Interstitial Retinol-binding Protein Research Articles

P92 Catechol-O-methyl transferase (COMT) expression in severe, early onset pre-eclampsia: is there a role in the pathophysiology of pre-eclampsia?

Biochemical and immunological techniques were used to determine the emergence of interstitial retinol binding protein (IRBP), rhodopsin, and stored retinyl esters (all-trans and 11-cis) during retinal development in normal and rd mice. IRBP could be demonstrated at embryonic Day 17 (E17), corresponding to an early stage of inner segment development. Although all-trans retinyl esters were present earlier, 11-cis retinyl esters did not appear until postnatal Days 6–7 (P6–P7), corresponding to rod outer segment (ROS) disc formation. Rhodopsin was detected at the same developmental stage. The proportion of 11-cis retinyl esters reached a maximum of 40–50% at P15–P20. Thereafter, the proportion dropped, due to more rapid accumulation of the all-trans isomer. Rhodopsin and IRBP increased in parallel with ROS elongation up to P25, when the ROS had reached their mature lengths. The increases then continued up to P40–P50. In rd (retinal degeneration) mice, IRBP and rhodopsin were identical with the controls until P12, but then dropped as the photoreceptors degenerated. Synthesis and secretion of IRBP in vitro was less than 10% of the controls in rd retinas at P26, when only 4–5% of the photoreceptors survived. The quantities of retinyl esters (mainly stearate and palmitate in the ratio of 6:1, respectively) stored in dark-adapted mouse eyes progressively increased as the animals aged, representing 0.5 mole eq. of the rhodopsin at 8 months. Although retinyl esters (11-cis and all-trans) also accumulated in rd mouse eyes up to P12, little further increase occurred. At P93, the retinyl esters (0.01 nmole · eye−1) were only 4% of the controls at P91. A peak in the proportion of 11-cis isomer occurred at P10–P20, but it averaged only 15% of the total ester and declined to 5% at P93. These findings support the hypothesis that IRBP is synthesized by the rods and cones, and suggest that its synthesis and secretion are initiated when the photoreceptor inner segments start to differentiate. 11-cis Retinoids and rhodopsin do not appear until the outer segments start to form. It is suggested that in the rd mouse the absence of photoreceptors, perhaps coupled with lack of normal interphotoreceptor matrix, leads to a loss in the ability of the pigment epithelium to store retinyl esters.

Phylogeny and species boundaries of the endemic species complex, Eliurus antsingy and E. carletoni (Rodentia: Muroidea: Nesomyidae), in Madagascar using mitochondrial and nuclear DNA sequence data

Current understanding of the taxonomy and species relationships within the genus Eliurus, a member of the endemic Malagasy rodent subfamily Nesomyinae, is largely based on morphological characters. Little has been published about the historical biogeography and diversification factors in members of this genus. Here we examined, using molecular data, the evolutionary relationships, and species boundaries within a species complex of Eliurus and the role of rivers in their evolution. Specifically, we tested the hypothesized monophyly of E. antsingy (west) and the recently described E. carletoni (north) using two mitochondrial genes, cytochrome b, the control region, and a nuclear marker, the Interstitial Retinol Binding Protein (IRBP). We also assessed the species boundaries of E. carletoni and examined phylogeographic patterns using two mitochondrial markers and an extensive geographic sampling. Molecular data show that members of the E. antsingy and E. carletoni complex together form a monophyletic group. Although samples provisionally assigned to E. carletoni based on morphological characters from different sites in northern Madagascar cluster in three separate phylogroups, they are monophyletic and assignable to this species. Rivers do not act as strict barriers to dispersal in E. carletoni. The demonstrated monophyly of both E. antsingy and E. carletoni contributes to the amounting evidence for a biogeographic link between the north and western regions of the island.

Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy

The aim of this study was to compare the protein profile of vitreous fluid from diabetic patients with proliferative diabetic retinopathy (PDR) with that from non-diabetic patients with idiopathic macular holes (MH). The mRNA of proteins differentially produced was also assessed in the retinas from diabetic and non-diabetic donors. Vitreous humour from type 1 diabetic patients with PDR (n = 8) and from non-diabetic patients with MH (n = 10) closely matched in terms of age were studied. The comparative proteomic analysis was performed using fluorescence-based difference gel electrophoresis (DIGE). Differentially produced proteins (abundance ratio >1.4, p < 0.05) were identified by mass spectrometry. Expressions of mRNA were measured by real-time RT-PCR in retinas from ten human eyes obtained at post-mortem (five eyes from diabetic subjects and five eyes from non-diabetic subjects). Eight proteins were highly produced in PDR patients in comparison with non-diabetic subjects: zinc-alpha(2)-glycoprotein (ZAG), apolipoprotein (apo) A1, apoH, fibrinogen A, and the complement factors C3, C4b, C9 and factor B). We found three proteins that were underproduced in PDR subjects: pigment epithelial derived factor (PEDF), interstitial retinol-binding protein (IRBP) and inter-alpha-trypsin inhibitor heavy chain (ITIH2). There was no overlap in the vitreous levels of the above-mentioned proteins between PDR patients and non-diabetic control subjects. The differential production of ZAG, C3, factor B, PEDF and IRBP was further confirmed by western blot, and was in agreement with mRNA levels detected in the retina. Proteomic analysis by DIGE, which permits an accurate quantitative comparison, was useful in identifying new potential candidates involved in the pathogenesis of PDR.

Taxonomy, evolutionary history and biogeography of the broad-toothed field mouse (Apodemus mystacinus) in the eastern Mediterranean area based on mitochondrial and nuclear genes

The broad-toothed field mouse (Apodemus mystacinus) is distributed throughout the Balkan Peninsula, Asia Minor and the Middle East. It is generally split into two different specific entities: Apodemus epimelas occurs on the Balkan Peninsula and A. mystacinus inhabits Asia Minor and the Middle East. This analysis, based on two mitochondrial regions (cytochrome b and the D-loop) and the interstitial retinol binding protein (IRBP) nuclear gene, confirms an important level of genetic divergence between the animals from these regions and their separation from each other at least 4.2–5.1 Mya, which is in favour of a distinct specific status. Finally, the broad-toothed field mice from south-western Turkey appear to be closely related to the animals from Crete but highly distinct from the populations of the other Oriental regions. This supports a distinct subspecific level (A. m. rhodius) for the insular animals and also for those from south-western Turkey. From a biogeographical point of view, it can be assumed that either late Pliocene or early Pleistocene cooling led to the isolation of two main groups of A. mystacinus, one in the Balkan region and the other one in Turkey and the Near East (Syria and Israel). In this region, it is suggested that a more recent event appeared during the Quaternary period, isolating broad-toothed field mice in Crete and leading to the appearance of two well-differentiated genetic groups: one in Crete and south-western Turkey, and the other widespread in northern and eastern Turkey as well as in the Near East.

Analysis of the IRBP gene as a cause of RP in 45 ARRP Spanish families

Autosomal recessive retinitis pigmentosa (ARRP) is a degenerative disorder of photoreceptors. Mutations in several genes encoding different proteins of the visual cascade are described. The inheritance of two intragenic markers within the interstitial retinol binding protein (IRBP) gene was established in 45 Spanish families with a history of ARRP. Homozygosity mapping and cosegregation analyses were positive in 19 families. Only one heterozygous T-C transition at position 3024 (exon 1) was detected in one consanguineous family. This variant was identified as a rare polymorphism and was present in 5% of the chromosomes analyzed.

Update on the phylogenetic systematics of New World monkeys: further DNA evidence for placing the pygmy marmoset (Cebuella) within the genus Callithrix.

We determined DNA sequences spanning the 1.8-kb long intron 1 of the interstitial retinol-binding protein nuclear gene (IRBP) for Callithrix geoffroyi, Callithrix humeralifer, and Callithrix argentata. With the 22 previously determined IRBP intron 1 sequences—21 from the 16 currently recognized genera of New World monkeys—the enlarged IRBP data represent for the marmoset genus Callithrix both its argentata and its jacchus species groups. Maximum-parsimony and neighbor-joining trees, constructed for the 25 aligned IRBP intron 1 sequences, support a provisional phylogenetic classification with three families: Atelidae, containing subfamily Atelinae; Pitheciidae, containing subfamily Pitheciinae; and Cebidae, containing subfamilies Cebinae, Aotinae, and Callitrichinae. In order to have taxa at the same hierarchical rank at equivalent age, this classification has all living callitrichines in a single tribe, Callitrichini, with four subtribes: Saguinina (Saguinus), Callimiconina (Callimico), Leontopithecina (Leontopithecus), and Callitrichina (Callithrix with the pygmy marmoset, Cebuella pygmaea, merged into it). The DNA evidence shows not only that Callithrix must include C. pygmaea to be monophyletic but also that the times of separation of pygmaea and the argentata and jacchus species groups from one another are to be expected (<5 Ma—million years ago) for species in a single genus. On relating the time course of the ceboid radiation to biogeographic information, it appears that in mid-Miocene times (10–11 Ma) a basal callitrichin stock branched into the ancestral population of Saguinus in one clade and the ancestral population of Leontopithecus and Callimico–Callithrix (or Leontopithecus–Callimico and Callithrix) in another clade. The proto-lion tamarins migrated south and eastward, where they were isolated in refugia, becoming the genus Leontopithecus. The stock remaining in Amazonia gave rise to present-day Callimico and Callithrix. The latter genus occupied a vast geographic area, giving rise to the argentata and pygmaea groups in Amazonia and to the jacchus group in central and eastern Brazil.

Molecular phylogeny of the New World monkeys (Platyrrhini, primates) based on two unlinked nuclear genes: IRBP intron 1 and epsilon-globin sequences.

Nuclear sequences of the 1.8 kilobase (kb) long intron 1 of the interstitial retinol-binding protein gene (IRBP), previously determined for 11 of the 16 extant genera of New World monkeys (superfamily Ceboidea, infraorder Platyrrhini), have now been determined for the remaining 5 genera. The maximum parsimony trees found, first with IRBP sequences alone and then with tandemly combined IRBP and epsilon-globin gene sequences from the same species, supported a provisional cladistic classification with the following clusters. Subtribes Callitrichina (Callithrix, Cebuella), Callimiconina (Callimico), Leontopithecina (Leontopithecus) and Saguina (Saguinus) constitute subfamily Callitrichinae, and subfamilies Callitrichinae, Aotinae (Aotus), and Cebinae (Cebus, Saimiri) constitute family Cebidae. Subtribes Chiropotina (Chiropotes, Cacajao) and Pitheciina (Pithecia) constitute tribe Pitheciini; and tribes Pitheciini and Callicebini (Callicebus) constitute subfamily Pitheciinae. Subtribes Brachytelina (Brachyteles, Lagothrix) and Atelina (Ateles) constitute tribe Atelini, and tribes Atelini and Alouattini (Alouatta) constitute subfamily Atelinae. The parsimony results were equivocal as to whether Pitheciinae should be grouped with Atelinae in family Atelidae or have its own family Pitheciidae. The cladistic groupings of extant ceboids were also examined by different stochastic evolutionary models that employed the same stochastic process of nucleotide substitutions but alternative putative phylogenetic trees on which the nucleotide substitutions occurred. Each model, i.e., each different tree, predicted a different multinomial distribution of nucleotide character patterns for the contemporary sequences. The predicted distributions that were closest to the actual observed distributions identified the best fitting trees. The cladistic relationships depicted in these best fitting trees agreed in almost all cases with those depicted in the maximum parsimony trees.

DNA Evidence on the Phylogenetic Systematics of New World Monkeys: Support for the Sister-Grouping of Cebus and Saimiri from Two Unlinked Nuclear Genes

Previous inferences from ϵ-globin gene sequences on cladistic relationships among the 16 extant genera of Ceboidea (the New World monkeys) were tested by strength of grouping and bootstrap values for the clades in the most parsimonious trees found: for this epsilon data set enlarged with additional Cebus and Saimiri orthologues; for another nuclear DNA sequence data set consisting of IRBP (interstitial retinol-binding protein gene) intron 1 orthologues; and for tandemly combined epsilon and IRBP sequences. Different ceboid species of the same genus always grouped strongly together as demonstrated by results on Cebus (capuchin monkeys), Saimiri (squirrel monkeys), Callicebus (titi monkeys), Aotus (night monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys). Other strong groupings that could be represented as monophyletic taxa in a cladistic classification were: Cebuella (pygmy marmoset) and Callithrix (marmoset) into subtribe Callitrichina; Callitrichina, Callimico (Goeldi′s monkey), Leontopithecus (lion tamarin), and Saguinus (tamarin) into subfamily Callitrichinae; Callitrichinae, Aotus, Cebus, and Saimiri into family Cebidae; Cacajao (uakari monkey) and Chiropotes (saki) into subtribe Chiropotina; Chiropotina and Pithecia (bearded saki) into tribe Pitheciini; Pitheciini and Callicebus into subfamily Pitheciinae; Brachyteles (woolly spider monkey), Lagothrix (woolly monkey), and Ateles into tribe Atelini; and Atelini and Alouatta into subfamily Atelinae. In addition the ϵ and IRBP results congruently grouped (but at lesser strengths) Brachyteles and Lagothrix into subtribe Brachytelina within Atelini, and also Cebus and Saimiri into subfamily Cebinae within Cebidae. Because the IRBP results weakly grouped Pitheciinae with Cebidae, whereas the epsilon results weakly grouped Pitheciinae with Atelinae, the present evidence is best represented in an interim cladistic classification of ceboids by dividing the superfamily Ceboidea into three families: Atelidae, Pitheciidae, and Cebidae.

Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53.

A transgenic mouse model for retinoblastoma was produced previously by directing SV40 T antigen expression to retinal photoreceptor cells using the promoter of the interstitial retinol-binding protein (IRBP) gene. This gene becomes active prior to the terminal differentiation of photoreceptors. Because T antigen-transforming activity is attributable, at least in part, to the inactivation of the retinoblastoma (pRb) and p53 tumor suppressor proteins, we addressed the role of p53 in the development of retinoblastoma in mice. Transgenic mice expressing HPV-16 E7 under the control of the IRBP promoter were generated to inactivate pRb in photoreceptors while leaving p53 intact. Rather than developing retinoblastomas, the retinas of these mice degenerate due to photoreceptor cell death at a time in development when photoreceptors are normally undergoing terminal differentiation. The dying cells exhibit the histological and ultrastructural features of apoptosis and contain fragmented DNA. p53 is required for the induction of apoptosis in this model, because mice expressing E7 in a p53 nullizygous background develop retinal tumors instead of undergoing retinal degeneration.

Characterization and comparative structural features of the gene for human interstitial retinol-binding protein.

We have cloned the gene for human interstitial retinol-binding protein (IRBP) and compared its nucleotide sequence with that of the corresponding cloned cDNA. The human IRBP gene is approximately 9.5 kilobase pairs (kbp) in length and consists of four exons separated by three introns. The introns are 1.6-1.9 kbp long. The gene is transcribed by photoreceptor and retinoblastoma cells into an approximately 4.3-kilobase mRNA that is translated and processed into a glycosylated protein of 135,000 Da. The amino acid sequence of human IRBP can be divided into four contiguous homology domains with 33-38% identity, suggesting a series of gene duplication events. In the gene, the boundaries of these domains are not defined by exon-intron junctions, as might have been expected. The first three homology domains and part of the fourth are all encoded by the first large exon, which is 3,180 base pairs long. The remainder of the fourth domain is encoded in the last three exons, which are 191, 143, and approximately 740 base pairs long, respectively. This unusual structure is shared with the bovine IRBP gene. A large (1.7 kbp) fragment appears to have been lost from the 3'-noncoding region of the last human exon. We conclude that the human and bovine genes have similar evolutionary histories.

Close linkage of MEN2A with RBP3 locus in Japanese kindreds

The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromosome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.

Retinoids bound to interstitial retinol-binding protein during light and dark-adaptation

High-performance liquid chromatography was used to determine the types and amounts of retinoids bound to interstitial retinol-binding protein (IRBP) during light- and dark-adaptation in frogs. IRBP was separated from CRBP and CRA1BP by ion-exchange chromatography and quantitated by determining the amount of Serva Blue R dye bound to it in stained sodium dodecyl sulfate polyacrylamide gels. The amount of IRBP was not significantly different in light- and dark-adapted eyes (0.15 ± 0.05 nmol/ eye compared with 0.18 ± 0.08 nmol/ eye). In the dark-adapted state, RBP bound mainly 11- cis retinol and 11- cis retinal in quantities that summed to about 1 mol/mol RBP. After the onset of light-adaptation, all-trans retinol increased from its very low dark-adapted evel, peaked at 0.2 mol/mol IRBP and then declined to the dark-adapted level again. Concomitantly, the otal retinoid bound to IRBP fell, mainly because there was a drop in the amount of 11- cis retinal. During dark-adaptation, the amount of 11- cis retinal increased. No significant changes were seen in the amount of 11- cis retinol in light and darkness. These findings support the hypothesis that when rhodopsin is bleached IRBP transports all- trans retinol from the retina to the pigment epithelium and that it delivers 11- cis retinal to the rod outer segments for rhodopsin regeneration.

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.

Internal quadruplication in the structure of human interstitial retinol-binding protein deduced from its cloned cDNA.

Interstitial retinol-binding protein (IRBP) is a glycoprotein that shuttles retinoids between the retina and pigment epithelium and is secreted by the photoreceptor cells of the vertebrate eye. Human retina cDNA libraries in lambda gt10 were screened with a previously isolated human IRBP probe (H.4 IRBP), yielding five overlapping cDNA clones generating a 4223-base sequence. A 17-kilobase pair clone (HGL.3) isolated by screening a human genomic library in EMBL3 with H.4 IRBP yielded a 2.5-kilobase pair SstI fragment that overlapped the 5' end of the cDNA sequence by 329 nucleotide residues. An open reading frame encoded the N-terminal sequence of human IRBP and predicted a protein consisting of 1262 amino acids with a molecular mass of 136,600. Two putative N-linked glycosylation sites were identified. The translated sequence suggests that there is a 16-amino acid presumptive signal peptide rich in hydrophobic residues and with a high alpha-helix probability preceding the N terminus of the mature protein. The amino acid sequence of human IRBP could be aligned with 87% identity with the amino acid sequences of 31 peptides (605 residues) purified from a tryptic digest of bovine IRBP. The protein sequence of human IRBP contains four duplicated segments (302-310 residues in length) with 33-38% identity. From the degree of identity between the bovine and human sequences, it is possible that IRBP evolved by several gene duplications that occurred 600-800 million years ago, before the emergence of the vertebrates.

IRBP-like proteins in the eyes of six cephalopod species—immunochemical relationship to vertebrate interstitial retinol-binding protein (IRBP) and cephalopod retinal-binding protein

SDS polyacrylamide gel electrophoresis and immunoblotting were used to examine soluble proteins from the eyes of six species of cephalopods i.e. Lolliguncula brevis, Sepia officinalis, Octopus maya, Octopus bimaculoides, Rossia pacifica and Loligo opalescens. All species had a protein (“IRBP”) with molecular weight virtually identical with vertebrate interstitial retinol-binding protein (IRBP) averaging 132,400 ± 700 (n = 6). “IRBP” reacted on nitrocellulose blot transfers with rabbit antibovine IRBP and rabbit antifrog IRBP antibodies. Unlike vertebrate IRBP, cephalopod “IRBP” (from L. brevis) did not bind exogenous retinol or concanavalin A. The N-terminal amino acid appeared to be blocked in samples electroeluted from SDS gels. The antifrog IRBP antibodies also reacted with a series of proteins with molecular weights between 46,000 and 47,000, identified as retinal-binding protein (RALBP) with anti-RALBP antibodies. Anti-IRBP also reacted with pure RALBP prepared from Todarodes pacificus. Occasionally, anti-RALBP antibodies were seen to react weakly with “IRBP” in some cephalopods. We conclude that RALBP, cephalopod “IRBP” and vertebrate IRBP share a common but distant ancestry, and that a protein resembling IRBP appeared before the vertebrates diverged from the invertebrates. Both RALBP and IRBP appear to have analogous functions in shuttling retinoids between rhodopsin and the corresponding isomerizing system, retinochrome in the cephalopods and retinol isomerase in the vertebrates. The function of cephalopod “IRBP” is unknown.

Levels of alpha- and gamma-tocopherol in human eyes: evaluation of the possible role of IRBP in intraocular alpha-tocopherol transport

Alpha-tocopherol was distributed almost equally between the retina and its underlying pigmented layers (pigment epithelium and choroid). Only 8.4% of the total alpha-tocopherol occurred in the iris and ciliary body. Alpha-tocopherol content was expressed as amount per eye, per cm2, and per 100 g wet weight. The combined retina and pigment epithelium-choroid contained 2.9 +/- 1.0 mg/100 g wet weight (means +/- SD, n = 30 donors). Gamma-tocopherol represented 20.9 +/- 12.2 mol % of the alpha-tocopherol. The anterior tissues contained 0.4 +/- 0.2 mg/100 g (n = 19 donors). No significant correlation with age was found. Purified bovine interstitial retinol-binding protein (IRBP) bound exogenous 3H-alpha-tocopherol, which could be displaced by unlabeled all-trans retinol (KD = 10(-6) M). Much higher concentrations of unlabeled alpha-tocopherol were required to achieve a partial displacement of bound 3H-all-trans retinol. No endogenous alpha-tocopherol could be detected in bovine interphotoreceptor matrix.

Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage.

Multiple endocrine neoplasis type 2A (MEN2A) is one of several kinds of cancers that appear to be inherited in an autosomally dominant fashion. We have assigned the MEN2A locus to chromosome 10 by linkage with a new DNA marker (D10S5). The linkage led us to investigate other chromosome 10 markers and demonstrate linkage between the disease locus and the interstitial retinol-binding protein (IRBP) gene. The D10S5 locus was sublocalized to 10q21.1 by hybridization in situ and the IRBP gene to p11.2----q11.2 with a secondary site at q24----q25. The linkages were established using 292 members of five families, three different restriction fragment length polymorphisms (RFLPs) at D10S5 and two RFLPs recognized by the IRBP probe. The recombination frequencies from pairwise linkage analysis between the disease and two marker loci D10S5 and IRBP were 0.19 and 0.11, with maximum lod scores of 3.6 and 8.0 respectively. Ordering of the three loci by multipoint analysis placed the IRBP gene approximately midway between the disease and D10S5 loci.

A linked genetic marker for multiple endocrine neoplasia type 2A on chromosome 10.

Multiple endocrine neoplasia type 2A (MEN2A) is an autosomal dominantly inherited cancer syndrome characterized by medullary carcinoma of the thyroid, phaeochromocytoma and hyperparathyroidism. Almost all gene carriers can be detected by screening tests before the age of 40, but the nature and location of the predisposing gene are unknown. Simpson et al. recently reported preliminary evidence for linkage between the DNA probe p9-12A on chromosome 10 and MEN2A. We now report linkage between the MEN2A locus and the interstitial retinol-binding protein gene, which is located on chromosome 10p11.2-q11.2.

Human interstitial retinol-binding protein (IRBP): cloning, partial sequence, and chromosomal localization.

A cloned 2184-bp cDNA coding for human interstitial retinol-binding protein (IRBP) has been isolated and sequenced. The probe hybridized to a 5.2-kb poly(A) RNA from human retinas. Nineteen tryptic peptides (363 amino acids) sequenced and purified from bovine IRBP could be aligned with 86-88% homology to the translated sequence. Two segments approximately 200 amino acids long were found to have a 41% residue identity, suggesting an internal duplication event. This cloned cDNA was used to probe DNA samples from a panel of 29 rodent-human somatic cell hybrids, mapping the structural gene for IRBP to chromosome 10. In situ hybridization suggested a regional localization near the centromere (p11.2----q11.2), although a secondary site of hybridization at q24----25 was also observed.

Interstitial retinol-binding protein and cellular retinal-binding protein in the mammalian pineal

Antibodies against bovine interstitial retinol-binding protein (IRBP) and cellular retinalbinding protein (CRA1BP) were used in immunochemical and immunocytochemical studies of the pineal glands of cattle, hamsters and rats (RCS and RCS-rdy +). On immunoblots, IRBP ( M r 144,000) was identified in cattle, hamster and rat pineal extracts. The abundance of IRBP in bovine pineals was33 ± 6mg·mg −1 ( x¯± SD, n = 12) soluble protein. RCS (Royal College of Surgeons) rat pineals gave a strong IRBP reaction on immunoblots, even when virtually no IRBP could be found in the eye due to photoreceptor degeneration. In the hamster retina IRBP immunostaining was distributed throughout the entire interphotoreceptor matrix and the outer segment layer. The pineal also showed strong IRBP-like immunostaining scattered uniformly throughout the gland. Other hamster brain regions showed no specific immunostaining; however, an immunoreactive protein with the same M r as IRBP was detected on Western blots of bovine cerebral cortex, spinal cord and brainstem soluble proteins. Immunoreactive proteins at lower M r were also detected in these tissues. CRA1BP immunoreactivity ( M r about 32,000) was observed in immunoblots of bovine, hamster and rat pineal proteins. These findings suggest that some mammalian pinealocytes are related to the retinal cells that contain CRA1BP (i.e. pigment epithelium, Muller cells) while others are related to the photoreceptors, which synthesize IRBP.