Studies of Viable T4 Bacteriophage Containing Cytosine-substituted DNA (T4dC Phage). III. Advantage of T4dC DNA as Substrate for Assay of Restriction Endonucleases

Cytosine-substituted phage T4 DNA (T4dC DNA) was demonstrated to be a splendid substrate for the assay of restriction endonucleases by agarose gel electrophoresis. For preparing those which cleave lambda phage DNA at few sites, T4dC DNA having appreciable number of cleavage sites was especially useful. As typical examples SalI and XbaI restriction endonucleases were chosen and an advantage of T4dC DNA for the enzyme unit determination was described. Screening of new restriction endonucleases from Streptomyces strains was facilitated by using T4dC DNA as a substrate for the assay.     

Physical mapping of the restriction fragments obtained from bacteriophage T4 dC-DNA with the restriction endonucleases SmaI, KpnI and BglII.

Summary The cytosine-containing DNA of a mutant of bacteriophage T4 was digested with restriction endonucleases SmaI, KpnI and BglII producing 5, 7 and 13 fragments respectively. Complete physical maps of the T4 genome were constructed with the enzymes SmaI and KpnI and an almost complete map with the enzyme BglII.     

A restriction map of the bacteriophage T4 genome

Summary We report a detailed restriction map of the bacteriophage T4 genome and the alignment of this map with the genetic map. The sites cut by the enzymes BglII, XhoI, KpnI, SalI, PstI, EcoRI and HindIII have been localized. Several novel approaches including two-dimensional (double restriction) electrophoretic separations were used.     

Physical mapping of bacteriophage T4

Summary The 134 positions of the cleavage sites of the restriction endonucleases XbaI, HaeII and EcoRI were determined for a cytosine-containing DNA of bacteriophage T4. This physical map was aligned with the genetic map. The T4 early regions were further identified by hybridization of RNA synthesized in vitro to the restriction fragments and two promoter regions were localized by filter binding tests and R-loop analysis.     

A physical map of bacteriophage T4 including the positions of strong promoters and terminators recognized in vitro

Summary We present a linearized physical map of the genome of bacteriophage T4. This map contains the cleavage sites for restriction enzymes SmaI, KpnI, SalI, BglII, XhoI, XbaI, ClaI, HaeII, EcoRI, and EcoRV. It also contains about 200 TaqI sites. The promoter sites recognized in vitro and a number of rho independent terminators have also been mapped.     

Phylogeny of the genera Trichophyton using mitochondrial DNA analysis

Diversity of mitochondrial DNA (mtDNA) was investigated in 92 Trichophyton rubrum strains, 2 T. mentagrophytes var. mentagrophytes, 2 T. m. vor. interdigitale, 2 T. m. var. goetzii, 1 T. m. var. erinacei, 2 T. quinckeanum, 2 T. schoenleinii, 1 T. tonsurans, 2 T. verrucosum var. album, 2 T. v. var. discoides, 1 T. violaceum var. violaceum, 1 Arthroderma benhamiae, and 1 A. vanbreuseghemii using endonucleases, Hae III, Msp I, Hind III, Xba I, and Bgl II. Trichophyton species were divided into 7 groups, and a phylogenetic tree was produced based on sequence divergence within mtDNA. The following results were obtained: (1) T. rubrum was divided into 2 groups Type I and Type II, and was suggested to be a complex. (2) A. benhamiae was closely related to T. m. var. erinacei. (3) T. rubrum Type II, T. tonsurans, and A. vanbreuseghemii showed identical restriction profiles, and were suggested to be closely related to each other or identical. (4) T. quinckeanum and T. schoenlenii showed identical restriction profiles, which differed slightly from those of A. vanbreuseghemii. (5) mtDNA analysis was useful in identifying pleomorphic strains.     

Suitability of hydroxyapatite and iron phosphate as P sources for Lupinus albus grown in nutrient solution

In the framework of efforts to introduce Tuber melanosporum as a cultivated crop to Israel, spores of the truffle, obtained from fruit-bodies procured in Italy and France, were used to inoculate oak seedlings and hazel suckers. Typical T. melanosporum mycorrhizas were observed 3 months after inoculation on roots of both plant species. One- to two-year-old mycorrhizal seedlings were outplanted at a number of experimental sites and irrigated regularly. Two sites characterized by alkaline soil but differing in soil composition and climatic conditions were chosen for the present study. DNA of ascocarps used for inoculation, DNA of re-isolated cultures and fungal DNA taken from tree mycorrhizas 4 years after outplanting were compared with T. melanosporum reference cultures by molecular methods. All T. melanosporum profiles proved to be identical except for one belonging to a reference culture, which exhibited an unusual HinfI ITS-RFLP pattern. A single base substitution, responsible for the different HinfI restriction site, distinguished the ITS region of this culture from a published T. melanosporum ITS sequence. ITS restriction polymorphism analyses determined that roots of all potted plants tested and many 4-year-old trees from the two experimental plots (irrespective of soil and climatic differences) were colonized by T. melanosporum.     

Studies of viable T4 bacteriophage containing cytosine-substituted DNA (T4dC phage)

Summary Digestion of non-glucosylated and cytosine-substituted T4 phage (T4dC) DNA with SalI restriction endonuclease showed that the DNA had nine SalI-sensitive sites. There were eight SalI sites in DNA from a strain which had a deletion in the rII-denB-ndd region. The comparison of two digestion patterns indicated that one of the SalI-sensitive sites was present in the deleted region and that the SalI-F fragment (8.4x10⁶ daltons) was located adjacent to the SalI-C or SalI-D fragment (15.5x10⁶ daltons) on the T4 chromosome. The DNA gave no detectable cleavage product when digested with BamHI endonuclease alone, while, when digested successively with BamHI and SalI, the DNA yielded two new digestion products in place of one fragment formed by SalI alone. The BamHI-sensitive site was in the SalI-A fragment (25.2x10⁶ daltons). The usefulness of this information for making cleavage maps of T4 phage chromosome is discussed.     

Transduction of Plasmid Antibiotic Resistance Determinants with PseudoT-Even Bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 fromEscherichia coli recA ⁺ and recA ^– donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc–segE–uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages , T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limitedin vivo by modification–restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification–restriction systemsEcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification–restriction system.     

Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).     

Localization of the dihydrofolate reductase gene on the physical map of bacteriophage T5

Summary Large quantities of dihydrofolate reductase are synthesized in bacteriophage T5 infected E. coli cells. Some evidence that this enzyme is the product of a viral gene was published by Mathews (1967). Further evidence is presented now by showing that the newly synthesized enzyme differs from the preexisting E. coli reductase in molecular weight and salt solubility.The expression of the T5 dihydrofolate reductase gene was not affected by deletions in the del region of the phage genome. The map position of the reductase gene was determined by marker rescue experiments designed as helped transfection procedure: When E. coli B cells were preinfected with T5 dihydrofolate reductase amber mutants, made competent, and transfected with T5 wild type DNA, viable phages were obtained. Wild type recombinant phages were observed, when the transfecting DNA had been digested with the restriction endonucleases EcoRI, HpaI, PstI, and SalI. No rescue occurred when the DNA had been digested with AluI, EcoRII, HindII, HindIII, MboII, Sau3A, and XbaI. Single EcoRI, HpaI, and SalI restriction fragments were isolated and found to rescue the dihydrofolate reductase gene. Their common overlapping sequence corresponds to 8.6% of the phage DNA, a segment of about 10,000 base pairs length, which extends from position 0.37 to position 0.46 of the physical map. After cleaving this segment at its single HindIII recognition site marker rescue no longer occurred. From these results it was concluded that the dihydrofolate reductase gene either lies at or very close to this site at position 0.4.The helped transfection method was also used to rescue T5 mutants with defects in the genes C2 and D9. Gene C2 was localized on an EcoRI fragment that covers the DNA from map position 0.08 to map position 0.25. By localizing the two genes B3 and C2 on the restriction map of the T5 DNA a correlation of the genetic and the physical maps of the T5 genome has been established. Abbreviations. The symbols for T5 phages follow those of McCorquodale (1975) and the nomenclature for restriction nucleases that of Smith and Nathans (1973). kb=kilo base pairs     

RFLP-based genetic relationships of Einkorn wheats

To study the relationships between different species of the Einkorn group, 55 different accessions ofTriticum monococcum,T. boeoticum,T. urartu,T. sinskajae,T. thaoudar andT. aegilopoides were analyzed. Fifteen anonymous probes and four clones corresponding to storage protein genes were used for detecting restriction fragment length polymorphisms (RFLPs). The DNA was restricted with the restriction enzymesAluI,HaeIII,RsaI andTaqI. The 25 probe/enzyme combinations employed yielded a total of 488 polymorphic fragments. Statistical analyses were performed using Jaccard's coefficient of similarity and principal coordinate analysis. Different values of similarity within the three main taxa,monococcum,boeoticum andurartu, were obtained; the grouping at the species level was quite well reflected by the RFLP analysis done here. The coincidence between RFLP data and the subspecies classification of theT. monococcum group was only partial. OneT. urartu accession is clearly different from all of the other 54 accessions. The need for an RFLP based revision of the Einkorn taxonomy is evident.     

Methylation of mitochondrial DNA in carrot (Daucus carota L.)

The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is discussed. Received: 16 April 1997 / Revision received: 4 July 1997 / Accepted: 30 July 1997     

Restriction Fragment Length Polymorphism Analysis of the Mitochondrial DNA of Fusarium oxysporum f. sp. ciceris

Seven isolates of Fusarium oxysporum f. sp. ciceris, representing pathogenic races 1 , 2, 3, and 4 from India and 0, 5, and 6 from Spain, were assayed for restriction fragment length polymorphisms (RFLPs) in the mitochondrial DNA,(mt DNA). The mt DNA fraction of total fungal DNA was purified and digested with the restriction endonucleases Bam HI, Bgl II, Eco RI. Kpn I, Sac I, Sal I, Sma I, and Xho I. The mt DNA is a circular molecule of 40.5 kb. No RFLP in the mt DNA was detected among the seven races of F. o. ciceris. The identical restriction patterns of mt DNA indicates an extensive conservation in the gene composition of mt DNA without sequence variation, and suggests that mt DNA of F. o. ciceris may not be responsible for pathogenic diversity. The restriction map of mt DNA from the race 6 isolate Fo 8272 was constructed by digestion of the mt DNA with five restriction enzymes: Eco RI, Kpn I, Sac I, Sal I, and Xho I, either singly or in selected pairs.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Restriction enzyme cleavage mapping of T7 virus early region

Summary DNA molecules of seven T7 mutants with overlapping deletions in the early region were cleaved by restriction enzymes HindII, HpaI and II, and HaeIII. The differences in the cleavage patterns after electrophoresis have been used to generate a cleavage map of the restriction sites of this enzyme. It covers the first 9% of the T7 DNA molecule. Cleavage points for HindII are at 0.60, 1.33, 1.59, 1.76, 5.26, 6.27, 7.4 and 8.38%; for HpaI and II at 1.36, 1.62, 4.46, 6.29, 6.62, 7.56, and 8.76%; for HaeIII at 3.85, 6.98, 7.88 and 8.26%. Some fragments have been located in the region containing the early promoters, others carry the complete sequences of gene 0.3.     

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.     

Development of a new host–vector system for colour selection of cloned DNA inserts using a newly designed β-galactosidase gene containing multiple cloning sites in Thermus thermophilus HB27

We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal⁻) on growth plates containing 3,4-cyclohexenoesculetin β-d-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites (Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host–vector system facilitates cloning procedures in T. thermophilus HB27.

     

Heterogeneity of the internal transcribed spacer 1 (ITS1) inTulipa (Liliaceae)

Heterogeneity of the internal transcribed spacer ITS1 of the rDNA within individuals ofTulipa gesneriana L.,T. kaufmanniana Regel, and their interspecific hybrids was analyzed by PCRRFLP, using the polymorphic restriction enzymesRsaI andHinfI, and by nucleotide sequence analysis. In most cases, the sum of the sizes of the restriction fragments was higher than the entire length of the undigested ITS fragment, indicating heterogeneity at the restriction sites within an individual. Differences in band intensities within the restriction patterns indicate the occurrence of variation in copy number of these different ITS1 variants within individuals. Automated sequencing without a visual inspection often failed to detect existing heterogeneity within sequences, resulting in a discrepancy between the sequencing and restriction analysis results. By visual interpretation of the sequences, the restriction patterns could mostly be predicted well. Fluorescence in situ hybridization (FISH) experiments in fourTulipa species revealed the occurrence of several rDNA spots. The number of rDNA loci varied from seven inT. gesneriana Christmas Marvel to ten inT. australis Link. This might explain the occurrence of heterogeneity in ITS sequences inTulipa, as homogenization of variants has to take place over different loci.     

Analysis of the C4 genes in baleen whales using a human cDNA probe

We have used a human C4 cDNA probe to investigate the complement component C4 gene in four members of the family Balaenopteridae: fin whale (Balaenoptera physalus), sei whale (B. borealis), minke whale (B. acutorostrata), and bryde's whale (B. edeni). Restriction mapping of genomic DNA from the first three species suggests the presence of only one locus in these species, and also shows that the C4 genes in the three species are very similar. We have used 14 restriction endonucleases to investigate the restriction fragment length polymorphism (RFLP) of fin whales, 13 enzymes for sei whales, and 8 enzymes for the minke whale. No polymorphism was seen in DNA from the five minke whale samples, but Rsa I and Taq I restriction enzymes gave polymorphism in fin and sei whales whereas Hind III and Msp I restriction enzymes showed polymorphism in sei whales only. Only one bryde's whale sample was available for investigation. The study of DNA available from mother-fetus pairs from the two polymorphic species demonstrated a simple, two-allele transmission of RFLP alleles.