell-surface class I MHC proteins expressed by cells from Ad12-transformed hamster and mouse lines. Moreover, an Ad2-transformed line that had become highly oncogenic after in vivo adaptation showed the same high level of MHC expression as the nononcogenic parent. The amounts of class I mRNA, analyzed by RNA blotting, were, in general, consistent with the levels of class I antigens expressed on the surfaces of these cells. These results indicate that there is no correlation between the tumorigenicity in immunocompetent syngeneic adult rodents of Ad2- and Ad5-transformed hamster and mouse cells and the level of class I MHC antigens expressed on the surfaces of these cells. Thus, the expression of different levels of class I MHC proteins does not seem to explain the differences in the oncogenicity between nononcogenic and highly oncogenic human Ad serotypes.
Maximum likelihood estimation for stationary point processes
Authors: Puri Madan L. , Tuan Pham D.
Published: May 2006

In this paper we derive the log likelihood function for point processes in terms of their stochastic intensities by using the martingale approach. For practical purposes we work with an approximate log likelihood function that is shown to possess the usual asymptotic properties of a log likelihood function. The resulting estimates are strongly consistent and asymptotically normal (under some regularity conditions). As a by-product, a strong law of large numbers and a central limit theorem for martingales in continuous times are derived.
A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome.
Authors: Sarnow P , Bernstein H D , Baltimore D
Published: May 2006

We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, representing a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature.
Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen.
Authors: Miyazaki J , Appella E , Ozato K
Published: May 2006

The third external domain of major histocompatibility class I antigens has a highly conserved disulfide bridge between cysteine-203 and cysteine-259. To elucidate the functional significance of this disulfide bridge, we have produced a mutant H-2Ld gene by site-directed mutagenesis in which the codon for cysteine-203 is changed to a codon for serine, which is unable to form a disulfide bridge. The mutant H-2Ld gene was introduced into mouse L cells and its expression has been studied. No measurable expression of the H-2Ld antigen was detected on the cell surface of the transformants by antibody-binding assays. However, a large quantity of the mutant H-2Ld antigen was found in the cytoplasm of the transformants as observed by immunoprecipitation of metabolically labeled lysate and by immunocytochemistry of membrane-permeabilized cells, using an antibody specific for the first external domain of the H-2Ld antigen. The mutant antigen was glycosylated and associated, at least in part, with beta 2-microglobulin. Subcellular fractionation experiments indicated that the transport of the antigen was blocked between the endoplasmic reticulum and the plasma membrane. It is concluded that structural integrity of the third external domain is a prerequisite for intracellular transport of class I antigens. On the basis of these findings we suggest that the domain structure containing the disulfide bridge serves as a signal structure necessary for receptor-mediated intracellular transport and that this requirement is the evolutionary basis for high conservation of similar structures present throughout the immunoglobulin supergene family.
Secondary activation of c-abl may be related to translocation to the nucleolar organizer region in an in vitro cultured rat leukemia cell line (K3D).
Authors: Takahashi R , Mihara K , Maeda S , Yamaguchi T , Chen H L , Aoyama N , Murao S , Hatanaka M , Sugiyama T
Published: May 2006

Localization of cellular oncogenes (c-onc) near the break points of translocations in tumor cells has indicated involvement of these genes in neoplastic growth. Enhanced transcription of the cellular homolog (c-abl) of the transforming sequence of Abelson murine leukemia virus was observed in K3D, which was one of the cloned cell lines of 7,12-dimethylbenz[a]anthracene-induced rat erythroblastic leukemia. Since the c-abl activation was not observed in the parent cell line (K2D) from which K3D was derived and the latter was different from the former in the presence of a new marker chromosome, t(3;12), this marker may play a role in the expression of c-abl in K3D cells. In contrast to the human c-onc assignments, few rat c-onc assignments have been reported. In situ molecular hybridization studies assigned c-abl to the 3q12 site of the normal chromosome 3 and to the break point of the translocation t(3;12) in K3D cells. Another break point in this translocation chromosome 12p11 involves the nucleolar region, and the 3;12 translocation may involve c-abl and nucleolar cistrons. These results provide evidence of secondary c-onc activation during karyotypic evolution of cloned malignant cells.
alpha-Transducin immunoreactivity in retinae and sensory pineal organs of adult vertebrates.
Authors: van Veen T , Ostholm T , Gierschik P , Spiegel A , Somers R , Korf H W , Klein D C
Published: May 2006

Antiserum against the alpha subunit of bovine rod-outer-segment transducin was used in an immunocytochemical study that identified the protein in retina (human, baboon, owl monkey, cow, rat, quail, newt, frog, salmon, eel, and lamprey), pineal organ (quail, newt, frog, salmon, eel, and lamprey), and parapineal organ (salmon and lamprey). No reaction was observed in the cow or rat pineal organ or the eel parapineal organ. The immunoreaction was very strong in outer segments but weak in perikarya. Immunoblots of crude tissue extracts of bovine rod-outer-segment membranes and frog and fish retina revealed a 39-kDa immunopositive band. The fish retina also contained two additional bands of mass 43 kDa and 25 kDa. Only the 43-kDa band was present in the fish pineal organ, which is photosensitive. This raises the possibility that the 43-kDa alpha transducin-immunopositive molecule present in the fish pineal organ and retina may be involved in phototransduction.
Lysozyme-induced fusion of liposomes with erythrocyte ghosts at acidic pH.
Authors: Arvinte T , Hildenbrand K , Wahl P , Nicolau C
Published: May 2006

Lysozyme that was covalently bound to the outer surface of sonicated vesicles induced fusion of the vesicles with human white erythrocyte ghosts. The kinetics of membrane mixing were evaluated by the resonance-energy-transfer method using L-alpha-dipalmitoyl phosphatidylethanolamine labeled at the free amino group with the energy donor 7-nitro-2,1,3-benzoxadiazol-4-yl or with the energy acceptor tetramethylrhodamine. The equilibrium state after fusion was characterized by using fluorescence photobleaching and recovery techniques. Rates and equilibrium percentages of fusion were maximal at the pH optimum of the enzyme, and rates were strongly reduced by the addition of N,N',N''-triacetylchitotriose, a competitive inhibitor of lysozyme. An apparent activation energy of 28 +/- kcal/mol was obtained for the lipid-mixing process. At 37 degrees C, the fusion half-time was 0.5 min. After 30 min at 37 degrees C, 40% of the labeled lipids initially present in the fusion mixture had a lateral diffusion constant, D, of 1.1 +/- 0.5 X 10(-9) cm2 X sec-1 in the ghost membrane. The strong induction of fusion at the lysozyme pH optimum was not observed in the absence of lysozyme or when free lysozyme was added to the solution. Bound lysozyme did not induce fusion of electrically neutral liposomes with each other. These observations indicate that it is the liposome-bound lysozyme that induces fusion between liposomes and erythrocyte ghosts.
Correlation between hormone dependency and the regulation of epidermal growth factor receptor by tumor promoters in human mammary carcinoma cells.
Authors: Roos W , Fabbro D , Küng W , Costa S D , Eppenberger U
Published: May 2006

The effects of the tumor promoter phorbol 12-tetradecanoate 13-acetate (TPA) on the epidermal growth factor (EGF) receptor levels were investigated in hormone-dependent (MCF-7, T-47-D, and ZR-75-1) and hormone-independent (MDA-MB-231, HBL-100, and BT-20) human mammary carcinoma cell lines. In the absence of TPA, hormone-independent cell lines contained high concentrations of low-affinity EGF receptors (apparent Kd = 8 X 10(-10) M), whereas hormone-dependent cell lines exhibited low concentrations of high-affinity receptors (apparent Kd = 1 X 10(-10) M). TPA causes a change of the receptor from a high- to the low-affinity state in hormone-dependent cell lines (MCF-7, T-47-D, and ZR-75-1), as well as in the hormone-independent HBL-100, whereas the affinity remained unchanged in MDA-MB-231 and BT-20 cells. In addition, progesterone receptor levels are decreased after TPA treatment in the hormone-dependent cell lines MCF-7, T-47-D, and ZR-75-1, whereas the estrogen receptor levels remained unchanged. Tumor promoters such as TPA or teleocidin inhibited the proliferation of these cell lines at concentrations above 10 microM with the exception of the T-47-D cells. The most sensitive cell line towards growth inhibition by tumor promoter was the hormone-dependent MCF-7 cell line. Evaluation of different TPA analogs indicated a positive correlation between the growth-inhibitory effects and their ability to stimulate the subcellular redistribution of protein kinase C activity in MCF-7 cells. These data suggest a protein kinase C-mediated down-regulation of the progesterone receptor concentration and of the EGF receptor affinity, which is supposed to mediate the mitogenic response. Furthermore, these results support the hypothesis that the tumor-derived growth factors induced by estradiol act via the EGF receptor in hormone-dependent mammary carcinoma cells.
A developmentally stable chromatin structure in the human beta-globin gene cluster.
Authors: Forrester W C , Thompson C , Elder J T , Groudine M
Published: May 2006

The DNase I-hypersensitive sites in the human embryonic beta-globin gene region have been mapped in erythroid-enriched fractions of disaggregated fetal livers, in adult nucleated red blood cells, and in fetal brain tissue. Our analysis of a region extending 11 kilobases (kb) 5' of the epsilon-globin gene reveals many minor nuclease-hypersensitive sites and one major site located 6.1 kb upstream of the epsilon-globin gene. All of these hypersensitive sites are erythroid-specific, and the major site is stable throughout erythroid development. As assayed by nuclear runoff transcription, little or no epsilon-globin gene expression is detectable in fetal or adult erythroid cells. Thus, the presence of the major hypersensitive site 5' of the epsilon-globin gene in both fetal and adult erythroid cells demonstrates that this site is not specifically correlated with transcription of the gene or with a particular stage of development. Rather, this site may reflect an early event in erythroid differentiation. In addition, DNase I has been used to probe the overall sensitivity of epsilon-globin chromatin in fetal erythroid cells. Our findings indicate that the epsilon-globin gene as well as the other genes in the beta-globin cluster reside within the chromatin domain that is more DNase I-sensitive than "bulk" chromatin.
cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts
Authors: Dawson William O. , Beck David L. , Knorr David A. , Grantham George L.
Published: May 2006

The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5′ and 3′ ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a
Pst
I site at the 5′ terminus, whereas a different primer was used to generate an
Nde
I site at the 3′ terminus. This strategy permitted removal of non-TMV sequences from cloned cDNA inserts by treatment with exonuclease VII following restriction endonuclease cleavage.
Pst
I linkers were added to TMV 3′ terminal cDNAs. Subgenomic cDNA fragments were ligated together into several independent full-genomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by
Pst
I digestion. Full-genomic TMV cDNA was ligated immediately downstream from the λ phage promoter from pPM1 and transcribed
in vitro
with
Escherichia coli
RNA polymerase. RNA transcripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3′ end. Transcripts from a construction with 6 extra nucleotides at the 5′ end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental virus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5′ termini of four independent cDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remarkably similar sequences.
Antibodies to synthetic joining segment peptide of the T-cell receptor beta-chain: serological cross-reaction between products of T-cell receptor genes, antigen binding T-cell receptors, and immunoglobulins.
Authors: Schluter S F , Marchalonis J J
Published: May 2006

Immunoglobulin light and heavy chains show sequence homology to one another and to the polypeptide chains of putative T-cell receptors in the J (joining) segment of the variable region. Antibodies produced against synthetic peptides corresponding to the entire JH1 region and part of the diversity segment region cross-react serologically with products of normal T cells and monoclonal T-cell lines. In this study we generate immune affinity-purified rabbit antibodies to a synthetic 16-mer peptide consisting of the entire JT sequence and part of the T-cell diversity sequence corresponding to these segments of the human putative T-cell receptor beta gene YT35. Both free peptide and peptide coupled to bovine serum albumin as carrier were found to stimulate the production of antibody. The immune affinity-purified anti-JT peptide antibodies bound to intact immunoglobulin and to light and heavy chain as detected by enzyme-linked immunosorbent assay and by immunoblot transfer. The antibody reacted by these techniques with membrane components of the human monoclonal amplifier T-cell MOLT-3 and the murine suppressor T-cell WEHI-7. The component detected in the MOLT-3 cell corresponded to the beta-chain of the alpha/beta heterodimer putative T-cell receptor; whereas the molecule detected in the WEHI-7 line had properties corresponding to those of antigen-specific T-cell suppressor receptors. The molecular size of this component under reducing conditions was approximately 68 kDa and the intact form had an apparent mass of 140 kDa. These results provide direct proof of serological cross-reaction among products of putative T-cell receptor genes, antigen-binding T-cell receptors, and immunoglobulins, thereby supporting the concept that antigen receptors of T lymphocytes all represent new immunoglobulin translocons.
Solution of Dirac's equation in Reissner-Nordström geometry
Authors: Pekeris C. L. , Frankowski K.
Published: May 2006

The nature of the singularity at the origin of the solution of Dirac's equation in Reissner-Nordström geometry is different from that in flat space. Here, both independent solutions are regular at the origin, and both give a convergent normalization integral. The choice between the two solutions is made by requiring that the variational integral, from which the differential equation can be derived, be convergent. The gravitational shift of the ground state of hydrogen in Reissner-Nordström geometry is estimated to be only of order 10
-36
MH.