Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5 and 3 ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) _n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) _n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.Paper 3082 of the Forest Research Laboratory, Oregon State University     

Identity of the RNA-binding protein K of hnRNP particles with protein H16, a sequence-specific single strand DNA-binding protein.

Protein H16, which we have identified previously in mammalian cell lines, binds in vitro to two single stranded DNA sites on the late strand of the early promoter of SV40. It has no other single strand binding site in the SV40 genome and does not bind to double stranded DNA. In vitro, H16 can be shown to stimulate strongly the activity of purified RNA polymerase II. Here we have purified this 70 kDa protein from cultured monkey cells and have sequenced three of its tryptic peptides. The analysis indicates that H16 is the simian homolog of human protein K, a nuclear RNA-binding protein found in heterogeneous nuclear ribonucleoprotein (hnRNP) particles, which contains a KH domain present in several proteins including the fragile X mental retardation gene product (FMR1). The binding affinities of protein K/H16 for RNA and DNA were subsequently compared in detail. They showed that under conditions where K/H16 binds strongly to its single stranded DNA site, it binds very weakly to the corresponding RNA sequence. This result suggests a possible shuttling of the protein from RNA to DNA during processes which involve opening of the DNA double helix.     

The alphaviruses: gene expression, replication, and evolution.

The alphaviruses are a genus of 26 enveloped viruses that cause disease in humans and domestic animals. Mosquitoes or other hematophagous arthropods serve as vectors for these viruses. The complete sequences of the +/- 11.7-kb plus-strand RNA genomes of eight alphaviruses have been determined, and partial sequences are known for several others; this has made possible evolutionary comparisons between different alphaviruses as well as comparisons of this group of viruses with other animal and plant viruses. Full-length cDNA clones from which infectious RNA can be recovered have been constructed for four alphaviruses; these clones have facilitated many molecular genetic studies as well as the development of these viruses as expression vectors. From these and studies involving biochemical approaches, many details of the replication cycle of the alphaviruses are known. The interactions of the viruses with host cells and host organisms have been exclusively studied, and the molecular basis of virulence and recovery from viral infection have been addressed in a large number of recent papers. The structure of the viruses has been determined to about 2.5 nm, making them the best-characterized enveloped virus to date. Because of the wealth of data that has appeared, these viruses represent a well-characterized system that tell us much about the evolution of RNA viruses, their replication, and their interactions with their hosts. This review summarizes our current knowledge of this group of viruses.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

Comparative analysis of multiple protein-sequence alignment methods [published erratum appears in Mol Biol Evol 1994 Sep;11(5):811]

We have analyzed a total of 12 different global and local multiple protein-sequence alignment methods. The purpose of this study is to evaluate each method's ability to correctly identify the ordered series of motifs found among all members of a given protein family. Four phylogenetically distributed sets of sequences from the hemoglobin, kinase, aspartic acid protease, and ribonuclease H protein families were used to test the methods. The performance of all 12 methods was affected by (1) the number of sequences in the test sets, (2) the degree of similarity among the sequences, and (3) the number of indels required to produce a multiple alignment. Global methods generally performed better than local methods in the detection of motif patterns.      

Multiple binding sites for cellular proteins in the 3' end of Sindbis alphavirus minus-sense RNA.

The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The first binding domain was found in the first 60 nucleotides, which represents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and the fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, Kr, of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Deletion mapping of the first domain showed that the first 10 nucleotides were critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides was deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40, or 41 to 55 had little effect on the binding, and deletion of nucleotides 15 to to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross River virus and Semliki Forest virus, and from rubella virus were also able to interact with the 50- and 52-kDa proteins. The Kr value for the Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times greater. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replication. We suggest that the binding of the 50- and 52-kDa proteins to the 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.