Physical mapping of plasmid pDB101: A potential vector plasmid for molecular cloning in streptococci

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.     

Molecular cloning in streptococci: Physical mapping of the vehicle plasmid pSM10 and demonstration of intergroup DNA transfer

Summary The streptococcal resistance plasmid pSM10 (8.3 kb), a deletion derivative of pSM10419 (22.9 kb) determining constitutive erythromycin and lincomycin resistance, was physically mapped with the restriction endonucleases AvaI, AvaII, EcoRI, HpaI, KpnI, PvuII (one site each), HindIII, HaeII (three sites each), HincII (four sites), and HhaI (five sites). Using the cryptic plasmid pVA318 as cloning vehicle, the largest HindIII fragment of pSM10 (3.3 kb) was shown to contain the erythromycin/lincomycin resistance gene(s) of the plasmid. The AvaII site of pSM10 proved to be suitable as a site for cloning AvaII-generated chromosomal DNA fragments from a group C streptococcal strain in the Challis strain of Streptococcus sanguis (group) H. A detailed physical map of the chimeric plasmid pSM10221 (12.8 kb), a fusion product of pSM10 and the staphylococcal chloramphenicol resistance plasmid pC221 (4.5 kb), is also presented. The plasmid chimera has properties making it potentially useful in development of a doubly selective streptococcal cloning vehicle by searching for insertional inactivation.     

A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance.

Summary This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, BglI, BglII, HindII, HindIII, HpaI, SalI, AvaI, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColE1::Tn5 plasmids, and a ColE1::Tn5 deletion derivative. BalI, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColE1::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a BglII site within this segment results in loss of the neomycin resistance phenotype. Since this BglII site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.     

Sites including those of origin and termination of replication are not freely available to single-cut restriction endonucleases in the supercompact form of simian virus 40 minichromosome.

Simian virus 40 minichromosomes, isolated from both virions (MV) and infected cells (MI), have highly compact structures in buffer containing 0.15 M NaCl and sediment with S values of about 90–100 and 115–130, respectively. Under the electron microscope, also, MI appear the more compact of the two. Only 30–35% of the sites of origin and termination of replication in MV are freely available to the restriction endonucleases $\text{Bgl}$ 1 and $\text{Bam}$ H1. MV are similarly resistant to $\text{Eco}$ R1 and $\text{Hpa}$ II. In contrast, almost no sites in MI are available to any of the above single-cut endonucleases. In 0.6 M NaCl, MV and MI change to relaxed structures of 45–55 S and 50–60 S, respectively, containing 20–24 nucleosomes per genome, and become more sensitive to $\text{Bgl}$ 1, $\text{Bam}$ H1, $\text{Eco}$ R1, and $\text{Hpa}$ II.     

Molecular cloning of part of the mitochondrial DNA of Drosophila melanogaster

We report the construction of recombinant plasmids containing part of the mitochondrial DNA of $\text{Drosophila}\text{melanogaster}$. Of the four fragments of this DNA generated by the restriction endonuclease HindIII, two were successfully cloned into the HindIII site of the plasmid pCM2. Unexpectedly the other two fragments could not be isolated by cloning into the HindIII site of either pCM2 or pBR322. Part of a third fragment, containing the gene for the large ribosomal RNA, was incorporated into the PstI site of pBR322. We show that this recombinant plasmid contains sequences complementary to an abundant RNA species which is present in $\text{Drosophila}$ embryos and which binds to oligo-dT-cellulose.     

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.     

Fine structure of polyoma virus DNA.

A fine structure map of polyoma DNA has been made based on cleavage with a number of restriction endonucleases (including HaeII and III, BamI, HindII and III, BumI, HpaII, and in part, HphI) and depurination of wild-type DNA, the eight HpaII restriction fragments and some HaeIII fragments. This analysis has made possible some correlation with simian virus 40 DNA, and has facilitated detailed examination of various polyoma strains and variants. Sequences from the region of the origin of DNA replication have been examined.     

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant

Summary Streptococcal plasmid pGB301 is an in vivo rearranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.7 kb) gave rise to the deletion derivative pGB301 (9.8 kb, copy number 10) which retained the multiple resistance phenotype of its ancestor (inducible MLS-resistance, chloramphenicol resistance). Among the eight restriction endonucleases used to physically map pGB301 were four that cleaved the plasmid at single sites yielding either sticky (HpaII, KpnI) or bluntends (HpaI, HaeIII/BspRI). Passenger DNA derived from larger streptococcal plasmids (pSF351C61, 69.5 kb; pIP800, 71 kb) was successfully inserted into the HpaII site and, by blunt-end cloning, into the HaeIII/BspRI site. The gentamicin/kanamycin resistance gene of pIP800 was expressed by recombinant plasmids carrying the insert in either orientation. Insertion of passenger DNA into the HaeIII/BspRI site (but not the HpaII site) caused instability of adjacent pGB301 sequences which were frequently deleted, thereby removing the chloramphenicol resistance phenotype. The vector pGB301 has a remarkable capacity for passenger DNA (inserts up to 7 kb) and the property of instability and loss of a resistance phenotype following insertion of passenger DNA into the HaeIII/BspRI site should facilitate the identification of cloned segments of DNA when using this plasmid in molecular cloning experiments.     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Cloning Vectors for Lactococci Based on a Plasmid Encoding Resistance to Cadmium

An 8.8-kb plasmid (pND302) was identified in Lactococcus lactis spp lactis M71 which encodes cadmium resistance (Cd^R). Most of the commercial lactococcal strains tested were sensitive to cadmium. Therefore, Cd^R should provide a useful selectable marker for constructing cloning vectors in lactococci. pND302 was mapped with a number of restriction enzymes and found to contain a unique EcoRI site suitable for cloning. Two E. coli/L. lactis shuttle cloning vectors, pND304 and pND624, were constructed by subcloning of the E. coli plasmids pBR322 and pGEM-7Zf(+) containing a 1.6-kb gene encoding nisin resistance (Nis^R) of lactococcal origin into the EcoRI site of pND302, separately. The E. coli DNA component of pND624 was removed and the resulting plasmid, pND625, consisted of only lactococcal DNA, expressing Nis^R and Cd^R, with two synthetic polylinkers that contain multiple restriction sites for versatile cloning. Both pND302 and pND625 can be transformed by electroporation into L. lactis LMO230 at 10³/μg DNA and maintained stably in LMO230. The results indicated that pND302 and pND625 are potential food-grade cloning vectors for lactococci. Received: 27 November 1995 / Accepted: 29 December 1995     

New cloning vectors using benomyl resistance as a dominant marker for selection inNeurospora crassa and in other filamentous fungi

We describe five new plasmid vectors derived from pBR322 that carry theNeurospora crassa β-tubulin gene conferring resistance to benomyl. The benomyl resistance gene has been modified to eliminate an internalEcoRI site to facilitate the cloning ofEcoRI restriction fragments. These plasmids allow rapid subcloning of fragments from one replicon to another without insert fragment purification due to the presence of different drug resistance markers (resistance to kanamycin, tetracycline, or chloramphenicol) carried on the plasmids. These vectors will allow rapid transformation ofN. crassa and other filamentous fungi to allow phenotypic characterization of subcloned fragments.     

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI-PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.     

Mapping of additional restriction enzyme cleavage sites on bacteriophage MB78 genome

A physical map of bacteriophage MB78 DNA indicating the cleavage sites for the enzymeBglII,ClaI,EcoRI,PvuII,SalI andSmaI comprising of a total of 34 cleavage sites have been constructed earlier. The cleavage sites for a few more restriction endonucleases likeApaI,AvaI,BglI,HindIII,KpnI andXhoI have now been mapped. A total of 72 cleavage sites on MB78 DNA are known by now. Relative positions ofEcoRI I and J fragments which could not be decided earlier has now been determined.     

Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2

Summary The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, Bam H-I, Sal I and Hpa I. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.     

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30°C these miniplasmids are present in 20–50 copies per cell of Escherichia coli, whereas at temperatures above 35°C the plasmids replicate without copy number control during 2–3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plasmid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded β-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.     

Transduction of Plasmid Antibiotic Resistance Determinants with PseudoT-Even Bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 fromEscherichia coli recA ⁺ and recA ^– donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc–segE–uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages , T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limitedin vivo by modification–restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification–restriction systemsEcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification–restriction system.     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.