Deletion of mouse MsrA results in HBO-induced cataract: MsrA repairs mitochondrial cytochrome c.

PURPOSE: Considerable evidence indicates a role for methionine sulfoxide reductase A (MsrA) in lens cell resistance to oxidative stress through its maintenance of mitochondrial function. Correspondingly, increased protein methionine sulfoxide (PMSO) is associated with lens aging and human cataract formation, suggesting that loss of MsrA activity is associated with this disease. Here we tested the hypothesis that loss of MsrA protein repair is associated with cataract formation. To test this hypothesis we examined the effect of MsrA deletion on lens opacity in mice treated with hyperbaric oxygen, identified lens mitochondrial proteins oxidized upon deletion of MsrA and determined the ability of MsrA to repair the identified proteins. METHODS: Wild-type and MsrA knockout mice were treated or not treated with 100 treatments of hyperbaric oxygen (HBO) over an 8 month period and lenses were examined by in vivo light scattering measurements documented by slit-lamp imaging. Co-immunoprecipitation of MsrA was conducted against five specific protein representatives of the five complexes of the electron transport chain in addition to cytochrome c (cyt c). Cyt c in lens protein from the knockout and wild-type lenses was subjected to cyanogen bromide (CNBr) cleavage to identify oxidized methionines. Methionine-specific CNBr cleavage was used to differentiate oxidized and un-oxidized methionines in cyt c in vitro and the ability of MsrA to restore the activity of oxidized cyt c was evaluated. Mass spectrometry analysis of cyt c was used to confirm oxidation and repair by MsrA in vitro. RESULTS: HBO treatment of MsrA knockout mice led to increased light scattering in the lens relative to wild-type mice. MsrA interacted with four of the five complexes of the mitochondrial electron transport chain as well as with cyt c. Cyt c was found to be aggregated and degraded in the knockout lenses consistent with its oxidation. In vitro analysis of oxidized cyt c revealed the presence of two oxidized methionines (met 65 and met 80) that were repairable by MsrA. Repair of the oxidized methionines in cyt c restored the activity of cytochrome c oxidase and reduced cytochrome c peroxidase activity. CONCLUSIONS: These results establish that MsrA deletion causes increased light scattering in mice exposed to HBO and they identify cyt c as oxidized in the knockout lenses. They also establish that MsrA can restore the in vitro activity of cyt c through its repair of PMSO. These results support the hypothesis that MsrA is important for the maintenance of lens transparency and provide evidence that repair of mitochondrial cyt c by MsrA could play an important role in defense of the lens against cataract formation.

Cloning, sequencing and differential expression of alphaB-crystallin in the zebrafish, Danio rerio.

Here we report the cloning and expression of alphaB-crystallin from the zebrafish. 5'- and 3'-RACE was used to isolate a 900-bp transcript that contained insertions and deletions that differentiate it from both alphaA-crystallin and HSP-27. The deduced amino acid sequence of zebrafish alphaB-crystallin revealed that it lacked four residues in the C-terminus implicated in protein-protein interactions in other vertebrate species. In addition, the sequence contained two substitutions at sites implicated in phosphorylation in other vertebrate species. Northern analysis and semi-quantitative RT-PCR indicate that zebrafish alphaB-crystallin is expressed at extremely low levels outside of the lens.

Chicken beta B1 crystallin: gene sequence and evidence for functional conservation of promoter activity between chicken and mouse.

The complete sequence was determined for the chicken beta B1-crystallin gene and 2.2 kbp of its 5' flanking region; the chicken gene was then compared to its rat ortholog. Although both have a 5' non-coding exon followed by 5 protein coding exons, the chicken gene is only 2.2 kbp while the rat gene is 13.6 kpb due to longer introns. The coding exons of the chicken beta B1-crystallin gene, like those of the rat and other beta-crystallin genes, each correspond to one of the four 'Greek key' motifs of the encoded protein. The only obvious similarity between the 5' flanking sequences of the chicken and rat beta B1-crystallin gene is associated with the TATA box. A CR1 repetitive element is present at positions -559 to -730 of the chicken beta B1-crystallin gene. In vivo footprinting using dimethyl sulfate/ligation mediated PCR showed that the PL-1 (-116/-102), PL-2 (-90/-76), OL-2 (-75/-68) and OL-1 (-125/-118) control elements identified previously (Roth et al. (1991) Mol. Cell. Biol. 11, 1488-1499) bind proteins within the chromatin of cultured embryonic chicken lens cells. Both -2448/+30 and -434/+30 promoter fragments from the chicken beta B1-crystallin gene directed lens-specific CAT gene expression in a copy number and position independent manner in transgenic mice. These data indicate that the structure and lens-specific expression of this gene are highly conserved although, like other crystallin genes, the 5' flanking sequences have diverged appreciably during evolution.

Protein-DNA interactions of the mouse alpha A-crystallin control regions. Differences between expressing and non-expressing cells.

Genomic footprinting, in vitro footprinting and mobility shift assays were used to investigate the molecular basis for expression of mouse alpha A-crystallin, a major structural protein of the transparent lens of vertebrates. The putative control region of the mouse alpha A-crystallin gene was footprinted by DNase I digestion in nuclear extracts, by dimethylsulfate treatment in cultured cells, and by micrococcal nuclease digestion in isolated nuclei. The resulting digestion patterns were compared between alpha TN4-1 lens cells, which express alpha A-crystallin, and L929 fibroblasts, which do not express alpha A-crystallin. Four regions of DNA were found occupied in both cell types. These included positions -111 to -97 (DE-1 region), positions -75 to -55 (alpha A-CRYBP1 region), positions -35 to -12 (TATA box and PE-1 region), and positions +23 to +43 (an AP-1 consensus sequence). The DNase I footprints of the DE-1 and alpha A-CRYBP1 regions, previously implicated as functional control elements, were substantially more pronounced using nuclear extract from the alpha TN4-1 cells than from the L929 fibroblasts, suggesting more stable protein binding with the former than with the latter. Numerous in vivo binding variations were noted between the two cell types in all four of the footprinted regions examined. Finally, two complexes (A and B) were formed specifically with nuclear extracts from the alpha TN4-1 cells and a synthetic deoxyoligonucleotide comprising the alpha A-CRYBP1 region. These data indicate that specific differences in protein-DNA interactions with putative control regions are associated with tissue-preferred expression of the mouse alpha A-crystallin gene.

Binding of tissue-specific forms of alpha A-CRYBP1 to their regulatory sequence in the mouse alpha A-crystallin-encoding gene: double-label immunoblotting of UV-crosslinked complexes.

The alpha A-CRYBP1 regulatory sequence (alpha A-CRYBP1RS), at nucleotides -66 to -57 of the mouse alpha A-crystallin-encoding gene (alpha A-CRY) promoter, is an important control element involved in the regulation of mouse alpha A-CRY expression. The gene encoding a protein (alpha A-CRYBP1) that specifically binds to the alpha A-CRYBP1RS sequence has been cloned from a cultured mouse lens cell line. In the present study, we have used an antibody (specific to the alpha A-CRYBP1 protein and made against a synthetic peptide) to directly identify UV-crosslinked protein-DNA complexes via a double-label immunoblotting technique. Multiple alpha A-CRYB1 antigenically related proteins interacted with alpha A-CRYBP1RS in nuclear extracts from both a cloned mouse lens cell line (alpha TN4-1) that expresses alpha A-CRY and a mouse fibroblast line (L929) that does not express the gene. Two sizes (50 kDa and 90 kDa) of proteins reacting with the alpha A-CRYBP1-specific Ab were detected in both cell lines and, in addition, a > 200-kDa protein reacting with the Ab was unique to the fibroblast line. Thus, alpha A-CRYBP1 antigenically related proteins interact with alpha A-CRYBP1RS regardless of alpha A-CRY expression. Moreover, differential processing of the alpha A-CRYBP1 protein and/or alternative splicing of the alpha A-CRY transcript may affect expression of alpha A-CRY.

Identification of negative-acting and protein-binding elements in the mouse alpha A-crystallin -1556/-1165 region.

The mouse alpha A-crystallin-encoding gene (alpha A-cry) is expressed in a highly lens-preferred manner. To date, it has been shown that this lens-preferred expression is controlled by four proximal positive-acting transcriptional regulatory elements: DE1 (-111/-97), alpha A-CRYBP1 (-66/-57), PE1/TATA (-35/-19) and PE2 (+24/+43). The present study extends our knowledge of mouse alpha A-cry transcriptional regulatory elements to the far upstream region of that gene by demonstrating that the -1556 to -1165 region contains negative-acting sequence elements which function in transfected lens cells derived from mouse, rabbit and chicken. This is the first negative-acting regulatory region identified in mouse alpha A-cry. The -1556 to -1165 region contains sequences similar to repressor/silencer elements identified in other genes, including those highly expressed in the lens, such as the delta 1-crystallin (delta 1-cry) and vimentin (vim) genes. The -1480 to -1401 region specifically interacts with nuclear proteins isolated from the alpha TN4-1 mouse lens cell line. Contained within this protein-binding region and positioned at -1453 to -1444 is a sequence (RS1) similar to the chicken delta 1-cry intron 3 repressor, and which competes for the formation of -1480 to -1401 DNA-protein complexes. Our findings suggest that lens nuclear proteins bind to the mouse alpha A-cry RS1 region. We demonstrate that the chicken delta 1-cry intron repressor binds similar nuclear proteins in chicken embryonic lens cells and mouse alpha TN4-1 lens cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and spatial analysis of metallothioneins expressed by the adult human lens.

PURPOSE: Metallothioneins (MTs) are a large family of proteins involved in multiple protective pathways including binding of toxic metals, free radical scavenging, and oxidative stress. Increased expression of the MT IIA gene in age-related cataractous relative to normal human lenses, suggesting a role for MTs in the maintenance of lens transparency, has previously been detected. The MT family consists of many closely related isoforms grouped into four classes (I-IV). As a first step toward defining the function of MTs in the lens, the range and expression patterns of those MT isoforms expressed by the adult human lens were established. METHODS: Normal human lenses were microdissected into epithelia and fiber cells. Primers specific for individual MT isoforms were designed. MT transcripts were monitored in whole lenses, epithelia, and fibers by RT-PCR and confirmed to be authentic by sequencing. MT protein levels were evaluated by immunoblotting and by immunostaining. RESULTS: Transcripts encoding MT classes I and II but not III or IV were detected in adult human lenses. In addition to MT IIA, five other MT transcripts were identified including IE, IF, IG, IH, and IL. MT IIA was detected almost exclusively in the lens epithelium, whereas the class I isoforms were detected at high levels in both lens epithelia and fibers. MT protein was detected almost exclusively in the lens epithelium. CONCLUSIONS: The present report establishes the spectrum of MTs expressed by the adult human lens, defines their spatial expression patterns in lens epithelia and fibers, and demonstrates that MT protein is abundantly present in the lens epithelium.

Expression of betaB(2)-crystallin mRNA and protein in retina, brain, and testis.

PURPOSE: To evaluate the expression of betaB(2)-crystallin mRNA and protein in rat, bovine, and human nonlens and nonocular tissues. METHODS: betaB(2)-crystallin mRNA levels were detected by RT-PCR. betaB(2)-crystallin protein was purified from rat and bovine tissues by FPLC chromatography. FPLC fractions were analyzed by immunoblotting. The identity of betaB(2)-crystallin protein, isolated from the retina, was confirmed by protein microsequencing. RESULTS: betaB(2)-crystallin transcript was detected in rat brain, rat testis, and human retina by RT-PCR. betaB(2)-crystallin transcript was not found in rat lung, heart, ovary, spleen, thymus, kidney, and liver or in human brain and testis. betaB(2)-crystallin protein was partially purified from and its identity confirmed in rat brain, rat testis, and bovine retina. The bovine retinal protein was further confirmed to be authentic betaB(2)-crystallin by protein microsequencing. CONCLUSIONS: These results establish that betaB(2)-crystallin mRNA and protein are expressed in tissues outside of the lens and outside of the eye including retina, brain, and testis. Extralenticular and extraocular expression of betaB(2)-crystallin, coupled with its participation in phosphorylation pathways, suggests that it has nonrefractive functions in these tissues.

Osteonectin/SPARC secreted by RPE and localized to the outer plexiform layer of the monkey retina.

PURPOSE: Osteonectin/SPARC is a secreted protein that has been implicated in ocular disease. Deletion of osteonectin/SPARC causes age-onset cataract in mice and the cataractous human lens has increased expression of osteonectin/SPARC. In this study, the expression and localization of osteonectin/SPARC in the monkey retina were determined as was secretion by cultured human retinal pigment epithelial (RPE) cells. METHODS: Adult Rhesus monkey eyes (Macaca mulatta) were dissected, and 5-mm macula and peripheral retina punches were obtained. Supernatants were collected from cultured human RPE cells. Subcellular fractionation of whole monkey retina was also performed. Osteonectin/SPARC expression and/or secretion was monitored by Northern and Western blot analyses, and localization was determined by immunocytochemistry. RESULTS: Outside of the retina osteonectin/SPARC mRNA is broadly expressed in many human tissues. Northern blot analysis shows that in the retina osteonectin/SPARC is expressed almost exclusively by the macular RPE/choroid. Western blot analysis revealed osteonectin/SPARC in both the macula and the peripheral neural retina but only in trace amounts in the RPE/choroid. In subcellular fractions of the whole retina, osteonectin/SPARC was detected, mainly in the soluble fraction but also in the membrane and nuclear fractions. Immunohistochemical analysis localized osteonectin/SPARC specifically to the outer plexiform layer. Western blot analysis of conditioned medium from human RPE cells cultured on porous substrates indicated that osteonectin/SPARC is secreted in large amounts from both the apical and basal sides of the RPE. CONCLUSIONS: Collectively these data provide evidence that osteonectin/SPARC is synthesized in the macular RPE, secreted, and subsequently transported to the outer plexiform layer. The expression pattern of osteonectin/SPARC in the subcellular retinal fractions is consistent with a soluble protein that is transported and internalized.

Differential display detects altered gene expression between cataractous and normal human lenses.

PURPOSE: To identify and analyze differentially genes expressed between lens epithelia dissected from age-related cataractous and noncataractous human lenses. METHODS: RNAs from 50 pooled cataractous and 25 pooled noncataractous epithelia were compared by reverse transcription-polymerase chain reaction differential display (RT-PCR-DD). Two differentially displayed bands were chosen for further study. These were reamplified, cloned, and sequenced. Expression of these genes was further evaluated in pooled and individual epithelia by RT-PCR with gene-specific primers. RESULTS: Significant differences in gene expression were detected between the cataractous and the noncataractous epithelia. Three mRNAs displayed higher levels and 12 mRNAs displayed lower levels of expression in the cataractous samples compared with that in the noncataractous samples. Of the mRNAs expressed at higher levels, one was identified as metallothionein IIa (METII). Of the mRNAs with decreased expression, one was identified as protein phosphatase 2A regulatory subunit (P2A-RS). Overexpression of METII and underexpression of P2A-RS were confirmed in pooled and individual epithelia. CONCLUSIONS: These results provide evidence that age-related cataract is associated with alterations in the expression of multiple epithelial genes including METII and P2A-RS. METII is a detoxification protein induced by oxidative stress, and P2A-RS is a mitotic suppressor involved in cell-cycle control. These results implicate these proteins and their associated functions in the maintenance of lens transparency.

Up-regulation of osteonectin/SPARC in age-related cataractous human lens epithelia.

PURPOSE: To characterize gene expression patterns between epithelia isolated from cataractous and normal human lenses. METHODS: Reverse transcriptase differential display was used to identify differential expression between cataractous and normal epithelia. RT-PCR was used to compare pooled and individual RNA samples. RESULTS: One transcript, up-regulated in cataractous as compared to normal epithelia, was identified as osteonectin which is also known as SPARC (secreted acidic protein rich in cysteines). RT-PCR confirmed over-expression of this RNA. High levels of osteonectin mRNA were also detected in six individual epithelia dissected from cataractous lenses. CONCLUSIONS: The present study provides evidence for up-regulation of osteonectin in human age-related cataract and suggests that osteonectin, a protein involved in cell-cycle control, extracellular matrix and Ca++ binding, plays an important role in human lens homeostasis and may be involved in processes leading to lens opacity.

Quantitation of PAX6 and PAX6(5a) transcript levels in adult human lens, cornea, and monkey retina.

PURPOSE: PAX6 is a critical regulator of the developing lens, other ocular tissues, central nervous system, and pancreas. There are two alternatively spliced forms of the protein, PAX6 and PAX6(5a), that may have different regulatory functions. This study was designed to determine the amounts of PAX6 and PAX6(5a) transcripts present in adult human lens epithelium and fibers, human cornea and monkey retina. METHODS: PAX6 and PAX6(5a) transcript levels were monitored in microdissected lens epithelia, lens fibers, whole lens, cornea, and retina by competitive RT-PCR. The levels of TBP/TFIID were examined in adult human lens epithelium and fibers as control. RESULTS: PAX6 and PAX6(5a) were expressed at equal levels in lens epithelium and fibers. Ninety-five times more PAX6 transcripts were detected in the epithelial cells than in the fibers. Adult human cornea and monkey retina expressed less PAX6/PAX6(5a) than lens epithelium but more than lens fibers. Correspondingly, 40 fold higher levels of TBP transcripts were detected in lens epithelium than fibers, suggesting reduced overall expression of transcription factors in the adult lens fibers. CONCLUSIONS: The presence of PAX6 and PAX6(5a) messages and proteins in adult lens epithelium suggest functions for both forms of PAX6 in the growth and maintenance of the adult human lens. The reduced levels of both forms of PAX6 in the lens fibers suggest down regulation of this gene during differentiation of epithelia into fibers. The lower level of TBP expression in lens fibers also suggests reduced transcriptional competence of adult lens fibers.

Increased expression of osteonectin/SPARC mRNA and protein in age-related human cataracts and spatial expression in the normal human lens.

PURPOSE: We have previously reported increased levels of Osteonectin/SPARC transcript in age-related cataractous compared to normal human lenses. The purpose of the present study was to evaluate the corresponding levels of osteonectin/SPARC protein in age-related cataractous relative to normal lenses and to evaluate the levels of osteonectin/SPARC transcript in specific types of age-related human cataracts. The spatial expression of osteonectin/SPARC was also evaluated in normal human lenses. METHODS: Specific types of age-related cataracts were collected and graded. Normal human lenses were microdissected into epithelia and fibers. Osteonectin/SPARC protein levels were monitored by Western immunoblotting, and transcript levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Osteonectin/SPARC expression patterns were examined by RT-PCR and by immunostaining. RESULTS: Higher levels of osteonectin/SPARC protein were detected in age-related cataractous relative to normal human lenses. Increased levels of osteonectin/SPARC transcript were also detected in posterior-subcapsular and nuclear cataractous lenses relative to normal lenses. Osteonectin/SPARC transcripts were detected in the lens epithelium but not fibers. Osteonectin/SPARC protein levels were highest in the peripheral lens epithelium. CONCLUSIONS: Consistent with our previous studies on osteonectin/SPARC mRNA levels, osteonectin/SPARC protein levels were also elevated in cataractous compared to normal human lenses. Increased levels of osteonectin/SPARC mRNA were also found in nuclear and posterior-subcapsular cataracts relative to normal lenses. Osteonectin/SPARC expression is confined to the lens epithelium, and osteonectin/SPARC levels are highest in the peripheral lens epithelium.

Recruitment of enzymes and stress proteins as lens crystallins.

The major water-soluble proteins--or crystallins--of the eye lens are either identical to or derived from proteins with non-refractive functions in numerous tissues. In general, the recruitment of crystallins has come from metabolic enzymes (usually with detoxification functions) or stress proteins. Some crystallins have been recruited without duplication of the original gene (i.e., lactate dehydrogenase B and alpha-enolase), while others have incurred one (i.e., argininosuccinate lyase and a small heat shock protein) or several (i.e., glutathione S-transferase) gene duplications. Enzyme (or stress protein)-crystallins often maintain their non-refractive function in the lens and/or other tissues as well as their refractive role, a process we call gene sharing. alpha-Crystallin/small heat shock protein/molecular chaperone is of special interest since it is the major crystallin of humans. There are two alpha-crystallin genes (alpha A and alpha B), with alpha B retaining the full functions of a small heat shock protein. Here we describe recent evidence indicating that alpha A and alpha B have kinase activity, which would make them members of the enzyme-crystallins. We also describe various regulatory elements of the mouse alpha-crystallin genes responsible for their expression in the lens and, for alpha B, in skeletal muscle. Delineating the control elements for gene expression of these multifunctional protective proteins provides the foundations for their eventual use in gene therapy. Finally, comparison of the mouse and chicken alpha A-crystallin genes reveals similarities and differences in their functional cis-acting elements, indicative of evolution at the level of gene regulation.

Phosphorylations of alpha A- and alpha B-crystallin.

In addition to being refractive proteins in the vertebrate lens, the two alpha-crystallin polypeptides (alpha A and alpha B) are also molecular chaperones that can protect proteins from thermal aggregation. The alpha B-crystallin polypeptide, a functional member of the small heat shock family, is expressed in many tissues in a developmentally regulated fashion, is stress-inducible, and is overexpressed in many degenerative diseases and some tumors indicating that it plays multiple roles. One possible clue to alpha-crystallin functions is the fact that both polypeptides are phosphorylated on serine residues by cAMP-dependent and cAMP-independent mechanisms. The cAMP-independent pathway is an autophosphorylation that has been demonstrated in vitro, depends on magnesium and requires cleavage of ATP. Disaggregation of alpha A-, but not alpha B-crystallin into tetramers results in an appreciable increase in autophosphorylation activity, reminiscent of other heat shock proteins, and suggests the possibility that changes in the aggregation state of alpha A-crystallin are involved in yet undiscovered signal transduction pathways. The alpha-crystallin polypeptides differ with respect to their abilities to undergo cAMP-dependent phosphorylation, with preference given to the alpha B-crystallin chain. These differences and complexities in alpha-crystallin phosphorylations, coupled with the differences in expression patterns of the two alpha-crystallin polypeptides, are consistent with the idea that each polypeptide has distinctive structural and metabolic roles.

Gene structure, localization and role in oxidative stress of methionine sulfoxide reductase A (MSRA) in the monkey retina.

MSRA (EC 1.8.4.6) is a member of the methionine sulfoxide reductase family that can reduce methionine sulfoxide (MetO) in proteins. This repair function has been shown to protect cells against oxidative damage. In this study we have assembled the complete gene structure of msrA and identified the presence of two distinct putative promoters that generate three different transcripts. These transcripts were cloned by 5'RACE and code for three MSRA isoforms with different N-termini. The different forms of MSRA target to distinct intracellular regions. The main MSRA transcript (msrA1) had been previously shown to target the mitochondria. MsrA2 and 3 originate from a second promoter and target the cytosol and nuclei. In the monkey retina msrA message was detected mainly in the macular RPE-choroid region while its activity was measured mainly in the soluble fractions of fractionated neural retina and RPE-choroid. The MSRA protein is found throughout the retina but is especially abundant at the photoreceptor synapses, ganglion and Müller cells. Interestingly, MSRA was not detected in the mitochondria of the photoreceptor inner segments. The RPE in the peripheral retina shows very low levels of expression but the RPE in the macular region is strongly labeled. Targeted silencing of msrA message rendered cultured RPE cells more sensitive to oxidative damage suggesting a role for MSRA in RPE protection against oxidative stress. Collectively these data suggest MSRA may play an important role in protecting macular RPE from oxidative damage.

Alpha-crystallin/small heat shock protein has autokinase activity.

The alpha-crystallins (alpha A and alpha B) are major water-soluble proteins of the transparent eye lens that are expressed in a variety of tissues and can function as molecular chaperones. alpha B-crystallin is also a small heat shock protein associated with numerous degenerative diseases and abnormal growth patterns. Previous experiments have shown that alpha A-and alpha B-crystallin are phosphorylated on specific serine residues by a cAMP-dependent pathway. Here we provide evidence that either total bovine alpha-crystallin or its isolated polypeptides can autophosphorylate serine by a cAMP-independent mechanism in the presence of Mg2+ and [gamma-32P]ATP; the autophosphorylated products isoelectrically focus with the authentic phosphorylated forms of the alpha-crystallin polypeptides. Thus, the alpha A- and alpha B-crystallin/small heat shock protein polypeptides are enzyme-crystallins which may be involved in metabolic pathways important for the development, maintenance, or pathology of the lens and other tissues.

Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation.

The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP. We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123. The mutations were made in wild-type crp as well as a cAMP-independent crp, crp*. The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon. These results provide genetic evidence to support that E72 and R82 are essential and S83 and R123 are important in the activation of CRP by cAMP.

Evidence for gelsolin as a corneal crystallin in zebrafish.

We have shown that gelsolin is one of the most prevalent water-soluble proteins in the transparent cornea of zebrafish. There are also significant amounts of actin. In contrast to actin, gelsolin is barely detectable in other eye tissues (iris, lens, and remaining eye) of the zebrafish. Gelsolin cDNA hybridized intensely in Northern blots to RNA from the cornea but not from the lens, brain, or headless body. The deduced zebrafish gelsolin is approximately 60% identical to mammalian cytosolic gelsolin and has the characteristic six segmental repeats as well as the binding sites for actin, calcium, and phosphatidylinositides. In situ hybridization tests showed that gelsolin mRNA is concentrated in the zebrafish corneal epithelium. The zebrafish corneal epithelium stains very weakly with rhodamine-phalloidin, indicating little F-actin in the cytoplasm. In contrast, the mouse corneal epithelium contains relatively little gelsolin and stains intensely with rhodamine-phalloidin, as does the zebrafish extraocular muscle. We propose, by analogy with the diverse crystallins of the eye lens and with the putative enzyme-crystallins (aldehyde dehydrogenase class 3 and other enzymes) of the mammalian cornea, that gelsolin and actin-gelsolin complexes act as water-soluble crystallins in the zebrafish cornea and contribute to its optical properties.

Murine transcription factor alpha A-crystallin binding protein I. Complete sequence, gene structure, expression, and functional inhibition via antisense RNA.

alpha A-crystallin binding protein I (alpha A-CRYBP1) is a ubiquitously expressed DNA binding protein that was previously identified by its ability to interact with a functionally important sequence in the mouse alpha A-crystallin gene promoter. Here, we have cloned a single copy gene with 10 exons spanning greater than 70 kb of genomic DNA that encodes alpha A-CRYBP1. The mouse alpha A-CRYBP1 gene specifies a 2,688-amino acid protein with 72% amino acid identity to its human homologue, PRDII-BF1. Both the human and the mouse proteins contain two sets of consensus C2H2 zinc fingers at each end as well a central nonconsensus zinc finger. The alpha A-CRYBP1 gene produces a 9.5-kb transcript in 11 different tissues as well as a testis-specific, 7.7-kb transcript. alpha A-CRYBP1 cDNA clones were isolated from adult mouse brain and testis as well as from cell lines derived from mouse lens (alpha TN4-1) and muscle (C2C12). A single clone isolated from the muscle C2C12 library contains an additional exon near the 5'-end that would prevent production of a functional protein if the normal translation start site were utilized; however, there is another potential initiation codon located downstream that is in frame with the rest of the coding region. In addition, we identified multiple cDNAs from the testis in which the final intron is still present. Finally, we used an antisense expression construct derived from an alpha A-CRYBP1 cDNA clone to provide the first functional evidence that alpha A-CRYBP1 regulates gene expression. When introduced into the alpha TN4-1 mouse lens cell line, the antisense construct significantly inhibited expression from a heterologous promoter that utilized the alpha A-CRYBP1 binding site as an enhancer.