Electronic publishing.
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Computer Communication Networks↗
Biomedical subjects
J M Nickerson
Publications and source records attributed to J M Nickerson.
Expression of retina-specific genes by mouse retinoblastoma cells.
PURPOSE: Two cell lines derived from ocular tumors of a transgenic mouse expressing the SV40 large T antigen have been established as models of human retinoblastoma. One line, TM, originated from a metastasis, and the other, TE, originated from the primary tumor. The authors compared these two lines with the normal adult mouse eye by analysis of the expression of five photoreceptor cell-specific proteins: IRBP, opsin, rod- and cone-specific transducins, and S-antigen. The authors sought to determine which of these proteins was expressed qualitatively and to examine semi-quantitatively for changes in the levels of expression in the cell lines. METHOD: Western blot analysis was used to detect photoreceptor-specific intracellular or secreted proteins. Total RNA was prepared from cultured cells or from mouse adult whole eye. Specific messenger levels in total RNA were determined either by northern hybridization analysis or by a semi-quantitative polymerase chain reaction (PCR), coupled to complementary DNA (cDNA) substrates prepared from total RNA. RESULTS: IRBP was present in the retinoblastoma cell lines and secreted into the medium. Neither S-antigen nor opsin were detectable by immunoblotting. IRBP and cone transducin mRNA were present in both cell lines. In contrast, opsin, rod transducin, and S-Antigen mRNAs were not detectable by PCR. beta-actin was present in the mRNA populations of whole eye and retinoblastoma. SV40 large T antigen mRNA was present only in retinoblastoma cells. CONCLUSIONS: IRBP and cone transducin expression in mouse retinoblastoma cells is independent of signaling provided directly or indirectly through large T antigen or Rb105 regulatory cascades. The pattern of photoreceptor-specific gene expression is similar to that seen in human retinoblastoma cell lines. These murine-derived cell lines may be useful as a tool to study IRBP and cone transducin expression in vitro and to determine early retinoblast expression patterns in the mouse.
Animals↗
Differential expression of interphotoreceptor retinoid-binding protein, opsin, cellular retinaldehyde-binding protein, and basic fibroblastic growth factor.
The mRNA for rat interphotoreceptor retinoid-binding protein (IRBP) consists of 5.2 kb and 6.4 kb transcripts which are expressed early during retinal development. Here, we characterized rat IRBP cDNA and genomic clones, determined the mechanism generating the two transcripts and compared their expression to mRNAs for opsin, cellular retinaldehyde-binding protein (CRAlBP) and basic fibroblast growth factor (bFGF). We found that human and rat IRBPs have a high degree of sequence homology (86% amino acid identity over the fourth repeat). RNA-PCR studies and Northern blot analysis, showed that the 6.4 kb mRNA has a longer 3'-untranslated region (UTR) than the 5.2 kb message. Both IRBP transcripts, but not the mRNA for opsin are present in the neonatal and adult pineal. During development, the mRNAs for IRBP and opsin reach one-half of their maximal levels by P5 and P11, respectively. The ratio of the two IRBP mRNAs remains constant throughout life. In contrast, the ratio of the longest to the shortest opsin mRNA decreases between E19 and 1 year of age. Immunohistochemistry demonstrated a marked increase in the amount of IRBP in the interphotoreceptor matrix between P1 and P9 corresponding to the enhanced expression of this mRNA. Up-regulation of opsin mRNA expression during the second postnatal week corresponds to the emergence of the outer segments. The temporal expression of CRAlBP is similar to IRBP while bFGF is not expressed until after photoreceptor differentiation is complete. In summary, two 3'-transcription termination sites explain the difference in IRBP mRNA sizes. The different temporal and tissue specific patterns of IRBP and opsin expression suggest that separate mechanisms control the expression of these two genes. The expression patterns of IRBP and CRAlBP are consistent with the role of vitamin A as a morphogen and bFGF in neuronal maintenance.
Age Factors↗
PCR detection of the MspI polymorphism in the human IRBP gene (RPB3).
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Base Sequence↗
Independent segregation of fundus albipunctatus and the transthyretin (prealbumin) gene.
A defect in the visual cycle has been suspected in patients with fundus albipunctatus (FALB), rendering genes encoding visual cycle components etiologic candidates. One such component is the retinoid and thyroxine transport protein transthyretin (TTR, prealbumin) which in the eye is synthesized only in the retinal pigment epithelium and is believed to play a role in retinal retinoid transport. The authors established polymerase chain reaction conditions that allow rapid assay of TTR alleles as defined by MspI and Fnu4HI restriction fragment length polymorphisms. In a candidate gene analysis of an affected family, they demonstrate independent segregation of the TTR and FALB disease loci. These results exclude the possibility that a TTR gene defect causes FALB in this family.
Alleles↗
Restriction fragment length polymorphism analysis using PCR coupled to restriction digests.
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Base Sequence↗
The molecular biology of IRBP: application to problems of uveitis, protein chemistry, and evolution.
In closing, molecular biology has helped us in the study of uveitis, in defining the parts of the IRBP molecule that cause EAU/EAP. Simple northern and Southern blotting experiments can answer significant questions in the study of gene structure and expression and in relating these to genetic eye diseases. Molecular biology has provided answers to several long-standing cell biology question such as: where is IRBP synthesized, where is the gene expressed, and how is the IRBP polypeptide processed? In considering how mother nature invented the IRBP gene, the gene structure suggests some interesting alternative models, and causes us to speculate about how large proteins may have evolved. Finally, the determination of the protein sequence helps to put us in the position to ask and answer questions about the function of IRBP. It also allows us to begin to determine the consequences of a nonfunctional IRBP. We don't know the answers to all these questions yet, but the structural analyses and the isolation of these genes and cDNAs, presented here, united with other powerful biological techniques should provide the answers.
Amino Acid Sequence↗
Repeated determinants within the retinal interphotoreceptor retinoid-binding protein (IRBP): immunological properties of the repeats of an immunodominant determinant.
Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes in the retina and pineal gland, induces inflammatory changes in these organs (EAU and EAP, respectively) when injected into various mammals. We have previously identified a determinant (residues 1169-1191) in bovine IRBP which is immunodominant and highly immunogenic and immunopathogenic in Lewis rats. IRBP exhibits a fourfold repeat structure and we report here on the comparison between the active sequence 1179-1191 and its three repeat peptides. Only one of the repeats, 271-283, cross-reacted with 1179-1191 and exhibited immunodominance, albeit of a low level. Peptide 271-283 was also immunogenic and immunopathogenic in Lewis rats, but with a minimal dose approximately 100 times higher than that of 1179-1191. Peptide 880-892, a nondominant determinant, resembled 271-283 in its immunogenicity, but was markedly less immunopathogenic. No immunological activity was detected in the fourth repeat peptide, 579-591. Peptide 1179-1191 was superior to the other repeats also in its antigenicity, i.e., the capacity to stimulate presensitized lymphocytes in culture: the minimal stimulatory concentrations of 1179-1191 was greater than 1000 times lower than those of 271-283 or 880-892. Furthermore, 1179-1191 was stimulatory at concentrations lower than those of 271-283 even when tested with lymphocytes sensitized against 271-283. A correlation was also found between the immunological activities of the repeat peptides and their amphipathicity. This study thus identifies two new immunopathogenic determinants of IRBP and provides additional data to show the association between immunodominance of peptides and their various immunological activities.
Amino Acid Sequence↗
Cloning of cDNAs encoding human interphotoreceptor retinoid-binding protein (IRBP) and comparison with bovine IRBP sequences.
We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.
Amino Acid Sequence↗
Synthesis of an immunopathogenic fusion protein derived from a bovine interphotoreceptor retinoid-binding protein cDNA clone.
We have extended the cDNA sequence of bovine interphotoreceptor retinoid-binding protein (IRBP) and subcloned one of the sequenced cDNA fragments into an expression vector. The nucleotide (nt) sequences of four bovine IRBP cDNA clones have been determined. These sequences when assembled cover the 3' proximal 3629 nt of the IRBP mRNA and encode the C-terminal 551 amino acids (aa) of IRBP. This cDNA sequence validates the intron: exon boundaries predicted from the gene. A 2-kb EcoRI insert from lambda IRBP2, one of the clones sequenced, encoding the C-terminal 136 aa of IRBP was subcloned into the expression vector pWR590-1. Escherichia coli carrying this plasmid construction, pXS590-IRBP, produced a fusion protein containing 583 N-terminal aa of beta-galactosidase, three linker aa residues, 136 C-terminal aa of IRBP and possibly a number of additional C-terminal residues due to suppressed termination. This 86-kDa fusion protein, purified by detergent/chaotrope extraction followed by reverse-phase high-performance liquid chromatography, cross-reacted with anti-bovine IRBP on Western blots. This protein induced an experimental autoimmune uveo-retinitis and experimental autoimmune pinealitis in Lewis rats indistinguishable from that induced by authentic bovine IRBP. Thus, it is evident that biological activity of this region of IRBP, as manifested by immuno-pathogenicity, is retained by the fusion protein.
Amino Acid Sequence↗
Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution.
The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.
Amino Acid Sequence↗
mRNA for interphotoreceptor retinoid-binding protein (IRBP): distribution and size diversity in vertebrate species.
Northern blots of total retinal RNA from a number of different vertebrate species were probed with a cDNA fragment corresponding to translated portions of bovine IRBP mRNA. A hybridizing band was detected in normal human retina (4.6 kb), Y-79 human retinoblastoma cells (4.4 kb), and in retinas of monkey (4.6 kb), guinea-pig (4.9 kb), mouse (6.1 kb), rat (two bands at 5.4 kb and 6.6 kb), rabbit (6.3 kb), cow (6.5 kb), and hamster (7.6 kb). Thus, the IRBP gene is expressed in the retinas of a wide variety of mammalian species. The mRNAs could be readily detected in about 20 micrograms of total RNA, suggesting that, in these species, IRBP message is relatively abundant. In contrast, only very weak hybridization was detected on northern blots of the three bird species examined (chicken, duck, quail). IRBP mRNA is thus relatively well expressed in mammals, but not in birds.
Animals↗
The isolation of a gene encoding interphotoreceptor retinoid-binding protein.
The gene locus for bovine IRBP, as well as several kilobases of sequence flanking the gene on either end, has been cloned. Two of the several clones seem to contain full-length copies of the protein encoding portion of the gene. Using these clones and cDNA clones, we have determined that there are one or perhaps two copies of the IRBP gene per haploid genome in several species. The gene is compact considering the large size of the protein (145,000 daltons) and its large mRNA (about 6.5 kb). Surprisingly, the gene is no more than 14 kb, being fully contained on lambda clones of maximum packaging size 20 kb. Small parts of the gene were sequenced for the purpose of proving the identity of the genomic clones. DNA sequencing of one of the IRBP gene clones demonstrates the existence of an intron in the gene. The sequence analysis of another fragment identified the N-terminus which has been sequenced at the protein level. The DNA sequence analysis showed the existence of a putative signal sequence and the potential existence of a short five amino acid sequence between the signal sequence and the authentic N-terminus of the secreted extracellular IRBP. This confirms and validates the finding of the extra five amino acid sequence that is present on 40-50% of the polypeptides in monkey and human IRBP which have been isolated from the subretinal space. The presence of the appropriate gene sequence for the pentapeptide but its absence in bovine IRBP indicates differences in processing among the vertebrate IRBPs.
Amino Acid Sequence↗
Gene sharing by delta-crystallin and argininosuccinate lyase.
The lens structural protein delta-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; L-argininosuccinate arginine-lyase, EC 4.3.2.1) have striking sequence similarity. We have demonstrated that duck delta-crystallin has enormously high ASL activity, while chicken delta-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken delta 1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken delta-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the delta 2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the delta 2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (delta-crystallin), although in chickens specialization and separation of functions may have occurred.
Amino Acid Sequence↗
Cyanogen bromide fragments of bovine interphotoreceptor retinoid-binding protein induce experimental autoimmune uveoretinitis in Lewis rats.
Interphotoreceptor retinoid-binding protein (IRBP), a retinal specific antigen, induces experimental autoimmune uveoretinitis (EAU) when injected into Lewis rats. Here we report that certain cyanogen bromide fragments of IRBP are capable of inducing EAU. Bovine IRBP, reduced and S-carboxymethylated, was subjected to cyanogen bromide cleavage. This CNBr digest was subjected to reversed-phase high performance liquid chromatography. Three fragments were purified to apparent homogeneity. These three fragments were subjected to gas-phase amino-terminal sequencing analysis. All three yielded single sequences, confirming their purity. On the basis of this amino-terminal sequencing and sequencing of cDNAs encoding bovine IRBP, two of these sequences, named CB-58 and CB-71, were localized to the C-terminal one-third of the IRBP molecule, whereas the third, a subfragment thought to result from cleavage at a tryptophan residue and named CB-47, was localized to the N-terminal one third of the protein. CB-71 and CB-47 shared a strong homology, suggesting a putative internal gene duplication event in the evolution of IRBP. All three of these fragments when injected into Lewis rats caused moderately severe EAU with early onset at relatively low doses. The histopathologic changes induced were indistinguishable from those caused by the intact protein. It would seem, therefore, that bovine IRBP contains multiple uveitogenic sites.
Amino Acid Sequence↗
Retinal photoreceptor neurons and pinealocytes accumulate mRNA for interphotoreceptor retinoid-binding protein (IRBP).
We have utilized cDNA probes and in situ hybridization techniques to define the subcellular localization of interphotoreceptor retinoid-binding protein (IRBP) mRNA in bovine and monkey retinas. Results suggest that the mRNA is mainly localized in rod photoreceptor neurons within the outer nuclear layer of the retina. IRBP mRNA is also abundant in cells of the pineal gland, strengthening the analogy between rod photoreceptor cells and pinealocytes.
Animals↗
Two delta-crystallin polypeptides are derived from a cloned delta 1-crystallin cDNA.
Previous studies have shown that there are 2 similar delta-crystallin genes (delta 1 and delta 2) and at least 2 delta-crystallin polypeptides in the chicken eye lens. We show here that both delta-crystallin polypeptides can be synthesized from mRNA transcribed in vitro from a cloned delta 1-crystallin cDNA. Both polypeptides co-migrate in SDS-urea-polyacrylamide electrophoresis with their authentic counterparts isolated from 15-day-old embryonic chicken lenses, and both react with sheep anti-chicken delta-crystallin serum. Screening nearly 900 delta-crystallin cDNA clones from a 15-day-old embryonic lens library with an oligonucleotide probe specific for exon 2 of the delta 2-crystallin gene failed to detect any delta 2 cDNA clones, indicating that the delta 2 gene produces little or no mRNA in the lens at this stage of development. Our results suggest that both of the observed delta-crystallin polypeptides are derived from mRNA transcribed from the delta 1 gene, with heterogeneity arising at the translational or co-translational level.
Amino Acid Sequence↗
Sequence of the chicken delta 2 crystallin gene and its intergenic spacer. Extreme homology with the delta 1 crystallin gene.
The chicken delta-crystallin locus consists of 2 nonallelic, tandemly arranged genes (5'-delta 1-delta 2-3'). Only the delta 1 gene is known to be expressed. The nucleotide sequence for the delta 1 gene has been reported recently (Nickerson, J.M., Wawrousek, E.F., Hawkins, J.W., Wakil, A.S., Wistow, G.J., Thomas, G., Norman, B.L., and Piatigorsky, J. (1985) J. Biol. Chem. 260, 9100-9105; Ohno, M., Sakamoto, H., Yasuda, K., Okada, T.S., and Shimura, Y. (1985) Nucleic Acids Res. 13, 1593-1606). We now report the sequence for the delta 2 gene and the 4-kilobase intergenic spacer between the two delta-crystallin genes. The delta 2 gene, like the delta 1 gene, has 17 exons and 16 introns. The homologous exons are remarkably similar: exons 3-17 are identical in size between delta 1 and delta 2, and the sequence homology ranges from 70% (exon 2) to 100% (exons 7, 12, and 15), with the remaining exons having 89-98% identity between the delta 1 and delta 2 genes. Consequently, the encoded delta 2 polypeptide is 91% identical to the delta 1 polypeptide. Considerable similarity also exists between homologous introns of delta 1 and delta 2, with most of the differences accounted for by insertions and/or deletions. The presence of a TATA box, consensus splice junctions (almost identical to the delta 1 gene), lariat branch sequences, and a polyadenylation signal strengthen the possibility that delta 2 is a functional gene.
Animals↗