The Chemical Fractionation of Rabbit and Swine Thymus

A chemical fractionation procedure, previously found applicable to bovine thymus and bovine and ovine palatine tonsils, was used to fractionate rabbit and hog thymus. With respect to the chemical fractionation steps, yields of fractions, and optical and electrophoretic properties, extracts from hog and rabbit thymus were indistinguishable from similar extracts prepared from calf thymus. The study provides composition and yield data applicable to the thymus of a small mammal readily available in most laboratories.     

Synthesis of DNA polymerase by in vitro translation of calf RNA

Synthesis of alpha-polymerase in translation mixtures containing calf thymus poly(A+) RNA was examined by activity gel analysis and by immuno-binding with a monoclonal antibody to calf thymus alpha-polymerase. Activity gel analysis indicated that a DNA polymerase catalytic polypeptide of Mr = approximately 120,000 had been synthesized. Immunobinding experiments indicated that an immunoreactive polypeptide of about the same size had been formed in vitro. Sucrose gradient centrifugation of calf thymus total RNA revealed that mRNA encoding the approximately 120,000-Mr DNA polymerase polypeptide sedimented at about 16S. This approximately 120,000-Mr catalytic polypeptide corresponds in size to an alpha-polymerase catalytic polypeptide found earlier in crude extracts of calf cells.     

Palatine tonsils and immunity. VI. Effect of thymosin and palatine tonsil extract on the immunologic reactivity of neonatally thymectomized and intact mice]

Experiments were conducted on Wistar rats, intact and neonatally thymectomized CBA mice; a study was made of the immunological activity of the extracts of the thymus and the palatine tonsils of calves obtained by the method of Goldstein et al (1966). The extract of the palatine tonsils obtained proved to contain a small amount of thymosin at whose expense an insignificant restoration of immunological reactivity occurs in its administration neonatally to thymectomized mice.     

Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.     

Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.     

Immunohistochemical and biochemical studies on DNA ligase from a rat liver parenchymal cell line

We have recently obtained a monospecific antibody against calf thymus DNA ligase composed of a single Mr = 130,000 polypeptide. Immunohistochemical studies using the antibody and immunoperoxidase detection methods indicated that DNA ligase in a rat liver parenchymal cell line (BB) is localized essentially in nucleus. The specific activity of DNA ligase from growing BB cells was more than 10-fold higher than that from rat hepatocytes. The molecular forms of DNA ligase in these cell-free extracts were also analyzed.     

Purification and immunological characterization of DNA polymerase-alpha from human acute lymphoblastic leukemia cells

DNA polymerase-alpha was purified from the cytosol of blast cells of a patient with acute lymphoblastic leukemia by ammonium sulfate fractionation and successive column chromatographies. The purified enzyme had a specific activity of 2943 units/mg protein with activated calf thymus DNA as a template. The enzyme sediments under high-salt conditions as a homogeneous band at 7.2 S and free from other DNA polymerases (beta, gamma) and terminal deoxynucleotidyl transferase activity. The native molecular weight of the enzyme from gel filtration and glycerol gradient centrifugation was found to be 175 000. The values of Stokes radius (53 A), diffusion coefficient (4.05 x 10(-7) cm2/s) and frictional ratio (1.42) determined by gel filtration suggest that the native enzyme is compact and globular. Antibodies to DNA polymerase-alpha were raised in rabbits. These antibodies, partially purified by 50% ammonium sulfate saturation and Sephadex G-200 chromatography, gave one precipitin band on immunodiffusion and inactivate DNA polymerase-alpha-. This antibody preparation also inhibited, in vitro, the activity of DNA polymerase-alpha from calf thymus, phytohemagglutinin-stimulated normal human lymphocytes, as well as that from other leukemic cells. Thus, DNA polymerase-alpha from calf thymus and human leukemic cells resemble each other in antibody specificity.     

Partial purification and characterization of poly(dC)-dependent DNA polymerase and its stimulating factor

Poly(dC)-dependent dGMP incorporating activity without a primer molecule and its stimulating factor were partially purified by successive column chromatographies. Polymerase activity that was highly dependent on the stimulating factor was separated from similar activity that was not stimulated by this factor by native DNA-cellulose column chromatography. The factor stimulating activity was strictly dependent on dGTP as substrate and incorporated dGMP into the 3′-OH terminus of poly(dC). However, no terminal deoxynucleotidyl transferase (EC 2.7.7.31) activity was detected in the preparation. The activity also responded to heat-denatured calf thymus DNA and poly(dT) as template, although to a lesser extent. The activity was inhibited by dideoxyGTP and N-ethylmaleimide, and was decreased significantly by aphidicolin and β-lapachon.     

The chemical fractionation of lymphatic organs

A method for chemically fractionating lymphatic organs has been described. The method has been shown to be applicable to bovine palatine tonsils, sheep palatine tonsils, and bovine thymus. Approximately 50 per cent of the dry weight of tonsils and about 30 per cent of thymus has been found to be soluble in the 0.15 M NaCl extract. Four components have been isolated which together account for 65 per cent by weight of the material in the extracts. Four other components have been identified and partially defined by means of electrophoretic mobility, solubility, or some other chemical or physical property.     

Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells     

RecA-like activity in mammalian cell extracts of different origin

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.     

Properties of purified calf thymus poly(adenosine diphosphate ribose) polymerase. Comparison of the DNA-independent and the DNA-dependent enzyme.

The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation.     

Stabilization of double-stranded DNA molecule by nonhistone peptidic effector from calf thymus

A peptidic effector from calf thymus causes a strong stabilization of DNA doublestranded molecule in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (<5000). The biological activity of the thymic factor cannot be attributed to a histone fragment. Melting data of the control DNA and of the DNA-active factor complex in various conditions of ionic strength and dielectric constant of the solution medium are recorded.     

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.     

Purification of a DNA polymerase-DNA primase complex from calf thymus glands

An immunoabsorbent column, prepared by covalently linking mouse monoclonal anti-calf thymus DNA polymerase-alpha to Protein A-Sepharose, was used as the primary purification step for rapid isolation of DNA polymerase-alpha from calf thymus-gland extracts. In a 4-step procedure consisting of the removal of nucleic acids by protamine sulfate precipitation, chromatography on the immunoabsorbent column, desalting on Sephadex G-50, and removal of bovine immunoglobulins on Protein A-Sepharose, DNA polymerase-alpha activity was purified about 5000-fold from the crude extract with greater than 40% recovery of total enzyme activity. The antibody column-purified DNA polymerase-alpha fraction contains a DNA primase activity that is efficient in replication of single-stranded DNA and poly(dT) when rNTPs are included in the replication reactions. Synthesis by calf thymus DNA polymerase-primase is totally dependent on added template. Complete replication of circular single-stranded phage DNA is achieved with polymerase-primase producing a nicked circular DNA containing oligoribonucleotide primer in the final product. Primers synthesized with single-stranded phage DNA as template were up to 10 nucleotides long when dNTPs were omitted from the reaction and 8 or less nucleotides long when dNTPs were present. Primers synthesized using poly(dT) consisted of three populations when dATP was absent from the reaction, averaging 20 nucleotides, 10 nucleotides, and 3-4 nucleotides. The 20-nucleotide population was not found when dATP was included in the reaction.     

Prospects of AIDS therapy by thymic humoral factor, a thymic hormone

Research performed in our laboratory has established that thymic humoral factor (THF), a peptide hormone isolated from calf thymus, leads to clonal expansion, differentiation and maturation of T-cell lymphocytes. THF augments the response to T-cell lectins, mixed lymphocyte reactions, graft-versus-host reactivity, antibody response to SRBC, cytotoxic responses and production of interleukin-2. Clinical data based on the use of THF of natural sources in about 200 patients indicate that it regulates differentiation of T-cell precursors, leading to normalization of the ratio between helper and suppressor/cytotoxic subsets. THF reconstitutes the defective cell-mediated immunity in patients affected by various types of neoplasms and suffering from secondary immune deficiencies, as a result of chemo and/or radiotherapy. THF-gamma 2 was purified and synthesized and found to be an octapeptide which retains all the biological activity of the thymus extracts. It is presently available for extensive clinical use.     

小牛胸腺新胸腺活性因子(TAF-Ⅰ)的纯化和鉴定

以小牛胸腺为原料 ,通过匀浆、冻融、超滤、SephadexG 15凝胶过滤、DEAE SephadexA 2 5阴离子交换层析和反相高效液相色谱等步骤 ,分离得到了胸腺活性因子Ⅰ (thymusactivityfactorⅠ ,TAF Ⅰ ) .5 0 0g小牛胸腺组织中纯化得到了 0 92mg胸腺活性因子Ⅰ .经ESI MS质谱鉴定 ,结果显示其分子量为 6 18 8.氨基酸组成分析显示它由Ala、Gly、Glu、Gln、Lys、Ser 6种氨基酸残基组成 .体外生物学活性实验证明 ,TAF Ⅰ能显著促进人外周血淋巴细胞的E 玫瑰花结的形成率和促进小鼠脾淋巴细胞的分裂增殖     

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes

An antibody was raised against high mobility group nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. — Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. — It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday.