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Purpose: Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. To increase our understanding of the cells types involved in pterygium, we have used expressed sequence tag analysis to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration., Methods: An unnormalized unamplified cDNA library was prepared from 15 pooled specimens of surgically removed pterygia as part of the NEIBank project. Gene expression patterns were compared with existing data for human cornea, limbus, and conjunctiva, and expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium., Results: Sequence analysis of 2,976 randomly selected clones produced over 1,800 unique clusters, potentially representing single genes. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Immunofluorescence of Krt12 and 13 in the normal ocular surface showed specificity of Krt12 in cornea and Krt13 in conjunctival and limbal epithelia, with a fairly sharp boundary at the limbal-corneal border. In the pterygium there was a patchy distribution of both Krt12 and 13 up to a normal corneal epithelial region specific for Krt12. Immunoglobulins were also among the prominently expressed transcripts. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. A number of genes highly expressed in cornea were not found in pterygium (several small leucine-rich proteoglycan family members) or were expressed at considerably lower levels (ALDH3A1 and decorin)., Conclusions: The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth.

Oxysterol-binding proteins (OSBPs) and oxysterol-binding-protein related proteins (ORPs) are encoded by most eukaryotic genomes examined to date; however, they have not yet been characterized in plants. Here we report the identification and characterization of PiORP1, an ORP of Petunia inflata that interacts with the cytoplasmic kinase domain of a receptor-like kinase, named PRK1, of P. inflata. PiORP1 is phosphorylated by PRK1 in vitro and therefore may be involved in PRK1 signaling during pollen development and growth. RNA gel blot analysis showed that PiORP1 and PRK1 had very similar expression patterns in developing pollen, mature pollen and pollen tubes. GFP fusion proteins of PiORP1 localized in the plasma membrane of pollen tubes at distinct foci and its PH domain alone was sufficient to mediate this localization. The sequence for the oxysterol-binding domain of PiORP1 was used to search the genome of Arabidopsis; 12 ORPs were identified and phylogenetic analysis revealed that they fell into two distinct clades, consistent with the ORPs of other eukaryotes. RT-PCR analysis showed that all 12 Arabidopsis ORPs were expressed; 10 were expressed in most of the tissues examined under normal growth conditions, but only three were expressed in pollen.

Purpose: Pterygium is a sunlight-related, ocular-surface lesion that can obscure vision. In order to identify specific genes that may play a role in pterygium pathogenesis, we analyzed the global gene expression profile of pterygium in relation to autologous conjunctiva., Methods: Oligonucleotide microarray hybridization was used to compare the gene expression profile between human whole pterygium and autologous conjunctiva. Selected genes were further characterized by RT-PCR, western blot, and immunohistochemistry, and comparisons were made with limbal and corneal tissues., Results: Thirty-four genes exhibited a 2 fold or greater difference in expression between human whole pterygium and autologous conjunctiva. Twenty-nine transcripts were increased and five transcripts were decreased in pterygium. Fibronectin, macrophage-inflammatory protein-4 (MIP-4), and lipocalin 2 (oncogene 24p3; NGAL) were increased 9, 5, and 2.4 fold, respectively, while Per1 and Ephrin-A1 were decreased 2 fold in pterygium. Western blots showed that fibronectin and MIP-4 were increased in pterygium compared to limbus, cornea, and conjunctiva. Immunohistochemical analysis showed fibronectin in the stroma; lipocalin 2 in the basal epithelial cells, basement membrane, and extracellular stroma; and MIP-4 in all areas of the pterygium., Conclusions: These data show both novel and previously identified extracellular-matrix-related, proinflammatory, angiogenic, fibrogenic, and oncogenic genes expressed in human pterygium. Comparisons of selected genes with limbal and corneal tissues gave results similar to comparisons between pterygium and normal conjunctiva. The increased expression of lipocalin 2, which activates matrix metalloproteinases (MMP), is consistent with our previous findings that MMP-9 and other MMPs are highly expressed in pterygium basal epithelium.

Oxysterol-binding proteins (OSBPs) have been described in a wide range of eukaryotes, and are often found to be part of a multi-gene family. We have used bioinformatics and data mining as a starting point for identifying new family members in humans based on the presence of the OSBP signature EQVSHHPP. In addition to OSBP and the recently reported OSBP2, we have found 10 other genes encoding oxysterol-binding domains. Here, we report cDNA and deduced peptide sequences of the previously unknown OSBPs and compare the peptides and genes. All of the genes encode a pleckstrin homology domain, except OSBPL2. However, two of the peptides, OSBPL2 and OSBPL1A, consist of the OSBP domain only. A second OSBPL1 transcript (OSBPL1B) contains 15 additional upstream exons, with a deduced peptide containing a pleckstrin homology domain. Cladistic analysis divides the human OSBP genes into five groups, whose members share similarities in sequence and gene structure; RT-PCR analysis indicates that expression patterns among group members vary widely.

A novel member of the neuron-specific protein (NSP) or newly named reticulon (RTN) gene family was isolated during a subtraction cloning between macula and peripheral retina. The mRNA for this NSP/RTN-like gene is approximately threefold more abundant in macula than in peripheral retina. The cDNA is 2527 bp long and contains an open reading frame of 236 amino acids. The deduced peptide shows a strong similarity to the NSP/RTN and tropomyosin-like gene families but it is clearly a novel member. The gene contains seven exons and spans more than 15 kb. The gene was localized to chromosome 11q13 between markers D11S4535 and D11S4627 using somatic cell hybrid panels. Southern blot analysis identified the presence of a pseudogene(s) that was subsequently localized to chromosome 4. Multitissue Northern blot analysis found this gene to be widely expressed in human tissues with the highest expression in the brain. We are calling this gene RTN3 to reflect the newly proposed nomenclature., (Copyright 1999 Academic Press.)

The MIP (major intrinsic protein) gene, a member of an ancient family of membrane channel genes, encodes the predominant fiber cell membrane protein of the ocular lens. Its specific expression in the lens fibers is temporally and spatially regulated during development. To study the regulation of expression of MIP and delineate the regulatory elements underlying its tissue specificity and ontogenic profile, we have cloned 2840 bp of the human MIP 5'-flanking sequence. The human MIP 5'-flanking sequence contains three complete Alu repetitive elements in tandem at position between nt -1699 and -2684 (nt -1699/-2684). These Alu elements appear to have had a complex evolutionary history with insertions at different times. We have fused DNA fragments containing MIP 5'-flanking sequences to the bacterial cat reporter gene encoding chloramphenicol acetyltransferase and assayed them in primary cultures of chicken lens cells. We have mapped two negative regulatory regions in the human MIP 5'-flanking sequences -1564/-1696 and -948/-1000. We demonstrated that the human MIP 5'-flanking sequence -253/+42 contains a functional promoter in lens cells but is inactive in kidney epithelial cells or mouse fibroblasts, suggesting that this sequence contains regulatory elements responsible for the lens-specific expression of MIP.

alpha A-crystallin, a major structural protein in the ocular lenses of all vertebrates, has been a valuable tool for molecular phylogenetic studies. This paper presents the complete sequence for human alpha A-crystallin derived from cDNA and genomic clones. The deduced amino acid sequence differs at two phylogenetically informative positions from that previously inferred from peptide composition. This led us to examine the same region of the alpha A-crystallin gene in 12 other mammalian species using direct sequencing of PCR-amplified genomic DNA. New sequences were added to the database, and corrections were made to all anthropoid sequences, defining clear synapomorphies for anthropoids as a clade distinct from prosimians. Within the anthropoids there are further synapomorphies delineating hominoids, Old World monkeys, and New World monkeys. Significantly, sequence revisions and the addition of new sequence for a prosimian, the sifaka, eliminate the previous support for the proposed anthropoid affinities of the tarsier inferred from alpha A-crystallin protein sequences. In addition, DNA sequences provide greater resolution of certain relationships. For example, although they are identical in protein sequence, comparison of DNA sequences clearly separates mouse and the common tree shrew, grouping the tree shrew closer to prosimians. These results show that adding DNA sequences to the existing alpha A-crystallin database can enhance its value in resolving phylogenetic relationships.

Approximately 2 kb of 5'-flanking sequences of the lens-specific alpha A-crystallin genes from human and mouse are presented and compared with similar regions of the chicken gene. A repetitive element was found approximately 1 kb upstream from the coding sequences of the alpha A-crystallin gene in all three species (Alu in human, B2 in mouse, and CR1 in chicken), suggesting that they may have an important functional or structural role. Despite the ability of alpha A-crystallin promoters to function across species, dot matrix analyses show only limited similarity among the 600 bp 5' to the structural genes of these three species. The human 5'-flanking sequence is more similar to that of the mouse and chicken than the mouse and chicken are to each other. Numerous short sequences (8-13 bp) are common to all three genes but are distributed differently in each species. The locations and conservation of these sequence motifs suggest functional roles, possibly as cis-regulatory elements of transcription. One motif is similar to the alpha A-CRYBP1 binding site implicated earlier in the transcriptional regulation of the mouse alpha A-crystallin gene, and other motifs correspond to sites previously mapped by methylation interference studies in the mouse alpha A-crystallin promoter. The modular arrangement of conserved sequence motifs is consistent with evolutionary changes occurring at the level of gene regulation.

The crystallin genes encode the major soluble proteins of the lens. Some of the crystallin genes are expressed exclusively in the lens while others are also expressed in different tissues. The two alpha-crystallin genes, alpha A and alpha B, differ in their tissue specificity. Transcription of the alpha A-crystallin gene occurs only in the lens, while the alpha B-crystallin gene is also expressed in other tissues, including heart, skeletal muscle, kidney, lung and brain. MIP (also called MP26), the major intrinsic protein of the lens fiber membranes, is also expressed exclusively in the lens. Correct expression of both alpha-crystallin and MIP are required for normal lens function. Here we review our studies on the molecular basis of expression of the alpha-crystallin and MIP genes in the lens. The 5' flanking sequences containing the initiation site of transcription of the alpha A-crystallin, alpha B-crystallin and MIP genes were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the expression of this reporter gene was studied in transient assays and transgenic mice. DNA sequences flanking the 5' end of the alpha A-crystallin gene contain regulatory elements responsible for the lens-specific expression and developmental regulation of the CAT gene in transgenic mice. Interestingly, although some of the murine alpha A-crystallin regulatory sequences are conserved in the human and chicken genes, different functional regulatory elements appear to control the expression of the murine and chicken alpha A-crystallin genes. The 5' flanking sequence of the alpha B-crystallin gene preferentially directs expression of the CAT gene to the lens and to skeletal muscle. Different regulatory elements of the alpha B-crystallin gene appear to be responsible for its transcription in various tissues. The 5' flanking sequence of the MIP gene also contains regulatory elements that direct expression of the CAT gene to lens cells; these sequences are not functional in transfected non-lens cells and are different from the cis regulatory elements controlling alpha-crystallin gene expression. The multiplicity of cis-regulatory elements controlling the transcription of these three genes indicates the complexity of the mechanisms that regulate gene expression in the lens.

Glucocorticoid treatment of cultured fibroblasts increases intracellular cyclic AMP accumulation induced by isoproterenol or cholera toxin. This increase in agonist activity is not a direct action of glucocorticoids on cyclic AMP metabolism since about 2 days are necessary for maximal effect. Basal cyclic AMP levels are not changed. In membrane preparations, GTP-dependent adenylate cyclase activity is increased, basal adenylate cyclase activity is unchanged, and NaF-stimulated activity is decreased. The number of beta-adrenergic receptors is unchanged, but the affinity of receptor for the antagonist dihydroalprenolol is increased about 3-fold. This change in affinity is probably not responsible for the increased response to isoproterenol since the augmented response is noted at 0.1 mM isoproterenol, a concentration much larger than the apparent KD (about 5 nM). The results suggest that an alteration in some component in the GTP-dependent regulatory complex is responsible for the increase in agonist response.

The frequent correspondence of exons to structural or functional domains in proteins has suggested that many proteins have evolved by modular assembly. This idea is supported by examples of apparent exon duplication and by shared domains among both alternatively spliced and completely separate genes. During this process it is probable that some combinations of exons would not prove advantageous and would therefore be lost. Here we report that within the active single-copy human gene for alpha A-crystallin there is a 'pseudo-exon' in the early stages of being extinguished, perhaps the result of a failed experiment in the evolution of this specialized, lens-specific protein.

The intracellular concentration of cyclic AMP regulates many cellular properties of normal rat, mouse, and human fibroblasts in culture. Thus it is important to elucidate how the activity of adenylate cyclase of fibroblasts might be altered and regulated by both intracellular and extracellular agents and events. In studies with several virally transformed fibroblast lines, as well as those transformed spontaneously or by 3-methylcholanthrene, a common feature of each type of transformation is a defective adenylate cyclase system. However, the means by which adenylate cyclase activity is altered differs with the cell system and the type of transformation. Here we concentrate on efforts to understand the regulatory properties of the adenylate cyclase from normal rat kidney fibroblasts. The modulation (increase or decrease) of the hormonal responsiveness of this enzyme may play an important role in its regulation. Of substantial interest is the isolation from serum of a high-molecular weight factor that selectively decreases the GTP and hormone-stimulated activities of adenylate cyclase. These findings are of value in our attempts at elucidation of the reasons for altered cyclase activity following virus transformation and during various stages of growth.