Comparative analysis of the MSAT-160 repeats in four species of common vole (Microtus, Arvicolidae)

The highly repeated tandemly arranged satellite DNA from the MSAT-160 family has been studied in 4 species of common vole (the Microtus arvalis group). All the monomer units analysed were classified into 4 subfamilies on the basis of similar nucleotide substitutions. The first 3 subfamilies do not show any species specificity since they combine monomers from several of the vole species examined; the fourth subfamily contains monomer units with substitutions specific to M. arvalis. Certain monomers of different species within the first 3 subfamilies display additional identical substitutions, making them more similar. Despite considerable similarity in monomer sequence within the subfamilies, specific features were found for each of the 4 species. A specific ratio of each type of monomer belonging to the corresponding subfamilies is typical of each species. In addition, the genomes of common vole species differ in the abundance of the MSAT-160 DNA, its pericentromeric location and organization. The mechanisms possibly involved in the evolution of the common vole MSAT-160 sequences are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

The STR120 satellite DNA of soybean: organization, evolution and chromosomal specificity

A highly repeated DNA sequence family, STR120, with tandemly arranged repetitive units (monomers) of approximately 120 bp, has been identified in soybean [Glycine max (L.) Merr.]. Five related clones showing tandem repeats of a 120-bp-long monomer were isolated from a soybean genomic library. Results of Southern blotting experiments using three of the clones as probes onto genomic DNA digested with different restriction enzymes were in agreement with a tandem arrangement of these sequences in the genome. A total of 12 monomers were sequenced, showing considerable sequence heterogeneity. A consensus sequence of 126 bp was obtained that exhibits an average similarity of 81% to the sequenced units. In three of the clones identified, neighbouring units are significantly more similar to each other than to units from different clones; in the remaining two clones, however, similarity between the two units observed is low (70%), while the overall similarity between the two clones is high (95%). This indicates that in these cases the repetitive unit may be the dimer rather than the monomer. Based on the presence of direct repeats within each monomer, we suggest that the 120-bp monomer may itself have evolved by duplication of an ancestral 60-bp unit. The STR120 family distribution is limited to annual soybeans and is not found, at least at high-copy number, in related perennial soybeans or other members of the tribe Phaseolae. Fluorescence in situ hybridization (FISH) to metaphase chromosomes using four of the clones as probes shows that the number of chromosomal locations differs depending on the stringency conditions and goes from two to eight when the stringency is progressively lowered. The estimated copy number for one of the clones is from 5000 to 10 000, but this may just represent a lower boundary for the whole family in consideration of the high sequence divergence observed within the family. FISH and sequence analysis therefore indicate that different subfamilies as well as higher-order repeat units are present in the STR120 family, very much like those in primate alpha satellite DNA, and that some of the subfamilies seem to exhibit divergence on a chromosomal basis.This revised version was published online in November 2005 with corrections to the Cover Date.     

Characterization and evolutionary dynamics of a complex family of satellite DNA in the leaf beetle <Emphasis Type="Italic">Chrysolina carnifex</Emphasis> (Coleoptera,Chrysomelidae)

The present study characterizes the complex satellite DNA from the specialized phytophagous beetle species Chrysolina carnifex. The satellite DNA is formed by six monomer types, partially homologous but having diverged enough to be separate on the phylogenetic trees, since each monomer type is located on a different branch, having statistically significant bootstrap values. Its analysis suggests a common evolutionary origin of all monomers from the same 211-bp sequence mainly by means of base-substitution mutations evolutionarily fixed to each monomer type and duplications and/or deletions of pre-existing segments in the 211-bp sequence. The analysis of the sequences and Southern hybridizations suggest that the monomers are organized in three types of repeats: monomers (211-bp) and higher-order repeats in the form of dimers (477-bp) or even trimers (633-bp). These repetitive units are not isolated from others, and do not present the pattern characteristic for the regular tandem arrangement of satellite DNA. In-situ hybridization with biotinylated probes corresponding to the three types of repeats showed the pericentromeric location of these sequences in all meiotic bivalents, coinciding with the heterochromatic blocks revealed by C-banding, indicating in addition that each type of repeat is neither isolated from others nor located in specific chromosomes but rather that they are intermixed in the heterochromatic regions. The presence of this repetitive DNA in C. haemoptera, C. bankii and C. americana was also tested by Southern analysis. The results show that this satellite DNA sequence is specific to the C. carnifex genome but has not been found in three other species of Chrysolina occupying similar or different host plants.     

Mechanism of high‐affinity abscisic acid binding to PYL9/RCAR1

Arabidopsis receptors of abscisic acid (ABA), the key plant hormone for adaptation to water stress, comprise 14 PYR/PYLs/RCARs proteins classified into three subfamilies I, II, and III, which suggests functional differentiation. Although their monomer–dimer equilibria may be correlated with differences in their ABA‐binding affinities, how the dimerization decreases the affinity is unclear. Comparative structural and binding studies between PYL9, which is a representative of high‐affinity subfamily I, and low‐affinity members of subfamily III reveals that the nonpolar triplet (Ile110, Val162, and Leu165) and Pro64 contribute to enhance ABA‐binding affinity by inducing a shift of the ABA carboxyl group to form additional direct hydrogen bonds with conserved Asn169. Our mutation studies of PYL1 successfully produced a monomeric mutant PYL1 exhibiting low ABA affinity and also a dimeric mutant PYL1 exhibiting high ABA‐binding affinity, suggesting that dimer formation of ABA receptors is not essential for their low ABA‐binding affinity. Our study contributes toward establishing the structural basis for the higher ABA‐binding affinity of the subfamily receptors and provides a clue for understanding the broad spectrum of hormone actions in plants manifested by the different hormone‐binding affinity of multiple receptors.     

Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens

Several repetitive sequences of the genome of Beta procumbens Chr. Sm., a wild beet species of the section Procumbentes of the genus Beta have been isolated. According to their genomic organization, the repeats were assigned to satellite DNA and families of dispersed DNA sequences.The tandem repeats are 229–246 bp long and belong to an AluI restriction satellite designated pAp11. Monomers of this satellite DNA form subfamilies which can be distinguished by the divergence or methylation of an internal restriction site. The satellite is amplified in the section Procumbentes, but is also found in species of the section Beta including cultivated beet (Beta vulgaris). The existence of the pAp11 satellite in distantly related species suggests that the AluI sequence family is an ancient component of Beta genomes and the ancestor of the diverged satellite subfamily pEV4 in B. vulgaris. Comparative fluorescent in-situ hybridization revealed remarkable differences in the chromosomal position between B. procumbens and B. vulgaris, indicating that the pAp11 and pEV4 satellites were most likely involved in the expansion or rearrangement of the intercalary B. vulgaris heterochromatin.Furthermore, we describe the molecular structure, and genomic and chromosomal organization of two repetitive DNA families which were designated pAp4 and pAp22 and are 1354 and 582 bp long, respectively. The families consist of sequence elements which are widely dispersed along B. procumbens chromosomes with local clustering and exclusion from distal euchromatic regions. FISH on meiotic chromosomes showed that both dispersed repeats are colocalized in some chromosomal regions. The interspersion of repeats of the pAp4 and pAp22 family was studied by PCR and enabled the determination of repeat flanking sequences. Sequence analysis revealed that pAp22 is either derived from or part of a long terminal repeat (LTR) of an Athila-like retrotransposon. Southern analysis and FISH with pAp4 and pAp22 showed that both dispersed repeats are species-specific and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. This was tested in the sugar beet hybrid PRO1 which contains a small B. procumbens chromosome fragment.     

Precipitin and mitogenic behavior of dimeric and tetrameric concanavalin A

The results of double diffusion and quantitative precipitin tests show that in contrast to the concanavalin A tetramer, the dimer is incapable of precipitating glycogen. The results support previous work suggesting that dimers composed of two fragmented monomer units possess only one binding site for carbohydrate (McKenzie and Sawyer, J. Biol. Chem. 1973. 248: 549). On the other hand, dimer and tetramer species had similar mitogenic activity as measured by the incorporation of radioactive thymidine by mouse spleen lymphocytes and induced cap formation to similar degrees. Results are discussed in terms of the affinity and valency of the polymeric forms and their stability at the cell surface.     

Structural and functional analysis of the origin of replication of mitochondrial DNA from Paramecium aurelia

Summary Replication of mitochondrial DNA in Paramecium aurelia involves the formation of a covalent crosslink at one end of this linear molecule and proceeds unidirectionally, producing a dimer consisting of two head to head monomers. The initiation regions within the dimer molecules have been sequenced and shown to be palindromic except for a central nonpalindromic A+T rich sequence, arranged in direct tandem repeats. This nonpalindromic region (see accompanying paper) has been identified as the cross-link which converts the initiation terminus into a continuous sequence. In this study, yeast transformation was used to assay the dimer initiation regions of P. aurelia mtDNA for the presence of autonomously replicating sequences. P. aurelia mtDNA fragments from species 1 and 4 were cloned into the yeast vector YIP5 and the hybrid plasmids (YPaM) were used to transform yeast. The dimer initiation regions from both species promoted high frequency transformation and extrachromosomal maintenance of YPaM plasmids. Subcloning analysis of the ARS-containing mtDNA fragments indicates, specifically, that the nonpalindrome, repetitive sequences are responsible for the autonomously replicating properties of YPaM plasmids.     

A novel active L1 retrotransposon subfamily in the mouse

Unlike human L1 retrotransposons, the 5' UTR of mouse L1 elements contains tandem repeats of approximately 200 bp in length called monomers. Multiple L1 subfamilies exist in the mouse which are distinguished by their monomer sequences. We previously described a young subfamily, called the T(F) subfamily, which contains approximately 1800 active elements among its 3000 full-length members. Here we characterize a novel subfamily of mouse L1 elements, G(F), which has unique monomer sequence and unusual patterns of monomer organization. A majority of these G(F) elements also have a unique length polymorphism in ORF1. Polymorphism analysis of G(F) elements in various mouse subspecies and laboratory strains revealed that, like T(F), the G(F) subfamily is young and expanding. About 1500 full-length G(F) elements exist in the diploid mouse genome and, based on the results of a cell culture assay, approximately 400 G(F) elements are potentially capable of retrotransposition. We also tested 14 A-type subfamily elements in the assay and estimate that about 900 active A elements may be present in the mouse genome. Thus, it is now known that there are three large active subfamilies of mouse L1s; T(F), A, and G(F), and that in total approximately 3000 full-length elements are potentially capable of active retrotransposition. This number is in great excess to the number of L1 elements thought to be active in the human genome.     

Side reactions during the initiation step in oligomerization of methacrylic esters by electron transfer reaction

Anionic oligomerization of methacrylic esters (methyl (MMA), ethyl (EMA), butyl (n-BMA), tert-butyl (t-BMA) methacrylates) was conducted in a one-step process by mixing, in tetrahydrofuran, the monomer, an alkali metal (sodium) and a deactivating agent (sulfur or tert-butyl alcohol). The formation of alcohol due to a nucleophilic attack of the ester group by the methacrylic carbanion was quantified and related to the monomer structure, temperature and nature of the living ends. It was shown that this side reaction is not limited to the propagation step but can occur during the initiation step. From mass spectra and nuclear magnetic resonance (NMR) spectroscopy of the protonated oligomers (tert-butyl alcohol as deactivating agent), it was concluded that this side reaction led to five-membered cyclic β-keto esters during the initiation step of MMA, EMA and n-BMA monomers only. This implies an intramolecular reaction between one carbanion of the dianionic dimer and the penultimate ester group. It was found that this reaction involves primarily dianionic tail-to-tail dimer, even though head-to-tail dimer was also formed.     

New insights into the karyotypic evolution in muroid rodents revealed by multicolor banding applying murine probes

Muroid rodents are composed of a wide range of species characterized by extensive karyotypic evolution. Even if this group includes such important laboratory animal models as domestic mouse (Mus musculus), Norway rat (Rattus norvegicus), Chinese hamster (Cricetulus griseus), and golden hamster (Mesocricetus auratus), comparative cytogenetic studies between rodents are difficult due to the characteristic rapid karyotypic evolution. Molecular cytogenetic methods can help resolve problems of comparing muroid chromosomes. Here, we used cross-species comparative multicolour banding with probes obtained from mouse chromosomes 3, 6, 18, and 19 to study the karyotypes of nine muroid species from the three subfamilies Murinae, Cricetinae, and Arvicolinae. Results from multicolour banding with these murine probes (mcb) allowed us to improve the comparative homology maps between these species and to obtain new insights into their karyotypic evolution. We identified evolutionary conserved chromosomal breakpoints and revealed four previously unrecognized homologous segments, four inversions, and 14 evolutionary new centromeres in the nine muroid species studied. We found Mus apomorphic rearrangements, not seen in other muroids, and defined several subfamily specific chromosome breaks, characteristic for Arvicolinae and Cricetinae. We show that mcb libraries are an effective tool both for the cytogenetic characterisation of important laboratory models such as the rat and hamster as well as elucidating the complex phylogenomics relationships of muroids.     

Interstitial telomeric repeats are enriched in the centromeres of chromosomes in Solanum species

Interstitial telomeric repeats (ITRs) were reported in a number of animal and plant species. Most ITRs are organized as short tandem arrays and are likely evolutionary relics derived from chromosomal rearrangements and DNA repairs. However, megabase-sized ITR arrays were reported in Solanum species. Here, we report a fluorescence in situ hybridization (FISH) survey of ITRs in all representative diploid Solanum species, including potato, tomato, and eggplant. FISH revealed massive amplification of ITRs in the centromeric regions of chromosomes from the Solanum species containing the B and P genomes. A significant proportion of the ITR FISH signals was mapped within the primary constrictions of the pachytene chromosomes of Solanum pinnatisectum (B genome). In addition, some ITR sites overlapped with St49, a satellite repeat enriched in centromeric DNA sequences associated with CENH3 nucleosomes, in both A and B genome Solanum species. These results show that some ITR subfamilies have been amplified and invaded in the functional centromeres of chromosomes in Solanum species.     

Homopolymerization and copolymerization of styrene and norbornene with Ni-based/MAO catalysts

The homopolymerization of styrene (Sty) and norbornene (NBE) was investigated in the presence of the nickel stearate (NiSt)/methylaluminoxane (MAO) catalytic system in toluene and in chlorobenzene at 20°C. The fully saturated structure of polynorbornene indicates that the two monomers polymerize by an ethylenic type addition reaction. The synthesis of true copolymers shows that one type of active species is operating for the tow monomers. Determination of reactivity ratios (r_NBE = 20.8 and r_Sty = 0.02) indicates a much higher reactivity of NBE, which is interpreted by a coordination mechanism. The styrene-norbornene copolymers exhibit glass transition temperatures (T_g) which range from 100°C to 320°C and follow Kovacs' law. The absence of crystallinity and the homogeneous repartition of monomer units along the chains yield highly transparent materials of high thermal stability.     

Radical polymerization reactivities of methacrylic acid coordinated to cobalt(III) complexes

Radical copolymerizations of methacrylic acid (MAA) coordinated to amminecobalt(III) complexes, methacrylatopentaamminecobalt(III) perchlorate ( 1 ) and cis-dimethacrylatotetraamminecobalt(III) perchlorate ( 2 ) with MAA were carried out in an aqueous medium at pH 3 and at pH 7. It was found that 1 becomes incorporated into the macromolecules more easily at pH 7 than at pH 3. This may relate to the electrostatic interactions between the reaction species (monomer and growing radical). It was also found that only 1 can be homo-polymerized at pH 3 although the product contains a certain amount of monomeric units of MAA. On the other hand, homo-polymerization of 1 at pH 7 or that of 2 at pH 3 gave no polymeric product. In terms of the data of monomer reactivity ratios, Q-e values and rates of copolymerization, reactivities of these metal complex monomers were discussed in connection with the effects of the transition metal complex attached to conjugated system of the methacrylate monomer.     

Solvent effects on the alternating copolymerization systems. Evaluation on equilibrium constants by NMR spectroscopy

The contributions of the charge-transfer complexes to the alternating copolymerizations (e.g., the styrene/maleic anhydride system) were studied. From the facts that the complex concentrations changed in various solvents and the rates of copolymerization depended on the solvent species, the behavior of the alternating copolymerization was considered to be controlled by the quantities of the reactive monomer complexes. In relatively strong donor solvents such as acetone and tetrahydrofuran, the concentrations of the monomer complexes were low because acceptor monomers also form complexes with the solvents. Therefore, the contributions of the monomer complexes to the copolymerization were neglectable. On the other hand, in benzene and carbon tetrachloride, which interact with acceptor monomers relatively weakly, the concentrations of the monomer complexes were relatively high. Then the copolymerizations were mainly controlled by the reactive monomer complexes. After all, it was suggested that the uncomplexed monomers as well as the complexed monomers should be taken into account on discussing the mechanisms of the alternating copolymerization between donor and acceptor monomers. In addition, the equilibrium constants or the complex concentrations could be determined in n-hexane by the use of NMR spectra.     

An approach to hyperbranched polymers with a degree of branching of 100%

General guidelines for the design of monomers for the synthesis of hyperbranched polymers without linear units are presented. The synthesis of one monomer fulfilling these requirements and the first results of the polymerization of this monomer are described. 4-(3-Maleimidopropoxy)-4′-methoxybenzaldehyde azine was used as a monomer of the AB₂-type in a “criss-cross” cycloaddition with the maleimide group as A-function and the azine as two B-groups. Melt condensation of this monomer gave a polymer (M _n = 5700) which showed ¹H and ¹³C NMR spectra corresponding well to the expected completely branched structure.     

18S rRNA gene sequences and phylogenetic relationships of European hard-tick species (Acari: Ixodidae)

The complete 18S rRNA gene sequences of the following six European hard-tick species were obtained by direct PCR cycle sequencing and silver-staining methods: Rhipicephalus pusillus, Boophilus annulatus, Dermacentor marginatus, Hyalomma lusitanicum, Haemaphysalis punctata, and Ixodes ricinus. Differences observed in the sequence alignment of these six species together with the 18S rRNA gene sequences of 13 other hard-tick species demonstrate that this gene is a good marker for supraspecific differentiation as well as genus grouping among hard ticks. Phylogenetic analyses strongly support that Hyalomma species share a common ancestor with Rhipicephalinae and, consequently, Hyalomminae should no longer be considered an independent subfamily. However, no definitive conclusion could be reached to support or oppose the separation of the subfamilies Haemaphysalinae and Amblyomminae. Accepted: 15 July 1997     

Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some triticeae species

The subtelomeric heterochromatin of rye (Secale cereale) chromosomes makes up 12–18% of the genome and consists largely of a small number of tandemly organized DNA sequence families. The genomic organization, chromosomal locations and the structural organization of monomer units of the major DNA sequences from these regions were investigated and compared in other Triticeae species from the generaSecale, Agropyron, Dasypyrum, Triticum andHordeum. Southern hybridization and polymerase chain reaction analysis established that all studied species preserve the tandem type of sequence organization but the copy number is altered drastically between species. In the pSc200 family, a fraction of the tandem arrays is present with a head-to-head orientation of dimers inS. cereale andS. montanum. Members of the same family are more heterogeneous and present as head-to-head monomers in theDasypyrum species andA. cristatum. In situ hybridization demonstrates different organization of the sequence families in the various species: pSc200 and pSc250 are concentrated in major blocks at the ends of most rye chromosome arms, whereas they are more dispersed and in smaller blocks inDasypyrum andAgropyron indicating that accumulation is not simply due to the sequence itself. In contrast to rye,D. villosum has large blocks of only pSc200 whereasD. breviaristatum shows greater amplification of pSc250. These data indicate that each repetitive family is an independent unit of evolution, and suggest that the twoDasypyrum species are not closely related. The data are discussed in terms of existing evolutionary models for repetitive DNA sequences. The contribution of random events, through molecular drive and selection, to the evolution of heterochromatic regions is considered.accepted for publication by S. Mizuno     

An intron loss in the chloroplast generpoC1 supports a monophyletic origin for the subfamily Cactoideae of the Cactaceae

The deletion of an approximately 700-bp intron in the chloroplast-encoded generpoC1 was shown in 21 representative species of the subfamily Cactoideae of the angiosperm family Cactaceae. Members of the subfamilies Pereskioideae and Opuntioideae were found to possess the intron, as did members of the related families Aizoaceae, Basellaceae, Didiereaceae, Phytolaccaceae, and Portulacaceae. These results support a monophyletic origin for the most-speciose subfamily of the cactus family, and represent a first report of the loss of this intron in dicots.     

Reciprocal chromosome painting between two South American bats: <Emphasis Type="Italic">Carollia brevicauda</Emphasis> and <Emphasis Type="Italic">Phyllostomus hastatus</Emphasis> (Phyllostomidae,Chiroptera)

The Neotropical Phyllostomidae family is the third largest in the order Chiroptera, with 56 genera and 140 species. Most researchers accept this family as monophyletic but its species are anatomically diverse and complex, leading to disagreement on its systematics and evolutionary relationships. Most of the genera of Phyllostomidae have highly conserved karyotypes but with intense intergeneric variability, which makes any comparative analysis using classical banding difficult. The use of chromosome painting is a modern way of genomic comparison on the cytological level, and will clarify the intense intergenus chromosomal variability in Phyllostomidae. Whole chromosome probes of species were produced as a tool for evolutionary studies in this family from two species from different subfamilies, Phyllostomus hastatus and Carollia brevicauda, which have large morphological and chromosomal differences, and these probes were used in reciprocal chromosome painting. The hybridization of the Phyllostomus probes on the Carollia genome revealed 24 conserved segments, while the Carollia probes on the Phyllostomus genome detected 26 segments. Many chromosome rearrangements have occurred during the divergence of these two genera. The sequence of events suggested a large number of rearrangements during the differentiation of the genera followed by high chromosomal stability within each genus.     

Kinetic Investigation of the Condensation Reaction of Activated Ester–Amides Containing Oligo(oxyethylene) Segments

The kinetics of polycondensation of activated ester–amides, namely pentachlorophenyl α‐amino‐ω‐oligo(ethyleneglycol)succinate hydrochlorides (PSPnNH) having a degree of oligomerization n = 1–3, were investigated in solution in dichloromethane in the presence of excess triethylamine. The large extinction coefficient of the pentachlorophenate formed during the reaction allowed the use of the stopped flow technique. Experimental data resulted in agreement with the existence of concurrent first‐ and second‐order processes, attributable to the intramolecular cyclization and intermolecular polycondensation reactions, respectively. The relative incidence of the cyclization reaction decreased on increasing the monomer concentration and on decreasing the number of oxyethylene units in the monomer. These hypotheses were confirmed by SEC, TLC, FT‐IR, and NMR characterization of the reaction products. The kinetics of PSP3NH are in very good agreement with the predictions of both the collision theory and the condensation theory of linear systems at equilibrium. However, the behavior of shorter chain monomers was opposite to that of the theoretical one, demonstrating that other parameters, such as steric hindrance and solvent interactions, must be taken into proper account. The reported findings clearly evidenced that polymerization of the investigated ester‐amides must be performed in bulk in order to suppress the cyclization reaction and to obtain high‐molecular‐weight polymers.

SEC traces of PSP3NH condensation products obtained at different monomer concentrations (Runs P7–P9).