Analysis Of Poly Research Articles

Studies on protein poly(ADP-ribosylation) using high resolution gel electrophoresis.

Analysis of poly(ADP-ribose) synthesized in cellular lysates or in isolated nuclei on 100-cm-long thin gels of 20% polyacrylamide, 2.5 M urea permits determination of the exact size of poly(ADP-ribose) molecules using labeled oligonucleotides as molecular weight markers. The size and concentration of poly(ADP-ribose) molecules increase at time intervals during its synthesis. Differences in the concentration of poly(ADP-ribose) size classes among cell lines are also shown. Inhibition of poly(ADP-ribose) degradation by ethacridine that directly interacts with the polymer and inhibits its hydrolysis by poly(ADP-ribose) glycohydrolase shows a dramatic increase in both polymer size and concentration. Use of alkaline conditions for the hydrolysis of poly(ADP-ribose)-protein linkages reveals a specific shortening of all size classes of poly(ADP-ribose) compared with its size in preparations obtained by extensive digestion of nuclei with nucleases, RNases, and proteases.

Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.

Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.

Phytochrome-mediated activation of the gene for cytosolic glutamine-synthetase (GS1) during imbibition of photosensitive lettuce seeds

A full-length cDNA encoding glutamine synthetase was isolated from a lambda gt11 library constructed from the poly(A)+ RNA isolated from lettuce seeds incubated under red light. The nucleotide sequence of the cDNA and the deduced sequence of amino acids showed a high degree of homology to those of the cytosol-type glutamine synthetase from other plants. Northern and dot-blot analyses of poly(A)+ RNA extracted from the seeds incubated under various light conditions showed that the activation of the gene for cytosolic glutamine-synthetase during imbibition of lettuce seeds is directly or indirectly regulated by phytochrome.

Post-transcriptional regulation of thymidine kinase gene expression during monocytic differentiation of HL60 promyelocytes

The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were performed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation.

S100 protein expression in human melanoma cells: Comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle

The synthesis of S100 protein in cultured human melanoma cells was examined using metabolic labeling with [ 35S]methionine, immunoprecipitation with anti-S100 protein antiserum, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Six of seven cell lines derived from melanomas synthesized relatively large amounts of S100 protein, whereas three cell lines derived from normal melanocytes synthesized lesser amounts. Synthesis of S100 protein was not detected in 10 human cell lines of non-neuroectodermal origin. Analysis of poly(A +) RNA from one melanoma cell line by Northern blot hybridization with a probe specific for the β subunit of rat S100 protein revealed a single mRNA species of 1.0 kb coding for the human protein. Flow cytometric analysis of individual cells of two melanoma cell lines and the rat glioma cell line C6 indicated that G0/G1 cells were heterogeneous with respect to S100 protein expression, while almost all the cells in S + G2 + M expressed S100 protein. These results suggest that expression of S100 protein in G0/G1 could be a prerequisite for progression of the cells through the cell cycle.

Analysis of poly(styrene—co-methyl acrylate) and poly(styrene—co-butyl acrylate) by high-performance liquid chromatography

Poly(styrene—co-methyl acrylate) and poly(styrene—co-butyl acrylate) were separated according to their chemical composition by gradient elution. The chromatographic separation on silica was optimized for a gradient ranging from n-heptane as a non-solvent to dichloromethane containing a small amount of methanol as a strong solvent. The influence of different stationary phases, the chemical composition and molecular mass on the separation of the copolymers was investigated. From the results of different chromatographic and turbidimetric experiments it is concluded that the copolymer separation is controlled by both precipitation and adsorption mechanisms. The contribution of adsorption processes to the separation is only advantageous when normal-phase gradients are applied.

α-Amylase gene transcription in tissues of normal dog

We studied the distribution of alpha-amylase mRNA in normal dog tissues by northern blotting (NB) and reverse transcription-polymerase chain reaction (RT-PCR) with human pancreatic (AMY2) and salivary (AMY1) alpha-amylase cDNA-specific primers. Analysis of poly(A+) RNA from various normal tissues by NB indicated the presence of detectable levels of alpha-amylase mRNA transcripts only in pancreas. Dot-blot analysis of DNA amplified with primers common to both (human) isoamylase mRNAs showed presence of alpha-amylase gene transcripts not only in pancreas but also in liver, small intestine, large intestine and fallopian tube. Traces of amylase gene transcripts were also observed in ovary, uterus and lung. Interestingly, amylase transcripts were not detectable in the parotid gland by NB or RT-PCR. We have also localized alpha-amylase mRNA transcripts to dog pancreas by in situ transcription and in situ hybridization. Our results suggest that there is high degree of homology between the alpha-amylase mRNA sequences in dog and human at least in the exon 3-4 regions of the human gene.

Human Gastric Cathepsin E: Predicted Sequence, Localization to Chromosome 1, and Sequence Homology with other Aspartic Proteinases

Abstract The predicted sequence of human gastric cathepsin E (CTSE) was determined by analysis of cDNA clones isolated from a library constructed with poly(A+) RNA from a gastric adenocarcinoma cell line. The CTSE cDNA clones were identified using a set of complementary 18-base oligonucleotide probes specific for a 6-residue sequence surrounding the first active site of all previously characterized human aspartic proteinases. Sequence analysis of CTSE cDNA clones revealed a 1188-base pair open reading frame that exhibited 59% sequence identity with human pepsinogen A. The predicted CTSE amino acid sequence includes a 379-residue proenzyme (Mr = 40,883) and a 17-residue signal peptide. The predicted CTSE amino acid composition was consistent with that of purified material from gastric mucosa and gastric adenocarcinoma cell lines. Additional evidence for the identification of the CTSE cDNA clones was obtained by analysis of poly(A+) RNA isolated from CTSE-producing and -nonproducing gastric adenocarcinoma cell subclones. Three RNA transcripts (3.6, 2.6, and 2.1 kilobases) were identified in poly(A+) RNA isolated from a gastric adenocarcinoma cell line that produced CTSE that were absent from nonproducing subclones. CTSE contains 7 cysteine residues, of which 6 were localized by comparative maximal alignment analysis with pepsinogen A to conserved residues that form intrachain disulfide bonds. The seventh cysteine residue of CTSE is located within the activation peptide region of the proenzyme. We suspect that this residue forms an interchain disulfide bond and thereby determines the dimerization of CTSE proenzyme molecules that is observed under native conditions. The CTSE gene was localized to human chromosome 1 by concurrent cytogenetic and cDNA probe analyses of a panel of human x mouse somatic cell hybrids.

Interspersion of the VHQ52 and VH7183 gene families in the NFS/N mouse.

Deletion mapping analysis has shown that members of the VH7183 and VHQ52 gene families are interspersed in the NFS/N mouse. To obtain direct evidence that members of these gene families are physically linked, an NFS/N liver library was constructed and genomic clones were analyzed for hybridization to both VHQ52 and VH7183 gene probes. Four clones were identified which contained both VHQ52 and VH7183 hybridizable restriction fragments. Two clones containing rearranged VHQ52 genes were also found to hybridize with the VH7183 gene probe. Sequence analysis of three of the VH7183-containing restriction fragments indicate that all are pseudogenes which contain interruptions at either the 5' and/or 3' ends of the VH coding region. Given the D-proximal location of at least a portion of the VHQ52 gene family relative to VH7183 in NFS/N mice, and the known correlation between D proximity and the frequency of VH gene utilization, 22 NFS/N-derived pre-B cell lines were analyzed for VHQ52 gene utilization. More than 40% of the identified H chain (VHDJH) rearrangements in this survey used members of this gene family. Furthermore, analysis of poly(A)+ RNA from NFS/N fetal liver and adult spleen also indicates preferential utilization of VHQ52 family in fetal liver. Kinetic studies show, however, that there are no changes in relative utilization throughout fetal ontogeny. The implications of these findings for the expression and randomization of the VH repertoire are discussed.

Expression of a potyvirus non-structural protein in transgenic tobacco

A cDNA fragment encoding the cytoplasmic inclusion protein of tobacco vein mottling virus was inserted into the plant expression cassette of a Ti plasmid-based binary vector. The vector was transferred to Agrobacterium tumifaciens, and following a modified leaf disc procedure, transformed tobacco plants were obtained. Analysis of poly(A) + RNA from transgenic plants revealed a novel RNA of approximately 2100 nucleotides possessing tobacco vein mottling virus sequences. Also, immunoprecipitation of protein extracts of [ 35S]methionine-labeled transformed callus using anti-cytoplasmic inclusion protein antiserum revealed a polypeptide of approximately 70 kDa. This size is consistent with that predicted from the inserted tobacco vein mottling virus coding sequences. Together these data demonstrate the expression of the cytoplasmic inclusion protein in the absence of viral infections.

Mutants of human colon adenocarcinoma, selected for thymidylate synthase deficiency.

GC3/c1 human colon adenocarcinoma cells were treated with the mutagen ethyl methanesulfonate, and three clones deficient in thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1. 45) activity were selected and characterized. Growth in medium deficient in thymidine caused cell death in two clones (TS- c1 and TS- c3), whereas one clone (TS- c2) showed limited growth. Growth correlated with thymidine synthase activity and 5-fluoro-2'-deoxyuridine 5'-monophosphate-binding capacity and with incorporation of 2'-deoxy[6-3H]uridine into DNA. In the presence of optimal thymidine, growth rates were only 5-18% that of the parental clone (GC3/c1), which grew equally well in thymidine-deficient or -replete medium. Analysis of poly(A)+ RNA showed normal levels of a 1.6-kilobase transcript in TS- c1 and TS- c2 but decreased levels (approximately 6% control) in TS- c3. Clone TS- c3 was 32-, 750-, and greater than 100,000-fold more resistant than the parental clone to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, and methotrexate, respectively. When inoculated into athymic nude mice, each TS- clone produced tumors, demonstrating continued ability to proliferate in vivo.

Analysis of the multiple 5' and 3' termini of poly(A)+ and poly(A)-deficient thymidylate synthase mRNA in growth-stimulated mouse fibroblasts.

Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the G0/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5' and 3' termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5' or 3' termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)- deficient cytoplasmic TS RNA showed that the 5' termini were the same as those found in poly(A) + mRNA. However, the 3' termini were extremely heterogeneous in length. Although some of the poly(A)- deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.

Isolation and characterization of a cDNA for mitochondrial manganese superoxide dismutase (SOD-3) of maize and its relation to other manganese superoxide dismutases

We describe the isolation of a cDNA clone for the nuclear-encoded manganese superoxide dismutase (SOD-3) of maize mitochondria. The cDNA, pSod3.1c, selects by hybridization an RNA which produces the SOD-3 precursor upon in vitro translation. The DNA sequence of pSod3.1c was determined from fragments subcloned in M13. The amino-acid sequence deduced from the nucleotide sequence displays significant homology with the amino-acid sequences of prokaryotic and eukaryotic Mn-SODs, but displays greater homology with mammalian Mn-SOD than it does with yeast or bacterial Mn-SOD. A 31 amino-acid transit peptide also is encoded by the pSod3.1c clone. Analysis of poly(A) + RNA indicates that Sod3 mRNA is approx. 1250 nucleotides in length. The amount of Sod3 transcript in seedling leaves is increased by light.

Processing of the primary transcript for the rat growth hormone gene in vivo.

Processing of the rat growth hormone (rGH) gene primary transcript and the effects of thyroid and glucocorticoid hormones on rGH pre-mRNA levels have been studied using subcloned radiolabeled DNA fragments from each of the four introns of this gene as probes. Blot-hybridization analysis of poly(A)+RNA from GC cells, GH3 cells, and normal pituitary gland indicates that processing of intron sequences from the precursor transcript takes place in a qualitatively similar fashion in each of these cell types. The data indicate that, in general, those introns closest to the termini of the primary transcript are removed first followed by removal of the internal introns. The suggested order of removal is IA, ID, IC, and IB. This process is unaffected qualitatively by thyroid or glucocorticoid hormones, both of which increase the rate of transcription of the gene. In addition to the primary transcript and the partially processed intermediate transcripts, GC and GH3 cells were found to contain a heterogenous group of intron-containing polyadenylated rGH gene transcripts which cannot be accounted for by any combination of intron deletions. These transcripts could arise either from internal start sites in the gene, premature termination of transcription, or inefficient processing of rGH mRNA precursors in the transformed cells. Thyroid hormone rapidly increases the levels of intron C-containing transcripts with kinetics that parallel the binding of thyroid hormone receptor to nuclei, but does not alter the ratio of primary to partially processed transcripts. These data suggest that most of the stimulatory activity of this hormone is due to effects on rGH gene transcription and not on pre-mRNA processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutron scattering from interfacially polymerized core-shell latexes

Small-angle neutron scattering (SANS) analyses of poly(methyl methacrylate) (PMMA) latexes yielded particle sizes that agree with those determined by a light-scattering technique. Emulsion polymerization of perdeuterated methyl methacrylate (d-MMA) and styrene (d-S) in the presence of preformed PMMA latexes resulted in particles with a core—shell morphology, as determined by SANS analysis for a hollow sphere. The locus of polymerization of the added deuterated monomer is therefore at the particle surface. Shells of d-S were less uniform in thickness than those of d-MMA.

Cell cycle regulation of poly(ADP-ribose) synthetase in FR3T3 cells

The properties of poly(ADP-ribose) synthetase were studied throughout the cell cycle progression of non-synchronized rat FR3T3 fibroblasts using an immunological and biochemical approach. Cells in the various phases of the cell cycle were sorted from an asynchronously growing population by using flow cytofluorometry. G 1, S and G 2 + M fractions were used for enzymatic assays in the presence of saturating concentrations of DNAase I for the analysis of total poly(ADP-ribose) synthetase; maximal enzyme activity was found in the G 2 + M phase. Purified IgG, specific for the FR3T3 poly(ADP-ribose) synthetase were used for the labelling of endogeneous synthetase in order to quantify the enzyme immunologically. Localization of nuclear immunofluorescence was observed and analysis of poly(ADP-ribose) synthetase content throughout the cell cycle were carried out using double fluorescent staining and cytofluorometry. Poly(ADP-ribose) synthetase content as measured immunologically was found to increase from G 1 to S and G 2 + M phases. Quiescent cells showed a lower content as measured immunologically of poly(ADP-ribose) synthetase than cells in the G 1 phase. In exponentially growing cells, the ratio between enzyme activity of poly(ADP-ribose) synthetase over the amount of enzyme measured immunologically was found to be higher in the G 2 + M phase. These results show that a cell-cycle specific event activates poly(ADP-ribose) synthetase in the G 2 + M phase.

Preparation and chromatographic analysis of poly(3-hydroxybutyrate) hydrolysis products

Preparation and chromatographic analysis of poly(3-hydroxybutyrate) hydrolysis products

HPTLC Separation of Homologous and Isomeric Poly(ethyleneamine)s

Abstract A simple, reliable HPTLC method has been developed for analysis of poly(ethyleneamine)s (PEAs). Ethylenediamine and all dimeric, trimeric, and tetrameric PEAs are separated without prior derivatization. Treatment with fluorescamine and visualization under ultraviolet light detects each PEA in quantities as low as 100 ng.

Préparation et caractérisation des poly(o‐chlorométhylstyrène), poly(m‐chlorométhylstyrène) et poly(p‐chlorométhylstyrène). Etudes des résonances magnétiques nucléaires 1H NMR et 13C NMR. Application à l'étude d'une résine de type merrifield

AbstractOrtho‐, méta‐ and para‐chloromethylstyrene were polymerized and their 1H NMR and 13C NMR spectra were studied. The knowledge of the chemical shifts, especially those in 13C NMR, allows the analysis of poly(o‐‐chloromethylstyrene‐co‐m‐‐chloromethylstyrene) and of a Merrifield‐type resin which was prepared by a terpolymerization reaction of styrene, chloromethylstyrene and divinylbenzene.

Model compounds for poly(oligomethylene glycol-4,4′-biphenyl dicarboxylates). An X-ray diffraction study

To provide the necessary parameters required for the conformational analysis of poly(oligomethylene glycol-4,4′-biphenyl dicarboxylates), two model compounds related to the above polyesters have been synthesized and their structures established by X-ray diffraction. The crystal structures of ethylene glycol di-para-phenylbenzoate (2DPPB) and 1,4-butylene glycol di-para-phenylbenzoate (4DPPB) have been solved by direct methods. 2DPPB is monoclinic with a = 7.174(2) Å, b = 8.101(2) Å, c = 19.183(4) Å, β = 102.48(2)°, Z = 2, space group P21/c. 4DPPB is triclinic with a = 9.978(2) Å, b = 11.172(3) Å, c = 11.437(2) Å, α = 85.46(2)°, β = 79.40(2)°, γ = 71.03(2)°, Z = 2, space group [Formula: see text]. The weighted least-squares refinements converged to Rw = 0.054 (2DPPB) and Rw = 0.057 (4DPPB). The values of ε, the tilt angle of the carboxylate group with respect to the phenylene ring, are 3.5° for 2DPPB and 1.5° and 1.4° for 4DPPB. A systematic study of this dihedral angle showed that the values of ε remain in the vicinity of 0°–10° and the value observed most frequently is 5°. The values of Φ, the dihedral angle between the two six-membered rings, are 36.2° in 2DPPB and 14.7° and 31.0° for 4DPPB. A systematic study with 71 biphenyl molecular fragments showed that distribution of Φ presents two maxima: one around 30° – 40° and the other in the range of 0°–10°. The conformation of the methylenic sequence is all-trans for 2BPPB and gtttt for 4DPPB.