A 37-residue cationic antimicrobial peptide named mesentericin Y 105(37) was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 105(37) contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 105(37) was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 105(37) is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 105(37) in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30-40% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17-31. To reveal the molecular basis of the specificity of mesentericin Y 105(37) targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum alpha-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14-18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 105(37) are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative alpha-helical domain 17-31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1-14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 105(37) is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation.

We have previously identified a 95-to 100-kDa cell surface glycoprotein, which we named BEN (for bursal epithelium and neurons), that is widely expressed during chicken embryonic development. In the central nervous system, it is restricted to subsets of neurons including the motoneurons and the inferior olivary nucleus neurons (which provide the cerebellum with the climbing fibers) where its expression occurs during the phase of axonogenesis and synaptogenesis. In the present work, we show that BEN expression extends to a variety of tissues originating from the three embryonic germ layers. We have found that BEN immunopurifled from neural, epithelial, and hemopoietic tissues is differently glycosylated and may or may not carry the HNK-1 epitope. We then cloned a full-length cDNA encoding this

The anti-&0 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the o70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-&0 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards &70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on o70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-o70 factor, and we therefore propose that the gene which we call asi4, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.During infection of Escherichia coli by phage T4, a large part of the program of viral gene expression is regulated at the transcriptional level. The host's RNA polymerase transcribes the 200 or so viral genes from different classes of T4-specific promoters (23). Some of these classes are recognized only after T4-coded functions modify this enzyme's specificity. The a subunits of RNA polymerase are covalently modified by ADP-ribosylation, and the enzyme binds a series of phage-coded proteins (13). Among these, the products of genes 33, 45, and 55 are required for late transcription (12). In particular, gp 55 (16) redirects RNA polymerase transcription initiation from T4 late promoters by replacing the E. coli c70 subunit. The product of gene rpbA (45) strongly binds to RNA polymerase core. A smaller RNA polymerase-binding gene product, the 10-kDa (10K) protein, copurifies with au7 on phosphocellulose and inhibits o70-directed transcription initiation by E. coli RNA polymerase holoenzyme (36). It is believed that the interaction of the T4 10K protein with bacterial c70 weakens o&0-core interaction; this, in turn, would allow gp 55 to successfully compete for core enzyme during T4 development (12). The 10K protein found in lysates from T4-infected cells strongly binds to RNA polymerase agarose affinity columns (27). Starting from this observation, we have previously used a biochemical approach to identify the gene encoding the T4 10K protein (25) T4 (36). This approach to the partial purification of gp asiA was chosen to minimize its progressive loss, observed during the purification of RNA polymerase from T4-infected cells (35). In Fig. 1, we show the autoradiography of an SDS-polyacrylamide gel illustrating the separation of the RNA polymerase-binding proteins coded by phage T4 after Bio-Rex 70 chromatography. The gp asiA is essentially found in...

Splicing of the chicken ␤-tropomyosin exon 6A is stimulated, both in vivo and in vitro, by an intronic pyrimidine-rich element (S4) located 37 nucleotides downstream of exon 6A. Several pyrimidine-rich sequences are able to substitute for the natural S4 enhancer with various stimulatory effects. We show that the different enhancer sequences recruit U1 small nuclear ribonucleoprotein (SnRNP) to the exon 6A 5 splice site, with an efficiency that correlates with the splicing activation. By using RNA affinity and two-dimensional gel electrophoresis, we characterized several proteins that bind to the different enhancer sequences. Heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP I (polypyrimidine track-binding protein, PTB) exhibit a higher level of interaction with the strong enhancer sequences (S4) than with the weakest enhancers. Functional analysis shows that hnRNP K is a component of the enhancer complex that promotes exon 6A splicing through the wild-type S4 sequence. The addition of recombinant hnRNP K to nuclear extracts preincubated with poly(rC) RNA competitor completely restores splicing efficiency to the original level. hnRNP I (PTB) was also found associated with the strong enhancer sequences. Its function in the splicing of exon 6A is discussed.Alternative splicing of pre-mRNAs generates different mRNAs from the same primary transcript and as such contributes to protein diversity (1-3). This process is thought to be important for regulation of the gene expression and has been enlightened by the discovery that human genome contains only 30,000 -40,000 genes. From these studies, it has been suggested that more than 50% of the genes are alternatively spliced (4). In many cases, alternative splicing is regulated in a tissue developmental stage or sex-specific manner and responds differently to metabolic stimuli (2, 3). Studies of different splicing events show that the bona fide regulation is orchestrated by multicomponent complexes that promote or repress the use of alternative splice sites through binding to regulatory sequences (1,5,6). The SR protein family is a well characterized class of proteins that are involved in both constitutive and alternative splicing (7,8). It has been shown that the binding of members of the SR proteins to purine enhancer sequences promotes the inclusion of the corresponding exon by facilitating the recognition of weak 3Ј splice sites (9). Another group of proteins that affect splice site selection are the hnRNPs.1 They constitute a large group of RNA-binding proteins that associate with nascent transcripts, and they can hinder or assist the splicing machinery (10, 11). Early experiments have shown that hnRNP A1 is able to antagonize the activity of ASF/SF2 by promoting a shift toward the selection of distal 5Ј splice sites (12, 13). More recently, it has been found that hnRNP A1 interacts with silencer elements to repress splicing of several pre-mRNAs (14 -18). In the case of human immunodeficiency virus type 1 tat splicing, it has been proposed that the binding o...