DNA topoisomerase I from rat brain neurons

The DNA topoisomerase I has been isolated from neurons of rat cerebral cortex. The most homogeneous fraction purified contains only one polypeptide of Mr approx. 100 000. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. The optimum monovalent cation concentration for the relaxation of superhelical DNA under conditions of DNA excess is found to be 175-200 mM. The neuron enzyme is similar to other mammalian type I DNA topoisomerases in that it links to the 3' ends of the broken DNA strands. Like calf thymus DNA topoisomerase I, the neuron topoisomerase can be selectively inhibited by poly(dG) but not by other homopolymerical deoxyribonucleotides.     

An extracellular endodeoxyribonuclease from Streptomyces aureofaciens

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.     

Intranucleosomal localization of the AP endodeoxyribonuclease of rat liver chromatin

The chromatin AP endodeoxyribonuclease of rat liver cells is located in the cores as well as in the linkers of the nucleosomes.     

A new site-specific endodeoxyribonuclease from Citrobacter freundii

Cfr10 I, a site-specific endonuclease from Citrobacter freundii strain RFL10, was isolated. It recognizes and cleaves the family of related sequences: 5'Pu decreases CCGGPy to generate DNA fragments with 5' tetranucleotide extensions. Cfr10 I may be useful in molecular cloning experiments, especially in conjunction with other enzymes which generate the same terminal extensions.     

Specific binding of muramyl peptides with rat brain membranes

A tripeptide analogue of N-acetylglucosaminyl-(beta 1-4)-N-acetylmuramyl dipeptide (GMDP) which contains C-terminal Lys residue (GMDP-Lys) was prepared. Its reaction with N-hydroxysuccinimidyl 3-(4-hydroxyphenyl)propionate (BH) followed by iodination gave the 125I-labelled derivative with specific activity ca. 2000 Ci/mmol. This compound was shown to bind specifically with rat brain membranes, dissociation constant Kd = 3.1 +/- 0.9 nM, binding capacity Bmax = 11.0 +/- 12 fmol/mg protein. Binding was inhibited by the non-radioactive iodinated derivative, unmodified GMDP-Lys and GMDP. Thus, the specific binding of immunoactive myramyl peptides with brain has been demonstrated for the first time.     

Specific inhibition of rat brain phospholipase D by lysophospholipids

Although the importance of phospholipase D (PLD) in signal transduction in mammalian cells is well documented, the negative regulation of PLD is poorly understood. This is primarily due to a lack of known specific inhibitors of PLD. We herein report that the activity of partially purified rat brain PLD is inhibited by certain lysophospholipids, such as lysophosphatidylinositol, lysophosphatidylglycerol, and lysophosphatidylserine in a highly specific manner. Inhibition of PLD by lysophospholipids was dose-dependent: the concentration of lysophosphatidylinositol required for half-maximal inhibition was about 3 micrometer. An analysis of the enzyme-kinetics suggested that lysophospholipids act as non-competitive inhibitors of PLD activity. As expected, PLD activity was stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). The inhibition of PLD by lysophospholipids, however, was not affected by the presence or absence of Arf or by an increase in PIP(2) concentration. A protein-binding assay suggested that lysophospholipids bind directly to PLD. These results indicate that the observed inhibition of PLD by lysophospholipids is due to their direct interaction rather than to an interaction between lysophospholipids and either Arf or PIP(2). The present study suggests that certain lysophospholipids are specific inhibitors of rat brain PLD in a cell-free system and may provide the new opportunities to investigate mechanisms by which PLD is regulated by lysophospholipids, presumably liberated by phospholipase A(2) activation, in mammalian cells.     

Specific processing of the thyrotropin-releasing prohormone in rat brain and spinal cord

Thyrotropin-releasing hormone (TRH) and TRH extended peptides were extracted from rat hypothalamus and spinal cord and resolved by gel exclusion chromatography under dissociating conditions. Peptides related to TRH were detected by trypsin digestion and radioimmunoassay with an antibody to TRH or an antibody raised against the pentapeptide Glp-His-Pro-Gly-Lys. In addition to the tripeptide hormone a series of C-terminally extended forms of TRH was shown to occur in both tissues; no N-terminally extended peptides were detected. The structure of the TRH-related peptides was confirmed by chromatographic identification of the N-terminal pentapeptide sequence released by trypsin. The TRH extended peptides, which accounted for 15-20% of the total TRH, were present in three groups of different molecular size corresponding to predicted fragments of the TRH prohormone. One of the peptides in the spinal cord was identified by chromatographic comparison with a synthetic 16-residue peptide representing residues 154-169 of the prohormone. In the spinal cord the TRH extended peptides differed in their relative concentrations from the corresponding peptides in the hypothalamus, possibly reflecting differences in processing. The finding of extended forms of TRH in which the extension occurs only on the C-terminal side of the hormone sequence shows that the prohormone undergoes highly specific processing.     

Non-homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.     

Deoxyribonucleases from rat brain

Two distinctly different DNases were isolated from rat brain and could be separated easily by ammonium sulphate fractionation. One of the DNases acts optimally at pH 5.0 hydrolysing preferentially native DNA and requiring an optimal Mg²⁺ concentration of about 0.03 m . The other DNase has its optimal pH between 7.4 and 8.9, acts preferentially on heat-denatured DNA and requires a lower Mg²⁺ concentration, the optimum being 1 × 10^?4m . Cerebellum from adult rat brain has a lower acid DNase activity and higher alkaline DNase activity, and therefore has a higher ratio of alkaline DNase to acid DNase than the other areas of brain studied. This unique activity ratio in cerebellum of adult rat brain was not observed in infant rat brain.     

DNA content of the mitochondria of the developing rat brain

Studies have been made on the content of DNA in the mitochondria from the brain of 1, 5, 10, 15, 21, 60, and 400 days old rats. Isolated DNA preparations contained 7% of RNA and 3% of proteins. It was shown that the amount of DNA in 21- and 60-day rats is 4 times higher that than in newborn ones. DNA content of the mitochondria increases during the first 3 weeks of postnatal life of rats.     

A new specific endodeoxyribonuclease from Escherichia coli RFL 72

A restriction endonuclease Eco72I with a novel substrate specificity has been isolated from Escherichia coli strain RFL 72. The enzyme recognizes (Formula: see text) hexanucleotide palindromic sequence and cleaves it, as indicated by the arrows, to produce blunt-ended fragments.