Differences In Restriction Patterns Research Articles

62-kb Plasmids Harboring rulAB Homologues Confer UV-tolerance and Epiphytic Fitness to Pseudomonas syringae pv. syringae Mango Isolates

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.

Cleaver: software for identifying taxon specific restriction endonuclease recognition sites

Cleaver is an application for identifying restriction endonuclease recognition sites that occur in some taxa but not in others. Differences in DNA fragment restriction patterns among taxa are the basis for many diagnostic assays for taxonomic identification and are used in procedures for removing the DNA of some taxa from pools of DNA from mixed sources. Cleaver analyses restriction digestion of groups of orthologous DNA sequences simultaneously to allow identification of differences in restriction pattern among the fragments derived from different taxa. Cleaver is freely available without registration from its website (http://cleaver.sourceforge.net/) and can be copied, modified and re-distributed under the terms of the GNU general public licence version2 (http://www.gnu.org/licences/gpl). The program can be run as a script for computers that have Python 2.3 and necessary extra modules installed. This allows it to run on Gnu/Linux, Unix, MacOSX and Windows platforms. Stand-alone executable versions for Windows and MacOSX operating systems are available.

Detection of mitochondrial DNA restriction site differences between the subspecies of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae: Meliponini)

The endemic range of the stingless bee Melipona quadrifasciata Lepeletier extends from the southern most state in Brazil, Rio Grande do Sul, up to the state of Paraíba, where it originally inhabited the Atlantic Rain Forest. Two subspecies have been morphologically recognized based on the tergal band pattern (yellow stripes across the abdomen), M. q. quadrifasciata and M. q. anthidioides. The objective of the present study was to characterize restriction site variation in the mitochondrial DNA of each subspecies. Four colonies of each subspecies were sampled. One individual per colony was used for DNA extraction and further analysis through amplification of nine fragments of the mitochondrial genome and digestion with 13 restriction enzymes (PCR+RFLP). Three enzymes showed restriction pattern differences between the subspecies. Those differences will be useful to determine the maternal origin of hybrids colonies, which may occur naturally or due to colony transportation among breeders.

Characterization of consecutive Streptococcus pyogenes isolates from patients with pharyngitis and bacteriological treatment failure: special reference to prtF1 and sic / drs.

To analyze bacteriological treatment failure in streptococcal pharyngitis, 40 consecutive Streptococcus pyogenes isolates from 18 patients were characterized. For 17 patients, isolates were indistinguishable with respect to emm type, random amplified polymorphic DNA pattern, and presence of prtF1 encoding the fibronectin-binding protein F1. prtF1 was detected only in the 11 isolates (4 patients) with emm12 and in the single isolate with emm6. Further analysis by vir(mga) regulon typing, sequencing of sic encoding the streptococcal inhibitor of complement from 19 isolates with emm1 (9 patients), and sequencing of drs (distantly related sic) from 11 isolates with emm12 revealed distinct sic alleles with insertions and/or deletions in sic that corresponded to differences in restriction patterns of the vir(mga) regulon only for paired isolates of 2 patients. Among isolates with emm12, 2 novel drs alleles were found. Analysis of these data suggests that neither the presence of prtF1 nor the diversification of sic / drs is required for the persistence of S. pyogenes in pharyngitis.

Recent and rapid amplification of the sperm basic nuclear protein genes in winter flounder

The high molecular weight basic nuclear proteins (HMrBNPs), which are tightly bound to sperm chromatin in winter flounder, are made up of imperfect reiterations of simple peptide sequences that contain phosphorylatable DNA-binding motifs. Genomic Southern blots hybridized with probes to the coding and non-coding regions of HMrBNP mRNA showed that HMrBNP sequences form a complex multi-gene family. Previously, one gene (2B) was used to establish an evolutionary link between histone H1 and the HMrBNPs. Further examination of this complex, multi-gene family has now revealed that the majority of the HMrBNP genes are linked as 4.5 kb direct tandem repeats that each contain a 2.8 kb coding region and a 1.7 kb intergenic region (IR). These findings, combined with the cloning of the IR, established that the tandemly repeated genes lack introns and code for the abundant 3 kb HMrBNP mRNAs that produce the prominent 110 kDa HMrBNP. Southern blotting of DNAs from other righteye flounder species showed that HMrBNP multi-gene families were present in closely related species, though with substantial differences in restriction patterns and band intensities, but were not detected in more distantly related flounders. These observations are consistent with recent and rapid elaboration of the HMrBNP gene family.

Replication of equid herpesvirus 4 in endothelial cells and synovia of a field case of viral pneumonia and synovitis in a foal

Equid herpesvirus 4 (EHV-4) infection was diagnosed as the cause of interstitial pneumonia in a 6-week-old conventionally reared Welsh pony foal, by cocultivation and immunolabelling with specific monoclonal antibodies, EHV-4 specific amplification of viral DNA, and immunohistological examination of infected tissues. The case was novel in that replication of the EHV-4 isolate in endothelial cells and in the synovial epithelium was a feature. Restriction digests of this isolate were compared with those of seven respiratory and one abortigenic EHV-4 isolate, and no differences in restriction pattern were evident. The implications of these findings for the pathogenesis of EHV-4 infection are discussed.

Ribosomal DNA internal transcribed spacer analysis supports synonomy of Scedosporium inflatum and Lomentospora prolificans

Scedosporium inflatum is a dematiaceous opportunistic pathogen originally described by D. Malloch and I.F. Salkin (Mycotaxon 21:247-255, 1984). However, E. Gueho and G. S. De Hoog (J. Mycol. Med. 118:3-9, 1991) recently suggested reducing this mold to synonomy with Lomentospora prolificans on the basis of their similar morphological and molecular characteristics. We have investigated the ribosomal DNA internal transcribed spacers (ITS), i.e., ITS I and ITS II, of 18 isolates, including these two fungi and a closely related pathogen, Scedosporium apiospermum, and its telemorph, Pseudallescheria boydii. Identical ITS restriction fragment length polymorphisms were found in eight isolates of S. inflatum and L. prolificans. These results support Gueho and De Hoog's proposal to combine S. inflatum and L. prolificans into the binomial Scedosporium prolificans. However, the ITS I sequence of S. apiospermum and the ITS restriction fragment length polymorphisms of S. apiospermum and P. boydii were found to be significantly different from those of S. inflatum and L. prolificans. The ITS restriction pattern differences may be valuable in clinical settings for distinguishing these fungi.

Human Papillomavirus DNA in Distant Metastases of Cervical Cancer

Eighteen distant metastases from cervical cancer to the extra-pelvic abdomen, extraabdominal lymph nodes, vulva, suburethral region, skin, and breast in 17 patients were analyzed by Southern blot hybridization under nonstringent and stringent conditions for the prevalence of human papillomavirus (HPV) type 11, 16, 18, 31, 33, and 35 DNA. Fourteen metastases in thirteen patients were HPV-positive. Thirteen tumors contained HPV-16 and one HPV-related sequences with varying copy number. In 9 of 11 cases, where the corresponding primary tumor could be studied, HPV positivity and type were identical. Two HPV-negative primary lesions had HPV-positive metastases; in three cases differences in restriction pattern or copy number were revealed. The HPV status showed no clear association with age of the patient, latency period between primary tumor and metastasis, histological findings, therapy, and clinical course of the disease after metastasis. The rather conserved presence of HPV DNA in distant metastases of cervical carcinoma underlines the importance of these viruses also for the maintenance of the malignant state.

Antigenic shifts in serotype determinants of Campylobacter coli are accompanied by changes in the chromosomal DNA restriction endonuclease digestion pattern.

Changes in somatic (O) lipopolysaccharide (LPS) antigenic specificities of Campylobacter coli serostrains were observed after continuous laboratory subculture. Two serostrains (C. coli O34 and C. coli O48) lost O specificity and did not react with homologous or any of the available heterologous antisera. The C. coli serostrain for serogroup O5, after subculture, yielded a variant that had acquired a new specificity which was detectable with a heterologous antiserum. In a repeat experiment with the original isolate of the O5 strain, a second variant was obtained which had not only acquired the same new determinant but had, unlike the first variant, lost reactivity with the homologous antiserum. Immunoblot experiments with homologous and heterologous antisera indicated that changes in antigenic specificity were associated with the O side chains of the LPS molecules. Results of restriction endonuclease analysis of chromosomal DNA of the variants and their parents revealed minor differences in restriction patterns which suggested that C. coli is capable of undergoing genomic re-arrangements that lead to changes in LPS specificity and structure.

Several groups among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction

Eight human herpesvirus 6 (HHV-6) strains were studied by Southern blot and polymerase chain reaction. DNA from infected cells was digested by a panel of restriction enzymes and hybridized with cloned BamHI fragments corresponding to about 30% of the HHV-6 strain SIE genome. In parallel, this DNA was amplified by polymerase chain reaction using pairs of primers derived from the strain SIE nucleotide sequence. Subsequently, amplification products were analyzed by hybridization, digestion with restriction endonucleases, and partial nucleotide sequencing. Overall results indicated that all strains were closely related to one another. However, concordant differences in restriction patterns allowed at least two groups to be distinguished, typified by strains SIE and HST, respectively. Differences between the two groups were found to reflect a limited number of punctual changes in nucleotide sequences. These results strengthen the idea of a unique HHV-6 species with genetic polymorphism. In addition, this study provides useful markers for the diagnosis and molecular epidemiology of HHV-6 infections.

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.

Restriction endonuclease analysis of varicella-zoster virus DNAs.

The DNAs of 14 strains of varicella-zoster virus (VZV) isolated from epidemiologically unrelated individuals were analyzed by restriction endonuclease cleavage and agarose gel electrophoresis. Small but distinct differences in restriction patterns between the isolates were observed when the DNAs were digested with each of four enzymes examined (EcoRI, SalI, PstI, and HindIII). Six out of 14 isolates (43%) were lacking the PstI cleavage site between K and N fragments; one isolate was lacking the HindIII cleavage site between M and N fragments. No changes were observed in restriction patterns in DNA of one of the strains after 33 passages in cultured cells.

Chloroplast DNA polymorphism in fertile and male-sterile cytoplasms of sorghum (Sorghum bicolor (L.) Moench)

Restriction endonuclease patterns of chloroplast DNA (cpDNA) were consistently distinguishable between fertile and male-sterile cytoplasms of sorghum [Sorghum bicolor (L.) Moench], whereas no differences in restriction patterns of cpDNA among male-sterile (A1) lines, including six isocytoplasmic strains, were revealed in this study. It is suggested that chloroplast DNA may contribute to the male sterility of A1 lines used currently in hybrid sorghum production.

Toxin production by strains of Pseudomonas syringae pv. tagetis

Strains of Pseudomonas syringae pv. tagetis were divided into three classes based on their capability to produce tagetitoxin: class 1 strains produce tagetitoxin in plants, but in-artificial media such small amounts are produced that normally none can be detected; class 2 strains however produce tagetitoxin in easily detectable amounts in vitro, as well as in plants; and class 3 strains do not produce the toxin. Class 1 and 2 strains from various hosts induced necrotic lesions and chlorosis of essentially equivalent severity in marigold; however mucoid isolates, which are class 2, produced increased amounts of tagetitoxin in vitro and caused earlier and more severe chlorosis. Class 3 strains were less virulent. When the DNA from 18 diverse strains representing all three classes were treated with each of three restriction endonucleases, no differences in restriction patterns were detected within each treatment, supporting the idea that they constitute a closely related group of strains.

Preliminary findings of restriction fragment differences in mitochondrial DNA among killer whales (Orcinus orca)

A preliminary assessment of mitochondrial DNA restriction patterns in the killer whale (Orcinus orca) was conducted using 10 captive North Atlantic killer whales from the southeastern coast of Iceland, a captive-born offspring of one of these whales, and 9 North Pacific killer whales. No restriction pattern variation was seen among these whales, using the enzymes BamH I, Bgl II, Hinf I, Kpn I, or Pvu II. Restriction pattern variation was found using the enzyme Hae III. This restriction endonuclease distinguished the North Atlantic killer whales (type 1) from the North Pacific killer whales. The North Pacific killer whales were further differentiated into two groups: those originating from the "resident" communities of the Vancouver Island region (type 2), and those from the "transient" community of Vancouver Island, as well as those stranded along the Oregon coast (type 3). The observed Hae III restriction pattern differences suggest that mitochondrial DNA analysis will be a valuable technique for investigating regional and local distributions of maternal lineages among killer whale pods, especially in the North Pacific.

Restriction pattern analysis of deoxyribonucleic acid isolated from callus and cell suspension of actinorhizal and non‐actinorhizal Betulaceae

Using cell suspensions, a method was elaborated to isolate high‐molecular‐weight genomic deoxyribonucleic acid (DNA; 65 MDa or more) from members of the Betulaceae: Alnus incana (L.) Moench, Alnus glutinosa (L.) Gaertn. and Betula papyrifera Marsh. The method was also effective for isolation of DNA from callus cells. Based on the chemical lysis of protoplasts, this procedure yielded 130 μg (callus) to 250 μg (cell suspension) of DNA (g fresh cells)−1, with a ratio A200/A28 of 1.7–2.0. The purified DNA obtained, formed distinct bands when restricted fragments were electrophoresed. Among the 10 endonucleases used for restriction analysis of Alnus glutinosa, Alnus incana and Betula papyrifera genomes, PvuI1 (EC 3.1.23.33) was unique in giving identical patterns for the two Ainus species. An unusual pattern occurred when Al‐2 DNA was restricted with Ava II (EC 3.1.23.4). It formed a ladder with a repeating fragment unit of 181 base pairs long. With the enzymes tested, no differences in restriction patterns were observed among clones of Alnus incana (AI‐2 vs AI‐2), Betula papyrifera (BP‐4 vs BP‐8) and subclones of Ainus glutinosa AG‐1 (PLFJ709 vs LF1709), suggesting genetic stability of the Betulaceae cultures.

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila

Using [ 32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.

Genotypic variation among wild isolates of two nuclear polyhedrosis viruses isolated from Spodoptera littoralis

Diseased Spodoptera littoralis larvae were collected from 21 different regions of Israel. Nuclear polyhedrosis viruses were isolated from these larvae, and viral DNAs were compared by restriction endonuclease analysis. Two distinct virus types, represented with approximately equal frequency, were found. Several wild isolates of each virus type exhibited minor restriction pattern differences. Plaque purification of the wild isolates revealed the presence of additional genotypic variants.

Remarkable restriction pattern differences between green and white branches of variegated ‘plastome mutator’ plants of Oenothera hookeri

Green-white variegated plants of the pm line of Oenothera hookeri were reported in the literature to be due to the action of a nuclear recessive ‘plastome mutator’ (pm). The plastid DNAs of green and white leaves have been studied by restriction analysis and Southern hybridization. Remarkable differences in restriction pattern have been found between them. The green plastids show the typical pattern of plastome I; the white plastids give the pattern of plastome III. The conclusion from our experiments is: variegated ‘pm’ plants contain two genetically different types of plastids. Their variegation is not due to nuclear gene-induced plastome mutations, but is the result of hybrid bleaching of the type III plastids, which were combined with type I plastids following — as we suppose-inadvertent pollination.

Classification of chloroplast DNA in Brassica napus possessing normal and male sterile cytoplasms.

Using restriction endonuclease analysis, chloroplast DNA (ctDNA) of normal or male sterile cytoplasms in Brassica napus were compared. There were some differences in restriction patterns of ctDNA from two types of cytoplasms digested with several restriction endonucleases. Based on the fragment pattern of Eco RI digested ctDNA, there were two types of ctDNAs in B, napus and one type of ctDNA was coincident with male sterile cytoplasm, whereas another type of ctDNA was observed only in the normal cytoplasm.