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M Rhoades

Publications and source records attributed to M Rhoades.

At least 19 recordsLinked to original sources
PubMed1987

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus).

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.

Animals↗
PubMed1986

Interruption-deficient mutants of bacteriophage T5: analysis of single-site mutants.

The properties of viable mutants of bacteriophage T5 that lack, singly, each of the four major sites at which single-chain interruptions normally occur in T5 DNA are described. The mutations responsible for loss of each interruption were mapped by analysis with HhaI, a restriction endonuclease with a cleavage site (pGCGC) that occurs at the 5' termini of the major interruptions (B. P. Nichols and J. E. Donelson, J. Virol. 22:520-526, 1977). For each mutant tested, loss of a specific interruption resulted in loss of a specific HhaI cleavage site. Multiple single-site mutants were constructed to determine the effect of loss of more than one interruption on phage viability. These recombinants, including a phage that lacks the four major interruptible sites, were fully viable and did not exhibit a compensating increase in the frequency of minor interruptions. The effect of loss of a specific interruption on genetic recombination was tested in two-factor crosses with markers that occur close to, but on opposite sites of, the interruption. Loss of the interruptible site did not affect recombination frequency.

Chromosome Mapping↗
PubMed1982

New physical map of bacteriophage T5 DNA.

The locations of 103 cleavage sites, produced by 13 restriction endonucleases, were mapped on the DNA of bacteriophage T5. Single- and double-digest fragment sizes were determined by agarose gel electrophoresis, using restriction fragments of phi X174 DNA and lambda DNA as molecular weight standards. Map coordinates were determined by a computer-based least-squares procedures (J. Schroeder and F. Blattner, Gene [Amst] 4:167-174, 1978). The fragment sizes predicted by the final map are all within 2% of the measured values. Based on this analysis, T5st(+) DNA contains 121,300 base pairs (Mr, 80.3 X 10(6) and has a terminal repetition of 10,160 base pairs (Mr, 6.7 X 10(6)). Restriction endonuclease analysis after treatment with exonuclease III and a single-strand-specific endonuclease allowed precise localization of five of the natural single-chain interruptions in T5 DNA. Revised locations for several T5 deletions were also determined.

Base Sequence↗
PubMed1980

New deletion mutant of bacteriophage T5.

Deletion mutants of bacteriphage T5 can be identified by analyzing the DNA content of single plaques, after alkaline denaturation, by agarose gel electrophoresis. Use of this procedure has resulted in the isolation of a new mutant, designated del2, that lacks a 1.6% segment located 68 to 70% from the left end of T5 DNA. This segment occurs between the early and late gene regions of T5 DNA.

DNA, Viral↗
PubMed1979

Interruption-deficient mutants of bacteriophage T5 I. Isolation and general properties.

Mutations of bacteriophage T5 were isolated which lack one or more of the natural single-chain interruptions that occur in the mature DNA of this virus. Interruption-deficient mutants were detected by screening survivors of hydroxylamine mutagenesis for altered DNA structure by electrophoresis in agarose slab gels. Over 60 independent mutants were isolated from a survey of approximately 800 phages particles. All of the mutants were viable and could be grouped into two classes. Mutants in one class lacked one of the localized sites where interruptions occur in T5 DNA. To date, mutants that affect five different sites have been obtained. Mutants in the other class were essentially free from interruptions or had a reduced frequency of interruptions throughout the genome. The members of this class included several amber mutants. Complementation tests indicated that at least two genes are required for the presence of interruptions in mature T5 DNA.

Coliphages↗
PubMed1979

Interruption-deficient mutants of bacteriophage T5. II. Properties of a mutant lacking a specific interruption.

An examination was made of the properties of T5HA4, a mutant of bacteriophage T5 that lacks the single-chain interruption that occurs at 7.9% from the left end of the genome. The DNAs of T5HA4 and the wild type were compared by electrophoresis in agarose gels of both single-stranded fragments produced by denaturation and duplex fragments generated by sequential treatment with exonuclease III and SI nuclease. These studies demonstrated that T5HA4 also lacks an interruption that occurs at 99.6% in wild-type DNA. The interruptions at 7.9 and 99.6% therefore occur within the 8.3% of T5 DNA that is terminally repetitious. Evidence on the location of other interruptions within the terminal repetition was also obtained. Analysis of T5HA4 with a restriction endonuclease indicated that the interruption deficiency is not due to a deletion or addition mutation. The injection of T5HA4 DNA into a host bacterium was found to occur, as with the wild type, in a two-step manner. The interruption at 7.9% is therefore not required for stopping DNA transfer after the initial 8% segment has been injected.

Bacteriophages↗
PubMed1979

Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions.

The DNA of bacteriophage BF23 possesses two structural features, localized single-chain interruptions and a large terminal repetition, previously described for T5, a closely related virus. As is the case for T5, single-chain interruptions occur with variable frequencies at a small number of fixed sites within one strand of the double-stranded BF23 genome. The sites where interruptions occur with the highest frequencies were napped by an electrophoretic analysis of the single-stranded fragments produced by denaturation of BF23 DNA. The positions of these fragments were determined by degrading BF23 DNA to various extents with lambda exonuclease and observing the relative order with which they were (i) degraded or (ii) released intact from the undenatured duplex. The exact locations of the interruptions were determined from analysis of analogous duplex fragments produced by degrading exonuclease III-treated BF23 DNA with a single-strand-specific endonuclease. BF23 has five principal sites (located at 7.9, 18.7, 32.4, 65.8, and 99.6% from the left end of the DNA) where interruptions occur in most molecules. The principal interruptions in T5 DNA occur at similar positions. The locations of eight secondary interruptions in BF23 DNA were also determined. In general, BF23 DNA has fewer secondary interruptions than t5 dna, although there is at least one location where an interruption occurs with a greater frequency in BF23. The presence of a terminal repetition in BF23 DNA was demonstrated by annealing ligase-repaired molecules that had been partially digested with lambda exonuclease. If the complementary sequences at both ends of the DNA were exposed by exonuclease treatment, the duplex segment that resulted from annealing could be released by digestion with a single-strand-specific endonuclease. This segment was analyzed by agarose gel electrophoresis and found to represent 8.4% of BF23 DNA.

Bacteriophage lambda↗
PubMed1979

Physical map of bacteriophage BF23 DNA: restriction enzyme analysis.

Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases SalI (3 fragments), BamHI (5 fragments), EcoRI, (8 fragments), BalI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with lambda exonuclease.

Chromosome Mapping↗
PubMed1977

Localization of single-chain interruptions in bacteriophage T5 DNA I. Electron microscopic studies.

Bacteriophage T5 DNA was examined in an electron microscope after limited digestion with exonuclease III from Escherichia coli. The effect of the exonuclease treatment was to convert each naturally occurring single-chain interruption in T5 DNA into a short segment of single-stranded DNA. The locations of these segments were determined for T5st(+) DNA, T5st(0) DNA, and fragments of T5st(0) DNA generated by EcoRI restriction endonuclease. The results indicate that single-chain interruptions occurr in a variable, but nonrandom, manner in T5 DNA. T5st(+) DNA has four principal interruptions located at sites approximately 7.9, 18.5, 32.6, and 64.8% from one end of the molecule. Interruptions occur at these sites in 80 to 90% of the population. A large number of additional sites, located primarily at the ends of the DNA, contain interruptions at lower frequencies. The average number of interruptions per genome, as determined by this method, is 8. A similar distribution of breaks occurs in T5st(0) DNA, except that the 32.6% site is missing. At least one of the principal interruptions is reproducibly located within an interval of 0.2% of the entire DNA.

Coliphages↗
PubMed1977

Localization of single-chain interruptions in bacteriophage T5 DNA. II. Electrophoretic studies.

Upon denaturation, T5 DNA yields a large number of discrete, single-chain fragments that can be resolved by agarose gel electrophoresis. The positions of the more prominent of these fragments in the T5 duplex were determined by analyzing their sensitivity to digestion with lambda exonuclease and their distribution among EcoRI fragments of T5 DNA. These experiments also provide firm evidence concerning the polarity of the strands in T5 DNA. An analogous study was carried out on the fragments produced by treating exonuclease III-degraded T5 DNA with the single-strand-specific SI endonuclease. This procedure yielded over 40 discrete duplex fragments that could be resolved with considerable precision by agarose gel electrophoresis. The positions of most of these fragments were determined by analyzing EcoRI fragments of T5st(+) and T5st(0) DNA. Over 20 sites where single-chain interruptions can occur in T5 DNA were identified, and the distribution of interruptions within the terminal repetition was shown to be identical at both ends of the molecule. A precise value for the size of the terminal repetition in T5 DNA was obtained by analyzing SI endonuclease digests of ligase-repaired, circular T5 DNA in agarose gels. The repeated segment represented 8.3% of the T5st(+) DNA. The results of this study also provide information concerning the properties of lambda exonuclease. Hydrolysis by this enzyme was not terminated when single-chain interruptions were encountered either in the strand being degraded or in the complementary strand.

Base Sequence↗
PubMed1977

Physical map of the bacteriophage T5 genome based on the cleavage products of the restriction endonucleases SalI, SmaI, BamI, and HpaI.

A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with lambda exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with lambda exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonucleases III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments.

Coliphages↗
PubMed1976

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA.

Four site-specific endodeoxyribonucleases have been partially purified from extracts of bacteriophage T5-infected Escherichia coli by gel filtration and affinity chromatography on single- and double-stranded DNA. The enzymes were detected and characterized by agarose gel electrophoresis of alkali-denatured digestion products. None of the four is found in uninfected cells. In the presence of a divalent cation, all four endonucleases make ligase-repairable, single-chain interruptions at specific sites in the duplex DNA of several bacteriophages (lambda, T7, and T5) and a mammalian virus (adenovirus 2). These activities are not stimulated by ATP. None of the four is active on single-stranded DNA. The fragments produced by each enzyme from ligase-repaired T5 DNA do not correspond to those derived from mature T5 DNA. Each of the enzymes is able to cleave the intact strand of T5 DNA.

Coliphages↗
PubMed1975

Heteroduplex mapping of heat-resistant deletion mutants of bacteriophage t5.

The bacteriophage T5 is known to spontaneously generate deletion mutants (st mutants) exhibiting enhanced resistance to heat inactivation in citrate buffer. A series of such mutants has been isolated and the deletions visualized by electron microscopy of heteroduplex molecules. The deletions are found to cluster in one region of the chromosome.

Journal Article↗