In vivo site-specific genetic recombination promoted by the EcoRI restriction endonuclease.

Site-specific genetic recombinations promoted in vivo by the EcoRI endonuclease has been demonstrated by using constructed hybrid plasmids in which the chloramphenicol resistance gene was inactivated by insertion of DNA fragments at an EcoRI site within the gene. Such recombination can involve either the joining of intracellularly generated cohesive termini of the same DNA fragment or intermolecular ligation of different DNA fragments. DNA cleavage and ligation in vivo are precise: recombinant DNA molecules show functional continuity of the gene sequence cleaved by the enzyme and regeneration of nucleotide recognition sites for both the EcoRI endonuclease and the EcoRI DNA methylase. In other experiments, EcoRI-generated fragments of eukaryotic DNA that had not been modified by the Escherichia coli K methylase were shown to be taken up by bacterial cells and to undergo intracellular ligation to segments of bacterial plasmid DNA.     

To neither bleed nor clot: That is the question

• Patients with atrial fibrillation (AF) when stented with thin‐strut colbalt chrome stents may have a similar outcome to patients without AF.
• Management of patients with AF potentially place patients at enhanced bleeding risk with triple antithrombotic therapy, or potential thrombotic risk if either oral anticoagulation is held or anti‐platelet therapy is truncated.
• Guideline‐based oral anticoagulation for AF is expanding this patient group presenting to the cardiac catheterization laboratory, yet management decisions are still based on limited studies and rely on expert concensus.

     

Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis.

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'.5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site. Recently, we reported the presence of two distinct and separable protein domains within this enzyme--one for the sequence-specific recognition and the other for endonuclease activity. Here, we report the construction of two insertion mutants of Fok I endonuclease. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence specificity as the wild-type enzyme. However, compared with the wild-type enzyme, they cleave one nucleotide further away from the recognition site on both strands of the DNA substrates. Thus, it is possible to alter the cleavage distance of Fok I by protein engineering.     

Evolution of sequence specificity in a restriction endonuclease by a point mutation

Restriction endonucleases (REases) protect bacteria from invading foreign DNAs and are endowed with exquisite sequence specificity. REases have originated from the ancestral proteins and evolved new sequence specificities by genetic recombination, gene duplication, replication slippage, and transpositional events. They are also speculated to have evolved from nonspecific endonucleases, attaining a high degree of sequence specificity through point mutations. We describe here an example of generation of exquisitely site-specific REase from a highly-promiscuous one by a single point mutation.     

The herpes simplex virus 1 segment inversion site is specifically cleaved by a virus-induced nuclear endonuclease.

Nuclear extracts from several tissue culture cell lines (human, primate, and murine) contain an endonuclease that specifically cleaves sequences at the herpes simplex virus 1 (HSV-1) segment inversion site. Mapping studies identified the preferential site of cleavage as a set of tandemly repeated dodecamers, the DR2 repeats. Endonuclease levels vary according to the proliferative state of the cell; little or no activity is detectable in extracts from quiescent cells, whereas high levels are expressed in dividing cells. Also, infection of density-arrested BSC-1 cells with HSV-1 induces a substantial increase (at least 35-fold) in endonucleolytic activity, which is first detectable at about 1 hr after infection at 32 degrees C. The elevated levels of enzyme activity then persist throughout the viral life cycle. In addition to the HSV-1 DR2 repeats, certain other G+C-rich sequences with an asymmetric distribution of purines and pyrimidines on the DNA strands and with appropriate sequences and lengths are substrates for the nuclease. These data indicate that target site recognition by the enzyme is conformation specific rather than sequence specific.     

In vitro adrenocorticotropin/beta-endorphin-releasing activity of vasopressin analogs is related neither to pressor nor to antidiuretic activity

Cells of the 7315c tumor released immunoreactive PRL (IR-PRL). Cholera toxin enhanced this release. Morphine and other opiate agonists inhibited IR-PRL release from both untreated and cholera toxin-treated tumor cells. The opiate-induced inhibition of IR-PRL release was concentration dependent and naloxone sensitive. Cholera toxin also enhanced the adenylate cyclase activity of 7315c tumor tissue. Opiates inhibited enzyme activity in both untreated and cholera toxin-treated 7315c tissue in a concentration-dependent and naloxone-sensitive manner. FK 33824 was more potent than [D-Ala2,D-Leu5]enkephalin in inhibiting IR-PRL release and adenylate cyclase activity. In cholera toxin-treated 7315c tumor tissue, GTP was required for opiate-induced inhibition of adenylate cyclase activity. Nonhydrolyzable analogs of GTP inhibited toxin-stimulated cyclase activity in the absence of an opiate. These results suggest that the 7315c tumor possesses a mu-opiate receptor; stimulation of this receptor inhibits both IR-PRL release and adenylate cyclase activity. An inhibitory guanyl nucleotide component may link the mu-opiate receptor to adenylate cyclase.     

Specificity of substrate recognition by the EcoRI restriction endonuclease.

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris-HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence, d(N-A-A-T-T-N)/d(N-T-T-A-A-N). The enzymatic activity responsible for this substrate recognition is referred to as EcoRI. Cleavage of pVH51 plasmid DNA under EcoRI conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI conditions at sites other than the standard EcoRI substrate.     

DNA from recombinogenic lambda bacteriophages generated by arl mutant of Escherichia coli is cleaved by single-strand-specific endonuclease S1.

When propagated on arl strains (a subclass of Escherichia coli hyper-rec mutants), lambda "Red-" duplication phages accumulated an enhanced potential for recombination. The physical properties of the recombinogenic phages thus obtained ("Arl-" phages) were similar to those of phages grown on arl+ bacteria. However, Arl- phage DNA was cleaved by endonuclease S1 under conditions such that the nuclease is specific for single-stranded DNA;DNA from control phages was S1-resistant. The number of S1 sites (defined by the apparent decrease in single-strand molecular weight) reached a maximum (seven to nine sites per strand of lambda DNA) after five or six rounds of growth on arl bacteria. Similarly, the recombinogenicity of Arl- phages reached a limiting value (recombination frequency, 15%) that was 5 times that of Arl+ phages. Recombinogenicity and S1 susceptibility were accumulated concomitantly during growth on arl+ bacteria. If all increased recombination occurred at the S1 sites, then these regions (about 40 bases each) were about 300 times as recombinogenic as normal DNA regions of the same size, and 1.5 times as recombinogenic as UV-induced lesions. Chromosomal DNA and plasmid DNA (pBR322) from arl cells were more susceptible to nuclease S1 than was DNA from arl+ bacteria. Analysis of the cleavage products suggests that the S1 sites on Arl- lambda phage DNA are located randomly.     

Amyloidogenesis is neither accelerated nor enhanced by injections of preformed fibrils in mice transgenic for wild-type human transthyretin: the question of infectivity.

It is possible to accelerate amyloid formation in both the Senescence Accelerated Mouse, where ApoAIIC is the precursor, and in murine Amyloid A (AA) by the injection of preformed fibrils in the former and amyloid enhancing factor, which appears to consist of AA fibril fragments, in the latter. These two observations have raised the question of whether murine amyloids, like scrapie, are infectious. Injection of preformed fibrils into mice transgenic for many copies of the human wild-type transthyretin gene do not result in acceleration or enhancement of the process of deposition or the conversion of non-Congophilic deposits to fibrils.     

Site-specific cleavage of single-stranded DNA by a Hemophilus restriction endonuclease.

Single-stranded viral DNA of bacteriophage f1 is cleaved into specific fragments by endo R-HaeIII, a restriction endonuclease isolated from Hemophilus aegyptius. The sites of the single strand cleavage correspond to those of the double strand cleavage. A single-stranded DNA fragment containing only one HaeIII site is also cleaved by this enzyme. This observation suggests that the reaction of single-stranded DNA cleavage does not require the formation of a symmetrical double-stranded structure that would result from the intramolecular base-pairing between two different HaeIII sites. Other restriction endonucleases may also cleave single-stranded DNA.     

Cleavage specificity of the restriction endonuclease isolated from Haemophilus gallinarum (Hga I).

The nucleotide sequences in the replicative form (duplex) of phiX174 DNA around six sites cut by Hga I, a restriction endonuclease from Haemophilus gallinarum, have been compared. The enzyme produces a staggered cleavage resulting in a pentanucleotide 5'-terminal extension. The sequences within and immediately surrounding the pentanucleotide cleavage site have no obvious relationship. However, the sequence 5'-G-A-C-G-C-3' 3'-C-T-G-C-G-5' occurs five nucleotide pairs to the left of the cut in the upper strand and 10 nucleotide pairs to the left of the cut in the lower strand and, therefore, is believed to constitute the recognition site. This is a member of the class of restriction endonucleases in which recognition and cleavage sites lack 2-fold rotational symmetry. The method used to define the cleavage site is of general applicability.     

Inactive X chromosome has the highest concentration of unmethylated Hha I sites.

A procedure enabling the highly sensitive detection of accessible restriction endonuclease sites on metaphase chromosomes is described. The procedure is based on the following: (i) a terminal deoxynucleotidyltransferase is used to add a biotinylated nucleotide (Bio-11-dUTP) tail to the 3' hydroxyl terminus generated by the action of a restriction enzyme and (ii) the biotinylated oligonucleotide is detected by a peroxidase-based immunocytochemical method. When used with the 5-methylcytosine-sensitive enzyme Hha I, it gives rise to a pattern close to R and T banding on autosomes. In addition, the staining of one X chromosome in females appears very unusual by its pattern and its strong intensity. This procedure, as applied on a case with a polysomy X chromosome, provides direct evidence of an overall hypomethylation of the inactive X chromosomes.     

Amyloidogenesis is neither accelerated nor enhanced by injections of preformed fibrils in mice transgenic for wild-type human transthyretin: the question of infectivity

It is possible to accelerate amyloid formation in both the Senescence Accelerated Mouse, where ApoAIIC is the precursor, and in murine Amyloid A (AA) by the injection of preformed fibrils in the former and amyloid enhancing factor, which appears to consist of AA fibril fragments, in the latter. These two observations have raised the question of whether murine amyloids, like scrapie, are infectious. Injection of preformed fibrils into mice transgenic for many copies of the human wild-type transthyretin gene do not result in acceleration or enhancement of the process of deposition or the conversion of non-Congophilic deposits to fibrils.     

Characterization of the restriction site of a prokaryotic intron-encoded endonuclease.

The 1016-base-pair (bp) intron in the T4 bacteriophage thymidylate synthase gene (td) contains a 735-bp open reading frame that encodes a protein product with endonucleolytic activity. The endonuclease shows specificity for the intronless form of the td gene. Highly purified endonuclease cleaves the DNA of the intronless form of the td gene in vitro at 24 bp upstream of the exon 1-exon 2 junction, generating a 2-base staggered cut with 3'-hydroxyl overhangs. Although the endonuclease cleaves in exon 1, it requires some exon 2 sequence for recognition. The maximum recognition sequence lies in an 87-bp stretch, from 52 bp upstream to 35 bp downstream of the cleavage site, ending at 11 bp into exon 2. The td intron endonuclease appears involved in the conversion of the intronless form of td to intron-containing td gene in the T-even phages. A role for intron mobility is discussed.     

Polymorphism in mitochondrial DNA of humans as revealed by restriction endonuclease analysis.

Mitochondrial DNA samples from each of 21 humans of diverse racial and geographic origin were digested with each of 18 restriction endonucleases. The sizes of the resulting DNA fragments were compared after gel electrophoresis. No differences among the samples were detected in digest with 7 of the enzymes. Analysis of digests with the remaining enzymes showed one or more differences. Each of the 21 samples could be characterized individually on the basis of these digests. All between-sample differences could be explained by single base substitutions. No evidence for sequence rearrangements (inversions, transpositions) was obtained. Fourteen of the site alterations were shared by two or more samples; six of these were shared between races. The data indicate that individuals differ from a postulated ancestral mtDNA sequence at 0.18% of their base pairs. On the basis of an estimated rate for base substitution of 1% per 10(6) years [Brown, W. M., George, M., Jr. & Wilson, A. C. (1979) Proc. Natl. Acad. Sci. USA 76, 1967-1971], Homo sapiens could have speciated or passed through a severe population constriction as recently as 180,000 years ago. The data suggest that group-specific patterns of cleavage exist. The high resolution and precision afforded by this method of analysis makes possible the investigation of many questions concerning human population genetics, evolution, and recent history.     

In inferior myocardial infarction,neither ST elevation in lead V1 nor ST depression in lead I are reliable findings for the diagnosis of right ventricular infarction

Objective

In the presence of inferior myocardial infarction (MI), ST depression (STD) in lead I has been claimed to be accurate for diagnosis of right ventricular (RV) MI. We sought to evaluate this claim and also whether ST Elevation (STE) in lead V1 would be helpful, with or without STD in V2.

Frequency of prolactin pulsatile release in normal men and in agonadal patients is neither coupled to LH release nor influenced by androgen modulation

OBJECTIVE: We wished to examine and characterize the prolactin pulsatile secretory pattern in both normal and agonadal males in order to assess whether there was any concordance with LH secretion. DESIGN: Patients were sampled every 5 minutes for 12 hours. PATIENTS: We studied five normal and four agonadal men, the latter group before and on testosterone enanthate (TE) (200 mg i.m. every 15 days) treatment. MEASUREMENTS: Prolactin and luteinizing hormone plasma levels were determined using commercial RIA systems. Pulse detection was performed using the DETECT program and the degree of concordance between luteinizing hormone and prolactin was established computing the specific concordance index. RESULTS: We demonstrated the presence of a frequent PRL secretory pattern in normal men (22.8 +/- 1.8 peaks/12h; mean +/- SEM) and in agonadal patients, both in basal conditions and during testosterone treatment (20.5 +/- 2.8 and 18 +/- 1.6 peaks/12h, respectively). The testosterone treatment in agonadal men significantly reduced luteinizing hormone pulse frequency (baseline: 27.5 +/- 2, testosterone administration: 18 +/- 1.3 peaks/12h, P < 0.01) but did not affect pulsatile prolactin release. Using a 10 and 15 minute sampling protocol, we observed that prolactin pulse frequency significantly decreased (P < 0.01) and was similar to the frequencies estimated in previous reports. When luteinizing hormone and prolactin time series were studied to evaluate the possible presence of a specific concordance (SC) between the secretory events of the two hormones, no significant degree of concomitancy was observed neither using the specific concordance index or the cross-correlation analysis. CONCLUSIONS: This report demonstrates (a) the presence of frequent pulsatile release of prolactin in both controls and agonadal patients (baseline and on testosterone enanthate), (b) the use of an appropriate sampling interval (5 minutes) to unmask the prolactin pulsatile release, (c) that in men, luteinizing hormone secretory events are not temporally linked to prolactin secretion, and (d) that androgens, even if reducing luteinizing hormone pulse frequency in agonadal patients, do not significantly affect prolactin pulsatile secretion, suggesting that testosterone and its metabolites do not affect lactotroph activity.     

Optical mapping of site-directed cleavages on single DNA molecules by the RecA-assisted restriction endonuclease technique.

Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves chromosomal DNA at a single EcoRI site within a given gene or selected sequence. We recently described a mapping technique, optical mapping, which uses fluorescence microscopy to produce high-resolution restriction maps rapidly by directly imaging restriction digestion cleavage events occurring on single deproteinized DNA molecules. Ordered maps are then constructed by noting fragment order and size, using several optically based techniques. Since we also wanted to map arbitrary sequences and gene locations, we combined RARE with optical mapping to produce site-specific visible EcoRI restriction cleavage sites on single DNA molecules. Here we describe this combined method, named optical RARE, and its initial application to mapping gene locations on yeast chromosomes.     

ATP-induced conformational changes in the restriction endonuclease from Escherichia coli K-12.

ATP induces a conformational change in the Escherichia coli K-12 restriction enzyme that allows it to discriminate between unmodified and modified DNA recognition sequences. This conformational change does not require ATP hydrolysis. However, ATP hydrolysis is a requirement for DNA cleavage.     

Estimation of genetic variation at the DNA level from restriction endonuclease data.

We consider the estimation of the genetic variation in a natural population when the data are obtained by the use of restriction endonucleases. Under the restriction endonuclease technique, a particular DNA segment is considered and cut wherever a recognition sequence appropriate to the endonuclease occurs. We consider data generated when a random sample of homologous DNA segments is treated in this way with one or a battery of restriction endonucleases. The numbers and sizes of the fragments that result indicate the locations and the frequencies of the recognition sequence (or, with a battery of restriction endonucleases, of each recognition sequence). These frequencies in the sample form the basis for an estimate of the amount of genetic variation in the population.