No abstract

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIll fragment revealed an open reading frame which could encode a protein of 407 amino acids with an Mr of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane ,(-sheet structures, implying a possible role for the protein in translocation of hydrophilic 0-antigen-containing materials.Salmonella strains of groups A, B, and Dl contained rfc-homologous DNA, but strains of groups Cl, C2, C3, D2, and E2 did not.Three major genetic regions have been implicated as dedicated to the biosynthesis of the polysaccharide component of the lipopolysaccharide (LPS) of Salmonella typhimurium. The rfa cluster of genes is involved in core synthesis, and the rfb region is involved in the synthesis of the 0-antigenic tetrasaccharide units (26). In addition, it is thought that genes of both the rfa and rfb clusters are involved in the transport and ligation of core-0-antigen complexes (26). The rfc region is thought to encode a polymerase responsible for the linking of the 0-antigen tetrasaccharide units into long chains, giving rise to typical smooth LPS (26). Mutants of S. typhimurium which are defective in the 0-antigen polymerase because of a mutation at the rfc locus produce LPS structures termed semirough (SR), which have at most one 0-antigenic tetrasaccharide unit attached to any given core unit (30,56). A bacteriophage sensitivity pattern characteristic of SR mutants has been described previously; such strains of S. typhimurium were resistant to the smooth LPS-specific phages 9NA and P22, sensitive to phage FO, and resistant to all phages specific for rough LPS (LPS without attached 0 antigen) except P22I (55).Mutants of the SR LPS phenotype have been identified in Salmonella groups B and E (22, 30). In S. typhimurium (group B), the rfc locus has been located between gal and trp (30, 44), corresponding to a map position between 18 and 34 min. An analogous locus in some Salmonella strains of group D has been postulated on the basis of the observation that in hybrids between Salmonella groups B (0-4, 5, 12) and D (0-9, 12), involving transfer of rib genes, the polymerase of one group could polymerize the 0 units of the other (24,31,51 This paper describes the cloning of the rfc gene of S. typhimurium by complementation of rfc defects in spontaneous and ISl0-derived SR mutants. A physical map of the DNA region encoding the rfc gene is presented, and attempts to visualize the gene product(s) are described. The distribution of rfc-homologous DNA in other Salmonella strains is also determined. The DNA fragment containing the gene is sequenced, and a putative rfc gene is ident...

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70).Substitution of Asp2o or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp2o was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.of the p55/p70 complex (i.e., the high-affinity IL-2R) that appears to be essential for a full proliferative response by T cells and for the late phase of effector lymphokine-activated killer functions.To understand the structure-function relationship ofhuIL-2 and the interactions with its receptor, we have endeavored to identify those amino acids that mediate binding to the high-affinity receptor as well as to its two component subunits. By using site-directed mutagenesis, we had engineered (11) a series of huIL-2 analogs by the introduction of specific deletions and substitutions to modify the protein. The mutant huIL-2 proteins were produced in Escherichia coli and assayed for biological activity. We have now extended these studies with additional analogs and assayed their ability to compete for binding to the IL-2R and its subunits. The identification of specific amino acids required for bioactivity and binding to the p55 and p70 subunits has allowed us to correlate the structure of IL-2 with its biological functions.Human interleukin 2 (huIL-2) exerts immunoregulatory effects on a variety of cells including T cells, activated B cells, natural killer cells, and lymphokine-activated killer cells. The biological effects of huIL-2 are mediated through specific interactions with cell surface receptors present on the target cells (1, 2). High-, intermediate-, and low-affinity forms ofthe IL-2 receptor (IL-2R) have been identified on these cells. The high-affinity receptor exhibits an apparent Kd of =10-1 M and exists as a complex of at least two distinct polypeptides, with molecular masses of 55 kDa (p55 or Tac) and 70-75 kDa (p70 or p75). Cells or cell lines expressing only p55 are capable of binding 4). These cells have been used to demonstrate that the p55 subunit corresponds to the lowaffinity form of the receptor (Kd = 1-3 x 10-8 M). It has been reported that the p70 protein, which is expressed at relatively high levels on the human YT-...

Insertion mutations were constructed in cloned pmi and rfc genes of Salmonella typhimurium, and these mutations were recombined (singly) into the chromosome of mouse-virulent S. typhimurium C5, displacing the wild-type alleles. Phage sensitivity profiles, lipopolysaccharide analysis, and DNA blotting all confirmed that the replacement events had occurred. The mutations were complemented by plasmid-borne wild-type alleles, as judged by the restoration of wild-type phage plaquing profiles and lipopolysaccharide production (both mutants) and the restoration ofpmi-encoded enzyme production (pmi mutant). The virulence, persistence, and immunizing capacities of the mutants fed to mice were compared with those of the wild-type strain and complemented mutants. Both mutants were much reduced in virulence, with the rfc mutant being avirulent even at 109 bacteria per mouse. This mutant was also avirulent at up to 106 bacteria per mouse when administered intraperitonealy. Both the rfc and pmi mutant strains persisted in the Peyer's patches of the gut after feeding and were capable of colonizing the deeper tissues of the mice from such initial infective foci. Both mutant strains were effective as live oral vaccines (107 bacteria or more) against oral S. typhimurium challenge