BamHI Restriction Sites Research Articles

Comparative genome mapping of bovine encephalitis herpesvirus, bovine herpesvirus 1, and buffalo herpesvirus

A clone library of 11 of 15 BamHI fragments representing 81% of the 140 kilobase DNA genome of the prototype bovine encephalitis herpesvirus strain N569 (BEHV.N569) was constructed. The clones were used to verify the BamHI, BstEII, EcoRI, and HindIII genomic maps for BEHV.N569 published by Engels et al. [Virus Res 6: 57-73 (1986)] for the same virus although some amendments/variations to the BamHI map were found in that 3 previously unidentified restriction sites were identified. Restriction site maps for BglII and KpnI were also derived for BEHV.N569. Southern blot analysis using 32P-labelled BEHV DNA as probe indicated that bovine herpesvirus 1 (BHV1), buffalo herpesvirus 1 (BuHV1) and caprine herpesvirus 1 (CaHV1) were similar and that the similarity occurred throughout the entire length of the genomes; CaHV1 was more distantly related to the other 3 viruses. Because of the similarities BEHV.N569 and BHV1. Cooper cloned DNA fragments were used to construct BamHI, BglII, BstEII, EcoRI, KpnI, and HindIII restriction site maps for the genome of BuHV1 and BamHI, BglII, and KpnI maps for the genome of BHV1.V155, a genital strain.

Ribosomal RNA genes inQuercus spp. (Fagaceae)

The taxonomy of the genusQuercus is still unclear. In order to elucidate the taxonomy of Mediterranean oaks we have analyzed ribosomal RNA genes ofQuercus cerris, Q. coccifera, Q. trojana, Q. ilex, Q. suber, andQ. macrolepis by means of Southern blot hybridization. Oak nuclear DNA was extracted from root tips of 300 acorns and from catkins of single plants. EcoRI and BamHI restriction endonucleases were used. DNA electrophoresis and rRNA/DNA hybridization were performed usingVicia faba rRNA 18 S and 25 S as probes. The rRNA genes of all the species studied have an identical restriction mapping in the 18 S and 25 S regions, while differences in length are present in the intergenic regions.Q. cerris possesses at least four types of genes of 12.1, 11.5, 8.5, and 8.3 kb;Q. coccifera at least three types of 12.4, 10.4, and 10.1 kb;Q. trojana possesses the same rRNA genes asQ. cerris plus another gene type 12.0 kb long, with EcoRI and BamHI restriction sites in the intergenic spacer;Q. ilex at least three types of 12.4, 10.85, and 9.5 kb;Q. suber at least five types of 11.5, 11.0, 8.6, 8.5, and 8.3 kb;Q. macrolepis, finally, at least seven types of 11.5, 11.0, 10.2, 8.6, 8.5, 8.3, and 8.15 kb.Q. coccifera andQ. ilex rDNA appears quite different respect to other species examined, while high similarity seems to exist betweenQ. cerris, Q. trojana, Q. suber, andQ. macrolepis. These results are in agreement with the taxonomic model proposed bySchwarz for the genusQuercus.

Polymorphism in ribosomal DNA repeat units of 12 Hordeum species

Total genomic DNA was isolated from leaves of individual accessions of 12 Hordeum species. SacI, HindIII, and BamHI restriction fragments hybridizing to the wheat rDNA probe pTA71 were compared. Thirteen rDNA repeat unit length variants were recovered, which combined to produce 11 distinct phenotypes. All rDNA repeat units had two SacI sites and no HindIII sites. The rDNA repeat units of H. secalinum, H. hrasdanicum, and H. distichum all had 3 BamHI sites, but in different configurations. The rDNA repeat units of the other species had at least 4 BamHI sites. Ladders of small hybridizing fragments in 8 of 9 of the latter accessions indicated the presence of occasional BamHI sites in some subrepeats within the intergenic spacer regions of the rDNA repeat units. Different distributions of BamHI restriction site maps within the Hordeum sections strengthen the concept of using variation in rDNA restriction sites as a taxonomic character.Key words: Hordeum, ribosomal DNA, polymorphism.

Insertional mutagenesis using a synthetic lac operator

We have developed a novel cassette for generating insertion mutants in multi-copy bacterial plasmids. The cassette consists of synthetic oligodeoxyribonucleotides (oligos) which form a DNA duplex following reconstitution in vitro, due to sequence complementarity. It contains a 21-bp segment of the lac operator ( lacZo), to provide a readily detectable phenotypic marker. Bacterial colonies ha boring plasmids with insertions of this cassette are blue due to constitutive expression of the lac operon resulting from titration of lac repressor molecules by plasmid-borne lacZo sequences. Synthetic oligos containing a desired sequence may be added to the cassette by complementary ends for targeted insertion into plasmids. Sequencing of the resulting insertion mutants is facilitated by using oligos within the cassette as primers for bidirectional sequencing. This allows a complete characterization of each insertion in terms of location, structure of flanking sequences, and orientation of the inserted oligo. We have used this system to construe a series of mutants in early region 1a genes of human adenovirus type 5. For this purpose we designed a cassette which had all three possible translational reading frames open when inserted in one orientation, and all reading frames closed in the other orientation. The cassette also had BamHI restriction sites at each end which could be used to ‘collapse’ mutants, reducing the size of each insert to 6 bp.

Ribosomal DNA repeat unit polymorphism in 25 Hordeum species

Tandemly repeated DNA sequences containing structural genes encoding ribosomal RNA (rDNA) were investigated in 25 species of Hordeum using the wheat rDNA probe pTA71. The rDNA repeat unit lengths were shown to vary between 8.5 and 10.7 kb. The number of length classes (1-3) per accession generally corresponded to the number of nucleolar organizing regions (NORs). Intraspecific variation was found in H. parodii, H. spontaneum and H. leporinum, but not in H. bulbosum. Restriction analysis showed that the positions of EcoRI, SacI and certain BamHI cleavage sites in the rRNA structural genes were highly conserved, and that repeat unit length variation was generally attributable to the intergenic spacer region. Five rDNA BamHI restriction site maps corresponded to the following groups of species: Map A - H. murinum, H. glaucum, H. leporinum, H. bulbosum, H. marinum, H. geniculatum; Map B - H. leporinum; Map C - H. vulgare, H. spontaneum, H. agriocrithon; Map D - H. chilense, H. bogdanii; and Map E - remaining 14 Hordeum species. The repeat unit of H. bulbosum differed from all other species by the presence of a HindIII site. The closer relationship of H. bulbosum to H. leporinum, H. murinum and H. glaucum than to H. vulgare was indicated by their BamHI restriction maps.

A novel vector allowing the expression of genes in a wide range of Gram-negative bacteria

The construction and use of a novel vector allowing the expression of genes in a wide range of Gram-negative bacteria is described. The vector utilizes the regulatory region from IS 50. The 70-bp promoter region was isolated from one of the terminal inverted repeats of Tn 5 by creating EcoRI and SalI or PstI restriction sites by in vitro mutagenesis. This 70-bp region was shown to direct the expression of cat and lacZ genes in different bacterial genera including Alcaligenes, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Different strains containing the cat gene behind the regulatory elements of IS 50 were able to tolerate high concentrations (300 μg/ml) ofchloramphenicol in the medium. The 70-bp promoter region was cloned into a broad-host-range plasmid behind multiple cloning sites to create pAV10, which has unique restriction sites for BamHI, KpnI, SstI, and XbaI. Genes cloned into pAV10 can be expressed in a variety of Gram-negative bacteria.

Indication for deletion of two introns in the oxi-3 gene of a respiratory-competent Saccharomyces cerevisiae strain.

Physical mapping of the mitochondrial DNA of the wild-type Saccharomyces cerevisiae strain RXII revealed that most of the restriction sites as well as the location of the apocytochrome b gene were identical in comparison with the known maps of the mitochondrial genome in other Saccharomyces cerevisiae strains. In the middle of the SalI linearized map of the RXII mitochondrial DNA, a deletion was detected which resulted in the loss of two EcoRI and one BamHI restriction sites. The corresponding region, however, exists in most other laboratory strains of Saccharomyces mapped so far. This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene. The nucleotide sequence of the subunit 1 gene showed that the BamHI site was located close to the aI3-A4 intron-exon junction and the distal EcoRI site close to the aI2-A2 boundary. I therefore conclude that these two introns are deleted in the mitochondrial genome of strain RXII. The exon A3 must have been conserved since this strain was respiratory competent. This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.

Structural organization and evolution of the plastid genome of Vaucheria sessilis (Xanthophyceae)

The plastid DNAs of 18 Vaucheria sessilis strains from various habitats in western Europe were digested with the restriction endonucleases Eco RI, Sal I, Bam HI and Pvu II. Their restriction patterns showed variable fragment divergencies. Two main groups of plastid genomes were recognized, which were substantiated by morphological features. The differences among the restriction patterns could be attributed to the loss or appearance of restriction sites and to minor size variations caused by deletions/insertions. The Sal I and Bam HI restriction sites which together discriminate six different plastid genomes were mapped on the circular molecule of 124 kilobase pairs (kbp). The plastid genomes contain two single copy regions separated by a duplicated set of rRNA genes in inverted orientation. The plastid genomes of several Vaucheria sessilis strains were shown to exist in two inversion isomers caused by intramolecular recombination within the inverted repeat segments.

Use of the X-chromosome linked hypoxanthine phosphoribosyl transferase gene as a marker of cell monoclonality in hemopoietic malignancies

The X-chromosome linked gene encoding hypoxanthine phosphoribosyl transferase (HPRT) has been reported to provide a novel approach to the investigation of cell monoclonality in females based on non-random methylation-sensitive restriction enzyme cleavage of the active vs inactive HPRT alleles and a polymorphic Bam HI restriction site to distinguish the maternal and paternal gene copies. In a survey of 80 females, which included 65 cases of hemopoietic malignancy and 15 normal individuals, we found only nine (11.3%) to be heterozygous (informative) for the Bam HI polymorphism. Monoclonality was demonstrable by HPRT gene analysis in five of six informative cases of leukemia, the exception being a case of acute myeloid leukemia which displayed anomalous methylation of the HPRT gene. Our studies suggest that the applicability of the HPRT gene probe strategy may be limited by (1) the low frequency of informative cases and (2) potential inappropriate methylation of the HPRT gene in a proportion of cases.

Bacteriophage lambda cloning system for the construction of directional cDNA libraries.

We have developed a bacteriophage lambda cloning vector, lambda ORF8, that can be used for the construction of cDNA libraries. The wild-type lambda genome contains five BamHI, five EcoRI, and seven HindIII restriction sites that have all been removed from the genome of lambda ORF8. Sites for these endonucleases are present within the multiple cloning site of lambda ORF8. We report a method for preparing cDNAs that can be cloned in a single orientation in our phage vector. The method utilizes the synthesis of double-stranded cDNA, including priming of first-strand synthesis by oligo(dT). After completion of second-strand synthesis, a bifunctional oligodeoxynucleotide linker is ligated to the cDNA fragments. This linker, which contains a BamHI restriction site, will create a HindIII restriction site when ligated to the 3' end of cDNA fragments. Subsequent treatment of methylated cDNA with HindIII and BamHI endonucleases allows these fragments to be cloned directionally into lambda ORF8. To demonstrate the utility of this cloning system, we prepared a library from 5 micrograms of mRNA isolated from phytohemagglutinin-stimulated human peripheral blood lymphocytes. The primary library contained 2 X 10(8) plaque-forming phage, at least 80% of which contain inserts. A portion of the library was examined for the presence of gamma-interferon-related clones to verify the method had generated a library that was representative of phytohemagglutinin-stimulated peripheral blood lymphocytes. This simple and efficient cDNA cloning system significantly reduces the amount of RNA and effort required for the preparation of large directionally cloned libraries.

A partial cDNA clone for porcine glucosephosphate isomerase: isolation, characterization and use in detection of restriction fragment length polymorphisms.

A cDNA library for porcine skeletal muscle was established in the vector pBR322. The library was screened with an oligonucleotide probe coding for a hexapeptide from glucosephosphate isomerase (Gpi). A positive clone with an insert of about 450 bp and restriction sites for PstI, BamHI and PvuII was isolated. A 362-bp PstI fragment was sequenced and shown to contain the codons for the hexapeptide as well as the remaining 29 amino acids of this Gpi peptide. The PstI fragment was used to probe pig genomic DNA. The restriction enzymes PvuII and SacI detected a set of polymorphisms with five bands, behaving as a set of insertion/deletion polymorphisms.

Restriction site map of the Chlorella virus PBCV-1 genome

The virus PBCV-1, which replicates in a Chlorella-like green alga, has a dsDNA genome. The DNA was mapped for BamHI, HindIII, and PstI restriction sites. The resulting map has a size of 333 kbp and is circular-indicating either covalently closed circular DNA or circularly permuted linear DNA. Several regions of repetitive DNA were also identified and located on the restriction map.

Positive selection vectors based on xylose utilization suppression

High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl − phenotype. This phenomenon was exploited for the development of a versatile positive selection system. The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site ofpUC9, yielding the prototype vector, pLX100. In this construct xylA expression is placed under the transcriptional control of the lac promoter. Transformation of any wild-type E. coli strain with pLX100 results in high levels of xylose isomerase and a Xyl − phenotype. Decreasing the activity below a critical level (approx. 100 u) restores the Xyl + phenotype. pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments. E. coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites. This makes the plasmid an ideal positive-selection cloning vector.

The 18- to 23-kb Deletion of the Macedonian δß-Thalassemia Includes the Entire δ and ß Globin Genes

Abstract Restriction endonuclease mapping analyses were made of DNA from a few members of a Macedonian family with hematological characteristics of delta beta-thalassemia, ie, microcytosis, normal HbA2 levels, and elevated levels of HbF (7% to 14%) with G gamma (average 40.5%) and A gamma T chains (average 59.5%). A large deletion of 18 to 23 kb was present with a 5′ breakpoint within a 670-bp segment of DNA between the HpaI and NcoI restriction sites 5′ to the delta globin gene, and a 3′ breakpoint between the BamHI and HpaI restriction sites located some 9 to 13 kb 3′ to the beta globin gene. This deletion is different from those present in other types of G gamma A gamma(delta beta)zero- thalassemia. The similarity of the hematological expression of these delta beta-thalassemic conditions which have somewhat comparable 5′ breakpoints supports the idea that an important fetal hemoglobin- controlling region lies between the psi beta and delta globin genes.

The 18- to 23-kb deletion of the Macedonian delta beta-thalassemia includes the entire delta and beta globin genes

Restriction endonuclease mapping analyses were made of DNA from a few members of a Macedonian family with hematological characteristics of delta beta-thalassemia, ie, microcytosis, normal HbA2 levels, and elevated levels of HbF (7% to 14%) with G gamma (average 40.5%) and A gamma T chains (average 59.5%). A large deletion of 18 to 23 kb was present with a 5' breakpoint within a 670-bp segment of DNA between the HpaI and NcoI restriction sites 5' to the delta globin gene, and a 3' breakpoint between the BamHI and HpaI restriction sites located some 9 to 13 kb 3' to the beta globin gene. This deletion is different from those present in other types of G gamma A gamma(delta beta)zero-thalassemia. The similarity of the hematological expression of these delta beta-thalassemic conditions which have somewhat comparable 5' breakpoints supports the idea that an important fetal hemoglobin-controlling region lies between the psi beta and delta globin genes.

Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra.

We compared the DNA sequence of the Autographa californica nuclear polyhedrosis virus polyhedrin gene with that of the polyhedrin gene from a morphology mutant called M5. A single point mutation was found at the BamHI restriction site within the polyhedrin coding sequence. This point mutation caused a substitution of leucine for proline at amino acid 58 in the M5 polyhedrin. This point mutation was shown to be responsible for both the appearance of cubic polyhedra and the altered mobility of the polypeptide on sodium dodecyl sulfate-polyacrylamide gels by transferring the M5 polyhedrin gene to the wild-type virus by cotransfection. Recombinants were found which assembled cubic polyhedra in infected cells, had the BamHI restriction site missing, and had an altered mobility of their polyhedrin polypeptide. Computed-predicted secondary-structure analysis indicated that the amino acid at position 58 could be critical to the proper folding of polyhedrin.

Human papillomavirus type 16 DNA in genital tumours: a pathological and molecular analysis.

The presence of human papillomavirus type 16 (HPV16) DNA in 34 genital tract tumours of Italian female patients was investigated by Southern blot hybridization in high stringency conditions. HPV16 DNA was detected in 16 neoplasias, including cervical invasive and intraepithelial lesions as well as vulvar intraepithelial neoplasias and, to a lesser extent, vulvar invasive carcinomas. Appropriate control tissues included in the study were negative. The data suggest that integration of viral DNA had occurred in most tumours, both in invasive and in intraepithelial lesions. HPV16 variants or defective genomes, lacking the BamHI restriction site, were detected in three tumours.

Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation.

By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16 alpha) encoding mouse liver microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice. mRNA selected by hybridization with clone p-16 alpha translated the P-450(16) alpha apoprotein in vitro. Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B. Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-450(16) alpha and P-450(15) alpha specifically inhibited testosterone 16 alpha-hydroxylase activity in microsomes. The cDNA insert of one recombinant plasmid (clone P-16 alpha-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I. 32P-labeled clone p-16 alpha-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice. This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-450(16) alpha in vitro as did the poly(A)+ RNA from females. Thus, the predominant expression of testosterone 16 alpha-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-450(16) alpha gene.

Effects of heterologous ribosomal binding sites on the transcription and translation of the lacZ gene of Escherichia coli

A vector (pKL203) was constructed which contains the promoter-operator region of the lacZ gene and the major part of the coding sequence of the lac operon. The lacZ translation initiation signals [Shine-Dalgamo (SD) sequence and AUG codon] were deleted, and in their place a synthetic linker sequence was inserted, providing single restriction sites for SmaI and BamHI. With this vector constructions were made in which initiation signals of other prokaryotic genes (phage MS2 maturation protein, phage QβA2 gene and tufB gene) were fused to the lacZ gene, giving rise to various fusion proteins. The introduction of N-terminal amino acids (aa) in β-galactosidase (β-gal) which differ from the wild-type aa invariably leads to an enzyme with a strongly reduced thermostability as compared to the wild-type enzyme. Therefore an immunoprecipitation method was used to measure the amount of fusion protein. It was found that these amounts varied strongly from one construction to another. Concomitant determinations of the amounts of lac-operon-specific mRNA showed an unexpectedly large variation among the clones. No strict correlation could be found between the level of lac mRNA and β-gal production. Per molecule of lac mRNA, translation appears to be most efficient when the homologous lacZ initiation signal is present.

The genome of choristoneura fumiferana nuclear polyhedrosis virus: Molecular cloning and mapping of the EcoRI, BamHI, SmaI, XbaI and Bg1II restriction sites

Abstract The DNA genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) was extracted from purified virus preparations and analyzed with restriction endonucleases EcoRI, BamHI, HindIII, SmaI, XbaI and Bg1II. The sizes of the fragments were calculated with respect to mobilities of standard adenovirus-2 DNA fragments. The total size of the genome was calculated to be about 125 kb. Most of the CfMNPV genome was then cloned as EcoRI fragments in the plasmid vector pBR322. The clones were authenticated by electrophoretic analysis in the presence of EcoRI-digested CfMNPV DNA and by hybridization of 32 P nick-translated clones to Southern blots of different enzyme digests of the genome. Data from such hybridization experiments complemented with other mapping procedures were used to construct restriction maps for BamHI, EcoRI, XbaI, Bg1II and SmaI enzymes.