bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of enzymatically active reverse transcriptase in Escherichia coli.
Authors: Tanese N , Roth M , Goff S P
Published: May 2006

Reverse transcriptase of murine retroviruses is a monomeric protein of approximately 80,000 daltons, which is encoded by the central portion of the viral pol gene. To prepare large quantities of the enzyme, we have constructed gene fusions between the trpE gene and portions of the pol gene of Moloney murine leukemia virus. The inserted pol gene sequences include the entire coding region for the mature enzyme and various amounts of additional coding sequences. Many of these constructs express high levels of reverse transcriptase activity even though the NH2 and COOH termini of the protein product only approximate the correct termini of the authentic protein.
Isolation of the gene encoding adenylate cyclase in Saccharomyces cerevisiae.
Authors: Casperson G F , Walker N , Bourne H R
Published: May 2006

By complementation of the cyr1-1 mutation in Saccharomyces cerevisiae, we have isolated yeast genomic DNA containing the structural gene that encodes the catalytic unit of adenylate cyclase (EC 4.6.1.1). The isolated DNA restored adenylate cyclase activity to cyr1-1 mutants and directed integration at the CYR1 locus. Wild-type strains transformed with CYR1 DNA on the high copy number vector YEp24 contained 4- to 6-fold more adenylate cyclase activity than strains carrying the plasmid with no insert. This result suggests that expression of the CYR1 gene product, rather than that of other polypeptide components of the adenylate cyclase system, limits total adenylate cyclase activity in S. cerevisiae. CYR1-containing plasmids also complemented the temperature-sensitive growth defect of the cell division cycle mutation cdc35-1, which confers a phenotype under restrictive conditions similar to that of cyr1-1 and maps to the same locus. Further, cdc35-1 cam mutants, which contain mutations that enable them to take up cAMP from the medium, grew at the restrictive temperature in the presence of exogenous cAMP. These observations support the view that CDC35 and CYR1 are allelic and confirm the hypothesis that cAMP synthesis is required for cells to pass through the "start" position of the cell division cycle.
Olfactory neuron-specific protein is translated from a large poly(A)+ mRNA.
Authors: Rogers K E , Grillo M , Sydor W , Poonian M , Margolis F L
Published: May 2006

Poly(A)+ mRNA was isolated from rat olfactory mucosa and translated in a rabbit reticulocyte cell-free protein synthesizing system. Olfactory marker protein (OMP) of Mr 18,500 was faithfully produced by this system upon addition of mucosal mRNA. The protein was identified by radioimmunoprecipitation with specific anti-OMP serum and by competitive displacement of the radioactive product with authentic OMP. In addition, the immunoprecipitated product comigrated with OMP on NaDodSO4/polyacrylamide gels and on HPLC. In vitro synthesized OMP represented 0.5% of the total translational products. Total olfactory mucosal poly(A)+ mRNA is approximately 1.5-21 kilobases in size, as determined by denaturing agarose gels. Translational assays of gel-fractionated poly(A)+ mRNA demonstrated that OMP mRNA occurs in the 2.5- to 3.4-kilobase range. An mRNA of this size could code for a protein significantly larger than OMP. Since the in vitro synthesized OMP is indistinguishable in size from OMP isolated from tissue, our data indicate that OMP is synthesized directly without the intermediate formation of a larger polypeptide precursor. Thus, OMP mRNA contains untranslated regions that are four to five times larger than the coding region.
Peripheral-type benzodiazepines influence ornithine decarboxylase levels and neurite outgrowth in PC12 cells.
Authors: Morgan J I , Johnson M D , Wang J K , Sonnenfeld K H , Spector S
Published: May 2006

A number of the benzodiazepines (BZDs) inhibit nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner in PC12 cell cultures. The rank order of potency of a series of BZDs for inhibition of neurite outgrowth does not correlate with the order of their affinity constants for the so-called peripheral BZD sites present on PC12 cells. Whereas the inhibition of neurite extension is stereospecific, the binding to the peripheral site is not. The inhibition of neurite outgrowth is not attributable to a blockade of NGF binding to either its fast or slow receptors. Additionally, several other characteristics of NGF stimulation of PC12 cells, such as autoadhesion and the induction of ornithine decarboxylase (OrnDCase), remain unaltered in the presence of BZDs. Many BZDs increase OrnDCase levels in PC12 cells in the absence of NGF. The OrnDCase response to BZDs is blocked by actinomycin D. Furthermore, the structural requirements of BZDs for induction of OrnDCase activity is not identical to that for the inhibition of NGF-induced neurite extension. Thus, unlike attenuation of neurite extension, BZD induction of OrnDCase is not stereospecific. Also, some BZDs block neurite growth but do not induce OrnDCase. We propose that there are at least three sites of action of BZDs on PC12 cells. The first is the well-characterized high-affinity peripheral site. The second distinct locus of action results in a blockade of NGF-induced neurite outgrowth. The structural requirements for the latter effect are essentially indistinguishable from those for BZD induction of hemoglobin synthesis in Friend erythroleukemia cells. The third site of action results in an induction of OrnDCase. Structural requirements for this activity are not identical to those for either the differentiation of Friend cells or the inhibition of neurite outgrowth.
Radiation inactivation of ricin occurs with transfer of destructive energy across a disulfide bridge.
Authors: Haigler H T , Woodbury D J , Kempner E S
Published: May 2006

The ionizing radiation sensitivity of ricin, a disulfide-linked heterodimeric protein, was studied as a model to determine the ability of disulfide bonds to transmit destructive energy. The radiation-dependent loss of A chain enzymatic activity after irradiation of either intact ricin or ricin in which the interchain disulfide bond was disrupted gave target sizes corresponding to the molecular size of dimeric ricin or monomeric A chain, respectively. These results clearly show that a disulfide bond can transmit destructive energy between protein subunits.
Nonrandom association of a type II procollagen genotype with achondroplasia.
Authors: Eng C E , Pauli R M , Strom C M
Published: May 2006

Achondroplasia is an autosomal dominant disorder that involves defective endochondral bone formation. Type II collagen is the predominant collagen of cartilage. We found a HindIII polymorphic site in the normal Caucasian population by using the type II procollagen gene probe pgHCol(II)A. The presence of this site yields a 7.0-kilobase (kb) band; its absence yields a 14.0-kb band. We found a significant deviation in genotype distribution and allele frequencies in a population of unrelated individuals with sporadic achondroplasia, compared with the normal control population. The HindIII genotype frequencies in 32 individuals with achondroplasia are 0.41 for the 7/7 genotype (controls, 0.08), 0.34 for the 7/14 genotype (controls, 0.54), and 0.25 for the 14/14 genotype (controls, 0.37). The apparent equilibrium excess of the "7" allele in individuals with achondroplasia may reflect either a predisposition for the mutation that causes achondroplasia or it could be the result of the achondroplasia-causing mutation. In either case, these findings suggest an association of the type II procollagen gene with achondroplasia.
Structural analysis of TL genes of the mouse.
Authors: Obata Y , Chen Y T , Stockert E , Old L J
Published: May 2006

Three Tla region-specific probes have been generated from the BALB/c genomic cosmid clone C6.3. One probe, pTL1, corresponds to 3' sequences of a thymus leukemia (TL)-encoding gene, whereas pTL2 and pTL3 detect noncoding flanking sequences. The TL specificity of pTL1 was demonstrated by studies of RNA from thymocytes of TL+ and TL- mouse strains and from TL+ and TL- leukemias; presence/absence of pTL1+ transcripts correlated with presence/absence of TL antigens detected serologically. Nine Tla haplotypes were defined by restriction fragment polymorphism with pTL1, and the number of TL genes has been estimated to be greater than or equal to 4 in Tlaa, Tlac and Tlae mice, greater than or equal to 3 in Tlad mice, and greater than or equal to 2 in Tlab and Tlaf mice. A TL-encoding gene (C25.1) from the C57BL/6TL+ leukemia ERLD has been cloned and sequenced, and the exon/intron organization of C25.1 has been deduced from the structure of pTL1+ cDNA clones and from the known organization of H-2 genes. The major structural differences between TL and H-2 genes are in exons coding for the cytoplasmic domain.
The I region of the C57BL/10 mouse: characterization and physical linkage to H-2K of an SB beta-like class II pseudogene, psi A beta 3.
Authors: Widera G , Flavell R A
Published: May 2006

In the C57BL/10 mouse, 140 kilobases (kb) of the I region (I-Ab, I-Eb) were isolated as recombinant cosmids. The class II genes A beta 2, A beta 1, A alpha,E beta 1, E beta 2, and E alpha are located from centromere to telomere in a region of approximately equal to 110 kb, which shows that the I region in the b haplotype has a similar overall organization to those described for the d, k, and wr7 haplotypes. In addition to these genes, we have also isolated a class II gene, psi A beta 3, which is physically linked to the class I H-2K region, 75 kb telomeric to the H-2Kb gene. This orients the H-2K region on the genetic map with the H-2Kb gene being located toward the I region. The sequence of the beta 2 domain of psi A beta 3 is similar to the immunoglobulin-like domain of other class II genes. Interestingly, it shows 83% nucleotide homology to the human SB beta gene, the same homology that was seen previously between the immunoglobulin-like exons of A beta 1 and DC beta and between E beta 1 and DR beta, respectively. It is likely, therefore, that psi A beta 3 represents a member of a third SB-like class II gene family present in addition to I-A and I-E genes and that the divergence of the SB family predates the speciation of rodents and primates. Comparison of the DNA sequence of the exon encoding the beta 2 domain of psi A beta 3 in the b or k haplotypes with functional class II genes shows that a deletion of eight nucleotides has occurred, such that the psi A beta 3 sequence cannot be translated into a functional class II protein. This suggests that psi A beta 3 is a pseudogene.
Regional distribution of calcium influx into bursting neurons detected with arsenazo III.
Authors: Graubard K , Ross W N
Published: May 2006

Absorbance changes of the metallochromic indicator arsenazo III were used in conjunction with an array of 100 photodiodes to measure changes in intracellular calcium concentration at many positions simultaneously in identified neurons of the crab stomatogastric ganglion. When stimulated with intrasomatically injected current, several of these neurons showed calcium changes all over the cell, indicating that calcium channels were distributed widely in the neuropil and on the soma. When the membrane potential was allowed to oscillate without stimulation, absorbance oscillations were detected all over the neuropil but not in the soma. A comparison between the membrane potential recorded in the soma and the calcium signal in the neuropil shows that calcium entry followed the slow voltage oscillation with the peak calcium signal detected 50-150 msec after the end of the voltage plateau.
A 5' spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei.
Authors: Sather S , Agabian N
Published: May 2006

The approximately 15 alpha- and 15 beta-tubulin genes of Trypanosoma brucei are arranged in a tandem array of alternating alpha- and beta-tubulin genes. We have examined the structure of mRNA transcripts from the tubulin gene family and have found at the 5' ends of both alpha- and beta-tubulin messages a 35-nucleotide spliced leader (SL) identical to that identified previously on the 5' ends of variant surface glycoprotein (VSG) mRNAs. No 35-nucleotide SL sequences were encoded in the tubulin intergenic regions; instead, the SL sequence apparently originates from small RNA transcripts encoded at sites that are unlinked to the tubulin genes. The tandem arrangement of the tubulin genes and the presence of the SL at the 5' end of both alpha- and beta-tubulin transcripts establishes that RNA joining between the SL and tubulin mRNA occurs in trans.
Complete structure of the alpha B-crystallin gene: conservation of the exon-intron distribution in the two nonlinked alpha-crystallin genes.
Authors: Quax-Jeuken Y , Quax W , van Rens G , Khan P M , Bloemendal H
Published: May 2006

We isolated bovine complementary DNA clones for the alpha A- and alpha B-crystallin subunits. The alpha B cDNA clone was used to isolate an alpha B-crystallin gene. This gene, derived from hamster, occurs as a single copy in the genome and is 3.2 kilobases long. The coding sequences are spread on three exons with a total length of 709 nucleotides. The exon-intron distribution of the hamster alpha B-crystallin gene is similar to that of the alpha A-crystallin gene except for the 69 nucleotides that specify the 23 "insert" residues of the alpha AIns chain by means of differential splicing. The 3' noncoding region of the alpha B mRNA (140 bases), which is short compared with the alpha A mRNA (520 bases), shows a remarkable homology between calf and hamster. Both alpha-crystallin cDNA clones have been used to assign the chromosomal location of the corresponding human genes with the aid of somatic cell hybrids. It is shown that the single-copy alpha A- and alpha B-crystallin genes are located on different chromosomes.
Anti-immunoglobulin augments the B-cell antigen-presentation function independently of internalization of receptor-antigen complex.
Authors: Casten L A , Lakey E K , Jelachich M L , Margoliash E , Pierce S K
Published: May 2006

All mouse splenic B cells, including small resting B cells, process and present the native globular protein antigens, pigeon and tobacco hornworm moth cytochromes c, to a cytochrome c-specific T-cell hybrid in a major histocompatibility complex-restricted fashion, in the micromolar to nanomolar antigen-concentration range. As is the case for macrophages, treatment with paraformaldehyde or the lysosomotropic agents chloroquine and ammonium chloride blocked processing of the native pigeon protein but did not affect the presentation of a carboxyl-terminal peptide fragment of pigeon cytochrome c (residues 81-104) which contained the T-cell antigenic determinant. However, in contrast to macrophages, whose antigen-processing and -presentation functions are insensitive to radiation, radiation blocked the processing of the native protein but not the presentation of the peptide fragment. The processing and presentation function of the B cells was augmented by F(ab')2 of rabbit anti-mouse Ig antibodies, in that 1/10th to 1/30th as many cells and 1/10th as much antigen were required to maximally activate the T-cell hybrid. This augmentation did not appear to be due to either crosslinking of the Ig receptors or to B-cell activation, as monovalent Fab fragments were nearly as effective as the bivalent reagent, and the concentrations of F(ab')2 anti-Ig used did not induce measurable proliferative responses. Furthermore, enhancement can occur in the absence of cytochrome c binding and internalization, since B cells that were fixed with paraformaldehyde after treatment with F(ab')2 anti-Ig were more effective in presenting the carboxyl-terminal peptide than were untreated fixed cells. The same phenomenon followed the binding of an irrelevant antigen (carboxydinitrophenylated bovine serum albumin) by antigen-binding B cells, resulting in enhanced processing and/or presentation of native pigeon cytochrome c. Thus, nonspecific enhancement of antigen processing and presentation can be obtained by either antigen or anti-Ig binding to the B-cell antigen receptor, both treatments presumably delivering the same signal without requiring internalization of the specifically bound antigen for subsequent processing.
Immunization with Leishmania receptor for macrophages protects mice against cutaneous leishmaniasis.
Authors: Handman E , Mitchell G F
Published: May 2006

The Leishmania major receptor for macrophages is a lipid-containing glycoconjugate that is recognized by the monoclonal antibody WIC-79.3. When L. major promastigotes were incubated with Fab fragments of WIC-79.3 prior to injection into genetically susceptible mice, their infectivity was decreased. Fab fragments from an irrelevant control antibody of the same class had no effect. The L. major glycolipid was purified from detergent-solubilized promastigotes by affinity chromatography on immobilized WIC-79.3 and used to vaccinate mice that are genetically resistant or susceptible to disease. Genetically resistant mice could be protected totally from cutaneous disease with as little as 5 micrograms of glycolipid. A high but not absolute level of resistance was also induced in the susceptible mice, in which the disease is otherwise fatal. No protection was obtained with the carbohydrate fragment of the glycolipid alone or by injection of the glycolipid in the absence of adjuvant. Genetically susceptible mice, immunized and protected from disease as a result of multiple injections of live avirulent cloned promastigotes of L. major, produced antibodies to the glycolipid of L. major. No antibodies were detected in serum from chronically diseased mice. The data suggest that this functionally important antigen of L. major is a candidate vaccine against cutaneous leishmaniasis.
Rat cells infected with anemia-inducing Friend leukemia virus contain integrated replication-competent but not defective proviral genomes.
Authors: Brown E H , Zajac-Kaye M , Pogo B G , Friend C
Published: May 2006

The integrated proviral DNA in five murine cell lines transformed by the anemic strain of Friend leukemia virus (FLV-A) was examined by Southern hybridization to a cloned Friend virus (F-MuLV) probe. Kpn I fragments 9 kilobases (kb) and 5.7 kb long were observed for each cell line. However, the number of copies of each fragment in the cell genome varied according to the cell type. As compared to the adherent epithelioid cell lines, the anchorage-independent erythroleukemic cell lines contained more copies of the 5.7-kb fragment than of the 9-kb fragment, suggesting that the former may be biologically significant and perhaps related to the growth of erythroid cells. The presence of Kpn I fragments of the same sizes, albeit in fewer copies, in normal mouse spleen DNA made it difficult to distinguish exogenous virus from endogenous viral sequences. Therefore, rat 3Y1 cells, which contained no murine endogenous viruses, were infected with FLV-A stock virus prepared directly from the spleens of leukemic mice. Only the 9-kb Kpn I fragment, representing replication-competent Friend virus component, was detected in the infected rat cell DNA. No hybridization was observed to a 0.6-kb fragment of the spleen focus-forming virus env gene that is specific for xenotropic and dual-tropic mink cell focus-forming viruses. Since the virus synthesized by the infected rat cells was leukemogenic in adult mice, these data suggest that the wild-type FLV-A is replicative and fully pathogenic in the absence of other competent virus components.
On the nature of the 5-lipoxygenase reaction in human leukocytes: enzyme purification and requirement for multiple stimulatory factors.
Authors: Rouzer C A , Samuelsson B
Published: May 2006

Arachidonate 5-lipoxygenase was purified 400-fold from homogenates of human peripheral blood leukocytes by a combination of ammonium sulfate fractionation, gel filtration chromatography, and HPLC on anion exchange and hydroxylapatite columns. NaDodSO4/polyacrylamide gel electrophoresis of the purified protein revealed the presence of a single major band (apparent Mr, 80,000). Densitometric analysis of the Coomassie blue staining pattern of the gels revealed that a 90-97% purity had been achieved. As has been reported for the 5-lipoxygenase from other mammalian sources, the human leukocyte enzyme required Ca2+ and ATP for maximal activity. In addition, a number of factors were isolated during the course of the purification, which possessed significant 5-lipoxygenase stimulatory activities. These were obtained in a high-speed pellet of leukocyte homogenate, a 60-90% ammonium sulfate precipitate fraction, and the unabsorbed protein from the first anion exchange HPLC step. In the absence of stimulatory factors, little activity was detected in the purified enzyme, even in the presence of Ca2+ and ATP. The specific function of these various factors is unknown, but their existence suggests that the human leukocyte 5-lipoxygenase is regulated by a complex mechanism that is likely to play an important role in the control of leukotriene and lipoxin biosynthesis.
BSC-1 growth inhibitor transforms a mitogenic stimulus into a hypertrophic stimulus for renal proximal tubular cells: relationship to Na+/H+ antiport activity.
Authors: Fine L G , Holley R W , Nasri H , Badie-Dezfooly B
Published: May 2006

Renal hypertrophy is characterized by an increase in cell size and protein content with minimal hyperplasia. The mechanisms of control of this pattern of cell growth have not been determined. The present studies examined whether the growth inhibitor elaborated by BSC-1 kidney epithelial cells (GI), which has nearly identical biological properties to transforming growth factor beta (TGF-beta), could transform a mitogenic stimulus into a hypertrophic stimulus for rabbit renal proximal tubular cells in primary culture. Insulin (10 micrograms/ml) plus hydrocortisone (50 nM) increased the amount of protein per cell, cell volume, and [3H]thymidine incorporation at 24 and 48 hr in these cells. GI/TGF-beta (10 units/ml) led to a minimal stimulation of [3H]thymidine incorporation. When added together with insulin plus hydrocortisone, GI/TGF-beta inhibited the stimulatory effect of these mitogens on [3H]thymidine incorporation but did not block the increase in protein per cell and cell volume--i.e., the cells underwent hypertrophy. The fact that this pattern persisted for 48 hr indicated that GI/TGF-beta exerted a prolonged inhibitory effect on mitogenic-stimulated DNA synthesis rather than delaying its onset. Amiloride-sensitive Na+ uptake (indicative of Na+/H+ antiport activity) correlated with protein per cell and cell volume rather than with DNA synthesis. P60 gel chromatographic fractionation of conditioned medium harvested from proximal tubular cells yielded a fraction that inhibited [3H]thymidine incorporation in BSC-1 cells and CCL 64 cells; the relative inhibitory activity on these cell lines and the chromatographic behavior were similar to those observed with GI/TGF-beta. These studies indicate that the control of cell size may be regulated by autocrine mechanisms mediated by the elaboration of growth inhibitory factors that alter the pattern of the growth response to mitogens.
Deletions of kappa chain constant region genes in mouse lambda chain-producing B cells involve intrachromosomal DNA recombinations similar to V-J joining.
Authors: Moore M W , Durdik J , Persiani D M , Selsing E
Published: May 2006

We isolated and characterized the germ-line counterpart of a DNA segment designated RS (for recombining sequence), that is frequently recombined in mouse lambda light chain-producing B lymphocytes. Using Southern blot analyses of myelomas and mouse-Chinese hamster fusion cell lines, we found that RS DNA sequences are located on mouse chromosome 6, evidently more than 15 kilobases downstream of the kappa light-chain locus. We find that a typical recognition site for Ig gene recombination is situated within germ-line RS sequences near the recombination points observed in at least two lambda chain-producing cell lines. This represents a complete and functional Ig recognition site that is not directly associated with Ig genes. We also characterized a recombined RS segment isolated from the cell line BM18-4.13.9. This recombined segment has a variable region kappa light chain gene (V kappa) joined directly to RS sequences. Our results suggest that the deletion of the kappa light chain constant region (C kappa) exon in many lambda chain-producing B cells is the result of RS recombination and that C kappa deletion may be mediated by the same processes as antibody gene V-J joining (J = joining segment gene). We discuss the potential biological significance of RS DNA recombination in B-cell maturation.
Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome.
Authors: Lidsky A S , Law M L , Morse H G , Kao F T , Rabin M , Ruddle F H , Woo S L
Published: May 2006

Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1). A cDNA clone for human PAH has previously been used to assign the corresponding gene to human chromosome 12. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3----qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of 125I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22----12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12.
Interleukin 2 receptor gene expression in normal human T lymphocytes.
Authors: Leonard W J , Krönke M , Peffer N J , Depper J M , Greene W C
Published: May 2006

We have used cDNAs for the human interleukin 2 (IL-2) receptor to study IL-2 receptor gene expression in normal activated T cells. Resting T cells do not contain detectable IL-2 receptor mRNA. Within 1 hr after stimulation with phytohemagglutinin (PHA), a large, presumably nuclear precursor RNA species is seen, which then gradually disappears. Mature IL-2 receptor mRNA forms appear within 8 hr after stimulation, reach peak levels between 8 and 24 hr, and then decline. Thus, in PHA-activated lymphocytes the rise and fall in IL-2 receptor mRNA levels precede by more than 24 hr the peak and decline of IL-2 receptor protein expression occurring at the cell surface. 4 beta-Phorbol 12-myristate 13-acetate (PMA) also stimulates IL-2 receptor mRNA and protein expression by T cells. Combinations of optimal concentrations of PHA and PMA produce an additive effect on IL-2 receptor mRNA levels, suggesting that PHA and PMA may induce IL-2 receptor gene expression through different, complementary mechanisms. Nuclease S1-protection assays indicate that IL-2 receptor mRNAs may differ in length due to the use of three different polyadenylylation signals. Further, these assays demonstrate the presence of transcripts that lack a 216-base segment within the protein-coding region and thus do not encode a functional IL-2 receptor. Nuclear transcription assays indicate that the increase in IL-2 receptor mRNA is reflected at the level of transcription. Thus, IL-2 receptor gene regulation controls IL-2 receptor expression at the cell surface and is intimately linked to the control of T-cell proliferation.
Genetic differences in oxygen toxicity are correlated with cytochrome P-450 inducibility.
Authors: Gonder J C , Proctor R A , Will J A
Published: May 2006

Susceptibility to oxygen toxicity was studied in three inbred and two hybrid strains of mice. Because in vitro studies have shown that the cytochrome P-450 enzymes can produce oxygen radicals and H2O2, we tested the hypothesis that inducibility of these enzymes might play a role in oxygen toxicity. Mice responsive to hepatic microsomal enzyme induction by aromatic hydrocarbons [C3H/HeJ, C3H/HeN, C3H/HeJ X DBA/2J (designated C3D2F1/J), C3H/HeN X DBA/2J (designated C3D2F1/N)] were more sensitive to the toxic effects of 100% oxygen exposure than were genetically unresponsive mice (DBA/2J). DBA/2J mice survived significantly longer exposure periods with less lung damage. Lung and liver cytochrome P-450 levels increased 2-to 3-fold in C3H and F1 mice during 100% oxygen exposure (maximum levels at 72-96 hr) and subsequently fell prior to death. No increases were seen in cytochrome P-450 levels in DBA/2J mice. Metabolic pathways involving cytochrome P-450 enzymes may initiate or modulate oxidative damage due to oxygen radicals. The difference in responsiveness of mice to microsomal enzyme induction may imply genetic differences in susceptibility to oxidative stress, may help to explain species differences in susceptibility, and may have long-term implications in therapeutics and patient care if similar inherited differences exist in humans.
A developmentally regulated mRNA from 3T3 adipocytes encodes a novel serine protease homologue.
Authors: Cook K S , Groves D L , Min H Y , Spiegelman B M
Published: May 2006

We previously have isolated cDNA clones for several mRNAs that increase in abundance during the differentiation of 3T3 adipocytes but whose physiological role is unknown. We show here that a mRNA that is complementary to one of these clones and encodes a protein of 28 kDa is expressed abundantly in mouse fat pads but not in several other mouse tissues. Sequence analysis of the corresponding cDNA clone indicated that the encoded protein shows 30% overall amino acid homology to several serine proteases including trypsin, chymotrypsin, and elastase. Homology is much higher (64%) between the 28-kDa protein and regions that are strongly conserved among the members of the serine protease family. The derived protein also has key features characteristic of active serine proteases, including the histidine, aspartic acid, and serine residues, which comprise the charge relay system, and a potential cleavage site for activation of the zymogen. Primer extension analysis performed to obtain the sequence of the 5' end of mRNA that encodes the 28-kDa protein indicates that two forms of this mRNA exist and probably arise through alternative splicing. The two mRNAs encode signal sequences that differ by the deletion of one amino acid near the predicted cleavage site of the signal peptide. These results demonstrate that adipocyte differentiation is accompanied by the expression of mRNA encoding a serine protease homologue that can be synthesized with two different signal peptides.
Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from
Anabaena
7120 expressed in
Escherichia coli
Authors: Gurevitz M. , Somerville C. R. , McIntosh L.
Published: May 2006

We have placed the genes encoding ribulose-bisphosphate carboxylase/oxygenase from the
Anabaena
7120 operon under transcriptional control of the
lac
promoter carried on the
Escherichia coli
plasmid pUC19. The genes encoding both the large and small subunit polypeptides (
rbcL
and
rbcS
) are transcribed and translated so that ≈0.6% of the soluble protein in
E. coli
extracts is a fully functional holoenzyme with a sedimentation coefficient of approximately 18S, which contains stoichiometric amounts of the two subunits. However, expression of the large subunit polypeptide vastly exceeds that of the small subunit because the majority of transcripts terminate in the intergenic region between the
rbcL
and
rbcS
genes. As a result, excess large subunit is synthesized and accumulates in
E. coli
as an insoluble and catalytically inactive form. Because small subunit is found only in the high molecular weight soluble form of ribulosebisphosphate carboxylase/oxygenase, we propose that the small subunit promotes assembly of the hexadecameric form of the enzyme via heterodimers of large and small subunits.
Isolation and chromosomal localization of the human fgr protooncogene, a distinct member of the tyrosine kinase gene family.
Authors: Tronick S R , Popescu N C , Cheah M S , Swan D C , Amsbaugh S C , Lengel C R , DiPaolo J A , Robbins K C
Published: May 2006

The cell-derived domain of Gardner-Rasheed feline sarcoma virus (GR-FeSV) consists of a gamma-actin- and a tyrosine-specific protein kinase-encoding sequence designated v-fgr. By utilizing a v-fgr probe, it was possible to detect related sequences present at low copy number in DNAs of a variety of mammalian species and to isolate a human fgr homologue. Comparative studies revealed that this human DNA clone represented all but 200 base pairs of v-fgr. Analysis of human genomic DNA demonstrated that the fgr protooncogene was distinct from the cellular homologues of other retrovirus onc genes. In addition, the fgr protooncogene was localized to the distal portion of the short arm of human chromosome 1 at p36.1-36.2 by in situ hybridization. Taken together, our findings establish that the fgr protooncogene is a unique member of the tyrosine kinase gene family.
Distribution of initial and persistent 2-acetylaminofluorene-induced DNA adducts within DNA loops.
Authors: Gupta R C , Dighe N R , Randerath K , Smith H C
Published: May 2006

The intranuclear distribution of initial and persistent DNA adducts induced in vivo after four weekly injections of the hepatocarcinogen 2-acetylaminofluorene was examined in rat liver by using a protocol that fractionates chromatin from various regions of each of the multiple nuclear DNA loops. Ten hours after the initial dose, two acetylated [(N-acetyl-N-(deoxyguanosin-8-yl)-2-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene] and one deacetylated [N-(deoxyguanosin-8-yl)-2-aminofluorene] adduct were detected by a 32P-labeling assay and were found to have a random genomic distribution, as evident by their relative concentrations in various chromatin fractions. These data suggest that all regions of the DNA loops are equally susceptible to adduct formation. A nonrandom persistence of the deacetylated adduct in the regions where the DNA loops are constrained by the nuclear matrix was evident by 6 days after the last dose and was markedly apparent by 60 days. In contrast, all chromatin fractions had equally inefficient removal of the N2-acetylated adduct by 6 days as well as 60 days but had complete removal of the C8-acetylated adduct. These findings suggest that pronounced regional differences in adduct repair along the DNA loops may play a role in chemically induced hepatocarcinogenesis.
Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes.
Authors: Falo L D , Sullivan K , Benacerraf B , Mescher M F , Rock K L
Published: May 2006

Several amino acid copolymers are potent immunogens under the control of major histocompatibility complex (MHC)-encoded Ir genes. We have further characterized their accessory-cell-dependent, MHC-restricted presentation to T lymphocytes. We initially characterized their processing requirements by investigating the ability of paraformaldehyde-fixed antigen-presenting cells (APC) to present these copolymers. Fixed APC can present poly(Glu56Lys35Phe9) and poly(Glu60Ala30Tyr10) provided that they have been incubated with antigen prior to fixation. The inability of these same fixed preparations to present soluble antigen indicates a fixation-sensitive antigen-processing step. In contrast, the antigens poly(Glu55Lys35Leu10) and poly(Glu55Lys35Tyr10) can be presented by APC fixed before antigen exposure. This differential requirement for antigen processing was exploited to analyze the events of antigen presentation in two related systems. First, the ability of isolated APC membranes to process and present antigen was assessed. APC membranes can present the antigens poly(GluLysLeu) and poly(GluLysTyr) in a specific and MHC-restricted manner. However, the isolated membranes fail to present either poly(GluLysPhe) or poly(GluAlaTyr), suggesting that such preparations can present but not process antigen. Second, the distinct properties of the various copolymers were used with fixed APC to test the effects of antigen processing on the phenomenon of antigen competition. APC that had processed poly(GluLysPhe) or poly(GluAlaTyr) were subsequently fixed and used to present antigen in the presence or absence of various antagonists. Under these conditions, poly(GluLysLeu) and poly(Glu50Tyr50) could effect specific inhibition, clearly indicating that antigen competition occurs distal to and does not require antigen processing. In contrast, native antigen with an absolute processing requirement is not capable of competing with preprocessed antigen on fixed APC. Taken together, these results suggest that processing is important for the molecular interactions between the copolymer antigens and the APC cell surface that are relevant to both antigen presentation and competitive inhibition.
Correlation of cell-surface phenotype with the establishment of interleukin 3-dependent cell lines from wild-mouse murine leukemia virus-induced neoplasms.
Authors: Holmes K L , Palaszynski E , Fredrickson T N , Morse H C , Ihle J N
Published: May 2006

The wild mouse ecotropic virus, Cas-Br-M murine leukemia virus, induces myeloid and erythroid leukemias as well as T-cell and B-cell lymphomas in NFS mice. The ability to establish long-term cell lines from these tumors in the presence or absence of the T-cell-derived lymphokine interleukin 3 (IL-3) was examined. IL-3-dependent cell lines were readily obtained from the majority of the myeloid or erythroid leukemias and immunoblastic lymphomas. In the absence of IL-3, only one long-term factor-independent cell line was obtained from a myelogenous leukemia. The majority of the thymic T-cell lymphomas or B-lineage lymphomas could not be cultured in the presence or absence of IL-3. The results suggest that transformation of hematopoietic lineages does not necessarily obviate the requirement for normal growth factors. The acquisition of independence from growth factors may require additional transforming events.
Expression of phaseolin cDNA genes in yeast under control of natural plant DNA sequences
Authors: Cramer Jane Harris , Lea Kristi , Slightom Jerry L.
Published: May 2006

We have constructed a strain of
Saccharomyces cerevisiae
that expresses two different members of the multigene family encoding phaseolin, the major seed storage glycoprotein from the French bean,
Phaseolus vulgaris
. Yeast vector plasmids have been engineered to include a
Phaseolus
DNA segment that contains the natural 5′ and 3′ plant genomic regulatory sequences flanking a cDNA copy of the protein-encoding region. Characterization of phaseolin transcripts isolated from transformed yeast cells revealed the presence of two classes of polyadenylylated RNA, approximately 1400 and 1800 bases, which initiate and terminate in plant DNA sequences. Protein extracts from transformants contain phaseolin-immunoreactive proteins similar in size to those isolated from plant tissue. These polypeptides are glycosylated in yeast and their molecular weights are consistent with the possibility that the phaseolin signal peptide has been cleaved to form the mature protein.
Imaging of optically active biological structures by use of circularly polarized light.
Authors: Keller D , Bustamante C , Maestre M F , Tinoco I
Published: May 2006

If an optically active (chiral) sample is placed in a microscope and illuminated with circularly polarized light, an image can be formed that is related to the circular dichroism of each feature of the sample. A theoretical investigation has been done for the circular differential image obtained by subtracting the images formed under right- and left-circularly polarized light. Two types of differential images are possible: (i) dark-field images formed from light reflected or scattered by the sample and (ii) bright-field images formed from light transmitted through the sample. The sign and magnitude of each feature in a circular differential image strongly depend on the structure of the sample. The dark-field circular differential images are most sensitive to large features with dimensions similar to the wavelength of illumination whereas the bright-field images are most sensitive to the short-range molecular order. Applications of circular differential imaging may include clinical fingerprinting of normal and transformed cells and structural analysis of individual cellular components.
Immunoglobulin JH, C mu, and C gamma gene rearrangements in human B lymphocytes clonally transformed by Epstein-Barr virus.
Authors: Brown N A , Liu C , Berenson J R , Garcia C R , Wang R , Calame K L
Published: May 2006

Somatic rearrangements and deletions of immunoglobulin gene segments have been demonstrated in several types of murine B cells. In addition, rearrangements of the JH, C mu, and light chain immunoglobulin gene segments have been reported in human pre-B-cell leukemias and B-cell lymphomas. We have used recombinant DNA probes for the human JH, C mu, and C gamma immunoglobulin gene loci to analyze the genetic events associated with heavy chain gene expression in human B cells clonally transformed by Epstein-Barr virus. Southern hybridization analysis of BamHI-digested cell clone DNAs shows that these human B-cell clones often have bi-allelic JH rearrangements and that heavy chain isotype switching is associated with bichromosomal C mu and C gamma gene rearrangements. Deletions of germ line C mu and C gamma segments were observed that were sometimes bi-allelic. Overall, the observed rearrangements and deletions of heavy chain constant region genes suggest that human heavy chain class switching proceeds in a general order consistent with the proposed order of the heavy chain gene classes along chromosome 14.
Purification and characterization of the mu opiate receptor from rat brain using affinity chromatography.
Authors: Maneckjee R , Zukin R S , Archer S , Michael J , Osei-Gyimah P
Published: May 2006

Opiate receptors have been solubilized from rat neural membranes and purified 500-fold (relative to the crude solubilized extract) by affinity chromatography. Active receptors were solubilized by using 3-[( 3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic derivative of cholic acid. Affinity chromatography was carried out using Affi-Gel 401, a sulfhydryl derivative of agarose to which "hybromet," a newly synthesized opioid ligand with high affinity for the mu receptor, had been attached. Scatchard analysis of [3H]etorphine binding to the purified receptor revealed a single class of high-affinity sites (Kd = 1.4 nM; Bmax = 2800 fmol/mg of protein). Half-maximal binding was achieved at approximately equal to 1 nM. Activity was markedly inhibited by protein modifying reagents, findings which suggest that the sites are proteinaceous. Opiate binding activity was also inhibited by the guanyl nucleotide GTP. Electrophoresis of the purified material under denaturing conditions revealed three subunits of molecular weights 94,000, 44,000, and 35,000. The inhibitory guanyl nucleotide binding protein (Ni) implicated in opiate action has been shown to be comprised of two subunits of molecular weights 42,000 and 35,000. Thus, the opiate receptor may be an aggregate of multiple protein components that may include a guanyl nucleotide binding protein.
Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene.
Authors: Miskimins W K , Roberts M P , McClelland A , Ruddle F H
Published: May 2006

We describe a procedure for detecting high-affinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.
Methionine-sensitive glycolysis in transformed cells.
Authors: Boerner P , Racker E
Published: May 2006

Glycolysis in several tumor cell lines grown in tissue culture was inhibited by methionine. Kirsten murine sarcoma virus-transformed rat kidney cells (K-NRK) were inhibited 60-75% by 10 mM methionine, whereas normal rat kidney (NRK-49F) cells showed little or no inhibition. Inhibition of glycolysis in K-NRK cells was manifest 2-4 hr after exposure to the amino acid. Glycolysis in a chemically transformed cell line of Madin-Darby canine kidney cells was also sensitive to methionine, but maximal inhibition (75%) required 18-24 hr of incubation with the amino acid. Under the same conditions glycolysis in the nontransformed canine cells was less than 20% inhibited by methionine. In Ehrlich ascites tumor cells grown in tissue culture, 10 mM methionine inhibited glycolysis by about 50%. Inhibition of glycolysis, even by 50 mM methionine, was rapidly reversible. Within 2 hr after removal of methionine the rate of glycolytic activity was restored to that observed in control cells. Furthermore, inhibition by methionine required a minimum level (7%) of serum in the growth medium and inhibition was not sensitive to cycloheximide. Only amino acids that are transported by system A (including the nonmetabolized analogue methylaminoisobutyric acid) specifically inhibited glycolysis in tumor cells. The only exception was phenylalanine, which was toxic to both transformed and normal cell lines.
Immunization of rabbits with purified RNA polymerase I induces a distinct population of antibodies against nucleic acids as well as anti-RNA polymerase I antibodies, both characteristic of systemic lupus erythematosus.
Authors: Stetler D A , Jacob S T
Published: May 2006

Rabbits were immunized with either RNA polymerase I or poly(A) polymerase that had been purified to apparent homogeneity and was devoid of nucleic acids. Sera from rabbits thus immunized were screened for antibodies against nucleic acids. All seven rabbits injected with RNA polymerase I but none of the four rabbits immunized with poly(A) polymerase produced anti-nucleic acid antibodies. Anti-RNA polymerase I antibodies were induced after a single injection of the enzyme. Anti-polynucleotide antibodies were not detectable until after the second immunization. Anti-RNA polymerase I antibodies could be detected with as little as 100 pg of purified RNA polymerase I in the radioimmunoassay. At least 50 ng of poly(A) or 200 ng of DNA was required to detect anti-nucleic acid antibodies. The immunoreactivity of anti-RNA polymerase I antisera was greater with synthetic polynucleotides than with DNA, particularly early in the immunization schedule. Alkaline phosphatase treatment of poly(A) to remove 5' phosphates nearly abolished its antigenicity with respect to the early sera and decreased antibody binding of later sera by 60%. These results indicate that the anti-nucleic acid antibodies produced early were primarily directed against determinants including the 5'-terminal phosphates while antibodies produced later were directed against other sites. The antinucleic acid antibodies and anti-RNA polymerase I antibodies formed two distinct populations that were not immunologically crossreactive. We suggest that after injection, RNA polymerase I becomes associated with the nucleic acids present in blood plasma which renders them immunogenic; thus, association of nucleic acids with autoimmunogenic RNA polymerase I may be one of the mechanisms by which anti-DNA antibodies are induced in systemic lupus erythematosus.