Evidence for Excision of Ultraviolet-Induced Pyrimidine Dimers from the DNA of Human Cells In Vitro

Within 12-24 hr after human cells were irradiated with ultraviolet light, approximately 50% of the ultraviolet-induced pyrimidine dimers were lost from the DNA. Pyrimidine dimers were found in the TCA-soluble fraction of ultraviolet-irradiated cells at 24 hr. Excess thymidine, caffeine, or hydroxyurea had no effect on the loss of pyrimidine dimers from the DNA of ultraviolet-irradiated cells.     

Defective Excision Repair of Pyrimidine Dimers in the Ultraviolet-Sensitive Escherichia coli ras− Mutant

The ras(-) mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras(-) mutant to UV has been studied biochemically. After low doses of UV, the ras(-) mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras(-) mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain.     

Thymine inhibits suppression by an Escherichia coli nonsense and frameshift suppressor

Summary A mutant strain of Escherichia coli suppressed a frameshift and some UAG, UAA, and UGA mutants of bacteriophage T4 at 37° C but not at 31° C. This suppression was inhibited by the addition of thymine or thymidine to the medium used to test phage growth. Furthermore, the suppressor strain required thymine or thymidine for growth on minimal medium at 43° C and if this auxotrophy was removed by reversion or recombination the strain no longer suppressed. These results suggest a link between thymidine nucleotide biosynthesis and suppression.     

In Vitro Evolution of an Archetypal Enteropathogenic Escherichia coli Strain

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nal^r) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nal^r strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Str^r) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK. Another, in gyrA, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD, the lysogenization regulator, to purB. The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nal^r clone has a lower growth rate than the Str^r isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nal^r clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro.     

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.     

Loss of Deoxyribonucleic Acid-Thymine During Thymine Starvation of Escherichia coli

A substantial loss of deoxyribonucleic acid-thymine occurs during thymine starvation of several thymine auxotrophs derived from Escherichia coli strains B, 15, and K-12.     

In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19-264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CP(19-264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.     

Cloning and Expression in Escherichia coli of the Polysaccharide Depolymerase Associated with Bacteriophage-Infected Erwinia amylovora

The bacteriophage-encoded polysaccharide depolymerase produced in Erwinia amylovora has been cloned and expressed in Escherichia coli. The bacteriophage ERA103 genome was observed to consist of five EcoRI fragments, labeled as follows: A, 7.5 kilobases (kb); B, 5.0 kb; C, 2.7 kb; D, 2.1 kb; and E, 1.8 kb. A restriction map for ERA103 was also prepared. Each of the fragments were cloned into the positive-selection vector pOP203(A₂⁺) and pBR322.     

In Vitro Translation of Cardiovirus Ribonucleic Acid by Mammalian Cell-free Extracts

Cell-free extracts prepared from Ehrlich ascites and mouse L cells synthesize viral proteins in response to encephalomyocarditis virus, mouse Elberfeld virus, and mengovirus ribonucleic acid. Although HeLa cell extracts are inactive, their ribosomes are functional in the presence of heterologous supernatant fractions. Synthesis depends upon the addition of adenosine triphosphate, guanosine triphosphate, an energy-generating system, and 4 mm Mg(2+). Initiation is completed during the first 10 to 20 min of incubation, but chain elongation continues for 1 hr or more. The products are of higher molecular weight than virion structural proteins and resemble polypeptides formed in virus-infected cells during a short pulse. Tryptic peptides of virion proteins and in vitro products are similar for all three cardioviruses.     

In Vitro Synthesis of Subunits of Bacterial Luciferase in an Escherichia coli System

RNA extracted from the luminescent bacterium Vibrio harveyi was translated in an Escherichia coli system. RNA from highly luminescent cells produced both α and β subunits of luciferase in vitro, as confirmed by immunoprecipitation and partial proteolysis. RNA from cells before induction of luminescence did not direct luciferase synthesis.     

Phospholipase Activity in Bacteriophage-Infected Escherichia coli I. Demonstration of a T4 Bacteriophage-Associated phospholipase

Phospholipase activity has been found to be associated with T4 phage and T4 ghost particles. The attachment of the phospholipase to the phage persists during purification through cesium chloride gradients and dialysis, indicating that it is firmly bound. The presence of the enzymatic activity on T4 ghosts suggests that it is not normally packaged within the head of the virus. The enzyme has specificity for phosphatidylglycerol and its activity is stimulated by 0.1% Triton X-100 and 20% methanol. It does not have a requirement for Ca²⁺ and is inactivated at temperatures above 60 C. The association of the phospholipase with T4 phage grown in a phospholipase-deficient host and its absence on unsuppressed T4amtA3 suggests that it may be phage gene specific.     

Base Excision by Thymine DNA Glycosylase Mediates DNA-Directed Cytotoxicity of 5-Fluorouracil

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU–induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU–based chemotherapy.     

植物提取物对霉菌的抑制作用

以生活环境中常见的霉菌(青霉、米曲霉和黑曲霉)为试验菌,选取可能对其有抑制作用的植物,通过对其有机成分进行粗提,用滤纸片法筛选对上述霉菌有抑制效果的植物。经查阅相关文献,得备选植物共25种,试验后发现黄连抑制作用最强,其次是丁香和黄芩,最后对这3种中草药植物的最低抑菌浓度进行定量测定。     

Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 mug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.