Degradation of RNA during the autolysis of Saccharomyces cerevisiae produces predominantly ribonucleotides

Autolytic degradation of yeast RNA occurs in many foods and beverages and can impact on the sensory quality of the product, but the resulting complex mixture of nucleotides, nucleosides and nucleobases has not been properly characterised. In this study, yeast autolysis was induced by incubating cell suspensions of Saccharomyces cerevisiae at 30–60 °C (pH 7.0), and at pH 4.0–7.0 (40 °C) for 10–14 days, and the RNA degradation products formed during the process were determined by reversed-phase HPLC. Up to 95% of cell RNA was degraded, with consequent leakage into the extracellular environment of mainly 3′-, 5′- and 2′-ribonucleotides, and lesser amounts of polynucleotides, ribonucleosides and nucleobases. The rate of RNA degradation and the composition of the breakdown products varied with temperature and pH. RNA degradation was fastest at 50 °C (pH 7.0). Autolysis at lower temperatures (30 °C and 40 °C) and at pH 5.0 and 6.0 favoured the formation of 3′-nucleotides, whereas autolysis at 40 °C and 50 °C (pH 7.0) favoured 5′- and 2′-nucleotides. The best conditions for the formation of the two flavour-enhancing nucleotides, 5′-AMP and 5′-GMP, were 50 °C (pH 7.0) and pH 4.0 (40 °C), respectively.     

Degradation of DNA during the autolysis of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The autolysis of yeast cells has practical implications in the production of fermented foods and beverages and flavourants for food processing. Protein and RNA degradation during yeast autolysis are well described but the fate of DNA is unclear. Yeast cells (Saccharomyces cerevisiae) were autolysed by incubating suspensions at 30–60°C (pH 7.0), and at pH 4.0–7.0 (40°C) for 10–14 days. Up to 55% of total DNA was degraded, with consequent leakage into the extracellular environment of mainly 3′- and 5′-deoxyribonucleotides, and lesser amounts of polynucleotides. The rate and extent of DNA degradation, composition of the DNA degradation products and DNase activity were affected by temperature and pH. The highest amount of DNA degradation occurred at 40°C and pH 7.0, where the highest DNase activity was recorded. DNase activity was lowest at 60°C and pH 4.0, where the proportion of polynucleotides in the degradation products was higher. Electronic Publication     

Structural consequences of a 3′ → 3′ DNA interstrand cross-link by a trinuclear platinum complex: unique formation of two such cross-links in a 10-mer duplex

Combined multidimensional nuclear magnetic resonance spectroscopy and electrospray mass spectrometry was used to analyze the platinated DNA adduct of the phase II anticancer drug [{trans-PtCl(NH₃)₂}₂-μ-{trans-Pt(NH₃)₂(NH₂(CH₂)₆NH₂)₂}](NO₃)₄ (BBR3464) with [5′-d(ACG*TATACG*T)-3′]₂. Two 1,2-interstrand cross-links were formed by concomitant binding of two trinuclear moieties to the oligonucleotide. The four DNA-bound platinum atoms coordinated in the major groove at N7 positions of guanines in the 3′ → 3′ direction and the central platinum unit is expected to lie in the DNA minor groove. This is the first report of such a DNA lesion. The melting temperature of the adduct is 76 °C and is 42 °C higher than that of the unplatinated DNA. The sugar residues of the platinated bases are in the N-type conformation and the G9 nucleoside is in the syn orientation, while the G3 nucleoside appears to retain the anti configuration. The secondary structure of DNA was significantly changed upon cross-linking of the two BBR3464 molecules. Base destacking occurs between A1/C2 and C2/G3 and weakened stacking is seen for the C8/G9 and G9/T10 bases. The lack of Watson–Crick base pairing is also seen for A1–T10 and C2–G9 base pairs, whereas Watson–Crick base pairs in the central sequence of the DNA (T4 → A7) are well maintained. While DNA repair proteins may “see” different platinated adducts as bulky “lesions”, the subtle differences involved in base pairing and stacking, as summarized here, may extend to their role as a substrate for repair enzymes. Thus, differences in protein recognition and repair efficiency among the various interstrand cross-links are likely and a subject worthy of detailed exploration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Kinetic analysis of the template effect in ribooligoguanylate elongation

We have undertaken a complete kinetic analysis of the template-directed oligoguanylate synthesis originated in Orgel's laboratory (Inoue and Orgel, 1982). The reaction of guanosine 5′-phospho-2-methylimidazolide, 2-MelmpG, with ribooligoguanylates all 3′–5′ linked, designatedn ³ withn=7−12, was studied in the presence/absence of the complementary template polycytidylic acid, poly(C). Conditions were chosen where poly(C) and 2-MelmpG are in large excess over the oligoguanylate. In the absence of the template at 37 °C the reaction leads to three isomeric oligomers that are elongated by one monomer unit. They are the 3′–5′ linked, (n+1)³, the 2′–5′ linked, (n+1)², and the pyrophosphate product, (n+1) ^p , formed in an approximate ratio 1:2:5. In the presence of the template the reaction is 20-fold faster and yields productsn+1,n+2,n+3 etc. as long as 2-MelmpG is available. Most importantly the formation of the natural, 3′–5′ linked isomer, is enhanced selectively by 140-fold at 37 °C. Qualitative observations allow the conclusion that this enhancement is temperature dependent and increases with decreasing temperature. For example, at 1 °C only the 3′–5′ linked isomers were detected. Initial rates for the disappearance of then ³ oligoguanylate were determined at 1, 23, and 37 °C. It was found that the pseudo-first order rate constant for oligoguanylate elongation was linearly proportional to the 2-MelmpG concentration. This implies that the reaction complex poly(C)·n ³·2-MelmpG does not accumulate under the reaction conditions, a conclusion which is also supported by infrared data (Miles and Frazier, 1982). The implication of the above results with respect to chemical evolution is that lower temperatures, i.e., close to freezing, enhance the regioselectivity of these template-directed reactions and that one way to improve replication models may be sought in finding conditions that favor stable reaction complexes. NASA — National Research Council Research Fellow.     

Continuous production of homopolynucleotides by immobilized polynucleotide phosphorylase

A polynucleotide phosphorylase was immobilized with glutaraldehyde, via an aminopropyl spacer, on porous glass. The specific activity of the immobilized enzyme was effectively increased by the addition of an appropriate ribonucleoside diphosphate on immobilization.A homopolynucleotide could be synthesized continuously by passing a nucleoside diphosphate solution through the immobilized enzyme column. The chain length of the product depended upon the temperature and the flow rate. Polyinosinic acid, poly(I), was continuously synthesized with the immobilized enzyme for about one month without appreciable loss of activity.Polyinosinic acid-polycytidylic acid, poly(I)·poly(C), prepared from poly(I) and poly(C) synthesized with the immobilized polynucleotide phosphorylase, induced interferon-β (IFN-β) in human cultured cells as effectively as that prepared from homopolynucleotides synthesized with the free enzyme.     

Selection for an optimal monovoltine life cycle in an unpredictable environment. Studies on the beetleCatops nigricans Spence (Col., Catopidae)

Summary Catops nigricans reproduces in the autumn. Pre-imaginal development is temperature-dependent and takes place during the winter, followed by aestivation in the early adult stage. This summer diapause is obligatory and temperature independent. It synchronizes the monovoltine life cycle with the annual cycle. In three populations collected near Kiel (54°22′ N, 10°6′ E), K?ln (50°54′ N, 7°6′ E), and Paris (49°25′ N, 2°20′ E), pre-imaginal development slowed and the duration of summer diapause decreased with increasing latitude. Synchronization of the critical breeding interval with the appropriate environmental conditions was achieved through temperature- and photoperiod-dependent sensitivity of ovipositing adults, through different thermal thresholds in eggs, larvae, and pupae, and through sensitivity to photoperiod in third-instars larvae.C. nigricans copes with the unpredictability of climatic conditions in different ways. The local populations have evolved a mean diapause length which probably adjusts the life cycle in most years to the optimal date for reproduction. The mean diapause length was 77 days for Kiel, 98 days for K?ln and 138 days for Paris at 10°C, short-day (=SD).C. nigricans also spreads the risk by varying diapause length. Amongthe progeny of single females the range of diapause duration covered about 70% of the total range of the whole population. The oviposition rate of females confined to subterranean life was the same as females confined to subterranean life was the same as in those living under the influence of a varying photoperiod.C. nigricans should therefore be able to live both in the litter layer of forests and also in the nests and galleries of small mammals.     

Adaptive latitudinal variation of the duration and thermal requirements for development in the ground beetle Amara communis (Panz.) (Coleoptera, Carabidae)

The data are obtained on development time at six constant temperatures (12, 14, 16, 18, 20, 22°C) and thermal requirements for preimaginal development in a ground beetle Amara communis from Arkhangelsk (64°34′N) and St. Petersburg (59°53′N). The larval and pupal development times were found to be significantly shorter in the Arkhangelsk than in the St. Petersburg population under all temperatures. As a result, total preimaginal development appeared to be shorter by 6.2–6.6% in the Arkhangelsk population. The regression lines of the larval, pupal and total (egg-to-adult) development rate on temperature for the Arkhangelsk population run above and steeper than the respective lines for the St. Petersburg population. Both populations share the similar values of the thermal thresholds (7.2–8.2°C). This explains faster preimaginal development in the northern population under all temperatures above the threshold. Thus, the slope of the regression lines increases, i.e., the sum of degree-days decreases, whereas the thermal threshold for development exhibited no distinctive changes from south to north in this species. Adults from Arkhangelsk reared in the experiments appeared heavier on the average in comparison with those from St. Petersburg, especially at 18–22°C. Temperature did not significantly affect adult weight, except the fact that the beetles were slightly heavier at 20 and 22°C. Consequently, the well-known “temperature-size rule” is violated in this species. Relative growth rate in larvae of A. communis increased considerably with temperature rise from 14 to 22°C. It was significantly higher in the beetles from Arkhangelsk at 18–22°C. There were no differences in larval growth rate between the two populations at 14 and 16°C.     

Nuclease Stn α from <Emphasis Type="Italic">Streptomyces thermonitrificans</Emphasis>: Characterization of the Associated Adenylic Acid Preferential Ribonuclease Activity

Nuclease Stn α from Streptomyces thermonitrificans hydrolyses DNA and RNA at the rate of approximately 10:l. The optimum pH and temperature for RNA hydrolysis were 7.0 and 45°C. The RNase activity of nuclease Stn α had neither an obligate requirement of metal ions nor was it activated in the presence of metal ions. The enzyme was inhibited by Zn²⁺, Mg²⁺, Co²⁺, and Ca²⁺; inorganic phosphate; pyrophosphate; NaCl; KCl; and metal chelators. It was stable at high concentrations of urea but susceptible to low concentrations of Sodium dodecyl sulfate and guanidine hydrochloride. The rates by which nuclease Stn α hydrolysed polyribonucleotides occurs in the order of poly A >> RNA >> poly U > poly G > poly C. The enzyme cleaved RNA to 3′ mononucleotides with preferential liberation of 3′AMP, indicating it to be an adenylic acid preferential endonuclease.     

Metal–bipyridine complexes in DNA backbones and effects on thermal stability

Modified oligonucleotides are showing potential for multiple applications, including drug design, nanoscale building blocks, and biosensors. In an effort to expand the functionality available to DNA, we have placed chelating ligands directly into the backbone of DNA. Between one and three nucleosides were replaced with 2,2′-bipyridine phosphates in 23-mer duplexes of DNA. An array of metal ions were added (Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Pt²⁺) and the influences on duplex stability were examined by melting temperature studies. Titrations and UV–vis absorption spectroscopy were used to provide insights into the nature of the metal complexes formed. We found that Ni²⁺ binding to 2,2′-bipyridine typically provided the greatest increase in duplex stability relative to the other metal ions examined. For example, addition of Ni²⁺ to one 2,2′-bipyridine–DNA duplex increased the melting temperature by 13 °C, from 65.0 ± 0.3 to 78.4 ± 0.9 °C. These studies show that metal ions and backbone ligands can be used to regulate DNA structure and stability.     

A probe for detection of G-rich target strands through fluorescence quenching

A modified fluorescent probe U^FAA AAT CTC CGC CGC was synthesized using the nucleoside analogue 3′-O-(N,N′-diisopropylamino-2-cyanoethoxyphosphinyl)-5′-O-(4,4′-dimethoxytrityl)-2′-O-(dansyl-1-sulfonamidohexylaminocarbonyl)uridine for hybridization studies with perfectly matched (U/A) complementary DNA and with a DNA strand having similar G-rich telomeric units at their 3′-ends. Data on the thermal stability and decrease in fluorescence intensity due to the presence of dG units clearly demonstrated the potential application of this approach in DNA diagnostics in homogeneous hybridization assays. The text was submitted by the authors in English.     

Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein: Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA

The effect that Escherichia coli single-stranded DNA binding (SSB) protein has on the single-stranded DNA-dependent ATPase activity of RecA protein is shown to depend upon a number of variables such as order of addition, magnesium concentration, temperature and the type of single-stranded DNA substrate used. When SSB protein is added to the DNA solution prior to the addition of RecA protein, a significant inhibition of ATPase activity is observed. Also, when SSB protein is added after the formation of a RecA protein-single-stranded DNA complex using either etheno M13 DNA, poly(dA) or poly(dT), or using single-stranded phage M13 DNA at lower temperature (25 °C) and magnesium chloride concentrations of 1 mm or 4 mm, a time-dependent inhibition of activity is observed. These results are consistent with the conclusion that SSB protein displaces the RecA protein from these DNA substrates, as described in the accompanying paper. However, if SSB protein is added last to complexes of RecA protein and single-stranded M13 DNA at elevated temperature (37 °C) and magnesium chloride concentrations of 4 mm or 10 mm, or to poly(dA) and poly(dT) that was renatured in the presence of RecA protein, no inhibition of ATPase activity is observed; in fact, a marked stimulation is observed for single-stranded M13 DNA. A similar effect is observed if the bacteriophage T4-coded gene 32 protein is substituted for SSB protein. The apparent stoichiometry of DNA (nucleotides) to RecA protein at the optimal ATPase activity for etheno M13 DNA, poly(dA) and poly(dT) is 6(±1) nucleotides per RecA protein monomer at 4 mm-MgCl₂ and 37 °C. Under the same conditions, the apparent stoichiometry obtained using single-stranded M13 DNA is 12 nucleotides per RecA protein monomer; however, the stoichiometry changes to 4.5 nucleotides per RecA protein monomer when SSB protein is added last. In addition, a stoichiometry of four nucleotides per RecA protein can be obtained with single-stranded M13 DNA in the absence of SSB protein if the reactions are carried out in 1 mm-MgCl₂. These data are consistent with the interpretation that secondary structure within the natural DNA substrate limits the accessibility of RecA protein to these regions. The role of SSB protein is to eliminate this secondary structure and allow RecA protein to bind to these previously inaccessible regions of the DNA. In addition, our results have disclosed an additional property of the RecA protein-single-stranded DNA complex: namely, in the presence of complementary base-pairing and at elevated temperatures and magnesium concentrations, a unique RecA protein-DNA complex forms that is resistant to inhibition by SSB protein.     

Hybridisation between South polar skua (Catharacta maccormicki) and Brown skua (C. antarctica lonnbergi) in the Antarctic Peninsula region

Hybridisation between South polar skua (C. maccormicki) and Brown skua (C. antarctica lonnbergi) in the area of the Antarctic Peninsula is known at least since the beginning of the last century but no survey has been done so far. This paper reviews information on the species composition of skua colonies of more than 10 pairs in the Antarctic Peninsula region, and the incidence of mixed pairs. Morphometrics, population size and breeding success were examined in detail at King George Island. The northward distribution of South polar skuas extended to King George Island (62°11′ S 59°00′ W), with a small outlying population on Signy Island (60°45′ S 45°36′ W), whereas Brown skuas did not breed further south than Anvers Island archipelago (64°46′ S 64°03′ W). The proportion of mixed pairs was highest at the northern end of the ∼500-km-wide hybrid zone. Body size distribution of sympatric skuas from King George Island is clearly bimodal but overlaps considerably and hybrids cannot be identified. Skua population sizes at Potter Peninsula/King George Island remained stable between 1994 and 2004. Numbers of mixed breeding pairs fluctuated more strongly than those of pure species pairs. Breeding success of Brown skuas varied the least.     

The relationship between environmental temperature and clothing insulation across a year

People adapt to thermal environments, such as the changing seasons, predominantly by controlling the amount of clothing insulation, usually in the form of the clothing that they wear. The aim of this study was to determine the actual daily clothing insulation on sedentary human subjects across the seasons. Thirteen females and seven males participated in experiments from January to December in a thermal chamber. Adjacent months were grouped in pairs to give six environmental conditions: (1) January/February = 5°C; (2) March/April = 14°C; (3) May/June = 25°C; (4) July/August = 29°C; (5) September/October = 23°C; (6) November/December = 8°C. Humidity(45 ± 5%) and air velocity(0.14 ± 0.01 m/s) were constant across all six experimental conditions. Participants put on their own clothing that allowed them to achieve thermal comfort for each air temperature, and sat for 60 min (1Met). The clothing insulation (clo) required by these participants had a significant relationship with air temperature: insulation was reduced as air temperature increased. The range of clothing insulation for each condition was 1.87–3.14 clo at 5°C(Jan/Feb), 1.62–2.63 clo at 14°C(Mar/Apr), 0.87–1.59 clo at 25°C(May/Jun), 0.4–1.01 clo at 29°C(Jul/Aug), 0.92–1.81 clo at 23°C (Sept/Oct), and 2.12–3.09 clo at 8°C(Nov/Dec) for females, and 1.84–2.90 clo at 5°C, 1.52–1.98 clo at 14°C, 1.04–1.23 clo at 25°C, 0.51–1.30 clo at 29°C, 0.82–1.45 clo at 23°C and 1.96–3.53 clo at 8°C for males. The hypothesis was that thermal insulation of free living clothing worn by sedentary Korean people would vary across seasons. For Korean people, a comfortable air temperature with clothing insulation of 1 clo was approximately 27°C. This is greater than the typical comfort temperature for 1 clo. It was also found that women clearly increased their clothing insulation level of their clothing as winter approached but did not decrease it by the same amount when spring came.     

Pyrimidine Deoxyribonucleosides Are Phosphorylated to Triphosphates at Low Temperatures Too,But Are Not Incorporated into DNA or Phospholipids.

Abstract

Thymidine (Thd) was phosphorylated to dTTP also at 0°C, both in Ehrlich ascites tumor cells and human tonsillar lymphocytes, but was not incorporated into DNA. The uptake and phosphorylation of ¹⁴C-Thd into the pool showed regular kinetics (Km 6, 6 uM), and the main metabolite was dTTP (75–84%) both at 0°C, and 37°C. Similarly, deoxycytidine (dCyd) was also transported and phosphorylated to nucleotides (76%) at low temperature, but no incorporation into DNA and phospholipid precursor liponucleotides could be detected at 0°C. Under the same conditions, at 37°C, when lymphocytes were labeled with 5-³H-dCyd, 51% of the total pool radioactivity was found in liponucleotides. Transport and phosphorylation of deoxynucleosides seem to be tightly coordinated at both temperatures, which processes are directly coupled to membrane-phospholipid and DNA biosynthesis, but only at physiological temperature while they seem “uncoupled” at low temperature. The fact that nucleoside phosphorylation occures also at low temperature has implications for several experimental techniques used in cell biology.     

Characterisation of a complex restriction/modification system detected in a Bifidobacterium longum strain

 Two type-II restriction endonucleases, BloI and BloII, have been detected in a Bifidobacterium longum strain. BloI is influenced by dam methylation: it cleaves dam ^- but not dam ⁺ DNA. It shows a temperature and pH optimum of 45°C and pH 7.5. Restriction analysis and cloning experiments showed that the recognition sequence is RGATCY and that the enzyme cuts 5′ to the guanine residue. It is an isoschizomer of commercial enzymes, BstYI and XhoII. The second activity is not inhibited by dam methylation. It has a temperature optimum between 25°C and 30°C and shows a broad pH optimum between 4.5 and 7.0. The activity is thermolabile and can be heat-killed by a 5 min incubation at 60°C. Cloning and sequencing experiments revealed that its recognition sequence is CTGCAG and that it cuts 5′ to the second guanine residue in the sequence. This enzyme is the first described isoschizomer of PstI. Received: 22 May 1995/Accepted: 26 July 1995     

Fractionation of plant Polyadenylated RNA on the basis of poly(A) size

Polyadenylated RNA from Vicia faba meristematic root cells was fractionated on the basis of mean poly(A) size by a thermal stepwise elution from poly(U) Sepharose. Such a procedure allowed the elimination of contaminating RNA at 30° and the collection of two populations of purified polyadenylated RNA at 40° and 50°, respectively. RNA eluting at the higher temperature carried a poly(A) segment (mean size of 100 nucleotides), twice as large as the RNA eluting at the lower temperature.     

An Extracellular S1-Type Nuclease of Marine Fungus Penicillium melinii

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5–8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3′–5′ mode to produce 5′-oligonucleotides and 5′-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.     

Thermal maxima for juvenile shortnose sturgeon acclimated to different temperatures

Many populations of shortnose sturgeon, Acipenser brevirostrum, in the southeastern United States continue to suffer from poor juvenile recruitment. High summer water temperatures, which may be exacerbated by anthropogenic activities, are thought to affect recruitment by limiting available summer habitat. However, information regarding temperature thresholds of shortnose sturgeon is limited. In this study, the thermal maximum method and a heating rate of 0.1°C min⁻¹ was used to determine critical and lethal thermal maxima for young-of-the-year (YOY) shortnose sturgeon acclimated to temperatures of 19.5 and 24.1°C. Fish used in the experiment were 0.6 to 35.0 g in weight and 64 to 140 days post hatch (dph) in age. Critical thermal maxima were 33.7°C (±0.3) and 35.1°C (±0.2) for fish acclimated to 19.5 and 24.1°C, respectively. Critical thermal maxima significantly increased with an increase in acclimation temperature (p < 0.0001). Lethal thermal maxima were 34.8°C (±0.1) and 36.1°C (±0.1) for fish acclimated to 19.5 and 24.1°C, respectively. Lethal thermal maxima were significantly affected by acclimation temperature, the log₁₀ (fish weight), and the interaction between log₁₀(fish weight) and acclimation temperature (p < 0.0001). Thermal maxima were used to estimate upper limits of safe temperature, thermal preferences, and optimal growth temperatures of YOY shortnose sturgeon. Upper limits of safe temperature were similar to previous temperature tolerance information and indicate that summer temperatures in southeastern rivers may be lethal to YOY shortnose sturgeon if suitable thermal refuge cannot be found.     

Characterization of MspNI (G/GWCC) and MspNII (R/GATCY), novel thermostable Type II restriction endonucleases from <Emphasis Type="Italic">Meiothermus</Emphasis> sp., isoschizomers of AvaII and BstYI

MspNI and MspNII, isoschizomers of prototype Type II restriction endonucleases AvaII and BstYI, were extracted from an extreme thermophile bacterium belonging to the genus Meiothermus, isolated from the hot sulphur springs in north Himalayan region of India where temperature and pH ranged from 60 to 80°C and 7.5 to 8.5, respectively. The two enzymes were purified to homogeneity using Cibacron-Blue 3GA Agarose, Q-Sepharose and SP-Sepharose chromatography and were homodimers with subunit molecular weights of 27 and 45 kDa, respectively. Restriction mapping and run-off sequencing of MspNI and MspNII cleaved pBR322 DNA showed that they recognized and cleaved 5′-G/GWCC-3′ and 5′-R/GATCY-3′ sites, respectively. MspNI and MspNII worked optimally at 60 and 70°C, 6 and 5 mM MgCl₂, respectively and showed no star activity in organic solvents. Both were resistant to sequence methylation and were stable up to 25 PCR cycles.