Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.     

Calcium in hippocampus following lidocaine induced seizures: an electron cytochemical study

The effect of lidocaine seizures on cellular accumulation of calcium was studied in hippocampal subfields CA1 and CA3 and the dentate gyrus of rats, using the combined oxalate-pyroantimonate method. The specificity of the reaction was ascertained by EGTA treatment and X-ray microanalysis. In control rats, calcium was visualized between myelin lamellae of axons, in synaptic vesicles and in some lysosomes. Two hours after onset of lidocaine seizures selective neuronal degenerations appeared in hippocampal subfields CA1 and CA3 but not in the dentate gyrus. Calcium deposits were present in numerous mitochondria of pyramidal cells and, occasionally, also of neuroglial cells. Many of these mitochondria exhibited ultrastructural alterations. Calcium uptake was most prominent in the CA3 sector but was also present in the CA1 subfield as well as the dentate gyrus. Intracellular calcium uptake, in consequence, is not the unique attribute of selectively vulnerable hippocampal neurons.     

Serum cortisol level changes in the maternal-fetoplacental unit between the 28th-40th weeks of pregnancy

Changes in the serum cortisol level of maternal venous, umbilical venous and umbilical arterial blood were studied separately between the 28th-36th weeks in cases of preterm deliveries (n = 74) and in the 40th week in cases of term delivery (n = 34). Results indicate that between the 28th-40th weeks of pregnancy the cortisol concentration increased only in the serum of the umbilical artery; the "umbilical arterial/umbilical venous concentration X100" quotient rose from a value of 86% measured at 28th-32nd weeks to 103% in the 40th week of pregnancy. Positive correlation was found between the cortisol concentration of the three samples. On the basis of these results the authors believe that fetal adreno-cortical activity increases before birth.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate into a synthetic DNA

The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).     

Isolation, purification and 1H-NMR characterization of a kringle 5 domain fragment from human plasminogen

A scheme is proposed for generating the intact Val-448-Phe-545 polypeptide of human plasminogen which contains the fifth kringle domain of the plasmin heavy chain. The procedure is based on a pepsin fragmentation of miniplasminogen and involves the purification of the kringle 5-containing fragment by gel filtration and ion-exchange chromatography. The final product is characterized by amino acid analysis, N- and C-terminal analyses, and high-resolution 1H-NMR spectroscopy at both 300 MHz and 611 MHz. We detect a (40:60%) Asp/Asn heterogeneity at site 452 of the Glu-plasminogen molecule. In the conventional kringle numbering system, the kringle 5 domain extends from Cys-1 to Cys-80, which corresponds to Cys-461 to Cys-540 in plasminogen. A preliminary 1H-NMR characterization of kringle 5 focuses on the global conformational features of the polypeptide. Assignments are given for a number of resonances, including the Tyr-72, the His imidazoles' and the Trp indoles' spin systems. Comparison with human plasminogen kringles 1 and 4 shows that the kringle 5 conformation is highly structured and very similar to that of the homologous domains. This conservancy is particularly striking in the environment surrounding Leu-46 and in the overall features of the aromatic spectrum. There are some differences, particularly in the buried His-33 imidazole group, whose H2 resonance is shifted to 9.67 ppm. A preliminary study of benzamidine-binding shows that the ligand interacts weakly (Ka approximately equal to 1.7 mM -1) mainly through the amidino functional group. Trp-62 and Tyr-72 are significantly perturbed by benzamidine, suggesting that these residues are part of the ligand-binding site.     

Substrate specificity of DNA polymerases. I. Enzyme-catalysed incorporation of 5-''1-alkenyl)-2''-deoxyuridines into DNA.

A series of (E)-5-(1-alkenyl)-dUTPs as well as 5-vinyl-and (Z)-5-(1-propenyl)-dUTP have been synthesized to study steric requirements in DNA polymerase reactions. Experiments were carried out in E. coli DNA polymerase I Klenow fragment enzyme system. Substrates were characterized by KM and Vmax-values, initial incorporation rates as well as by total extent of incorporation of the analogues into poly(dA-dT) as a template-primer. Incorporation of the analogues could be best correlated with Vmax-values as well as the very similar initial incorporation rate values. Reactivity (Vmax/KM) showed no correlation with the extent of incorporation. 5-Vinyl-dUTP proved to be as good a substrate of the enzyme as dTTP, whereas (E)-5-(1-heptenyl)-and (E)-5-(1-octenyl)-dUTPs were very poor substrates, their incorporation was strongly limited and they also proved to be very efficient inhibitors of DNA replication, as shown by Ki-values. Substrate specificity of the Klenow enzyme can be explained by the steric hindrance of C-5 substituent, by the "orientational steric substituent effect" concept.     

G2 effects of DNA-repair inhibitors on chromatid-type aberrations in root-tip cells treated with maleic hydrazide and mitomycin C

In recent years the existence of a DNA-repair process in G2 has been proposed to explain the potentiating effects of DNA-repair inhibitors given in G2 on chromatid aberrations (CA) induced by S-dependent as well as S-independent DNA-damaging agents. In the present report, root-tip cells of Allium cepa were exposed to maleic hydrazide (MH) or mitomycin C (MMC) and post-treated in G2 with caffeine (Caff) and various inhibitors of DNA synthesis. No enhancement of chromosome damage was observed when Caff was present in G2, but hydroxyurea (HU) or 5-fluorodeoxyuridine (FdUrd) potentiated the frequencies of CA. A slight additional increase of CA frequencies was observed following treatment with Ara C and excess thymidine in G2. When MH-damaged cells were pulse-treated with Caff earlier during recovery, the yield of CA was enhanced. The earlier Caff was present following MH treatment, the stronger was the potentiation.     

Aging and cerebral aminopeptidase activity

A comparative study of brain aminopeptidase activity between 18 month old male rats and young adults of 3 months has been carried out utilizing the arylamides Leu-, Arg-, Lys-, Tyr- and Asp-beta-naphthylamide as substrates. Statistical analysis of results showed no significant differences either in areas studied or for enzymatic activities detected when both ages were compared. Two different patterns of regional distribution of enzymatic activity were observed: One came to be as a result of the use of Lys-, Arg-, Leu- or Tyr-beta-naphthylamide and the other as a result of the use of Asp-beta-naphthylamide.