Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Construction of plasmid vectors that facilitate subcloning and recovery of yeast and Escherichia coli DNA fragments

We have constructed a plasmid vector (pNF2) which is a derivative of the multicopy yeast cloning vehicle YEp24. This derivative contains a single BamHI site flanked immediately on each side by SalI sites. The latter site was selected because it appears to be infrequent in yeast nuclear DNA. Thus, DNA fragments produced by partial digestion with enzymes (such as Sau3A) that cut at frequent intervals and leave single-stranded ends that have sequence homology with BamHI sites, can be conveniently subcloned into this site. Such fragments can then be excised intact by digestion with SalI enzyme. Plasmid pNF2 also contains the kanamycin-resistance (kanR) gene derived from Tn903 and confers resistance in yeast to the antibiotic G418. pNF2 was converted into an integrating vector (pNF3) by deleting a 2.2-kb EcoRI fragment containing a sequence that determines autonomous replication in yeast. Further deletion of a HindIII fragment containing the yeast URA3 gene converts the plasmid into one containing only pBR322 sequences plus the kanR gene (pNF4).     

基于同尾酶技术构建CCL3L1 基因串联重组质粒的方法

目的:利用同尾酶技术将CCL3L1基因重复连续插入pcDNA6.2-GW/miR载体,构建含有CCL3L1基因串联体的重组质粒,实现小片段CCL3L1有效延长。方法:PCR扩增CCL3L1基因并在引物的两端设有同尾酶BamHI和BglII限制性内切酶位点,纯化PCR产物插入pMD18-T载体,阳性克隆命名为pMD18T-CCL3L1。BamHI和BglII双酶切pMD18T-CCL3L1和pcDNA6.2-GW/miR载体后将第一个CCL3L1片段插入pcDNA6.2-GW/miR载体命名为pcDNA6.2-CCL3L1-1。由于载体本身在BglII位点后带有XhoI酶切位点利用BamHI和XhoI切割pcDNA6.2-CCL3L1-1回收CCL3L1片段,BglII和XhoI切割pcDNA6.2-CCL3L1-1回收大片段做载体重组形成含有两个连续CCL3L1片段的质粒命名为pcDNA6.2-CCL3L1-2,重复此步骤可得到含有N个CCL3L1基因串联体的重组质粒pcDNA6.2-CCL3L1-X。结果:经酶切和测序证实成功构建含有4个CCL3L1基因串联体的重组质粒pcDNA6.2-CCL3L1-4,并同时产生含有1个和2个CCL3L1基因串联体的重组质粒。结论:利用同尾酶技术可以快速有效地构建CCL3L1基因串联重组质粒,实现目的片段的无限扩大,为小片段基因表达的研究奠定基础。     

Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli.

The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth.     

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.     

Restriction endonuclease mapping of the proviral DNA of the exogenous RIII murine mammary tumor virus

Cellular DNA containing integrated murine mammary tumor virus (MuMTV) was isolated from FeI/C6 feline kidney cells and CCL64 mink lung cells infected with milkborne RIII MuMTV. By using restriction enzyme HpaI, intact RIII MuMTV provirus (length, 8.7 kilobases [kb]) was excised from the cellular DNA. Subsequent restriction endonuclease analysis of this HpaI fragment with KpnI, HindIII, EcoRI, BamHI, BglII, PstI, SstI, SalI, and XhoI enabled us to construct a map of the RIII virus genome. A comparison of this map with the maps of the GR and C3H MuMTV's revealed that there are greater sequence differences between the RIII virus and the GR and C3H MuMTV proviruses than there are between the GR and C3H proviruses. The following are features of the restriction map unique to the RIII provirus: the presence of three BamHI and two EcoRI cleavage sites, a HpaI cleavage site in the terminal 3'-5' repeat unit of the provirus, and the absence of an XhoI cleavage site. Another distinguishing feature of the RIII provirus is that the sizes of some of the restriction fragments produced by cleavage of the RIII provirus with PstI are different from the sizes of the fragments obtained by PstI cleavage of the GR and C3H proviruses. Like the GR proviral DNA, the RIII proviral DNA has three SstI (SacI) cleavage sites, whereas the C3H provirus has only two SstI sites. HpaI digestion of MuMTV-infected mink lung cell DNA revealed only one class of provirus (an 8.7-kb fragment); however, we observed several minor classes of RIII proviral DNA in addition to the major class of provirus DNA in infected cat kidney cells. PstI digestion of the HpaI 8.7-kb fragments from both feline and mink cells generated a 3.7-kb DNA fragment identical in size to a PstI-generated fragment that has been found in GR and C3H milkborne virus-infected cells. Although a fragment similar in size to the milkborne 3.7-kb PstI fragment has been found as an endogenous component in many C3H and GR mouse tissues, we did not observe such an endogenous fragment in the RIII mouse strain. Therefore, the 3.7-kb fragment may be useful as a marker for the milkborne RIII MuMTV provirus in RIII mice.     

Genetic characteristics and physical organization of the R-plasmid pBS52 with a broad range of bacterial hosts

The genetic and physical data on Pseudomonas aeruginosa plasmid pBS52 coding for the resistances to ampicillin, streptomycin and sulfonamids have been obtained. This conjugative plasmid is transferable to a broad range of gram-negative bacterial hosts and compatible with the broad host-range plasmids from all known incompatibility groups. The plasmid size has been determined (38 Kb) and a physical map has been constructed using restriction endonucleases EcoRI, EcoRV, BamHI, BglII, PstI, PvuII, SalI, SlaI. The presence of a fragment, approximately 200 bp in size, which contains the sites for many of widely used restriction endonucleases is a characteristic feature of the plasmid pBS52.     

Identification and characterization of a new replication region in the Neisseria gonorrhoeae beta-lactamase plasmid pFA3.

The 7.1-kilobase-pair (kbp) plasmid pFA3 specifies TEM beta-lactamase production in Neisseria gonorrhoeae. We studied the minimal region required for replication of this plasmid in Escherichia coli by constructing a set of nested deletions of the 3.4-kbp PstI-HindIII fragment. The smallest fragment capable of maintenance in E. coli when ligated to a streptomycin-spectinomycin resistance cassette was 2.0 kbp in size and was different from another autonomously replicating fragment of pFA3 reported by K. H. Yeung and J. Dillon (Plasmid 20:232-240, 1988). The fragment contained single BamHI and XbaI sites and specified a 39-K protein. Fragments subcloned from the minimal region or constructed by deletion from the 3' or 5' ends were not capable of autonomous replication. Mutants constructed by end filling and religating DNA cleaved at the BamHI or XbaI sites were not capable of autonomous replication and no longer produced the 39K protein. These results suggest that replication is dependent on the 39K protein. DNA sequence analysis of the region showed an A-T-rich region followed by four 22-bp direct repeats followed by an open reading frame encoding a 39K basic protein.     

Restriction map, partial cloning and localization of 9S and 12S kinetoplast RNA genes on the maxicircle component of the kinetoplast DNA of Leishmania tarentolae.

We have constructed a restriction map of the maxicircle component of the kinetoplast DNA of Leishmania tarentolae for the enzymes EcoRI, Bam HI, HaeIII, HpaII, SalI, BglII and HindIII. The 9 and 12S kinetoplast RNAs were localized on this map. Two fragments of this maxicircle molecule were cloned in the bacterial plasmid, pBR322, including a 4.4 . 10(6) dalton EcoRI/BamHI fragment which contains the 9 and 12S RNA genes.     

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.     

Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

Cloning the origin of transfer region of the resistance plasmid R1

The insertion of a 7.7-kb EcoRI fragment of the resistance plasmid R1 into pBR325 yielded a plasmid which is mobilizable by pDB12, a multicopy derivative of R1drd-19 lacking most of the resistance determinants. The vector alone was not mobilizable in this system. From this observation we conclude that we have cloned the origin of transfer (oriT) of R1. After inserting a 5.3-kb PvuII-EcoRI fragment of the 7.7-kb region into pUC9 the DNA was cleaved randomly with DNaseI and BamHI linkers were attached to the ends. A subsequent BamHI digestion and electrophoretic separation of the resulting DNA molecules by their size allowed us to generate an ordered series of stepwise shortened plasmids. Plasmids with a deletion of approximately 3400 bp could no longer be mobilized. Since the next larger plasmid with 284 additional base pairs could be mobilized, we are able to confine the oriT location within this extra nucleotide stretch. The DNA sequence of this region was determined. Dominant features within the DNA region are a high AT content and five inverted repeats, which might function as recognition or substrate sites for proteins of the conjugational transfer system.     

Dysfunctional alpha-globin gene in hemoglobin H disease in blacks. A dinucleotide deletion produces a frameshift and a termination codon

alpha-Thalassemia trait is common in Black Americans; the (-alpha) haplotype occurs in 30% of that population. However, hemoglobin H disease (genotype:- -/-alpha) is very uncommon due to the rarity of the (- -) haplotype. A subject with HbH-HbG Philadelphia (alpha 2(68)Asn----Lys) synthesized only alpha G and no alpha A. Digestion of DNA with BamHI produced a single 10-kilobase (kb) alpha-specific fragment. Her son had alpha-thalassemia trait, did not make HbG Philadelphia, and demonstrated 14- and 10-kb alpha fragments upon BamHI digestion. Since the 14-kb fragment could not have been inherited from the mother, the son apparently received from her a chromosome bearing a single nonfunctional (alpha T) gene. Therefore, the two genotypes are: mother (-alpha G/-alpha T), son (-alpha T/alpha alpha). A 16-kb BglII fragment, containing the gene of interest from the son, was cloned into the BamHI site of phage EMBL 3 followed by subcloning of a 1.5-kb PstI alpha-specific fragment into plasmid pBR322. The mutant alpha gene demonstrated a deletion of an AG dinucleotide from the tandem repeat normally occurring in the Glu-Arg codons 30 and 31 at the junction of the first exon with intervening sequence 1. The loss of two nucleotides leads to a reading frameshift and a totally novel amino acid coding sequence in the second exon from codons 31-54 followed by the appearance of a chain termination codon (TAA) at position 55. No complete globin chain can be produced from this gene. HbH disease in this Black family is thus due to the combination of gene deletion and a nonfunctional alpha gene.     

Plasmid vehicles for direct cloning of Escherichia coli promoters.

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.     

A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae

A new plasmid, pSP2, was constructed as a cloning vector for use in Streptococcus pneumoniae. It allows direct selection of recombinant plasmids, even for DNA fragments not homologous to the S. pneumoniae chromosome, as based on the failure to maintain long inverted repeats (LIRs) hyphen-free in bacterial plasmids. Plasmid pSP2 contains a 1.4-kb BamHI fragment ("hyphen") flanked by 1.9-kb LIRs. The removal of the 1.4-kb BamHI fragment followed by ligation creates a plasmid containing a 1.9-kb insert-free LIR; plasmids with such non-hyphenated LIRs were not established when transferred into S. pneumoniae. Replacement of the original 1.4-kb insert by other restriction fragments restored plasmid viability. Investigation of plasmid transfer by transformation suggests that intrastrand synapsis between the LIRs could occur, thus facilitating plasmid establishment (a process we call self-facilitation). Such an intrastrand synapsis could also account for rare occurrences of insert-inversion noticed upon transfer as well as for the formation of palindrome-deleted derivatives at low frequency. Plasmid pSP2 carries two selectable genes, tet and ermC, and can be used for cloning of fragments produced by a variety of restriction enzymes (BamHI, Bg/II, Bc/I or Sau3A, and Sa/I or XhoI).     

Characterization of the phospholipase C gene of Pseudomonas aeruginosa cloned in Escherichia coli

We have cloned a 4.9-kb fragment of Pseudomonas aeruginosa DNA containing the structural gene of phospholipase C (PLC), by inserting it into the BamHI site of plasmid pBR322. Strains of Escherichia coli carrying this recombinant plasmid produce PLC, but expression of the gene differs from that in P. aeruginosa in two respects: (i) synthesis of the enzyme appears to be constitutive, i.e., not repressible by the presence of inorganic phosphate in the growth medium, and (ii) most of the enzyme remains associated with the outer membrane instead of being secreted. Insertion mutagenesis at a unique restriction site within the PLC gene destroyed the ability of the plasmid to code, in maxicells, for phospholipase C activity and for an Mr 80000 polypeptide.     

Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1.

The conjugative transfer (tra) genes of a 52-kilobase (kb) staphylococcal plasmid, pGO1, were localized by deletion analysis and transposon insertional inactivation. All transfer-defective (Tra-) deletions and Tn551 or Tn917 transposon insertions occurred within a 14.5-kb BglII fragment. Deletions and insertions outside this fragment all left the plasmid transfer proficient (Tra+). The tra region was found to be flanked by directly repeated DNA sequences, approximately 900 base pairs in length, at either end. Clones containing the 14.5-kb BglII fragment (pGO200) and subclones from this fragment were constructed in Escherichia coli on shuttle plasmids and introduced into Staphylococcus aureus protoplasts. Protoplasts could not be transformed with pGO200E (pGO200 on the staphylococcal replicon, pE194) or subclones containing DNA at one end of the tra fragment unless pGO1 or specific cloned tra DNA fragments were present in the recipient cell. However, once stabilized by sequences present on a second replicon, each tra fragment could be successfully introduced alone into other plasmid-free S. aureus recipients by conjugative mobilization or transduction. In this manner, two clones containing overlapping fragments comprising the entire 14.5-kb BglII fragment were shown to complement each other. The low-frequency transfer resulted in transconjugants containing one clone intact, deletions of that clone, and recombinants of the two clones. The resulting recombinant plasmid (pGO220), which regenerated the tra region intact on a single replicon, transferred at frequencies comparable to those of pGO1. Thus, all the genes necessary and sufficient for conjugative transfer of pGO1 are contained within a 14.5-kb region of DNA.