Peripherin, a 39-kDa membrane protein, has been previously localized to the rim region of the vertebrate rod photoreceptor disk membrane by use of monoclonal antibodies and immunocytochemical labeling techniques. As an initial step in determining the structure and function of this protein, we have cloned and sequenced cDNA containing its complete coding sequence. A bovine retinal lambda gt11 expression library was screened with the antibodies, and a 583 base pair clone was initially isolated. The remaining part of the coding sequence was obtained from subsequent rescreenings of the same library and an independent lambda gt10 library. A C-terminal CNBr fragment of peripherin was purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography. The amino acid sequence of the isolated C-terminal peptide and the N-terminal sequence analysis of immunoaffinity-purified peripherin are in agreement with the cDNA sequence. The cDNA sequence predicts that there are possibly four transmembrane domains. On the basis of immunocytochemical studies and sequence analysis, the hydrophilic C-terminal segment containing the antigenic sites for the antiperipherin monoclonal antibodies has been localized on the cytoplasmic side of the disk membrane. There are three consensus sequences for asparagine-linked glycosylation. Deglycosylation studies have indicated that at least one of these sites is utilized. The possible function of peripherin in relation to its primary structure is discussed.

Arginine-binding RNA motifs are important to protein-RNA interaction and perhaps also for Archean biochemistry. Selection-amplification was used to isolate three RNAs that are eluted by free arginine from an L-arginine affinity column (Kd approximately 0.2-0.4 mM). The binding sites contain specific internal and bulge loops, whose sequences can include arginine coding triplets. Binding is highly specific for arginine, but all three motifs, like the self-splicing group I intron, also bind guanosine 5'-monophosphate. One site is stereoselective, somewhat preferring D-arginine.

Retinal degeneration slow (rds) is a retinal disorder of an inbred strain of mice in which the outer segment of the photoreceptor cell fails to develop. A candidate gene has recently been described for the rds defect [Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. & Sutcliffe, J. G. (1989) Nature (London) 338,[70][71][72][73]. Neither the identity of the normal gene product nor its intracellular localization had been determined. We report here that the amino acid sequence of the bovine photoreceptor-cell protein peripherin, which was previously localized to the rim region of the photoreceptor disk membrane, is 92.5% identical to the sequence of the mouse protein encoded by the normal rds gene. The differences between the two sequences can be attributed to species variation. Monoclonal antibodies were used with Western blot analysis to localize the wild-type mouse peripherin/rds protein to isolated mouse rod outer segments and to show that it, like bovine peripherin, exists as two subunits linked by one or more disulfide bonds. The relative amounts of peripherin/rds protein and rhodopsin in retinal extracts of normal and rds mutant mice were also compared. Identification of peripherin as the protein encoded by the normal rds gene and its localization to membranes of rod outer segments will serve as a basis for studies directed toward defining the role of this protein in the morphogenesis and maintenance of the outer segment and toward understanding the mechanism by which the rds mutation causes retinal degeneration.Retinal degeneration slow (rds) is an inherited retinal degeneration that has been identified in an inbred strain of mice (1). Mice that are homozygous for the rds gene fail to develop the outer segment of photoreceptor cells (2-4). Other retinal cells and other parts of the photoreceptor cells, including the inner segment, cell body, synaptic region, and cilium, develop normally during the 3-week postnatal period of development. After this time, however, the photoreceptor cells lacking the outer segments begin to undergo a slow progressive degeneration, and after 12 months few photoreceptor cells remain. Demant et al. (5) have localized the rds mutation to mouse chromosome 17. More recently, Travis et al. (6) have identified a candidate gene in which an insertion of 10 kilobase pairs offoreign DNA into an exon is thought to be responsible for the rds defect. Neither the normal gene product nor its localization within the photoreceptor cell was reported.Outer segments of bovine rod photoreceptor cells contain the membrane protein peripherin (7). Analysis by SDS/ polyacrylamide gel electrophoresis in the presence and absence of a disulfide-reducing agent indicates that this protein consists of two subunits of apparent molecular mass 33 kDa linked together by one or more disulfide bonds. Immunogold labeling studies using monoclonal antibodies indicate that peripherin is localized along the rim region of rod outer segment (ROS) disks (7,8). Recently, the cDNA sequence of bovine periphe...

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage‐ligations or transesterifications, or from the 3′ end of the gRNA by the same mechanisms. We have demonstrated gRNA‐dependent U insertions into a specific editing site of a pre‐edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA‐mRNA anchor duplex was necessary for activity. UTP and alpha‐beta bond hydrolysis of ATP were required, and the activity was inhibited above 50–100 mM KCl. A gRNA‐independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA‐independent U insertion activity and had no effect on the gRNA‐dependent activity. Blocking the 3′ OH of the gRNA had little effect on the gRNA‐dependent U insertion activity. The data are consistent with a cleavage‐ligation model in which the Us are derived directly from UTP.

The biological role of RNA is delimited by its possible reactions, which can be explored by selection. A comparison of selected RNAs that bind one ligand with those that bind two related ligands suggests that a single nucleotide substitution can expand binding specificity. An RNA site with dual (joint) specificity has adenine and cytosine bases whose pKa's appear shifted upward, thereby mimicking an efficient general acid-base catalyst. The joint site also contains two conserved, looped arginine-coding triplets implicated in arginine site formation. Two selected joint RNAs are identical in some regions and distinct in others. The distinct regions, like some peptides, seem to function similarly without being similar in primary structure.