Biomedical subjects

K W Minton

Publications and source records attributed to K W Minton.

At least 19 recordsLinked to original sources

PubMed1992

Ultraviolet-induced dimerization of non-adjacent pyrimidines in poly[d(A-T)].

The DNA photoproduct responsible for the ultraviolet (UV) light-induced -1 frameshift mutation remains unknown. We recently identified a UV photoproduct consisting of a cyclobutane dimer occurring between non-adjacent thymine residues in the same strand, [sequence: see text] and proposed that replication across this unrepaired photoproduct might result in a -1 frameshift mutation since the intervening base is extrahelical. Until now this novel photoproduct has only been identified in single-stranded DNA polymers and does not occur in UV-irradiated double-stranded polymers due to conformational restraint. This observation suggested that this photoproduct could only occur in vivo in chromosomal sites that were single-stranded. In the current work the cis-syn dithymine cyclobutane dimer has been identified in the self-complementary polymer poly[d(A-T)] when UV irradiated in solution conditions (concentrated manganese chloride or 60% ethanol plus trace salts) wherein this polymer remains double-stranded but the double-helix is partially destabilized. Taken together, the current findings suggest that dipyrimidine photoproducts between non-adjacent residues on the same strand could occur in vivo in double-stranded, but partially destabilized, DNA.

Base Sequence↗

PubMed1992

Promoter probe and shuttle plasmids for Deinococcus radiodurans.

Two improved Deinococcus radiodurans-Escherichia coli shuttle vectors have been constructed. pI3 is a 16-kb plasmid that confers chloramphenicol resistance in D. radiodurans (CmR, cat) and ampicillin resistance in E. coli (ApR) and contains a multiple cloning site that does not interrupt sequences necessary for replication or drug resistance in either host. pI304 is a promoter-probe plasmid that is similar to pI3, but lacks the D. radiodurans promoting sequence for the cat gene, while retaining sequences necessary for replication.

Ampicillin Resistance↗

PubMed1992

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

Cell Survival↗

PubMed1991

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.

Cloning, Molecular↗

PubMed1991

Partial complementation of the UV sensitivity of Deinococcus radiodurans excision repair mutants by the cloned denV gene of bacteriophage T4.

Deinococcus radiodurans has 2 endonucleases that incise UV-irradiated DNA. UV endonuclease-alpha and UV endonuclease-beta, that are believed to functionally overlap. Both endonucleases must be mutationally inactivated to yield an incisionless, markedly UV-sensitive phenotype. denV, the bacteriophage T4 gene encoding pyrimidine dimer-DNA glycosylase (PD-glycosylase), was introduced and expressed via duplication insertion in D. radiodurans wild-type, and single and double UV endonuclease mutants. The strain deficient in UV endonuclease-alpha has wild-type UV resistance, and the expression of PD-glycosylase exerted no survival effect on this strain or wild-type. Expression of denV increased survival of both the markedly UV-sensitive double mutant and the moderately UV-sensitive strain deficient only in UV endonuclease-beta. In endonuclease-beta-deficient cells phenotypic complementation by denV was almost complete in restoring UV resistance to wild-type levels. These results suggest that UV endonuclease-alpha (which is present in the endonuclease-beta-deficient cells) does not recognize one or more types of cyclobutane dimer incised by the PD-glycosylase or UV endonuclease-beta.

Chromosomes↗

PubMed1991

AP endonuclease and uracil DNA glycosylase activities in Deinococcus radiodurans.

An endonuclease specific for apurinic/apyrimidinic (AP) sites was identified and purified from extracts of Deinococcus radiodurans. The enzyme is 34.5 kD, has no activity towards normal, alkylated, uracil-containing, or UV-irradiated DNA, and is active in the presence of EDTA. The addition of up to 10 mM Mg2+ or Mn2+ did not affect activity, but higher concentrations were inhibitory. There is no associated exonuclease activity, either in the presence or absence of divalent cation. Optimal reaction conditions were 150 mM NaCl and pH 7.5. A uracil DNA glycosylase was also detected, active in the presence of EDTA, selectively removing uracil from DNA without generating other byproducts. The optimal reaction conditions were 50 mM NaCl and pH 7.5. Implications for base excision repair in D. radiodurans are discussed.

DNA Damage↗

PubMed1991

Analysis by pulsed-field gel electrophoresis of DNA double-strand breakage and repair in Deinococcus radiodurans and a radiosensitive mutant.

Double-strand break (dsb) induction and rejoining after ionizing radiation was analysed in Deinococcus radiodurans and a radiosensitive mutant by pulsed-field gel electrophoresis. Following 2 kGy, migration of genomic DNA (not restriction cleaved) from the plug into the gel was extensive, but was not observed after 90 min postirradiation recovery. By this time D. radiodurans chromosomes were intact, as demonstrated by restoration of the Not I restriction cleavage pattern of 11 bands, which we found to be the characteristic pattern in unirradiated cells. Following the higher exposure of 4 kGy, dsb rejoining took approximately 180 min, twice as long as required following the 2 kGy exposure. Restoration of dsb in the radiosensitive mutant strain 112, which appears to be defective in recombination, was markedly retarded at both 2 and 4 kGy. The Not I restriction fragments of wild-type D. radiodurans and the radiosensitive mutant were identical, totaling 3.58 Mbp, equivalent to 2.36 x 10(9) daltons per chromosome.

DNA↗

PubMed1991

DNA polymorphisms in new isolates of 'Deinococcus radiopugnans'.

Nineteen new Deinococcus isolates from soil, 18 of which were from samples immediately adjacent to a lake in Nottingham, UK, were characterized by conventional criteria, and found to be identical to each other and to conform most closely to the species D. radiopugnans. However, we detected three different restriction enzyme activities in three different isolates. Because of this suggestion of heterogeneity, we examined the isolates for restriction fragment length polymorphisms (RFLPs). RFLP analysis of the 19 original isolates, using three different probes, distinguished 17 divergent groups. This extraordinary diversity cannot be attributed to geographical differences, nor to the method of isolation, which employed UV-radiation selection.

Blotting, Southern↗

PubMed1991

Formation of single and double strand breaks in DNA ultraviolet irradiated at high intensity.

The induction of single-strand breaks (SSB) by two quantum processes in DNA is well established. We now report that biphotonic processes result in double-strand breaks (DSB) as well. pUC19 and bacteriophage M13 RF DNA were irradiated using an excimer laser (248 nm) at intensities of 10(7), 10(9), 10(10) and 10(11) W/m2 and doses up to 30 kJ/m2. The proportion of DNA as supercoil, open circular, linear and short fragments was determined by gel electrophoresis. Linear molecules were noted at fluences where supercoiled DNA was still present. The random occurrence of independent SSB in proximity to each other on opposite strands (producing linear DNA) implies introduction of numerous SSB per molecule in the sample. If so, supercoiled DNA that has sustained no SSB should not be observed. A model accounting for the amounts of supercoiled, open circular, linear and shorter fragments of DNA due to SSB, DSB and Scissions (opposition of two independently occurring SSB producing an apparent DSB) was developed, our experimental data and those of others were fit to the model, and quantum yields determined for SSB and DSB formation at each intensity. Results showed that high intensity laser radiation caused an increase in the quantum yields for both SSB and DSB formation. The mechanism of DSB formation is unknown, and may be due to simultaneous cleavage of both strands in one biphotonic event or the biased introduction of an SSB opposite a preexisting SSB, requiring two biphotonic events.

Coliphages↗

PubMed1991

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.

Chromosomes, Bacterial↗

PubMed1991

A highly conserved repeated chromosomal sequence in the radioresistant bacterium Deinococcus radiodurans SARK.

A DNA fragment containing a portion of a DNA damage-inducible gene from Deinococcus radiodurans SARK hybridized to numerous fragments of SARK genomic DNA because of a highly conserved repetitive chromosomal element. The element is of variable length, ranging from 150 to 192 bp, depending on the absence or presence of one or two 21-bp sequences located internally. A putative translational start site of the damage-inducible gene is within the reiterated element. The element contains dyad symmetries that suggest modes of transcriptional and/or translational control.

Amino Acid Sequence↗

PubMed1991

Evidence against enhancement of the radioresistance of Escherichia coli by cloned Deinococcus radiodurans DNA.

Deinococcus radiodurans genomic DNA, introduced to Escherichia coli in cloning vectors, has been reported to produce radioresistant E. coli that can be selected by gamma irradiation. In this report prior results are reassessed experimentally, and additional studies are presented. Results to date suggest that the acquired radioresistance of E. coli selected by gamma irradiation does not stem from expression of stable plasmid-encoded D. radiodurans sequences, and that acquired radioresistance is not readily transmitted to naive (unirradiated) E. coli by transformation of plasmid recovered from the radioresistant isolates. Several interpretations are discussed.

DNA, Bacterial↗

PubMed1990

Paraquat toxicity is increased in Escherichia coli defective in the synthesis of polyamines.

We have shown that toxicity of paraquat for Escherichia coli is increased over 10-fold in strains defective in the biosynthesis of spermidine compared to isogenic strains containing spermidine. The increased sensitivity of these spermidine-deficient mutants to paraquat is eliminated by growth in medium containing spermidine or by endogenous supplementation of spermidine by the use of a speE+D+ plasmid. No paraquat toxicity is seen in the absence of oxygen, even in amine-deficient strains, indicating that superoxide is the agent responsible for the increased toxicity. However, the specific mechanisms responsible for the increased paraquat toxicity in the spermidine-deficient mutants remain to be determined. The marked sensitivity to paraquat of E. coli deficient in spermidine is of particular interest, since such mutants have no other phenotypic properties that can be easily assayed. This increased sensitivity has been used as the basis of a convenient method for scoring for mutants in polyamine biosynthesis and for the detection of plasmids containing the biosynthetic genes.

Aerobiosis↗

PubMed1990

Gene fusions with lacZ by duplication insertion in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is the most-studied species of a eubacterial family characterized by extreme resistance to DNA damage. We have focused on developing molecular biological techniques to investigate the genetics of this organism. We report construction of lacZ gene fusions by a method involving both in vitro splicing and the natural transformation of D. radiodurans. Numerous fusion strains were identified by expression of beta-galactosidase. Among these fusion strains, several were inducible by exposure to the DNA-damaging agent mitomycin C, and four of the inducible fusion constructs were cloned in Escherichia coli. Hybridization studies indicate that one of the damage-inducible genes contains a sequence reiterated throughout the D. radiodurans chromosome. Survival measurements show that two of the fusion strains have increased sensitivity to mitomycin C, suggesting that the fusions within these strains inactivate repair functions.

Cloning, Molecular↗

PubMed1990

18 S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells.

The effect of ionizing radiation (137Cs) on processing and transport of ribosomal RNA (rRNA) was studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. This approach permits kinetic analysis of processing of 45 S rRNA (radiolabeled predominantly prior to irradiation) into its 28 S and 18 S rRNA daughter species following irradiation. By this technique, we have recently demonstrated an increase in the normal 28 S:18 S rRNA stoichiometric ratio of 1:1 to as high as 1.6:1 during the interval 5 to 20 h following irradiation of HeLa cells at greater than or equal to 7.5 Gy. Alterations in 28 S:18 S ratio were evaluated in greater detail at early times following irradiation, up to 2 h. The 28 S:18 S ratio was found to be maximal at 1 h after radiation, at about 2:1, following 5 or 10 Gy. Using a method for rapid separation of nucleus from cytoplasm, transport of rRNA from nucleus to cytoplasm was also evaluated during this period. Despite an increase in the rate of 45 S rRNA processing, as well as an increased 28 S:18 S ratio, no alterations in transport from nucleus to cytoplasm were detected. This lack of transport alteration suggests that accumulation of excess 28 S rRNA is restricted to the nucleus, where it may represent an early step in the process of radiation-induced cell killing.

Biological Transport↗

PubMed1989

Extensive photodimerization of non-adjacent pyrimidines.

In a prior study we found that non-adjacent thymidyl residues in the single-stranded alternating copolymer poly[d(G-T)] are subject to photodimerization by germicidal lamp irradiation (lambda max 254 nm). The maximum yield of this photoproduct was 1% of the total thymine of poly[d(G-T)]. We now report that dimer formation in this polymer is increased to 10 to 40% thymine as dimer between non-adjacent pyrimidines, using near-ultraviolet irradiation (lambda max 310 nm) with or without acetone triplet-sensitization. As previously observed for 254 nm irradiation, dimer formation was nearly absent in double-stranded poly[d(G-T).d(C-A)]. These observations extend prior findings by demonstrating high-yield dimerization between non-adjacent pyrimidines via direct irradiation at environmentally relevant wavelengths (greater than or equal to 280 nm), and are potentially relevant to the mechanism of the ultraviolet light-induced targeted -1 frameshift mutation.

Acetone↗

PubMed1989

Reduced formation of bipyrimidine photoproducts in DNA UV irradiated at high intensity.

DNA was irradiated using an excimer laser (248 nm) at low intensity (3.15 x 10(7) watts/m2) or high intensity (1.25 x 10(11) watts/m2). Fluences up to 30 kJ/m2 were delivered at either intensity. Following irradiation, DNA damage products were measured, yielding the following findings: 1) the rate of formation of thymine-thymine and thymine-cytosine cyclobutane dimers and the bipyrimidine photoadduct 6-4'-[pyrimidine-2'-one]thymine were reduced at high intensity by about 2-fold and 2) extensive release of free thymine and thymine decomposition fragments occurred at high intensity, but not at low intensity. The effects of high intensity UV are due to promotion of low-lying excited state(s) by absorption of a second photon, producing higher excited state(s) with consequent ionization and base loss. Possible excited state intermediates in this process are the lowest triplet state of DNA bases and prolonged singlet states associated with excimer formation. The depletion of these excited states via promotion may be the cause of the diminished yield of bimolecular pyrimidine photoproducts, suggesting that these photoproducts are formed at low UV intensity in part from long-lived excited states. Long-lived excited states present at conventional UV intensities may contribute to formation of some photoproducts that occur rarely, but are of potential biologic importance, such as dimers between nonadjacent pyrimidines on the same strand and interstrand dimers forming DNA cross-links.

DNA↗

PubMed1989

Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids.

An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.

Cloning, Molecular↗