Loss of Octarepeats in two processed prion pseudogenes in the red squirrel, Sciurus vulgaris.

The N-terminal region of the mammalian prion protein (PrP) contains an 'octapeptide' repeat which is involved in copper binding. This eight- or nine-residue peptide is repeated four to seven times, depending on the species, and polymorphisms in repeat number do occur. Alleles with three repeats are very rare in humans and goats, and deduced PrP sequences with two repeats have only been reported in two lemur species and in the red squirrel, Sciurus vulgaris. We here describe that the red squirrel two-repeat PrP sequence actually represents a retroposed pseudogene, and that an additional and older processed pseudogene with three repeats also occurs in this species as well as in ground squirrels. We argue that repeat numbers may tend to contract rather than expand in prion retropseudogenes, and that functional prion genes with two repeats may not be viable.

Why proteins without an alpha-crystallin domain should not be included in the human small heat shock protein family HSPB.

The presence of an alpha-crystallin domain documents the evolutionary relatedness of the ubiquitous family of small heat shock proteins. Sequence and three-dimensional structure provide no evidence for the presence of such a domain in HSPC034, recently proposed as the 11th member of the human HSPB family. Also, phylogenetic analyses detect no relationship between HSPC034 and the human HSPB1-10 sequences. Arguments are provided as to why inclusion in the HSPB family of proteins like HSPC034, which resemble small heat shock proteins in being heat-inducible and having chaperone-like properties and a low monomeric mass, but are evolutionarily unrelated, is misleading and confusing.

The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio.

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 degrees C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or alphaB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.

Evolution of closely linked gene pairs in vertebrate genomes.

The orientation of closely linked genes in mammalian genomes is not random: there are more head-to-head (h2h) gene pairs than expected. To understand the origin of this enrichment in h2h gene pairs, we have analyzed the phylogenetic distribution of gene pairs separated by less than 600 bp of intergenic DNA (gene duos). We show here that a lack of head-to-tail (h2t) gene duos is an even more distinctive characteristic of mammalian genomes, with the platypus genome as the only exception. In nonmammalian vertebrate and in nonvertebrate genomes, the frequency of h2h, h2t, and tail-to-tail (t2t) gene duos is close to random. In tetrapod genomes, the h2t and t2t gene duos are more likely to be part of a larger gene cluster of closely spaced genes than h2h gene duos; in fish and urochordate genomes, the reverse is seen. In human and mouse tissues, the expression profiles of gene duos were skewed toward positive coexpression, irrespective of orientation. The organization of orthologs of both members of about 40% of the human gene duos could be traced in other species, enabling a prediction of the organization at the branch points of gnathostomes, tetrapods, amniotes, and euarchontoglires. The accumulation of h2h gene duos started in tetrapods, whereas that of h2t and t2t gene duos only started in amniotes. The apparent lack of evolutionary conservation of h2t and t2t gene duos relative to that of h2h gene duos is thus a result of their relatively late origin in the lineage leading to mammals; we show that once they are formed h2t and t2t gene duos are as stable as h2h gene duos.

Molecular phylogeny and divergence times of Malagasy tenrecs: influence of data partitioning and taxon sampling on dating analyses.

BACKGROUND: Malagasy tenrecs belong to the Afrotherian clade of placental mammals and comprise three subfamilies divided in eight genera (Tenrecinae: Tenrec, Echinops, Setifer and Hemicentetes; Oryzorictinae: Oryzorictes, Limnogale and Microgale; Geogalinae:Geogale). The diversity of their morphology and incomplete taxon sampling made it difficult until now to resolve phylogenies based on either morphology or molecular data for this group. Therefore, in order to delineate the evolutionary history of this family, phylogenetic and dating analyses were performed on a four nuclear genes dataset (ADRA2B, AR, GHR and vWF) including all Malagasy tenrec genera. Moreover, the influence of both taxon sampling and data partitioning on the accuracy of the estimated ages were assessed. RESULTS: Within Afrotheria the vast majority of the nodes received a high support, including the grouping of hyrax with sea cow and the monophyly of both Afroinsectivora (Macroscelidea + Afrosoricida) and Afroinsectiphillia (Tubulidentata + Afroinsectivora). Strongly supported relationships were also recovered among all tenrec genera, allowing us to firmly establish the grouping of Geogale with Oryzorictinae, and to confirm the previously hypothesized nesting of Limnogale within the genus Microgale. The timeline of Malagasy tenrec diversification does not reflect a fast adaptive radiation after the arrival on Madagascar, indicating that morphological specializations have appeared over the whole evolutionary history of the family, and not just in a short period after colonization. In our analysis, age estimates at the root of a clade became older with increased taxon sampling of that clade. Moreover an augmentation of data partitions resulted in older age estimates as well, whereas standard deviations increased when more extreme partition schemes were used. CONCLUSION: Our results provide as yet the best resolved gene tree comprising all Malagasy tenrec genera, and may lead to a revision of tenrec taxonomy. A timeframe of tenrec evolution built on the basis of this solid phylogenetic framework showed that morphological specializations of the tenrecs may have been affected by environmental changes caused by climatic and/or subsequent colonization events. Analyses including various taxon sampling and data partitions allow us to point out some possible pitfalls that may lead to biased results in molecular dating; however, further analyses are needed to corroborate these observations.

Tissue transglutaminase catalyzes the deamidation of glutamines in lens betaB(2)- and betaB(3)-crystallins.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including alphaB-crystallin and several beta-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked betaB(2)- and betaA(3)-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of betaB(2)- and betaB(3)-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the beta-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.

Arrival and diversification of caviomorph rodents and platyrrhine primates in South America.

Platyrrhine primates and caviomorph rodents are clades of mammals that colonized South America during its period of isolation from the other continents, between 100 and 3 million years ago (Mya). Until now, no molecular study investigated the timing of the South American colonization by these two lineages with the same molecular data set. Using sequences from three nuclear genes (ADRA2B, vWF, and IRBP, both separate and combined) from 60 species, and eight fossil calibration constraints, we estimated the times of origin and diversification of platyrrhines and caviomorphs via a Bayesian relaxed molecular clock approach. To account for the possible effect of an accelerated rate of evolution of the IRBP gene along the branch leading to the anthropoids, we performed the datings with and without IRBP (3768 sites and 2469 sites, respectively). The time window for the colonization of South America by primates and by rodents is demarcated by the dates of origin (upper bound) and radiation (lower bound) of platyrrhines and caviomorphs. According to this approach, platyrrhine primates colonized South America between 37.0 +/- 3.0 Mya (or 38.9 +/- 4.0 Mya without IRBP) and 16.8 +/- 2.3 (or 20.1 +/- 3.3) Mya, and caviomorph rodents between 45.4 +/- 4.1 (or 43.7 +/- 4.8) Mya and 36.7 +/- 3.7 (or 35.8 +/- 4.3) Mya. Considering both the fossil record and these molecular datings, the favored scenarios are a trans-Atlantic migration of primates from Africa at the end of the Eocene or beginning of the Oligocene, and a colonization of South America by rodents during the Middle or Late Eocene. Based on our nuclear DNA data, we cannot rule out the possibility of a concomitant arrival of primates and rodents in South America. The caviomorphs radiated soon after their arrival, before the Oligocene glaciations, and these early caviomorph lineages persisted until the present. By contrast, few platyrrhine fossils are known in the Oligocene, and the present-day taxa are the result of a quite recent, Early Miocene diversification.

Site-specific transamidation and deamidation of the small heat-shock protein Hsp20 by tissue transglutaminase.

Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q31 and the C-terminal K162 are involved in inter- and intramolecular crosslinking (transamidation). Q31 is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q66, which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.

The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians.

Morphological data supports monotremes as the sister group of Theria (extant marsupials + eutherians), but phylogenetic analyses of 12 mitochondrial protein-coding genes have strongly supported the grouping of monotremes with marsupials: the Marsupionta hypothesis. Various nuclear genes tend to support Theria, but a comprehensive study of long concatenated sequences and broad taxon sampling is lacking. We therefore determined sequences from six nuclear genes and obtained additional sequences from the databases to create two large and independent nuclear data sets. One (data set I) emphasized taxon sampling and comprised five genes, with a concatenated length of 2,793 bp, from 21 species (two monotremes, six marsupials, nine placentals, and four outgroups). The other (data set II) emphasized gene sampling and comprised eight genes and three proteins, with a concatenated length of 10,773 bp or 3,669 amino acids, from five taxa (a monotreme, a marsupial, a rodent, human, and chicken). Both data sets were analyzed by parsimony, minimum evolution, maximum likelihood, and Bayesian methods using various models and data partitions. Data set I gave bootstrap support values for Theria between 55% and 100%, while support for Marsupionta was at most 12.3%. Taking base compositional bias into account generally increased the support for Theria. Data set II exclusively supported Theria, with the highest possible values and significantly rejected Marsupionta. Independent phylogenetic evidence in support of Theria was obtained from two single amino acid deletions and one insertion, while no supporting insertions and deletions were found for Marsupionta. On the basis of our data sets, the time of divergence between Monotremata and Theria was estimated at 231-217 MYA and between Marsupialia and Eutheria at 193-186 MYA. The morphological evidence for a basal position of Monotremata, well separated from Theria, is thus fully supported by the available molecular data from nuclear genes.

Asynchronous colonization of Madagascar by the four endemic clades of primates, tenrecs, carnivores, and rodents as inferred from nuclear genes.

Madagascar harbors four large adaptive radiations of endemic terrestrial mammals: lemurs, tenrecs, carnivorans, and rodents. These rank among the most spectacular examples of evolutionary diversification, but their monophyly and origins are debated. The lack of Tertiary fossils from Madagascar leaves molecular studies as most promising to solve these controversies. We provide a simultaneous reconstruction of phylogeny and age of the four radiations based on a 3.5-kb data set from three nuclear genes (ADRA2B, vWF, and AR). The analysis supports each as a monophyletic clade, sister to African taxa, and thereby identifies four events of colonization out of Africa. To infer the time windows for colonization, we take into account both the divergence from the closest non-insular sister group and the initial intra-insular radiation, which is a novel but conservative approach in studies of the colonization history of Madagascar. We estimate that lemurs colonized Madagascar between 60 million years ago (Mya) (split from lorises) and 50 Mya (lemur radiation) (70-41 Mya taking 95% credibility intervals into account), tenrecs between 42 and 25 Mya (50-20 Mya), carnivorans between 26 and 19 Mya (33-14 Mya), and rodents between 24 and 20 Mya (30-15 Mya). These datings suggest at least two asynchronous colonization events: by lemurs in the Late Cretaceous-Middle Eocene, and by carnivorans and rodents in the Early Oligocene-Early Miocene. The colonization by tenrecs may have taken place simultaneously with either of these two events, or in a third event in the Late Eocene-Oligocene. Colonization by at least lemurs, rodents, and carnivorans appears to have occurred by overseas rafting rather than via a land bridge hypothesized to have existed between 45 and 26 Mya, but the second scenario cannot be ruled out if credibility intervals are taken into account.

Nuclear import of {alpha}B-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G.

Phosphorylation modulates the functioning of alphaB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of alphaB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of alphaB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of alphaB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated alphaB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing alphaB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of alphaB-crystallin R120G.

Evolutionary diversity of vertebrate small heat shock proteins.

All vertebrates express multiple small heat shock proteins (sHsps), which are important components of the cellular chaperoning machinery and display a spectacular diversity of functions. This ranges from remodeling the cytoskeleton and inhibiting apoptosis to serving as structural proteins in eye lens and sperm tail. Most information is available for the 10 known mammalian sHsps, formally named HspB1-B10. Only three of them (Hsp27/B1, alphaA-crystallin/B4, alphaB-crystallin/B5) have been reported from nonmammalian vertebrates, while an apparent paralog, Hsp30/B11, is found in frogs and teleost fish. To reconstruct the evolutionary diversification of the sHsps in vertebrates, we searched for additional sHsps in genome, protein, and EST databases and sequenced some avian and amphibian sHsps (HspB2, Hsp30/B11). The urochordate Ciona intestinalis was included in the search, as the outgroup of vertebrates. Orthologs of seven mammalian sHsps were now found in other vertebrate classes. Two novel sHsps, named HspB11 and HspB12, were recognized in birds, and four novel sHsps, named HspB12-B15, in teleost fish. Secondary structure predictions of orthologous sHsps from different vertebrate classes indicate conservation of the beta-sandwich structure of the functionally important C-terminal "alpha-crystallin domain," while the N-terminal domains generally have alpha-helical structures, despite their pronounced sequence variation. The constructed chordate sHsp tree is supported by shared introns, indels, and diagnostic sequences. The tree distinguishes putative orthologous and paralogous relationships, which will facilitate the functional and structural comparison of the various vertebrate sHsps. The 15 recognized paralogous vertebrate sHsps reflect the period of extensive gene duplications early in vertebrate evolution. Eleven of these sHsps are grouped in a clade that might be specific for chordates. It is inferred that at least 13 intron insertions have occurred during the evolution of chordate sHsp genes, while a single ancient intron is maintained in some lineages, in line with the general trend of massive intron gain before or during early vertebrate radiation. Interesting is the occurrence of several head-to-head located pairs of chordate sHsp genes.

Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1.

Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappé et al., Biochim. Biophys. Acta 1520, 1-6, 2001). Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues. Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR. Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining. We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens. To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins. TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9. Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells. The possible functional significance of this interaction is discussed.

Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.

Transglutaminase catalyzes differential crosslinking of small heat shock proteins and amyloid-beta.

Crosslinking of proteins by tissue transglutaminase (tTG) is enhanced in amyloid (Abeta) deposits characteristic of Alzheimer's disease and sporadic inclusion body myositis. Small heat shock proteins (sHsps) also occur in amyloid deposits. We here report the substrate characteristics for tTG of six sHsps. Hsp27, Hsp20 and HspB8 are both lysine- and glutamine-donors, alphaB-crystallin only is a lysine-donor, HspB2 a glutamine-donor, and HspB3 no substrate at all. Close interaction of proteins stimulates crosslinking efficiency as crosslinking between different sHsps only takes place within the same heteromeric complex. We also observed that alphaB-crystallin, Hsp27 and Hsp20 associate with Abeta in vitro, and can be readily crosslinked by tTG.

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity.

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.

Sequence and functional conservation of the intergenic region between the head-to-head genes encoding the small heat shock proteins alphaB-crystallin and HspB2 in the mammalian lineage.

An unexpected feature of the large mammalian genome is the frequent occurrence of closely linked head-to-head gene pairs. Close apposition of such gene pairs has been suggested to be due to sharing of regulatory elements. We show here that the head-to-head gene pair encoding two small heat shock proteins, alphaB-crystallin and HspB2, is closely linked in all major mammalian clades, suggesting that this close linkage is of selective advantage. Yet alphaB-crystallin is abundantly expressed in lens and muscle and in response to a heat shock, while HspB2 is abundant only in muscle and not upregulated by a heat shock. The intergenic distance between the genes for these two proteins in mammals ranges from 645 bp (platypus) to 1069 bp (opossum), with an average of about 900 bp; in chicken the distance was the same as in duck (1.6 kb). Phylogenetic footprinting and sequence alignment identified a number of conserved sequence elements close to the HspB2 promoter and two farther upstream. All known regulatory elements of the mouse alphaB-crystallin promoter are conserved, except in platypus and birds. The lens-specific region 1 (LSR1) and the heat shock elements (HSEs) lack in birds; in platypus the LSR1 is reduced to a Pax-6 site, while the Pax-6 site in LSR2 and a HSE are absent. Most likely the primordial mammalian alphaB-crystallin promoter had two LSRs and two HSEs. In transfection experiments the platypus alphaB-crystallin promoter retained heat shock responsiveness and lens expression. It also directed lens expression in Xenopus laevis transgenes, as did the HspB2 promoter of rat or blind mole rat. Deletion of the middle of the intergenic region including the upstream enhancer affected the activity of both the rat alphaB-crystallin and the HspB2 promoters, suggesting sharing of the enhancer region by the two promoters.

Molecules consolidate the placental mammal tree.

Deciphering relationships among the orders of placental mammals remains an important problem in evolutionary biology and has implications for understanding patterns of morphological character evolution, reconstructing the ancestral placental genome, and evaluating the role of plate tectonics and dispersal in the biogeographic history of this group. Until recently, both molecular and morphological studies provided only a limited and questionable resolution of placental relationships. Studies based on larger and more diverse molecular datasets, and using an array of methodological approaches, are now converging on a stable tree topology with four major groups of placental mammals. The emerging tree has revealed numerous instances of convergent evolution and suggests a role for plate tectonics in the early evolutionary history of placental mammals. The reconstruction of mammalian phylogeny illustrates both the pitfalls and the powers of molecular systematics.

Influence of hormones and growth factors on lens protein composition: the effect of dexamethasone and PDGF-AA.

PURPOSE: To investigate the effect of hormones and ocular growth factors on the expression of alpha-, beta-, and gamma-crystallins in rat lens epithelial and fiber cells. METHODS: PDGF-AA, EGF, NGF, M-CSF, BMP-2, BMP-4, dexamethasone, and estrogen were tested for their ability to alter the spectrum of crystallins in explanted newborn rat lens epithelial cells or in vitro differentiating newborn rat lens fiber cells. The accumulation of alphaA-, aB-, betaA3/1-, betaB2-, and gamma-crystallin was measured by western blot and dot blot analysis. The morphology of the rat lens explants after culture was examined by hematoxylin-eosin staining, while crystallins were localized by immunofluorescence. RESULTS: Only dexamethasone and PDGF-AA showed an effect on relative crystallin levels. In the presence of dexamethasone the amount of alphaB-crystallin was increased in lens epithelial cells, but dexamethasone did not affect the crystallin spectrum in fiber cells. In rat lens epithelial explants cultured with PDGF-AA an increase in beta- and gamma-crystallin expression was seen. The spectrum of beta- and gamma-crystallins synthesized differed from that present in lens fiber cells. The cells expressing beta- and gamma-crystallin after culture with PDGF-AA were scattered in the epithelial cell layer and retained an epithelial morphology. PDGF-AA did not change the spectrum of crystallins synthesized in lens fiber cells but did enhance the rate of fiber cell differentiation, in agreement with results of others. CONCLUSIONS: Both dexamethasone and PDGF-AA influence crystallin gene expression in cultured rat lens epithelial cells. Dexamethasone enhances the expression of alphaB-crystallin while culturing in the presence of PDGF-AA caused an increase in beta- as well as gamma-crystallin synthesis. Since at least the gamma-crystallin genes are known to be silenced in epithelial cells by DNA methylation, PDGF-AA may be able to induce one of the steps towards fiber cell differentiation in some epithelial cells.