Restriction Enzyme Recognition Sites Research Articles

Direct induction of G1-specific transcripts following reactivation of the Cdc28 kinase in the absence of de novo protein synthesis.

In Saccharomyces cerevisiae, the genes encoding the HO endonuclease, G1-specific cyclins CLN1 and CLN2, as well as most proteins involved in DNA synthesis, are periodically transcribed with maximal levels reached in late G1. For HO and the DNA replication genes, cell cycle stage-specific expression has been shown to be dependent on the Cdc28 kinase and passage through START. Here, we show that cells released from cdc28ts arrest in the presence of cycloheximide show wild-type levels of induction for HO, CLN1, and CDC9 (DNA ligase). Induction is gradual with a significant lag not seen in untreated cells where transcript levels fluctuate coordinately with the cell cycle. This lag may be due, at least in part, to association of the Cdc28 peptide with G1 cyclins to form an active kinase complex because overexpression of CLN2 prior to release in cycloheximide increases the rate of induction for CDC9 and HO. Consistent with this, release from pheromone arrest (where CLN1 and CLN2 are not expressed) in cycloheximide shows no induction at all. Transcriptional activation of CDC9 is likely to be mediated through a conserved promoter element also present in genes for other DNA synthesis enzymes similarly cell cycle regulated. The element contains an intact MluI restriction enzyme recognition site (consensus approximately 5'-A/TPuACGCGTNA/T-3'). Insertion of a 20-bp fragment from the CDC9 promoter (containing a MluI element) upstream of LacZ confers both periodic expression and transcriptional induction in cycloheximide following release from cdc28ts arrest. High levels of induction depended on both the MluI element and CDC28. These results suggest that the activity of trans-acting factors that operate through the MluI element may be governed by phosphorylation by the Cdc28 kinase.

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.

Detection of the G to A mitochondrial DNA mutation at position 11778 in German families with Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy (LHON) is characterized by acute or subacute bilateral (usually permanent) loss of central vision, caused by neuroretinal degeneration. The maternal inheritance is explained by the mitochondrial origin of the disease. Recently, a single mitochondrial DNA (mtDNA) mutation, a G to A substitution at position 11778 that converts a highly conserved arginine to histidine, has been associated with LHON. The mutation eliminates an SfaNI restriction enzyme recognition site and thus provides a method for detection of the mutation by amplification, enzyme digestion and agarose gel electropheresis of polymerase chain reaction (PCR) products. Leukocyte mtDNA from 7 German families with LHON, diagnosed by clinical criteria, was tested for the presence of the G to A mutation at bp 11778. The mtDNa mutation, detected as a loss of the SfaNI site, was seen in one family. The G to A mtDNA mutation is the only known gene alteration associated with LHON so far. It has been identified in patients of different ethnic origin and recent reports strongly support the hypothesis that it represents the most frequent cause of LHON. Identification of the mtDNA replacement mutation using PCR and restriction enzyme digestion requires only a small amount of blood and can be performed rapidly. This method is thus a useful tool in the diagnosis of LHON.

Long-range physical mapping of the α-amylase-1 (α-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Long-range physical maps of the small multigene family of the malt alpha-amylase genes (alpha-Amy-1) located on the long arms of wheat chromosomes 6A (the alpha-Amy-A1 locus) and 6B (alpha-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an alpha-Amy-1 cDNA probe and four gene-specific genomic probes from the alpha-Amy-B1 locus, the size of the alpha-Amy-A1 locus was estimated to be about 700 kb and of the alpha-Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of 'CpG islands' are apparent in alpha-Amy-B1, and three in alpha-Amy-A1. Correlation between recombination frequency and physical distance within the alpha-Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.

Rapid methods in bacterial DNA fingerprinting.

The characterization and comparison of isolates of bacterial species by comparing restriction enzyme digests of their chromosomal DNA (fingerprints) is highly discriminatory for different strains and allows similarities between them to be readily determined. However, the utility of the technique is dependent on the selection of appropriate restriction enzyme(s) and on the method of determining the similarities between the fingerprints generated. We report here a system which circumvents these two problems. The restriction enzyme is selected from amongst those which have a suitable frequency of restriction for given enzyme-genome combinations. The frequencies of restriction enzyme recognition sites are calculated from the frequencies of di- and trinucleotides in sequenced genes from the species of interest using Markov chain analysis. Fingerprints are compared by dividing them up into sections with DNA size standards, scoring the number of bands in a few of these sections, and comparing these scores (numerical profiles) to establish similarities. In this way a single electrophoretic gel yields easily analysable data which can be compared with data from other gels. The time from the acquisition of bacterial isolates to their final characterization is much reduced in comparison to existing methods.

A cell cycle-responsive transcriptional control element and a negative control element in the gene encoding DNA polymerase alpha in Saccharomyces cerevisiae.

Transcription of the POL1 gene of Saccharomyces cerevisiae, which encodes DNA polymerase alpha, the DNA polymerase required for the initiation of DNA replication, has previously been shown to be cell cycle regulated. To understand how the POL1 gene senses cell cycle position, we have investigated the cis-acting elements that respond to the factors that govern cell cycle progression. In this report we demonstrate that a region of 54 nucleotides containing the repeated element ACGCGT, which conforms to an Mlu I restriction endonuclease recognition site, contains all information necessary for transcriptional activation and cell cycle responsiveness. Although oligonucleotides lacking either one or both of the repeated Mlu I sites can function as an upstream activating sequence, the presence of at least one Mlu I site stimulates expression and, moreover, is absolutely essential for cell cycle regulation. A synthetic oligonucleotide corresponding to a 19-base-pair sequence in the POL1 promoter containing one Mlu I site can function as an autonomous cell cycle-responsive upstream element (upstream activation sequence) with temporal regulation indistinguishable from that previously described for the POL1 gene. Thus, the Mlu I site is an essential part of a cis-acting element responsible for the observed periodic activation. This sequence differs from previously defined cell cycle-responsive transcriptional control elements in the yeast HO endonuclease and histone genes. We also present evidence for a negative regulatory element in the 5' flanking region of the Mlu I upstream activation sequence.

Effect of an adjacent base on detection of a point mutation by restriction enzyme digestion

While routinely mapping point mutations within the arginase locus of a collection of hyperargininemic patients, we discovered that a base immediately outside a restriction endonuclease recognition site (TaqI) can eliminate cleavage of this site by this enzyme. The genetic lesion lay in a base immediately flanking a TaqI recognition site within exon 8 of the arginase locus and abolished cutting by approximately 80%. We wish to emphasize the necessity of heeding subtle cues frequently encountered while generating restriction enzyme data, because neither Southern blot maps nor endonuclease digestion of polymerase chain reaction amplified products of exon 8 accurately predicted where the point mutation lay. To our knowledge, this is the first instance of inhibition of cleavage by flanking bases occurring on natural (nonsynthetic) DNA substrates, i.e., within the clinical setting of characterization of a human genetic disorder.

Crystal and molecular structure of a DNA fragment: d(CGTGAATTCACG)

The crystal structure of the dodecanucleotide d(CGTGAATTCACG) has been determined to a resolution of 2.7 A and refined to an R factor of 17.0% for 1532 reflections. The sequence crystallizes as a B-form double helix, with Watson-Crick base pairing. This sequence contains the EcoRI restriction endonuclease recognition site, GAATTC, and is flanked by CGT on the 5'-end and ACG on the 3'-end, in contrast to the CGC on the 5'-end and GCG on the 3'-end in the parent dodecamer d(CGCGAATTCGCG). A comparison with the isomorphous parent compound shows that any changes in the structure induced by the change in the sequence in the flanking region are highly localized. The global conformation of the duplex is conserved. The overall bend in the helix is 10 degrees. The average helical twist values for the present and the parent structures are 36.5 degrees and 36.4 degrees, respectively, corresponding to 10 base pairs per turn. The buckle at the substituted sites are significantly different from those seen at the corresponding positions in the parent dodecamer. Step 2 (GpT) is underwound with respect to the parent structure (27 degrees vs 36 degrees) and step 3 (TpG) is overwound (34 degrees vs 27 degrees). There is a spine of hydration in the narrow minor groove. The N3 atom of adenine on the substituted A10 and A22 bases are involved in the formation of hydrogen bonds with other duplexes or with water; the N3 atom of guanine on G10 and G22 bases in the parent structure does not form hydrogen bonds.

The molecular basis and diagnosis of familial hypercholesterolaemia in South African Afrikaners

Three different point mutations were recently identified in South African familial hypercholesterolaemics. These mutations result in the modification of recognition sites of specific restriction endonucleases. This study describes rapid methods for presymptomatic detection of these defects based on restriction enzyme analysis or allele-specific hybridization of enzymatically amplified genomic DNA. These methods were used to determine the frequencies of the three known low-density lipoprotein (LDL) receptor gene mutations in 138 chromosomes of Afrikaner FH patients. It has been shown that a common mutation at the 3' end of exon 4 (base 681) of the LDL receptor gene is present in about 70% of alleles, while the mutations in exons 9 (base 1285) and 4 (base 523) of the gene are present in about 20 and 10% respectively of the genes studied. These mutations were found in approximately 95% of Afrikaner familial hypercholesterolaemic patients studied, indicating at least three founder members for the disease in this population of South Africa.

Cloning and characterization of the ribosomal RNA gene repeat from Ostertagia ostertagi

Clones of the rRNA genes of Ostertagia ostertagi were selected from a library prepared from the genomic DNA of adult worms of strain LA-2. A 13.4-kb insert in a clone, λOOR78, is comprised of one complete 7.5-kb rDNA unit and portions of two adjacent units. The rDNA unit is directly repeated in a head-to-tail fashion and represents approximately 0.9% of the total genomic DNA. This repeating unit appears to be the only long tandemly repeated sequence in the genome. Restriction enzyme recognition sites in the rDNAs of four strains of O. ostertagi were fully conserved with the exception of one PstI site present in the large rRNA gene which was absent from a proportion of the genes of the LA-2 strain. The rDNA of O. ostertagi is more similar to that of Caenorhabditis elegans in unit length and arrangement than to parasitic helminths previously examined.

Molecular genetics of human cancer predisposition and progression

The development of human cancer is generally thought to entail a series of events that cause a progressively more malignant phenotype. Such a hypothesis predicts that tumor cells of the ultimate stage will carry each of the events, cells of the penultimate stage will carry each of the events less the last one and so on. A dissection of the pathway from a normal cell to a fully malignant tumor may thus be viewed as the unraveling of a nested set of aberrations. In experiments designed to elucidate these events we have compared genotypic combinations at genomic loci defined by restriction endonuclease recognition site variation in normal and tumor tissues from patients with various forms and stages of cancer. The first step, inherited predisposition, is best described for retinoblastoma in which a recessive mutation of a locus residing in the 13q14 region of the genome is unmasked by aberrant, but specific, mitotic chromosomal segregation. Similar mechanisms involving the distal short arm of chromosome 17 are apparent in astrocytic tumors and the events are shared by cells in each malignancy state. DNA sequencing indicates that these events accomplish the homozygosis of mutant alleles of the p53 gene. Copy number amplification of the epidermal growth factor receptor gene occurs in intermediate and late-stage tumors whereas loss of heterozygosity for loci on chromosome 10 is restricted to the ultimate stage, glioblastoma multiforme. These results suggest a genetic approach to defining degrees of tumor progression and the locations of genes involved in the pathway as a prelude to their molecular isolation and characterization.

Host‐controlled modification and restriction as a criterion of evaluating the therapeutical potential of Pseudomonas phage

The recently isolated phages phi ST3 and phi ST1 were compared as to their lysis behaviour in about 100 different P. aeruginosa strains. The growth of phi ST3 varies greatly in different host strains. We demonstrated one case of "non-classical", host-dependent modification and restriction. Here the capability to adsorb, and consequently to reproduce in a given host strain differs, depending on which modification the phage acquired in its former host. The DNA-containing phage phi ST1 displays stable lysis properties in the majority of the host strains. This makes phi ST1 a candidate for therapeutic phage preparations. One of the reasons for stable lysis properties is the apparent selection against recognition sites of restriction enzymes in its genome.

Method of artificial DNA splicing by directed ligation (SDL).

An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.

Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase

The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad poi). Ad poi and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad poi a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad poi genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.

Site-Specific Cleavage of natural mRNA sequences by newly designed hairpin catalytic RNAs

The negative strand of tobacco ringspot virus satellite RNA is a self-cleaving RNA. Its catalytic domain and substrate domain have been identified, and the catalytic domain has been named hairpin catalytic RNA. Here we report the construction of a plasmid containing a modified hairpin catalytic RNA sequence that can be transcribed in vitro. Because this plasmid has two specific restriction enzyme recognition sites at both ends of the substrate binding site in the catalytic RNA sequence, it is possible to construct new plasmids by substituting different sequences in the substrate binding site. Using this plasmid, synthetic DNA, and in vitro transcription, we obtained three ribozymes designed to cleave Escherichia coli prolipoprotein signal peptidase (lsp) mRNA at specific sites. All three ribozymes cleaved the lsp mRNA sequence in vitro at the specific sites, and two of them cleaved it efficiently. Kinetic analyses showed that one had a higher kcat/Km value than that of the well-known hammerhead ribozyme. Problems associated with attaining the goal of expressing these ribozymes in vivo also are discussed.

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

Patterns of digestion of human chromosomes by restriction endonucleases demonstrated byin situ nick translation

A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.

Rabbit Ig kappa 1b6 gene structure.

Previous studies employing Southern blot analyses have detected multiple kappa-homologous sequences within EcoRI-digested DNA isolated from kappa 1b6 homozygous rabbits and kappa 1b6 L chain secreting RMH H158 cell line. These results are very unexpected because the published partial protein sequence for the kappa 1b6 C region is incompatible with an EcoRI restriction endonuclease recognition sequence at the nucleotide level for this allotype. To determine their identity, the kappa-homologous sequences were isolated from DNA extracted from a kappa 1b6 L chain secreting RMH H158 cell line by molecular cloning. Structural analyses demonstrated these sequences to contain genetic information encoding the majority of the kappa 1b6 L chain gene locus. The protein sequence deduced from the kappa 1b6 C region gene was shown to differ from the published partial kappa 1b6 C region protein sequence at five amino acid positions. One of these differences results in a glycine to serine interchange that introduces an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene. Subsequent genomic Southern blot analyses confirmed this structural assignment. Based on these data, the EcoRI-sensitive kappa-homologous fragments present within the genomes of the RMH H158 cell line and kappa 1b6 homozygous rabbits represent the nominal kappa 1 gene and not an alternative kappa isotype or kappa pseudogene. Rabbit Ig kappa 1 allelic nucleotide sequence homology comparisons have shown the isolated kappa 1b6 J-C gene locus to display common structural features previously identified in other kappa 1 alleles.

Solution conformations of protein-binding DNA sequences: Characterization by vibrational spectroscopy and energy minimization procedures

The solution structures of the oligomeric DNAs d(GGGGCCCC) 2 (I), d(CGCGCGCG) 2 (II), d(GGGTACCC) 2 (III), d(GGGATCCC) 2 (IV), d(CGCTAGCG) 2 (V), d(GGAATTCC) 2 (VI), d(GGATCC) 2 (VII) and d(GCATGC) 2 (VIII) were analyzed. In low-salt solutions the overall conformation of these oligomers is predominantly B-like but not A-like as observed in crystals of (I), (III) and (IV) by others. The conformation of (I), (III) and (IV) containing homo (dG) · (dC) tracts is different from that of mixed-sequence DNAs like in (II) and (V) or in poly(dG-dC) · poly (dG-dC). Also (IV), (VI), (VII) and (VIII) involving restriction enzyme recognition sites reveal spectral differences. Attempts to correlate these spectral differences with structural differences within the B-family on the basis of calculated fine structures were started.

Transcriptionally active genome regions are preferred targets for retrovirus integration

We have analyzed the transcriptional activity of cellular target sequences for Moloney murine leukemia virus integration in mouse fibroblasts. At least five of the nine random, unselected integration target sequences studied showed direct evidence for transcriptional activity by hybridization to nuclear run-on transcripts prepared from uninfected cells. At least four of the sequences contained multiple recognition sites for several restriction enzymes that cut preferentially in CpG-rich islands, indicating integration into 5' or 3' ends or flanking regions of genes. Assuming that only a minor fraction (less than 20%) of the genome is transcribed in mammalian cells, we calculated the probability that this association of retroviral integration sites with transcribed sequences is due to chance to be very low (1.6 x 10(-2]. Thus, our results strongly suggest that transcriptionally active genome regions are preferred targets for retrovirus integration.