Developmentally regulated expression of specific tau sequences

Tau protein undergoes a shift in its molecular mass and its electrophoretic complexity during early postnatal development. We have sequenced a tau cDNA from an adult rat brain expression library and have found two inserted sequences. One of these inserts predicts a fourth repeated sequence homologous to the other three in the carboxyl end of tau that have the property of microtubule binding. Oligonucleotide probes directed against the insert hybridized only to tau mRNA at postnatal time points, even though tau is first expressed as early as embryonic day 13. A probe directed against the junction revealed expression of non-insert-containing tau mRNA from embryonic day 14 until postnatal day 8, after which time there was an abrupt decline in the expression of this immature form. Comparison of the developmentally expressed tau sequences with those sequences obtained directly from Alzheimer paired helical filaments revealed the presence of both the mature and the immature tau mRNA sequences.     

Developmentally regulated phosphoproteins associated with chromosome complexes in yeast

Previous studies on our laboratory have shown that nuclear DNA from Saccharomyces cerevisiae can be isolated in the form of fast-sedimenting chromosome complexes (FSCC). In cycling cells, three FSCC forms, denoted g1 and g2, can be distinguished by their characteristic sedimentation velocities and are found correspondingly in cells in G1, S and G2 of the cell cycle, respectively. A fourth form, denoted go, is found exclusively in stationary-phase and nitrogen-starved cells and defines the non-cycling state, Go. We used the differing sedimentation velocities of the FSCC of proliferating and non-cycling cells as a way to isolate and examine their associated proteins. We report here a two-dimensional polyacrylamide gel electrophoresis analysis of [35S]methionine and 32PO4-labelled proteins extracted from FSCC isolated from cycling cells (g1, r and g2, collectively denoted 'cycling FSCC'), and nutritionally arrested cells (go FSCC). Among the 120 35S-labelled FSCC-associated polypeptides detected, 25 were unique to go FSCC and 7 were unique to cycling FSCC. Among the 84 32P-labelled FSCC-associated polypeptides detected, 52 were unique to go FSCC and 7 were unique to cycling FSCC. Comigrating 35S and 32P-labelled polypeptides were matched in 34 of the 84 phosphorylated polypeptides, and 21 of these showed the same specificity of association to either cycling or go FSCC. This analysis demonstrates that there are major differences in the proteins associated with FSCC from cycling and nutritionally arrested cells, and indicates that a relationship exists between the growth state of the yeast cell, protein phosphorylation and chromosome-complex structure.     

Developmentally controlled telomere addition in wild-type and mutant paramecia.

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.     

Developmentally regulated conversion of mesenchyme to epithelium

Polarized epithelial cells perform many critical physiological functions in multicellular organisms. Recent embryological studies of the conversion of nonpolar mesenchymal cells to epithelium in the developing mouse kidney have provided vital information on the molecular mechanisms that initiate epithelial cell polarization. To become polar, the cells first attach to the basement membrane that is produced by the developing epithelial cells themselves. Of the basement membrane components, laminin has a key role in the development of epithelial cell polarity. Laminin is a multidomain glycoprotein composed of three subunits: A, B1, and B2. One binding site for epithelial cells is found in the carboxyl-terminal part of the A chain of laminin. Antibodies reacting with this part of laminin inhibit polarization of developing epithelial cells in organ cultures of embryonic kidneys. Expression studies also suggest that the A chain of laminin is important for epithelial cell polarization; the A chain appears when the cells begin to polarize, whereas B chains are expressed at an earlier stage of development. The studies of conversion of mesenchyme to epithelium suggest that morphogenesis can be controlled by differential expression of laminin chains.     

Developmentally regulated alterations in Polycomb repressive complex 1 proteins on the inactive X chromosome

Polycomb group (PcG) proteins belonging to the polycomb (Pc) repressive complexes 1 and 2 (PRC1 and PRC2) maintain homeotic gene silencing. In Drosophila, PRC2 methylates histone H3 on lysine 27, and this epigenetic mark facilitates recruitment of PRC1. Mouse PRC2 (mPRC2) has been implicated in X inactivation, as mPRC2 proteins transiently accumulate on the inactive X chromosome (Xi) at the onset of X inactivation to methylate histone H3 lysine 27 (H3-K27). In this study, we demonstrate that mPRC1 proteins localize to the Xi, and that different mPRC1 proteins accumulate on the Xi during initiation and maintenance of X inactivation in embryonic cells. The Xi accumulation of mPRC1 proteins requires Xist RNA and is not solely regulated by the presence of H3-K27 methylation, as not all cells that exhibit this epigenetic mark on the Xi show Xi enrichment of mPRC1 proteins. Our results implicate mPRC1 in X inactivation and suggest that the regulated assembly of PcG protein complexes on the Xi contributes to this multistep process.     

Developmentally regulated sphingolipid synthesis in African trypanosomes

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.     

Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus.

The hypotrichous ciliated protozoa undergo a massive genome rearrangement process after their sexual cycle. One frequent type of rearrangement is the removal of DNA sequences (internal eliminated sequences; IESs) from internal regions of DNA molecules. In this study, we characterized the removal of IESs in Euplotes crassus. Southern hybridization analyses combined with cytological observations indicated that IES removal is an early event in macronuclear development, occurring during the polytene chromosome stage and prior to the chromosome fragmentation process. The results are consistent with IES removal occurring via an intramolecular DNA breakage and rejoining process.     

Developmentally regulated DNA methylation in Dictyostelium discoideum

Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.     

Developmentally regulated sphingolipid degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.     

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.     

Developmentally regulated proteases in Allomyces arbuscula

A calcium-requiring neutral protease has been detected in the vegetative mycelia of Allomyces arbuscula. The half maximum activation of the enzyme required 0.7 mM and 2.8 mM Ca²⁺ in the crude and partially-purified preparation, respectively. Coinciding with differentiation of zoosporangia, there is a massive induction of another neutral protease which does not require Ca²⁺ for its activity and is of the serine type.     

Developmentally regulated telomere addition in Tetrahymena thermophila.

To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure. Two developmentally independent examples of one macronuclear telomere had a 5 base pair difference in the position of the junction between the telomeric repeats and the adjacent sequences. A telomere-adjacent sequence, in the form of a synthetic oligonucleotide, was unable to prime the addition of telomeric repeats in vitro. The implications of these results for the mechanisms underlying developmentally programmed chromosome fragmentation and telomere addition in Tetrahymena are discussed.     

Developmentally regulated expression of hemoglobin subunits in avascular tissues

We investigated the spatio-temporal profile of hemoglobin subunit expression in developing avascular tissues. Significant up-regulation of hemoglobin subunits was identified in microarray experiments comparing blastocyst inner cell masses with undifferentiated embryonic stem (ES) cells. Hemoglobin expression changes were confirmed using embryoid bodies (derived from in vitro differentiation of ES cells) to model very early development at pre-vascular stages of embryogenesis; i.e. prior to hematopoiesis. We also demonstrate, using RT-PCR, Western blotting and immunocytochemistry, expression of adult and fetal mouse hemoglobin subunits in the avascular ocular lens at various stages of development and maturation. Hemoglobin proteins were expressed in lens epithelial cells (cytoplasmic) and cortical lens fiber cells (nuclear and cell-surface-associated); however, a sensitive heme assay demonstrated negligible levels of heme in the developing lens postnatally. Hemoglobin expression was also observed in the developing eye in corneal endothelium and retinal ganglion cells. Gut sections showed, in addition to erythrocytes, hemoglobin protein staining in rare, individual villus epithelial cells. These results suggest a paradigm shift: hemoglobin subunits are expressed in the avascular lens and cornea and in pre-hematopoietic embryos. It is likely, therefore, that hemoglobin subunits have novel developmental roles; the absence of the heme group from the lens would indicate that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of hemoglobin subunits in the perinuclear region during lens fiber cell differentiation, when denucleation is taking place, may indicate involvement in the apoptosis-like signaling processes occurring in differentiating lens fiber cells.     

Developmentally regulated compartmentalization of adenylate cyclase in Dictyostelium discoideum

Adenylate cyclase of aggregation phase Dictyostelium discoideum is activated by extracellular adenosine 3', 5'-cyclic monophosphate (cAMP), and the cAMP synthesized is secreted. The distribution of the enzyme was determined in sucrose gradients loaded with whole cell lysates. Cell lysates prepared after 4.5 hr of starvation revealed membranes containing adenylate cyclase at 44% and 33% sucrose. The activity of the latter peak was detected in the presence of the detergent (CHAPS), 3-(3-cholamidopropyl) dimethylammonio-3-propanesulfonate, which inhibited the activity of the former to some extent. Adenylate cyclase activity of the 2 peaks differed with respect to solubility in CHAPS and their kinetics. The 44% sucrose region of the gradient contained the bulk of the plasma membranes, as judged by a cell surface glycoprotein marker (contact site A). The 33% peak is composed of small vesicular structures, as determined by electron microscopy. The distribution of adenylate cyclase activity detected in sucrose gradients shifted from the 33% to the 44% sucrose peak during development. In addition, the 44% peak became increasingly resistant to the inhibitory effect of CHAPS. Both changes were accelerated by extracellular cAMP, but only the latter was abolished when the production of endogenous cAMP was inhibited by caffeine. Pulsing cells with cAMP overcame the inhibitory effect of caffeine.     

In situ hybridization of two cloned chromosome 7 sequences tightly linked to the cystic fibrosis locus

Two DNA sequences closely linked to the cystic fibrosis locus have been sublocalized to 7q31.3----q32 by in situ hybridization. These findings are consistent with previously published maps of that region of human chromosome 7. The cystic fibrosis locus therefore maps to the 7q31.3----q32 region, a more distal location that had been inferred from previous data.