Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.     

A system for shotgun DNA sequencing.

A multipurpose cloning site has been introduced into the gene for beta-galactosidase (beta-D-galactosidegalactohydrolase, EC 3.21.23) on the single-stranded DNA phage M13mp2 (Gronenborn, B. and Messing, J., (1978) Nature 272, 375-377) with the use of synthetic DNA. The site contributes 14 additional codons and does not affect the ability of the lac gene product to undergo intracistronic complementation. Two restriction endonuclease cleavage sites in the viral gene II were removed by single base-pair mutations. Using the new phage M13mp7, DNA fragments generated by cleavage with a variety of different restriction endonucleases can be cloned directly. The nucleotide sequences of the cloned DNAs can be determined rapidly by DNA synthesis using chain terminators and a synthetic oligonucleotide primer complementary to 15 bases preceeding the new array of restriction sites.     

Construction of a gene coding for the precursor of human tumor necrosis factor]

The cDNA sequence for human tumor necrosis factor (hTNF) was reconstructed in vitro from genomic sequence. Using the oligonucleotide directed mutagenesis, a site for restriction endonuclease ClaI was introduced into the end of the first exon. The nucleotide sequence representing the second and third exons flanked with restriction sites ClaI and XhoI was obtained by means of chemical enzymatic synthesis. Assembly of the total gene coding for precursor of hTNF was accomplished in pTNF33 plasmid containing semisynthetic gene for mature hTNF with appropriate restriction sites.     

Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization

We have chemically synthesized a DNA duplex of 560 nucleotides that codes for the hepatitis B virus (HBV) core protein. The synthetic gene contains 27 unique internal restriction sites. Thereby, it can easily be mutagenized by replacement of rather short restriction fragments. A number of restriction recognition sequences are in common between the synthetic and the authentic gene, thus allowing for the transfer of synthetic segments into the cloned viral genome. Several unexpected mutations in the synthetic gene were readily corrected utilizing the multiple unique restriction sites. In Escherichia coli, the expression level of the synthetic gene product amounts to about 4% of the total soluble protein. It forms particles closely resembling native HBV cores. After transfer of the synthetic gene into the viral genome, transient expression in a hepatoma cell line yields proteins indistinguishable from the native gene products. The synthetic gene thus provides a useful tool for studies on the structure and function of the isolated HBV core protein as well as the gene and its various products in the viral life-cycle.     

The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation

The versatility of insertional inactivation of β-galactosidase activity for subcloning and sequencing has been enhanced by combining a chemically synthesized oligonucleotide which specifies nine 6-bp-cutter restriction sites including BglII, XhoI, NruI, ClaI, SacI and EcoRV in various configurations with existing polylinkers to create a set of highly versatile cloning sites. These improved polylinkers have been inserted into plasmids (the pICs) for routine cloning of double-stranded DNA, and into chimeric phage/plasmids (the pICEMs) for biological production of single stranded DNA. The most versatile Polylinker specifies 17 restriction sites in the β-galactosidase α-complementing gene fragment. One of the new polylinkers was inserted into M 13 DNA to produce a vector (M13mIC7) with nine cloning sites.     

New shuttle-type vectors for cloning in Escherichia coli and Agrobacterium tumefaciens

The vectors capable of replication in Escherichia coli and Agrobacterium tumefaciens have been constructed on the basis of the plasmid pUB5502. The constructed vectors pVA12, pVA12-2, pVA12-4 contain the mini-replicon and trimethoprim resistance gene (Tp) of a broad host-range plasmid R388 (IncW). The pVA12 vector (8.8 kb) has been constructed by insertion of a kanamycin resistance gene (Km) from the plasmid pUC-4K into a Psti site. It possesses 7 unique restriction sites for XhoI, SmaI, PvuI, PvuII, HindIII, EcoRI, BamHI and the markers for kanamycin and trimethoprim resistance (Km and Tp). The pVA12-2 and pVA12-4 vectors were obtained as a result of changing of the PvuII-EcoI fragment of pVA12 carrying the Tp gene for the PvuII-EcoRI fragment of pBR322 carrying the Tc gene. These plasmids have the same size of 9.7 kb and 8 unique sites for restriction endonucleases XhoI, SmaI, PvuI, PvuII, EcoRI, EcoRV, SalI, BalI and Km and Tc genes. No difference has been registered between the two plasmids by restriction analysis, but pVA12-4 has the dramatically increased copy number in Escherichia coli cells. All three vectors are transferable to Agrobacterium tumefaciens with the same frequencies by transformation or conjugation and do not affect the oncogenicity of pTi.     

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.     

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.     

Synthesis of DNA coding for human proinsulin

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.     

应用限制性显示PCR技术构建细菌poly(A)化mRNA的cDNA文库

针对细菌mRNA poly(A)化位点的高度多态性,利用oligo(dT)与poly(A)特异结合的特性,以oligo(dT)一纤维素纯化mRNA,并以oligo(dT)18为引物逆转录合成cDNA,用限制性内切酶消化cDNA,所得的限制性内切酶片段与通用接头相连,通过10个选择性引物组合进行选择性PCR,使各片段得以扩增并分布于10个亚组中,并进行克隆,成功地克隆了100多个基因片段,已对其中40个进行了测序分析,探讨了限制性显示PCR技术在细菌poly(A)化mRNA cDNA库构建中的应用价值。     

Versatile cassettes designed for the copper inducible expression of proteins in yeast

A series of yeast expression vectors and cassettes utilizing the CUP1 gene of Saccharomyces cerevisiae have been constructed. The cassettes contain multiple cloning sites for gene fusions and were created by inserting a 27-bp polylinker at the +14 position of the CUP1 gene. The cassettes are portable as restriction fragments and enable copper-regulated expression of foreign proteins in S. cerevisiae. In copper sensitive yeast, multiple copies of the CUP1 cassettes confer copper resistance due to the production of the copper metallothionein. Genes cloned into the CUP1 cassettes, however, usually prevent translation of the metallothionein leading to a loss of resistance. This could be useful for one-step cloning into yeast.     

Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses

Plasmid vectors containing multiple cloning sites suitable for the rapid insertion of protein-coding sequences into poxviruses have been constructed. They are based on pUC plasmids and carry the thymidine kinase (TK) gene of vaccinia virus interrupted by a vaccinia virus promoter. Six unique restriction enzyme sites (BamHI, SalI/HincII, PstI, HindIII, EcoRI), located within 40 bp of vaccinia virus promoters transposed from the HindIII-F or HindIII-C fragment of the vaccinia virus genome, allow rapid insertion of foreign-protein-coding sequences into these plasmids. Such plasmids can be used to construct recombinant poxviruses expressing foreign proteins using marker-rescue recombination techniques and selection for TK negative viruses. Vaccinia viruses expressing the haemagglutinin (HA) gene of swine influenza virus, A/NJ/11/76 (H1N1), have been constructed.     

Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes

An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites. This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region. In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK). Selection for loss of the galK gene, but for retention of the plasmid in E. coli, results in a plasmid in which the two fragments have undergone homologous recombination. Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites. These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E. coli.     

A new vector for insertion of any DNA fragment into the chromosome of transformable Neisseriae

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.     

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.     

Refinement of diagnostic assays for a probable causal mutation for porcine and human malignant hyperthermia.

The substitutions of T for C1843 in the porcine ryanodine receptor (RYR1) gene, which deletes a HinPI restriction endonuclease site and creates a HgiAI site, and of T for C1840 in human RYR1, which deletes a RsaI site, lead to Cys for Arg substitutions in the ryanodine receptors and are probable causal mutations for malignant hyperthermia (MH). To improve the restriction endonuclease assay of these sites, thereby providing an accurate, reliable diagnosis for MH, introns flanking the exon containing the mutation were sequenced, permitting identification and PCR amplification of a 659-bp porcine gene sequence that contains both constant and variant HgiAI sites and a 922-bp human gene sequence that contains both constant and variant RsaI sites. As a result, these PCR-amplified sequences contain constant internal controls for the reliable differentiation by restriction endonuclease digestion of normal, heterozygous, and MH genotypes.