A selective λ phage cloning vector with automatic excision of the insert in a plasmid

A bacteriophage λ cloning vehicle has been constructed for the generation of cDNA libraries. The vector has the following properties. (1) It has a unique BamHI site engineered into the λ gam gene. Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spi⁻ phenotype). (2) When the recombinant phage infects a Cre-producing E. coli strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert. (3) Single-stranded DNAs can be recovered by growing helper M13 phages on bacteria harboring such plasmids. The vector, λMGU2, has been used to construct a nematode (Caenorhabditis elegans) cDNA library.     

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.     

Immunological detection of cloned antigenic genes of Vibrio cholerae in Escherichia coli

Antibodies have been used as probe to detect cloned genes coding for toxin and surface antigens of Vibrio cholerae E1 Tor strain KB207. Eco RI-digested chromosomal DNA of KB207 was cloned in plasmid pBR325 and transformed in Escherichia coli HB 101(λcI857). Transformants were grown at 32° C on plates containing antibodies. Lysogen was induced at 42 °C to release expressed antigens. Antigen-antibody reaction produced a halo around positive clones.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Hymenoic acid, a novel specific inhibitor of human DNA polymerase lambda from a fungus of Hymenochaetaceae sp

Hymenoic acid (1) is a natural compound isolated from cultures of a fungus, Hymenochaetaceae sp., and this structure was determined by spectroscopic analyses. Compound 1 is a novel sesquiterpene, trans-4-[(1′E,5′S)-5′-carboxy-1′-methyl-1′-hexenyl]cyclohexanecarboxylic acid. This compound selectively inhibited the activity of human DNA polymerase λ (pol λ) in vitro, and 50% inhibition was observed at a concentration of 91.7 μM. Compound 1 did not influence the activities of the other seven mammalian pols (i.e., pols , γ, δ, ε, η, ι, and κ), but also showed no effect even on the activity of pol β, which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. This compound also did not inhibit the activities of prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 1 could be a selective inhibitor of eukaryotic pol λ. This compound had no inhibitory activities against two N-terminal truncated pol λ, del-1 pol λ (lacking nuclear localization signal (NLS), BRCA1 C-terminus (BRCT) domain [residues 133–575]), and del-2 pol λ (lacking NLS, BRCT, domain and proline-rich region [residues 245–575]). The compound 1-induced inhibition of intact pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

The Cleavage of Nucleic Acids in Reversed Micelles Using Site Specific Endonucleases

Plasmid and λ DNA molecules of between 2.2 and 48.5 kb pairs can be solubilised in n-hexane containing the surfactant sodium dioctyl sulfosuccinate (AOT) and aqueous buffers. Linear λ phage DNA fragments (2.2-23.1 kb pairs) and intact λ bio 1 DNA (48.5 kb pairs) are efficiently cleaved by Bam HI and Em RI in systems containing 100 mM AOT. Under these conditions, λ bio 1 DNA undergoes regioselective restriction by Hind III at only one site but is completely cleaved when the surfactant concentration is lowered to 50 mM. Covalent closed circular plasmid DNA (pUC8, 2.73 kb pairs) is only partially linearised by Eco RI and Bam HI in reversed micelles; Hae II cleavage affords both complete and partial restriction fragments. The results suggest that the tertiary structures adopted by substrate DNA in reversed micelles influence the availability of restriction sites.     

The maturation of coliphage lambda DNA in the absence of its packaging

In vivo, λ DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize λ terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of λ gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos.

Since the only λ genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe 2 maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another λ gene that maps to the right of gene B.     

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.     

Inactivation of bacteriophage λ and λ DNA by nitrogen mustard

Bacteriophage λ and λ DNA were treated with alkylating agents. The survival of phage was assayed by infectivity and that of DNA by infectivity of phage particles assembled from the DNA in vitro. Phage λ were more sensitive to nitrogen mustard (Cl(CH₂)₂NMe(CH₂)₂Cl; HN2) than was λ DNA. The inactivation of λ DNA was biphasic; the second component of the inactivation was sensitive to mutations allelic for recA, polA and uvrB. This behaviour was not shown by pBR322 plasmid DNA treated with HN2 nor by λ DNA treated with monofunctional alkylating agents (or HN2 if the second alkylation reaction was stopped by addition of a mercaptan). From Arrhenius plots, the activation energy for the reactions with DNA and intact phage were found to be different. The activation energy for the inactivation of intact phage was the same as that (measured independently) for the predominant reaction (or class of reactions) in which HN2 cross-links DNA to protein in λ particles. From these data we conclude that the inactivation of λ by HN2 is due, primarily, to DNA-protein cross-linking. The implications for the mode of action of DNA-reactive bifunctional anti-viral and cytotoxic compounds are discussed.     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

The thiol-specific antioxidant protein from human brain: gene cloning and analysis of conserved cysteine regions

The complete cDNA encoding human thiol-specific antioxidant protein (PRP) was isolated from a human brain cDNA library in the λZap expression vector. An open reading frame (ORF) was identified and found to encode a polypeptide of 197 aa with a M_r of 21 729. The cDNA contained 98 bp of 5′-untranslated sequence (UTR) and 259 bp of 3′-UTR containing a poly(A) signal, AATAAA. Expression of the human PRP cDNA in Escherichia coli yielded a functionally active protein. The observed local sequence homologies between human PRP and other homologous proteins whose functions have not yet been defined give important insight into elucidating the biochemical function of a new protein family which has highly conserved regions containing cysteine.     

Gas chromatography–mass spectrometry with high-performance liquid chromatography prepurification for monitoring the endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from γ-irradiated DNA

The endonuclease III from Escherichia coli is a repair enzyme which exhibits both a glycosylase and an endonuclease functions. The activity of the enzyme can be assayed by measuring the released targeted bases in solution from a sample of modified DNA. In the present study, gas chromatography–mass spectrometry was used together with an HPLC prepurification step in order to single out the released bases. The prepurification was found to enhance the specificity and the sensitivity of the assay. Thus, the overall method allowed us to analyze separately 5-hydroxy-5,6-dihydrothymine from the cis and trans isomers of 6-hydroxy-5,6-dihydrothymine. Examples of application of the assay are provided with the measurement of the E. coli endonuclease III-mediated excision of 5-hydroxy-5,6-dihydrothymine and 5,6-dihydrothymine from samples of γ-irradiated DNA in the presence of cysteine.     

Genetic control of the UV-induced SOS mutator effect in single- and double-stranded DNA phages

The SOS hypothesis postulated that the mutator effect on undameged DNA that generates phage-untargeted mutagenesis (UTM) results directly from the mechanism of targeted mutagenesis. RecA protein, which stimulates the cleavage of both the LexA repressor and UmuD protein, and the UmuDC gene products are required for UV-induced targeted mutagenesis. The use of phage λ for analyzing UV-induced mutagenesis has permitted a distinction to be made between the mechanisms of targeted and untargeted mutagenesis, in that the two processes differ with respect to their genetic requirements for recA⁺ and umuDC⁺ genes. In this paper, we show thet (i) proficiency for excision repair is required for UTM in double-stranded DNA phage but not in single-stranded DNA phage; (ii) the umuC function, which is not required for UTM of the double-stranded DNA phage λ, is necessary for untargeted mutagenesis of the single-stranded DNA phages M13 and φX174; (iii) for both single-stranded and double-stranded DNA phage, UV irradiation of the host increases the level of recA730-induced UTM. Our results are also consistent with the interpretation that the expression of untargeted mutagenesis in phage λ and in M13 depends on the polymerase and to a lesser extent on the exonuclease 5′ → 3′, activities of Po1I. These results suggest that the involvement of the RecA and UmuDC proteins may be related to more than the presence of base damage in the DNA substrate.     

Simultaneous expression of glutaryl-7-aminocephalosporanic acid acylase gene and lysis genes of phage λ in a recombinant E. coli

Glutaryl-7-aminocephalosporanic acid acylase (GLA), recommended for use in the form of immobilized-enzyme, is one of the two key enzymes in the two-step synthesis of 7-aminocephalosporanic acid. For simplifying the process of cell disruption and immobilization, the lysis genes of phage λ (S⁻RRz) with the S amber mutation were designed to introduce into the over-expression system of GLA. A novel recombinant strain, E. coli TB1/pMKC-AS, simultaneously containing the maltose binding protein gene (malE), the lysis genes (S⁻RRz) and the target GLA gene (Acy) in a same operon, was successfully constructed. Under neutral pH conditions, cell growth and GLA activity of TB1/pMKC-AS was not affected by the presence of the lysis genes, however, autolysis phenomenon was observed under weak alkaline conditions. Through pH control and fed-batch culture, the GLA activity of TB1/pMKC-AS reached as high as 6810 U/L with 24.8 g/L dry cell density (OD₆₀₀ = 67.9) in a 5 L fermentor. In contrast to the cells of E. coli TB1/pMKC-Acy without the lysis genes, the mild EDTA/Tris buffer (pH 8.0) can cause the lysis of the cells of TB1/pMKC-AS containing the lysis genes. Correspondingly, a mild pH 9.0/42 °C incubation method was developed for conveniently degrading the recombinant cells of TB1/pMKC-AS, based on the expression of the lysis genes. Further experiments showed that the cell lysate after the mild incubation disruption can be directly immobilized by 10% polyacrylamide to make the immobilized enzymes. In comparison with the immobilized GLA from TB1/pMKC-Acy, the immobilized cell lysate of TB1/pMKC-AS has the similar characteristics of catalysis stability, implying a great potential for industrial application of the lysis genes-assisted cell disruption.