N-Terminal sequences of α-crystallin

The amino acid sequences at the N-terminal ends of the chains of the lens protein, alpha-crystallin, were studied. Both the main kinds of chain in bovine alpha-crystallin (A chains and B chains) have an N-terminal methionine residue, and the amino group is acetylated. Selective purification of the peptides in a tryptic digest of bovine alpha-crystallin gave a preparation consisting largely of the N-terminal peptide from the A chains, and the sequence of this peptide was elucidated. Subsequently, the N-terminal peptides were prepared from separated A and B chains. The proposed sequences are: A chain, acetyl-Met-Asp-Ile-Ala-Ile-Gln-His-Pro-Trp-Phe-Lys; B chain, acetyl-Met-Asp-Ile-Ala-Ile-His-(Pro,Trp)-Ile-Arg. The similarity between the sequences supports the hypothesis that the A and B chains are derived evolutionarily from a common precursor.     

Hinge sequences as signaling agents?

We report an unexpected finding of common structural principles in two unrelated signaling systems: the FAS death domain transformation that initializes the extrinsic apoptotic pathway and signaling by calmodulin bending. The location and design of the hinge is postulated to be a general principle for creating potential signaling event. We suggest that already existing tool can predict the existence of such a hinge and formulate the hypothesis that the internal instabilities designed into the hinge sequences are necessary devices in effective signaling events.     

Are introns in-series error-detecting sequences?

The probability of the accurate transmission of a message sequence can be increased by the addition of non-message sequences which permit errors in the message sequence to be detected and corrected. It is proposed that sequences in introns (or in other non-message genomic regions) serve this function with respect to the transmission of genetic information.     

How accurately can we discriminate G-protein-coupled receptors as 7-tms TM protein sequences from other sequences?

The group of 2502 transmembrane (TM) protein sequences with seven TM segments (7-tms) registered in SWISS-PROT 46.0 contains 2200 G-protein-coupled receptors (GPCRs), indicating that GPCR candidates can be detected with a reliability of 87.9% in the eukaryotic genomes merely by correctly predicting the number of TM segments as 7-tms. The predictive accuracies of TM topology-prediction methods proposed so far are not as high as expected; even the best method, HMMTOP 2.0, can only achieve a capture rate of 7-tms sequences of 77.6%. It is necessary to improve this performance as much as possible, even if by only a few percentage points, in order to identify as many novel GPCR candidate genes as possible among the increasing number of newly sequenced genomes. In this study, we propose a simple but useful prediction method for detecting as many 7-tms TM protein sequences as GPCR candidates in eukaryotic genomes as possible. This is achieved by employing a two-step prediction procedure. The first step involves collecting 7-tms sequences by the best prediction method (HMMTOP 2.0), and the second involves picking up the remaining 7-tms sequences by the second-best method (TMHMM 2.0). By this procedure, the capture rate of 7-tms TM protein sequences in SWISS-PROT can be improved considerably from 77.6% to 84.5%, and the number of GPCR candidate sequences predicted as 7-tms in the human genome (Build 35) is increased from 790 (by HMMTOP 2.0) to 903. These 790 and 903 candidate sequences include, respectively, 587 and 636 of the known human GPCRs of the 717 registered in SWISS-PROT 46.0, demonstrating that the proposed combinatorial method is effective in detecting GPCR candidate genes in eukaryotic genomes.     

Preliminary methylation analysis of prothymosin α genomic sequences

Prothymosin α is a mammalian nuclear protein involved in cell proliferation and differentiation. Here, we carried out the first study of the methylation status of ProTα genomic sequences in cell lines during differentiation as well as in tumoral tissues. We found that there is hypermethylation in all cell lines analyzed with a pattern that is characteristic of each cell type revealing specific genomic reorganizations. The decrease of ProTα mRNA during differentiation was not accompanied by changes in the methylation status. Remarkably, we found that there is hypomethylation in gastrointestinal tumors when compared with the peritumoral tissue. The biological implications of these findings are discussed.     

Nucleotide sequences of HS-α satellite DNA from kangaroo rat dipodomys ordii and characterization of similar sequences in other rodents

The most common repeated nucleotide sequences of the highly repetitive satellite HS-α fraction from kangaroo rat Dipodomys ordii was determined using ribosubstitution methods. This sequence was α nucleotides long and represented about 25% of the total HS-α satellite DNA, while the remaining DNA was composed of sequence variants related to the most common sequence. The sequences of the commonest of these variants are reported. Furthermore, the most common repeated sequence was identical to that reported for the α satellite of guinea pig Cavia porcellus. The α satellites of guinea pig, Cavia porcellus, pocket gopher, Thomomys bottae and antelope ground squirrel, Ammospermophilus leucurus, are shown to have sequences in common with the kangaroo rat. This implies that the simplest repeated sequences of mammalian satellite DNAs may persist over much longer evolutionary times than previously thought.Attempts to explain the very rapid quantitative changes in satellites whose sequence is strongly conserved have led us to consider that they might have a role in sympatric speciation. Among the novel features of the model presented is that fluctuations in satellites could be due to “speciation genes.” Such genes would confer a strong selective advantage in certain situations, and could explain the many puzzling instances in which large numbers of new related species have appeared over a short evolutionary span.     

Genetic variability in Fomes fomentarius reconfirmed by translation elongation factor 1-α DNA sequences and 25S LSU rRNA sequences

The existence of two cryptic species within strains of the wood-decaying fungus Fomes fomentarius was revealed recently based on the internal transcribed spacer (ITS) sequence variability. In this study for the first time the sequences of another molecular markers, partial translation elongation factor 1-α (efa) region and partial 25S large subunit ribosomal RNA gene were obtained and used to evaluate genetic variability of F. fomentarius. Congruent phylogeny was observed for all three markers used confirming the presence of two cryptic species within F. fomentarius. Surprisingly, ITS sequence variability within F. fomentarius was significantly lower compared to the variability of efa sequences (0.023 versus 0.036 nucleotide substitutions per site) questioning the discriminatory power of ITS sequences for fungal species identification.     

Comparison of amino acid sequences of eleven different α-amylases

Summary A comparison was made of the amino acid sequences of 11 different -amylases. The 6 animal -amylases tested were found to be highly homologous (about 80 to 90%, depending on the species compared). Amino acid sequence of Bacillus stearothermophilus -amylase was fairly homologous (about 60%) with that of a thermostable -amylase from Bacillus amyloliquefaciens. Homology was least among the thermolabile amylases from Bacillus subtilis, Aspergillus oryzae, plants and animals. Nevertheless, four highly homologous regions were found in the amino acid sequences of all the enzymes, despite their widely different origins. It was inferred that these four homologous regions were likely to be the active and/or substrate-binding sites.     

Location of β-amylase sequences in wheat and its relatives

Summary A -amylase cDNA clone isolated from barley has been used to locate -amylase encoding sequences on wheat, rye, and Aegilops umbellulata chromosomes by hybridisation to restriction endonuclease digested DNA obtained from wheat aneuploid and wheat-alien addition lines. Structural genes were identified on homoeologous group 4 and 5 chromosomes, confirming the results of isozyme studies. In addition, a further set of structural genes was found on homoeologous group 2 chromosomes. It is proposed that there are two homoeoallelic series, -Amy-1 on group 4 or 5 chromosomes, and -Amy-2 on group 2 chromosomes. Evidence is presented that each locus contains one or two -amylase structural genes, and it is suggested that the large number of isozymes seen upon IEF are due to post-translational modifications.     

Archaea: what can we learn from their sequences?

Gene-by-gene and traditional biochemical approaches continue to reveal surprising molecular features in the archaeal domain. In addition, the complete sequencing of several archaeal genomes has further confirmed the phenotypic coherence of these micro-organisms at the molecular level. Nevertheless, the phylogeny of Archaea and the nature of the last universal common ancestor are still matters for debate.     

DNA expansions generated by human Polμ on iterative sequences

Polµ is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polµ is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polµ-mediated dinucleotide expansion as a function of the sequence context. In this model, Polµ requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5′-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polµ residues His³²⁹ and Arg³⁸⁷ as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polµ at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.     

Phylogenetic analysis of the Glomeromycota by partial β-tubulin gene sequences

The 3’ end of the β-tubulin gene was amplified from 50 isolates of 45 species in Glomeromycota. The analyses included a representative selection of all families except Pacisporaceae and Geosiphonaceae. Phylogenetic analyses excluded three intron regions at the same relative positions in all species due to sequence and length polymorphisms. The β-tubulin gene phylogeny was similar to the 18S rRNA gene phylogeny at the family and species level, but it was not concordant at the order level. Species in Gigasporaceae and Glomeraceae grouped together but without statistical support. Paralogous sequences in Glomus species likely contributed to phylogenetic ambiguity. Trees generated using different fungal phyla as out-groups yielded a concordant topology. Family relationships within the Glomeromycota did not change regardless if the third codon position was included or excluded from the analysis. Multiple clones from three isolates of Scutellospora heterogama yielded divergent sequences. However, phylogenetic patterns suggested that only a single copy of the β-tubulin gene was present, with variation attributed to intraspecific sequence divergence.