Genome-wide evolutionary conservation of N-glycosylation sites

Although posttranslational protein modifications are generally thought to perform important cellular functions, recent studies showed that a large fraction of phosphorylation sites are not evolutionarily conserved. Whether the same is true for other protein modifications, such as N-glycosylation is an open question. N-glycosylation is a form of cotranslational and posttranslational modification that occurs by enzymatic addition of a polysaccharide, or glycan, to an asparagine (N) residue of a protein. Examining a large set of experimentally determined mouse N-glycosylation sites, we find that the evolutionary rate of glycosylated asparagines is significantly lower than that of nonglycosylated asparagines of the same proteins. We further confirm that the conservation of glycosylated asparagines is accompanied by the conservation of the canonical motif sequence for glycosylation, suggesting that the above substitution rate difference is related to glycosylation. Interestingly, when solvent accessibility is considered, the substitution rate disparity between glycosylated and nonglycosylated asparagines is highly significant at solvent accessible sites but not at solvent inaccessible sites. Thus, although the solvent inaccessible glycosylation sites were experimentally identified, they are unlikely to be genuine or physiologically important. For solvent accessible asparagines, our analysis reveals a widespread and strong functional constraint on glycosylation, unlike what has been observed for phosphorylation sites in most studies, including our own analysis. Because the majority of N-glycosylation occurs at solvent accessible sites, our results show an overall functional importance for N-glycosylation.     

Inferring protein-protein interacting sites using residue conservation and evolutionary information

This paper proposes a novel method using protein residue conservation and evolution information, i.e., spatial sequence profile, sequence information entropy and evolution rate, to infer protein binding sites. Some predictors based on support vector machines (SVMs) algorithm are constructed to predict the role of surface residues in protein-protein interface. By combining protein residue characters, the prediction performance can be improved obviously. We then made use of the predicted labels of neighbor residues to improve the performance of the predictors. The efficiency and the effectiveness of our proposed approach are verified by its better prediction performance based on a non-redundant data set of heterodimers.     

Cloning of the human activin receptor cDNA reveals high evolutionary conservation.

The entire coding region of the human activin receptor was obtained from a human testis cDNA library. Analysis of the 1539 nucleotide (513 amino acid) sequence of the receptor reveals that there are only 83 nucleotide differences compared to the coding sequence of the mouse activin receptor. Similar to its ligands, the amino acid sequence of the activin receptor is highly conserved with only two conservative amino acid differences (Lys-39 and Val-92 in human versus Arg-39 and Ile-92 in the mouse). This high degree of conservation of the activin receptor illustrates a strong evolutionary selection and confirms that activin and its receptor play an important role in development.     

Nucleotide sequences and expression of cDNA clones for boar and bull transition protein 1 and its evolutionary conservation in mammals

During spermatogenesis, the nucleoproteins undergo several dramatic changes as the germinal cells differentiate to produce the mature sperm. With nuclear elongation and condensation, the histones are replaced by basic spermatidal transition proteins, which are themselves subsequently replaced by protamines. We have isolated cDNA clones for one of the transition proteins, namely for TP1, of bull and boar. It turned out that TP1 is a small, but very basic protein with 54 amino acids (21% arginine, 19% lysine) and is highly conserved during mammalian evolution at the nucleotide as well as at the amino-acid level. Gene expression is restricted to the mammalian testis, and the message first appears in round spermatids. Thus production of TP1 is an example of haploid gene expression in mammals. The size of the mRNA for TP1 was found to be identical in 11 different mammalian species at around 600 bp. Hybridization experiments were done with cDNAs from boar and bull, respectively. The positive results in all mammalian species give further evidence for the conservation of the TP1 gene during mammalian evolution and its functional importance in spermatid differentiation.     

Leader region of mdg1 Drosophila retrotransposon RNA contains 3''-end processing sites.

Transient expression of the mdg1 deletion mutants revealed sites of 3'-end processing in the leader region of the transcribed RNA. The efficiency of the processing is regulated in different types of cells. The sequences within the mdg1 body and the 3'-LTR are involved in its regulation. We have also shown, that one of the small open reading frames in the mdg1 leader region in principle might be translated.     

The rabbitCD1 and the evolutionary conservation of theCD1 gene family

A comparison of the genes encoding the CD1 leucocyte differentiation antigens in man and mouse shows important differences which prompted us to analyze theCD1 genes of the rabbit. We have found that the rabbit genome contains multipleCD1 loci. Upon cloning and sequencing, one of these loci was found to encode the known rabbit CD1-like antigen (R-Ta) and to be closely related to the humanCD1b gene, which is absent in the mouse, while a second rabbit gene is closely related to both the humanR3 and the mouseCD1 genes. The data reinforce the notion of the existence of two classes ofCD1 genes, one of which is conserved in all species, while the other, albeit also evolutionarily old, has been deleted in mice as well as in other rodents. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M26248 and M26249.     

Ecological preferences of neotropical cave bats in roost site selection and their implications for conservation

Bats frequently use caves as roosts due to higher environmental stability and protection. However, species-specific ecological and physiological requirements and conditions of roosts and their surroundings can influence species presence. Little is known on cave choice by bats in the Neotropics, a species- and cave-rich region. Understanding how bats, cave characteristics and the surrounding landscape are related with each other helps the management and conservation of bats and caves. Based on sampling 19 caves using both diurnal observations and captures, we tested (1) whether bat richness was positively related to cave temperature, humidity, size, stability, and heterogeneity of microhabitats, and the preservation and heterogeneity of the surrounding landscape in central Brazil; (2) whether cave and landscape characteristics influenced on species composition; (3) how species responded to cave and landscape variables; and (4) whether these relationships changed between seasons. Temperature was a limiting factor, whereas environmental stability, humidity, and structural attributes of caves favored the occurrence of more species. Larger caves, rich in microhabitats – but more stable than the external environment – supported more species, especially in the dry season. Landscape context did not influence species richness and composition in the caves, but the percentage of vegetation around caves was important for certain species. Our results highlight the influence of environmental variables in the process of cave selection by Neotropical bat species. Moreover, we emphasize the importance of cave protection for bats and provide useful information for decision-making in processes of environmental licensing.     

A long terminal repeat retrotransposon of fission yeast has strong preferences for specific sites of insertion

The successful dispersal of transposons depends on the critical balance between the fitness of the host and the ability of the transposon to insert into the host genome. One method transposons may use to avoid the disruption of coding sequences is to target integration into safe havens. We explored the interaction between the long terminal repeat retrotransposon Tf1 and the genome of the yeast Schizosaccharomyces pombe. Using techniques that were specifically designed to detect integration of Tf1 throughout the genome and to avoid bias in this detection, we generated 51 insertion events. Although 60.2% of the genome of S. pombe is coding sequence, all but one of the insertions occurred in intergenic regions. We also found that Tf1 was significantly more likely to insert into intergenic regions that included polymerase II promoters than into regions between convergent gene pairs. Interestingly, 8 of the 51 insertion sites were isolated multiple times from genetically independent cultures. This result suggests that specific sites in intergenic regions are targeted by Tf1. Perhaps the most surprising observation was that per kilobase of nonrepetitive sequence, Tf1 was significantly more likely to insert into chromosome 3 than into one of the other two chromosomes. This preference was found not to be due to differences in the distribution or composition of intergenic sequences within the three chromosomes.     

The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation

The base substitution fidelity of DNA polymerase-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) has been determined in vitro, for all 12 possible mispairs at 96 sites in a forward mutational target. Averaging all errors over all known detectable sites, pol-gamma is the most accurate enzyme, producing one error for every 10,000 bases polymerized. Pol-beta is much less accurate, with an error rate of 1/1,500, while pol-alpha has an intermediate accuracy of 1/4,000. The relative differences in fidelity between the DNA polymerases are strongly influenced by the nature of the mispair. For example, G(template):dATP mispairs and G:dGTP mispairs are formed with about equal frequency by all three classes of DNA polymerases, yet pol-gamma produces T:dGTP mispairs at a 100-fold lower frequency than does pol-beta. The DNA polymerases exhibit distinct differences in template site preferences as well as substrate insertion preferences. The increase in accuracy apparent in proceeding from the least selective to the most accurate enzyme results primarily from a decrease in mispair formations at template A and T residues and a decrease in misinsertion of pyrimidine deoxynucleotides. These data clearly demonstrate a major role for eucaryotic DNA polymerases in modulating base mispair frequencies at the level of insertion. In addition to direct mispair formation due to an incorrect incorporation event, an examination of the errors produced by each of the three classes of DNA polymerases at two particular sites in the target sequence suggests that some base substitution errors result from transient misalignment of the primer-template. A model is presented to explain this phenomenon, termed "Dislocation Mutagenesis." The data are discussed in relation to the extensive literature on base substitution errors and to the origin of spontaneous base substitutions in animal cells.     

Drosophila 5 S RNA processing requires the 1-118 base pair and additional sequence proximal to the processing site.

Using an in vitro processing system, we have identified a required sequence surrounding the Drosophila melanogaster 5 S RNA processing site at nucleotide 120. Mutations in this region vary the processing rate from complete inhibition to a level equal to or greater than wild type. Analysis of mutants at +1 and in the region 118-122 separates the inhibitory effect into two parts. 1) Nucleotide 118 C, the base-paired nucleotide in helix I proximal to the processing site, plays an essential role. Changing it to a purine inhibits processing. The +1-118 base pair must be intact, but this alone is not sufficient for processing, since compensatory changes at +1 do not restore down-processing mutants at 118 to the wild type level. 2) The processing site has to be pyrimidine rich; multiple contiguous purines inhibit processing. On the other hand, multiple pyrimidines can largely negate the inhibitory effect of a mutation at position 118. Thus a base-paired C at 118 followed by a stretch of pyrimidines is the processing signal, which may be recognized by the processing enzyme and/or a required accessory factor.     

Prediction of C-to-U RNA editing sites in plant mitochondria using both biochemical and evolutionary information

Although cytidine-to-uridine conversions in plant mitochondria were discovered 18 years ago, it was still an enigmatic process. Since the sequencing projects of plant mitochondrial genomes are providing more and more available sequences, the requirements of computationally identifying C-to-U RNA editing sites are also increasing. By incorporating both evolutionary and biochemical information, we developed a novel algorithm for predicting C-to-U RNA editing sites in plant mitochondria. The algorithm has been implemented as an online service called CURE (Cytidine-to-Uridine Recognizing Editor). CURE performs better than other methods that are based on only biochemical or only evolutionary information. CURE also provides the ability of predicting C-to-U RNA editing sites in non-coding regions and the synonymous C-to-U RNA editing sites in coding regions that are impossible for other methods. Furthermore, CURE can carry out prediction directly on the entire mitochondria genome sequence. The prediction results of CURE suggest the functional importance of synonymous RNA editing sites, which was neglected before. The CURE service can be accessed at http://bioinfo.au.tsinghua.edu.cn/cure.     

Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes

A partial library of cloned human DNA was screened for sequences represented on and specific to the X chromosome. The library was constructed from Bam HI-digested human DNA from cells with X chromosome polyploidy, and was cloned in pBR322. The screening was performed by individually hybridizing ³²P-labeled cloned plasmids to Southern blots containing Bam HI-digested DNA from mouse-human hybrid cells having the human X chromosome and from derivative hybrids lacking the human X. Of 45 clones assayed, 33 contained sequences homologous to ones represented many times on the X. In situ hybridization to metaphase chromosomes demonstrated that at least four of these clones were homologous to autosomes as well. Only one of the 18 clones of this kind tested cross-hybridized with another. Two recombinant plasmids were shown to be derived from the X chromosome and to be X chromosome-specific by three criteria: they hybridized to a single band in the Southern blots of Bam HI-digested DNA from hybrid cells containing the X chromosome; they hybridized to a band of the same molecular weight as did the inserted DNA fragment; and they showed a dosage effect when hybridized to Southern blots of Bam HI-digested DNA from XY and XXX cells. One of these hybridized as a single-copy or low-order reiterated sequence in a Cot analysis using male DNA as driver. Our methods can be applied to the identification of any chromosome-specific clone. The two X-specific clones identified here should be useful in investigating the mechanism of X inactivation and in isolating a Barr body.     

Rabbit neuromedin U-25: lack of conservation of a posttranslational processing site

The rabbit small intestine contains neuromedin U-like immunoreactivity (22 pmol/g wet tissue weight) that was resolved into a single major molecular form by reversed-phase HPLC. The primary structure of the peptide was established as: Phe-Pro-Val-Asp-Glu-Glu-Phe-Gln-Ser-Pro10-Phe-Gly-Ser-Arg-Ser-Arg- Gly-Tyr-Phe- Leu20-Phe-Arg-Pro-Arg-Asn.NH2. In rabbit neuromedin U, the Arg16-Arg17 dibasic residue processing site that is found in pig and dog neuromedin U-25 is replaced by Arg-Gly, but this potential monobasic processing site is not utilized by cleavage enzyme(s) in the intestine.     

Distribution of substitution rates and location of insertion sites in the tertiary structure of ribosomal RNA

The relative substitution rate of each nucleotide site in bacterial small subunit rRNA, large subunit rRNA and 5S rRNA was calculated from sequence alignments for each molecule. Two-dimensional and three-dimensional variability maps of the rRNAs were obtained by plotting the substitution rates on secondary structure models and on the tertiary structure of the rRNAs available from X-ray diffraction results. This showed that the substitution rates are generally low near the centre of the ribosome, where the nucleotides essential for its function are situated, and that they increase towards the surface. An inventory was made of insertions characteristic of the Archaea, Bacteria and Eucarya domains, and for additional insertions present in specific eukaryotic taxa. All these insertions occur at the ribosome surface. The taxon-specific insertions seem to arise randomly in the eukaryotic evolutionary tree, without any phylogenetic relatedness between the taxa possessing them.