Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.     

SST versus EST in gene recognition

The expressed sequence tag (EST) data provide a powerful tool for identification of transcribed DNA sequences. However, as EST are relatively short, many exons are poorly covered by EST, thus reducing the utility of EST data. Recently, signature sequence tag (SST) fingerprints were proposed as an alternative to EST fingerprints. Given a fingerprint set of probes, SST of a clone is a subset of probes from the fingerprint set that hybridize with the clone. We demonstrate that besides being a powerful technique for screening cDNA libraries, SST technology provides for very accurate gene predictions. Even with a small fingerprint set (600-800 probes), SST-based gene recognition outperforms many conventional and EST-based methods. The increase in the size of the fingerprint set to 1500 probes provides almost perfect gene recognition. Even more importantly, SST-based gene predictions miss very few exons and, therefore, provide an opportunity to bypass the cDNA sequencing step on the way from finished genomic sequence to mutation detection in gene-hunting projects. Because SST data can be obtained in a highly parallel and inexpensive way, SST technology has a potential of complementing EST technology for gene hunting.     

Finding subtle motifs by branching from sample strings

Many motif finding algorithms apply local search techniques to a set of seeds. For example, GibbsDNA (Lawrence et al. 1993, Science, 262, 208-214) applies Gibbs sampling to random seeds, and MEME (Bailey and Elkan, 1994, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology (ISMB-94), 28-36) applies the EM algorithm to selected sample strings, i.e. substrings of the sample. In the case of subtle motifs, recent benchmarking efforts show that both random seeds and selected sample strings may never get close to the globally optimal motif. We propose a new approach which searches motif space by branching from sample strings, and implement this idea in both pattern-based and profile-based settings. Our PatternBranching and ProfileBranching algorithms achieve favorable results relative to other motif finding algorithms. Availability: http://www-cse.ucsd.edu/groups/bioinformatics/software.html     

Reconstructing tumor genome architectures

Although cancer progression is often associated with genome rearrangements, little is known about the detailed genomic architecture of tumor genomes. The attempt to reconstruct the genomic organization of a tumor genome recently resulted in the development of the End Sequence Profiling (ESP) technique, and the application of this technique to human MCF7 tumor cells. We formulate the ESP Genome Reconstruction Problem, and develop an algorithm to solve this problem in the case of sparse ESP data. We apply our algorithm to analyze human MCF7 tumor cells, and obtain the first reconstruction of the putative architecture of human MCF7 tumor genome. Our results assist in the ongoing ESP analysis of MCF7 tumors by suggesting additional ESP experiments for the completion of a reliable reconstruction of the MCF7 tumor genome, and by focusing BAC re-sequencing efforts.     

Performance-Guarantee Gene Predictions via Spliced Alignment

An important and still unsolved problem in gene prediction is designing an algorithm that not only predicts genes but estimates the quality ofindividualpredictions as well. Since experimental biologists areinterested mainly in the reliability of individual predictions (rather than in the average reliability of an algorithm) we attempted to develop a gene recognition algorithm that guarantees a certain quality of predictions. We demonstrate here that the similarity level with a related protein is a reliable quality estimator for thespliced alignmentapproach to gene recognition. We also study the average performance of the spliced alignment algorithm for different targets on a complete set of human genomic sequences with known relatives and demonstrate that the average performance of the method remains high even for very distant targets. Using plant, fungal, and prokaryotic target proteins for recognition of human genes leads to accurate predictions with 95, 93, and 91% correlation coefficient, respectively. For target proteins with similarity score above 60%, not only the average correlation coefficient is very high (97% and up) but also the quality of individual predictions isguaranteedto be at least 82%. It indicates that for this level of similarity the worst case performance of the spliced alignment algorithm is better than the average case performance of many statistical gene recognition methods.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.