A phylogenetic study of krill (Crustacea: Euphausiacea) reveals new taxa and co-evolution of morphological characters

The first comprehensive phylogenetic study of Euphausiacea (all 86 valid species) is presented. It is based on four molecular markers and 168 morphological characters (including 58 characters of the petasma). Phylogenetic analyses support the monophyly and robustness of the families Bentheuphausidae and Euphausiidae and reveal three major clades for which we erect three new subfamilies: Thysanopodinae, Euphausiinae and Nematoscelinae. All genus-level clades are statistically supported (except Thysanopoda in molecular analyses), deeply nested within the subfamily-level clades, and encompass 14 new species groups. Copulatory structures have a major impact on tree topology in the morphological analysis, the removal of which resulted in only half the number of supported clades and genera. We revealed three groups of morphological characters, which are probably coupled with the same biological role and thus interlinked evolutionarily: (i) antennular peduncle and petasma (copulation); (ii) eyes and anterior thoracopods (feeding); and (iii) shape of carapace and pleon (defence). We analysed the evolutionary pathways of the clades into main oceanic biotopes and compared them with morphological adaptations most likely to be coupled with this process.     

Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut

Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.     

Zebra: a web server for bioinformatic analysis of diverse protein families

During evolution of proteins from a common ancestor, one functional property can be preserved while others can vary leading to functional diversity. A systematic study of the corresponding adaptive mutations provides a key to one of the most challenging problems of modern structural biology – understanding the impact of amino acid substitutions on protein function. The subfamily-specific positions (SSPs) are conserved within functional subfamilies but are different between them and, therefore, seem to be responsible for functional diversity in protein superfamilies. Consequently, a corresponding method to perform the bioinformatic analysis of sequence and structural data has to be implemented in the common laboratory practice to study the structure–function relationship in proteins and develop novel protein engineering strategies. This paper describes Zebra web server – a powerful remote platform that implements a novel bioinformatic analysis algorithm to study diverse protein families. It is the first application that provides specificity determinants at different levels of functional classification, therefore addressing complex functional diversity of large superfamilies. Statistical analysis is implemented to automatically select a set of highly significant SSPs to be used as hotspots for directed evolution or rational design experiments and analyzed studying the structure–function relationship. Zebra results are provided in two ways – (1) as a single all-in-one parsable text file and (2) as PyMol sessions with structural representation of SSPs. Zebra web server is available at http://biokinet.belozersky.msu.ru/zebra.     

Role of inter-domain cavity in the attachment of the orange carotenoid protein to the phycobilisome core and to the fluorescence recovery protein

Using molecular modeling and known spatial structure of proteins, we have derived a universal 3D model of the orange carotenoid protein (OCP) and phycobilisome (PBS) interaction in the process of non-photochemical PBS quenching. The characteristic tip of the phycobilin domain of the core-membrane linker polypeptide (L_CM) forms the attachment site on the PBS core surface for interaction with the central inter-domain cavity of the OCP molecule. This spatial arrangement has to be the most advantageous one because the L_CM, as the major terminal PBS-fluorescence emitter, accumulates energy from the most other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, in cyanobacteria, the small fluorescence recovery protein is wedged in the OCP’s central cavity, weakening the PBS and OCP interaction. The presence of another one protein, the red carotenoid protein, in some cyanobacterial species, which also can interact with the PBS, also corresponds to this model.     

Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids

Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho‐Tyr, meta‐Tyr (m‐Tyr), and so on. Cytosolic phenylalanyl‐tRNA synthetase (PheRS) exerts control over the translation accuracy, hydrolyzing misacylated products, while monomeric mitochondrial PheRS lacks the editing activity. Recently we showed that “teamwork” of cytosolic and mitochondrial PheRSs cannot prevent incorporation of m‐Tyr and l ‐Dopa into proteins. Here, we present human mitochondrial chimeric PheRS with implanted editing module taken from EcPheRS. The monomeric mitochondrial chimera possesses editing activity, while in bacterial and cytosolic PheRSs this type of activity was detected for the (αβ)₂ architecture only. The fusion protein catalyzes aminoacylation of tRNA^Phe with cognate phenylalanine and effectively hydrolyzes the noncognate aminoacyl‐tRNAs: Tyr‐tRNA^Phe and m‐Tyr‐tRNA^Phe.     

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to “normal” DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15, 59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20, 22, 26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. ​(Fig.1).1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism. Open in a separate windowFIG. 1“Classical” model for the generation of microsatellite alterations by DNA polymerase slippage. Two single strands of a replicating DNA molecule are shown, with each repeat unit indicated by a rectangle. Arrows indicate the 3′ ends of the strand, and the top and bottom strands represent the elongating primer strand and the template strand, respectively. Step 1, the primer and template strand dissociate; step 2, the primer and template strands reassociate in a misaligned configuration, resulting in an unpaired repeat on either the template strand (left side) or primer strand (right side); step 3, DNA synthesis is completed. If the unpaired repeats are not excised by the DNA mismatch repair system, after the next round of DNA synthesis one DNA molecule will be shortened by one repeat (left side) or lengthened by one repeat (right side).A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the instability of a dinucleotide microsatellite. The most likely explanation of this result is that the DNA mismatches (unpaired repeats) resulting from DNA polymerase slippage events are efficiently removed from the newly synthesized strand by the DNA mismatch repair system. Thus, in the absence of mismatch repair, tract instability is elevated. From genetic studies, it has been found that mismatch repair in yeast efficiently corrects DNA mismatches involving 1- to 14-base loops (the size of the repeat units in microsatellites) but fails to correct mismatches involving loops larger than 16 bases (the size of the repeat units in minisatellites) (3, 41, 53). An inefficient mechanism, not involving the classical DNA mismatch repair system, is capable of correcting large DNA loops formed during meiotic recombination (19).In addition to mutations affecting DNA mismatch repair, some mutations affecting DNA replication in yeast destabilize microsatellites. Yeast strains bearing a null mutation in the RAD27 (RTH1) gene have high levels of instability of the dinucleotide poly(GT) and the trinucleotide CAG, specifically elevating single-repeat insertions (18, 39). RAD27 encodes the homolog of the mammalian FEN-1 protein, a 5′-to-3′ exonuclease (10, 11, 33). This nuclease activity is required for removing the terminal ribonucleotide residue from the 5′ end of the Okazaki fragment (9, 14, 35, 54, 55, 57); this step is necessary for the two adjoining fragments to be ligated together. FEN-1 appears to be active as either an exonuclease in the presence of a single-stranded gap upstream of the 5′ terminus or an endonuclease on a 5′ flap structure (13, 34). Since yeast strains that contain a null mutation in RAD27 grow poorly but are viable (38, 43), it is likely that less efficient nuclease activities that are also capable of 5′ Okazaki fragment processing are present in yeast. In addition to destabilizing dinucleotide microsatellites, rad27 strains have high levels of spontaneous mitotic recombination, elevated rates of forward mutation, and increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) (18, 38, 43). In contrast to the mutations normally seen in mismatch repair mutants, i.e., point mutations or small frameshifts, the types of mutations observed in the absence of Rad27p are duplications of sequences flanked by short direct repeats (4 to 7 bp in length) (49). These duplications were not affected by the DNA mismatch repair system.The same class of sequences that are duplicated in the rad27 strains show an elevated rate (up to 1,000-fold) of deletion in strains containing a temperature-sensitive allele (pol3-t) of the yeast gene encoding DNA polymerase δ (52, 53). This mutant (initially named tex1) was isolated in a strain that exhibited an increased excision rate of a bacterial transposon with long terminal repeats inserted within a yeast gene (7). The pol3-t allele, which encodes a mutation (Gly₆₄₁ to Ala₆₄₁) (51) located near the putative nucleotide binding and active-site domains of the enzyme (58), is thought to diminish the rate of lagging-strand synthesis resulting in long stretches of single-stranded DNA on the lagging-strand template (8). This single-stranded DNA may have the potential to form intrastrand base-paired structures, creating interactions between short direct repeats. These interactions would result in an increased frequency of deletions caused by DNA polymerase slippage.Since rad27 and pol3-t mutations elevate the rates of duplications and deletions associated with short separated repeats in nonrepetitive DNA sequences, Kunkel et al. (22) suggested that these mutations could also destabilize minisatellites. In this paper, we examine the effects of rad27 and pol3-t mutations on the stability of simple repeats in which the repeat unit length varies between 1 and 20 bp. Our results show that both mutations destabilize both microsatellites and minisatellites, but that the mechanisms involved in the destabilization are different for the two mutations.