Difference between RP2 and RP3 phenotypes in X linked retinitis pigmentosa.

AIM: X linked retinitis pigmentosa (XLRP) has two genetic loci known as "RP2" and "RP3". Clinical features reported to differentiate RP2 from RP3 include a higher prevalence of myopia and primary cone dysfunction in RP2, and late onset night blindness and tapetal reflex in RP3. Members from 14 XLRP families were examined in an attempt to verify these differences. METHODS: 16 affected males and 37 females from 14 XLRP families assigned as either RP2 or RP3 by haplotype analysis and/or by heterogeneity analysis were examined. Members of all 14 families who were willing to participate but unavailable for examination were contacted and detailed interviews carried out. RESULTS: No clear phenotypic differences were found that could be used to reliably differentiate RP2 from RP3 with respect to myopia and onset of night blindness. The tapetal reflex was also found to be present in carriers of both RP2 and RP3. CONCLUSIONS: XLRP is a heterogeneous class of rod degenerative disorders with no clear phenotypic differentiation between the two genetic loci RP2 and RP3. There is a continuum of clinical presentations which can be seen in both RP2 and RP3, but the features within a given family tend to be consistent. However, interfamilial variability is prevalent leading to a wide range of clinical presentations and more than one abnormal allele at each gene locus cannot be excluded.

Genomic organization of the human TIMP-1 gene. Investigation of a causative role in the pathogenesis of X-linked retinitis pigmentosa 2.

PURPOSE: To evaluate the role of TIMP-1 in inherited retinal degeneration. METHODS: The genomic structure of the TIMP-1 gene was established and male patients with x-linked retinitis pigmentosa 2 from five families were screened for sequence alterations by direct sequencing in all exons, exon-intron boundaries, and the 5' upstream region of the gene. RESULTS: TIMP-1 appears to be expressed in the retina at low levels and consists of six exons spanning a genomic region of approximately 4.5 kb on Xp11.23. No disease-specific sequence alterations were identified. A site substitution in exon 5 was observed in samples from control subjects and patients, but it did not alter the amino acid sequence of the protein product. CONCLUSIONS: The results of this study exclude mutations in the TIMP-1 coding sequence, splice sites, and the 5' upstream region as a cause of retinal degeneration in x-linked retinitis pigmentosa 2. However, an as yet unidentified regulatory element that lies outside these intervals may be implicated. The role of this tightly regulated protein in the normal functioning of the retina has yet to be determined.

Mapping the RP2 locus for X-linked retinitis pigmentosa on proximal Xp: a genetically defined 5-cM critical region and exclusion of candidate genes by physical mapping.

Genetic linkage studies have implicated at least two loci for X-linked retinitis pigmentosa (XLRP) on proximal Xp. We now report a defined genetic localization for the RP2 locus to a 5-cM interval in Xp11.3-11.23. Haplotype analysis of polymorphic markers in recombinant individuals from two XLRP families has enabled us to identify DXS8083 and DXS6616 as the new distal and proximal flanking markers for RP2. Using STS-content and YAC end-clone mapping, an approximately 1.2 Mb YAC contig has been established encompassing the proximal RP2 boundary and extending from T1MP1 to DXS1240 in Xp11.23. Several ESTs have been positioned and ordered on this contig, one of which is novel to the region, identified by sequence data-base match to a physically mapped YAC insert terminal STS. Integration of the genetic and physical data has placed four retinally expressed genes proximal to DXS6616, and thereby excluded them from a causitive role in RP2. This work now provides a much needed focus for positional cloning approaches to isolation of the defective gene.

Structure of sesbania mosaic virus at 3 A resolution.

BACKGROUND: Sobemoviruses are a group of RNA plant viruses that have a narrow host range. They are characterized in vitro by their stability, high thermal inactivation point and longevity. The three-dimensional structure of only one virus belonging to this group, southern bean mosaic virus (SBMV), is known. Structural studies on sesbania mosaic virus (SMV), which is closely related to SBMV, will provide details of the molecular interactions that are likely to be important in the stability and assembly of sobemoviruses. RESULTS: We have determined the three-dimensional structure of SMV at 3 A resolution. The polypeptide fold and quaternary organization are very similar to those of SBMV. The capsid consists of sixty icosahedral asymmetric units, each comprising three copies of a chemically identical coat protein subunit, which are designated as A, B and C and are in structurally different environments. Four cation-binding sites have been located in the icosahedral asymmetric unit. Of these, the site at the quasi-threefold axis is not found in SBMV. Structural differences are observed in loops and regions close to this cation-binding site. Preliminary studies on ethylene diamine tetra acetic acid (EDTA) treated crystals suggest asymmetry in removal of the quasi-equivalent cations at the AB, BC, and AC subunit interfaces. CONCLUSIONS: Despite the overall similarity between SMV and SBMV in the nature of the polypeptide fold, these viruses show a number of differences in intermolecular interactions. The polar interactions at the quasi-threefold axis are substantially less in SMV and positively charged residues on the RNA-facing side of the protein and in the N-terminal arm are not particularly well conserved. This suggests that protein-RNA interactions are likely to be different between the two viruses.

Primary structure of sesbania mosaic virus coat protein: its implications to the assembly and architecture of the virus.

Sesbania mosaic virus (SMV) is a plant virus that infects Sesbania grandiflora plants in Andhra Pradesh, India. The amino acid sequence of the coat protein of SMV was determined using purified peptides generated by cleavage with trypsin, chymotrypsin, V8 protease and clostripain. The 230 residues so far determined were compared to the corresponding residues of southern bean mosaic virus (SBMV), the type member of sobemoviruses. The overall identity between the sequences is 61.7%. The amino terminal 64 residues, which constitute an independent domain (R-domain) known to interact with RNA, are conserved to a lower extent (52.5%). Comparison of the positively charged residues in this domain suggests that the RNA-protein interactions are considerably weaker in SMV. The residues that constitute the major domain of the coat protein, the surface domain (S-domain, residues 65-260), are better conserved (66.5%). The positively charged residues of this domain that face the nucleic acid are well conserved. The longest conserved stretch of residues (131- 142) corresponds to the loop involved in intersubunit interactions between subunits related by the quasi 3-fold symmetry. A unique cation binding site located on the quasi 3-fold axis contributes to the stability of SMV. These differences are reflected in the increased stability of the SMV coat protein and its ability to be reconstituted with RNA at pH 7.5. A major epitope was identified using monoclonal antibodies to SMV in the segment 201-223 which contains an exposed helix in the capsid structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-copy-number repeat sequences flank the DiGeorge/velo-cardio-facial syndrome loci at 22q11.

DiGeorge syndrome and velo-cardio-facial syndrome are associated with deletions within 22q11. In attempting to refine the shortest region of overlap for these syndromes we have employed fluorescence in situ hybridisation. The results obtained for some probes indicate the presence of low-copy-number repeat families dispersed through proximal 22q. Several primate species have been examined for the presence or absence of two sequences mapping to pter-22q11. The results suggest a relatively recent evolutionary origin for these sequences and the loss of one sequence during the course of primate evolution.

Structure of Sesbania mosaic virus at 4.7 A resolution and partial sequence of the coat protein.

Sesbania mosaic virus (SMV) is a plant virus infecting Sesbania grandiflora plants in Andhra Pradesh, India. Amino acid sequence of the tryptic peptides of SMV coat protein were determined using a gas phase sequenator. These sequences showed identical amino acids at 69% of the positions when aligned with the corresponding residues of southern bean mosaic virus (SBMV). Crystals diffracting to better than 3 A resolution were obtained by precipitating the virus with ammonium sulphate. The crystals belonged to rhombohedral space group R3 with a = 291.4 A and alpha = 61.9 degrees. Three-dimensional X-ray diffraction data on these crystals were collected to a resolution of 4.7 A, using a Siemens-Nicolet area detector system. Self-rotation function studies revealed the icosahedral symmetry of the virus particles, as well as their precise orientation in the unit cell. Cross-rotation function and modelling studies with SBMV showed that it is a valid starting model for SMV structure determination. Low resolution phases computed using a polyalanine model of SBMV were subjected to refinement and extension by real-space electron density averaging and solvent flattening. The final electron density map revealed a polypeptide fold similar to SBMV. The single disulphide bridge of SBMV coat protein is retained in SMV. Four icosahedrally independent cation binding sites have been tentatively identified. Three of these sites, related by a quasi threefold axis, are also found in SBMV. The fourth site is situated on the quasi threefold axis. Aspartic acid residues, which replace Ile218 of SBMV from the quasi threefold-related subunits are suitable ligands to the cation at this site.

Genetic mapping in Methylophilus methylotrophus AS1.

Prime plasmids have been isolated from Methylophilus methylotrophus AS1 using the IncP-1 plasmid pMO172. Such plasmids can complement mutant function when transferred to appropriate strains of Pseudomonas aeruginosa PAO. From the range of particular functions complemented by each prime plasmid, a preliminary map of the M. methylotrophus AS1 genome with four groups of linked markers was obtained. Physical examination of two of these prime plasmids showed that each had acquired an additional DNA segment. Southern hybridization experiments showed there was sequence homology between the additional DNA segments and M. methylotrophus AS1 DNA, confirming that these prime plasmids carried a segment of the M. methylotrophus AS1 genome. Mapping by complementation may be applicable to other bacteria in which mutants suitable for selecting recombinants are not readily available.