Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

Degradation of transforming and transfecting DNA by the restriction endonucleases of type I and type II isolated from Haemophilus influenzae.

The restriction endonucleases of type I and II from Haemophilus influenzae were studied for their activity on transforming and transfecting DNA. Type I restriction enzyme from Haemophilus influenzae Rf, which requires adenosine 5'-triphosphate, reduced the size of unmodified bacterial DNA from 66x106 daltons to approximately 18x106 daltons and did not attack modified DNA. The action of this enzyme gives only a low level of inactivation of single and linked markers in the transforming DNA. In contrast the HP1c1 phage DNA was drastically inactivated by this enzyme. The endoR.Hind III degrades the ummodified bacterial DNA but the segments generated by this enzyme are still capable of being integrated in transformation. The enzyme has no activity on HP1c1 phage DNA.     

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.     

Restriction endonucleases and their applications

Restriction endonucleases are deoxyribonucleases which cleave double-stranded DNA into fragments. With only one exception, all restriction endonucleases recognize short, non-methylated DNA sequences. Restriction endonucleases can be divided into two groups based on the position of the cleavage site relative to the recognition sequence. Class I restriction endonucleases cleave double-stranded DNA at positions outside the recognition sequence and generate fragments of random size. The cleavage sites of Class II restriction endonucleases are located, in most cases, within the recognition sequence. Most of the Class II restriction endonucleases recognize 4, 5, or 6 base pair palindromes and generate fragments with either flush ends or staggered ends. DNA fragments with staggered ends contain 3, 4, or 5 nucleotide single-stranded tails called ‘sticky ends’. DNA fragments produced by Class II restriction endonuclease cleavage can be separated on gels according to their molecular weight. The fragments can be isolated from the gel and used for sequence analysis to elucidate genetic information stored in DNA. Further, an isolated fragment can be inserted into a small extrachromosomal DNA, e.g. plasmid, phage or viral DNA, and its replication and expression can be studied in clones of prokaryotic or eukaryotic cells. Restriction endonucleases and cloning technology are powerful modern tools for attacking genetic problems in medicine, agriculture and industrial microbiology.     

Repair of Deoxyribonucleic Acid in Haemophilus influenzae I. X-Ray Sensitivity of Ultraviolet-sensitive Mutants and Their Behavior as Hosts to Ultraviolet-irradiated Bacteriophage and Transforming Deoxyribonucleic Acid

Seven mutants of Haemophilus influenzae were isolated by the criterion of sensitivity to ultraviolet (UV) inactivation of colony formation. These mutants and the wild type were characterized with regard to X-ray inactivation of colony formation, UV induction of division inhibition, the ability of the eight strains to act as recipients to UV-irradiated H. influenzae phage and transforming deoxyribonucleic acid (DNA), and the influence of acriflavine on the survival of UV-irradiated transforming DNA with these strains as recipients. The photoreactivable sector of transforming DNA with yeast photoreactivating enzyme was measured for the most UV-sensitive mutant and was found to be greater than that of wild type. Judged by the above criteria, the order of the strains' sensitivities shows some, but by no means complete, correlation from one type of sensitivity characterization to another, indicating that a minimum of two variables is needed to explain the differences in the strains. Acriflavine increases the UV sensitivity of transforming DNA except in the most sensitive mutant. This effect is usually, but not always, more pronounced in the case of the more UV-resistant marker. The acriflavine effect is postulated to be the result of at least two factors: (i) interference with repair of transforming DNA in the host cell, and (ii) interference with the probability of recombination between transforming DNA and host DNA.     

Action of Haemophilus Endodeoxyribonuclease on Biologically Active Deoxyribonucleic Acid

An enzyme similar to that described by Smith and Wilcox (15) for Haemophilus influenzae which attacks foreign deoxyribonucleic acid (DNA) but not its own has been isolated and purified from H. parainfluenzae. The enzyme degrades foreign DNA to limited sizes and can destroy the transforming activity of H. influenzae and Bacillus subtilis DNA. The enzyme can also destroy the biological activity of H. influenzae phage and prophage DNA. On the other hand, the H. influenzae endodeoxyribonuclease can destroy the transforming activity of H. parainfluenzae DNA but not its own DNA. It also attacks B. subtilis DNA and its transforming activity.     

Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae

Voll, Mary Jane (University of Pennsylvania, Philadelphia), and Sol H. Goodgal. Loss of activity of transforming deoxyribonucleic acid after uptake by Haemophilus influenzae. J. Bacteriol. 90:873-883. 1965.-Transforming deoxyribonucleic acid (DNA) which has been irreversibly removed from solution by competent cells undergoes a progressive loss in marker activity when tested by lysis of the cells and exposure to new recipient cells. The loss of activity is limited and marker-specific, with greater inactivation of those markers with lower efficiencies of transformation. Recipient factors or donor factors which have undergone recombination, as measured by the appearance of linked markers, do not undergo inactivation. The efficiency of transformation can be correlated with the sensitivity of a marker to inactivation after DNA uptake. A mutation which affects the efficiency of transformation is found to increase sensitivity to postuptake inactivation. The rate of inactivation is temperature-dependent. At temperatures of 20 and 45 C, marker inactivation can occur without concomitant recombination. During the uptake process, DNA is retained in an acid-insoluble form, indicating that the fate of Haemophilus influenzae DNA differs from the fate of transforming DNA in pneumococcus.     

Multiplex-endonuclease genotyping approach (mega): a tool for the fine-scale detection of unlinked polymorphic DNA markers

Restriction enzyme-detectable polymorphisms have been used for assessing genetic differences and generating informative genetic markers. The most detailed fingerprinting analyses have been obtained using the AFLP (amplified fragment length polymorphism) technique, which accesses subsets of polymorphisms at one or two restriction sites. To combine increased discriminatory power with the stringency of polymerase chain reaction amplification, it would be beneficial to access additional independent restriction sites per analysis, and to amplify subsets of DNA restriction fragments with only one pair of oligonucleotide primers. We have now developed a unique approach that permits the simultaneous use of four or more endonucleases in combination with one pair of adapters/primers, and applied it to genotype 21 trypanosome populations to subspecific level. The approach takes advantage of the fact that some endonucleases create cohesive ends that are compatible with the overhang sites created by other endonucleases. We demonstrate the greater resolution of identifiable polymorphic fragments over the conventional ligation-mediated restriction analysis method, and discuss the value of the approach as a tool for fine genetic mapping of Trypanosoma brucei. Finally, we propose use of the method for fine characterisation and for identifying co-dominant genetic markers in a variety of other taxa. Edited by: W. HennigAn erratum to this article can be found at     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.     

Biological properties of a Haemophilus influenzae restriction enzyme, Hind I.

A type I restriction enzyme from Haemophilus influenzae, Hind I, which requires adenosine 5' -triphosphate and 5-adenosyl methionine, was studied for its activity on transfecting and transforming deoxyribonculeic acid (DNA). The enzyme reduced the size of unmodified bacteriophage S2 DNA from 37 X 10(6) daltons to approximately 10 X 10(6) daltons, but did not affect modified S2 DNA. Unmodified transforming DNA was attacked in vitro by Hind I; however, relatively low levels of inactivation were obtained for single markers, and linked transformants were inactivated as a function of the distance between markers. In contrast, unmodified bacterial DNA was not inactivated in vivo for either single or linked markers by the Hind I restriction system, probably because the segments generated by Hind I were still capable of being integrated in vivo. The lack of preferential inactivation of markers by the enzyme suggests that it makes random breaks in the DNA.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Relation between the transforming activity of a marker and its proximity to the end of the DNA particle

Summary Tranforming pneumococcal DNA is inactivated by treatment with restriction enzymes. For mutations belonging to the same locus (amiA locus), the extent of inactivation depends strongly upon the mutations and the enzymes. Two EcoRI and one BamHI restriction sites have been located within the amiA locus. After treatment of donor DNA with either one of these enzymes, the lowest transforming activity is observed for mutations that map near restriction sites. This effect of proximity to the nearest end of the DNA fragment extends over a distance of 1,400 nucleotides. The curve of transforming activity versus DNA size obtained with endonuclease-generated DNA fragments is very similar to that obtained previously with mechanically sheared DNA. Both curves show a striking slope change for donor DNA size around 2,700 base pairs, i.e. twice the length found for the extent of the end effect. We suggest that for donor DNA fragments larger than 2,700 base pairs the transforming activity depends mainly upon the size of donor whereas for donor DNA fragments shorter than 2,700 base pairs both a size-dependent phenomenon and the end effect contribute to reduce drastically the transforming activity.     

Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1c.

A 26-kilobase BamHI restriction endonuclease DNA fragment was cloned from Pseudomonas pickettii PKO1, a strain isolated from a soil microcosm that had been amended with benzene, toluene, and xylene. This DNA fragment, cloned into vector plasmid pRO1727 and designated pRO1957, allowed Pseudomonas aeruginosa PAO1c to grow on phenol as the sole source of carbon. Physical and functional restriction endonuclease maps have been derived for the cloned DNA fragment. Two DNA fragments carried in trans and derived from subclones of pRO1957 show phenol hydroxylase activity in cell extracts of P. aeruginosa. Deletion and subcloning analyses of these fragments indicated that the gene encoding phenol hydroxylase is positively regulated. Phenol and m-cresol were shown to be inducers of the enzyme. o-Cresol and p-cresol did not induce enzymatic activity but could be metabolized by cells that had been previously exposed to phenol or m-cresol; moreover, the enzyme exhibited a rather broad substrate specificity and was sensitive to thiol-inhibiting reagents. A novel polypeptide with an estimated molecular mass of 80,000 daltons was detected in extracts of phenol-induced cells of P. aeruginosa carrying plasmid pRO1959.     

Simian adenovirus genome. II. Identification of specific fragments of viral DNA possessing transforming activity

The antigenic variant of simian adenovirus 7 (SA7) DNA was cleaved by restriction endonucleases EcoRI, XbaI, BamHI, SalI. The resulted digests of viral DNA were tested for transforming activity using the "calcium" technique. It was shown that BamHI. XbaI and SalI digests transformed primary baby rat kidney cells as well as native viral DNA. The transforming activity of separated BamHI and SalI fragments was tested also. The viral DNA fragments with transforming activity (BamHI-B and SalI-B) were localised on the left of the physical map of the viral genome. It was also shown that fragment-transformed cell lines were able to form colonies in 0.33% agarose medium.     

Restriction and Modification of Bacteriophage S2 in Haemophilus influenzae

The major conclusion from these studies is that variants of Haemophilus influenzae Rd which restrict and modify phage S2 are metastable and capable of giving rise to one another with high frequency. Nonrestrictive RdS cells segregate spontaneously to the restricting, modifying phenotype in about 5% of the progeny of a single clone. The restrictive cells derived from RdS revert to the nonrestrictive phenotype in 15 to 25% of the progeny of a single clone. These frequencies are not appreciably affected by treatment with acriflavine or ethidium bromide, compounds which affect plasmid stability, or by nitrosoguanidine, a powerful mutagen. The genetic locus for restriction and modification of bacteriophage S2 is found to have a chromosomal position between the biotin and proline loci. Restriction-modification of phage S2 has been shown to be a function of its deoxyribonucleic acid (DNA) in that transfection with S2 phage DNA or prophage DNA is subject to host restriction and modification. An enzyme preparation, which contains endodeoxyribonuclease but no appreciable exonuclease activity, from mutant H. influenzae com(-10) did not restrict phage S2.RdS DNA or prophage DNA transfecting activity, indicating that this endodeoxyribonuclease is not responsible for phage restriction. A new restriction enzyme isolated from H. influenzae Rd was found to be the major enzyme involved in the restriction of bacteriophage S2. The enzyme inactivated the transfecting activity of unmodified phage DNA but did not attack modified phage DNA. Unlike endodeoxyribonuclease R, this enzyme requires adenosine triphosphate and S-adenosylmethionine.     

Location of the 5-Methylcytosine Group on the Bacteriophage φX174 Genome

Bacteriophage phiX174 DNA was labeled in vivo with [methyl-(3)H]methionine. The methyl-labeled progeny DNA was extracted from purified bacteriophage phiX174 particles and was used as template for in vitro synthesis of the complementary strand in the presence of the nucleoside triphosphates and Escherichia coli polymerase I. The resultant replicative form DNA was then cleaved, in separate experiments, with restriction endonucleases from Haemophilus influenzae and H. aegyptius. The DNA fragments were analyzed by polyacrylamide gel electrophoresis. It is concluded that the single methylcytosine in the viral DNA is located in a specific region of the phiX174 genome, very likely in gene H.