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PubMed1996

[Suppression of experimental autoimmune uveitis by energy restriction].

The effects of energy restriction on experimental autoimmune uveitis (EAU) were investigated. Seven-week-old, male Lewis rats were divided into three groups. Experiment A) A control group was fed ad libitum, a second group was given 50% calorie restricted food from the day of immunization, and a third group was given 50% calorie restricted food from 2 weeks before immunization. Experiment B) A control group was fed ad libitum, a second group was given 25% calorie restricted food from 4 weeks before immunization, and a third group was given 50% calorie restricted food 4 weeks before immunization. All rats were immunized with interphotoreceptor retinoid-binding protein (IRBP). In experiment A), in the group given 50% calorie restricted food from 2 weeks before immunization, and in experiment B), in the group given 50% calorie restricted food from 4 weeks before immunization, the onset of EAU was significantly delayed, the clinical symptoms were lessened, and delayed hypersensitivity against IRBP was suppressed. Clinically and histologically, the severity was also low. In the group given 50% calorie restricted food from 4 weeks before immunization, the titer of anti-IRBP antibody was also significantly lower than in the control group.

Animals↗

PubMed1997

Effect of postweaning nutritional and environmental restrictions on discrimination learning in albino rats.

Charles Foster littermate albino rats (72) were randomized into 9 independent groups in a 3 x 3 factorial design by varying nutrition and environment each at three levels: normal nutrition, 25% nutritional restriction and 50% nutritional restriction; normal environment, 3-week environmental restriction and 6-week environmental restriction. The nutritional and environmental restrictions were imposed from the 30th day of life for 6 weeks. The animals were subsequently tested for black/white original learning followed by reversal discrimination learning in a T-maze. The results indicated significant main effects for nutrition and environment on indices of learning ability. However, the nutrition x environment interaction was not significant. Post hoc analysis revealed that moderate and severe nutritional restriction as well as long-term environmental restriction caused significant discrimination learning deficits. Short-term environmental restriction failed to produce significant effect on learning behaviour. The results are discussed in context of environmental stress effects, altered brain mechanisms and psychological changes such as increased drive level and emotionality of the animal.

Animal Nutritional Physiological Phenomena↗

PubMed1991

The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay.

The genomic variability of poliovirus was examined by analyzing the restriction fragment length polymorphism of a reverse-transcribed genomic fragment amplified by the polymerase chain reaction. The fragment was a 480-nucleotide sequence of the poliovirus genome coding for the N-terminal half of the capsid protein VP1, including antigenic site 1. The identification of a pair of generic primers flanking this fragment allowed its amplification in practically all the poliovirus strains tested so far (more than 150). By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles could be generated for the amplified genomic fragment of each of the six reference poliovirus strains tested: one representative wild poliovirus of each of the three serotypes (P1/Mahoney, P2/Lansing, and P3/Finland/23127/84) and the three Sabin vaccine strains. When 21 poliovirus field isolates previously identified as Sabin vaccine-related were tested, they showed restriction profiles identical to those of the originating homotypic Sabin virus, demonstrating the conservation of these profiles during virus replication in humans. These profiles could thus be used as markers for Sabin-derived genotypes. To compare the distribution of poliovirus genotypes in nature before and after the introduction of poliovirus vaccines, the restriction profiles of the amplified genomic fragment of a total of 72 strains of various geographic and temporal origins were determined. Strains isolated before the introduction of polio vaccines displayed a wide diversity of genotypes. In contrast, wild (Sabin unrelated) strains isolated after vaccine introduction, during a single epidemic in a particular geographic area, showed identical or very similar restriction profiles, indicating the circulation of predominant regional genotypes. Our results indicate that the assay we developed for the analysis of the restriction fragment length polymorphism of the poliovirus genome may be used to identify and characterize poliovirus genotypes circulating in nature.

Base Sequence↗

PubMed2006

Mitochondrial oxidative stress, aging and caloric restriction: the protein and methionine connection.

Caloric restriction (CR) decreases aging rate and mitochondrial ROS (MitROS) production and oxidative stress in rat postmitotic tissues. Low levels of these parameters are also typical traits of long-lived mammals and birds. However, it is not known what dietary components are responsible for these changes during CR. It was recently observed that 40% protein restriction without strong CR also decreases MitROS generation and oxidative stress. This is interesting because protein restriction also increases maximum longevity (although to a lower extent than CR) and is a much more practicable intervention for humans than CR. Moreover, it was recently found that 80% methionine restriction substituting it for l-glutamate in the diet also decreases MitROS generation in rat liver. Thus, methionine restriction seems to be responsible for the decrease in ROS production observed in caloric restriction. This is interesting because it is known that exactly that procedure of methionine restriction also increases maximum longevity. Moreover, recent data show that methionine levels in tissue proteins negatively correlate with maximum longevity in mammals and birds. All these suggest that lowering of methionine levels is involved in the control of mitochondrial oxidative stress and vertebrate longevity by at least two different mechanisms: decreasing the sensitivity of proteins to oxidative damage, and lowering of the rate of ROS generation at mitochondria.

Aging↗

PubMed2006

Energy restriction but not protein source affects antioxidant capacity in athletes.

The primary purpose of this study was to examine the effect of energy restriction on antioxidant capacity in trained athletes. Secondly, our study determined whether dietary protein source influenced the antioxidant response, performance, and immunity. Twenty male cyclists consumed either whey or casein supplement (40 g/day) in addition to their diet for 17 days. All subjects subsequently underwent 4 days of energy restriction using a formula diet (20 kcal/kg) while continuing protein supplementation. Energy restriction caused 2.7 +/- 0.3 kg weight loss, increased lymphocyte total glutathione (tGSH) 37%, red blood cell glutathione peroxidase 48%, plasma cysteine 12%, and decreased whole blood reduced to oxidized GSH (rGSH/GSSG) ratio by 52%. The only immunity factor altered by energy restriction was an increase in stimulated phagocytosis (65%). Acute submaximal exercise reduced blood tGSH but increased glutathione peroxidase. Performance of a high intensity cycle test following 45 min of moderate exercise tended to be reduced by energy restriction (P = 0.06) but was unaffected by protein source. Energy restriction caused a negative nitrogen balance with no difference from dietary protein source. In conclusion, acute energy restriction increased plasma cysteine and several markers of the glutathione antioxidant system in trained athletes. A high cysteine dietary protein source did not influence these responses.

Adult↗

PubMed2006

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system.

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.

Amino Acid Sequence↗

PubMed2002

Complex restriction enzymes: NTP-driven molecular motors.

Survival is assuredly the prime directive for all living organisms either as individuals or as a species. One of the main challenges encountered by bacterial populations is the danger of bacteriophage attacks, since infection of a single bacterium may rapidly propagate, decimating the entire population. In order to protect themselves against this acute threat, bacteria have developed an array of defence mechanisms, which range from preventing the infection itself via interference with bacteriophage adsorption to the cell surface and prevention of phage DNA injection, to degradation of the injected phage DNA. This last defence mechanism is catalysed by the bacterial restriction-modification (R-M) systems, and in particular, by nucleoside 5'-triphosphate (NTP)-dependent restriction enzymes, e.g. type I and type III R-M systems or the modification-dependent endonucleases. Type I and type III restriction systems have dual properties. They may either act as methylases and protect the host's own DNA against restriction by methylating specific residues, or they catalyse ATP-dependent endonuclease activity so that invading foreign DNA lacking the host-specific methylation is degraded. These defence mechanism systems are further complemented by the presence of methylation-dependent, GTP-dependent endonucleases, that restricts specifically methylated DNA. Although all three types of endonucleases are structurally very different, they share a common functional mechanism. They recognise and bind to specific DNA sequences but do not cleave DNA within those target sites. They belong to the general class of DNA motor proteins, which use the free energy associated with nucleoside 5'-triphosphate hydrolysis to translocate DNA so that the subsequent DNA cleavage event occurs at a distance from the endonuclease recognition site. Moreover, DNA cleavage appears to be a random process triggered upon stalling of the DNA translocation process and requiring dimerisation of the bound endonucleases for a concerted break of both DNA strands. In this review, we present a detailed description and analysis of the functional mechanism of the three known NTP-dependent restriction systems: type I and type III restriction-modification enzymes, as well as the methylation-dependent McrBC endonuclease.

Binding Sites↗

PubMed1997

Characterization of avian Chlamydia psittaci strains using omp1 restriction mapping and serovar-specific monoclonal antibodies.

In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.

Animals↗

PubMed1997

Comparison of three restriction endonucleases in IS1245-based RFLP typing of Mycobacterium avium.

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.

DNA Transposable Elements↗

PubMed1994

Response to UV damage by four Escherichia coli K-12 restriction systems.

To understand the role of restriction in regulating gene flow in bacterial populations, we would like to understand the regulation of restriction enzyme activity. Several antirestriction (restriction alleviation) systems are known that reduce the activity of type I restriction enzymes like EcoKI in vivo. Most of these do not act on type II or type III enzymes, but little information is available for the unclassified modification-dependent systems, of which there are three in E. coli K-12. Of particular interest are two physiological controls on type I enzymes: EcoKI restriction is reduced 2 to 3 orders of magnitude following DNA damage, and a similar effect is seen constitutively in Dam- cells. We used the behavior of EcoKI as a control for testing the response to UV treatment of the three endogenous modification-dependent restriction systems of K-12, McrA, McrBC, and Mrr. Two of these were also tested for response to Dam status. We find that all four resident restriction systems show reduced activity following UV treatment, but not in a unified fashion; each response was genetically and physiologically distinct. Possible mechanisms are discussed.

Bacterial Proteins↗

PubMed1994

Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli.

RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. glutamicum clones was developed. By using this procedure, clones of the restriction-deficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orf1 and orf2) on the complementing DNA fragment. The region comprising orf1 and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orf1 is essential for complementation, but inactivation of orf2 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterium lilium ATCC 15990.

Adaptation, Biological↗

PubMed1996

Restriction analysis of actinomycetes chromosomal DNA.

Actinomycetes DNAs were digested with restriction enzymes to study the presence of methylated bases. Analysis showed that the enterobacterial Dam and Dcm systems are absent. Methylation at the internal cytosine in CCGG sequences, typical of eukaryotes, was also absent. We also tested 18 restriction endonucleases recognizing six base pair sequences (all of which were inhibited by methylation). Results showed a higher number of restriction sites for enzymes recognizing CG-rich sequences (CG endonucleases) than for enzymes recognizing AT-rich sequences (AT endonucleases). Restriction patterns with CG endonucleases were quite uniform, with the remarkable exception of XhoI, which yielded a small number of DNA bands. The study performed with AT endonucleases allowed differentiation of three groups of enzymes based on different degrees of chromosomal sensitivity. One group (BclI and BglII) produced restriction patterns with more abundant restriction sites than expected, a second group (ClaI, EcoRI, and EcoRV) yielded the predicted number of DNA fragments, and the third group (HpaI, HindIII, XbaI, and DraI) produced an unexpectedly low number of fragments. Some individual cases of resistance to particular enzymes could be explained by the presence of restriction-modification systems with the same specificity.

Actinomycetales↗

PubMed2003

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.

Animals↗

PubMed1991

[Analysis of leptospiral deoxyribonucleic acids by restriction endonucleases].

For the application of restriction endonuclease analysis in typing and identifying leptospira, we selected some serovars and isolates, and analysed preliminarily their DNA with four restriction enzymes, EcoR I, Bgl II, Hha I, and Hind III. The DNA samples were isolated from the reference strains and isolates as follows: Serovar lai 56601, 017 (the virulent strain for PDH model of guinea pig), Serovar autumnalis 56606, Serovar manhao II 67020, and isolates 87112 and 87369. Each 2 micrograms of DNA was digested with 20mu of restriction enzyme at 37 degrees C for 2h and electrophoresed in 0.8% agarose gel. The gels were stained in ethidium bromide and photographed with UV light. In our experiments, apparently different restriction patterns of serovar lai 017 were observed with four restriction enzymes. Serovar lai, serovar autumnalis and serovar manhao II showed different patterns with EcoR I, especially in high molecular regions. We also observed in serovar lai 017 a distinct 10.5kb band which was obscure in 56601, the reference strain of serovar lai, after EcoR I digestion. The three serovars showed some delicate differences in Hind III restriction pattern. The two isolates from Apodemus agrarius in Sichuan (1987) 87112 and 87369 had patterns identical to those of serovar lai 56601, 017 with EcoR I, and 87112 also had a pattern identical to 56601, 017 with Hind III. Our results indicate that selected three serovars can be identified by analysis of their DNA with EcoR I and Hind III. It is suggested that restriction endonuclease analysis be a good method in typing and identify leptospira and in studying the differences of special DNA molecules.

DNA Restriction Enzymes↗

PubMed2003

Energy restriction and the risk of spontaneous mammary tumors in mice: a meta-analysis.

Our meta-analysis was aimed at providing a systematic review of the literature regarding the effect of energy restriction on spontaneous mammary tumors in mice and at providing a more precise pooled (summary) estimate of the risk of mammary tumors. A sensitivity analysis was conducted to obtain insight in potential heterogeneity between the animal studies. A literature search was conducted with the following terms to identify relevant articles: animal studies, mammary tumors, fat restricted, dietary carbohydrates, energy restriction and calorie restriction. A criteria list for the assessment of quality items (i.e., study characteristics) in animal experiments was developed that was intended to quantitatively assess potential factors that underlie heterogeneous results of different animal experiments. Incidence figures were used to calculate the risk difference. The pooled risk difference was calculated by random effects meta regression analysis. Fourteen animal experiments were included in this meta-analysis. Publication bias could not be identified. The pooled risk difference for the 14 studies was -0.55 with a narrow 95% confidence interval (-0.69; -0.41), implying that the energy-restricted animal groups developed 55% less mammary tumors than the control groups. No heterogeneity could be detected between the studies based on study characteristics that included the age of mice at the start of intervention, duration of intervention, allocation of the mice, use of ad libitum control group, fertility of the mice and the type of energy-providing nutrient (fat, carbohydrate or protein). This meta-analysis confirms that energy restriction in itself consistently protects against the development of mammary tumor in mice, irrespective of the type of restricted nutrient or other study characteristics.

Animals↗

PubMed2002

Immunomodulation of experimental colitis via caloric restriction: role of Nk1.1+ T cells.

Inflammatory bowel diseases are immune-mediated disorders. Dietary restriction and NK1.1+ liver-associated lymphocytes (LAL) are considered to be involved in immunomodulation of autoimmune diseases. Our aim was to evaluate the effect of caloric restriction on experimental colitis and to determine NK1.1+ LAL function in immunoregulation. Experimental colitis was induced in C57 black mice by intracolonic instillation of trinitrobenzene sulfonic acid. Caloric restriction to 60% of the daily requirement was started 2 weeks prior to, or simultaneously with, colitis induction and continued throughout the study. Control mice were fed ad libitum. Colitis was assessed by standard clinical and macroscopic scores. To determine the mechanism involved in immunomodulation, liver lymphocytes were isolated and analyzed for NK1.1+ T-cell markers by FACS. T-cell function was evaluated by T-cell proliferation. Serum cytokines were measured by ELISA. Dietary restriction to 60% markedly ameliorated experimental colitis in both groups. These mice gained weight and showed improved macroscopic parameters of colitis. NK1.1+ LAL numbers increased fourfold and NKT cytotoxicity twofold in caloric-restricted mice. The antigen-specific T-cell proliferation index decreased (from 4.45 in controls to 1.15), and IFN-gamma and IL-12 serum levels decreased (from 290 to 200 pg and from 122 to 53 pg, respectively) in caloric-restricted mice. Our conclusion was that dietary restriction induced immunomodulation of experimental colitis and ameliorated the disease. This effect was mediated via an increase in NK1.1+ T lymphocytes, which may play a critical role in keeping the T-cell balance in immunoregulation.

Animals↗

PubMed1999

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and H37Ra.

IS6110-based restriction fragment length polymorphism (RFLP) analysis of Mycobacterium tuberculosis H37Rv and its avirulent mutant H37Ra was performed by a number of restriction enzymes, including Nru I, EcoN I, Pst I, and Pvu II. No differences were found in the IS 6110-fingerprints of the study strains by Nru I. One differential IS6110-positive restriction fragment was detected by EcoN I in strain H37Ra, while analysis by Pst I revealed that two fragments of the strain H37Rv were replaced by four novel IS6110-positive fragments in the strain H37Ra. By using Pvu II, a restriction enzyme that cleaves IS 6110 once, and by probing for an IS6110 specific target sequence located to the right of the Pvu II site, we found that the strains H37Rv and H37Ra share 13 IS6110-positive restriction fragments and that one IS6110-positive restriction fragment of H37Rv is replaced by four novel fragments in H37Ra; by probing for an IS6110-specific target sequence to the left of the Pvu II site, 13 shared restriction fragments and 2 differential bands in strain H37Ra were detected. These findings demonstrate that novel insertions of the IS6110 element exist in the avirulent strain H37Ra and raise the question of the role, if any, of IS6110-insertional mutagenesis in the establishment of the avirulent M. tuberculosis H37Ra phenotype.

DNA Transposable Elements↗

PubMed1978

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: classification of petites, and deletion mapping of mitochondrial genes.

We have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HPaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit- markers to regions of the grande restriction map as follows: C (99.3--1.4 map units)--OXI-1 (2.5--15.7)--OXI-2 (18.5--25)--P (28.1--34.2)--OXI-3 (32.2--61.2--OII (60--62)--COB (64.6--80.8--0I (80.4--85.7)--E (95--98.9).

DNA Restriction Enzymes↗