Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.     

Regulation of gene expression in rat prostate by androgen and beta-adrenergic receptor pathways

Denervation of rat ventral prostate has been accomplished by excising prostatic tissue fragments and implanting them under the renal capsules of intact syngeneic rats. This resulted in a substantial reduction of expression of a major organ-specific secretory protein, prostatic binding protein (PBP). The depressed level of PBP and its subunits and mRNAs could be restored, however, to as much as 80% of control levels by the administration of a pharmacological dose of exogenous androgen, testosterone propionate (TP), and/or a beta-adrenergic agonist, isoproterenol (ISO). Furthermore, compared to ascorbate-treated controls, TP and ISO increased the synthesis of total cellular protein and PBP by the prostatic renal implants. TP and/or ISO also remodelled the luminal epithelial structure and elevated secretory functions. ISO alone had no effect, however, in castrated animals, indicating that androgen plays a dominant role in the restoration of tissue PBP content. Concomitant to increased PBP content and remodelling of prostatic histomorphology, androgen was also found to raise the depressed levels of beta 2-adrenergic and androgen receptors in the prostatic isografts maintained in intact hosts. In contrast, although an established rat prostatic epithelial cell line (NbE-1) contains high affinity androgen receptor, androgen failed to restore beta-adrenergic receptor as well as PBP content in this cultured cell line. These results, taken together, suggest that a tight coupling between androgen receptor and beta 2-adrenergic receptor pathways may be a prerequisite for PBP expression and functional differentiation in the rat ventral prostate gland.     

Species-specific TNF induction of thymocyte proliferation

Summary Recombinant murine (rMu) tumor necrosis factor (TNF), in a standard comitogenic assay with phytohemagglutinin, induced murine thymocyte proliferation, while up to 10,000-fold higher concentrations of recombinant human TNF did not. The induction of thymocyte proliferation was dependent upon TNF concentration in a biphasic manner. Thus, 100 to 1000 units/ml TNF were near optimal while concentrations 1,000 units/ml caused apparent down regulation. The effect was abrogated by neutralizing antibody to rMu-TNF but not by neutralizing antibody to rMu-interleukin 1 or . The rMu-TNF did not induce proliferation of the mature murine T-helper cell line, D10.G4.1, in the presence of mitogen. Taken together the results indicate that TNF, in a strictly species-specific manner, can regulate thymocyte proliferation independently of interleukin 1.Supported in part by Asahi Chemical Industry Co., Inc. and by USPHS Grants CA-24538, CA-15142 and CA-09072 awarded by the National Cancer Institute, Department of Health and Human Services     

Estimation of oxygen penetration depth in immobilized cells

Summary A simple method is proposed for calculating oxygen pentration depth in immobilized cells by assuming zero order kinetics in the presence of several external oxygen transport resistances. Calculations indicate that typical penetration depths of oxygen for immobilized microbial cells are in the range of 50–200 and those for immobilized or encapsulated animal and plant tissue culture are about 500–1000 . Based on calculations, oxygen transport in microencapsulation and microcarriers for tissue cultures are not transport-limited, but a slight limitation is expected for those in a hollow fiber reactor.Nomenclature a_s specific area of a support (cm) - Bi Biot number - dimensionless - C_b oxygen concentration in the bulk liquid (mM) - C _b C _b ^* -C_cr (mM) - C _b ^* bulk oxygen concentration in equilibrium with air (mM) - C_cr critical oxygen concentration (mM) - C_s oxygen concentration in the solid phase (mM) - d_p diameter or thickness of a support (cm) - D_eff effective diffusivity of oxygen in the solid phase (cm²/s) - k_m membrane permeability of oxygen (cm/s) - k _m ^* D_eff/_m - k_La_L liquid phase mass transfer rate coefficient (1/s) - k_sa_s solid phase mass transfer rate coefficient (1/s) - (OUR)_v volumetric oxygen uptake rate (mmol O₂/l) - p geometry parameter, p=0 for slab, p=1 for cylinder, p=2 for sphere - P_d oxygen penetration depth (cm) - P _d oxygen penetration depth in the absence of external diffusion limitation (cm) - Q volumetric oxygen uptake rate, (mmol O₂/l·h) - specific oxygen uptake rate (mmol O₂gm biomass (dry)·h) - r length coordinate (cm) - r_c oxygen penetration depth for sphere (cm) - r _c r_c in the absence of external diffusion limitation (cm) - r _c ^* oxygen penetration depth for cylinder (cm) - r _c ^* r _c ^* in the absence of external diffusion limitation (cm) - r_com combined mass transfer rate resistance (s) - r_d location where C_s becomes zero or C_cr (cm) - r_i radius of cylinder or sphere, half thickness of slab (cm) - U_sg superficial gas velocity (cm/s) - X cell concentration (g/l) Greek letters Thiele modulus, dimensionless - _L, _s liquid and solid phase volume fraction, respectively, dimensionless - effectiveness factor On sabbatical leave from KAIST, Seoul, Korea     

Effects of subacute treatment with cocaine on activities of N-demethylase, UDP-glucuronyltransferase and sulfotransferase in WKY and SHR rat liver--sex and strain differences

The effects of subacute treatment with cocaine on activities of cocaine N-demethylase, UDP-glucuronyltransferase (GT) toward 4-nitrophenol and phenolphthalein and sulfotransferase (ST) toward androsterone and 4-nitrophenol in livers from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated. Hepatic metabolism of cocaine was different between the sexes (with males having higher N-demethylase activity) and the strains (with WKY rats having higher activity). The effects of subacute cocaine administration on the activity of cocaine N-demethylase were also sex- and strain-related. Whereas cocaine administration increased activity of hepatic N-demethylase in both female strains, it decreased activity in male WKY and had no effect on activity in male SHR. Sex and strain-related as well as cocaine-induced differences were also found in activities of hepatic GT toward 4-nitrophenol and phenolphthalein as well as in activity of hepatic ST towards andersterone and 4-nitrophenol. These results suggest that some of the individual variation in the effects of cocaine may be due to sex and genetic differences in the hepatic metabolism of cocaine and/or in sexually and/or/genetically-determined differences in how cocaine affects hepatic metabolism of other xenobiotics.     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)