bluuebunny/crossref_metadata_2025 huggingface dataset:
Segregation of polymorphic T-cell receptor genes in human families.
Authors: Robinson M A , Kindt T J
Published: May 2006

Polymorphism in the genes encoding the constant (C) region of the beta chain of the T-cell antigen receptor (CT beta, also called C beta) has been detected by molecular genotyping analyses. In initial screenings, a panel of restriction endonucleases was used to digest DNA samples from two individuals; the digested samples were subjected to Southern blot analyses using a CT beta probe. The enzyme Bgl II revealed restriction-fragment-length polymorphism in these samples and was subsequently used to test 59 individual members of eight different families. Polymorphic fragments detected in six of the families could be used to follow the segregation of T-cell receptor genes; in many cases maternal and/or paternal haplotypes could be assigned. All members of two additional families displayed a single CT beta hybridizing fragment. In one family the DNA sample from one of the children lacked an expected Bgl II restriction fragment. On the basis of analyses with other restriction enzymes, the most likely explanation is that the lymphoblastoid B-cell line used as a source of genomic DNA for this individual had rearranged or altered CT beta genes. Restriction-fragment-length polymorphisms used to discriminate CT beta haplotypes in families provide useful markers that will facilitate linkage studies and genetic analyses of T-cell function.
Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.
Authors: Zavoico G B , Halenda S P , Sha'afi R I , Feinstein M B
Published: May 2006

The tumor-promoting phorbol diester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2+ in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; IC50 vs. thrombin = 2 ng/ml, 3.4 nM: greater than 90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2+ mobilization was evident within 30 sec of pretreatment with PMA and was essentially complete by 6-10 min at 10-20 ng of PMA per ml. Thrombin-induced secretion was initially accelerated in the presence of PMA, but after 1 min it was progressively inhibited when Ca2+ mobilization was depressed by greater than 60%. PMA did not inhibit Ca2+ mobilization or secretion caused by A23187. Thrombin-induced phosphatidylinositol 4,5-[32P]bisphosphate breakdown and [32P]phosphatidic acid production were also initially increased by PMA and then progressively depressed. Inhibition of thrombin-induced lipid metabolism required higher concentrations of PMA (IC50 = 10 ng/ml), and it was not overcome by A23187. 4 alpha-Phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit any responses to thrombin. These results suggest that activation of protein kinase C, which initially fosters secretion and aggregation, may subsequently exert negative feedback on the receptor-mediated mobilization of intracellular Ca2+ and the hydrolysis of phosphatidylinositol 4,5-bisphosphate.
Immunoaffinity purification of factor IX (Christmas factor) by using conformation-specific antibodies directed against the factor IX-metal complex.
Authors: Liebman H A , Limentani S A , Furie B C , Furie B
Published: May 2006

Factor IX is a vitamin K-dependent blood clotting zymogen that is functionally defective or absent in patients with hemophilia B. A method of immunoaffinity chromatography has been developed for a one-step high yield purification of factor IX directly from plasma. The technique utilizes conformation-specific antibodies that bind solely to the metal-stabilized factor IX conformer, but not to the conformer of factor IX found in the absence of metal ions. Anti-factor IX-Ca(II) antibodies were immobilized on an agarose matrix. Human plasma in the presence of 7.5 mM MgCl2 was applied to the antibody-agarose column. The factor IX that binds to these antibodies was specifically eluted by metal chelation with EDTA. This immunopurification resulted in a 10,000-fold one-step purification of the fully functional zymogen. Purified factor IX yielded a single band upon gel electrophoresis in Na-DodSO4 and had a specific activity of 120-150 units/mg. The purified factor IX was separated from other vitamin K-dependent blood clotting proteins and hepatitis virus; no activated factor IX was detected. This method has application for the large scale purification of factor IX for the treatment of hemophilia B.
Purification and characterization of a human T-lymphocyte-derived glial growth-promoting factor.
Authors: Benveniste E N , Merrill J E , Kaufman S E , Golde D W , Gasson J C
Published: May 2006

Antigen- or lectin-stimulated T lymphocytes and human T-cell leukemia virus (HTLV)-infected cell lines secrete lymphokines that can influence the growth and function of a variety of cell types. We recently demonstrated that supernatants from the HTLV-II-infected Mo-T-cell line stimulate the proliferation of rat brain oligodendrocytes and astrocytes. We have now purified a glial growth-promoting factor (GGPF) from these supernatants. Purification from serum-free conditioned medium was accomplished by sequential concentration, ammonium sulfate precipitation, lentillectin affinity chromatography, gel filtration, and reversed-phase high-performance liquid chromatography. GGPF is assayed by its ability to stimulate DNA synthesis in oligodendrocytes, as measured by [3H]thymidine uptake. The purified GGPF has an apparent Mr of 30,000 when analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, however, GGPF appears as a single band of Mr 18,000. Both reduced and unreduced forms have biological activity, suggesting that GGPF exists in both a functional monomeric and dimeric form. Purified GGPF appears to be a biochemically and functionally distinct lymphokine.
A sequence downstream of A-A-U-A-A-A is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes.
Authors: Conway L , Wickens M
Published: May 2006

The 3' terminus of simian virus 40 late mRNA is formed by nucleolytic cleavage of an mRNA precursor. In this report we show that efficient cleavage requires a sequence in the 3' flanking region, "downstream" of the highly conserved A-A-U-A-A-A sequence. This sequence is not required for polyadenylylation itself or for accurately positioning the 3' terminus that is formed.
Sequence determination of the capsid protein gene and flanking regions of tobacco etch virus: Evidence for synthesis and processing of a polyprotein in potyvirus genome expression
Authors: Allison Richard F. , Sorenson John C. , Kelly Mary E. , Armstrong Frank B. , Dougherty William G.
Published: May 2006

The nucleotide sequence of the 3′-terminal portion of the tobacco etch virus (TEV) genome was determined. The 2324-nucleotide sequence represented approximately one-fourth of the TEV genome and included the capsid protein gene and flanking regions. An open reading frame of 2135 nucleotides and an untranslated region of 189 nucleotides adjacent to a polyadenylate tract were identified. The sequence began within an open reading frame, indicating that the initiation codon was upstream of the available sequence data. The sequence of the 20 NH
2
-terminal amino acids of the TEV capsid protein was established chemically. An identical amino acid sequence, predicted from the nucleotide sequence, was located, commencing at amino acid - 263. These data indicated that maturation of the capsid protein required a post-translational cleavage of a larger protein precursor, with a probable cleavage site between the amino acids glutamine and glycine.
In vitro expression of chloroplast genes in lysates of higher plant chloroplasts.
Authors: Bard J , Bourque D P , Hildebrand M , Zaitlin D
Published: May 2006

A DNA-dependent in vitro-coupled transcription-translation system has been prepared from lysates of isolated chloroplasts. These lysates are comparable to those of Escherichia coli in transcriptional and translational fidelity and efficiency in response to a given template DNA. When Nicotiana tabacum chloroplast DNA is used as template with chloroplast lysates (N. tabacum or spinach) or E. coli lysates, NaDodSO4 gel analysis reveals similar polypeptide patterns that are distinct from the patterns obtained with E. coli DNA. Genes in recombinant plasmids containing chloroplast DNA are also expressed in these in vitro systems. DNA . RNA hybridization experiments show that transcripts are synthesized from most of the chloroplast genome. Newly synthesized large subunit of ribulosebisphosphate carboxylase/oxygenate and a transcript of the large subunit gene (rbcL) are observed in chloroplast lysates using as template chloroplast DNA or cloned fragments of tobacco chloroplast DNA that contain the large subunit gene. Results suggest that differential expression of chloroplast genes occurs in vitro. By using cloned chloroplast DNA templates in this homologous system, it is possible to identify and map structural genes for chloroplast proteins.
Calmodulin genes in trypanosomes are tandemly repeated and produce multiple mRNAs with a common 5' leader sequence.
Authors: Tschudi C , Young A S , Ruben L , Patton C L , Richards F F
Published: May 2006

In Trypanosoma brucei gambiense, the Ca2+ binding protein calmodulin is encoded by three identical tandemly repeated genes. The transcripts of these genes consist of several RNA species similar in size. A 35-nucleotide spliced leader sequence is present at the 5' end of each mRNA but is not encoded by DNA contiguous to these genes. We have identified two different sites for the fusion of the leader to the mRNA. These results strongly support the idea that a novel, possibly discontinuous, transcription mechanism is used by these parasites.
Secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes.
Authors: Caulfield M P , Furlong D , Tai P C , Davis B D
Published: May 2006

The S complex of Bacillus subtilis, a set of four proteins that appears to be involved in protein secretion, is shown to be attached to 70S ribosomes: antibody to its 64-kDa component can aggregate these ribosomes, and the complex can be chemically crosslinked to ribosomal proteins. Low Mg2+ or prolonged high-speed centrifugation in a sucrose gradient releases the S complex from the ribosomes, and it is recovered as an aggregate with an S value of 76. Electron microscopy shows that these aggregates have a regular structure, somewhat resembling clathrin cages, with a diameter of about 45 nm. If these aggregates are physiological, their function would differ significantly from that of the signal recognition particle of eukaryotes.
Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28.
Authors: Reed S I , Hadwiger J A , Lörincz A T
Published: May 2006

Antibodies raised against the protein encoded by a lacZ-CDC28 in-frame fusion were shown to immunoprecipitate the CDC28 product from yeast cell lysates. The polypeptide p36CDC28 is a phosphoprotein of apparent Mr 36,000. Immune complexes prepared from yeast cell lysates by using anti-CDC28 antibody were found to possess a protein kinase activity, as determined by the transfer of label from [gamma-32P]ATP to a coprecipitated Mr 40,000 protein of unknown identity or function (p40). This activity was absent or thermolabile when extracts were prepared from several different cdc28 temperature-sensitive strains. The protein kinase activity was dependent on Zn2+ and transferred phosphate specifically to serine and threonine residues.
Nucleotide sequence of the G protein gene of human respiratory syncytial virus reveals an unusual type of viral membrane protein.
Authors: Wertz G W , Collins P L , Huang Y , Gruber C , Levine S , Ball L A
Published: May 2006

The major surface glycoprotein (G) of human respiratory syncytial (RS) virus has an estimated mature Mr of 84,000-90,000. Among a library of cDNA clones prepared from RS virus mRNAs, we identified clones that hybridized to a message that encoded a Mr 36,000 polypeptide that was specifically immunoprecipitated with anti-G antiserum. The amino acid sequence of the G protein backbone was determined by nucleotide sequence analysis of several of the cDNA clones. It contains a combination of structural features that make it unique among the known viral glycoproteins. The G mRNA is 918 nucleotides long and contains a single major open reading frame that encodes a polypeptide having 298 amino acid residues with a Mr of 32,587, a finding consistent with the Mr 36,000 estimate for the in vitro translation product of the G mRNA. This suggests that greater than 50% of the molecular weight of the mature glycoprotein may be contributed by carbohydrate. Glycosylation of G is largely resistant to tunicamycin, an inhibitor of the attachment of N-linked oligosaccharides, suggesting that the majority of the carbohydrate residues are attached via O-glycosidic bonds. In accordance with this, serine and threonine residues, the acceptor sites for O-linked oligosaccharides, comprise 30.6% of the total amino acid composition. There are also four potential acceptor sites for N-linked oligosaccharides. The amino acid sequence lacks both an NH2-terminal hydrophobic signal sequence and a COOH-terminal hydrophobic region. Instead, a strongly hydrophobic region is located between amino acid residues 38 and 66. This region may serve as both the signal to insert the nascent polypeptide through the membrane and as the membrane anchor site.
Structure and sequence divergence of two archaebacterial genes.
Authors: Cue D , Beckler G S , Reeve J N , Konisky J
Published: May 2006

The DNA sequences of a region that includes the hisA gene of two related methanogenic archaebacteria, Methanococcus voltae and Methanococcus vannielii, have been compared. Both organisms show a similar genome organization in this region, displaying three open reading frames (ORFs) separated by regions of very high A + T content. Two of the ORFs, including ORFHisA, show significant DNA sequence homology. As might be expected for organisms having a genome that is A + T-rich, there is a high preference for A and U as the third base in codons. Although the regions upstream of the structural genes contain prokaryotic-like promoter sequences, it is not known whether they are recognized as promoters in these archaebacterial cells. A ribosome binding site, G-G-T-G, is located 6 base pairs preceding the ATG translation initiation sequence of both hisA genes. The sequences upstream of the two hisA genes show only limited sequence homology. The M. voltae intergenic region contains four tandemly arranged repetitions of an 11-base-pair sequence, whereas the M. vannielii sequence contains both direct and inverted repetitive sequences. Based on the degree of hisA sequence homology, we conclude that M. voltae and M. vannielii are less closely related taxonomically than are members of the enteric group of eubacteria.
Biosynthetic 20-kilodalton methionyl-human growth hormone has diabetogenic and insulin-like activities.
Authors: Kostyo J L , Cameron C M , Olson K C , Jones A J , Pai R C
Published: May 2006

The anterior pituitary gland produces a 20-kilodalton (kDa) variant of human growth hormone (hGH) that differs from the predominant 22-kDa form of hGH in that amino acid residues 32-46 are deleted. Previous work has suggested that the 20-kDa variant possesses the full growth-promoting and lactogenic activities of 22-kDa hGH but lacks its intrinsic diabetogenic and insulin-like activities. In the present study, recombinant DNA techniques were used to prepare biosynthetic 20-kDa hGH, and some of the biological properties of the purified hGH variant were examined. The biosynthetic 20-kDa hGH variant was found to share the propensity for aggregation exhibited by its native counterpart. Moreover, like the native variant, biosynthetic 20-kDa hGH possessed full growth-promoting activity in the weight gain test in hypophysectomized rats. However, contrary to previous work suggesting that native 20-kDa hGH lacks diabetogenic and insulin-like activities, biosynthetic 20-kDa hGH was found to have substantial diabetogenic activity when administered chronically to ob/ob mice and to possess approximately 20% the in vitro insulin-like activity of biosynthetic 22-kDa hGH on isolated epididymal adipose tissue of hypophysectomized rats. The diabetogenic and insulin-like activities of biosynthetic 20-kDa hGH cannot be ascribed to contamination of the hormone preparation with the 22-kDa form of hGH or with other diabetogenic or insulin-like pituitary peptides. Therefore, the results strongly suggest that diabetogenic and insulin-like activities are also intrinsic properties of the 20-kDa variant of hGH.
Specific proteins associated with Creutzfeldt-Jakob disease and scrapie share antigenic and carbohydrate determinants.
Authors: Manuelidis L , Valley S , Manuelidis E E
Published: May 2006

Small amounts of brain tissue (2 g) infected with Creutzfeldt-Jakob disease (CJD) can be fractionated by using a simple 1-day method that includes lysis with N-lauroylsarcosine. Unique fibrils have been identified previously in scrapie- and CJD-infected tissue. These fibrils were abundant in final fractions. Preparations from human CJD autopsy material and from experimental hamster and guinea pig CJD all displayed readily identifiable fibrils that were not seen in control preparations. Thus, these methods appear to be of value in biopsy diagnosis of suspected human cases of CJD. Lysis with N-lauroylsarcosine quantitatively solubilized infectivity from membrane-rich fractions. Significant infectivity was recovered in microfractionations. After proteinase K digestion, a diffuse band at 29 kDa was detectable on NaDodSO4/PAGE. This 29-kDa material was not present in uninfected control brain and was similar to that seen in scrapie. Protein blots of human, guinea pig, and hamster CJD fractions were tested with an antibody raised against a 29-kDa band from mouse scrapie; 29-kDa proteins were labeled in all CJD and scrapie fractions but not in controls. These results indicate that specific proteins in both these diseases share common antigenic determinants. Ricin and wheat germ agglutinin, but not concanavalin A, also labeled a portion of the 29-kDa band from hamster CJD and hamster scrapie fractions, but they did not label any bands in normal hamster fractions at the same gel protein loads. When proteinase K treatment was omitted, specific bands of approximately equal to 35 kDa were detected in CJD samples. These results are consistent with the idea that some CJD- and scrapie-specific proteins are glycoproteins or sialoglycoproteins that can reside in or possibly derive from cell membranes.
Cloning and expression of cDNA for salmon growth hormone in
Escherichia coli
Authors: Sekine Susumu , Mizukami Tamio , Nishi Tatsunari , Kuwana Yoshihisa , Saito Akiko , Sato Moriyuki , Itoh Seiga , Kawauchi Hiroshi
Published: May 2006

cDNA clones encoding chum salmon (
Oncorhynchus keta
) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)
+
RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in
Escherichia coli
carrying a plasmid in which the sGH cDNA was under control of the
E. coli trp
promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from
E. coli
stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.
Biochemical rationale and the cardiac response of patients with muscle disease to therapy with coenzyme Q10.
Authors: Folkers K , Wolaniuk J , Simonsen R , Morishita M , Vadhanavikit S
Published: May 2006

Cardiac disease is commonly associated with virtually every form of muscular dystrophy and myopathy. A double-blind and open crossover trial on the oral administration of coenzyme Q10 (CoQ10) to 12 patients with progressive muscular dystrophies and neurogenic atrophies was conducted. These diseases included the Duchenne, Becker, and limb-girdle dystrophies, myotonic dystrophy, Charcot-Marie-Tooth disease, and Welander disease. The impaired cardiac function was noninvasively and extensively monitored by impedance cardiography. Solely by significant change or no change in stroke volume and cardiac output, all 8 patients on blind CoQ10 and all 4 on blind placebo were correctly assigned (P less than 0.003). After the limited 3-month trial, improved physical well-being was observed for 4/8 treated patients and for 0/4 placebo patients; of the latter, 3/4 improved on CoQ10; 2/8 patients resigned before crossover; 5/6 on CoQ10 in crossover maintained improved cardiac function; 1/6 crossed over from CoQ10 to placebo relapsed. The rationale of this trial was based on known mitochondrial myopathies, which involve respiratory enzymes, the known presence of CoQ10 in respiration, and prior clinical data on CoQ10 and dystrophy. These results indicate that the impaired myocardial function of such patients with muscular disease may have some association with impaired function of skeletal muscle, both of which may be improved by CoQ10 therapy. The cardiac improvement was definitely positive. The improvement in well-being was subjective, but probably real. Likely, CoQ10 does not alter genetic defects but can benefit the sequelae of mitochondrial impairment from such defects. CoQ10 is the only known substance that offers a safe and improved quality of life for such patients having muscle disease, and it is based on intrinsic bioenergetics.
Beta 2-adrenergic receptors contribute to catecholamine-stimulated shortening of action potential duration in dog atrial muscle.
Authors: Liang B T , Frame L H , Molinoff P B
Published: May 2006

beta 1- and beta 2-adrenergic receptors coexist in atria of humans and a variety of other species. The significance of these receptors and the quantitative extent to which beta 2-adrenergic receptors contribute to the electrophysiologic effects of catecholamines acting at beta-adrenergic receptors have not been determined. In the present study, the beta 1-selective antagonist ICI 89,406 and the beta 2-selective antagonist ICI 118,551 were used to determine the relative densities of beta 1- and beta 2-adrenergic receptors on membranes prepared from dog atria and the contribution that each subtype makes to isoproterenol-induced shortening of action potential duration measured at 75% repolarization (APD75). Computer-aided nonlinear regression analysis of the inhibition of the specific binding of [125I]iodopindolol and the inhibition of isoproterenol-stimulated APD75 shortening by these antagonists showed that a two-site model fits the data better than a one-site model (P less than 0.0001). The affinity constants for each selective antagonist determined by inhibition of APD75 shortening were similar to those determined in studies of the inhibition of the specific binding of [125I]iodopindolol to beta 1- and beta 2-adrenergic receptors. beta 2-Adrenergic receptors made up approximately equal to 25% of the total number of beta-adrenergic receptors but mediated approximately equal to 50% of the electrophysiologic effect of isoproterenol. The inhibition of the binding of [125I]iodopindolol and of the APD75 shortening by propranolol, a nonselective antagonist, was best fit by a one-site model. In other experiments, dogs were treated with reserpine and atropine to eliminate complications caused by the presence of endogenous norepinephrine and acetylcholine. Results obtained with atria from these animals were similar to those obtained in studies with control atria. These data suggest that beta 2-adrenergic receptors as well as beta 1-adrenergic receptors are involved in mediating the electrophysiologic effects of catecholamines in dog atrial muscle and that both beta 1- and beta 2-adrenergic receptors are present on atrial muscle cells.
Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.
Authors: Klarsfeld A , Changeux J P
Published: May 2006

In vitro blocking the spontaneous activity of primary cultures of chicken embryo myotubes with tetrodotoxin increases approximately equal to 2-fold their content in surface acetylcholine receptor. To investigate this effect at the level of gene expression, chicken genomic DNA sequences coding for the acetylcholine receptor alpha subunit were isolated and characterized. They were shown to belong to a single-copy, polymorphic gene with at least two alleles in the chicken strain utilized. Probes derived from these genomic clones were used to quantitate levels of alpha-subunit mRNA. In culture, a 2-day exposure to tetrodotoxin increased these mRNA levels up to 13-fold, a value similar to that observed after denervation of chick leg muscle (approximately equal to 17-fold). Actin mRNA levels varied little in any of these experiments. These results support the notion that membrane electrical activity affects acetylcholine receptor expression by regulating the accumulation of the corresponding mRNAs.
Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen.
Authors: Yonemoto W , Jarvis-Morar M , Brugge J S , Bolen J B , Israel M A
Published: May 2006

We have examined the in vitro phosphorylation of cellular src protein (pp60c-src) molecules associated with the polyoma virus middle-sized tumor antigen in polyoma virus-transformed cells. These pp60c-src molecules possessed an enhanced tyrosyl kinase activity, migrated aberrantly on NaDodSO4/polyacrylamide gels, and contained a novel site of tyrosine phosphorylation within the amino-terminal region of the molecule. The pp60c-src molecules not associated with the middle-sized tumor antigen were phosphorylated exclusively on a tyrosine residue within the carboxyl-terminal domain of pp60c-src. A similar modified form of the middle-sized tumor antigen-associated pp60c-src protein was detected in lysates from polyoma virus-transformed cells labeled in vivo with [32P]orthophosphate in the presence of sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatases.
Fusion of influenza virus membranes with liposomes at pH 7.5.
Authors: Haywood A M , Boyer B P
Published: May 2006

Influenza virus X-31 (H3N2) membranes fuse with liposomes containing ganglioside GD1a at pH 7.5. Fusion was demonstrated by electron microscopy and also can be measured by counting the labeled virus proteins incorporated into liposomes after bound virus has been removed. Liposomes composed of lipids that have no net charge behave as reported by other investigators and do not fuse with influenza X-31 membranes at neutral pH, but they do fuse at low pH. Therefore, the liposomal composition is a factor in whether liposomes fuse with influenza virus membranes at neutral pH, probably by determining whether binding occurs. The liposomal composition necessary for fusion at neutral pH needs to be individualized for each influenza subtype. To establish that a virus requires low pH for membrane fusion, it is first necessary to establish that fusion does not occur at neutral pH under conditions where adequate binding occurs.
Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation.
Authors: Kamps M P , Buss J E , Sefton B M
Published: May 2006

p60src, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the alpha-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotide-directed mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p60src with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p60src, the inability of this mutant p60src protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.
Regulation of aspartate transcarbamoylase synthesis in Escherichia coli: analysis of deletion mutations in the promoter region of the pyrBI operon.
Authors: Levin H L , Schachman H K
Published: May 2006

The catalytic and regulatory polypeptide chains of Escherichia coli aspartate transcarbamoylase are encoded by the pyrB and pyrI genes, respectively, which constitute a single transcriptional unit in the pyrBI operon. The DNA sequence immediately preceding the first structural gene, pyrB, contains a short open reading frame that could encode a 44-amino acid leader peptide and a (G+C)-rich region of dyad symmetry followed by eight thymidine residues. Synthesis of the enzyme is negatively controlled at the level of transcription depending on the cellular level of UTP, and an attenuation mechanism has been proposed to account for the 70-fold increase in pyrBI expression on pyrimidine starvation. The potential role of the dyad and eight thymidines as an attenuator was tested with a plasmid containing the promoter region of the pyrBI operon upstream of the galK coding sequence. When cells containing this plasmid, pPYRB10, were grown in a medium low in uracil, there was an 83-fold increase in galactokinase activity compared with the same cells grown at high uracil levels. This regulation is similar to that for aspartate transcarbamoylase synthesis in cells depleted of pyrimidines. Deletions constructed in the promoter region of pPYRB10 from the 3' side produced one plasmid that retained normal control of galK expression and five that exhibited greatly reduced regulation. Nucleotide sequence determination showed that the one deletion mutation that was functionally similar to the wild-type plasmid contained the entire region of dyad symmetry, including the eight thymidines. The plasmids with more extensive deletions lacked the region with dyad symmetry and the eight thymidines. One of the deletion mutants that exhibited very low levels of regulation lacks the entire sequence coding for the putative leader peptide up to the major promoter. The results demonstrating the crucial role of a 19-nucleotide sequence (from -33 to -15) support an attenuation model but indicate that other mechanisms also contribute to the regulation of the pyrBI operon.
Inherently radioresistant cells exist in some human tumors.
Authors: Weichselbaum R R , Dahlberg W , Little J B
Published: May 2006

We investigated the survival of 29 human tumor cell lines after treatment with ionizing radiation during exponential growth. The radiosensitivity of 20 tumor cell lines was within the range of normal fibroblasts. However, 9 cell lines derived from radio-incurable tumors appeared inherently radioresistant. When the ability to repair potentially lethal x-ray damage was studied in plateau phase cultures, some tumor cell lines derived from radio-incurable tumors were more efficient in this process. These results indicate that the presence of radioresistant and/or repair-proficient cells, which exist in some human tumors, may be responsible for failure to obtain local control following therapeutic x-ray treatment.
DNA supercoiling affects in vitro transcription of two maize chloroplast genes differently.
Authors: Stirdivant S M , Crossland L D , Bogorad L
Published: May 2006

Two adjacent, divergently transcribed, developmentally regulated genes of the maize chloroplast chromosome have different superhelical density/transcriptional activation profiles when transcribed in vitro by the homologous DNA-dependent RNA polymerase. Promoter-specific transcription of the gene for the beta and epsilon subunits of coupling factor 1 (cf1BE) increases and plateaus from templates of increasing negative superhelicity, while transcription of the gene for ribulose bisphosphate carboxylase large subunit (rcL) rises and then falls. Maximal transcription from the two promoters occurs at different template negative superhelical densities and transcription of the two genes is stimulated to different degrees. The different superhelicity profiles alter the molar ratios of the two transcripts over an order of magnitude. Changes in DNA conformation represent one possible mechanism for the differential regulation of the genes.
Molecular mechanism of codon recognition by tRNA species with modified uridine in the first position of the anticodon.
Authors: Yokoyama S , Watanabe T , Murao K , Ishikura H , Yamaizumi Z , Nishimura S , Miyazawa T
Published: May 2006

Proton NMR analyses have been made to elucidate the conformational characteristics of modified nucleotides as found in the first position of the anticodon of tRNA [derivatives of 5-methyl-2-thiouridine 5'-monophosphate (pxm5s2U) and derivatives of 5-hydroxyuridine 5'-monophosphate (pxo5U)]. In pxm5s2U, the C3'-endo form is extraordinarily more stable than the C2'-endo form for the ribose ring, because of the combined effects of the 2-thiocarbonyl group and the 5-substituent. By contrast, in pxo5U, the C2'-endo form is much more stable than the C3'-endo form, because of the interaction between the 5-substituent and the 5'-phosphate group. The enthalpy differences between the C2'-endo form and the C3'-endo form have been obtained as 1.1, -0.7, and 0.1 kcal/mol (1 cal = 4.184 J) for pxm5s2U, pxo5U, and unmodified uridine 5'-monophosphate, respectively. These findings lead to the conclusion that xm5s2U in the first position of the anticodon exclusively takes the C3'-endo form to recognize adenosine (but not uridine) as the third letter of the codon, whereas xo5U takes the C2'-endo form as well as the C3'-endo form to recognize adenosine, guanosine, and uridine as the third letter of the codon on ribosome. Accordingly, the biological significance of such modifications of uridine to xm5s2U/xo5U is in the regulation of the conformational rigidity/flexibility in the first position of the anticodon so as to guarantee the correct and efficient translation of codons in protein biosynthesis.
Gene expression during terminal differentiation: dexamethasone suppression of inducer-mediated alpha 1- and beta maj-globin gene expression.
Authors: Kaneda T , Murate T , Sheffery M , Brown K , Rifkind R A , Marks P A
Published: May 2006

Previous studies demonstrated that hexamethylenebisacetamide (HMBA)-mediated murine erythroleukemia cell (MELC) commitment to terminal division could be suppressed by dexamethasone. A rapid (less than 2 hr) increase (step-up) in commitment to terminal cell division was observed if, after 60-70 hr in culture with inducer and steroid, MELC were transferred to medium with HMBA alone. This step-up commitment was not inhibited by actinomycin or cordycepin but was blocked by cycloheximide. In this study, we show that dexamethasone blocks HMBA-mediated activation of alpha 1- and beta maj-globin gene transcription but not the induced chromatin change characterized by appearance of DNase I-hypersensitive regions upstream from the 5' cap sites of the alpha 1- and beta maj-globin genes. A rapid (less than 2 hr) activation (step-up) of alpha 1-globin gene transcription was observed if, after 48-60 hr in culture with HMBA and dexamethasone, MELC were transferred to medium with HMBA alone. Activation of transcription of the beta maj-globin gene requires 12-24 hr of further culture. Cycloheximide inhibits step-up transcription of both globin genes. Thus, dexamethasone blocks HMBA-mediated modulation of transcription of several nonlinked genes whose expression is altered in a coordinated manner during induced MELC terminal differentiation. Further, the steroid blocks at a late step, a step after that which is rate-limiting to HMBA-mediated MELC differentiation.
Structural organization of the DR subregion of the human major histocompatibility complex.
Authors: Spies T , Sorrentino R , Boss J M , Okada K , Strominger J L
Published: May 2006

Two clusters of overlapping cosmid and lambda phage clones comprising 205 kilobases (kb) have been isolated from the DR subregion of the human major histocompatibility complex from a DR4 haplotype. A single DR alpha and three DR beta genes were identified. In one cluster (135 kb), the DR alpha gene is 90 kb distant from the DR beta gene encoding a molecule that carries the MT3 serological specificity. In the second cluster (70 kb), the DR beta gene determining the DR4 specificity is located 22 kb apart from a DR beta pseudogene (DR beta psi). A 3- to 4-kb sequence located at the 5' end of the DR beta (MT3) gene is common to all three DR beta-chain genes. In addition, three more copies of this sequence are spaced between the DR alpha and the DR beta (MT3) genes in the first cluster and one of these, at least, is associated with a DR beta 1 exon, suggesting that additional genes could be encoded in this region and that multiple duplication events have led to its evolution.
Hemin inhibits internalization of transferrin by reticulocytes and promotes phosphorylation of the membrane transferrin receptor.
Authors: Cox T M , O'Donnell M W , Aisen P , London I M
Published: May 2006

Addition of hemin to reticulocytes inhibits incorporation of iron from transferrin [Ponka, P. & Neuwirt, J. (1969) Blood 33, 609-707]. Heme also regulates protein synthesis in immature erythroid cells through its effects on phosphorylation of the initiation factor eIF-2. We have therefore examined its effects on endocytosis of iron-transferrin and phosphorylation of the transferrin receptor. Hemin (10-50 microM) reduced iron transport but increased cell-associated transferrin. When intracellular iron delivery was inhibited by NH4Cl, no such increase in cell-associated transferrin was seen. During uptake of 125I-labeled transferrin in the steady state, the use of a washing technique to dissociate bound transferrin on the cell membrane showed that radioligand accumulated on the surface of hemin-treated cells. Hemin reduced the initial influx of transferrin, thereby diminishing incorporation of iron. Receptor phosphorylation was investigated by immunoprecipitation of reticulocyte extracts after metabolic labeling with [32P]Pi. In the absence of ligand, phosphorylated receptor was chiefly localized on cell stroma. Exposure to transferrin increased cytosolic phosphorylated receptor from 15-30% to approximately 50% of the total, an effect overcome by hemin treatment. Addition of hemin in the presence of transferrin enhanced net phosphorylated receptor in the reticulocyte in association with a redistribution of phosphorylated receptor to stromal membranes. The findings suggest a possible relationship of phosphorylation to endocytosis of the transferrin receptor in reticulocytes.
Potential role of the src gene product in inhibition of gap-junctional communication in NIH/3T3 cells.
Authors: Chang C C , Trosko J E , Kung H J , Bombick D , Matsumura F
Published: May 2006

The effects of the src gene on the activity of protein kinase C and intercellular communication have been studied in transformed NIH/3T3 clones isolated from soft agar following transfection with the plasmid carrying the v-src gene (psrc-11). Six transformed clones that were studied contained newly incorporated v-src genes in the genome, had an increased amount of pp60src, and showed enhanced activities of protein kinase C. Intercellular communication, studied by observing with autoradiography the transfer of [3H]uridine nucleotide from prelabeled donor cells to recipient cells in contact, was found to be reduced in transformed clones as compared to parental NIH/3T3 cells. Treatment with phorbol 12-myristate 13-acetate was also found to increase protein kinase C activity and to reduce intercellular communication in normal NIH/3T3 cells. These results suggest that the v-src gene product, in a manner similar to some of the powerful tumor promoters, may directly or indirectly affect cell-cell communication.
Differentiation of a human leukemia cell line and expression of collagenase inhibitor.
Authors: Bar-Shavit Z , Teitelbaum S L , Stricklin G P , Eisen A Z , Kahn A J , Welgus H G
Published: May 2006

A human collagenase inhibitor (CI) of Mr 28,500 has been extensively characterized in skin fibroblasts and identified in a variety of connective tissues. Because human alveolar macrophages synthesize and secrete both a collagenase and CI that are immunologically and functionally identical to their counterparts in fibroblasts, we studied the production of such proteins by an immature human cell line (HL60) that can be induced to differentiate along monocytic or granulocytic pathways. The cells failed to synthesize collagenase under any culture condition tested. However, upon exposure to 1,25-dihydroxyvitamin D3 or phorbol esters (PMA), both of which promote monocytic differentiation of HL60, these cells synthesized and released CI in a dose-dependent manner. Furthermore, the extent of CI expression was paralleled by the acquisition by such cells of the monocytic marker 63D3, indicating that inhibitor production and differentiation are closely correlated. This CI was immunologically and functionally identical to that produced by human macrophages and human skin fibroblasts. The quantity of CI synthesized by PMA-stimulated cells was 3- to 5-fold greater than produced by human alveolar macrophages, approximately equal to 1 microgram per 10(6) cells per day. In contrast, undifferentiated HL60 cells produced little or no detectable CI (less than or equal to 10-20 ng per 10(6) cells per day). Interestingly, when HL60 cells were stimulated to undergo granulocytic differentiation by dimethyl sulfoxide or retinoic acid, they also produced the "monocytic" CI.
Transposable element sequences involved in the enhancement of yeast gene expression.
Authors: Roeder G S , Rose A B , Pearlman R E
Published: May 2006

The his4-917 mutation of yeast results from the insertion of a Ty element, Ty917, into the 5' regulatory region of the HIS4 gene. Ty917 prevents HIS4 transcription, thus rendering the cell histidine requiring. Recombination between Ty917 and a Ty element elsewhere in the yeast genome can result in the replacement of part or all of the Ty917 element by sequences from the Ty element. Recombinant derivatives display a variety of phenotypes including His-, weakly His+, and strongly His+. In most of the His+ derivatives, the expression of HIS4 is controlled by genes at the mating type locus. To identify the Ty sequences important in controlling the expression of an adjacent gene, we used Ty elements that have different effects on gene expression to construct hybrid Ty elements in vitro. The effects of these hybrid elements on HIS4 expression were examined. These experiments indicate that the critical sequence differences between Ty elements that permit HIS4 expression and those that prevent its expression lie in the rightmost (HIS4-proximal) 730 base pairs of the element. The DNA sequence of this region was determined for three elements: Ty917, which prevents HIS4 expression; Ty917(467), which confers a weak His+ phenotype; and Ty917(480), which confers a strong His+ phenotype. Within this region, Ty917(467) differs from Ty917 by a single base-pair change that is in the internal (epsilon) region of the Ty element. Ty917(480) differs from Ty917 by this same base-pair change and by 10 changes in the terminal delta sequence. The sequence change common to Ty917(467) and Ty917(480) lies in a region of the Ty element that is homologous to the simian virus 40 enhancer of transcription.
Genes encoding the T-cell receptor alpha and beta subunits are transcribed in an ordered manner during intrathymic ontogeny.
Authors: Royer H D , Ramarli D , Acuto O , Campen T J , Reinherz E L
Published: May 2006

To further characterize sequential events involved in activation of genes encoding the T-cell receptor (a complex of T3 molecules and a disulfide-linked heterodimer designated Ti, T3-Ti) for antigen and the major histocompatibility complex during intrathymic ontogeny, cDNA probes specific for Ti alpha and Ti beta subunits were used for transcriptional analysis. Ti beta transcript levels were minimal in stage I thymocytes, maximal in stage II thymocytes, and intermediate in state III thymocytes. In contrast, Ti alpha transcriptional activity was virtually undetectable in stage I, was low in stage II, and achieved high levels only in the stage III compartment. Analysis of tumor populations derived from individual stages of thymic differentiation confirmed these observations and demonstrated that clonal stage I-II cells often express Ti beta RNA in the absence of Ti alpha RNA. The latter was not a consequence of functional Ti alpha-subunit isotypy because each of 13 interleukin 2-dependent T-cell clones, including inducer, suppressor, and cytotoxic T cells, contain transcripts that hybridize with the Ti alpha probe. From these data it is concluded that Ti beta gene activation precedes Ti alpha gene activation. Moreover, the high level of Ti beta mRNA preceding Ti alpha mRNA expression implies that Ti beta mRNA and/or its protein products may regulate Ti alpha gene transcription.
Regulated expression of mammalian histone H4 genes in vivo requires a trans-acting transcription factor.
Authors: Capasso O , Heintz N
Published: May 2006

Mouse L cells containing integrated copies of a human histone H4 gene have been obtained by cotransfection with the herpesvirus thymidine kinase gene. Nuclease S1 assays of RNA from several independent cell lines show that the expression of the introduced H4 gene is regulated during the cell cycle. One of these cell lines (line 6-8) contains more than 60 human H4 gene copies per haploid genome and does not express the endogenous mouse histone H4 mRNA. In contrast, the expression of the mouse H2a and H3 mRNAs in this cell line is not perturbed. In cell revertants that have lost the majority of the human H4 gene copies, the expression of the mouse H4 mRNA is restored, demonstrating that the mouse genes remain functional although not expressed. The rate of transcription of the histone H4 genes in clone 6-8 is at least 10-fold greater than that of the parental cell line and it is regulated during traversal of the cell cycle. These results show that the expression of mammalian histone H4 genes involves both a trans-acting transcriptional regulatory factor and an H4-specific activity. We propose that cell cycle regulation of histone gene expression may be effected through subtype-specific transcriptional regulatory proteins.