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Reports on metronidazole resistance of Trichomonas vaginalis strains have been on the increase. This study investigated the in vitro metronidazole resistance patterns in T. vaginalis isolates obtained from South African pregnant women and the genotypes of these isolates. This study included 362 pregnant women recruited from a hospital in Durban, South Africa. The women provided self-collected vaginal swabs for the detection of T. vaginalis by culture in Diamonds media. Cultured isolates were then subjected to anaerobic susceptibility assays to metronidazole. For the genotyping assays, the actin gene was digested by HindII, MseI, and RsaI. The banding patterns obtained after digestion was used to determine the genotypes. A total of 21/362 (5.8%) pregnant women tested positive for T. vaginalis infection. Of the 21 T. vaginalis isolates tested for metronidazole susceptibility, 9.5% (2/21) had a minimum inhibitory concentration (MIC) of 4 μg/ml (resistant), 38.1% (8/21) had a MIC of 2 μg/ml (intermediate), and 52.4% (11/21) had a MIC ≤ 1 μg/ml (susceptible). The dominant genotype that was identified across the isolates was genotype G. There was no correlation between genotype harboured and metronidazole susceptibility patterns. In this study, resistance to metronidazole was observed in clinical isolates of T. vaginalis. This study did not find a correlation between genotype harboured and metronidazole susceptibility patterns. Despite the lack of association, our study provides data on an area of research that is currently lacking in our setting.

Summary Fasciolosis caused by Fasciola hepatica is a common parasitic disease of livestock especially sheep and cattle. In this study molecular characterization of β-tubulin isotype 3 gene in Fasciola hepatica isolates from cattle and sheep in Turkey was carried out. For this purpose a total of 80 adult Fasciola hepatica isolates were collected from 20 sheep and 20 cattle in Kayseri and Erzurum provinces. PCR-RFLP was performed on β-tubulin isotype 3 gene and MboII revealed two fragments of approximately 350 bp and 390 bp, whereas HphI enzyme yielded 210, 340 and 540 bp bands, HindII yielded 380 and 450 bp bands in all samples. A total of 80 isolates were tested by SSCP and all of them presented the same band profiles. Six samples (4 sheep and 2 cattle) were randomly selected and DNA sequence of a 935 bp coding fragment of β-tubulin isotype 3 was performed. Sheep samples were more polymorphic than the cattle. This β-tubulin isotype 3 gene polymorphism of F.hepatica isolates from sheep and cattle of two distinct geographical areas of Turkey have been investigated for the first time.

Background: Trichomonas vaginalis is a prevalent sexually transmitted infection cause trichomoniasis. In this study prevalence and genotype of Iranian isolates of T. vaginalis infected (dsRNA) viruses were evaluated by PCR-RFLP and obtained patterns were then confirmed by sequence analysis and genotype of these Iranian isolates confirmed again. Methods: Ten strains of T.vaginalis were collected from 1700 vaginal samples of women referred to hospitals associated with Iran University of Medical Sciences in Tehran, Iran during Feb 2016 to Jul 2017, evaluated in points of infection to T. vaginalis Virus (TVV-1) were used in a PCR-RFLP. All of ten isolates of T. vaginalis were examined by designed nested PCR for actin gene and then digestion patterns of three endonuclease enzymes of HindII, MseI and RsaI were evaluated and genotype of these isolates was defined. Results: By combination of fragments pattern of three enzymes of HindII, RsaI and MseI, three genotypes were found; six genotypes E, two genotypes G and two genotypes I. The most dominant genotypes were genotype E. Among four TVV infected isolates two genotype E, one genotype G and one genotype I were found, however among six uninfected T. vaginalis isolates to TVV-1, all of three genotypes were also found. Conclusion: Three genotypes E, G and I in T. vaginalis infected with dsRNA isolates were found, however, these three genotypes in T. vaginalis without virus were also observed. Further study is needed to evaluate genotypes of T. vaginalis, which infected virus in more great T. vaginalis population

Background: PCR/RFLP of Human growth hormone gene was used to differentiate among ten athletes. Results: AatI, AviII, BsaI, HindII, and SacI restriction endonucleases gave high levels of intraspecific profile for all the studied gene of the ten athletes. Other enzymes including AvaI, Age and DraI were tested for their ability to differentiate these individuals. Conclusion: The study demonstrated that the HGH gene contains useful genetic markers for the identification of athletes.

The nucleotide sequence of the second part of the simian virus 40 DNA HindII + III restriction fragment A is presented. The sequence extends from map position 0.533 to 0.424 and together with the first part of Hind-A [Volckaert et al. Proc. Natl Acad. Sci. U.S.A. 75, 2160–2164 (1978)] completes the total Hind-A sequence, comprising 1169 base pairs. The second half of Hind-A includes the region corresponding to the second splicing boundary common to small tumor antigen (small-t) and large tumor antigen (large-T) mRNA and it contains coding information for an internal portion of large-T antigen. Two similar secondary structures of reasonable thermodynamic stability can be proposed for the nucleotide sequence of the pre-mRNA corresponding to the region reported here. Their possible relevance to the splicing of the SV40 early mRNAs is discussed. The deduced amino acid sequence is 188 residues long and contains a Lys-Lys-Lys-Arg-Lys stretch which may be involved in the DNA binding capacity of large-T. A presumptive phosphorylation site is also present.

PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif r region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714–1720, 1999). Digestion with four restriction enzymes ( Hae III, Hin dII, Mva I, and Acc II), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by Hae III digestion of the amplified rpoB DNA. By Hin dII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri , and other closely related species, were distinguished by simultaneous digestion of Mva I and Acc II. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.

Weston, Peter H. and Johnson, L.A.S. (National Herbarium of New South Wales, Royal Botanic Gardens, Sydney, N.S.W. 2000, Australia) 1996. Persoonia hindii (Proteaceae), a new species from the Newnes Plateau, New South Wales. Telopea 7(3): 199–203. Persoonia hindii P.H. Weston & L.A.S. Johnson, a new species from the Newnes Plateau in the Blue Mountains, New South Wales is described and distinguished from its closest relatives. The keys to Persoonia that have been published recently in the Flora of New South Wales and Flora of Australia are amended to include this species.

Restriction fragment length polymorphisms of the heat-shock protein 70 gene have been used for discriminating Leishmania species. Here, we validated HindII as a much cheaper alternative to EcoRII and SduI for discriminating Leishmania (Viannia) braziliensis from Leishmania (Viannia) naiffi and an atypical Leishmania (V.) braziliensis group, which was previously not possible. (C) 2013 Elsevier Inc. All rights reserved.

Swiss microbial geneticist, Werner Arber shared the 1978 Nobel Prize in Physiology or Medicine with Hamilton Smith and Daniel Nathans for their discovery of restriction endonucleases. Werner Arber was born in Granichen, Switzerland in 1929. Following a public school education, he entered the Swiss Polytechnical School in Zurich in 1949, working toward a diploma in natural sciences. There, his first research experience involved isolating and characterizing an isomer of chlorine. Following graduation in 1953, Arber joined a graduate program at the University of Geneva, taking on an assistanceship in electron microscopy (EM), in which he studied gene transfer in the bacterial virus (bacteriophage) lambda. Eventually encountering limitations with EM as a tool, he began using microbial genetics as a methodology for his studies. The study of microbial genetics had been possible for a relatively short time: DNA had been discovered to carry genetic information only a decade before he d entered the field. After earning his Ph.D. in 1958, Arber continued to develop skills in microbial genetics, working with colleagues in the United States for a short time before returning to Geneva at beginning of 1960. There, he continued working on lambda transduction in E. coli, but found that the virus would not efficiently propagate. Recalling research done seven years earlier by Joe Bertani and Jean Weigle on "host-controlled restriction-modification", he realized there must be a host-controlled modification of the invading DNA, and sought to identify the mechanism. Based on Grete Kallengerger s work that demonstrated degradation of both irradiated and non-irradiated phage lambda following injection in a host, Arber and his graduate student, Daisy Dussoix further investigated the fate of DNA, and found that restriction and modification (later determined to be postreplicative nuclotide methylation) directly affected DNA, but did not cause mutations. They also found that theses were properties of the bacterial strains, and that both viral and cellular DNA were degraded. Together, Arber and Dussoix reported their findings to scientific community in 1961 at the First International Biophysics Congress in Stockholm. Aber also presented the research to the Science Faculty of University of Geneva in 1962, earning the Plantamour-Prevost prize. Based on his work and the work of others, he hypothesized that an enzyme in the host bacterium cut DNA into smaller pieces at specific sites, and methylase modified the host DNA to protect it from the digestive enzyme. These theories were later confirmed by Urs Kuhnlein, who found that mutation of specific sites rendered the phage resistant to cleavage; Hamilton smith, who identified Type II endonuclease HindII; and Daniel Nathans, who used HindII to break the SV40 virus into 11 fragments, allowing him to determine its method of replication. Since the discovery of restriction endonucleases, researchers have used them as tools to study the functions of genes of all types of organisms. Restriction enzymes have also facilitated the study of gene functions and enabled production of substances of medical and nutritional importance. Arber feels that in the next few decades we will learn much from the study of epigentics --factors that can affect the phenotype of an organism without changing the genetic information--. He is proud that, in that studying restriction degradation and DNA methylation in the 1960s, he was among the first in studying epigenetic phenomenon.

The definitive version is available at www3.interscience.wiley.com, Human trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a highly prevalent sexually transmitted infection. However, little is known about the degree of strain variability of T. vaginalis. A reliable classification method for T. vaginalis strains would be a useful tool in the study of the epidemiology, pathogenesis and transmission of T. vaginalis. A PCR-restriction fragment length polymorphism typing method was designed and evaluated using T. vaginalis isolates obtained after culture of vaginal specimens collected in the Democratic Republic of Congo and in Zambia. The variation of the actin gene of T. vaginalis was determined for three ATCC reference strains and 151 T. vaginalis isolates. Eight different types were identified, on the basis of the digestion patterns of the amplified actin gene, with each of the restriction enzymes HindII, MseI and RsaI. It was determined that the ATCC reference strains 30001, 30240 and 50141 were of actin genotypes G, H and E, respectively. The actin genotype type E was more common in the Democratic Republic of Congo, whereas type G was the commonest type in Zambia. Translation of the nucleotide sequence showed up to three amino acid substitutions. We developed a reproducible, sensitive and specific typing method for T. vaginalis, and were able to distinguish at least eight T. vaginalis actin genotypes. Further studies are needed to evaluate the method using clinical specimens and to determine the utility of the typing method for the genotypic characterization of T. vaginalis.

In a native osteosarcoma specimen from a patient cured of bilateral retinoblastoma eight years ago, we found a deletion of a 7.5 kb HindIII fragment within the retinoblastoma gene. Our results contribute further evidence that the 7.5 kb fragment harbors a Breakpoint Cluster Region (BCR) in osteosarcoma. In tumor tissue of another osteosarcoma patient the retinoblastoma gene did not reveal any defect on DNA or mRNA level, suggesting different transforming events in this patient.

A polymerase chain reaction with the restriction fragment length polymorphism (PCR-RFLP) method using universal primers complementary to the conserved region of the cytochrome b gene (cyt b) of the mitochondrion DNA (mtDNA) of vertebrates was applied to the identification of the origin of blood meals in tsetse flies. Blood samples from ten potential tsetse hosts of the family bovidae (cattle, water buffalo, red buffalo, waterbuck, springbok, goat, sheep, sable antelope, oryx and dik-dik) were included in this study. Sites for appropriate restriction endonucleases cuts were chosen by pairwise alignment of the amplified 359 bp fragments. A flow chart of endonucleases digestion using three restriction enzymes (e.g. TaqI, AluI and HindII) for the unequivocal identification of the respective bovid species was developed. A number of additional non-specific DNA fragments attributed to the co-amplification of cytochrome b pseudogenes were observed in some species (e.g. in red buffalo and dik-dik after digestion with AluI) but did not hamper assignment of bovid species. The detection rate of host DNA in tsetse by PCR-RFLP was 100, 80, 60 and 40% at 24, 48, 72 and 96 h after in vitro feeding, respectively. Identification of the last blood meal was possible even when tsetse had previously fed on different hosts.

Local strains of Haemophilus ducreyi were characterized by ribosomal DNA fingerprinting in order to develop a typing scheme for this organism. Chromosomal DNAs from 30 strains were extracted, and restriction fragments were separated by agarose gel electrophoresis after cleavage with BglII, HindIII, HindII, and BstEII. Of the 30 local strains studied, nine BglII, eight HindIII, six HindII, and five BstEII ribotype patterns were observed. In order to develop a typing scheme, we correlated the ribotype patterns obtained with each of the four enzymes for each strain. By this scheme, 13 ribotypes designated b to n were defined among the local strains. This study demonstrates that there is a diversity of ribotypes among H. ducreyi strains in our area, which emphasizes that there are many strains responsible for chancroid. This novel typing scheme provides a useful tool for further epidemiological studies of chancroid.

In this study, polymorphism in ipa genes was found in five out of nine EIEC serotypes studied. When SalI and HindII were used in RFLP-PCR assays many EIEC serotypes showed polymorphism in ipaB and ipaD. On the other hand, no polymorphism was observed in ipaA and ipaC in these strains. The polymorphism present in EIEC strains is serotype-dependent, since restriction patterns were conserved amongst strains belonging to the same serotype. When IpaB deduced amino acid sequences of S. flexneri M90T and FBC124-13 were compared, ten amino acids changes could be observed mainly in the amino-terminal region. The deduced EIEC IpaD amino-acid sequence presented 91% similarity with the Shigella strain. In this case, amino acid changes were spread out through the whole structure, except in the carboxyl-terminal region. No presente estudo, foi encontrado polimorfismo no gene ipa em cinco sorotipos de EIEC, de nove estudados. Quando enzimas de restrição SalI e HindII foram utilizadas no ensaio de PCR-RFLP, amostras de EIEC apresentaram polimorfismo em ipaB e ipaD. Por outro lado, não foram observados polimorfismos nos genes ipaA e ipaC nestas cepas, quando diversas enzimas de restrição foram utilizadas. O polimorfismo presente em cepas de EIEC é sorotipo-dependente, uma vez que os padrões de restrição foram conservados entre as cepas pertencentes ao mesmo sorotipo. Quando a seqüência deduzida de aminoácidos de IpaB de S. flexneri M90T e FBC124-13 foram comparadas, mudanças foram observadas em dez aminoácidos na região amino-terminal. A seqüência deduzida de aminoácidos de IpaD de EIEC apresentou similaridade de 91% com a cepa de Shigella. Neste caso, mudanças de aminoácidos ocorreram em toda a estrutura da molécula de IpaD, exceto na região carboxi-terminal.

The outer membrane proteins (OMPs) of Pasteurella multocida are potential immunogens. The 16 kDa OMP of P. multocida P52, serotype B:2, was identified as one of the major immunodominant antigens. The gene omp16, encoding a 16 kDa outer membrane protein, was amplified, cloned into a pBluescript SK(–) vector and sequenced. Complete sequence homology was observed between the 16 kDa OMP gene of P. multocida P52 (serotypes B:2) and P. multocida T16 (somatic serotype 3,4). This gene was distributed among different serotypes of P. multocida and found to localize in a 6.0 kb HindII fragment of the P. multocida genome.

Fifty-eight isolates of Shigella sonnei from three outbreaks in school children and eight control isolates from epidemiologically unrelated sporadic clinical infections in Taiwan were compared by antibiotic susceptibility testing and molecular typing. Antibiotic susceptibility testing showed that all strains except one sporadic isolate were multi-resistant. Ribotyping after restriction endonuclease digestion with SalI, PvuII and HindII generated the same ribosomal pattern in 65 of the 66 isolates. Plasmid profile analysis and pulsed-field gel electrophoresis (PFGE) produced eight and nine distinct patterns, respectively, and were in agreement with the epidemiological relationship of the outbreak strains. Nevertheless, some of the sporadic isolates could be discriminated only by a combination of these two methods. This study showed that plasmid profiling in combination with PFGE may be superior to ribotyping in molecular epidemiological investigations of S. sonnei.

Five clinical isolates of Gardnerella vaginalis known to carry the tetracycline resistance determinant Tet M were examined by hybridization and nucleotide sequencing. Four of the strains carried tet(M) genes with identical sequences. The two versions of the tet(M) gene found in G. vaginalis did not show complete identity with other published tet(M) sequences, but showed mosaic structures with regions of homology to tet(M) gene sequences from Tn916, Tn1545 and the American type plasmid found in Neisseria gonorrhoeae. Hybridization studies showed that all isolates carried the tet(M) gene on a single HindII restriction fragment of variable length. No evidence was found for the presence of sequences homologous to the transposition functions of Tn916.

Polymerase chain reaction was applied to the rapid identification and detection of mousepox virus. This was accomplished by selection of primers targeting the A-type inclusion body protein gene. By investigating 20 strains belonging to five different species of the genus Orthopoxvirus, amplification was achieved only with the seven mousepox virus strains examined. The size of the resulting DNA fragment accounted for 116 base pairs and contained a recognition site for the restriction enzyme HindII, thus confirming its viral origin. Amplification of mousepox virus specific sequences was also possible from infected mouse lung tissue and serum.

Patient human immunodeficiency virus type 1 (HIV-1) isolates that are resistant to protease inhibitors may contain amino acid substitutions L10I/V, M46L/I, G-48V, L63P, V82A/F/T, I84V, and L90M in the protease gene. Substitutions at positions 82 and/or 90 occur in variants that display high levels of resistance to certain protease inhibitors. Nucleotide substitutions at these two sites also lead to the loss of two HindII restriction enzyme digestion sites, and these changes make possible a rapid procedure for the detection of drug-resistant variants in patients on protease inhibitor therapy. This procedure was used to detect the emergence of mutated viruses at various times after the initiation of therapy with the HIV-1 protease inhibitor indinavir. The method includes viral RNA isolation from plasma and reverse transcription PCR amplification of the protease gene with fluorescence-tagged primers. The PCR product is digested with HindII, the cleavage products are separated on a urea-acrylamide gel in a DNA sequencer, and the extent of cleavage is automatically analyzed with commercially available software. In viruses from 34 blood samples from four patients, mutations leading to an amino acid change at residue 82 appeared as early as 6 weeks after the start of therapy and persisted throughout the course of the study period (48 weeks). Mutations leading to double substitutions at residues 82 and 90 were seen at a lower frequency and appeared later than the change at position 82. The changes detected by restriction enzyme cleavage were confirmed by DNA sequencing of the cloned protease genes by reverse transcription PCR amplification of viral RNA from isolates in plasma. In addition to the changes at positions 82 and 90, we have identified M46L/I, G48V, and I54V substitutions in isolates derived from indinavir-treated patients. HindII analysis of uncloned, PCR-amplified DNA offers a rapid screening procedure for the detection of virus isolates containing mutations at amino acid residues 82 and 90 in the HIV-1 protease gene. By using other restriction enzymes, the same method can be used to detect additional protease drug-resistant variants and is generally applicable for the detection of mutations.

In 1962, Hamilton Smith abandoned a career in medicine to follow his passion for the emerging field of molecular biology; within six years, he had made the discovery of a lifetime. As a new Johns Hopkins faculty member, Smith, together with his first graduate student, Kent Wilcox, geared up to study recombination in vitro but instead discovered the restriction enzyme “R” in Haemophilus influenzae. By cobbling together crude techniques, Smith, along with Wilcox and later Tom Kelly, showed that R cleaves DNA at a specific recognition sequence, a palindromic site, yielding blunt-ended DNA fragments. Now known as HindII, R proved to be the first of an enormous class of Type II restriction enzymes, and as such, presaged gene cloning, allowed DNA to be reproducibly fragmented and then sequenced, and enabled physical mapping of genomes. Smith went on to discover DNA methylases that constitute the other half of the bacterial host restriction and modification systems, as hypothesized by Werner Arber of Switzerland. Together with Arber and his Hopkins colleague Daniel Nathans, who first used the enzyme on SV40 DNA and demonstrated discrete bands on a tube gel, Smith shared the Nobel Prize for Physiology or Medicine in 1978.

The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34950 Da); the MTase is predicted to be 309 aa (35550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible λ, pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.

Parameters of the polynucleotide kinase-catalyzed exchange reaction between [gamma-32P]ATP and 5'-phosphoryl DNAs have been measured with the termini generated by the following endonucleases: EcoRI (Berkner, K. L., and Folk, W. R. (1977) J. Biol. Chem. 252, 3176-3184), Hpa II, BamHI, and HindIII (external termini); HindII and Hpa I (blunt terminal); Hae II and Hha I (internal termini). In every case, exchange is reproducible and proportional to the number of termini. However, in most cases, the exchange reaction does not proceed to the theoretical maximum. External termini and single-stranded DNAs are labeled more rapidly and to approximately 5-fold higher levels than blunt or internal termini. Concentrations of 100 to 200 microns ADP and 12 microns ATP are optimal for labeling all types of termini with the exchange reaction.

It is shown here that distamycin A and actinomycin D can protect the recognition sites of endo R.EcoRI, EcoRII, HindII, HindIII, HpaI and HpaII from the attack of these restriction endonucleases. At proper distamycin concentrations only two endo R.EcoRI sites of phage lambda DNA are available for the restriction enzyme--sRI1 and sRI4. This phenomenon results in the appearance of larger DNA fragments comprising several consecutive fragments of endo R.EcoRI complete cleavage. The distamycin fragments isolated from the agarose gels can be subsequently cleaved by endo R.EcoRI with the yield of the fragments of complete digestion. We have compared the effect of distamycin A and actinomycin D on a number of restriction endonucleases having different nucleotide sequences in the recognition sites and established that antibiotic action depends on the nucleotide sequences of the recognition sites and their closest environment

The action of a number of restriction enzymes on DNA.RNA hybrids has been examined using hybrids synthesised with RNAs of cucumber mosaic virus as templates. The enzymes EcoRI, HindII, SalI, MspI, HhaI, AluI, TaqI and HaeIII cleaved the DNA strand of the hybrids (and possible also the RNA strand) into specific fragments. For four of these enzymes, HhaI, AluI, TaqI and HaeIII, comparison of the restriction fragments produced with the known sequences of the viral RNAs confirmed that they were recognising and cleaving the DNA strand of the hybrids at their correct recognition sequences. It is likely that the ability to utilise DNA.RNA hybrids as substrates is a general property of Type II restriction enzymes.

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.

DNA restriction fragments generally elute from RPC-5 in order of their size. However, some fragments elute later than predicted. Chromatographic studies were performed on five different restriction digests (Hae III, Hha I, Alu I, Taq I, and Hae III + HindII) of pRZ2 DNA in an effort to localize the regions which have the delayed properties. Also, the magnitude of the delay was quantitated in each case. Most of the delayed fragments were localized in one major (931 bp) and one minor (approximately 210 bp) region of the genome. The fragments exhibiting a greater extent of delay were in the major region. The results described herein and in the following paper show that, in most cases, this effect can be explained by the base composition, or sequence of the fragments, or both.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.

Variation in mitochondrial DNA (mtDNA) in a sample of 110 Finns was analyzed with six restriction enzymes, AvaII, BamHI, HaeII, HindII, HpaI, and MspI, by using total blood cell DNA probed with mouse mtDNA. Two new enzyme morphs were observed, one for HaeII and one for HindII. Double-digestion experiments indicated that the BamHI morphs 2 and 3 result from base changes leading to AvaII morphs 3 and 9, respectively. Of the ten different mtDNA types observed, defined by restriction fragment patterns, seven have been previously described in Caucasoid populations. The three new "Finnish" mtDNA types can be derived from Caucasoid lineages by single restriction site changes. The results were used to reconstruct a phylogenetic tree for Caucasoid mtDNA types defined by the enzymes used. The frequencies of mtDNA types were used to compute genetic distances between Finns, Italians, and Israeli Jews. The frequencies of both enzyme morphs and mtDNA types show that the Finnish population is highly homogeneous.

A 1.7 × 103 base-pair SalI fragment of mouse ribosomal gene spacer undergoes recA-independent deletions of DNA in units of approximately 126 base-pairs when cloned in λ or bacterial plasmids. When we examined the structure of the 1.7 × 103 base-pair piece with PvuII we found it to be composed of about equal numbers of copies of each of two subrepeating units, 120 and 130 base-pairs in size. The correlation between the size of the structural subunits and the functional genetic unit of this fragment as expressed in Escherichia coli led us to study the organization of these sequences in mice. SalI (or HindII) digests of DNA samples from wild and inbred strains revealed extensive heterogeneity in the size of fragments homologous to this 1.7 × 103 base-pair piece. A total of 15 different size classes were detected in our samples. We found that these fragments were also organized in PvuII repeating units about equal in size to the PvuII repeats in the cloned 1.7 × 103 base-pair piece. Using an objective analytical procedure (see the Appendix) we determined that the 15 different fragments found in our mouse DNA samples probably originated as a result of genetic events based on a 135 base-pair structural unit. We consider the similarity between the size of the PvuII structural unit and the unit of genetic behavior in both the cloned and uncloned DNA samples to be significant. We suspect that there are aspects of the nucleotide structure or organization of the PvuII repeating units that play a dominant role in its genetic behavior, regardless of whether these sequences are present in E. coli or mice. We believe that the clones containing this mouse sequence may provide an experimental system for studying the nature of the genetic events that are involved in multigene evolution.

1. Degradation of highly purified kinetoplast DNA (kDNA) networks with restriction endonucleases yields "extra" bands in agarose gels that are absent from digests of mini-circles. Each of the five endonucleases tested, i.e. AluI, HapII, EcoRI, Hsu and HindII + III, yields a unique set of "extra" bands. The "extra" bands consist of linear DNA; they are not mini-circle oligomers and their added molecular weight, calculated from mobility in gels, are around 2 X 10(7). Double digests with two restriction endonucleases yield a new set of "extra" bands, showing that the "extra" bands obtained with different enzymes are all derived from the same complex component of kDNA. In digests of 32P-labelled kDNA an average of 2.3% of the radioactivity is recovered in the "extra" bands. 2. Treatment of kDNA networks with the single-strand-specific S1 nuclease of Aspergillus oryzae preferentially releases a linear DNA with a molecular weight of 26 X 10(6), calculated from mobility in gels. We present evidence that the 'extra' bands obtained with restriction endonucleases are derived from this component. 3. DNA-DNA renaturation analysis of fragmented kDNA shows the presence of a minor complex component with a complexity of about 3 X 10(7), making up less than 10% of the total kDNA. 4. From these results we conclude that 3--5% of the kDNA consists of a homogeneous class of maxi-circles catenated in the mini-circle network. The molecular weight of these maxi-circles is about 26 X 10(6) and they contain a unique, non-repetitive, non-mini-circle nucleotide sequence. This component is a prime candidate for the true mitochondrial DNA of trypanosomes.

The HindII + III restriction enzyme fragmentation pattern of various lambda-phi80trp deoxyribonucleic acid molecules is presented. An analysis of deoxyribonucleic acid molecules carrying deletions ending within the trp regulatory elements and a deoxyribonucleic acid molecule carrying a deletion within trpE indicates that a fragment of 8.3 X 10(5) daltons contains at least part of the trp promoter, the entire trp leader region, and part of the trpE gene. The observation that ribonucleic acid polymerase, when present in the HindII + III digestion mixture, results in the fusion of this 8.3 X 10(5)-dalton fragment to the preceding bacterial fragment suggests that HindII + III cuts within trpP.

Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double-stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed.

The nucleotide sequence of the simian virus 40 (SV40) genome region between the cleavage sites for restriction endonucleases EcoRI (map position 0) and HindII (map position 0.05) has been determined mainly by the partial chemical DNA degradation procedure of Maxam and Gilbert. This fragment represents 5.3% of the genome of SV40 and is located in the late region, internally in the VP1 gene. The message strand shows only one open reading frame for translation into protein, which connects to the one for the preceding fragment. On this basis part of the amino acid sequence of the VP1 protein is presented.

The order of G4 Haemophilus influenzae Rd (HindII) DNA fragments was mapped by electron microscopy. G4 HindII DNA fragments were annealed to φX174 viral singles-tranded DNA circles and found to partially hybridize. Eight to eighteen percent of the φX174 DNA circles had a G4 DNA fragment attached, depending upon the fragment and the time of annealing. The G4 HindII restriction enzyme cleavage map was aligned with the φX174 restriction enzyme cleavage map by analyzing under the electron microscope partial heteroduplex molecules consisting of φX174 single-stranded viral DNA circles annealed with φX174 Arthrobacter luteus (AluI) DNA fragments as markers and G4 HindII DNA fragments. These results indicate that the gene order of the bacteriophages G4 and φX174 is the same. Despite the high frequency of φX174/G4 heteroduplex DNA molecules observed under the electron microscope, heterologous marker rescue experiments with G4 DNA fragments and φX174 mutant viral DNA strand circles were not successful, except in the case of a φX174 amber mutation in gene E (am3).

The origin for bacteriophage M13 single-strand synthesis has been localized by two different experimental approaches: (1) localization of the discontinuity in late life-cycle RFII ‡ , one of the initial product molecules during single-strand synthesis, and (2) determination of a gradient of radioactive label in RFI pulselabeled during single-strand synthesis. M13 RFII formed during single-strand synthesis contains a single discontinuity in the viral strand. Cleavage of this RFII species with the HindII restriction enzyme yields two discrete viral strand fragments that locate the discontinuity at a distance approximately 10% of the genome from the HindII restriction site. RFII molecules were also used as template for limited repair synthesis with Escherichia coli DNA polymerase I and 32P-labeled deoxynucleoside triphosphates. Upon treatment of these repaired RFII with HpaII restriction endonuclease and electrophoresis in an agarose gel, the discontinuity was localized in a specific restriction fragment, the F fragment. The existence of a gradient of pulse label in RFI isolated late in infection was determined by mixing [3H]thymidine pulselabeled RFI and uniformly-labeled [32P]RFI, treating this mixture with HpaII endonuclease, separating the fragments by electrophoresis in an agarose gel, and examining the ratio of pulse label to uniform label in the restriction fragments. The ratio varied with the position of the fragment on the cleavage map indicating the temporal order of synthesis. Both experimental approaches place the origin of M13 viral-strand synthesis within the HpaII fragment F. This fragment is approximately 400 base-pairs in length and is within an intergenic space between genes II and IV on the genetic map. This region was previously shown to contain the origin for complementary-strand synthesis in vitro.

Human papillomavirus (HPV) DNA form I (supercoiled) was prepared from plantar warts. HPV DNA was cleaved with restriction enzymes obtained from the following sources: escherichia coli (EcoRI), Hemophilus influenzae strain Rd (both unfractionated Hind and aeparated HindII and HindIII enzymes) and Hemophilus parainfluenzae (HpaI). The cleavage products were analyzed by polyacrylamide gradient slab gel electrophoresis and electron microscopy. HPV DNA was cleaved into two fragments by EcoRI (87% and 13% of the genome) and into six fragments, ranging in size from 33.5 to 1.2% of the genome, by Hind endonucleases. The six Hind fragments result from the cleavage of three sequences recognized by HindII, two of which are also cleaved by HpaI, and of three sequence recognized by HindIII. The order of these fragments was determined by comparing their size with that of the fragments obtained with HindII, HindIII, HpaI, and the mixture of HindIII + Hpal. The two EcoRI cleavage sites were located on two adjacent Hind fragments and one of these sites has been taken for the zero point to construct a physical map. The treatment of superhelical HPV DNA with bacteriophage T4 gene 32 protein yields circular structures with a denaturation loop. The cleavage of these complexes with EcoRI and HindIII has shown two easily denatured regions which were located on the cleavage map.

Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal DNA (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast rDNA genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.

Four mutants of polyoma virus lacking endonuclease HindII site 1 were isolated and characterized with respect to the VP1 coding sequence. Three of these mutants had deletions that removed 0.2 to 0.3% of the genome. All three deletion mutants encoded VP1 proteins that were smaller than wild type and that lacked one or more tryptic peptides normally found in the wild-type VP1 protein. Our results suggest the HindII site 1 is at, or very near, the carboxy terminal end of the coding sequence for VP1. A model for the peptide organization in that region is presented.

1) We have constructed independent physical maps of the mtDNAs from three different wild-type Saccharomyces strains by double-digestion analysis and hybridization analysis, using restriction endonucleases EcoRI, HindII, HindIII, PstI, BamHI, Aval, HhaI, SalI and XhoI. Twentynine restriction enzyme sites have been localized on the mtDNA of Saccharomyces carlsbergensis, 47 on the mtDNA of Saccharomyces cerevisiae strain JS1-3D and 38 on the mtDNA of Saccharomyces cerevisiae strain KL14-4A. 2) Although the three DNAs show considerable differences in their fragmentation patterns with most nucleases tested, the overall sequence organization of the three maps of 30–40 fragments is identical. Differences in the maps can be explained by extra restriction enzyme recognition sites, possibly located on inserted pieces of DNA and by insertions and deletions. 3) Four major insertions (900, 1,500, 2,600 and 3,000 bp long) are found in KL14-4A mtDNA relative to Saccharomyces carlsbergensis mtDNA. These insertions are clustered in one quadrant of the mtDNA and account for the difference in size of these two mtDNAs (75,750 and 68,000 bp, respectively).

1 We have used restriction enzyme analysis of petite mtDNAs to construct a detailed physical map of the 21S region on the mtDNA of the Saccharomyces cerevisiae strain JS1-3D. The map covers a segment of about 20,000 bp, on which the recognition sites of the enzymes HapII, HindII, HindIII, SalI, XhoI and HhaI have been localized (22 sites in total). This map has been checked in various ways against the independently constructed overall physical map of the mtDNA of strain JS1-3D. In addition, we have constructed a physical map with a resolution of about 200 bp of a HapII fragment of 1850 bp long, which carries the loci ω, RIB-1 and probably RIB-2. 2. The 21S rRNA hybridizes with the five adjacent HindII+III fragments TD9, DT19, TD15, DT14 and TT1, which lie in that order on the physical map of the 21S region. Of these, the two non-adjacent fragments TD9 and DT14 show a much stronger hybridization with 21S rRNA than DT19, TD15, and TT1. 3. The fragment DD5 (=DT19+TD15) and part of DT14 belong to a sequence of about 1000 bp, which is absent from Saccharomyces carlsbergensis mtDNA. Although DD5 and DT14 show (very weak, respectively stronger) hybridization with 21S rRNA, the 1000 bp insert probably does not code for the 21S rRNA: the 21S rRNA of S. carlsbergensis comigrates with the 21S rRNA of JS1-3D on polyacrylamide gels under denaturing conditions. 4. Fragment DT14 hybridizes with the HindII +III fragment TD9, which shows the strongest hybridization with 21S rRNA. The presence of these sequence homologies has hampered the precise mapping of the 21S rRNA cistron. Our results are compatible, however, with the hypothesis that the sequences, coding for 21S rRNA, are located on HindII+III fragments that are not adjacent on JS1-3D mtDNA, namely TD9, DT14 and TT1.

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.

The physical structure of the genome of feline herpesvirus-1, a major upper respiratory tract pathogen of cats, was studied. Purified FHV-1 DNA was analyzed by restriction endonuclease and gamma 5' exonuclease digestion, blot hybridization, and electron microscopy. To facilitate further studies, nine bacteriophage clones were isolated which contained 85% of the viral genome as SalI inserts, and DNA from these clones was used in blot hybridization experiments and as substrates for restriction digest analysis. Data from these studies permitted construction of a SalI and partial HindII and EcoRI restriction maps of the viral genome. FHV-1 DNA is approximately 134 kb in size and is composed of a long (L) and a short (S) segment. The long segment (U1) is 104 kb in size and is composed of unique DNA. The adjacent S segment is approximately 30 kb in size and contains a central portion of unique DNA (Us) which is approximately 8 kb in size. The Us region is bounded by inverted repeat sequences which are 11 kb in size. Therefore, the physical structure of the FHV-1 genome is similar to the genomes of other alpha-herpesviruses.

DNA synthesized in vitro using deoxynucleoside phosphorothioates as substrates is quite similar to normal DNA in its biochemical properties (Vosberg, H.P., and Eckstein, F. (1977) Biochemistry 16, 3633-3640). In order to investigate the effect of phosphorothioate groups in DNA on the cleavage pattern of restriction endonucleases phosphorothioate double-stranded, circular, replicative form of fd DNA was synthesized in vitro with Escherichia coli DNA polymerase I using native single-stranded DNA as template and mixtures of three normal nucleotides and one nucleoside phosphorothioate analogue as substrates. The double-stranded products were hybrids with respect to their phosphorothioate content. Restriction analysis of normal and phosphorothioate DNA with the restriction endonucleases Hae III, Bam HI, Hpa II, HindII, Alu I, and Taq I showed that the enzymes were inhibited to different degrees depending on which of the nucleotides was replaced by the phosphorothioate. Most significant, inhibition was seen throughout with those DNAs which contained a phosphorothioate exactly at the cleavage site. Phosphorothioate substitutions at other positions, but still within the recognition sequences, were, except for Alu I, not or weakly inhibitory. Phosphorothioate nucleotides not present in the recognition sequences did not affect at all the fragment patterns. The results show that recognition sequences of restriction endonucleases can be selectively protected against cleavage by base-specific introduction of phosphorothioate groups into DNA.

Synthetic hexadecanucleotide duplexes containing a single purine nucleotide analogue in the recognition sites of the restriction endonucleases HindII, SalI and TaqI were used to investigate the restriction site determinants required by these enzymes for sequence recognition and phosphodiester bond cleavage. The enzymes were, in general, unaffected by changes introduced into the minor groove of the helix. SalI was found to be inhibited by the major groove modifications introduced into the fourth position of its recognition sequence GTCGAC. HindII and TaqI were, by contrast, able to cleave the sites containing the analogues at this position. TaqI and, to a lesser extent, HindII could also be shown to tolerate "mismatch analogues" at this site.

A physical map of the group A streptococcal bacteriophage A25 genome was constructed using data obtained from restriction enzyme digestion analysis of purified DNA. Phage A25 was 34.6 kb in size and was digested with several enzymes which generated the following number of fragments: AvaI (3), EcoRI (2), HindIII (4), HaeII (10), HindII (22), HpaI (11), and PvuII (8). Restriction enzymes BamH1, PstI, and SaI did not cleave phage A25 DNA. A physical map of the group C streptococcal phage C1 genome was also constructed. Phage C1 was 16.9 kb in size and was cleaved by the following enzymes to give the indicated number of fragments: EcoRI (8), HaeII (10), HaeII (4), HaeII (10), HindIII (5), HpaI (8), PvuII (3), and SalI (2). Restriction enzymes AvaI, AvaII, BamH1, EcoRV, HpaII, and PstI did not cleave phage C1 DNA.

the cleavages produced in S13 by the restriction enzymes HindII, HaeIII, HpaI, HpaII, Pstl, AvaI, MboI, AluI, HphI, HhaI, HaeII, MboII, XhoI, and Hinf have been compared with those produced in φX174. Some restriction enzymes produce, from φX174 and S13, very similar sized fragments while others produce totally dissimilar sized fragments. S13 have several new cleavage sites that are not present in φX174. For approximately every five cleavage sites that φX174 and S13 have in common, S13 has gained one extra cleavage site.

A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L. M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA). M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT. Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.

Earlier papers from our laboratory reported the nucleotide sequence of the beginning of the simian virus 40 HindII + III fragment D (Hind-D, which contains the information for the presumed initiation codon for the VP2 protein), HindII + III fragment E (HindII + III fragment K (Hind-K, which contains the information for the termination signal for the VP2 and VP3 proteins and the initiation signal for the VP1 protein). We report here the nucleotide sequence of the remainder of fragment Hind-D as determined by the partial, chemical degradation procedure of Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. (1977) 74, 560–564]. As the aforementioned restriction fragments cover the total of the overlapping VP2 and VP3 genes, the entire nucleotide sequence of these genes, as well as the derived amino acid sequence of the VP2 and VP3 proteins, is now known. Assuming that eukaryotic protein synthesis always starts with an AUG codon (and not with a GUG) and considering various evidence about the position of the VP3 gene within the VP2 gene, we conclude that the second AUG codon in phase coded by fragment Hind-D serves as the initiation codon for the translation of VP3. The first part of VP2 (98 residues), which is not present in VP3, is very hydrophobic and contains no cysteine or basic amino acids; possibly it serves for binding to, or penetrating through, membranes. The very basic carboxyl terminus of both VP2 and VP3 may possibly be involved in attachment to the DNA.

We have analysed limit digests of mini-circles from kinetoplast DNA of Crithidia luciliae by gel electrophoresis. Endonucleases HapII and AluI cut the circles into at least 37 and 21 fragments, respectively, and leave no circles intact. In both cases the added molecular weights of the fragments, estimated from mobility in gels, exceeds 18 × 106, i.e. more than 12 times the molecular weight of the mini-circle DNA. Endonucleases HindII + III, EcoRI and HpaI cut only part of the circles. These results show that the mini-circles are heterogeneous in base sequence. Different sequence classes are present in different amounts. DNA-DNA renaturation analysis of mini-circle DNA yields a complexity of about 3 × 106, i.e. twice the molecular weight of one mini-circle. The Δtm of native and renatured duplexes is about 1 °C, showing that the sequence heterogeneity is a micro-heterogeneity. Electron microscopy, gel electrophoresis and sedimentation analysis show that the circles that are not cut by endonucleases HindII + III remain catenated in very large associations. These associations lack the ‘rosette’ structures and the long edge loops characteristic of intact kinetoplast DNA. This suggests that the mini-circle classes cut by endonucleases HindII + III are present throughout the network and that the maxi-circle component of the network (see accompanying paper) is not essential to hold the network together. Prolonged electrophoresis on 1.5% or 2% agarose gels resolves the open mini-circles into three and linearized mini-circles into four bands, present in different amounts. We conclude that the minicircles are also heterogeneous in size, the difference in size between the two extreme size classes being 4% of the contour length. Digestion with endonuclease HapII shows that at least three out of these four bands differ in sequence. Possible mechanisms that could account for the micro-heterogeneity in sequence of minicircles are discussed.

A physical map of the COB region in mtDNA of yeast has been established. This region harbours a split gene coding for apocytochrome b. It includes restriction sites of seven endonucleases (EcoRI, HindII, HindIII, HaeIII, HpaII, AluI and BamHI). Various mtDNA sequences of this region, retained in a series of genetically characterized rho(-) clones have been allocated to this map. The combination of this physical map with a genetic map of the rho(-) clones revealed that 1) cob(-) mutational sites spread over 8,400 bp, 2) mutations in sequences coding for apocytochrome b map in five distinct segments which are separated by intervening sequences with minimum lengths from 350 to 1,900 bp.