A physical map of belomycin-specific fragmentation sites on PM2 bacteriophage DNA

Bleomycin treatment of PM2 DNA results in fragmentation of the genome at several specific sites. Application of restriction endonuclease digestion followed by bleomycin treatment has provided the basis for constructing a physical map of bleomycin fragmentation sites. Eleven sites have been located on the physical map relative toHpa II,Pst I, andHindIII cleavage sites. The fragmentation sites are not clustered in a particular region of the PM2 genome but 3 of the 11 sites do occur between theHpa II andPst I cleavage sites, a segment of DNA which comprises 14% of the PM2 DNA length.     

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.     

Plasmid vectors derived from phage Mu allow direct selection of transformants containing cloned HindIII and PstI fragments

Summary The vector plasmids pKN001 and pKN80 both contain the EcoRI.C fragment of E.coli phage Mu DNA which codes for a killing function that is efficiently expressed upon transformation into Mu-sensitive bacteria. By in vitro insertion of HindIII fragments at the single HindIII site of pKN80 or of PstI fragments at the single PstI site of pKN001 the killing function is inactivated. The resulting plasmids have a selective advantage over the religated vector when transformed into Mu-sensitive bacteria. More than 90% of the transformants contain hybrid plasmids. These results show the usefulness of Mu DNA containing plasmids pKN001 and pKN80 as vectors that allow the direct selection for recombinant plasmids.     

Towards an integrated linkage map of common bean

Summary Two genomic libraries were established to provide markers to develop an integrated map combining molecular markers and genes for qualitative and quantitative morpho-agronomic traits in common bean. Contrasting characteristics were observed for the two libraries. While 89% of the PstI clones were classified as single-copy sequences, only 21% of the EcoRIBamHI clones belonged in that category. Clones of these two libraries were hybridized against genomic DNA of nine genotypes chosen according to their divergent evolutionary origin and contrasting agronomic traits. Eight restriction enzymes were used in this study. PstI clones revealed 80–90% polymorphism between the Andean and Middle American gene pools and 50–60% polymorphism within these gene pools. However, under the same conditions only 30% of the EcoRI-BamHI clones showed polymorphism between the Middle American and Andean gene pools. Hybridization with PstI clones to EcoRI-, EcoRV-, or HindIII-digested genomic DNA resulted in a cumulative frequency of polymorphism of approximately 80%. Hybridizations to BamHI-, HaeIII-, HinfI-, PstI-, and XbaI-digested genomic DNA detected no additional polymorphisms not revealed by the former three enzymes. In the PstI library, a positive correlation was observed between the average size of hybridizing restriction fragments and the frequency of polymorphism detected by each restriction enzyme. This relationship is consistent with the higher proportion of insertion/deletion events compared with the frequency of nucleotide substitutions observed in that library.     

Cloning and sequencing the HinfI restriction and modification genes

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).     

Cloning of the yeast ribosomal DNA repeat unit in SstI and HindIII lambda vectors using genetic and physical size selections.

The size of DNA fragments complementary to ribosomal RNA was determined in SstI and HindIII restriction spectra from totally and partially cleaved yeast (Saccharomyces cerevisiae) DNA. The results indicated that the yeast ribosomal RNA gene cluster consists of 9000 base-pair long tandemly repeated units. Three different repeating units, which are overlapping with respect to their sequences, were cloned as SstI and HindIII fragments with λ vectors. The isolation of these clones was facilitated by genetic or physical preselection for those recombinant phage which contained DNA inserts in the expected size range. Both preselection methods gave about a 30-fold purification with respect to the λ-rDNA clones. A heteroduplex analysis of the clones obtained with a three-component HindIII vector showed that the center part of the λ genome carrying λ recombination and regulation genes (57 to 77% λ) can become inverted without apparent decrease of growth capacities.     

Cloning of the carbohydrate-binding portion of the toxin A gene ofClostridium difficile

A portion of the toxin A gene ofClostridium difficile was cloned into pBR322 withEscherichia coli Chi 1776 as the host. Five identical clones, each containing a 4.7-kbPstI restriction endonuclease fragment and producing toxin A antigens, were detected with affinity-purified, monospecific antibodies against toxin A. Digestion of the cloned DNA withPstI revealed as internal restriction site that resulted in two fragments (2.1 and 2.6 kb in size). Probe DNA from either of these fragments hybridized with DNA in the 4.7 kb region ofPstI-digested, high-molecular-weight DNA from the sourceC. difficile strain, indicating that the internalPstI site is protected. The probe DNA also hybridized with restriction-digested DNA from five additional toxigenic strains, but it did not hybridize with DNA from four nontoxigenic strains. In addition, a DNA fragment from a toxigenic strain ofClostridium sordellii, whose toxin cross-reacts with antibody toC. difficile toxin A, hybridized with the clonedC. difficile DNA. Unlike native toxin A, the cell lysate from the recombinant clone was not cytotoxic to Chinese hamster ovary cells or enterotoxic in hamsters. It did agglutinate rabbit red blood cells, a characteristic of toxin A. The cell lysate also exhibited a line of partial identity when compared with purified toxin A in Ouchterlony assays, and it reacted with monoclonal antibody to toxin A in an enzyme-linked immunosorbent assay. The cloned DNA appears to code for a nontoxic binding portion of toxin A, which is responsible for binding to galactose-1-3-galactose-1-4-N-acetylglucosamine.A preliminary report of this work has been presented by S.B. Price and J.L. Johnson, Abstracts of the Annual Meeting of the American Society for Microbiology, 1986:67.     

Nucleotide Sequence of Polymerase Chain Reaction Product Amplified from Rickettsia japonica DNA Using Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190-kDa antigen gene was cloned into M13mp19 RF DNA at the EcoRI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer-flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii, demonstrating 35 nucleotide substitutions in R. japonica. The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR-amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease AfaI but two PstI sites giving three fragments of 121, 159, and 253 bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii. The cleavage sites for endonucleases AluI, HinfI, and MunI that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did PstI. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

Cloning of human mitochondrial DNA in Escherichia coli

In order to determine its nucleotide sequence, human mitochondrial DNA (mtDNA) purified from term placentae was cloned in Escherichia coli using the plasmid vector pBR322. The products of an mtDNA MboI digestion (23 fragments ranging in size from 2800 to 25 base-pairs (bp)) were ligated with BamHI-cut pBR322. The ampicillin-resistant tetracycline-sensitive colonies obtained upon transformation of E. coli χ1776 were screened by agarose gel electrophoresis of colony lysates, colony hybridization and restriction analysis. All but MboI fragment 2 were obtained in this way. MboI fragments 5 and 8 were each found only once among the 705 clones screened. All other MboI fragments were approximately equally represented in the population of clones except for a slight bias towards smaller fragments. MboI fragment 2 overlaps with the mtDNA BamHI/EcoRI (1.7 kb³) and the 0.9 kb HinIII fragments. These were cloned in similarly restricted pBR322 to provide a set of clones covering most of the mtDNA molecule. Clones representative of each MboI fragment were shown to be complementary to mtDNA by hybridization to Southern blots of mtDNA digests and were thereby partially mapped. Further mapping was obtained by restriction analysis of mtDNA sequentially degraded by exonuclease III. A collection of recombinant clones has thus been obtained using the mtDNA isolated from a single placenta and is now being used to obtain a complete nucleotide sequence of human mtDNA.     

Molecular cloning and sequencing of lysozyme gene of bacteriophage SF6 ofBacillus subtilis

Digestion of bacteriophage SF6 (ofBacillus subtilis) DNA with restriction endonuclease Pst I generates seven fragments (A-G). These fragments were cloned in pBR322 DNA, and the recombinant clones carrying the lysozyme gene were identified by measuring lysozyme activity. The complete nucleotide sequence of 996 bp fragment D, containing lysozymze gene, was determined. One open reading frame was present at 13 bp, and the termination codons were present at 961 bp. The deduced amino acid sequence of the lysozyme gene was also determined.     

Molecular cloning and physical map of bacteriophage PM2 DNA

The entire genome and the DNA fragments of the lipid-containing bacteriophage pM2 were cloned in the pBR322 plasmid vector. A physical map including the sites for the following restriction enzymes was obtained: HpaII, HaeIII, TthI, Sau96I, AvaII, PstI, BstNI, AccI, HincII, HpaI and HindIII. No restriction sites on PM2 DNA were found for BalI, BamHI, BclI, BglI, BglII, BstEII, KpnI, PvuII, SacI, SalI, Sau3A, XbaI and XhoI.     

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI

Summary The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI.The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI.Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.     

RFLP analysis in chrysanthemum. I. Probe and primer development

In order to study genetic variability at the DNA level in chrysanthemum (Dendranthema grandiflora Tzvelev) PstI and HindIII genomic libraries were constructed. Probes from both libraries were tested for the presence of restriction fragment length polymorphisms (RFLPs). Of the probes from the PstI library 91% appeared to hybridize to low-copy genes, while only 35% of those from the HindIII library appeared to do so. The PstI probes were used in further analyses as 79% of them showed RFLPs, whereas the HindIII low-copy number probes gave only 14% polymorphic patterns. Because of the hexaploid character of chrysanthemum, complex patterns generally consisting of 6–12 fragments were visible on a Southern blot after hybridization. To simplify the genetic analysis, locus-specific polymerase chain reaction (PCR) primers were developed that gave simple polymorphic patterns in a number of cases. The RFLP probes and primers developed will be used in future marker-assisted selection in this polyploid crop.     

A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA

Summary The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.     

Restriction cleavage maps of coliphages 186 and P2

Summary The restriction enzymes BamHI, BglII, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, BglII-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and XhoI-1; b) P2, BamHI-3, BglII-2 EcoRI-3, HindIII-0, PstI-3, XbaI-1 and XhoI-0. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

Organization of the interferon-inducible 2′,5′-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse

Interferons (IFNs) induce a 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to ³²P-labeled mouse RNase L cDNA and the 5′-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5′-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.     

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Summary A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20.DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.     

Recombination between chloroplast genomes of Trachystoma ballii and Brassica juncea following protoplast fusion

We document here the presence of a recombinant plastome in a cytoplasmic male sterile (CMS) line of Brassica juncea developed from the somatic hybrid Trachystoma ballii + B. juncea. Restriction endonuclease digestion of the chloroplast (cp) DNA has revealed that the recombinant plastome gives rise to novel fragments in addition to the parent-specific fragments. Analysis of the 16S rRNA region by Southern hybridization shows no variation between B. juncea, T. ballii and the CMS line. The rbcL gene region of the recombinant plastome is identical to that in T. ballii. Analysis with probes for psbA and psbD using single and double DNA digests indicates that the hybridization patterns of the recombinant plastome are identical to those of the parents in digests obtained with some restriction enzymes, while novel bands hybridize to probes in other digests. In the psbA region, a B. juncea-specific PstI site and a T. ballii-specific EcoRI site are found in the recombinant plastome. The psbD region of the recombinant plastome contains a B. juncea-specific HindIII site and T. ballii-specific BamHI and HpaII sites. These results indicate the occurrence of intergenomic recombination between the chloroplasts of T. ballii and B. juncea in the somatic hybrid from which the CMS line was developed. The recombined plastome appears to be a mosaic of fragments specific to both parents and the recombination event has occurred in the single-copy regions. These recombinational events have not caused any imbalance in the recombinant plastome in terms of chloroplast-related functions, which have remained stable over generations. Received: 3 March 1998 / Accepted: 5 August 1998