Louis P. Visentin

An acidic, alanine-rich 50 S ribosomal protein from Halobacterium cutirubrum: Amino acid sequence homology with Escherichia coli proteins L7 and L12

being identified by two-dimensional electrophoresis [2] and amino acid composition [5]. Tryptic peptides of L20, prepared by incubation at 37°C for 3 hr with a 1% (weight enzyme: weight protein) trypsin solution, were fractionated by gel fil- tration chromatography on a Sephadex G-50 super- fine column (1.5 X 270 cm) with distilled water as the eluant. The resulting peaks, which were monitored at 230 nm, were analysed for amino acid composition with a Durrum D-500 amino acid analyser after hy- drolysis at 110°C for 18 hr with 6 N HCl in vacua. Peptide L20-T4 was eluted soon after the void volume and was well separated from other smaller tryptic pep- tides. The amino-terminal sequence of protein L20 (6 mg) and L20-T4 (2 mg) was determined by automatic Edman degradation (91 using a Beckman Model 890C sequencer with the quadral protein program. The thia- zolinone derivatives, converted to their PTH-deriva- tives, were identified by thin-layer chromatography on silica gel plates as described by Wittmann-Liebold [lo] The thiazolinone or PTH-derivatives were hydrolysed separately with 6 N HCl and Hl [ 1 I] at 130°C for 20 hr, and the amino acid formed was analysed with a Durrum D-500 amino acid analyser. 127

A direct-selection vector derived from pColE3-CA38 and adapted for foreign gene expression ☆

The construction of a plasmid vector, pVT25, which allows an efficient and direct selection for transformed cells carrying recombinant plasmids is described. In this vector, the replicon and ApR gene from plasmid pBR327 are fused to the colE3 gene of pColE3-CA38, whereby positive selection is based on the inactivation of the lethal colicin E3 by the insertion of a foreign DNA fragment. However, pVT25 can be maintained within the Escherichia coli cells when complemented with another plasmid, pVT26, which expresses the colicin E3 immunity (imm) and the TcR phenotypes. Furthermore, pVT25 was used to regulate the expression of the synthetic human proinsulin gene fused to the colE3 gene at the single ClaI site. The production of the characteristic C-peptide of proinsulin, monitored by radioimmunoassay, was shown to be under the control of the inducible promoter of the colE3 gene.

Restriction endonuclease mapping of Co1E2-P9 and Co1E3-CA38 plasmids

Using single and double restriction-endonuclease digestions, 16 and 17 cleavage sites have been mapped for the ColE2-P9 and ColE3-CA38 plasmids, respectively. One or more sites for AvaI, BglI, EcoRI, HincII, PvuI, PvuII, SmaI and XhoI endonucleases were found in both plasmids, two BglII sites were found only in ColE2-P9, and one KpnI site was unique to ColE3-CA38. ColE2-P9 was found to be slightly smaller than ColE3-CA38, 4.4 Md compared to 4.6 Md. Eleven restriction sites are common to both plasmids in that they are identically placed relative to each other. These sites define a continuous DNA segment equal to over 60% of each plasmid. The remaining portions of the plasmids, which contain the non-homologous regions identified by Inselburg and Johns (1975) have no restriction sites in common, and differ in size by about 0.2 Md.

Correlation between 30S ribosomal proteins of Bacillus stearothermophilus and Escherichia coli.

SummaryThe 30S ribosomal proteins from Bacillus stearothermophilus strains 799 and 10 were purified and correlated with those from E. coli by comparing their two-dimensional electrophoretic mobility, immunological cross-reaction, molecular weight, amino acid composition and partial amino acid sequence. A high degree of similarity was observed among the proteins from these taxonomically distant bacterial species.

Ion effects on protein-nucleic acid interactions: The disassembly of the 50-S ribosomal subunit from the halophilic bacterium, Halobacterium cutirubrum

The 50-S ribosomal subunits from the extreme halophilic bacterium, Halo-bacterium cutirubrum, stable structurally and functionally in concentrated salt solutions were subjected to ionic environments depleted in either K+ or Mg2+ or both. Under these conditions specific classes of proteins are released from the subunit along with the 5 S RNA. Two-dimensional electrophoretic analysis of the resultant split protein fractions indicate some mutually exclusive effects of specific ions on the binding of specific proteins to the 23 S RNA as well as on the retention of 5 S RNA within the ribosomal macrostructure.

Homologies in procaryotic ribosomal proteins: Alanine rich acidic proteins associated with polypeptide translocation

Abstract : Translocation of the growing peptide chain in the synthesis of proteins is mediated to some extent by certain novel acidic ribosomal proteins. These proteins, designated L7 and L12 in Escherichia coli, are localized on the 50 S ribosomal subunit and differ only in the acetylation state of the N-terminal serine. The isolation, chemical characterization and N-terminal sequence analysis of an L7-L12 equivalent from B. stearothermophilus is reported, and the structural features are compared with the E. coli polypeptides, a comparison that is of particular note in view of the considerable interest in the structure, function and evolution of the genetic translation apparatus.

Procaryotic ribosomal proteins: N‐terminal sequence homologies and structural correspondence of 30 S ribosomal proteins from Escherichia coli and Bacillus stearothermophilus

Abstract : In attempting to evidence the evolution and the structure--function relationships of the ribosome in procaryotes the authors have undertaken a comparative amino acid sequence analysis of ribosomal proteins from Escherichia coli, and Bacillus stearothermophilus and Halobacterium cutirubrum, organisms which differ substantially in their physiological tolerances and taxonomic relationships. Previous structural studies have indicated a high degree of homology in some of the 30 S ribosomal proteins from E. coli and B. stearothermophilus. Reported in this paper is a summary of results on the study of the amino terminal regions of 19 ribosomal proteins E. coli strain Q13 and 21 from B. stearothermophilus strain 10.

Isolation and structure of the replicon of the promiscuous plasmid pCU1

Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.

Ribosomal protein S1/S1A in bacteria.

Recently, Escherichia coli ribosomal protein Sl has been shown to have important and diverse functions in the logistics of mRNA processing during in vitro protein synthesis [l-9] and Qfl replication [lo-121. In the course of an RNA-protein interaction study with Sl, we discovered that there are two distinct large molecular weight ribosomal proteins, both of which have been designated as Sl by different laboratories. The two proteins have been purified, characterized, and designated as Sl and Sl A. In view of the functions ascribed to Sl in the interaction with mRNA [l-9] , 16 S RNA [13] and other proteins (a replicase) [ 11,141, the question naturally arises as to the universality of the Sl type protein in various organisms. Indeed, it has been stated that certain specialized bacterial types lack

The immunity genes of colicins E2 and E8 are closely related.

We have determined the nucleotide sequence of the newly characterized colicin E8imm gene which exists in tandem with the colicin E3imm gene in the: ColE3-CA38 plasmid. Comparison of these immunity structures reveals considerable sequence divergence) but the ColE8imm gene is markedly homologous to the colicin E2imm gene from the ColE2-P9 plasmid.