ABSTRACT The E2 protein (422 amino acid residues long) of Semliki Forest virus is a spanning membrane protein which is made in the rough endoplasmic reticulum of the infected cell and transported to the cell surface. The cytoplasmic domain of this protein comprises 31 amino acid residues. We introduced deletions of various sizes into the gene region encoding this part of the protein molecule and analyzed the transport behavior of the mutant proteins. The deletions were made using exonuclease digestions of cloned cDNA encoding the E2 protein. When the mutated DNA molecules, engineered into an expression vector, were introduced into nuclei of baby hamster kidney 21 cells, membrane proteins with cytoplasmic deletions were expressed and routed to the cell surface in the same way as the wild-type protein. This suggests that the cytoplasmic domain of the E2 protein does not carry information that is needed for its transport from the rough endoplasmic reticulum to the cell surface. Proteins with many different destinations are synthesized on polysomes bound to the endoplasmic reticulum (RER) (22). Some proteins become secreted from the cell, while others are routed to the cell surface or to various intracellular organelles.

Abstract. Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla,

EFFICIENT ALGORITHMS FOR SEQUENCE ANALYSIS WITH CONCAVE AND CONVEX GAP COSTS David A. Eppstein We describe algorithms for two problems in sequence analysis: sequence alignment with gaps (multiple consecutive insertions and deletions treated as a unit) and RNA secondary structure with single loops only. We make the assumption that the gap cost or loop cost is a convex or concave function of the length of the gap or loop, and show how this assumption may be used to develop e#cient algorithms for these problems. We show how the restriction to convex or concave functions may be relaxed, and give algorithms for solving the problems when the cost functions are neither convex nor concave, but can be split into a small number of convex or concave functions. Finally we point out some sparsity in the structure of our sequence analysis problems, and describe how we may take advantage of that sparsity to further speed up our algorithms. CONTENTS 1. Introduction ............................1 ...

Abstract. The 20-kD regulatory light chain (RLC) plays a central role in the regulation of smooth muscle contraction. Little is known about the structure or expression of smooth muscle myosin light chain (MLC) genes. A cDNA library was constructed in the expression vector, Lgt-ll, with mRNA derived from cultured rat aortic smooth muscle cells. Using antibody generated against tracheal smooth muscle myosin, three cDNA clones encoding a RLC were isolated, one of which, SmRLC-2, represents a full-length transcript of the RLC mRNA. The derived amino acid sequence shows 94.2 % homology with the chicken gizzard RLC, and 70 and 52 % homology with the rat skeletal and cardiac muscle MLC-2 proteins, respectively. Thus, the gene encoding the putative smooth muscle

Abstract. Ribophorins I and II are two transmembrane glycoproteins that are characteristic of the rough endoplasmic reticulum and are thought to be part of the apparatus that affects the co-translational translocation of polypeptides synthesized on membrane-bound polysomes. A ribophorin I eDNA clone containing a 0.6-kb insert was isolated from a rat liver lambda gtll eDNA library by immunoscreening with specific antibodies. This cDNA was used to isolate a clone (2.3 kb) from a rat brain lambda gtU eDNA library that contains the entire ribophorin I coding sequence. SP6 RNA transcripts of the insert in this clone directed the in vitro synthesis of a polypeptide of the expected size that was immunoprecipitated with anti-ribophorin I antibodies. When synthesized in the presence of microsomes,

Abstract. A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oli-gonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratificationrelated cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed. PITHELIAL differentiation is usually characterized by the formation of intermediate-sized filaments (IFs) ~ of the cytokeratin type (20, 28, 85, 86). The early embryonal epithelia, i.e., ecto- and endoderm, are simple polar epithelia and possess IFs of the most simple polypeptide composition, i.e., one representative of the acidic (type I) subfamily, i.e., cytokeratin 18, and one representative of the more basic (type II) cytokeratin subfamily, i.e., cytokeratin

Abstract. This report describes the isolation and characterization of genomic and cDNA clones which define a subfamily of type I keratins in Xenopus laevis whose expression is restricted to embryonic and larval stages. The XK81 subfamily, named after the prototype cDNA clone DG81, contains four members arranged in two pairs of closely homologous loci; they were named 81A1, A2, B1, and B2. Genomic clones were obtained representing all of these regions. The A1 gene has been completely sequenced together with ~1 kb of flanking sequences at each end; this gene corresponds to the previously reported cDNA clone 8128 (Jonas, E., T. D. Sargent, and I. B. Dawid, 1985,

Abstract. We report the sequence of a cDNA clone that codes for the carboxy-terminal portion of the peroxisomal protein, acyl-CoA oxidase, from the yeast, Candida tropicalis. This is a newly identified acyl-CoA oxidase sequence, most likely a second allele of POX4. The cDNA clone was expressed by in vitro transcription followed by translation. The major product, a 43-kD protein, associated with isolated peroxisomes in an in vitro import assay. More than half of the peroxisome-associated protein was protected from added protease, implying that it was internalized within the organelle. These findings indicate that there is sufficient information in the carboxy-terminal portion of the protein to target it to peroxisomes.

Abstract. The soluble cytoplasmic protein pyruvate kinase (PK) has been expressed at the cell surface in a membrane-anchored form (APK). The hybrid protein contains the NH2-terminal signal/anchor domain of a class II integral membrane protein (hemagglutinin/ neuraminidase, of the paramyxovirus SV5) fused to the PK NH2 terminus. APK contains a cryptic site that is used for N-linked glycosylation but elimination of this site by site-specific mutagenesis does not prevent cell surface localization. Truncated forms of the APK molecule, with up to 80 % of the PK region of APK removed, can also be expressed at the cell surface. These data suggest that neither the complete PK molecule nor its glycosylation are necessary for intracellular transport of PK to the cell surface, and it is possible

Understanding the regulation of adhesins defines a pathogenic bacterium’s interaction with the local environment within the host. In certain strains of Streptococcus pyogenes, transcription of prtF, the gene which encodes the fibronectin-binding adhesin protein F, is activated by RofA under anaerobic conditions. RofA binds specifically to DNA in its target promoters and autoregulates its own expression. In this study, we have used DNase I protection assays to further investigate the interaction of RofA with its target promoters. In the region between rofA and the gene which encodes protein F (prtF), RofA binds to two distinct sites: a smaller site (17 bp) adjacent to the rofA promoter, and a larger site (40 bp) adjacent to the prtF promoter. Analysis of fusions to a novel reporter gene whose product consists of the fusion of the N-terminal secretion domain of protein F with the C-terminal enzymatic domain of the enterococcal alkaline phosphatase (PhoZ) revealed that the small RofA binding site had no direct role in control of prtF transcription but contributed to regulation of rofA. Comparison in several strains representing different patterns of prtF expression indicated that the larger site was required for activation of rofA and of prtF in all strains by both RofA-dependent and-independent pathways. Thus, it would appear that a common recognition sequence provides separate entries to a final common pathway in S. pyogenes virulence gene expression. The identification of multiple RofA-like proteins and promoters with RofA binding sites implies the existence of a widespread interacting regulatory network.