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PubMed1985

Demonstration of the colinearity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments.

In this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid-cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available.

Base Sequence↗

PubMed1988

Restriction mapping of Lymantria dispar nuclear polyhedrosis virus DNA: localization of the polyhedrin gene and identification of four homologous regions.

The genome of the multiple-embedded nuclear polyhedrosis virus (MNPV) of Lymantria dispar (LdMNPV) was partially characterized by restriction endonuclease analysis and a physical map was constructed using cosmid cloning and Southern cross blot hybridization. Using BamHI, BglII, EcoRI and HindIII, the size of the genome was estimated to be 88.5 x 10(6) Mr or 134.04 kbp. LdMNPV DNA was also analysed using methylation-sensitive restriction enzymes. The resulting restriction profiles suggested that extensive methylation did not occur at the nucleotide sequence recognized by HpaII and MspI. A BamHI restriction map was constructed by comparing overlapping BamHI fragments between cosmid clones containing partial digests of viral DNA. The positions of the BglII, EcoRI and HindIII sites were determined by Southern cross blot hybridizations and aligned to the BamHI restriction map. At least four homologous regions were identified by cross blot hybridizations of BglII-digested LdMNPV DNA and such regions were found to be interspersed along the genome in a fashion similar to that reported for other baculoviruses. Using recombinant plasmids containing the HindIII-V fragment of Autographa californica MNPV to probe Southern blots of LdMNPV DNA, the restriction fragment(s) that contain the polyhedrin gene were identified. Based on these findings the map was oriented with the polyhedrin gene of LdMNPV as the zero point.

Chromosome Mapping↗

PubMed1991

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci.

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.

Animals↗

PubMed1993

Sensitivity of naturally occurring coliphages to type I and type II restriction and modification.

Protection against lethal infections by bacteriophage may seem the most likely role of restriction-modification (R-M) systems in bacteria and the reason for their evolution. There are, however, phenomena which question this phage-mediated selection hypothesis for the maintenance of extant R-M systems. Most prominent among these are the mechanisms phage have to avoid or otherwise limit the effects of the restriction endonucleases produced by their host bacteria. To evaluate the importance of these antirestriction mechanisms in Escherichia coli, we have examined the sensitivity of coliphage from natural and laboratory sources to a series of type I and II R-M systems. The results of our study indicate that, in vivo, restriction endonucleases have no effect on a substantial fraction of naturally occurring coliphage. The absence of restriction sites appears to be the most common reason why these phage are unaffected by type II restriction endonucleases, but other antirestriction mechanisms also operate. On the other hand, the frequency of naturally occurring coliphage sensitive to restriction appears sufficiently great for phage-mediated selection to be a viable hypothesis for the maintenance of R-M in E. coli and its accessory elements.

Biological Evolution↗

PubMed1988

Characterisation of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA.

Clinical isolates of Staphylococcus aureus from the London Hospital were characterised by genetic analysis of antibiotic-resistance determinants and by restriction endonuclease digestion of chromosomal DNA and compared with isolates from elsewhere in the UK. Restriction enzyme digestion of chromosomal DNA confirmed that a single strain of methicillin-resistant S. aureus (MRSA) persists at the London Hospital, although its antibiotic-resistance profile and plasmid carriage are not constant. Methicillin-sensitive isolates, on the other hand, each had readily distinguishable and unique DNA restriction patterns. The DNA restriction digest pattern of the London Hospital MRSA isolates was identical to that of "epidemic" (E) MRSA isolates from the Thames regions. By contrast, other MRSA isolates had DNA restriction patterns which differed from those of EMRSA isolates and from each other. These results confirm the discriminatory value of restriction pattern analysis as a typing method.

Chromosomes, Bacterial↗

PubMed1994

Are point mutations or DNA rearrangements responsible for the restriction fragment length polymorphisms that are used to type bacteria?

Restriction fragment length polymorphisms (RFLPs) detected in total DNA or in rRNA genes are widely used to differentiate strains of bacteria. The changes accounting for these polymorphisms and the extent of genomic difference that they reflect are generally unknown. In this report, several methods have been used to examine the DNA differences between nine Enterococcus faecalis isolates. Restriction fragments from total DNA and from rRNA genes were compared between isolates using four and five different restriction enzymes, respectively. The proportion of polymorphic fragments detected was greater with total DNA than with rRNA gene patterns, but depended considerably on the restriction enzyme used. DNA changes underlying nine RFLPs were investigated by using the polymorphic fragments as probes to test for alteration in the position of recognition sites of other enzymes. Two polymorphisms were deduced to result from point mutation in a restriction site. Six were judged to result from DNA rearrangements, five of which involved deletion/insertion of the entire probe fragment. The results demonstrate that DNA rearrangements may be responsible for a high proportion of RFLPs used to differentiate and type strains of bacteria. While this does not limit the utility of such methods, it does preclude calculation of overall DNA sequence conservation from similarities in restriction pattern between isolates. DNA sequence determination of the 16S-23S rRNA intergenic spacer of three isolates revealed minimal base substitutions (less than 1%), suggesting that overall sequence divergence between the isolates may be low.

Bacterial Typing Techniques↗

PubMed2005

Relationship between protein O-linked glycosylation and insulin-stimulated glucose transport in rat skeletal muscle following calorie restriction or exposure to O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate.

AIMS AND BACKGROUND: Protein O-linked glycosylation is regulated in vivo by the concentration of hexosamine substrates. Calorie restriction (60% of ad libitum intake) for 20 days causes decreased UDP-N-acetylhexosamine levels and increased insulin-mediated glucose transport in rat skeletal muscle. Conversely, prolonged incubation (19 h) of muscle with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenyl-carbamate (PUGNAc; an inhibitor of N-acetyl-beta-D-glucosaminidase) is characterized by increased O-linked glycosylation and insulin resistance. We aimed to determine the calorie restriction effect on O-linked glycosylation and characterize the temporal relationship between PUGNAc-induced O-linked glycosylation and insulin resistance. HYPOTHESIS: A calorie restriction protocol characterized by decreased muscle hexosamine levels will result in a global reduction in O-linked glycosylated proteins in muscle, and PUGNAc-induced insulin resistance will coincide with increased O-linked glycosylation. METHODS: Plantaris muscle and liver from rats (ad libitum or calorie restricted) were analysed for O-linked glycosylation using two antibodies against different O-linked N-acetylglucosamine epitopes. Also, rat epitrochlearis muscles were incubated for 8.5 h +/- 100 mum PUGNAc prior to measurement of [(3)H]-3-O-methylglucose transport and O-linked glycosylation. RESULTS: Calorie restriction did not alter protein O-linked glycosylated levels in muscle or liver. Incubation with PUGNAc for 8.5 h resulted in increased in O-linked glycosylation but unaltered basal or insulin-stimulated glucose transport. CONCLUSIONS: The delay between O-linked glycosylation and insulin resistance in muscle incubated with PUGNAc suggests an indirect, relatively slow mechanism for insulin resistance. The effect of calorie restriction on insulin action in muscle is unlikely to be the direct result of a global change in protein O-linked glycosylation.

Acetylglucosamine↗

PubMed2004

Unchanged fasting and postprandial adiponectin levels following a 4-day caloric restriction in young healthy men.

OBJECTIVE: Adiponectin is an adipocyte-secreted protein that has been shown to promote fat oxidation and insulin sensitivity in animals. Whether acute energy restriction is associated with modulation of adiponectin in humans remains largely unknown. The purpose of this study was to examine the effects of a short-term caloric restriction on fasting and postprandial adiponectin levels in young healthy men. RESEARCH DESIGN AND METHODS: Fifteen young healthy men were subjected to a 4-day energy restricted diet (-800 kcal/day). Before and after the intervention, anthropometric measurements, fasting and postprandial glucose, insulin and adiponectin were measured at 0, 30 and 60 min after a fixed breakfast. RESULTS: While fat mass remained stable, minor but significant changes in weight were observed after the 4-day energy-restricted diet. Glucose and insulin levels increased postprandially, and patterns did not differ before and after the intervention. Fasting levels of adiponectin remained unchanged after the energy restriction, and postprandial levels were not significantly different from fasting levels either before, or after, the intervention. CONCLUSIONS: Fasting and postprandial adiponectin levels are not acutely modulated by a short-term energy restriction in young healthy men.

Adiponectin↗

PubMed1989

Specificity of HLA restricting elements for human nickel reactive T cell clones.

In order to study the fine specificity of HLA class II restriction, we have established nickel specific T cell clones from a nickel allergic patient. Cells were cloned by limiting dilution after primary stimulation and selection of nickel specific blasts. Several clones were established which were all shown to carry the CD4 marker. All clones were shown to be completely blocked by monoclonal antibodies directed against DR antigens, but unaffected by antibodies against DQ or DP, thus demonstrating their DR specificity. For the study of HLA class II restriction, a panel of cell donors was carefully HLA typed by including the use of DRB and DQB cDNA probes. Specificity analysis, using allogeneic antigen presenting cells, revealed that the clones were either restricted to DR3- or DR4-like molecules, which is consistent with the fact that the donor was DR3, DR4 positive. However, the studies also revealed that the fine specificity of the DR3 and DR4 restriction could not be completely assessed by serological and genomic typing of panel cells. This indicates that cellularly defined HLA restriction elements recognized by T cells cannot be defined properly with available class II typing methods, and the results of these experiments documented the additional polymorphism of class II restriction elements. The clonal specificity analysis has shed further light on the biologically relevant level of DR polymorphism.

Clone Cells↗

PubMed2004

GATC-specific restriction and modification systems in treponemes.

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.

Adenine↗

PubMed2004

Programming of adult blood pressure by maternal protein restriction: role of nephrogenesis.

BACKGROUND: Modest maternal protein restriction leads to hypertension and a reduced number of glomeruli in adult male but not female offspring. This study determined whether a more severe protein restriction has equivalent effects on male and female rat offspring, and examined the role of nephrogenesis in this programming. METHODS: Sprague-Dawley rats were fed a protein-restricted (5% protein) diet throughout (LLP), or during the first (LLP/NP) or second (NP/LLP) half of pregnancy. Controls ate a normal diet (NP, 19% protein). Adult offspring were chronically instrumented at 22 weeks; glomerular number and volume were estimated using stereologic techniques. RESULTS: Mean arterial pressures in male offspring were significantly higher in LLP (136 +/- 2 mm Hg) or NP/LLP (137 +/- 2 mm Hg) than in LLP/NP (125 +/- 1 mm Hg) or NP (125 +/- 2 mm Hg). Moreover, the hypertension was salt-sensitive (increase of 16 +/- 4 mm Hg in LLP on a high Na(+) diet compared to 2 +/- 2 mm Hg in NP). Glomerular number (per kidney) was reduced (15,400 +/- 2,411 in LLP vs. 27,208 +/- 1,534 in NP) but average individual glomerular volume was not different (1.98 +/- 0.18 106 micro(3) in LLP vs. 2.01 +/- 0.14 106 micro(3) in NP). Female offspring showed qualitatively similar results. CONCLUSION: Severe maternal dietary protein restriction reduces glomerular number and programs for salt-sensitive adult hypertension in both female and male offspring. The window of sensitivity of adult blood pressure to prenatal protein restriction falls within the period of nephrogenesis in the rat. These data are consistent with the hypothesis that maternal protein restriction causes adult hypertension in the offspring through impairment of renal development.

Aging↗

PubMed1981

Plasma aldosterone response to angiotensin II in sodium-restricted elderly subjects with essential hypertension.

The plasma aldosterone (PA) response to sodium restriction (25 mEq daily for 4 days) and to graded infusions of angiotensin II (AII, 2, 4 and 8 ng/kg/min each for 30 min) during a low-sodium intake were studied in 15 elderly subjects with mild essential hypertension versus 10 elderly normotensive subjects. The PA response to sodium restriction relative to changes in plasma renin activity (PRA) was estimated by the ratio of PA increment to PRA increment after sodium restriction (delta PA/delta PRA). THe PA response to graded AII infusions was determined by the increment of PA above the basal level after each dose of AII. In 10 of the 15 elderly hypertensive subjects whose PRAs responded normally to sodium restriction, the delta PA/delta PRA ratios and PA increments during the graded AII infusions were similar to those in the elderly normotensive subjects. However, in the remaining 5 elderly hypertensive subjects whose PRAs responded subnormally to sodium restriction, the delta PA/delta PRA ratios were high and the PA increments greater during the graded AII infusions. THe increments of mean blood pressure during the graded AII infusions were similar in the foregoing 10 of 15 hypertensive subjects, and significantly greater during the AII infusion rates of 4 and 8 ng/kg/min in the remaining 5 hypertensive subjects when compared with those in the normotensive subjects. Apparently some subjects with essential hypertension, whose PRAs response subnormally to sodium restriction, have an abnormally enhanced adrenal responsiveness to AII under the conditions of low-sodium intake.

Aged↗

PubMed2005

Conjugal TOL transfer from Pseudomonas putida to Pseudomonas aeruginosa: effects of restriction proficiency, toxicant exposure, cell density ratios, and conjugation detection method on observed transfer efficiencies.

The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10(-3) for PAO1162N and 2 x 10(1) for PAO2002N) and total cell densities (10(5) cells/mm(2) for PAO1162N and 10(6) cells/mm(2) for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10(-7) (events/mm(2))(-1) for PAO1162N and 10(-11) (events/mm(2))(-1) for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.

Colony Count, Microbial↗

PubMed1978

Demonstration of three different subtypes of adenovirus type 7 by DNA restriction site mapping.

Restriction site mapping of the genomes of eight different isolates of adenovirus serotype 7 (Ad7) has been performed with six different restriction endonucleases. In this analysis, 37 different restriction sites were localized. Three distinctly different cleavage patterns of the genomes of the Ad7 strains were observed. These strains could not be distinguished by serological techniques. The following three subtypes were defined on the basis of their restriction site patterns: the Ad7 prototype, represented by strain Gomen originally isolated from a case of pharyngitis; subtype Ad7a, represented by the Ad7 vaccine strain and strains isolated from undifferentiated respiratory disease and from a healthy carrier; and a third subtype of Ad7, represented by three strains which were isolated from Swedish patients, all having pronounced clinical symptoms indicating severe systemic infection. A comparison of the restriction site maps of the protype of Ad3 and the three subtypes of Ad7 indicated greater differences in the position of restriction sites between strains of Ad7 than between strains of the two serotypes. This technique is consequently recommended to obtain a more precise definition of distinct entities of viruses.

Adenovirus Infections, Human↗

PubMed1978

Biological characteristics of a type I restriction-modification system in Staphylococcus aureus.

Two restriction-modification systems, S1 and S2, are present in Staphylococcus aureus RN450 (S. Iordanescu and M. Surdeanu, J. Gen. Microbiol., 96:277-281, 1976). System S2 affects phage multiplication after both infection and transfection. Unmodified plasmid and chromosomal DNAs are also not expressed following transduction and transformation into a restrictive host. Restricted phages are, however, capable of conferring phage-mediated competence, although the state of competence does not affect the restriction-modification system. The restricting activity of system S2 is inactivated by heat treatment of the cells. An enzymatic activity that restricts unmodified phage DNA in the presence of ATP, Mg2+, and S-adenosylmethionine was recovered from cell-free extracts of a strain RN450 derivative.

DNA Restriction Enzymes↗

PubMed1979

Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane.

The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E. coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes. For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA. From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped. No restriction fragments of DNA from the E. coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene.

Bacterial Proteins↗

PubMed1980

Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli.

Expression of the gene gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, is regulated by growth rate. Using deoxyribonucleic acid from the specialized transducing phage lambda h80 dgnd his as the source of gnd, we cloned restriction fragments carrying the complete gene and portions of it on the plasmid vector pBR322. A hybrid plasmid carrying a 3.7-megadalton HindIII restriction fragment from the phage was prepared and found to be gnd+. Through restriction mapping of this fragment and subcloning segments of it, we prepared a gnd+ hybrid plasmid which carried only 1.85 megadaltons of E. coli deoxyribonucleic acid. A cleavage site for the restriction endonuclease PstI was located on the genetic map of gnd by cloning adjacent EcoRI-PstI restriction fragments and crossing the resulting hybrid plasmids with previously mapped gnd deletion and bacteriophage Mu insertion mutants. A maxicell experiment was used to determine the direction of transcription of gnd, to identify which EcoRI-PstI fragment contains the gnd promote, and to localize th beginning of the structural gene to a region about 850 +/- 150 base pairs from the PstI cleavage site. A fine-structure restriction map surrounding the PstI cleavage site was prepared for endonucleases KpnI, HincII, HaeIII, HpaII, and TaqI.

Chromosome Mapping↗

PubMed2002

A DNA methyltransferase can protect the genome from postdisturbance attack by a restriction-modification gene complex.

In prokaryotic genomes, some DNA methyltransferases form a restriction-modification gene complex, but some others are present by themselves. Dcm gene product, one of these orphan methyltransferases found in Escherichia coli and related bacteria, methylates DNA to generate 5'-C(m)CWGG just as some of its eukaryotic homologues do. Vsr mismatch repair function of an adjacent gene prevents C-to-T mutagenesis enhanced by this methylation but promotes other types of mutation and likely has affected genome evolution. The reason for the existence of the dcm-vsr gene pair has been unclear. Earlier we found that several restriction-modification gene complexes behave selfishly in that their loss from a cell leads to cell killing through restriction attack on the genome. There is also increasing evidence for their potential mobility. EcoRII restriction-modification gene complex recognizes the same sequence as Dcm, and its methyltransferase is phylogenetically related to Dcm. In the present work, we found that stabilization of maintenance of a plasmid by linkage of EcoRII gene complex, likely through postsegregational cell killing, is diminished by dcm function. Disturbance of EcoRII restriction-modification gene complex led to extensive chromosome degradation and severe loss of cell viability. This cell killing was partially suppressed by chromosomal dcm and completely abolished by dcm expressed from a plasmid. Dcm, therefore, can play the role of a "molecular vaccine" by defending the genome against parasitism by a restriction-modification gene complex.

Animals↗