Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.
A clone containing an 18S ribosomal RNA (rRNA) gene has been isolated from a human genomic library constructed in lambda Charon 4A. This gene was sequenced and found to be 1868 bp long. The sequence divergencies in the human 18S rRNA gene and the previously sequenced mouse and rat genes are found in one G + C-rich region of 110 bp located in the 5' domain of the molecule. Except for this variable region, extensive homology exists among these three mammalian genes. Overall, the human 18S rRNA gene is 98.8% homologous with those of rat and mouse.
The complete nucleotide sequence of the rat 18S ribosomal RNA gene has been determined. A comparison of the rat 18S ribosomal RNA gene sequence with the known sequences of yeast and frog revealed three conserved (stable) regions, two unstable regions, and three large inserts. (A,T) leads to (G,C) changes were more frequent than (G,C) leads to (A,T) changes for three comparisons (yeast leads to frog, frog leads to rat, and yeast leads to rat). GC pairs were inserted preferentially over AT pairs for the same three comparisons. These two factors contribute to the progressively higher GC content of 18S ribosomal RNA of yeast, frog, and rat.
Various catenated and replicating molecules of colicin El isolated from minicells have been identified in regions of neutral sucrose density gradients or cesium chloride-ethidium bromide density gradients. These colicin molecules have been found in those regions of the gradient where pulse-labeled DNA accumulates.Adler et al.(1) isolated a mutant of Escherichia coli, P678-54, that produced significant numbers of small, DNA-less progeny ("minicells") under normal growth conditions. Subsequent work has shown that if plasmid or episomal DNAs are carried by that mutant, they can segregate into (2-6) and replicate in (2, 6, 7) the minicells. In this report minicells containing colicin factor El (Col El) DNA have been used to examine DNA replication. Two principal advantages of studying DNA replication in minicells are first, the small size of the Col El DNA monomers [4 X 108 daltons (8)1 and second, the absence of chromosomal DNA, which otherwise interferes with the identification of extra-chromosomal DNA. The study of the replication of Col El DNA in minicells has shown that (a) all of the observed DNA in minicells carrying Col El DNA is Col El DNA (2, 7); (b) the Col El DNA principally exists as covalently-closed circular and open circular molecules, though linear monomers are also found (2, 7); (c) the Col El DNA can undergo at least two complete rounds of replication (2); and (d) replicating structures identical to the forked circular molecules originally found by Cairns (9), as well as those similar to the structure of rolling circles as postulated by Gilbert and Dressler (10), can be identified (7). These observations have led to a further examination of Col El DNA replication, a part of which is the subject of this report. MATERIALS AND METHODSAll experiments were performed with purified minicell preparations (1 viable bacterium per 106-107 minicells) isolated from log-phase cultures of E. coli strain P678-54 (Col El) (2) grown in Tris-Casamino acid-glucose medium supplemented with 20 ug/ml of threonine and leucine and with 1 jig/ml of vitamin B1 (2). The detailed procedures for minicell purification, [3Hlthymidine labeling of Col El DNA in the above medium supplemented with 250,ug/ml of deoxyadenosine and 0.5 jg/ml of thymidine, and DNA extraction from minicells treated with spheroplast medium [lysozyme-EDTA-pancreatic ribonuclease] are as described (2, 11). Pronase treatment (1 mg/ml of predigested Pronase for 15 min at 37°C) of minicells previously incubated in spheroplast medium was introduced into the DNA extraction procedure before the addition of Sarkosyl NL30 and subsequent phenol extraction (2, 11). Neutral sucrose density gradient sedimentation analysis (2) and cesium chloride-ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridium bromide) density-gradient analysis of DNA (2, 12) and electron microscope techniques (7, 13, 14) were also described in detail. Buffer solutions used are Tris-saline (TS) 0.05 M Tris-0.05 M NaCl (pH 8.0); Tris-EDTA-Saline (TES), which is TS + 5 mM EDTA; and s...
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