Restriction Endonuclease Cleavage Sites Research Articles

Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase.

A gene coding for the Nereis sarcoplasmic calcium-binding protein (NSCP) was synthesized and expressed in Escherichia coli. The sequence of the gene was derived from the protein sequence by reverse translation. It possesses a number of unique, regularly spaced, restriction endonuclease cleavage sites to facilitate future site-directed mutagenesis. For the cloning strategy the gene sequence was divided into four parts. Three parts were cloned by ligation of hybridized oligomers and one part by inverse PCR. The protein was expressed as a fusion protein with the bacterial chloramphenicol acetyl-transferase (CAT), which could be easily purified by affinity chromatography. At the junction of the CAT and NSCP moieties a recognition site for the proteolytic enzyme factor Xa was built in. However, the distance between the moieties appeared to be crucial to warrant cleavage. A kinetic analysis showed that NSCP prepared from the sandworm and the one expressed by E. coli behaved in the same way. This system provides a basis for site-specific mutagenesis studies, in order to elucidate the molecular mechanism of cation binding and concomitant conformational changes.

Translation of reovirus RNA species m1 can initiate at either of the first two in-frame initiation codons.

The m1 species of reovirus RNA, which encodes the minor protein component mu 2, possesses two initiation codons, one "strong" according to Kozak rules and preceded by 13 residues (IC1), the other "weak" and located 49 codons downstream of the first (IC2). In reovirus-infected cells only IC2 is used, but initiation from IC1 can be activated, and efficiency of initiation from either initiation codon modulated over a wide range, by coupling unrelated sequences to either or both ends of m1 RNA. For example, when the M1 genome segment is cloned into the thymidine kinase gene of vaccinia virus in such a way that various "irrelevant" stretches of nucleotides comprising restriction endonuclease cleavage sites or promoter remnants are coupled to the 5' end of m1 RNA, translation of the resultant transcripts is also initiated at IC2, with frequencies controlled by the nature of the attached sequences. However, in rabbit reticulocyte lysates these same transcripts are translated from IC1 as well as from IC2, and transcripts in which m1 RNA is preceded by long sequences of encephalomyocarditis virus RNA (from the T7 polymerase-controlled pTM1 vector) are translated exclusively from IC1. By contrast, m1 RNA itself is translated only from IC2. It appears that the most important factor that controls the extent to which translation is initiated from IC1 and IC2 is their "availability," which is likely to be a function of the extent to which the regions on either side of them interact with each other (and also, to a lesser extent, with the 3' untranslated region) either directly or via interaction with host cell proteins. The effects described here are of considerable potential significance when genetic material is rearranged as a result of translocations, insertions, deletions, and amplifications--that is, when sequences that are normally separated are brought into apposition.

Inhibition of Restriction Endonuclease Cleavage Site via Triple Helix Formation by Homopyrimidine Phosphorothioate Oligonucleotides

The ability of pyrimidine rich oligonucleotide phosphorothioate to form stable triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme Ksp 632-I was examined. First, we synthesized double strand oligonucleotides corresponding to the SV4O sites and studied their interaction with homopyrimidine oligodeoxyribonucleotides including replacement of the other chain either PS group (SO-ODNs) in second nucleotide position (from 5′-terminus) and end capped with the PS group at both 3′- and 5′-ends (S 2O-ODNs). The resulting perfect DNA triplexes were detected by gel.mobility shift. The phosphorothioate oligonucleotide analogues (SO-ODNs) and (S 2O-ODNs) were shown to inhibit enzymatic cleavage under conditions that allow for triple helix formation. Inhibition is sequence.specific and occurs in the micromolar concentration range. Of particular interest is the Sp-phosphorothioate analogue (Sp-SO-ODNs) which inhibited endonuclease more than the other phosphorothioate oligonuckotide analogues (Rp-SO-ODNs or S 2O-ODNs).

Construction of a vector for site-specific frameshift mutagenesis containing the mutable hotspot of Salmonella typhimurium TA98 on an M13 bacteriophage.

Frameshift mutations demonstrate a high degree of sequence specificity. In order to provide a vector for site-specific frameshift mutagenesis experiments, a recombinant M13 phage (M13MB102) was constructed by substitution of 27 base pairs of the Salmonella typhimurium hisD3052 sequence for 27 base pairs of the polylinker region of M13mp19. The inserted sequence contains most of the hotspot for frameshift mutations in hisD3052 and its derivative strain TA98. Structural elements of the insert include reiterated bases, direct repeats, and palindromes, and four unique restriction endonuclease cleavage sites. The recombinant phage produced blue plaques when grown in Escherichia coli strain JM105 on X-Gal indicator plates and exhibited a spontaneous mutation frequency similar to that of M13mp19. Methodology is described for preparation, isolation, and purification of closed circular duplex M13MB102 genomes containing an adduct between the SphI and BssHII cleavage sites in the (-)-strand and uracil residues in the (+)-strand. The latter modification decreases replication of the (+)-strand by 4 orders of magnitude and maximizes use of the adducted (-)-strand for in vivo replication. The structure of M13MB102 and the procedures described for introducing adducts at defined positions in its hisD3052 insert provide a convenient approach for evaluating the potential of individual carcinogen-DNA adducts to induce frameshift mutations.

Characterization of a plasmid-encoded urease gene cluster found in members of the family Enterobacteriaceae.

Plasmid-encoded urease gene clusters found in uropathogenic isolates of Escherichia coli, Providencia stuartii, and Salmonella cubana demonstrated DNA homology, similar positions of restriction endonuclease cleavage sites, and manners of urease expression and therefore represent the same locus. DNA sequence analysis indicated that the plasmid-encoded urease genes are closely related to the Proteus mirabilis urease genes.

PCR priming from the restriction endonuclease site 3' extension.

During the course of extending the sequence of the 5' flanking region of a gene involved in drug resistance from the intestinal protozoan parasite Giardia duodenalis, the region was found to be unstable in Escherichia coli. As an alternative to cloning we elected to amplify the region by PCR using the linker-adapter method of Kalman etal. (1), one of the 'vectorette' (Cambridge Research Biochemicals) or 'anchored-PCR' systems (2, 3, 4). The linker—adapter principle depends upon the covalent attachment of the primer to the template at a distal restriction endonuclease cleavage site; the double-stranded linker-primer containing the appropriate restriction endonuclease 5' extension is therefore ligated to the template. A known internal primer is used to amplify by PCR back through the 5' terminus-ligated primer on the other strand, thereby generating a complementary copy from which conventional PCR amplification can be continued to generate copies of the unknown region. For the internal primer we utilised a double-stranded DNA segment from the cloned portion of the gene which we had already sequenced and knew was stable in E. coli, similar to the method described by Sarkar and Sommer (5). However, we encountered a number of amplification artifacts which included the generation of apparently very high molecular weight DNA and anomalous bands. This was partly ascribed to self-priming of the double stranded DNA primer (also see ref. 5) and could be prevented by removing 37 bp from one end by restriction endonuclease cleavage. A second cause of the observed anomalies was the use of the linker—adapter, modified from that described by Kalman et al. (1) to incorporate a Sad terminus and ligated to a distal upstream Sad restriction site. The adapter has a 3' extension instead of the 5' described by Kalman et al. The priming activity of this combination caused the generation of a high molecular weight spread of DNA which eventually would not enter a 1% agarose gel. Standard changes in boundary conditions did not prevent the anomalies.

CDNA cloning and expression of aTalaromyces emersonii amylase encoding genetic determinant inEscherichia coli

Intact mRNA has been isolated from the thermophilic fungusTalaromyces emersonii following growth on starch containing media. This has been used as template to synthesise cDNA. The cDNA was cloned into theEscherichia coli expression vector system, pUC18 and this was used to transformE. coli. Transformed colonies were screened for production of amylase activity and a number of positive recombinants were detected. One of those was found to contain a plasmid named pMH1, which harboured a 1.2 kb insert. Sub-cloning experiments verified that the amylase phenotype was encoded for by this fragment. The fragment was characterised using restriction enzyme cleavage site analysis. The origin of the insert was verified using both Northem and Southem blotting hybridisation analysis ofT. emersonii RNA and DNA respectively.

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.

Geographic populations of the medfly may be differentiated by mitochondrial DNA variation

Restriction enzyme cleavage sites of mitochondrial DNA (mtDNA) from the Mediterranean fruit fly were found to vary among introduced populations in the Neotropics. The survey included samples from 15 established natural populations and 5 laboratory cultures from Hawaii, Central America, South America and West Africa and samples from recent California infestations (1989, 1991). Based on restriction fragment length polymorphisms from 2 enzymes, Hawaii is an unlikely source for the 1989 and 1991 California infestations. Interpopulational variation in mtDNA demonstrates the potential for the technique to trace the process of colonization (geographic spread) by this insect.

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.

Genetic and molecular analysis of pIP417 and pIP419: Bacteroides plasmids encoding 5-nitroimidazole resistance

This report describes a genetic and molecular analysis of two transferable Bacteroides plasmids, pIP417 and pIP419, which carry genetic determinants conferring low-level resistance to 5-nitroimidazoles. The restriction endonuclease cleavage sites for each plasmid were localized. The Ni R genetic determinants of pIP417 and pIP419 plasmids have been cloned into the Bacteroides cloning vector pBI191 (C.J. Smith, J. Bacteriol. 164, 294–301, 1985) as PvuII and Sau3A fragments, respectively. Both inserts had different restriction sites and did not cross-hybridize by Southern blot analysis. Genetic data obtained by cloning into pBI191 clearly show that the PvuII-generated fragments A (Rep) and B (Mob) of pIP417 are involved in plasmid replication and transfer, respectively. Although encoding resistance to the same antibiotic, both plasmids appeared different with regard to the 5-nitroimidazole resistance and replication genetic determinants. However, they share a homology in a region involved, at least in one case, in plasmid transfer. Considering the spontaneous high level of resistance to 5-nitroimidazole in Escherichia coli, this work, based on direct gene cloning into Bacteroides, demonstrates the value of such an approach.

A novel variant of transthyretin (prealbumin), Thr119 to Met, associated with increased thyroxine binding.

A group of patients with prealbumin associated hyperthyroxinemia possess a common single base substitution in the fourth exon of their transthyretin gene. This cytosine to thymine substitution occurs in the codon for residue 119 and results in the predicted replacement of a threonine residue with a methionine at this position. A new NcoI restriction endonuclease cleavage site is created by the point mutation and can be detected by a rapid and simple assay based on the polymerase chain reaction. This variant transthyretin is inherited in an autosomal dominant manner and is apparently not amyloidogenic but is associated with increased thyroxine binding. As healthy heterozygous individuals have normal serum thyroxine concentrations, the hyperthyroxinemia sometimes found may not be primarily due to the variant.

Genetic heterogeneity among human papillomaviruses (HPV) associated with epidermodysplasia verruciformis: evidence for multiple allelic forms of HPV5 and HPV8 E6 genes

In order to get some insight into modifications of human papillomavirus (HPV) genomes which could play a role in tumor progression in epidermodysplasia verruciformis (EV), we studied three EV patients infected by HPV5 and one by HPV8, with cancers containing mostly or only episomal viral genomes with a deletion. The mutants were compared with the full-length genomes present in the benign lesions of each patient. Deletions affected the L1 and/or L2 open reading frames (ORFs), and extended in the 5′ end of the long control region in two cancers. The isolates studied showed a polymorphism of restriction endonuclease cleavage sites and variations in the nucleotide sequence of the E6 ORF and the regions flanking the deletions. However, except for one patient infected by two distinct HPV5 variants, no difference was observed in the nucleotide sequence of isolates cloned from the benign lesions and the cancer of the same patient. This may suggest that point mutations are not involved in tumor progression. Comparison of nucleotide sequence data revealed an unexpectedly high number of nucleotide substitutions among the four HPV5 variants and the HPV8 variant, as compared with HPV5 and HPV8 published sequences. Changes involved 49 of the 457 nucleotides of HPV5 E6 ORF and 14 of the 465 nucleotides of HPV8 E6 ORF. This corresponds to amino acid substitutions affecting 17 of the 157 amino acids of HPV5 E6 proteins and 7 of the 155 amino acids of HPV8 E6 proteins. Half of the substitutions represent nonconservative changes. The variants showing the highest degree of sequence variation were detected in additional EV patients by PCR. This points to the existence of a set of HPV5 and HPV8 stable variants, encoding for multiple allelic forms of the transforming E6 gene.

Generation of hybrid human immunodeficiency virus utilizing the cotransfection method and analysis of cellular tropism

Human immunodeficiency viruses (HIV) isolated from infected individuals show tremendous genetic and biologic diversity. To delineate the genetic determinants underlying specific biologic characteristics, such as rate of replication, cytopathic effects, and ability to infect macrophages and T4 lymphoid cells, generation of hybrid HIV using viruses which exhibit distinct biologic features is essential. To develop methods for generating hybrid HIV, we constructed truncated HIV proviral DNA plasmids. Upon digestion with restriction enzymes, these plasmid DNAs were cotransfected into human rhabdomyosarcoma cells to generate hybrid HIV. The hybrid HIVs derived by this method were infectious upon transmission to both phytohemagglutinin-stimulated peripheral blood lymphocytes and established human leukemic T-cell lines. The virus derived from molecular clone pHXB2 (HIVHTLV-III) productively infected CEMx174 cells. On the other hand, molecular clone pARV (HIVSF2)-derived virus did not show productive infection of CEMx174 cells when used as a cell-free virus. The hybrid HIV containing the 3' end of the genome from pARV and the 5' end of the genome from pHXB2 was effective in infecting CEMx174 cells, but the converse hybrid containing 5' pARV and 3' pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef play a role in the ability of the virus to infect a certain cell type. The intracellular ligation method should be useful in the analysis of related and unrelated HIV-1 isolates with common restriction enzyme cleavage sites.

Molecular Diagnosis of Alcelaphine Herpesvirus (Malignant Catarrhal Fever) Infections by Nested Amplification of Viral DNA in Bovine Blood Buffy Coat Specimens

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.

Repeated DNA in Pneumocystis carinii

A 16-kb DNA fragment designated Rp3-1 and cloned from the genome of rat-derived Pneumocystis carinii was found to contain sequences that were repeated approximately 70 times per genome. The repeated sequences in Rp3-1 spanned at least 10.4 kb. Sequences in Rp3-1 were present on each of the 16 P. carinii chromosomes resolved by field inversion gel electrophoresis. Most of the P. carinii genomic sequences homologous to those in the Rp3-1 clone appeared to be as long as those in the Rp3-1 clone but were highly polymorphic with respect to restriction enzyme cleavage sites. The Rp3-1 DNA fragment appears to be a member of a family of large, degenerate, dispersed repeats.

Mitochondrial DNA variation in geographic populations of Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera; Chrysomelidae)

This study demonstrates variability in restriction enzyme cleavage sites of mitochondrial DNA (mtDNA) among four popalations of Colorado potato beetle (CPB). A suite of three enzymes (EcoRI,HpaI,PstI) was sufficient to discriminate among the populations tested. Individuals heteroplasmic for restriction enzyme patterns were found in some populations. Variability in CPB mtDNA should prove useful in efforts to trace the origin and dispersal of the species in North America.

Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli.

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.

Chloroplast DNA Restriction Site Variation, Phylogenetic Relationships, and Character Evolution Among Sections of North Americn Coreopsis (Asteraceae)

Restriction enzyme cleavage site variation in the chloroplast DNA's (cpDNA's) of 25 of the 44 recognized species representing all 11 sections of North American Coreopsis was employed to assess phylogenetic relationships among the sections. Cladistic analysis of the data produced a phylogeny that is similar to, yet distinct in several aspects from, other hypotheses generated from morphological characters. The phylogeny based on cpDNA consists of two primary lineages within North American Coreopsis. One lineage includes sects. Calliopsis, Coreopsis, Eublepharis, Palmatae, and Silphidium from the eastern and southeastern United States. The other evolutionary line comprises sects. Anathysana, Electra, Leptosyne, Pseudoagarista, Pugiopappus, and Tuckermannia from California and Mexico. These two basic lineages have been recognized in other phylogenies except the rela- tionship of the monotypic sect. Silphidium has been problematical; the cpDNA data are conclusive in showing that sect. Silphidium belongs to the eastern lineage. Only the two sections of annual species from California, Pugiopappus and Leptosyne, are not monophyletic based on cpDNA data. Sects. Electra and Anathysana, which consist of woody species from Mexico, form a monophyletic assemblage based on cpDNA whereas this was not the case for the cladistic analysis of morphological data. Data from flavonoid chemistry and number of isozymes of phosphoglucose isomerase are more supportive of relationships between sects. Electra and Anathysana as inferred from cpDNA as com- pared to morphological data. A paucity of restriction site mutations precludes resolving relationships among the eastern U.S. sections; however, the available cpDNA data are in agreement with the phylogeny generated from morphological features.

Identification of the base substitution responsible for the Ag(x/y) polymorphism of apolipoprotein B-100.

The identification of the base substitution responsible for Ag(x/y) completes the description of the antigen group polymorphisms associated with the apolipoprotein B polypeptide. Surprisingly, all five antigen group polymorphisms alter restriction endonuclease cleavage sites and have associated restriction fragment length polymorphisms, thereby providing a convenient alternative for antigen group phenotyping.