PstI Cleavage Site Research Articles

Heat shock protein gene 70‐2 polymorphism is differentially associated with the clinical phenotypes of ulcerative colitis and Crohn's disease

A single nucleotide polymorphism in heat shock protein 70-2 (HSP70-2) has been shown to be associated with a severe clinical course in Crohn's disease (CD), but it is not known if such a relationship exists in ulcerative colitis (UC). The aim of the present study was to identify associations between the HSP70-2 polymorphism and the clinical courses of CD and UC in Koreans. Restriction fragment length polymorphism analysis was performed for HSP70-2 polymorphisms using the PstI-cleavage site present in the B allele but not in the A allele of the DNA obtained from 101 patients with CD, 144 patients with UC, and 245 age- and sex-matched healthy controls. Study subjects were classified by disease behavior, severity and extent of disease. In CD, multivariate analysis showed that the AA genotype of HSP70-2 polymorphisms was associated with non-perforating disease (OR 10.10, 95% CI 1.66-15.38) and male sex (OR 3.56, 95% CI 1.04-12.23), and that the BB genotype was associated with severe CD (OR 12.03, 95% CI 1.60-101.56). In contrast, multivariate analysis for UC showed that the AA genotype was associated with severe UC (OR 2.02, 95% CI 1.34-3.03). CD patients with BB genotype of HSP70-2 polymorphism tend to experience a more severe clinical course and allele A is associated with more severe UC. HSP70-2 polymorphism may be used to predict CD and UC phenotypes, which can illuminate immunological differences in CD and UC.

Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

Detection of Varicella-Zoster Virus Genome in the Vitreous Humor From Two Patients With Acute Retinal Necrosis; Lacking or Having a PstI Cleavage Site

Using polymerase chain reaction, we detected the varicella-zoster virus genome in the vitreous humor of two patients with clinically diagnosed acute retinal necrosis. One of the two cases was thought to be caused by infection with a varicella-zoster virus lacking a PstI cleavage site. We could not find any clinical differences between the two substrains. The presence of a PstI cleavage site on the varicella-zoster virus genome might not be associated with the occurrence of acute retinal necrosis.

Restriction endonuclease analysis of varicella-zoster virus DNAs.

The DNAs of 14 strains of varicella-zoster virus (VZV) isolated from epidemiologically unrelated individuals were analyzed by restriction endonuclease cleavage and agarose gel electrophoresis. Small but distinct differences in restriction patterns between the isolates were observed when the DNAs were digested with each of four enzymes examined (EcoRI, SalI, PstI, and HindIII). Six out of 14 isolates (43%) were lacking the PstI cleavage site between K and N fragments; one isolate was lacking the HindIII cleavage site between M and N fragments. No changes were observed in restriction patterns in DNA of one of the strains after 33 passages in cultured cells.

Detection of human parvovirus B19 DNA by using the polymerase chain reaction

The polymerase chain reaction (PCR) was investigated for detecting human parvovirus B19 (B19) DNA in sera. Three pairs of oligonucleotides were evaluated as primers. The best oligonucleotide pair spanned 699 nucleotides, including the region common to VP1 and VP2. After PCR amplification of B19 DNA in serum, a 699-nucleotide DNA fragment was detected on agarose gels. This DNA fragment was B19 DNA, because after Southern transfer it hybridized to a 19-nucleotide internal probe and contained a single PstI cleavage site. Dot blot hybridization with a radiolabeled cloned portion of the B19 genome as a probe was compared with PCR. PCR was 10(4) times more sensitive than dot blot hybridization and, with an internal radiolabeled probe, 10(7) times more sensitive than dot blot hybridization. Of 29 serum specimens from 18 patients with proven B19 infections, 24 were PCR positive. None of 20 serum samples from uninfected controls were positive. Of 22 serum samples positive for immunoglobulin M to B19, PCR detected B19 DNA in 17. Seven serum samples lacking immunoglobulin M were PCR positive. PCR detected B19 DNA in urine, amniotic fluid, pleural fluid, ascites, and leukocyte extracts. PCR is a rapid and simple method for diagnosing infections with human parvovirus B19 but must be combined with serologic tests for immunoglobulin M to B19, especially when testing only a single serum sample.

The baboon gene for apolipoprotein A-I: characterization ofacDNA clone and identification of DNA polymorphisms for genetic studies of cholesterol metabolism

We have isolated and sequenced a baboon apolipoprotein A-I (ApoA-I) clone from a liver cDNA library using a human cDNA hybridization probe. This baboon cDNA contains the entire ApoA-I coding region (801 bp, 267 aa), a 3' untranslated region, and a poly(A) tail. Among comparisons with apoAI sequences from other species, the baboon cDNA is most similar to that of the cynomolgus macaque (99.2% homologous) and least similar to the rat sequence (72.6% homologous). A high frequency of nonsynonymous substitutions are observed by alignment of baboon and human apoAI cDNAs, but comparisons ofhydrophilicity profiles show that protein structure is conserved by substitutions of aa with similar properties. A polymorphic PstI cleavage site was identified by Southern blot analysis and subsequently mapped to the 5' end of the baboon apoAI gene. To identify effects of apoAI allelic variation on cholesterol metabolism, we used immunoblotting to compare the distributions of ApoA-I among lipoprotein size classes in baboons from each genotype under basal and atherogenic diets. We observed an increase of ApoA-I in high density lipoprotein (size class 1) particles after atherogenic diets in homozygotes for one allele, as compared to slight decreases in the other genotypes.

Structure of three spliced mRNAs from region E3 of adenovirus type 2

A cDNA library representing early adenovirus type 2 (Ad2) mRNA was constructed. The cDNA copies were inserted into the PstI cleavage site of the pBR322 plasmid, and clones containing sequences from region E3 of the Ad2 genome were identified by colony hybridization. Selected clones were characterized by restriction enzyme cleavage, hybridization, and partial DNA sequence analysis. The precise structure of three spliced mRNAs was established by comparing the results with the DNA sequence of region E3 from Ad2 (Herissé et al., Nucl. Acids Res. 8 (1980) 2173–2191; Herissé and Galibert, Nucl. Acids Res. 9 (1981) 1229–1249). One of the characterized mRNA species encodes the E3/19K glycoprotein, whereas the other two most likely encode the E3 14K protein. The results demonstrate, moreover, that certain splice points which are used to generate the major E3 mRNAs are also used to splice the supplementary leader segments to the fibre mRNA at late times after infection. Two separate poly(A)-addition sites were identified in region E3 by analysis of the cDNA clones; one is preceded by the hexanucleotide sequence AAUAAA, whereas the other is preceded by an altered hexanucleotide, having the sequence AUUAAA.

Cloning of Xenopus laevis nuclear poly(A)-rich RNA sequences. Evidence for post-transcriptional control.

cDNA/RNA hybridization experiments of polysomal and nuclear poly(A)-rich RNA from early tadpole stages of Xenopus laevis revealed that part of the nuclear poly(A)-rich RNA sequences are not present within the polysomal polyadenylated RNA. For a more detailed analysis of these sequences we have cloned double-stranded cDNA derived from tadpole nuclear poly(A)-rich RNA in the PstI cleavage site of pBR 322. By colony screening with 32P-labelled cDNA from polysomal and nuclear poly(A)-rich RNA of the tadpole stage we could identify and isolate some of the cloned sequences, which are present only within the nuclear RNA. However, hybridization with cDNA from polysomal poly(A)-rich RNA of the gastrula stage indicated that at least one of those sequences which are confined to the nucleus at tadpole stage may serve as mRNA at gastrula stage. We present evidence that nuclear and polysomal poly(A)-rich RNA molecules containing the same nucleotide sequence differ in size and that size reduction at the level of processing precedes and may enable cytoplasmic export. We conclude that, besides stage-specific regulation of transcription, post-transcriptional control mechanisms are also involved in gene expression during embryonic development.

Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli.

Expression of the gene gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, is regulated by growth rate. Using deoxyribonucleic acid from the specialized transducing phage lambda h80 dgnd his as the source of gnd, we cloned restriction fragments carrying the complete gene and portions of it on the plasmid vector pBR322. A hybrid plasmid carrying a 3.7-megadalton HindIII restriction fragment from the phage was prepared and found to be gnd+. Through restriction mapping of this fragment and subcloning segments of it, we prepared a gnd+ hybrid plasmid which carried only 1.85 megadaltons of E. coli deoxyribonucleic acid. A cleavage site for the restriction endonuclease PstI was located on the genetic map of gnd by cloning adjacent EcoRI-PstI restriction fragments and crossing the resulting hybrid plasmids with previously mapped gnd deletion and bacteriophage Mu insertion mutants. A maxicell experiment was used to determine the direction of transcription of gnd, to identify which EcoRI-PstI fragment contains the gnd promote, and to localize th beginning of the structural gene to a region about 850 +/- 150 base pairs from the PstI cleavage site. A fine-structure restriction map surrounding the PstI cleavage site was prepared for endonucleases KpnI, HincII, HaeIII, HpaII, and TaqI.

Deletions and an inversion induced by a resident IS1 of the lactose transposon Tn951.

DNA-DNA filter binding tests, "Southern" blotting experiments and DNA heteroduplex analysis clearly show that Tn951 contains an IS1 element. This IS1-951 sequence is peculiar in that it does not contain the PstI cleavage site which is usually observed on E. coli derived IS1 elements. Nonetheless, IS1-951 induces deletions. This process is temperature dependent. One instance of an IS1-951 induced inversion was observed, the structure of which is compatible with the current models of transposition of IS elements.

Bacteriophage φX174 RF DNA replication in vivo: A study by electron microscopy

We have investigated bacteriophage φX174 RF † † Abbreviations used: RFI, replicative form DNA with both strands closed; RFII, replicative form DNA with one or more discontinuities in either strand: PrI 2, propidium diiodide. DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point. Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 ( PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication. Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained. The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA. Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo. A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9).

In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions

The construction of a plasmid carrying the right end PstI . B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI.C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrates that Mu-specific functions can be analyzed from cloned fragments.

The insertion element IS1 is a natural constituent of coliphage P1 DNA

The presence of one copy of the insertion element IS1 in P1 DNA at map unit 20 of the physical genome map is revealed by restriction enzyme cleavage patterns and electron microscopy. This IS1 element is cleaved once by the restriction endonuclease PstI and extends about 500 to 600 base pairs to the left and 200 to 300 base pairs to the right of the unique PstI cleavage site of P1 DNA. Two P1Cm derivatives, P1Cm246 and P1Cm89, carrying a chloramphenicol resistance determinant contain DNA insertions with two terminal directly repeated IS1 elements. Insertion of such IS1-mediated transposition elements may occur at the IS1 site in the P1 genome or at other sites. The significance of IS1 as a natural constitutent of P1 DNA is discussed.

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system ( HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R· PstI site, (5′)C-T-G-C-A ↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r −m − recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.