Cloning, nucleotide sequence and high level expression of the gene coding for the connector protein of Bacillus subtilis phage φ 29

The φ 29 DNA restriction fragment HindIII-D, shown to contain gene 10 coding for the connector protein, has been cloned in plasmid pPLc28 under the control of the p_L promoter of phage λ. After heat induction to inactivate the λ repressor, a protein with the electrophoretic mobility of the connector protein p 10 was synthesized, accounting for about 30 % of the total Escherichia coli protein after 3 h of induction. The 2205 nucleotide-long sequence of the cloned HindIII-D fragment has been determined. The sequenced region has an ORF coding for a protein of M_r 35881 that was shown to correspond to the connector protein by determination of the ammo-terminal sequence of purified protein p10. Features of the nucleotide sequence and the amino acid sequence of protein p10 are discussed.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the -galactosidase leader peptide, under the control of the -galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Two gene duplication events in the evolution of the human heat-stable alkaline phosphatases

There are at least three alkaline phosphatase (AP) isoenzymes in man: a heat-stable placental enzyme (PLAP), a less heat-stable intestinal form (IAP), and the very heat-labile AP enriched in liver, bone and kidney. In addition to these enzymes, there is a heat-stable activity in the thymus and testis that is similar but not identical to the PLAP (the PLAP-like enzyme). Previous work has demonstrated a close structural relatedness among the IAP, PLAP and PLAP-like enzymes. Thus, it is possible that there are three human genes encoding heat-stable AP enzymes. To test this hypothesis, we have used a PLAP cDNA clone to screen a human genomic library cloned into the phage vector 1EMBL-3. Three sets of clones were isolated, each bearing a distinct coding region homologous to the PLAP cDNA probe. Nucleotide sequence analysis of the 5′ ends of these genes allowed comparison of their derived peptide sequences and positive identification of two of the genes. One of the genes encodes the PLAP (the PLAP-1 gene), another encodes the IAP, and a third closely resembles the PLAP-1 gene, but is distinct from it (the PLAP-2 gene). The PLAP-2 gene is highly homologous (> 95%) with the PLAP-1 except in the first exon, where sequences encoding the hydrophobic signal peptide are nearly identical with the same region of the IAP gene. These results demonstrate the existence of a small family of PLAP-related genes which is the result of at least two duplication events during the descent of man.     

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.     

Isolation of low-copy-number sequences that neighbor satellite DNA in mammals

To investigate the role of satellite DNA in eukaryotic genomes, we isolated from an African green monkey (Cercopithecus aethiops) genomic library cloned segments containing the previously described deca-satellite linked to low-copy-number genomic sequences. Three such clones were obtained. The low-copy-number sequences in the three clones do not cross-hybridize suggesting that they derive from different genomic loci. The structure of one of the clones, λAMkA, is described in detail. Subcloned segments containing the low-copy-number sequences from λAMkA anneal to monkey, human and mouse genomic DNA. The subcloned probes were used to select clones containing homologous sequences from a second, independent monkey library as well as from human and mouse genomic libraries. Several of the newly isolated monkey clones hybridized to probes containing the species-specific deca- and -satellites, confirming the genomic association of the low-copy-number sequence in λAMkA with satellite DNA. Moreover, several of the human and mouse clones hybridized to species-specific human and mouse satellite DNAs, respectively. These experiments indicate that the low-copy-number sequence in λMkA and its association with satellite DNA is conserved in primates and rodents.     

The coding region of the human c-mos pseudogene contains Alu repeat insertions

We have determined the nucleotide sequence of an 841-bp fragment derived from a segment of the human genome previously cloned by Chumakov et al. [Gene 17 (1982) 19–26] and Zabarovsky et al. [Gene 23 (1983) 379–384] and containing regions homologous to the viral mos gene probe. This sequence displays homology with part of the coding region of the human and murine c-mos genes, contains several termination codons, and is interrupted by two Alu-family elements flanked by short direct repeats. Probably, the progenitor of the human c-mos gene was duplicated approximately at the time of mammalian divergence, was converted to a pseudogene, and acquired insertions of two Alu elements.     

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Avian retrovirus nucleocapsid protein, pp12, produced in Escherichia coli has biochemical properties identical to unphosphorylated viral protein

The avian sarcoma and leukosis viruses contain an RNA binding nucleocapsid protein, pp12, in the phosphorylated form. An Escherichia coli expression vector carrying the Rous sarcoma virus Prague C strain gene coding for this protein has been constructed using a site-directed deletion mutagenesis method to place start and stop codons into translational reading frame with the N- and C-terminal coding sequences of the protein, respectively. The protein is produced efficiently in bacteria (rp12), is soluble and readily purified. It is also indistinguishable from the unphosphorylated viral pp12 protein in its (1) migration on SDS-polyacrylamide gels, (2) reactivity with rabbit antisera directed against purified viral pp12, (3) low RNA-binding affinity, and (4) ability to serve as a substrate for in vitro phosphorylation at serine-40 by protease-activated kinase I. The ability to analyze the biochemical activities of the normal, modified, and mutant forms of this protein is an essential step in elucidating its role in the retroviral life cycle. This expression clone will be especially useful in testing for the effects of mutations before they are reconstructed into retroviral genomes for analyses.     

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.     

Application of partial restriction procedure in both shotgun and non-random strategies for nucleotide sequencing

Partial digestion of a target DNA fragment with 4-bp-recognition restriction enzymes followed by a forced ligation to an M13 vector was employed for the construction of a subfragment library. The library can be used for either shotgun or non-random nucleotide sequencing. Application of the partial digests generated with the 4-bp recognition restriction enzymes instead of DNase I in the improved non-random strategy for nucleotide sequencing (Li and Wu, 1987) made the procedure as easy as that of the random strategy. The library can also be used in shotgun nucleotide sequencing directly, and few self-ligated subfragments were found. The usefulness of this procedure was demonstrated by the sequencing of a goat 6.5-kb EcoRI fragment, which is located 5' to the globin gene.     

Cloning and expression of FimA-c3d recombinant protein

Recombinant protein consisting of an antigen fused to C3d may elicit a more robust immune response than the antigen alone. The objective of the present study was to clone FimA-c3d recombinant DNA into pCold-TF vector and successfully express in prokaryotic expression vector. FimA subunit of type I fimbriae from Salmonella enterica serovar Enteritidis was conjugated to mouse complement fragment molecule C3d. FimA and FimA-mC3d, FimA-mC3d₂ and FimA-mC3d₃ were cloned into the expression vector pCold-TF. Constructions of the recombinants pCold-TF-fimA with different copies of C3d were confirmed by digestion with restriction enzymes and sequencing. Soluble fusion proteins of FimA with different copies of C3d were induced by IPTG and were expressed in Escherichia coli BL21 (DE3) under optimal conditions. The results showed that the proteins induced from recombinants pCold-TF-fimA, pCold-TF-fimA-mC3d, pCold-TF-fimA-mC3d₂ and pCold-TF-fimA-mC3d₃ were 70, 100, 130 and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC). Finally we conclude that antigen conjugated with mC3d can be functionally expressed in prokaryotic vectors.     

A method for construction of specialized transducing phage rho 11 of Bacillus subtilis.

DNA from a temperate phage rho 11 and chromosomal DNA of Bacillus subtilis 168 were digested with endonuclease EcoRI and then ligated with T4 polynucleotide ligase. The ligated DNA fragments were used to transform a lysogenic strain, B. subtilis spoA12 lys21 hisA1 leuA8 p11, and Lys+, His+ or Leu+ transformants were selected. The cells of each type were then mixed, grown and treated with mitomycin C; the induced phages were tested for abilities abilities to form plaques and to tranduce the auxotrophic marker. Various types of plaque-forming or defective phages which transduce hisA or lys marker at considerably high frequencies were thus obtained.     

The cis-acting 3''-element of rubella virus RNA has DNA promoter activity.

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.