Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Growth of Escherichia coli in the presence of trimethoprim-sulfamethoxazole facilitates detection of Shiga-like toxin producing strains by colony blot assay

Abstract Subinhibitory concentrations of trimethoprim-sulfamethoxazole increased the total yield of Shiga-like toxin (SLT), produced by Shigella dysenteria 1 and by enterophathogenic and enterohemorrhagic strains of Escherichia coli . Stimulation of SLT synthesis by trimethoprim-sulfamethoxazole was demonstrated by an increase in cytotoxic activity for HeLa cells and the diameter of the zone formed around bacterial colonies probed with monoclonal antibodies to SLT. Thus, supplementation of culture media with trimetroprimsulfamethoxazol will facilitate SLT purification and detection of SLT-producing bacteria.     

Molecular cloning of the F8 fimbrial antigen from Escherichia coli

Abstract The genetic determinant coding for the P-specific F8 fimbriae was cloned from the chromosome of the Escherichia coli wild-type strain 2980 (O18:K5:H5:F1C, F8). The F8 determinant was further subcloned into the Pst I site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned counterpart was demonstrated. The cloned F8 fimbriae and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepithelial cells. The cloned F8 determinant was well expressed in a variety of host strains.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the -galactosidase leader peptide, under the control of the -galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.