Enzymatic syntheses of DNA-silicas using DNA polymerase.

Oligothymidylic acids couple to an activated ester silica (N-hydroxysuccinimidyl-silica) only when they contain an added aminoalkyl group. Heteropolymeric oligomers containing other nucleotide bases were shown to also couple by way of the nucleotide base (adenine, cytosine, or guanine); however, when a heteropolymeric oligonucleotide also contains a 5'-aminoalkyl moiety, coupling by way of the latter is the favored reaction. When duplex hybrids of oligonucleotides are formed, the nucleotide bases are protected from chemical coupling. Coupling by way of nucleotide bases would be detrimental to some chromatography experiments. On the basis of these observations, two different procedures were developed to produce DNA-silicas in which a single strand of the DNA is coupled by only its 5'-terminus. In the first of these, the polymerase chain reaction was used with a 5'-aminoalkyl primer to make a duplex DNA with one strand containing the 5'-aminoalkyl group and the duplex DNA is then coupled to the activated ester silica. This yielded a silica containing about 0.17 nmol of a 242-mer per gram silica which bound only probes specific for the coupled strand. In the other procedure, a template DNA strand was poly(A) tailed and hybridized to (dT)18-silica. DNA polymerase I (Klenow large fragments) was then used to copy the template-specified sequence directly onto the 3'-terminus of the (dT)18. This procedure yielded about 1.2 to 2.7 nmol DNA copied/g of silica of a specific 21-mer sequence. The DNA-silica produced selectively hybridized only with complementary sequences and not with DNA lacking that sequence. Either of these procedures thus produces DNA-silicas from heteropolymeric DNA sequences with a predetermined, specific 5'-terminal site of attachment.     

In vitro biosynthesis of plasminogen in a cell-free system directed by mRNA fractions isolated from monkey liver

mRNA was isolated from total RNA of monkey liver by oligo(dT)-cellulose chromatography and was translated in a rabbit reticulocyte cell-free system. Analysis of the translation products immunoprecipitated with specific antibodies to monkey plasma plasminogen revealed a molecule with characteristics similar to those of native plasminogen. The purification of the mRNA by centrifugation on sucrose gradients indicated the presence of plasminogen mRNAs in both the 23S and 18S RNA fractions. Both plasminogen mRNAs can be further purified by chromatography on Sepharose 4B. Affinity chromatography of the proteins synthesized in vitro by total mRNA from liver, as well as by the purified mRNAs, on L-lysine-substituted Sepharose revealed that both major plasma plasminogen forms (1 and 2) are synthesized, as precursors, in the system. The in vitro synthesized plasminogen is similar in its physical and chemical properties to native plasma plasminogen as determined by its ability to bind to L-lysine-substituted Sepharose and its molecular interaction with streptokinase. The purified mRNAs were also translated in the presence of dog pancreas microsomal membranes, and and fractionated on concanavalin A-Sepharose. The 23S mRNA directed the synthesis of a plasminogen molecule similar to the circulating plasma plasminogen form 1, whereas the 18S mRNA directed the synthesis of a molecule similar to the circulating plasma plasminogen form 2. Our evidence indicates that the synthesis of the two major circulating plasma plasminogen forms is directed in the liver by separate mRNAs.     

Diadenosine 5′,5‴-P1,P4-tetraphosphate- (Ap4A-) mediated stimulation of DNA synthesis in extracts from Xenopus laevis oocytes

Diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) stimulates DNA synthesis in Xenopus laevis oocytes in the presence of activated DNA as template. Besides Ap₄A, other analogues such as Ap₃A, ATP and other derivatives are able to stimulate DNA polymerase activity. The effect of Ap₄A on DNA synthesis is observed with poly(dT) and poly(dT)-poly(dA) as templates, while no effect is found with poly(dA)(dT)_12–18 and poly(dC)(dG)_12–18. In the presence of a poly(dT) template, the oocyte extract is able to utilize Ap₄A as primer and to form a covalent bond between this dinucleotide and the nascent poly(dA) chain. An Ap₄A-binding protein present in the system has been purified and separated from DNA polymerase α-primase after phosphocellulose chromatography. After this separation, Ap₄A is no longer able to stimulate the polymerase activity, or to be utilized as primer by DNA polymerase α-primase.     

Preparation of ferric adsorbent paper and its interaction with phosphate-containing biomolecules.

A procedure for the synthesis of a selective adsorbent for phosphate-containing biomolecules is described. The sorbent is based on Whatman chromatography paper, which is activated with epichlorohydrine, followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups are saturated with ferric ions. The synthesized adsorbent is a counterpart to Chelating Sepharose and makes it possible to extend the use of immobilized ferric chelating groups for analytical purposes. It displays a high affinity towards compounds containing free terminal phosphate groups (phosphopeptides, nucleotides). The results of the binding experiments are compared to the corresponding data obtained with Chelating Sepharose gels.     

猪生长激素cDNA的分子克隆

用改进的氯化锂沉淀沉法和寡聚(dT)一纤维素亲和层析法由猪垂体制得总mRNA。以此总mRNA为模板,合成cDNA。钝端连接重组到质粒pUC19的SmaI位点,转化E.coli JM107,筛选出阳性克隆。用限制性内切酶酶切签定及5’端部分核苷酸序列分析证明:克隆了全长猪生长激素cDNA,其长度约为896bp。     

Preparation of Ferric Adsorbent Paper and Its Interaction with Phosphate-Containing Biomolecules

Abstract

A procedure for the synthesis of a selective adsorbent for phosphate-containing biomolecules is described. The sorbent is based on Whatman chromatography paper, which is activated with epichlorohydrine, followed by the coupling of iminodiacetic acid to the active surface of the sorbent. The immobilized complex-forming chelating groups are saturated with ferric ions. The synthesized adsorbent is a counterpart to Chelating Sepharose and makes it possible to extend the use of immobilized ferric chelating groups for analytical purposes. It displays a high affinity towards compounds containing free terminal phosphate groups (phosphopeptides, nucleotides). The results of the binding experiments are compared to the corresponding data obtained with Chelating Sepharose gels.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

Synthesis of site-specifically modified oligoribonucleotides for studies of the recognition of TAR RNA by HIV-1 tat protein and studies of hammerhead ribozymes.

Synthetic oligoribonucleotides having single uracil residues replaced by dU, dT, 2'-O-methylU or 5-bromodU have been prepared and used in the study of the interaction of HIV-1 tat protein with an RNA stem-loop. The preparation of phosphoramidites of 5-bromouridine and purine riboside suitable for use in solid-phase oligoribonucleotide synthesis is also described. The effect of adenine replacement by purine in a hammerhead ribozyme has also been determined.     

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.     

The selective isolation of the uterine oestradiol-receptor complex by binding to oligo(dT)-cellulose. The mediation of an essential activator in the transformation of cytosol receptor.

The [3H]oestradiol-receptor complex was selectively isolated from rat uterus cytosol by column chromatography on oligo(dT)-cellulose. Optimal conditions are described for the binding of the complex to oligo(dT)-cellulose, which is shown to be similar to its binding to DNA-cellulose. The cytosol complex has an apparent mol. wt. of 50,000-60,000 in high salt concentrations, as determined by Sephadex G-100 chromatography. This corresponds to the 4S cytoplasmic oestradiol receptor. In binding to oligo(dT)-cellulose the receptor is transformed into a form with an apparent mol.wt. of 100,000-120,000, corresponding to the 5S nuclear receptor complex. This transformation mimics the conversion in vivo of the cytoplasmic oestradiol receptor into the nuclear form. The binding of the complex to oligo(dT)-cellulose as a 5S nuclear form is unequivocally demonstrated to require the mediation of an activating present in the cytosol. The requirement for an activating factor is discussed in relation to reports that nuclear binding of the oestradiol-receptor complex is not dictated solely by the availability of the cytoplasmic oestradiol receptor.     

Rapid isolation of human plasma anti-alpha-galactoside antibody using sugar-specific binding to guar galactomannan or agarose.

A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Hybridization of Oligo(dT) to RNA on nitrocellulose

Quantitation of mRNA immobilized on nitrocellulose filters is an essential aspect of some studies in molecular biology. Hybridization of oligo(dT)₁₈ to the poly(A) tails of mRNA can be used to measure filter-bound mRNA and thus provides a basis for comparing abundance of specific mRNAs. Hybridization rate of ³²P-labeled oligo(dT)₁₈ in 0.75 M NaCl, 75 mM sodium citrate, pH 7 (5 × SSC) to immobilized RNA was maximal at 25°C. Filters were fully hybridized under these conditions within 1 hr when the oligo(dT)₁₈ concentration was 10 pmol/ml or higher. Salt dependence of the dissociation temperature (T_d) of oligo(dT)₁₈:RNA duplex on filters was described by the equation T_d = 42 − 20log₁₀[molar Na⁺] (°C). With stringent washing of the duplex (four 5-min washes in 2 × SSC at room temperature), oligo(dT)₁₈ gave no signal with plasmid DNA, rRNA, or tRNA. We have found that olig(dT)₁₈ can be used to normalize signal strengths rapidly and conveniently from total or oligo(dT)-selected eukaryotic RNA.     

Inhibition of growth of hair follicles by a lectin-like substance from rat skin

A small molecular weight (5 000-10 000) substance has been isolated from rat skin by affinity chromatography on a column of acid-hydrolysed Sepharose. The substance agglutinates rabbit red blood cells, inhibits DNA synthesis in rat hair follicles, and causes the appearance of autophagic vacuoles in the epithelial cells of the lower follicle bulb.     

Induction, isolation and a characterization of the lipid content of plasma vitellogenin from two Salmo species: rainbow trout (Salmo gairdneri) and sea trout (Salmo trutta)

Vitellogenin synthesis is induced in juvenile rainbow trout (Salmo gairdneri) and juvenile sea trout (Salmo trutta) by estradiol-17 beta. A purification procedure for vitellogenin from trout plasma by precipitation with MgCl2-EDTA and subsequent anion exchange chromatography on DEAE-Sephacel is described. The total lipid contents of purified rainbow trout and sea trout vitellogenins are 18 and 19%, respectively. Approximately 2/3 of the lipids are phospholipids, while the remainder consists of triglycerides and cholesterol. Phosphorus determinations on delipidated vitellogenin yield a phosphorus content of 0.63% in rainbow trout and 0.58% in sea trout vitellogenin. Native (dimeric) vitellogenins from rainbow trout and sea trout both have an apparent molecular weight of 440,000, when estimated by gel filtration on Sepharose 6B.     

Rapid synthesis of long-chain deoxyribooligonucleotides by the N-methylimidazolide phosphotriester method.

A modified phosphotriester method has been employed for the efficient chemical synthesis of long-chain deoxyribooligonucleotides. During the course of this work, a general and rapid procedure was developed for the preparation of 24-62-mers in solution. Preparative reversed phase column chromatography on silanized silica gel was used to purify triester intermediates starting from 10-mers. The rapid synthesis of 32-mer and 42-mer on glass and silica gel supports using suitably protected 2-8-mer blocks as coupling units has been also accomplished. In particular, a convenient procedure for the solid-phase synthesis of oligonucleotide blocks bearing 3'-terminal phosphodiester groups is described.