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PubMed1985

The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes.

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [14C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding. It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.

Chromosome Banding↗

PubMed1985

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction.

The host-controlled K restriction of unmodified phage lambda was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA-, recA-, and recB- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage lambda ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI systems, (2) ard gene(s) did not mediate EcoK type modification of lambda DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage lambda.

Bacterial Proteins↗

PubMed1991

Multiple restriction fragment length polymorphisms at the GLUT2 locus: GLUT2 haplotypes for genetic analysis of type 2 (non-insulin-dependent) diabetes mellitus.

The liver/islet glucose transporter (GLUT2) is expressed in the liver and in the Beta cells of pancreatic islets and is a candidate gene for the inherited defect in Type 2 (non-insulin-dependent) diabetes mellitus. A series of restriction fragment length polymorphisms have been identified using a GLUT2 cDNA probe with five restriction enzymes in a British white Caucasian population. Five independent restriction fragment length polymorphisms detected by restriction enzymes EcoRI (two restriction fragment length polymorphisms termed EcoRI-1, EcoRI-2), TaqI (two restriction fragment length polymorphisms termed TaqI-1, TaqI-2), and BclI (BclI-2) were used to construct GLUT2 haplotypes. Significant linkage disequilibrium was observed between four polymorphic sites EcoRI-2, TaqI-1, TaqI-2 and BclI-2 but linkage disequilibrium was not observed with EcoRI-1 polymorphic site and the other four sites. The frequencies of GLUT2 restriction fragment length polymorphisms and haplotypes in 50 Type 2 diabetic subjects and 50 non-diabetic control subjects show no significant differences suggesting that it is unlikely that there is a single major defect of this gene contributing to the inherited susceptibility to Type 2 diabetes in a Caucasian population.

Alleles↗

PubMed1995

Molecular characterization and identification of Saprolegnia by restriction analysis of genes coding for ribosomal RNA.

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species of Saprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except for S. hypogyna, S. delica, S. lapponica, and S. mixta. The rDNA data indicate that S. lapponica and S. lapponica and S. mixta are conspecific with S. ferax, whereas there is no support for the proposed synonymies of S. diclina with S. delica and of S. mixta with S. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in the S. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains of S. parasitica originating from the U.K. and Japan. HhaI and BsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonuclease BstUI was diagnostic for S. parasitica, generating identical fingerprints for all stains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved in S. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed that S. diclina and S. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification of Saprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.

Base Sequence↗

PubMed1996

Subtyping of human T-lymphotropic virus type I by amplification of long terminal repeat sequences and restriction fragment length polymorphism analysis in carriers with multiple transfusions.

Five major subtypes of human T-lymphotropic virus type I (HTLV-I) have been proposed: cosmopolitan, Japanese, West African, Central African, and Melanesian. Based on nucleotide variations specific to particular subtypes, it was possible to genotype HTLV-I rapidly by restriction fragment length polymorphism (RFLP) studies following polymerase chain reaction (PCR). In this study, the restriction patterns of two LTR fragments were analyzed using eight restriction endonucleases (AvaI, Eco57I, BsoFI, NdeI, SacI, DraI, MaeII, and MaeIII). Genotyping of HTLV-I was done in nine patients with adult T-cell leukemia or HTLV-I-associated myelopathy/tropical spastic paraparesis, in three prostitutes, and in 19 carriers with multiple transfusion in Taiwan. The subtyping results of RFLP studies using these eight restriction endonucleases were in accordance with those of phylogenetic analysis. A substitution of G by A at nucleotide position 503, which creates the DraI site but suppresses the SacI site, was found not only in the Japanese subtype but also in a minority of the cosmopolitan subtype. A mutation near the position of subtype-specific nucleotide variations might suppress the restriction site and lead to unexpected restriction patterns. Amplification of more than one proviral fragment and RFLP studies with a group of appropriate restriction endonucleases may provide rapid and accurate genotyping of HTLV-I. More carriers are required to evaluate the possibility of mixed infection with different HTLV-I subtypes.

Base Sequence↗

PubMed2007

Transient caloric restriction and cancer risk (The Netherlands).

Over the past century, many animal experiments have shown that caloric restriction can reduce the risk of cancer, a finding that proved to be highly reproducible. Many papers have been published on its potential for human health, but until know little evidence is available on its actual effects in humans. In Utrecht, The Netherlands, we have been investigating the effects of the 1944-1945 Dutch famine on breast cancer risk factors and breast cancer risk, and paradoxically the relatively short-term famine seemed to be related to increased breast cancer risk in later life. One of the differences between the famine situation and the large body of evidence from animal experiments is the duration of caloric restriction. Almost all animal experiments investigated sustained caloric restriction and information on the effects of short-term transient caloric restriction is very scarce. A search in the literature identified some animal experiments on short-term transient caloric restriction and these seemed to be at least supportive to the famine findings. Because caloric restriction in humans for preventive health measures would be mostly short-term, it is important to extend animal research on short-term caloric restriction.

Animals↗

PubMed1993

Altered gustatory development in Na(+)-restricted rats is not explained by low Na+ levels in mothers' milk.

Placing pregnant rats on a sodium-deficient diet (0.03% NaCl) very early in gestation and then weaning the offspring (sodium-restricted rats) to the same diet precludes development of amiloride-sensitive sodium taste transduction pathways in the offspring. However, normal amiloride-sensitive sodium taste responses can be restored by permitting sodium ingestion by sodium-restricted rats. The present study tested the hypothesis that the concentration of sodium in sodium-restricted mothers' milk must be abnormally low in order to preserve altered gustatory function in the offspring. Other milk electrolyte and total protein concentrations were determined as well. Milk sodium was similar between sodium-restricted and control rat mothers at 10-13 and 16-20 days postpartum, as were levels of potassium and chloride. At 10-13 days postpartum, total protein was higher in milk from sodium-restricted mothers. Sodium-restricted mothers' milk calcium concentrations were higher versus controls at 16-20 days postpartum. These results indicate that the lack of gustatory amiloride sensitivity in sodium-restricted rats cannot be attributed to deficient dietary sodium levels during the suckling period.

Amiloride↗

PubMed1992

Evidence for the existence of a restriction-modification system common to several species of the family Vibrionaceae.

A broad-host-range vibriophage KVP40 originally isolated on Vibrio parahaemolyticus 1010 was restricted and modified by strains of at least five Vibrio and one Photobacterium species. 1010 was a non-restricting host. An anti-restriction mutant KVP40 aar1 was isolated after propagating the phage on a restricting host, V. anguillarum VIB36. KVP40 aar1 grown on either 1010 or VIB36, as well as the parental phage grown on VIB36, showed much higher efficiencies of plating on all the restricting hosts as compared with the parental phage grown on 1010, indicating that these restricting hosts probably share a common restriction-modification system active in vivo on KVP40.

DNA Restriction-Modification Enzymes↗

PubMed1986

Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments.

A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers). These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site. The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer. They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end. This was evidenced by mobility shifts of the splinker-ligated restriction fragments. The splinker-ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus. The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase. As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence.

Base Composition↗

PubMed1986

Specificity of restriction endonucleases and methylases--a review.

The properties and sources of all known restriction endonucleases and methylases are listed. The enzymes are cross-indexed (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the double-stranded DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (integrated into Table II), the structure of the generated fragment ends (Table III), and the sensitivity to different kinds of DNA methylation (Table V). In Table IV the conversion of two- and four-base 5'-protruding ends into new recognition sequences is compiled which is obtained by the fill-in reaction with Klenow fragment of the Escherichia coli DNA polymerase I or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments. Table VI classifies the restriction methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises restriction endonucleases which are known to be inhibited or activated by the modified nucleotides. The detailed sequences of those overlapping restriction sites are also included which become resistant to cleavage after the sequential action of corresponding restriction methylases and endonucleases [N11, M21]. By this approach large DNA fragments can be generated which is helpful in the construction of genomic libraries. The data given in both Tables IV and VI allow the design of novel sequence specificities. These procedures complement the creation of universal cleavage specificities applying class IIS enzymes and bivalent DNA adapter molecules [P17, S82].

Base Sequence↗

PubMedDate not supplied

Application of restriction endonuclease analysis of chromosomal DNA in the study of Staphylococcus aureus colonization in continuous ambulatory peritoneal dialysis patients.

The relationship between nasal and skin colonization with Staphylococcus aureus and subsequent infection in continuous ambulatory peritoneal dialysis (CAPD) patients in Brazil has been documented by restriction endonuclease analysis of plasmid DNA. However, S. aureus strains without detectable plasmids have been identified. Using restriction endonuclease analysis of chromosomal DNA hybridized with an rRNA gene probe, we document the diversity of S. aureus strains without detectable plasmids colonizing CAPD patients. Ten paired strains without detectable plasmids from five patients were studied by restriction endonuclease analysis of chromosomal DNA and by phage typing. Five different profiles were obtained by restriction endonuclease analysis of chromosomal DNA. Although four of the ten paired isolates were nontypeable by phage typing, all were discriminated by restriction endonuclease analysis of chromosomal DNA. These results demonstrate that restriction endonuclease analysis of chromosomal DNA is a useful epidemiologic tool and complements the restriction endonuclease analysis of plasmid DNA of S. aureus by providing a means of typing strains without detectable plasmids.

Bacteriophage Typing↗

PubMed1991

Primary restrictive cardiomyopathy: clinical and pathologic characteristics.

Twenty-four patients with restrictive cardiomyopathy were identified at St. Thomas' Hospital during a 17-year period. All had endomyocardial biopsy, but in two patients the biopsy specimens were small and nondiagnostic. Seven patients had amyloidosis and five had other specific heart muscle diseases. The remaining 10 patients with primary restrictive cardiomyopathy had myocyte hypertrophy or interstitial fibrosis, or both. Patients with primary restrictive cardiomyopathy presented earlier but survived longer after presentation than did those with amyloidosis. In each group, survival after cardiac catheterization was related to cardiac index but not to filling pressures. Primary restrictive cardiomyopathy was associated with complete heart block in four patients, two of whom had skeletal myopathy. One had a family history of dominantly inherited skeletal myopathy. Primary restrictive cardiomyopathy was present in a mother and daughter. Two other patients had a family history of heart failure, sudden death or complete heart block, alone or in combination, at a young age. Restrictive hemodynamics and complete heart block were present in patients even in the absence of significant fibrosis. The data suggest that primary restrictive cardiomyopathy may be a distinct myopathy with dominant inheritance and incomplete penetrance that is expressed morphologically as myocyte hypertrophy and interstitial fibrosis. Skeletal myopathy may be associated with the cardiomyopathy.

Adolescent↗

PubMed1996

Differentiation of constrictive pericarditis from restrictive cardiomyopathy: assessment of left ventricular diastolic velocities in longitudinal axis by Doppler tissue imaging.

OBJECTIVES: We sought to determine the utility of left ventricular expansion velocities in differentiating constrictive pericarditis from restrictive cardiomyopathy. BACKGROUND: Several studies have shown that left ventricular diastolic expansion is influenced by the elastic recoil forces of the myocardium. These forces are affected by intrinsic myocardial disease but should be preserved when diastole is impaired as a result of extrinsic causes. METHODS: Using Doppler tissue imaging, we measured peak early velocity of longitudinal axis expansion (Ea) in 8 patients with constrictive pericarditis, 7 patients with restriction and 15 normal volunteers. Transmitral early (E) and late (A) Doppler flow velocities, left ventricular systolic and diastolic volumes, ejection fraction and mitral annular M-mode displacement were also compared between the groups. RESULTS: The Ea value was significantly higher in normal subjects (14.5 +/- 4.7 cm/s [mean +/- SD]) and in patients with constriction (14.8 +/- 4.8 cm/s) than in those with restriction (5.1 +/- 1.4 cm/s, p < 0.001 constriction vs. restriction). There was weak correlation between Ea and the extent of annular displacement (r = 0.55, p = 0.004) and the E/A ratio (r = 0.44, p = 0.03). There was no correlation between Ea and E (r = 0.33, p = 0.07) or ejection fraction (r = 0.21, p = 0.26). By multivariate analysis, Ea was the best variable for differentiating constriction from restriction. CONCLUSIONS: Our study indicates that longitudinal axis expansion velocities are markedly reduced in patients with restrictive cardiomyopathy. The poor correlation found with transvalvular flow velocities suggests that Ea may be relatively preload independent. The measurement of longitudinal axis expansion velocities provides a clinically useful distinction between constrictive pericarditis and restrictive cardiomyopathy and may prove to be valuable in the study of diastolic function.

Adult↗

PubMed2005

Maternal gestational protein-calorie restriction decreases the number of glomeruli and causes glomerular hypertrophy in adult hypertensive rats.

OBJECTIVE: This work analyzed the renal function and structure in offspring rats that were submitted to maternal protein-calorie restriction during prenatal or lactation periods. STUDY DESIGN: Kidneys from adult offspring were studied. Animals from mothers that were submitted to food restriction were separated in 3 groups: control, prenatal restriction, and lactation restriction. Blood pressure, microalbuminuria, and glomerular filtration rate were determined. Kidney cortical remodeling was analyzed with stereology; volume-weighted glomerular volume and the number of glomeruli were estimated. RESULTS: Adult prenatal restriction offspring showed enhanced microalbuminuria, decreased glomerular filtration rate, and hypertension; their kidneys showed a smaller number of hypertrophied glomeruli than control and lactation restriction animals. CONCLUSION: Maternal prenatal protein-calorie restriction in rats causes kidney disease in adult offspring, which is characterized by hypertension and renal dysfunction and suggests secondary kidney remodeling because of an impairment of glomerulogenesis.

Albuminuria↗

PubMed2004

Differentiation of constrictive pericarditis from restrictive cardiomyopathy using mitral annular velocity by tissue Doppler echocardiography.

This study evaluated the diagnostic role of early diastolic mitral annular velocity (E') by tissue Doppler echocardiography for differentiating constrictive pericarditis from restrictive cardiomyopathy (primary restrictive cardiomyopathy and cardiac amyloidosis). The study group consisted of 75 patients (53 men, 22 women; mean age 62 years, range 27 to 87). Of these, 23 patients had surgically confirmed constrictive pericarditis, 38 had biopsy-proved systemic amyloidosis and typical echocardiographic features of cardiac involvement, and 14 had primary restrictive cardiomyopathy. Standard mitral inflow characteristics were measured. Tissue Doppler echocardiography was used to measure E' at the septal annulus. E' was significantly higher in patients with constrictive pericarditis than in those with primary restrictive cardiomyopathy or cardiac amyloidosis (12.3 vs 5.1 cm/second, p <0.001). An E' cut-off value > or =8 cm/second resulted in 95% sensitivity and 96% specificity for the diagnosis of constrictive pericarditis. There was no overlap of E' between patients who had constrictive pericarditis and those who had cardiac amyloidosis. In a subgroup analysis of restrictive cardiomyopathy, E' of patients who had cardiac amyloidosis was significantly lower than that of patients who had primary restrictive cardiomyopathy (4.6 vs 6.3 cm/second, p <0.001). Thus, E' velocity can distinguish between constrictive pericarditis and restrictive cardiomyopathy with a specific cut-off value in patients with clinical and echocardiographic evidence of diastolic heart failure.

Aged↗

PubMed2004

Lack of direct effect of moderate hyperleptinemia to improve endothelial function in lean rat aorta: role of calorie restriction.

Leptin stimulates vascular sympathetic activity and NO release. The resulting effect of hyperleptinemia on endothelial function is unknown. Reduction of food intake associated with hyperleptinemia may mediate some of the vascular effects of leptin. This study was designed to assess the relative effects of short-term moderate caloric restriction and hyperleptinemia on vascular function in lean rat. Endothelium-dependent and -independent vasorelaxation, nitric oxide synthase (eNOS) expression and nitrite production were measured in lean rats receiving leptin subcutaneous infusion (-26% caloric intake, -4% body weight versus control) or moderate caloric restriction (pair-feeding) for one week. Plasma leptin was increased (P < = 0.05) by approximately 300% in the leptin-infused group, while it decreased (P < or = 0.05) by approximately 40% in calorie-restricted animals. Both leptin infusion and calorie restriction resulted in improved (P < 0.05) endothelium-dependent vasorelaxation to acetylcholine (EC50: -6.1 +/- 0.2 and -6.2 +/- 0.2 versus -5.4 +/- 0.2 in control) and unaltered endothelium-independent vasodilation to DEA-NONOate. Furthermore, in aortas from leptin-infused and calorie-restricted rats, expression of eNOS and nitrite production were increased (P < = 0.05) to similar extent compared to control animals. These findings suggest that moderate short-term calorie restriction with adaptive hypoleptinemia has independent beneficial effects on endothelial function in lean animals by enhancing eNOS expression and function. In addition, physiological hyperleptinemia does not independently contribute to improved vascular function above and beyond the effect of calorie restriction.

Animals↗

PubMed2006

Influence of lifetime food restriction on physiological variables in Labrador retriever dogs.

Effects of lifetime food restriction on erythrocytes and numerous clinical chemistry, thyroid, parathyroid, and acid-base variables are reported from a paired-feeding study of 25% diet restriction in dogs. The 48 dogs were paired by gender and weaning weight within litter, and 1 dog in each pair was fed 25% less than its pair mate, from age 8 weeks until death. Erythrocyte and serum biochemistry profiles were evaluated by annual sampling intervals and longitudinally. Erythrocyte variables were slightly higher among control-fed dogs, a possible reflection of the need to support both higher body fat mass and lean mass that uses energy less efficiently. Among serum biochemistry variables, glucose and triglyceride were lower among diet-restricted dogs, while creatinine was slightly higher in the absence of renal disease or failure, over the life spans of the dogs. Glucose outcomes reflect improved glucose tolerance that has been demonstrated with diet restriction protocols in several species, while triglyceride data may reflect the difference in total body fat cells between feeding groups. Creatinine outcomes may reflect lean mass responses to diet restriction or more efficient function of the intracellular proteasome. Serum triiodothyronine levels were lower among diet-restricted dogs. Other clinical chemistry and thyroid variables, parathyroid variables, and acid-base variables were not strongly influenced by diet restriction but revealed age-related effects.

Aging↗

PubMed2005

Caloric restriction in humans: potential pitfalls and health concerns.

To date, the only intervention that has consistently been shown to slow the rate of aging, and to increase mean and maximum lifespan in short-lived species, is life-long calorie restriction. It is yet unclear whether long-term calorie restriction in longer lived species (i.e. primates and humans) will have a similar effect. In humans, several studies investigating short-term calorie restriction or "weight loss" programs suggest beneficial outcomes on parameters of cardiovascular disease. Studies on long-term calorie restriction are performed on a self-selected group of human subjects and show similar effects. However, few studies are currently investigating the quality of life and potential pitfalls of long-term calorie restriction in humans. It is likely that some of the physiological and psychological effects of caloric restriction that occur in animals may impact the human life very differently. For certain, calorie restriction has a plethora of health benefits in mammals, such as a reduction in age-related diseases such as cancer. However, despite the "magic" of CR, this intervention in humans may present itself with a number of health concerns, which may not be applicable to or impact the life of experimental animals, but may do so in humans. These potential pitfalls and "side effects" are not clearly addressed in the literature and will be a focus of this review.

Aging↗