Deamidation of the asparaginyl-glycyl sequence

The deamidation of Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe has been studied as a model for the facile deamidation of the Asn-Gly sequence in proteins. At alkaline pH, the product in each case is an identical mixture of Ac-alpha-Asp-Gly-NHMe (approximately 22%) and Ac-beta-Asp-Gly-NHMe (approximately 78%) as determined by n.m.r. spectroscopy. Because this same ratio is obtained from both Ac-Asn-Gly-NHMe and Ac-Isn-Gly-NHMe, the postulated mechanism, that deamidation proceeds through a cyclic imide intermediate, is confirmed. Unlike peptides of aspartyl esters, cyclization does not occur under nonaqueous conditions or at low pH in aqueous solution.     

Development of expressed sequence tag derived-simple sequence repeats in the genus Lilium

Although lily is the second largest flower crop in cutting flower commodity, only six simple sequence repeats SSRs have been reported. Thus, we developed expressed sequence tag derived-SSRs (EST-SSRs) for the Lilium genus. Among 2,235 unique ESTs, 754 ESTs contained SSR motifs, among which 165 ESTs were amenable to primer design. Among these 165 EST-SSRs, 131 EST-SSRs showed amplification in at least one Lilium species, and 76 EST-SSRs showed amplification in at least nine species. Of the 76 EST-SSRs, 47 showed amplification in all Lilium species analyzed. Using 10 breeding lines, we selected 21 EST-SSRs that had the highest number of alleles and polymorphism information content. The polymorphism information content values of these selected EST-SSRs ranged from 0.49 to 0.94 with an average of 0.76, which are higher than other plant species. The phylogenetic dendrogram derived from the amplification profiles of the 21 high polymorphic EST-SSRs was congruent with the genetic background of the 84 selected lily accessions and hybrids, which are available in commerce. Thus, the developed EST-SSRs will be very useful in germplasm management, genetic diversity analysis, cultivar finger printing, and molecular breeding in the lily.     

Identification of the DNA sequence required for transposition immunity of the gamma delta sequence.

A plasmid containing the transposon gamma delta sequence was immune to further insertion of gamma delta (transposition immunity). Plasmids carrying a fragment containing either 0.2 kilobase pairs of the gamma end or 0.4 kilobase pairs of the delta end of the gamma delta sequence were immune, and other parts of the gamma delta sequence did not confer immunity. The terminal 38-base-pair (bp) sequence of the delta end of the gamma delta was sufficient to confer immunity, the 38-bp sequence of the gamma end conferred only moderate immunity, and the terminal 35-bp sequence, which was completely identical at both the gamma and delta ends, was insufficient to confer immunity.     

Importance of the propeptide sequence of human preproparathyroid hormone for signal sequence function

The function of amino-terminal pro-specific peptides (propeptides), sequences often found on intermediate precursor forms of secreted proteins, is poorly understood. Human preproparathyroid hormone (prepro-PTH), a precursor protein containing such a propeptide, is initially synthesized as a precursor containing a 25-amino acid signal sequence, a 6-amino acid propeptide, and the 84-amino acid mature secreted peptide. Cloned cDNA encoding prepro-PTH and synthetic oligonucleotides were used to generate a mutant missing precisely the pro-specific sequences. The effects of this deletion on signal sequence function and on secretion per se were assessed after expression of the mutant cDNA in intact cells and in a cell-free translation system using synthetic mRNA in the presence of microsomal membranes. The mutant precursor protein was inefficiently translocated and cleaved, and cleavage occurred both at the normal site and within the signal sequence. Thus, for the eukaryotic protein prepro-PTH, sequences immediately downstream and separate from the classically defined signal sequence facilitate accurate and efficient signal function.     

Comparison of sequence masking algorithms and the detection of biased protein sequence regions

MOTIVATION: Separation of protein sequence regions according to their local information complexity and subsequent masking of low complexity regions has greatly enhanced the reliability of function prediction by sequence similarity. Comparisons with alternative methods that focus on compositional sequence bias rather than information complexity measures have shown that removal of compositional bias yields at least as sensitive and much more specific results. Besides the application of sequence masking algorithms to sequence similarity searches, the study of the masked regions themselves is of great interest. Traditionally, however, these have been neglected despite evidence of their functional relevance. RESULTS: Here we demonstrate that compositional bias seems to be a more effective measure for the detection of biologically meaningful signals. Typical results on proteins are compared to results for sequences that have been randomized in various ways, conserving composition and local correlations for individual proteins or the entire set. It is remarkable that low-complexity regions have the same form of distribution in proteins as in randomized sequences, and that the signal from randomized sequences with conserved local correlations and amino acid composition almost matches the signal from proteins. This is not the case for sequence bias, which hence seems to be a genuinely biological phenomenon in contrast to patches of low complexity.     

The nucleotide sequence of the tnpA gene completes the sequence of the Pseudomonas transposon Tn501.

The nucleotide sequence of the gene (tnpA) which codes for the transposase of transposon Tn501 has been determined. It contains an open reading frame for a polypeptide of Mr = 111,500, which terminates within the inverted repeat sequence of the transposon. The reading frame would be transcribed in the same direction as the mercury-resistance genes and the tnpR gene. The amino acid sequence predicted from this reading frame shows 32% identity with that of the transposase of the related transposon Tn3. The C-terminal regions of these two polypeptides show slightly greater homology than the N-terminal regions when conservative amino acid substitutions are considered. With this sequence determination, the nucleotide sequence of Tn501 is fully defined. The main features of the sequence are briefly presented.     

Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase

Complementary DNA clones for human glucocerebrosidase were isolated from a human hepatoma library in lambda gt11. The complete nucleotide sequence of the 1805-base pair cDNA insert has been determined. In addition to 5' and 3' untranslated regions (51 and 206 base pairs, respectively), the cDNA insert contains 1548 base pairs that completely encode human glucocerebrosidase. All possible N-linked glycosylation sites are identified. Examination of the 19 amino acids of the leader polypeptide beginning with the ATG at position 52 revealed a hydrophobic core and a carboxyl-terminal glycine at the peptidase cleavage site, features consistent with the leader sequences described for other human translocated proteins. The Mr of 57,000 calculated from the 516 amino acids deduced from cDNA sequence is in good agreement with that identified by immunoprecipitation following in vitro translation of human placental mRNA.     

Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene

The amino acid sequence of the egg yolk storage protein phosvitin has been deduced from the nucleotide sequence of part of the chicken vitellogenin gene. Of the phosvitin sequence, 210 amino acids including the N-terminal residue are contained on one large exon, whereas the remaining six amino acids are encoded on the next exon. Phosvitin contains a core region of 99 amino acids, consisting of 80 serines, grouped in runs of maximally 14 residues interspersed by arginines, lysines, and asparagines. The serines of the core region are encoded by AGC and AGT codons exclusively and the arginines by AGA and AGG, which results in a continuous stretch of 99 codons with adenine in the first position. The N-terminal quarter of the phosvitin sequence contains 16 serines grouped in a cluster with alanines and threonines and coded mainly by TCX triplets. The C-terminal part includes 27 serines, preferentially coded by AGC and AGT, 13 histidine residues, and the sequence ...Asn-Gly-Ser... at which the carbohydrate moiety of phosvitin is attached. Heteroduplex formation between cloned DNAs from chicken and Xenopus vitellogenin genes shows that the phosvitin sequence contains a stretch of highly conserved sequence.     

In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.     

Predicting residue solvent accessibility from protein sequence by considering the sequence environment

The solvent accessibility of each residue is predicted on the basis of the protein sequence. A set of 338 monomeric, non-homologous and high-resolution protein crystal structures is used as a learning set and a jackknife procedure is applied to each entry. The prediction is based on the comparison of the observed and the average values of the solvent-accessible area. It appears that the prediction accuracy is significantly improved by considering the residue types preceding and/or following the residue whose accessibility must be predicted. In contrast, the separate treatment of different secondary structural types does not improve the quality of the prediction. It is furthermore shown that the residue accessibility is much better predicted in small than in larger proteins. Such a discrepancy must be carefully considered in any algorithm for predicting residue accessibility.     

The effect of sequence quality on sequence alignment

Motivation: The nucleotide sequencing process produces not onlythe sequence of nucleotides, but also associated quality values.Quality values provide valuable information, but are primarilyused only for trimming sequences and generally ignored in subsequentanalyses. Results: This article describes how the scoring schemes of standardalignment algorithms can be modified to take into account qualityvalues to produce improved alignments and statistically moreaccurate scores. A prototype implementation is also provided,and used to post-process a set of BLAST results. Quality-adjustedalignment is a natural extension of standard alignment methods,and can be implemented with only a small constant factor performancepenalty. The method can also be applied to related methods includingheuristic search algorithms like BLAST and FASTA. Availability: Software is available at http://malde.org/~ketil/qaa. Contact: ketil.malde{at}imr.no Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: Limsoon Wong     

Searching the protein sequence database

As the volume of protein sequence data grows, rapid methods for searching the protein sequence database become of primary importance. Rigorous comparison of sequences is obtained with the well-known dynamic programming algorithms. However, these algorithms are not rapid enough to use for routinely searching the entire database. In this paper we discuss some methods that can be used for rapid searches.     

Nucleotide sequence of the rice cytochrome f gene and the presence of sequence variation near this gene

The rice (Oryza sativa L. var. Labelle) chloroplast (cp) gene encoding cytochrome f has been isolated and sequenced. The coding region of this rice gene displays 95.1%, 85.3% and 85.2% nucleotide sequence homology with that of wheat, pea and spinach, respectively. To examine the cpDNA sequence variation in rice, cpDNA from Labelle and its parents, Belle Patna and Dawn was compared. Using the cytochrome f gene as the probe for hybridization, we found several differences in the size and number of restriction fragments in the cp genome of three rice varieties. An additional restriction fragment found in the Belle Patna cp suggests that this cp genome is either heterogeneous or contains two copies of cytochrome f gene per cpDNA.