Improvement and application of a promoter-probe vector bearing the PHO5 gene as the indicator marker in Saccharomyces cerevisiae

A promoter-probe plasmid vector, pVC701, in Saccharomyces cerevisiae having the repressible acid phosphatase (rAPase; EC 3.1.3.2) gene, PHO5, of S. cerevisiae as the selection marker (Hwang et al., Appl. Microbiol. Biotechnol., 28, 155–159, 1988) was improved by inserting multiple cloning sites in the 5′ non-transcribed region of PHO5 and by replacing a 93-bp sequence containing three ATG sequences out of the frame but directly flanked by the translation initiation codon ATG with a 5′-CACC-3′ sequence. The resulting YCp type plasmid, pVC727, is replicable in both S. cerevisiae and Escherichia coli hosts. The function of the vector was confirmed by inserting a DNA fragment bearing a promoter region of the GAP gene encoding glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae at the cloning site. Using the vector, we could analyse the cis-acting upstream regulatory region of the HIS5 gene of S. cerevisiae and also isolate Gcd⁻ (general control derepressed) mutants occurring in the trans-acting loci of the general amino acid control.     

Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

The cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17 was cloned on separatePstI,BamHI, andEcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host,Escherichia coli DH5. High level constitutive expression of the gene product was also detrimental to theE. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kbEcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the hostB. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in theB. subtilis host; however, expression was at a low level. Subcloning of the 3-kbEcoRI fragment into pUC18 and transformation intoE. coli XL1-Blue (FlacI^q) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from theBacillus temperate phage SPO2 promoter of pPL708 may increase expression of this gene.Florida Agricultural Experiment Station Journal Series No. R-02177     

Isolation and characterization of a genomic sequence of maize coding for a zein gene

Summary An EcoRI restriction endonuclease fragment of maize DNA coding for the 19,000 dalton zein protein was cloned in phage gt WES. The zein gene was identified by the electron microscopic analysis of RNA-DNA hybrids (R-loops) and DNA-DNA hybrids (D-loops). The R-loops were formed with poly(rA)-containing RNA isolated from 18 days post-pollination maize endosperm and showed no intervening non-hybridizing sequences (introns) within their 800 base length. A cDNA clone specific for the 19,000 dalton zein protein formed D-loops in the same position and orientation as the R-loops. The cloned fragment measured 4.4 kilobases (kb), the same size as an EcoRI fragment of maize DNA revealed by Southern analysis.     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979     

Lambdoid phages that simplify the recovery of in vitro recombinants

Summary Derivatives of phage λ are described for use as vectors for fragments of DNA generated with theHindIII andEcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the λ gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of thelacZ gene ofE. coli able to complement alacZ host. The appropriatelacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.     

Molecular cloning of the tolC locus of Escherichia coli K-12 with the use of transposon Tn10

Summary We have cloned the tolC gene of E. coli K-12 into pSF2124 by using transposon Tn10 as the marker to first isolate the relevant DNA fragment. The gene is on a 10.5 kb EcoRI fragment, and Tn5 insertion mutagenesis locates the gene near one end of this EcoRI fragment. An EcoRI-PstI fragment has been subcloned into pBR322 to facilitate further analysis of the gene.Abbreviations Tris Tris (hydroxymethyl) aminomethane - EDTA Ethylenediamine tetra-acetic acid - DOC Sodium deoxycholate - DNA Deoxyribonucleic acid - SDS Sodium dodecyl sulphate - kb kilo base pairs     

Maintenance and expression of heterologous genes in chloroplast ofChlamydomonas reinhardtii

The chloroplast genome ofChlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplastatpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). TheatpA-aadA cassette has been placed within the chloroplast DNAEcoRI restriction enzyme fragment 14, or within the chloroplastBamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DNA then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstrate that the chloroplastatpA promoter inatpA-aadA routinely recombines with its endogenous counterpart, resulting in heteroplasmic chloroplast DNA populations that may persist for many generations. The heterologous gene does not require a 3 inverted repeat sequence for its expression. TheatpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady state level ofatpA-aadA mRNA. However, neither genomic position, gene copy number, or mRNA level have a significant effect on cellular resistance to spectinomycin, nor activity of theaadA gene productin vitro. These results suggest that, in the case ofaadA, the limiting step for expression of this gene is at the translational or post-translational level. TheatpA-aadA cassette should prove a useful model for future studies on the maintenance and expression of heterologous genes inC. reinhardtii chloroplasts.     

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage

Summary The position of the metJBLF gene cluster in the transducing phage dmet102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the BCDEF (nin5) EcoRI fragment of gtl (BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.     

Characterization of a Rhizobium etli chromosomal gene required for nodule development on Phaseolus vulgaris L.

A chromosomal gene, required for nodule development on Phaseolus bean, was characterized from Rhizobium etli strain TAL182. MLC640 is a Tn5 insertion mutant of TAL182 which shows decreased motility in soft TY agar and is defective in nodule development. The site of Tn5 insertion in MLC640 mapped to a 3.6-kb EcoRI chromosomal fragment. The 3.6-kb fragment was subcloned from the cosmid pUHR80 which complemented MLC640. Further subcloning and site-directed Tn5 mutagenesis localized the gene for nodule development to a 1.7-kb region within the 3.6-kb EcoRI fragment. Southern hybridization using the 3.6-kb EcoRI fragment as the probe against genomic DNA of several Rhizobium spp. indicated that this gene is conserved in different rhizobia.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gimore 402, Honolulu, Hawaii 96822. USA;     

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome

To date, vertebrate DNA has been found methylated at the 5 position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5-GGCC-3 sequences in the teleost Sparus aurata EcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.     

Indication for deletion of two introns in theoxi-3 gene of a respiratory-competentSaccharomyces cerevisiae strain

Physical mapping of the mitochondrial DNA of the wild-typeSaccharomyces cerevisiae strainRXII revealed that most of the restriction sites as well as the location of the apocytochromeb gene were identical in comparison with the known maps of the mitochondrial genome in otherSaccharomyces cerevisiae strains. In the middle of theSalI linearized map of theRXII mitochondrial DNA, a deletion was detected which resulted in the loss of twoEcoRI and oneBamHI restriction sites. The corresponding region, however, exists in most other laboratory strains ofSaccharomyces mapped so far. This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene. The nucleotide sequence of the subunit 1 gene showed that theBamHI site was located close to the aI3-A4 intron-exon junction and the distalEcoRI site close to the aI2-A2 boundary. I therefore conclude that these two introns are deleted in the mitochondrial genome of strainRXII. The exon A3 must have been conserved since this strain was respiratory competent. This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Size and organization of a multigene family encoding phaseolin, the major seed storage protein of Phaseolus vulgaris L.

Summary Solution hybridization kinetics and genomic nitrocellulose blot hybridization analyses show that the Phaseolus vulgaris L. (French bean) storage proteins (phaseolins) are encoded as a small, homologous, multigene family consisting of approximately seven members. Restriction endonuclease site mapping (EcoRI, BamHI, and BglII) of DNA regions flanking the phaseolin genes has shown that the gene family can be divided into at least three characteristic fragment size classes. Clones representative of two of these phaseolin gene classes have been isolated from a 1059 phage library.     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Construction of recombinant K88 DNA with Ptac promoter

The gene encoding K88ab was localized on the 11.6 kbHindIII-HindIII fragment of 74 kb plasmid DNA ofE. coli 7301. The smallest recombinant DNA producing the K88ab antigen was obtained by excision of the 5.15 kbEcoRI-EcoRI fragment from recombinant DNA composed of the 11.6 kb K88ab fragment in the pBR322 vectro. The size of the smallest fragment was 6.5 kb. Expression of the K88ab antigen was controlled by the P1 promoter of the pBR322 vector. Substitution of promoter P_tac for promoter P1 made it possible to achieve expression of the K88ab antigen byE. coli MT. Substitution of promoter P_L for promoter P1 failed to achieve expression of the K88 ab antigen in the recipient strains used.     

Construction and physical mapping of plasmids containing the MetA gene of Escherichia coli K-12

Summary Plasmids containing the metA gene of E. coli K-12 were constructed in vitro using pBR322 as the cloning vehicle and metA transducing phage as the source of metA DNA. EcoRI digests of pBR322 and metA20 were joined by ligase and plasmids carrying the metA gene were selected after transformation in a metA deletion strain. Recombinant DNA molecules contained one pBR322 fragment and one metA20 fragment of 12.2 kb which was present in either of two possible orientations. Plasmids constructed by BamH1 digestion of metA2 contained a single bacterial DNA fragment of 5.8 kb inserted in the tet gene. Insertion of the metA fragment led to loss of resistance to tetracycline in one orientation and partial resistance in the opposite orientation.     

HindIII/Eco RI RFLP of the human immunoglobulin IGHE gene detected with a Cɛ probe in the French and Tunisian populations

Summary Three differentHindIII/EcoRI restriction fragment alleles of the human immunoglobulin IGHE gene have been detected with a C probe in the Tunisian population. Two of these alleles can be explained by a Negroid contribution in their gene pool.     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

A DNA segment within the colicin E1 structural gene on ColE1 affecting immunity to colicin

Summary Insertion of DNA at the EcoRI site of ColE1 results in increase of immunity to colicin killing in E. coli harboring such recombinant ColE1 plasmid as compared to E. coli (ColE1). This effect is neither due to cis or trans interactions originating from the inserted foreign DNA fragment, nor to changes in plasmid copy number. This defect in the immunity mechanism is not trans complemented for by wild type ColE1. Increase in immunity can also be obtained by deleting a DNA segment from the ColE1 genome. This segment is 120 bp left to the EcoRI site within the colicin structural gene. It is concluded that the structure of DNA per se, around the EcoRI site, within colicin structural gene, is the structure which affects immunity expression.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.