Restriction Endonucleases Bam Research Articles

Differentiation of the Vaccine F-strain from other strains of Mycoplasma Gallisepticum by restriction endonuclease analysis

The electrophoretic patterns of the nucleic acids (DNA) of Mycoplasma gallisepticum strains digested with the restriction enzymes Bam HI, Eco RI and Hind III were useful for differentiating the vaccine F-strain from other strains of M. gallisepticum. The procedure was more sensitive than the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. The vaccine F-strain, represented by cultures designated F-K810 and F-F2F10 was clearly differentiated from other strains of M. gallisepticum. This procedure may be useful in field studies to determine if the vaccine strain will replace wild-type M. gallisepticum in commercial layers.

Chloroplast Genome Mapping and Plastid Ulrastructure Analysis of Chlorophyll Deficient Mutants of Alfalfa

Physical maps of chloroplast genomes allow comparison of genome structure and organization and can provide physical markers useful for evaluating plastid inheritance. Physical maps of the chloroplast DNA (ctDNA) from phenotypically normal alfalfa (Medicago sativa L.) genotypes and two independently isolated cytoplasmically and biparentally inherited chlorophyll‐deficient mutants were developed and compared. Both mutants were observed as somatic sectors but each had a different phenotype, one producing albino and the other yellow‐green tissue. Maps of overlapping restriction fragments were constructed using both purified chloroplast DNA and total DNA digested with the restriction endonucleases Bam HI, Xho I, and Hind III and utilizing a complete set of chloroplast probes. A total of four points of variation, two changes in restriction endonuclease recognition sites and two small addition/deletion events were observed among the genotypes studied. Combinations of these polymorphisms defined four distinct ctDNA genotypes. Only one addition/deletion polymorphism was unique to the mutant chloroplast DNA (ctDNAs) and the albino and yellow‐green chloroplast genomes were indistinguishable from each other by this analysis. Electron microscopy revealed differences in development between the two chlorophyll‐deficient mutants. Plastids from albino tissue showed envelope degradation, no internal membrane development, sparse stroma with vacuoles, and osmiophyllic globuli. Plastids from yellow‐green tissue showed limited thylakoid membrane development, no grana, relatively dense stroma with ribosome‐like particles, osmiophyllic globules, and various degrees of vacuolization. This study identified molecular markers with which to assess plastid inheritance in alfalfa and demonstrated that the dramatic arrest in plastid development found in both mutants was not paralleled by large changes in their chloroplast genomes.

Class II human leukocyte antigen genes and T cell receptor polymorphisms in patients with rheumatoid arthritis

Human leukocyte antigen (HLA) typing was performed in 174 patients with rheumatoid arthritis and 222 white control subjects. Increases in HLA-DR4 and HLA-DR1 were observed as in previous studies. Each of these appeared to be inherited as dominant risk factors. Southern blotting with a DR-beta probe after digestion of genomic DNA with the restriction enzyme Bam HI showed seven bands. Three of them correlated with DR4 and were increased in rheumatoid arthritis patients. Subsets of DR4 were determined by polymerase chain reaction amplification followed by hybridization with oligonucleotide probes. Dw4 was increased in rheumatoid arthritis patients, and the frequency of the other subsets appeared to be similar in rheumatoid arthritis patients and control subjects. A polymorphism associated with the T cell receptor V-beta-8 gene family was significantly increased in rheumatoid arthritis patients.

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining.

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.

Definition of the RFLP alleles in the human immunoglobulin IGHG gene locus.

In order to define more precisely the polymorphism of the human immunoglobulin IGHG (C gamma) genes and, consequently, to understand the structure and evolution of this multigene family, we have investigated the Restriction Fragment Length Polymorphisms (RFLP) of the IGHG genes in 113 unrelated individuals and 18 families. Using the restriction enzymes Bam HI, Sac I and Eco RI, and hybridization to a IGHG probe and to a specific IGHG3 (C gamma 3) probe, we describe 47 different restriction fragments (RF) in the IGHG locus, allowing us to define at least 15 RFLP alleles for the different IGHG genes. Our data demonstrate that the restriction fragment length polymorphism is occasionally due to the presence or absence of a given restriction site as a consequence of a point mutation, such as the creation of an Eco RI site, at the codon 3 of exon 2 in one IGHG4 allele (IGHG4*D2). However, most of the RFLP we observed seem to result from the insertion or deletion of a few hundred base pairs as illustrated by the IGHG3 polymorphism.

Isolation and characterization of a variant of human papillomavirus type 11 from a nasal inverting (Schneiderian) papilloma.

We have previously reported the presence of a variant of human papillomavirus (HPV) type 11 in a nasal inverting papilloma [Respler et al., 1987]. In the present study, we have cloned molecularly the DNA of this variant at its unique restriction enzyme Bam HI site into lambda BF101 phage. Restriction enzyme mapping and DNA sequencing revealed that the genome of this virus contained an extra 531 base pair (bp) which was the repeat of most of the noncoding region (ncr) of HPV 11. Insertion of transcriptional control elements, including the repeated sequence, in front of the chloramphenicol acetyltransferase (CAT) gene resulted in a 5- to 30-fold increase in expression in transfected cells, as compared to the constructs containing a single ncr of HPV 11. This increased expression was due to enhanced levels of CAT RNA the synthesis of which is initiated by the viral promoter element.

Plasmids of Yersinia enterocolitica and Yersinia pseudotuberculosis: Analysis with restriction endonucleases

The plasmids of Yersinia enterocolitica and Yersinia pseudotuberculosis tested differed in regard to the following aspect: there were some of a molecular size of 41 to 47 Mdal which could be correlated with autoagglutination of the host bacteria, and the others with a smaller size of 19 to 34 Mdal that lacked the capacity to code for autoagglutination. Analysis of both types of plasmids with the restriction endonucleases Bam HI, Eco RI and Hind III besides of some similarities revealed also considerable serogroup-specific differences in their DNA fragmentation pattern.

Isolation of canine adenovirus-2 from the faeces of dogs with enteric disease and its unambiguous typing by restriction endonuclease mapping

Fifty-four faecal specimens obtained from kennelled dogs with diarrhoeal disease were used to inoculate a range of cell types in tissue culture. Particles resembling adenovirus virions were seen in three specimens and 22 stools yielded an adenovirus upon culture. Viral DNA from each isolate was digested with the restriction endonucleases Bam H1 and Pst 1. Agarose gel electrophoresis revealed identical restriction patterns for all isolates. One isolate, 9228, was selected as a prototype and was compared with reference strains of canine adenovirus-2 ( cav-2) (Manhattan and Toronto A26/61) and cav-1. Isolate 9228 was clearly distinct from cav-1 but identical to both Manhattan and Toronto (A26/61) strains of cav-2. However, restriction site polymorphism was observed in 9228 following digestion with Hpa II. Isolate 9228 was similarly compared with all commercially available vaccinal isolates of cav-2 and was shown to be clearly distinct from these.

Physical analysis of PB2, a temperate bacteriophage of Agrobacterium tumefaciens.

We have constructed a detailed physical map of PB2, a temperate bacteriophage of Agrobacterium tumefaciens. The restriction endonucleases Bam HI (3 sites), Eco RI (22 sites), Hind III (19 sites), Hpa I (5 sites), Xba I (1 site), and Xho I (1 site) were used to elucidate the map of infectious PB2 DNA. The map was generated by reciprocal sequential digestions and analysis of partial digestion products of isolated restriction fragments. We have determined the genome size as 66.35 +/- 1.71 kb. The potential of PB2 as a phage-based shuttle vector for the A. tumefaciens-plant transformation system is discussed.

Changes in Restriction Enzyme Pattern of the Equine Herpes Virus Type 1 (EHV‐1) Strain Rac H DNA During Attenuation

SummaryEquine herpesvirus type 1 (EHV‐1) strain Rac H is used after attenuation (256 passages in embryonic porcine kidney cells) as a live vaccine against rhinopneumonitis and abortion. The genome of this strain shows a different DNA pattern after digestion with the restriction endonuclease Bam HI from that of the abortigenic virus isolates. A virus subpopulation revealing this pattern existed already at passage 182. This pattern is likely to serve as an in vitro marker for differentiation of field virus from the vaccine strain.ZusammenfassungÄnderungen im Restriktionsenzymschnittmuster der DNS des equinen Herpesvirus Typ 1 (EHV‐1) Stammes Rac H während der AttenuierungDer durch 256 Passagen auf embryonalen Schweinenierenzellen attenuierte Stamm Rac H wird als Lebendvakzine gegen Rhinopneumonitis/Stutenabort (EHV‐1) eingesetzt. Das Genom dieses Stammes wies nach Verdau mit der Restriktions‐Endonuclease Bam HI ein von Abortisolaten deutlich unterscheidbares DNS‐Schnittmuster auf. Es wird dargestellt, daß bereits zum Zeitpunkt der 182. Passage eine Subpopulation mit diesem Schnittmuster existierte.Dieses Muster ist im Sinne eines “in vitro markers” zur Unterscheidung von Impfstamm und Feldvirusisolaten geeignet.

Haplotypes of alpha-globin-gene in the Saudi population--the triplicated gene (alpha alpha alpha anti 3.7/).

Using restriction endonucleases Bam HI, Bg1 II, Hpa I, Hind III and Xba I, the alpha alpha alpha anti 3.7/ haplotype was studied in the Saudi population. This paper presents the frequency of the alpha gene haplotype alpha alpha alpha anti 3.7/ in different regions of Saudi Arabia, where thalassaemia occurs at a high frequency.

Leftward deletion ?-thalassaemia in the Saudi Arabian population

Restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of the deletion type of alpha-thalassaemia in the Saudi population. Four homozygous cases and six heterozygous cases of the leftward deletion type of alpha-thalassaemia (-alpha) were identified. So far, the leftward type of alpha-thalassaemia has been identified mainly in the Asiatic population, while the rightward deletion is universally distributed. This paper reports for the first time the presence of leftward deletion in the Saudi population and discusses the possibility of a more universal distribution of leftward deletion.

Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells

Treatment of Chinese hamster ovary (CHO) cells with the restriction endonuclease Bam H I (recognition site: G/GATCC) leads to high frequencies of chromosomal aberrations. Experiments with bromodeoxyuridine-labelled chromosomes show that the aberrations occur nearly exclusively in first post-treatment metaphases. The results are interpreted to mean that only some of the cells take up the enzyme and that these cells are the ones showing the aberrations. Cells which do not take up the enzyme show up as differentially stained metaphases and have no aberrations. Why some cells take up the restriction enzyme and others not is not known, possibly this is dependent on the physiological condition of the cells.

Effect of glutaraldehyde on the activity of some DNA restriction endonucleases.

The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI, Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above. Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed.

Cloning of pEA3, a large plasmid ofEnterobacter agglomerans containing nitrogenase structural genes

A clone bank of an indigenous plasmid ofEnterobacter agglomerans containing structural nitrogen-fixation (nif) genes was established in a non-mobilisable, multicopy derivative of the cosmid vector pHC79. The restriction enzyme Bam HI was used to establish the clone bank and it was found that 96% of the clones contained inserts. The clones containingnif-genes were identified by Southern hybridisation usingKlebsiella pneumoniae nif DNA (KpnifHDKY) as the radioactive probe. Thenif-genes ofE. agglomerans showed extensive homology to those ofK. pneumoniae but the restriction enzyme fragment patterns of thenif-genes ofE. agglomerans were different. The plasmid bornenif-genes ofE. agglomerans are clustered as inK. pneumoniae.

Recognition schemes for protein-nucleic acid interactions

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d-GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a3E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.

Restriction Endonuclease Map of pTi Bo542, a Potential Ti Plasmid Vector for Genetic Engineering of Plants

We report here a physical map of pTi Bo542, a wide-host-range tumor-inducing plasmid of Agrobacterium tumefaciens. Forty-two fragments generated by the restriction endonuclease Bam HI were arranged in a circular array using electrophoretic analysis of clones containing overlapping segments of pTi Bo542. The T-DNA region (the part of the Ti plasmid transferred to the plant) was predicted by its homology to T-DNAs from heterologous octopine and nopaline Ti plasmids. Southern hybridizations against soybean tumor DNA using clones from this region as probes confirmed this prediction.

Fragment comparison of hamster mitochondrial DNA: General conclusions about the evolution of mitochondrial DNA

1. 1. Mitochondrial DNA from heart, liver and kidney of two hamster species, Mesocricetus auratus and Cricetulus griseus has been digested with the restriction endonucleases Bam HI, Bgl I, Eco RI, Hae III, Hind III, Hpa II and Xba I. 2. 2. Cleavage patterns for Hpa II are identical for both species, while only two of seven bands are common with Hae III. All other endonucleases give a species-specific cleavage pattern. 3. 3. The results suggest a fairly high phylogenetic differentiation of the mitochondrial genome between the two hamster species. 4. 4. The large differences in mitochondrial DNA variability between different species is discussed as a function of mutation rate and species-specific generation time.

Changes in mitochondrial DNA during aging

Mitochondrial DNA (mtDNA) was isolated from liver mitochondria of rats between 2 and 24 months of age. The mtDNA was purified by cesium chloride—ethidium bromide isopycnic density gradient centrifugation. In the gradients, in addition to the two expected bands of ethidium-DNA complex, there was observed a third, more dense band ( d = 1.69 g cm 3 ). This novel band, rarely observed in preparations from younger animals, was present in most preparations from older animals. The latter was characterized using the diphenylamine assay(s) and ascertained to contain DNA and carbohydrate components. Agarose gel electrophoresis revealed the DNA of the novel band to have a migration identical to form I mtDNA. Digestion of the novel band with the restriction endonuclease Bam HI yielded products identical to those obtained upon treatment of form I mtDNA with Bam HI. The observation of mtDNA at a density of 1.69 g cm 3 indicates the presence, predominantly in older animals, of a subclass of mtDNA molecules with altered ethidium binding properties. The significance of this mtDNA and its position in the gradient is unclear at this time.

Structural analysis of rodent growth hormone genes: application to genetic forms of hypopituitarism.

Analysis of GH gene structure in the mouse permits evolutionary comparisons with GH gene structure in the rat and provides information about the mechanism responsible for heritable deficiencies of anterior pituitary hormones. The little mutation in mice is analogous to autosomal recessive, isolated, partial deficiency of GH in man, whereas the Snell dwarf mutation is a model for autosomal recessive deficiency of GH, TSH, and PRL. Mouse cellular DNA was digested with the restriction enzymes Eco RI, Bam HI, Bgl II, Hind III, and Kpn I, singly and in combination. Gene sequences containing coding information for GH were detected by hybridization to a radiolabeled recombinant DNA probe complementary to a rat GH mRNA. The results of genomic restriction analysis indicate that there is a single type of mouse GH gene sequence per haploid genome with a length equal to or less than 32000 base pairs. The distances separating 6-base restriction sites close to the mouse GH gene differ from those close to the rat GH gene. A Kpn I site in the codons for amino acids 103 and 104 of rat GH is absent in the mouse gene. Restriction patterns of DNA from little and Snell dwarf mice did not differ from those of normal mice, indicating that these mutations do not involve deletions of the mouse GH gene.