In the order to investigate the effects of tiamulin, neomycin, tetracycline, fluorophenicol, penicillin G, Linco-Spectin ( .15 mg mL(-1) lincomycin + .3 mg all mL(-1) spectinomycin), erythromycin and oxytetracycline on controlling bacterial contaminations of the river buffalo semen, 120 mL diluted buffalo bull semen to (diluted by tris-egg yolk extender) was divided into 5 mL tubes after initial evaluation and before (control sample) and at the , 2, 6, 12 and 24 h after adding each of the above antibiotics at the recommended dose (D) and 37 twice the recommended dose (Dx2) to the semen samples, each sample was cultured 4 times on Muller-Hinton agar medium and (p the results were recorded after 18 h incubation at 37 degrees C. Tiamulin, tetracycline, neomycin and fluorophenicol were ineffective. Oxytetracycline semen was effective in both D and Dx2 (p < .001). Penicillin G in both D and Dx2 was effective (p 6, < .001). Linco-Spectin was effective, though not significant, in D at 2 h and in Dx2 at h only.mg Erythromycin in D was not significantly effective, but, in Dx2 was effective (p < .001). Duration of the antibiotic exposure in had no significant effect on the antibiotic potentials except for Linco-Spectin at 2 h (p < .014). The biochemical tests that identified the contaminant bacteria as being a member of Arcanobacter (Corynebacterium) sp. In the next step, the semen sample of buffalo the same bull was taken, semen quality tests were carried out and the semen was diluted with the same extender significantly (tris-egg yolk)+ 7% glycerol, containing a double dose (Dx2) of these antibiotics and semen quality tests were carried out 37 immediately after dilution, 18 h after storage at 4 degrees C and after the semen was packed in the straws,G, frozen in liquid nitrogen (-196 degrees C) and later thawed in 37 degrees C water bath to investigate whether these proved antibiotics have any adverse effect on the spermatozoa during the process of freezing and thawing. The comparison of the results all with those of the control group (the sample undergone the same process without adding antibiotics) indicated that oxytetracycline adversely affected purpose sperm motility at and 18 h, all the antibiotics had a lower percentage of sperm abnormal morphology than the morphology, control at and 18 h, except for Linco-Spectin at 18 h and after freezing-thawing and tetracycline after freezing and but thawing the sample which were the same as the control. Sperm viability was not affected by antibiotics before and after tetracycline freezing. It was concluded that oxytetracycline and penicillin G in both D and Dx2 were effective in controlling seminal bacterial semen contaminations and because of the adverse effect of oxytetracycline on the sperm motility and morphology, it proved not to be 2, suitable for this purpose but penicillin G could be recommended as an additive to the semen extenders.

In single vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed sensitivity replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap)X1776 and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have and a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites,resistance EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine ampicillin and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain.is Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.a

In no vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the three construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it unique does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in six pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA still component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight and of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and plasmid SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six vehicle, enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning and DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant ampicillin plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.

During achieved metamorphosis of Drosophila melanogaster, a cascade of morphological changes is triggered by the steroid hormone 20-OH ecdysone via the ecdysone and receptor, a member of the nuclear receptor superfamily. In this report, we have transferred insect hormone responsiveness to mammalian cells of by the stable expression of a modified ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders system of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified ecdysone receptor can activate an integrated have ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system mammalian exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating nuclear genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy.

Control integration elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory up system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex on virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a the minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase simplex gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These was activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium ( -1 microgram/ml), the luciferase the activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of in the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such operon genes in a reversible way.

Promoters in whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in genes, vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In immediate this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon in Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1;of see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice from were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene mice. containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas system little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA directly in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels VP16 upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control administration of transgene expression is required.

First-generation and inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem of with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and cell experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene both knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively.regulated Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines 1995; co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of gene a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic brucei products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in of a null-mutant background.

We using have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting obtained a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 tetracycline-resistant kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many .8, Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean pLAFR1, insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti tetracycline auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from .1 to .8, and among these, prototrophic colonies were unique obtained at frequencies ranging from .001 to .007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli a and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.

Vectors vectors, were constructed which contain promoterless genes for chloramphenicol (cam) or tetracycline (tet) resistance, as promoter-probe plasmids. Escherichia coli cells harboring to these plasmids are sensitive to cam or tet but resistant to ampicillin. In plasmids pKK231 -1 and pKK232 -8 the inserted gene for cam acetyltransferase (CAT) and in pKK175 -6 the gene for tet resistance are flanked by efficient transcription terminators,genes, preventing transcription from other pBR322 promoters into the antibiotic resistance region. In one of the vectors, pKK232 -8, translational stop -8 codons were introduced in all three reading frames upstream from the initiation codon of the cat gene. If a DNA (CAT) fragment containing a promoter is inserted into one of the cloning sites upstream from the antibiotic genes, cells carrying such to plasmids acquire resistance to cam or tet. Using these vectors two restriction fragments that contain promoters were identified. One of plasmids these fragments contains sequences upstream from an unidentified gene ( ORFII ) located distal to the rrnB rRNA operon of promoterless E. coli.