Transgenosis with the participation of plasmid RP1: Indication of the presence of a “composite plasmid” in an intergeneric hybrid ofEscherichia coli

One of the transconjugants (1–7) previously obtained by the writers by conjugation ofEscherichia coli J-62 withPseudomonas aeruginosa 1822, in addition to plasmid RP1 had acquired the ability to grow without proline and tryptophan. A careful study showed that during conjugation of the transconjugant 1–7 with various strains ofE. coli the plasmid RP1 and the chromosomal genes are transmitted together, whereas during transduction with the aid of bacteriophage P1 they are transmitted independently; fertility is found only in transductants carrying the plasmid RP1. The results of these experiments suggest that during intergeneric conjugation chromosomal genes can be transferred even without any firm link with the plasmid (as in the case of composite plasmids). Corresponding fragments of the chromosome ofPs. aeruginosa in cells ofE. coli evidently form small nontransmissible replicons.Laboratory of Extrachromosomal Inheritance, All-Union Research Institute of Protein Biosynthesis. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 9, pp. 360–362, September, 1977.     

Plasmid cloning and expression of the E. coli polA + gene in S. cerevisiae

Summary The E. coli polA ⁺ gene has been subcloned from a specialised transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomally-encoded protein. It was able to counteract the radio sensitivity of polA1, polAex1, polAex2 and polA12 mutants, but no complementation of polA107 mutants occurred, even though the plasmid polA⁺ gene was expressed. S. cerevisiae ars-1 or 2 replicative sequences were introduced into the polA⁺ plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2–20 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA⁺ gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA⁺ gene.     

Transformation of Cochliobolus lunatus with pUT 720 changes the steroid hydroxylating ability of the fungus

Summary The filamentous fungus Cochliobolus lunatus, a known 11-hydroxylator of steroids, was transformed to bleomycin resistance using the heterologous plasmid pUT 720. This plasmid contains the Sh ble gene expressed under the control of the Aspergillus nidulans gpd and trpC expression signals. The bleomycin-resistant colonies appeared with a frequency of six per g of DNA. All colonies were real transformants and no abortive growth was observed. In all transformants tested the plasmid molecules became stably integrated into the genome of the host, and one of the plasmid molecules integrated in a site-specific manner. Transformants retained the ability to hydroxylate the steroid ring, but the hydroxy group was inserted at the 15 position.     

Killer toxin production in Pichia acaciae is associated with linear DNA plasmids

Summary We have identified a strain of the yeast Pichia acaciae which produces a killer toxin active against the yeast Debaryomyces tamarii. The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs). P. acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain. A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity. Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5 protected ends. The P. acaciae system differs from that of the well-studied Kluyveromyces lactis killer system both in the range of susceptible strains and in the sizes of the plasmids involved. Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.     

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae

Summary [2 m⁺ and [2m°] yeast were transformed to stable leucine prototrophy with the hybrid yeast — E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 m yeast DNA plasmid in addition to the yeast nuclear LEU2 ⁺ gene and the Co1E1 derivative, pMB9. In the [2 m⁺] transformants, a new wholly yeast LEU2 ⁺ plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 m DNA. The plamid, pYX, in the absence of 2 m DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 m DNA portion of the plasmid. pJDB219 was found to require the presence of 2 m DNA to undergo this intramolecular recombination. The results suggest that 2, m DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.     

Highly-efficient electrotransformation of the yeast Hansenula polymorpha

A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×10⁶/g plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per g DNA) for H. polymorpha remained high when large amounts (up to 10g) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.     

A new,rapid and efficient transformation procedure for Neurospora

Summary A new, rapid, efficient and reliable method for transforming Neurospora crassa is described. In this procedure, germinated conidia are treated with lithium acetate, then incubated with DNA, followed by exposure to polyethylene glycol and then a brief heat shock, prior to plating on selective medium. Optimal conditions to achieve a high transformation rate are reported. Transformation can be obtained with both circular and linear plasmid DNA and also with genomic DNA. Although the rate is substantially decreased, transformation was also obtained with relatively impure DNA preparations, such as that made via rapid miniprep procedures. This transformation technique is simple and reliable and provides a considerable savings in time and materials.     

Molecular cloning of the DAC2/FUS3 gene essential for pheromone-induced G1-arrest of the cell cycle in Saccharomyces cerevisiae

Summary Mating pheromones, known as a and -factors, arrest the division of cells of opposite mating types, and a respectively, in Saccharomyces cerevisiae. I have cloned the DAC2 gene, which is required for both pheromone-induced division-arrest and cell-fusion during conjugation. The constructed dac2::LEU2 null mutation leads to defects in both pheromone-induced divisionarrest and cell-fusion during conjugation; it also suppresses the growth defect caused by the gpal mutation (a mutation in the subunit of the S. cerevisiae G protein). These results indicate that DAC2 may be the same gene as FUS3, which was recently isolated by Elion et al. (1990) as a gene essential for cell-fusion during conjugation. The dac2::LEU2 null mutant also showed morphological alterations in response to mating pheromones. I show here that the DAC2 product plays an essential role in both the division-arrest signalling pathway of the yeast pheromone response and in cell-fusion during conjugation.     

A nucleotide sequence involved in replicative transformation of a filamentous fungus

Replicative plasmids generated through in-vivo recombination have been identified among transformants of the fungus Pleurotus ostreatus. In addition to sequences from a standard selection vector (pAN7-1), these recombinant plasmids contain recombined sequences of chromosomal origin conferring replicative potential upon the vector. One such recombined sequence, an 1148-bp insert into plasmid pP01, has been characterized. This sequence has been analyzed for secondary structural features as well as for consensus sites affiliated with origins of replication (ori) in other eukaryotic systems. The 1148-bp insert lacks an ORF and does not contain an acceptable match to the commonly identified 11-bp ars consensus sequence (A/TTTTATA/GTTTA/T) for autonomous replication in the yeast Saccharomyces cerevisiae. The analysis, however, revealed a cluster of three hairpin-loop-forming subsequences with individual G_25°C free energy values of-7.6,-6.4 and-5.2 kcal mol^-1. Also found were two 7-bp analogues to centromere-affiliated sequences recognized in other fungi, as well as several putative gyrase recognition sites comparable to the 9-bp S. cerevisiae/E. coli gyrase-binding consensus sequence. Sequences comparable to the ori of the yeast 2-m plasmid or to various sequences associated with ori of yeast/fungal mitochondrial DNAs (mtDNA) were not present in the 1148-bp insert. Replication of pP01 appears rather to involve a replication of chromosomal derivation devoid of an ars-type consensus.     

Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.)

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2mmol/L hydroxyurea for 18h, a 4.5-h recovery in hydroxyurea-free medium, 2h incubation with 10µmol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4×10⁵ morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The purity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.     

High-efficiency electrotransformation of the yeast Schwanniomyces occidentalis

A method has been developed for introducing heterologous DNA rapidly and efficiently by electroper-meabilization into the yeast Schwanniomyces occidentalis. A transformation efficiency as high as 2×10⁵ transformants/g of plasmid DNA was obtained with a squarewave electric pulse of 2.17 kV/cm during 18 ms. Small quantities of DNA (5 ng) can be used to transform 3×10⁸ cells. The main parameters which have been optimized are: presence of adenine in the culture medium, pretreatment of the cells with dithiothreitol during the exponential growth phase of the cells, amount of cells treated, and pulse-field strength and duration. Competent cells can be stored to allow electrotransformation whenever needed.     

Elevated monocyte interleukin-6 (IL-6) production in immunosuppressed trauma patients. I. Role of FcγRI cross-linking stimulation

This study demonstrates that immunodepressed trauma patients' monocytes produce elevated interleukin-6 to adherence, bacterial, and cytokine stimulation compared to immunocompetent trauma patients' or normals' monocytes, suggesting theirin vivo preactivation possibly mediated by the hyperimmunoglobulinemia which characterizes these patients. Furthermore, stimulation of monocytes through cross-linking their FcRI induces and augments interleukin-6 (IL-6) production to subsequent stimulation both in trauma patients' (P<0.001) and in normals' (P<0.001) monocytes. As we reported earlier, immunodepressed trauma patients have an increased proportion of FcRI-bearing monocytes in their total monocyte population and here we show that those FcRI⁺ monocytes produce significantly elevated interleukin-6, suggesting a relationship between elevated monocyte interleukin-6 production and FcRI triggering. Interleukin-6 induction by FcRI stimulation is not mediated solely by FcRI-induced MØ tumor necrosis factor alpha, IL-1, or IL-1 production and is independent of MØ prostaglandin E₂ levels. Therefore, FcRI stimulation-induced elevated MØ IL-6 might contribute to the increased immunoglobulin levels posttrauma.     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Unexpectedly long 145 ribosomal RNA gene in Tetrahymena mitochondria

Summary Extraction of RNA from Tetrahymena mitochondrial ribosomes yields several RNA species, including a large 21S molecule, a small 14S molecule, a 7S molecule, and other smaller RNAs. The molecular weight of the 14S rRNA indicates that it is about 1,300 bases in length. We have sequenced the 14S rRNA gene and, by aligning our sequence with that of the corresponding small rRNA from E. coli, find that the 14S rDNA is at least 1,635 bases in length. We propose, based on the results of hybridization studies, that this unexpected length is due to the presence of 7S RNA sequence within the 14S gene sequence. The 7S region is apparently lost from the 14S rRNA, yet is still a component of the ribosome.     

High frequency FLP-independent homologous DNA recombination of 2 μ plasmid in the yeast Saccharomyces cerevisiae

Summary The purpose of this work is to identify and quantitate in vivo 2 plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 DNA recombination events occurring in FLP⁺ cells is the contribution of both FLP-mediated and FLP-independent events.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

An efficient cell-free translation system from Aspergillus nidulans and in vitro translocation of prepro-a-factor across Aspergillus microsomes

Summary We describe the preparation of an in vitro translation system from heat shock-treated Aspergillus nidulans, capable of supporting efficient and faithful synthesis of proteins from natural and in vitro transcribed eukaryotic messages. In vitro synthesized prepro--factor was translocated across Aspergillus nidulans microsomal membranes in either the homologous A. nidulans or a yeast cell-free system. The translocated prepro--factor was protected from digestion by protease and glycosylated to higher MW forms.     

Cloning of a DNA fragment from Cephalosporium acremonium which functions as an autonomous replication sequence in yeast

Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization.PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/g DNA. Transformation frequencies of 715 transformants/g DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast.The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.