Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis. Additional detection of an exon potentially coding for a new peptide

From a genomic library of Xenopus laevis, two genes coding for different preprocaeruleins have been isolated and sequenced. These correspond to the type I and type III precursors analyzed previously at the cDNA level [Richter, K., Egger, R. and Kreil, G. (1986) J. Biol. Chem. 261, 3676-3680]. The type III gene comprises eight exons; the type I apparently contains eight exons as well, of which six have been sequenced. The genetic information for the dekapeptide caerulein is present on small exons of 45 base pairs. The two genes are highly homologous in their 5'-flanking region, the exon/intron boundaries, and long stretches of intron sequences. A possible scheme for the evolution of this small family of genes through exon and gene duplications is presented. In the type I gene, in place of one of the caerulein exons, a potential exon with conserved splice sites was discovered. If expressed in some frog cells, this exon would code for a new peptide 60% homologous to caerulein.     

Action of phospholipases on the cytoplasmic membrane of Escherichia coli. Stimulation by melittin

The emission maximum of the single tryptophan residue of melittin was measured in the presence of phosphatidylethanolamine liposomes and Escherichia coli cytoplasmic membranes. In both cases, the fluorescence maximum was shifted to shorter wavelengths indicating a transfer of the indole ring to an apolar environment. E. coli membranes were labelled in position 2 of their phospholipids with [¹⁴C]oleic acid. These membranes were used for measuring the activity of an endogenous phospholipase A₂. A slow hydrolysis is observed, which can be accelerated by adding melittin. The extent of the stimulation depends on the molar ratio of melittin to membrane phospholipid. Under suitable conditions, the initial rate of hydrolysis is six to seven times higher in the presence than in the absence of melittin. The action of the phospholipase A₂ from bee venom is also stimulated by melittin. An identical stimulation was observed with either E. coli membranes or pure phosphatidylethanolamine liposomes as substrate.     

Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum

The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined. The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli. The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria. The derived amino acid sequence corresponds to a protein of Mr 40,439. Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data. A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E. coli. Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism. However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.     

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Biosynthesis of thyrotropin releasing hormone in the skin of Xenopus laevis: partial sequence of the precursor deduced from cloned cDNA

Skin of Xenopus laevis contains relatively large quantities of thyrotropin releasing hormone (TRH). Total mRNA isolated from skin was cloned in the plasmid pUC8. Among 1400 cDNA clones, one was found with an insert of 478 nucleotides coding for the amino-terminal part of prepro-TRH. This clone was detected using a mixture of two synthetic undecanucleotides for colony hybridization. The single open reading frame starts with a methionine residue and a stretch of hydrophobic amino acids, as is typical for signal peptides, and terminates at the poly(C) tail without a stop codon. The deduced polypeptide of 123 amino acids contains three copies of the sequences Lys-Arg-Gln-His-Pro-Gly-Lys Arg-Arg and a fourth incomplete copy at the carboxyl end. Typical pro-hormone processing at this sequence would yield pGlu-His-Pro.NH2,i.e. TRH. It is concluded that the cloned part of the mRNA codes for prepro-TRH and that the TRH precursor from skin of X. laevis is a polyprotein containing at least four copies of the end product in its amino acid sequence.