6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P . patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'-and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Succharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS-and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. putulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2',4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in , I gtll yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucinezipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.

After digestion of protein S4 with trypsin, all 32 tryptic peptides were isolated. Their amino acid compositions were analyzed and the sequence of the amino acids within the tryptic peptides was determined by means of a solid-phase peptide sequenator and by exopeptidases. Alignment of the tryptic peptides was established by analyzing and partially sequencing peptides isolated after digestion of the S4 protein with chymotrypsin, thermolysin and a glutamic-acid-specific protease. Further information about the alignment of peptides came from treatment of S4 with CNBr and with a lysine-modifying reagent.Protein S4 consists of 203 amino acids (Asp,, ASn8, Thr,, Ser,,, Glu,,, Gln,,, Pro,, Gly,,, Ahl8, Val,,, Cys,, Met,, Ile8, Leu,,, Tyr,, Phe,, His,, Lys,,, Arg,, and Trp,) and has a molecular weight of 22550. The basic amino acids are clustered in five regions. Many short repetitions mainly with charged amino acids occur. A prediction is made for regions with a-helices and with B-sheets.Protein S4 [l] is one of the most intensively studied proteins of Escherichia coli ribosomes. It is required for ribosome reconstitution in vitro [2,3] and forms a stoichiometric site-specific complex with 16-S RNA [2,4-81. The portion of the RNA involved in the binding has been identified as part of the 5' terminus with about 400 nucleotides [9-111. On the other hand the part of the protein that binds to the RNA has been investigated using S4 molecules altered by mutations [12] or by chemical modifications [13,14].The functional role of S4 in protein synthesis is less clear, but studies on mutants which have an altered S4 protein [15 -231 indicate that S4 can interfere with the selection of the correct tRNA at the acceptor site.

No abstract

Recovery of the host after infection by the intracellular pathogen Listeria monocytogenes is dependent on cell-mediated immunity. Little is known of the nature of listerial antigens that induce cell-mediated responses in the infected host. In this study we report on the identification and cloning of an Escherichia coli recombinant encoding a listerial antigen, designated ImaA, capable of eliciting a specific delayed-type hypersensitivity response in Listeria-immune mice. Nucleotide sequencing of the Listeria DNA insert in plasmid pLM10 showed that the ImaA gene product consisted of 170 amino acids with a molecular weight of 17,994. The predicted amino acid sequence suggests that the protein is localized to the bacterial plasma membrane or cell wall. The ImaA gene was unique to the pathogenic species L. monocytogenes and Listeria ivanovii; it was not present in any other species of the genus Listeria.