Versatile kanamycin-resistance cartridges for vector construction in Escherichia coli

To generate polylinker sequences which can be transferred together with an adjacent selectable marker, two plasmids (pWW-84 and pWW-97) were constructed which contain a kanamycin-resistance gene (KmR) flanked by various restriction sites. From these plasmids KmR-cartridges can be obtained as EcoRI, BamHI, SalI, AccI or HincII fragments for insertion into the appropriate restriction site of any plasmid. The following restriction sites can be introduced with these cartridges: BamHI, SalI (AccI, HincII), EcoRI, SacI, SphI and KpnI (Asp718) all adjacent to KmR, XhoI and HindIII, both within KmR. If desired, KmR can be removed by PstI digestion and religation, creating a single PstI site and leaving all adjacent sites intact.     

Restriction endonuclease cleavage of 5-methyl-deoxycytosine hemimethylated DNA at high enzyme-to-substrate ratios.

We have investigated the ability of a large number of restriction enzymes to digest non-canonically hemimethylated DNA at high enzyme-to-substrate ratios. A single-stranded unmethylated phagemid was used as a template to complete synthesis of the second strand using 5-methyl-dCTP to substitute for all the deoxycytosine residues. A fragment of this double-stranded hemimethylated DNA which contains the multiple cloning site region was used as a substrate. For all the enzymes tested, at least some degree of protection from digestion is observed. Sites completely protected from digestion by their cognate enzymes are SalI, BstXI, SacI, SacII, SmaI, SstI, XhoI, PstI, HinfI, BamHI and AccI. Sites partially protected from digestion by their cognate enzymes are XbaI, HindIII, KpnI, SpeI, ClaI, EcoRI and PvuII. Knowledge of the sensitivity of commonly used restriction enzymes to hemimethylated substrates is useful for several applications, which will be discussed.     

Heterogeneity in the structural basis of the human complement C4A null allele (C4A Q0) as revealed by HindIII restriction fragment length polymorphism analysis

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes. C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes, 21-OHA and 21-OHB, and is also remarkable in the high frequency of the 'null' alleles, C4A Q0 and C4B Q0. The molecular basis for the C4A Q0 allele was studied in 26 families through restriction fragment length polymorphism (RFLP) analysis with C4 and 21-OH cDNA probes after digestion of the DNA with the endonuclease HindIII. The individuals expressing the extended haplotype HLA-A1 (of A2) Cw7 B8 C2C BfS C4AQ0B1 DR3 have a large deletion taking off the C4A and 21-OHA genes.     

Properties of the plasmid pFT15/10-1 isolated from the vaccine strain of Francisella tularensis

Plasmid, designated pFT15/10-1, was isolated from Francisella tularensis vaccine strain 15/10. The plasmid is presented by the homogeneous 5.02 +/- 0.054 Md monomeric circular DNA molecules in electron microscopic preparations. Plasmid size is 7-7.3 kb as defined by electrophoresis in agarose gel. The restriction analysis has revealed that plasmid pFT15/10-1 possesses a single specific cleavage site for restriction endonuclease EcoRI, two sites for restriction endonucleases BamHI, BgIII, HincII, HindIII, PstI, three sites for BglI and SalI, some for AluI, TagI, MvaI, CfrI. Plasmid is not digested by restriction endonucleases SmaI, XmaI, KpnI, MluI. Restriction map of the plasmid was constructed for most frequently used restriction endonucleases.     

Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs.

Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.     

3 micron DNA - an extrachromosomal ribosomal DNA in the yeast Saccharomyces cerevisiae

A new class of extrachromosomal DNA which consists predominantly of covalently closed molecules with lengths around 3 micron, has been detected in Saccharomyces cerevisiae strain 6-1G-P188 from the Peterhof collection. Restriction analysis of the 3 micron DNA as well as of recombinant plasmids carrying HindIII fragments of the 3 micron DNA permitted construction of a physical map of the new extrachromosomal DNA species, and detection of two types differing by one EcoRI restriction site. Molecular hybridization, as well as comparison of the restriction maps, revealed the complete structural identity of the 3 micron DNA with a chromosomal repetitive unit of rDNA containing the genes for 25 S, 18 S, 5.8 S and 5 S rRNAs.     

Identification and characterization of polymorphisms at the HAS alpha1-acid glycoprotein (ORM*) gene locus in Caucasians

Human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a group of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Nonrandom associations were found between the BamHI, EcoRI and BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the individuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms should prove useful for other population and forensic studies.     

Identification of unintegrated forms of Kirsten murine sarcoma viral DNA and restriction endonuclease cleavage map of linear DNA.

We detected unintegrated linear 7.0-kilobase pair DNA and covalently closed circular DNA species in NIH3T3 cells recently infected with Kirsten murine sarcoma virus. Using the linear DNA, we constructed a restriction endonuclease cleavage map and compared it with the map of Harvey murine sarcoma virus. The restriction endonuclease maps of two segments, one 1.2 kilobase pairs (SmaI site) to 3.7 kilobase pairs (HindIII site) from the right end (corresponding to the viral 3' side) and the other 0.5 kilobase pair (SmaI and KpnI sites) to 0.9 kilobase pair (KpnI site) from the left end, were identical in the two virus types.     

An insertion within the factor IX gene: hemophilia BEl Salvador.

A patient with moderate to severe hemophilia B has been found to have a large insertion within his factor IX gene. The site of insertion is located in a DNA segment of approximately 0.8 kb between exon IV and an EcoRI site within intron D. The size of the DNA insertion is approximately 6 kb, and it contains at least two TaqI sites, two EcoRI sites, and one HindIII site. The insert probably originates from outside the FIX gene and does not represent an internal duplication. We propose that this abnormal FIX gene be called FIX El Salvador in recognition of the birthplace of the patient.     

Cloning of the complete human cytomegalovirus genome in cosmids

Purified virion DNA (155 X 10(6) Mr) of human cytomegalovirus (CMV) strain Ad169 was partially cleaved with restriction endonucleases HindIII and EcoRI and cloned in the respective cleavage sites of cosmid pHC79. A complete gene library was established in a set of clones containing the viral DNA in long overlapping segments. Restriction maps for HindIII (29 fragments) and EcoRI (36 fragments) were constructed from the linkage of cosmid-cloned fragments, from double digestions of cloned DNA, and from blot hybridization of labeled cloned viral DNA with restriction fragments of virion DNA and singly or doubly cleaved cosmid clones.     

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.     

The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41

Restriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temperate phage 16-3 have been established. Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, 'evenly' distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome. All the restriction maps have been fitted to each other by computer optimalization. Beyond for conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.     

Molecular cloning of circular unintegrated DNA of two types of the SEATO strain of gibbon ape leukemia virus.

Closed circular unintegrated DNA of the SEATO strain of gibbon ape leukemia virus (GaLV-S) was isolated from canine thymus fibroblasts after cocultivation with chronically infected bat lung fibroblasts. Restriction endonuclease HindIII cleaves GaLV-S DNA once, thus allowing isolation and cloning of HindIII-digested unintegrated DNA in a permitted form. Two clones isolated in the vector, Charon 21A, were nearly identical by restriction enzyme mapping to each of the two types of GaLV-S previously observed. These two types differ at a single SalI site. Unlike previous maps of GaLV-S proviral DNA, however, both clones lack SstI sites in the long-terminal-repeat units. Both the GaLV-S clones and the major species of GaLV-S proviral DNA contain an EcoRI site in the long-terminal-repeat units. The presence of this EcoRI site and the absence of an SstI site in the GaLV-S long-terminal-repeat units differentiate it from all other known GaLV strains and from the closely related nononcogenic simian sarcoma-associated virus. Heteroduplex comparisons of each of the two clones to clones of simian sarcoma-associated virus show no obvious deletion or substitution loops. This suggests that the ability of GaLV-S to induce myeloid leukemia in gibbon apes in not due to an acquired onc gene.     

Dual evolutionary origin for the rat genetic sequences of Harvey murine sarcoma virus.

Detailed restriction endonuclease maps were developed for Harvey murine sarcoma virus (Ha-MuSV) DNA (clone H-1), molecularly closed at its unique EcoRI site in pBR322, for three nonoverlapping subgenomic HindIII clones which together span the entire H-1 clone and for a molecularly cloned DNA copy of a portion of rat 30S RNA (which represents the majority of the rat genetic sequences in Ha-MuSV). Molecular hybridization of the 30S clone to small restriction fragments of clone H-1 revealed a 0.9-to-1.0-kilobase pair region in the 5' half of the Ha-MuSV genome not homologous to the 30S clone, although the 30S clone did contain related sequences in Ha-MuSV on both sides of this nonhomologous region. By using cloned sequences from a segment of the Ha-MuSV nonhomology region as a probe for hybridization to Southern blots of DNA from rat, mouse, bat, and chicken cells, one to three bands were detected in DNA of each species. By contrast, the 30S clone DNA was highly related to many sequences in rat DNA, partially related to fewer mouse DNA sequences, and homologous only to one to three bands in bat and chicken DNA. Earlier work had shown that the 5' half of the Ha-MuSV genome coded for transformation and for the viral p21 protein (Chang et al., J. Virol. 35: 76--92, 1980; Wei et al., Proc. Natl. Acad. Sci. U.S.A., in press). We used two subgenomic HindIII clones whose shared HindIII site mapped within the 5' region of clone H-1 nonhomologous to the 30S clone to test whether the nonhomologous segment might encode the transforming and p21 functions. Although neither of the subgenomic HindIII fragments by themselves induced transformation, ligation of these two nontransforming DNAs to each other did restore p21-mediated transformation. A conclusion consistent with these results is that a region in the 5' half of the Ha-MuSV genome evolutionarily distinct from and not present in rat 30S RNA is essential for transformation and for p21 encoding.     

Plasmid vector for cloning, determination of nucleotide sequence and directed assembly of DNA fragments

A plasmid vector pNIMB has been constructed (starting) from the pUR222 plasmid as a result of substitution of the polylinker containing restriction sites: PstI, SalGI, AccI, HindII, BamHI EcoRI and by other synthetic linkers with additional sites for HindIII and HgaI. Plasmid pNIMB does not differ from the parent one phenotypically. Compared to pUR222 the vector contains an additional site for cloning HindIII fragments of DNA and allows to clone SalGI/BamHI- and PstI/SalGI-fragments. Cloning of DNA fragments in all seven unique sites of pNiMB gives the possibility for sequencing the fragments avoiding their isolation from the gel. Moreover, this vector may be useful for cloning and directed assembly of chemically synthesised DNA fragments when the endonuclease HgaI sites are used.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Chimeric streptococcal plasmids and their use as molecular cloning vehicles in Streptococcus sanguis (Challis).

Chimeric plasmids, which were useful as cloning vehicles in a Streptococcus sanguis (Challis) host vector system, have been constructed. By using three different strategies of restriction endonuclease digestion and ligation, a deoxyribonucleic acid (DNA) fragment bearing an erythromycin resistance determinant was ligated in vitro to a phenotypially cryptic plasmid from Streptococcus ferus. Recombinant plasmids could be recovered after transformation of S. sanguis (Challis) with these preparations. Three useful chimeras were constructed. pVA680, 5.5 megadaltons in size, contained a single KpnI site into which passenger DNA may be spliced. pVA736, 5.0 megadaltons in size, contained single EcoRI, HindIII, and KpnI sites into which passenger DNA may be spliced. The EcoRI and KpnI sites of pVA736 may be used in combination with one another when ligating DNA into this plasmid. pVA738, 3.7 megadaltons in size, contained single HindIII and AvaI sites into which passenger DNA may be spliced. pVA680, pVA736, and pVA738 were stably maintained as multicopy plasmids in S. sanguis (Challis). None of them continued to replicate (amplify) in chloramphenicol-treated cells. By using pVA736 as a vector, we have cloned a chloramphenicol resistance determinant obtained from a large, conjugative streptococcal R plasmid. In addition, chromosomal DNA sequences from Streptococcus mutans have been inserted into pVA736 by using the KpnI-EcoRI site combination.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

Restriction map of corynebacteriophages beta c and beta vir and physical localization of the diphtheria tox operon

The BamHI, EcoRI, HindIII, and KpnI restriction endonuclease maps of corynebacteriophage beta c and beta vir were constructed. beta vir appeared to be identical to beta c, except for an approximate 1-kilobase deletion that removed a BamHI site, two KpnI sites, and three EcoRI restriction sites. The diphtheria tox operon was located by hybridizing in vitro 32P-labeled tox messenger ribonucleic acid to blots of endonuclease-digested beta deoxyribonucleic acids. The messenger ribonucleic acid probe was found to hybridize to a 2.1-kilobase region of the beta genome. Since approximately 1.9 kilobases is required to encode prodiphtheria toxin, the data presented strongly suggest that the tox operon of beta is monocistronic.     

Characterization of avian myeloblastosis-associated virus DNA intermediates.

The major species of unintegrated linear viral DNA identified in chicken embryonic fibroblasts infected with either the avian myeloblastosis-associated viruses (MAV-1, MAV-2) or the standard avian myeloblastosis virus complex (AMV-S) has a mass of 5.3 X 10(6) daltons. An additional minor DNA component observed only in AMV-S-infected cells has a mass of 4.9 X 10(6) daltons. The unintegrated linear viral DNAs and integrated proviruses of MAV-1 and MAV-2 have been analyzed by digestion with the restriction endonucleases EcoRI and HindIII. MAV-2 lacks a HindIII site present in MAV-1. These fragments have been compared to those generated by EcoRI and HindIII digestion of linear viral DNAs of AMV-S. Restriction enzyme digestion of AMV-S viral DNA produced unique fragments not found with either MAV-1 or MAV-2 viral DNAs. The major viral component present in AMV-S stocks has the HindIII restriction pattern of MAV-1. Restriction enzyme analysis of the 5.3 X 10(6)-dalton unintegrated MAV viral DNAs and their integrated proviruses suggests that the DNAs have a direct terminal redundancy of approximately 0.3 megadaltons and integrate colinearly with respect to the unintegrated linear DNA.