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A Swedish family with choroideremia and a deletion of the CHM gene has been studied with ophthalmological examination, full-field electroretinography, and DNA analysis in order to characterize the phenotype of the disease. Although all four patients studied had a complete deletion of the gene, they showed a considerable variability regarding the phenotype, including the electroretinogram tracings. Two of the affected males demonstrated a severe form of choroideremia with low or nondetectable ERG recordings, while the other two affected males showed a less severe phenotype with only a slight reduction of the ERG amplitudes. The variation of the clinical phenotype among family members carrying the same mutation indicates that the severity of choroideremia is not solely a function of the CHM gene.

X-linked deafness with stapes fixation (DFN3) is caused by mutations in the POU3F4 gene at Xq21.1. By employing pulsed field gel electrophoresis (PFGE) we identified a chromosomal aberration in the DNA of a DFN3 patient who did not show alterations in the open reading frame (ORF) of POU3F4. Southern blot analysis indicated that a DNA segment of 150 kb, located 170 kb proximal to the POU3F4 gene, was duplicated. Fluorescence in situ hybridization (FISH) analysis, PFGE, and detailed Southern analysis revealed that this duplication is part of a more complex rearrangement including a paracentric inversion involving the Xq21.1 region, and presumably the Xq21.3 region. Since at least two DFN3-associated minideletions are situated proximal to the duplicated segment, the inversion most likely disconnects the POU3F4 gene from a regulatory element which is located at a distance of at least 400 kb upstream of the POU3F4 gene.

In a previous study, we have developed a panel of chromosomal rearrangements for the physical mapping of the q13-q21 region of the human X chromosome (Philippe et al., Genomics 17: 147-152, 1993). Here, we report the physical localization of 36 additional polymorphic markers by polymerase chain reaction analysis. The high density of chromosomal breakpoints in Xq21 allows us to map 58 DNA loci in 22 intervals. As a result, this segment of the X chromosome is saturated with approximately three sequence tagged sites per megabase of DNA, which will facilitate the construction of a YAC contig of this region.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so-called SH2 domains. The signal transduction pathways activated by EGF include the phosphatidylinositol pathway, leading to activation of protein kinase C and to increase in the intracellular Ca2+ concentration, and to the ras pathway leading to MAP kinase activation. Recently the cytoplasm has been implicated as playing an important role in EGF induced signal transduction. The EGF receptor has been demonstrated to be an actin-binding protein. In addition EGF causes a rapid actin depolymerisation and the formation of membrane ruffles. In particular these membrane ruffles have been shown to act as the first site of signal transduction after EGF binding, and thus may be considered as signal transduction structures. Finally evidence has been presented suggesting a positive role for EGF and/or the receptor in the nucleus.

Deafness with fixation of the stapes (DFN3) is the most frequent X-linked form of hearing impairment. The underlying gene has been localized to a 500-kilobase segment of the Xq21 band. Here, it is reported that a candidate gene for this disorder, Brain 4 (POU3F4), which encodes a transcription factor with a POU domain, maps to the same interval. In five unrelated patients with DFN3 but not in 50 normal controls, small mutations were found that result in truncation of the predicted protein or in nonconservative amino acid substitutions. These findings indicate that POU3F4 mutations are a molecular cause of DFN3.

Pre-embedding and post-embedding in situ hybridization techniques were compared for the localization of RNAs in the nucleus. 28S rRNA and transcripts of the epidermal growth factor receptor (EGF-receptor) were localized with both hybridization methods. Pre-embedding hybridizations were performed on cells permeabilized with Triton X-100, whereas post-embedding hybridizations were carried out on Lowicryl K4M sections. From these studies it was concluded that, for labelling of 28S rRNA, the post-embedding in situ hybridization is preferred, whereas EGF-receptor transcripts were successfully detected only after pre-embedding in situ hybridization. Furthermore, the detection of the hybrids with ultra-small gold particles was compared to the detection with 6 nm gold particles in both pre- and post-embedding in situ hybridization studies. From our results it is concluded that the use of ultra-small gold particles results in higher label efficiency. Therefore, ultra-small gold particles are preferable to 6 nm gold particles for the detection of hybrids in high-resolution in situ hybridization experiments.

Linkage analysis has been performed in a large Dutch pedigree with X-linked recessive congenital stationary night blindness (CSNB) by utilizing 16 DNA markers from the proximal short arm of the human X chromosome (Xp21.1-11.2). Thirteen polymorphic markers are at least partially informative and have enabled pairwise and multipoint linkage analysis. For three loci, i.e. DXS228, the monoamine oxidase B gene and the Norrie disease gene (NDG), multipoint linkage studies have yielded maximum lod scores of > 3.0 at a recombination fraction of zero. Analysis of recombination events has enabled us to rule out the possibility that the underlying defect in this family is allelic to RP3; the gene defect could also be excluded from the proximal part of the region known to carry RP2. Linkage data are consistent with a possible involvement of the NDG but mutations in the open reading frame of this gene have not been found.

Positional cloning has previously resulted in the identification of a gene which is disrupted by deletions in patients with the classic choroideremia (CHM) phenotype. More subtle mutations had been identified in 4 exons of the 3' portion but not elsewhere in the CHM gene. We have now isolated and characterized the complete open reading frame of the CHM gene and determined its exon-intron structure. The CHM gene encodes a protein of 653 amino acids, which is highly homologous to the mouse and rat CHM proteins, and, to a slightly lesser extent, to the human CHM-like (CHML) protein. The open reading frame (ORF) of the human CHM gene consists of 15 exons, spanning at least 150 kb of Xq21.2, and it is possible that there is an additional exon corresponding to the 5' non-coding region of the gene. Cloning of the 5' end of the CHM gene and the elucidation of its intron-exon structure enabled us to localize the X-chromosomal breakpoint in a CHM female with an X;7 translocation between exons 3 and 4.

Rab escort proteins (REPs) bind to newly synthesized Rab proteins and remain bound during and after the attachment of a geranylgeranyl (GG) group by the catalytic component of the Rab GG transferase. Transfer of the GG group is absolutely dependent on the participation of a REP. REP-1, the first characterized REP, is produced by a gene on the X chromosome that is defective in patients with choroideremia, a form of retinal degeneration. Cremers et al. (Cremers, F.P.M., Molloy, C. M., van de Pol, D. J. R., van den Hurk, J. A. J. M., Bach, I., Geurts van Kessel, A. H. M., and Ropers, H.-H. (1992) Hum. Mol. Genet. 1, 71-75) isolated a related gene, designated choroideremia-like, which encodes a protein that closely resembles REP-1. In the current studies, we produced REP-1 and REP-2 by recombinant DNA methods and showed that both proteins were approximately equal in facilitating the attachment of GG groups to several Rab proteins, including Rab1A, Rab5A, and Rab6. However, REP-2 was only 25% as active as REP-1 in supporting GG attachment to Rab3A and Rab3D. The low activity toward Rab3A was increased to that of Rab1A when the COOH-terminal 12 amino acids of Rab3A were replaced with the corresponding residues of Rab1A. We suggest that REP-2 substitutes for the absent function of REP-1 in nonretinal cells of patients with choroideremia, thus preventing cellular dysfunction throughout the body. In the retina, REP-2 may be only partially effective, leading eventually to retinal degeneration and blindness.

We have defined a radiation hybrid panel for Xq11-q22 characterized with 20 DNA markers and shown how the panel can be used in conjunction with Alu-PCR and a gridded X-specific cosmid library to isolate cosmids from a preselected subregion. We used Alu-PCR products derived from two radiation hybrids, IHB2-B30 and IHB1-A12, sharing a segment in proximal Xq13.1, containing DXS453 to CCG1, as the only human component in common. Used as probes, these Alu-PCR products identified 39 cosmids on the gridded X-cosmid libraries that were positive for both probes. The target specificity of the derived cosmids was very high, 11 of 13 cosmids tested mapped to the region of fragment overlap in the hybrids, as was determined by two translocation breakpoints bordering most of the target interval. Accounting for a redundancy of four in the libraries, the isolated 39 cosmids should correspond to about 10 independent loci derived from the region of fragment overlap defined by these radiation hybrids. In addition, a subset of five radiation hybrids having breakpoints in the target region could be used to further subdivide the 11 cosmids into an ordered set of five submapping intervals of Xq13.1.

DNA probe screening of somatic cell hybrids derived from females with X;autosome translocations has enabled us to define eight new breakpoints within the Xq13-q22 region. Together with other X-chromosome rearrangements that have been described earlier, these breakpoints subdivide the Xq21-q22 region into 20 intervals. Our panel refines the physical assignment of 40 probes in the Xq21-q22 segment. Thus, these X-chromosome rearrangements are useful tools for ordering X-linked markers and genes on the proximal long arm of the human X chromosome.

cDNA cloning of component A of rat Rab geranylgeranyl transferase confirms identity of the protein with the human choroideremia gene product and its resemblance to Rab3A guanine nucleotide dissociation inhibitor (GDI), which binds prenylated Rabs. In biochemical assays we demonstrate that component A binds unprenylated Rab1A, presents it to the catalytic component B, and remains bound to it after the geranylgeranyl transfer reaction. In the absence of detergents, the reaction terminates when all of component A is occupied with prenylated Rab. Detergents allow multiple rounds of catalysis, apparently by dissociating the component A-Rab complex and thus allowing recycling of component A. Within the cell, component A may be regenerated by transferring its prenylated Rab to a protein acceptor, such as Rab3A GDI. In view of its function in escorting Rab proteins during and presumably after the prenyl transfer reaction, we propose to rename component A as Rab escort protein (REP). A genetic defect in REP underlies human choroideremia, a disease of retinal degeneration.

We have localized the mRNA of the epidermal growth factor receptor (EGF-receptor) in nuclei of A431 cells by non-radioactive in situ hybridization at the light microscopical level using digoxigenin-labelled DNA probes. Both formaldehyde and glutaraldehyde fixations were tested before the hybridization was performed. Glutaraldehyde, compared with formaldehyde fixation, gives a less diffuse hybridization signal, which is easier to localize. Therefore, glutaraldehyde was used as a fixative in the hybridization experiments. It is demonstrated that the mRNA of the EGF-receptor is present in restricted domains mainly located around the nucleolus. This location of the EGF-receptor mRNA was unaltered after extraction of chromatin. Therefore it is concluded that the messenger RNA of the EGF-receptor is attached to the nuclear matrix. A possible biological role for the location of mRNA of the EGF-receptor around the nucleolus is discussed.

We have searched for mutations in the choroideremia gene (CHM) in patients from 12 Danish families in which CHM is segregating. Employing polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis, and direct DNA sequencing, different mutations have been identified in 6 patients. All the mutations will interfere with the correct translation of the mRNA predicting a truncated protein or no gene product at all.

A candidate gene for Norrie disease, an X-linked disorder characterized by blindness, deafness and mental disturbances, was recently isolated and found to contain microdeletions in numerous patients. No strong homologies were identified. By studying the number and spacing of cysteine residues, we now detect homologies between the Norrie gene product and a C-terminal domain which is common to a group of proteins including mucins. Three newly-characterized missense mutations, replacing evolutionarily conserved cysteines or creating new cysteine codons, emphasize the functional importance of these sites. These findings and the clinical features of this disorder suggest a possible role for the Norrie gene in neuroectodermal cell-cell interaction.

Recently, we and others have isolated a candidate gene for X linked Norrie disease (ND) which was found to be deleted or disrupted in several patients. As a prerequisite for the identification of point mutations in the ND gene we have established the exon-intron structure of this gene. In 17 unrelated patients and 15 controls, PCR products derived from the promoter region, exons 1 and 2 as well as the coding part of exon 3 were analysed with the single strand conformation polymorphism (SSCP) technique. In 12 patients altered PCR fragments were detected which were studied in detail by direct sequencing. Eleven different mutations were found, and all but one are likely to give rise to significant structural changes in the predicted protein. These findings, and the absence of functionally relevant base changes in healthy controls, emphasize the causal role of this candidate gene in Norrie disease and pave the way for reliable diagnosis and carrier detection.

Linkage studies and cytogenetically visible deletions associated with nonspecific X-linked mental retardation (XLMR) and a specific form of deafness (DFN3) have indicated that the genes responsible for these disorders are located at Xq21. Using DNA probes from this region, we have studied several overlapping deletions spanning different parts of Xq21. This has enabled us to assign the DFN3 gene and a gene for nonspecific XLMR to an interval that encompasses the locus DXS232 and that is flanked by DXS26 and DXS121.

Homology with the mouse bare patches mutant suggests that the gene for the X-linked dominant chondrodysplasia punctata/ichthyosis/cataract/short stature syndrome (Happle syndrome) is located in the human Xq28 region. To test this hypothesis, we performed a linkage study in three families comprising a total of 12 informative meioses. Multiple recombinations appear to exclude the Xq28 region as the site of the gene. Surprisingly, multiple crossovers were also found with 26 other markers spread along the rest of the X chromosome. Two-point linkage analysis and analysis of recombination chromosomes seem to exclude the gene from the entire X chromosome. Three different mechanisms are discussed that could explain the apparent exclusion of an X-linked gene from the X chromosome by linkage analysis: (a) different mutations on the X chromosome disturbing X inactivation, (b) metabolic interference, i.e. allele incompatibility of an X-linked gene, and (c) an unstable pre-mutation that can become silent in males. We favour the last explanation, as it would account for the unexpected sex ratio (M:F) of 1.2:1 among surviving siblings, and for the striking clinical variability of the phenotype, including stepwise increases in disease expression in successive generations.

Employing various probes from the proximal part of the Xq21 region, which is known to harbor the DFN3 gene, we have investigated 13 unrelated male probands with X-linked deafness, to detect possible deletions. For two of these patients, microdeletions could be detected by using probe pHU16 (DXS26). One of these deletions also encompasses locus DXS169, indicating that it extends farther toward the centromere. The presence of normal hybridization patterns in the DNA of 25 unrelated control males suggests that these deletions are the primary cause of progressive mixed deafness in these patients. If so, their molecular characterization may pave the way for the identification and isolation of the corresponding gene.

Employing the mouse homologue of the human choroideremia cDNA as a probe, we have identified a homologous human gene. The consensus cDNA of this gene, designated human choroideremia-like (hCHML) gene, encompasses an open reading frame of 1968 base pairs. The deduced polypeptide of hCHML displays several regions of homology to smg p25A GDI, a bovine protein known to regulate the GDP/GTP exchange of the GTP-binding protein smg p25A. hCHML is located at 1q31-qter, a chromosomal region which, by means of linkage analysis, was previously shown to carry a gene locus for Usher syndrome type II. The colocalization of hCHML and Usher syndrome type II, as well as the clinical similarities between choroideremia and Usher syndrome type II, make hCHML a candidate gene for this disorder.

Choroideremia (CHM) is an X-linked progressive degeneration of the choroid and retina. 12% of unrelated male patients carry deletions of the partially cloned CHM gene. In Finland, there are more than 120 living CHM patients belonging to eight apparently unrelated pedigrees. Molecular deletions involving the CHM gene have been detected in three families. We have screened the remaining five families for point mutations. In one large family a single nucleotide (T) insertion into the donor splice site of exon C leads to two aberrantly spliced mRNAs both producing a premature stop codon. The mutation can be assayed easily by amplification and digestion with Msel. Our findings provide additional evidence for the pathogenetic role of CHM mutations and provide a diagnostic tool for one fifth of the world's known CHM patients.

Coagulase-negative staphylococci (CoNS) have evolved into important agents of foreign body-related infections. Adhesion of causative bacteria to biomaterials is considered to be an essential step in these infections. We and others have shown that adhesion of CoNS to biomaterials may be mediated by protease-sensitive surface constituents. In the present study we expanded on these investigations by characterizing a biomaterial adhesin of Staphylococcus epidermidis 354 by using a strain-specific monoclonal antibody (MAb 36.4). MAb 36.4 was strongly and exclusively reactive with strain 354 in an enzyme-linked immunosorbent assay in which whole bacteria were used as antigens. Immunoblotting of cell wall polypeptides of strain 354 revealed strong reactivity with a 200- to 220-kDa band and a weaker reaction in the 100- to 110-kDa range. Preincubation of strain 354 with MAb 36.4 resulted in a 54 to 91% (mean +/- standard deviation, 74% +/- 14%; n = 10) inhibition of adhesion to polystyrene spheres. Fab fragments prepared from MAb 36.4 also inhibited adhesion effectively, indicating specific blocking of an adhesion antigen rather than aspecific inhibition. Immunogold electron microscopy with MAb 36.4 revealed deposition of gold particles on the cell surface and possibly also on fimbrialike surface projections. It is concluded that a surface-located protein antigen of S. epidermidis 354 recognized by MAb 36.4 acts as an adhesin mediating attachment to uncoated foreign material. It is speculated that this type of adhesion to biomaterials may play an important role in the pathogenesis of foreign body-related infections caused by CoNS.

We report linkage studies in 18 choroideremia (TCD) families using four closely linked polymorphic markers. Probe pZ11, which is known to be deleted in several unrelated patients with TCD, showed no recombinations (zeta max 15.63 at theta = 0.00). In contrast, one recombination was observed with DXS367, which is also physically very close to TCD. Loci DXS95 and DXYS69 each showed more than one recombination with TCD. Moreover, these analyses revealed a double crossover between TCD and DXYS1, changing the previously reported very close linkage to a recombination fraction of 0.04 with a lod score of 9.93. Multipoint linkage analysis placed TCD proximal to DXS95-DXYS69 and very close to DXS367-pZ11 with almost identical multipoint lod score maxima either proximal to DXS367 (zeta max = 23.43) or proximal to pZ11 (zeta max = 23.36). These results provide a refined linkage map around TCD and will also be useful in DNA diagnostics of the disease.

In order to characterize a previously described submicroscopic deletion encompassing (part of) the choroideremia (tapetochoroidal dystrophy: TCD) gene, we have cloned a 10.5-kb EcoRI fragment from the patient's DNA; this fragment carries the junction between both deletion endpoints ("junction fragment"). The distal portion of this fragment defines a new marker within, or just distal to, the TCD gene. This marker has been employed to confirm the diagnosis in several affected family members, and to rule out carriership in a female at risk with conspicuous clinical signs.

Choroideraemia (tapetochoroidal dystrophy, TCD), a common form of X-linked blindness, is characterized by progressive dystrophy of the choroid, retinal pigment epithelium and retina. Previous studies have assigned the TCD gene to a small segment of the Xq21 band. By making use of reverse genetics strategies we have isolated eight overlapping complementary DNA clones from the same chromosomal region. The corresponding gene is expressed in retina, choroid and retinal pigment epithelium. The cDNAs encompass an open reading frame of 948 base pairs that is structurally altered in eight TCD patients with deletions, and in a female patient with a balanced translocation involving Xq21. These findings provide strong evidence that we have cloned the gene underlying choroideraemia. Elucidation of its function should provide new insights into the molecular mechanisms responsible for this disorder and other hereditary retinopathies.

Making use of the p1bD5 probe (DXS165), we have isolated several markers from the choroideremia locus by chromosomal jumping, preparative field-inversion gel electrophoresis, and cloning of a deletion junction fragment. With these clones we were able to identify and characterize eight deletions in 69 choroideremia patients investigated. The deletions are heterogeneous, in both size and location. The smallest deletion (patient LGL1134) comprises approximately 45 kb of DNA, whereas the largest ones (patients 25.6 and LGL2905) span a DNA segment of at least 5 megabases, which is comparable in size to the smallest deletion detected in a TCD patient (patient XL45) showing a complex phenotype. The TCD deletions encompass variable parts of 150-200-kb DNA segment that is flanked by p1bD5 (DXS165) at the centromeric side and by pZ 11 at the telomeric side. The deletions in patients 33.1, LGL1101, and LGl1134 do not span a translocation breakpoint which was previously mapped on the X chromosome of a female with TCD. The clones isolated from the TCD locus are valuable diagnostic markers for deletion analysis of patients or carrier females. In addition, they should be useful for the isolation of expressed sequences that are part of the TCD gene.

X-linked albinism-deafness syndrome (ADFN) was described in one Israeli Jewish family and is characterized by congenital nerve deafness and piebaldness. The ADFN mutation probably affects the migration of neural crest-derived precursors of the melanocytes. As a first step toward identifying the ADFN gene, a linkage study was performed to localize the disease locus on the X chromosome. The family was found to be informative for 11 of 107 RFLPs along the X, and two-point analysis showed four of them--factor 9 (F9), DXS91, DXS37, and DNF1--to have definite or suggestive linkage with ADFN. Multipoint linkage analysis indicated two possible orders within this cluster of loci, neither of which was preferable. In both orders F9 was the most distal, and the best estimate for the location of ADFN was between F9 and the next proximal marker (8.6 cM from F9 [Z = 8.1] or 8.3 cM from F9 [Z = 7.9]). These results suggest that the ADFN is at Xq26.3-q27.1. Disagreement between our data and previous localization of DXS91 at Xq11-q13 was resolved by hybridization of the probe pXG-17, which detects the DXS91 locus, to a panel of somatic cell hybrids containing different portions of the X chromosome. This experiment showed that this locus is definitely at Xq24-q26. Together with the linkage data, our results place DXS91 at Xq26 and underscore the importance of using more than one mapping method for the localization of molecular probes.

By making use of preparative field inversion gel electrophoresis, we have constructed a lambda ZAP library that is highly enriched for sequences from the choroideremia locus. In vivo excision of pBluescript SK(-) constructs from lambda ZAP obviates the subcloning of DNA inserts and allows for rapid processing of several hundred recombinants. From a 625 kb Sfil fragment we isolated 7 clones that were physically mapped using microdeletions associated with the disease. One of these clones is located within, or just telomeric to, the choroideremia gene and detects two restriction fragment length polymorphisms (RFLPs). Another clone detects a RFLP which maps centromeric to the disease locus. Together these probes should improve the reliability of linkage analysis in choroideremia families and should pave the way for the isolation of the choroideremia gene.

Characterization of several male-viable deletions and duplications with 20 random DNA probes has enabled us to subdivide the Xq21 region into seven discernible intervals. Almost all of the deletions spanning part of Xq21 are associated with choroideremia and mental retardation, with deafness being another common feature. The gene locus for choroideremia was assigned to interval 3 spanning the loci DXS95, DXS165, and DXS233. Genes for X-linked deafness and mental retardation were tentatively assigned to interval 2. Deletions of intervals 4 through 7 were not associated with any clinical abnormality. We have constructed a preliminary long-range restriction map of intervals 2 and 3 using field-inversion gel electrophoresis. The DXS232, DXS121, and DXS233 loci are located on the same SfiI fragment, whereas the DXS165 and DXS95 loci could not be linked to this cluster using SfiI and SalI.

While performing a systematic search for chromosomal microdeletions in patients with clinically complex X-linked syndromes, we have observed that large male-viable deletions and duplications are clustered in heterochromatic regions of the X chromosome. Apart from the Xp21 band, where numerous deletions have been found that encompass the Duchenne muscular dystrophy gene, an increasing number of deletions and duplications have been observed that span (part of) the Xq21 segment. To refine the molecular and genetic map of this region, we have employed 52 cloned single-copy DNA sequences from the Xcen-q22 segment to characterize two partly overlapping tandem duplications and two interstitial deletions on the proximal long arm of the human X chromosome. Together with a panel of somatic cell hybrids that had been described earlier, these four rearrangements enabled us to order the 52 probes into nine different groups and to narrow the regional assignment of several genes, including those for tapetochoroidal dystrophy and anhidrotic ectodermal dysplasia.

Using various probes from the Xq21 region which is known to carry the choroideremia (tapetochoroideal dystrophy, TCD) locus, we have screened the DNAs from eight unrelated male choroidermia patients for microdeletions. In two of these patients, but not in any of 45 males tested as controls, lack of hybridization signals with probe plbD5 suggested a deletion encompassing the DXS165 locus and (part of) the TCD gene. Absence of additional clinical features in these patients and the fact that two closely linked, and probably flanking, TCD markers (DXYS1 and DXS72) are not deleted may indicate that the physical distance between the DXS165 locus and the TCD gene is small.

A human genomic cosmid clone, pHcos gamma-1, has been isolated containing two closely linked gamma-crystallin genes, oriented in the same direction. The sequence of these genes and their 5' and 3' flanking regions has been determined. The coding regions of both genes are interrupted by two introns. The first introns (94 and 100 bp, respectively) are located in the 5' region of the genes. The second introns (2.82 and 0.95 kb, respectively) divide the genes into two halves, each encoding a structural domain of the gamma-crystallin protein. The coding regions of the two genes show 80% homology. Due to a mutation in the splice acceptor site of the second intron of the first gene, the coding region of its third exon is 3 bp longer than that of the second gene. In the flanking regions several conserved sequence elements were found, including those elements that are known to be necessary for the correct expression of eukaryotic genes. The flanking and intronic regions of the genes contain 'simple sequence' DNA and Alu repeats. The Alu repeats are usually clustered, contain truncated elements, and are often located near simple sequence DNA.

In order to contrast anionic sites in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules. After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl2 concentration used during staining. At 0.4 M MgCl2, the length was mostly within the range 100-180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.

An insertional translocation into the proximal long arm of the X chromosome in a boy showing muscular hypotony, growth retardation, psychomotor retardation, cryptorchidism, and Pelizaeus-Merzbacher disease (PMD) was identified as a duplication of the Xq21-q22 segment by employing DNA probes. With densitometric scanning for quantitation of hybridization signals, 15 Xq probes were assigned to the duplicated region. Analysis of the duplication allowed us to dissect the X-Y homologous region physically at Xq21 and to refine the assignments of the loci for DXYS5, DXYS12, DXYS13, DXS94, DXS95, DXS96, DXS111, and DXS211. Furthermore, we demonstrated the presence of two different DXYS13 and DXS17 alleles in genomic DNA of our patient, suggesting that the duplication resulted from a meiotic recombination event involving the two maternal X chromosomes.

The sequences of a 51-kb region containing the cluster of five rat gamma-crystallin-coding genes (CRYG) and of a 7-kb region surrounding the sixth rat CRYG gene were determined. Approximately 78% of the total sequence represents intergenic DNA. We also sequenced 22 kb of DNA from the human CRYG gene cluster. All CRYG genes are associated with CpG-rich regions. The sequence similarity between the human and rat gene regions drops sharply (to 65%) in intronic and 3'-flanking regions but decreases only gradually in the 5'-flanking region. Highly conserved regions (greater than 80%) are found as far upstream as 1.5 kb. Overall intergenic distances are conserved. The human region contains much more repetitive DNA (24% vs. 10%) but less simple-sequence (sps) DNA (0.7% vs. 4%) than the rat region. Almost all repeats and spsDNA elements are located in the intergenic region. The location of repetitive and spsDNA differs between the orthologous regions and these elements were probably inserted after the evolutionary separation of rat and man. The Alu repeats in man and the B3 repeats in the rat are close copies of their respective consensus sequences and bordered by virtually perfect repeats. In contrast, the B1 and B2 repeats in the rat have diverged considerably from the consensus sequence and the surrounding direct repeats are usually imperfect. Thus the dispersion of the B1 and B2 repeats in the rat probably preceded that of the B3 repeats. Within the rat genomic region the spacing of Z-DNA elements is surprisingly regular, they are located about 12 kb apart. A search for putative matrix-associated regions suggests that the rat CRYG gene cluster is organized into two chromosomal domains.

The gene encoding ferredoxin I (petF1) from the filamentous cyanobacterium Anabaena sp. PCC 7937 (Anabaena variabilis ATCC 29413) was cloned by low stringency hybridization with the ferredoxin cDNA from the higher plant Silene pratensis. The petF1 gene from the unicellular cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) was cloned by low stringency hybridization with the petF1 gene from Anabaena sp. PCC 7937. One copy of the petF genes was detected in both organisms, and a single transcript of about 630 b was found for Synechococcus sp. PCC 7942. Both the Synechococcus sp. PCC 7942 and the Anabaena sp. PCC 7937 petF1 genes contain a 297 bp open reading frame coding for a small acidic protein, consisting of 98 amino-acid residues, with a molecular mass of about 10.5 kDa.The ferredoxin content of Synechococcus sp. PCC 7942 is strongly reduced under ironlimited growth conditions. The slight decrease in the amount of ferredoxin transcript found under iron limitation does not account for the more severe reduction in ferredoxin protein observed. The main regulation of the ferredoxin content probably is effected at the level of translation and/or degradation. Although ferredoxin expression can be strongly reduced by iron stress, the ferredoxin function seems to be indispensable, as Synechococcus sp. PCC 7942 appeared refractory to yield mutants lacking the petF1 gene.

In order to localize and characterize proteoglycans in human lung alveoli, we have used the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. After staining, five types of Cuprolinic Blue-positive filaments become apparent: two types in the basement membranes of type I and type II epithelial cells respectively and lying in one or two layers; one type, more scattered, localized in the basement membrane of the endothelial cells and another kind associated with collagen fibrils and separated from each other according to the main banding period (+/- 60 nm) of these fibrils. Finally, there was a type of filament which was only locally present at a variety of places. The basement membrane filaments were sensitive to heparinase, heparitinase, pronase (without prefixation) and nitrous acid treatment, but not to Streptomyces hyaluronidase, neuraminidase, chondroitinase ABC, chondroitinase AC, pronase (after prefixation) and 2.0 M MgCl2 treatment. The basement membrane filaments, therefore, represent heparan sulphate-containing proteoglycans. On the other hand, the collagen fibril associated filaments were sensitive to treatment with heparinase, chondroitinase ABC and pronase (without prefixation), but insensitive to Streptomyces hyaluronidase, neuraminidase, nitrous acid, heparitinase, chondroitinase AC, pronase (after prefixation) and 2.0 M MgCl2 (after prefixation) treatment. These filaments thus represent iduronic acid-rich dermatan sulphate-containing proteoglycans. Several physiological functions for these proteoglycans are discussed.

Transgenic tomato plants that constitutively express a foreign plastocyanin gene were used to study protein transport in different tissues. Normally expression of endogenous plastocyanin genes in plants is restricted to photosynthetic tissues only, whereas this foreign plastocyanin protein is found to be present in all tissues examined. The protein is transported into the local plastids in these tissues and it is processed to the mature size. We conclude that plastids of developmentally different tissues are capable of importing precursor proteins that are normally not found in these tissues. Most likely such plastids, though functionally and morphologically differentiated, have similar or identical protein import mechanisms when compared to the chloroplasts in green tissue.

The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, nor Streptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0 M MgCl2 (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase or Streptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0 M MgCl2 (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.

Choroideremia (tapeto-choroidal dystrophy, TCD), an X chromosome-linked disorder of retina and choroid, causes progressive nightblindness and central blindness in affected males by the third to fourth decade of life. Recently, we have been able to map the TCD gene to a small region of overlap between five different, male-viable Xq21 deletions that were found in patients with TCD and other clinical features. Two families were identified in which classical, nonsyndromic TCD is associated with small interstitial deletions that are only detectable with probe p1bD5 (DXS165). To characterize these and two other deletions that were identified more recently, we have used the chromosome walking and jumping techniques to generate a set of five chromosomal-jumping clones flanking the DXS165 locus at various distances. With these clones, we could localize four of the eight deletion endpoints and the breakpoint on the X chromosome of a female with a de novo X/13 translocation and choroideremia. These studies assign the TCD gene, or part of it, to a DNA segment of only 15-20 kilobases.