Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

The high genetic homology of threeMacaca fascicularis and twoMacaca mulatta subspecies on the basis of their highly repeated DNA restriction patterns

We have studied highly repeated DNA sequences of three subspecies ofM. fascicularis (M.f. philippinensis M.f. mordax, M.f. fusca) and of two subspecies ofM. mulatta (M.m. lasiotus, M.m. mulatta). Restriction patterns were obtained after digestion with 9 restriction endonucleases and evidenced after southern blotting and hybridization with Bam HI satellite DNA fragments fromM. fascicularis subspecies. M. fascicularis andM. mulatta subspecies studied, present morphological differences but indistinguishable karyotypes: highly repeated DNA analysis, resulting in the same restriction patterns for all the restriction sites studied with highly repeated DNA probes characteristic of the threeM. fascicularis subspecies, gave arguments in favour of the high genetic homology ofM.f. philippinensis, M.f. mordax, M.f. fusca on one side, andM.m. lasiotus andM.m. mulatta on the other, which can be distinguished only on the basis of morphological criteria.     

Highly repeated DNA analysis and systematics of the Lemuridae,a family of Malagasy prosimians

Hybridization of highly repeated DNA sequences ofEulemur fulvus mayottensis, Lemur catta, andVarecia has been performed on blots of different species of Lemuridae (L. catta, Hapalemur griseus, Varecia variegata variegata, V. v. rubra, E. macaco macaco, E. coronatus, E. mongoz, andE. rubriventer). The probe ofE. fulvus only hybridized with the differentEulemur species, whereas that ofVarecia hybridized with the two subspecies ofVarecia and that ofL. catta with bothL. catta andHapalemur. These results were used to confirm the classification ofVarecia in a separate genus and to review the separation of theL. catta/Hapalemur group from the other species ofEulemur. Comparison of the migration patterns from DNA fragments of these different species has been used to propose a cladogram of the differentEulemur species.     

Relationships between species ofLeymus,Psathyrostachys, andHordeum (Poaceae,Triticeae) inferred from Southern hybridization of genomic and cloned DNA probes

We have used total genomic DNA as a probe to size-fractionated restriction enzyme digests of genomic DNA from a range ofTriticeae species from the generaLeymus Hochst.,Psathyrostachys Nevski, andHordeum L., and hybrids betweenHordeum andLeymus to investigate their taxonomic relationships. Genomic Southern hybridization was found to be an effective and simple way to assess the distribution and diversity of essentially species-specific and common, repetitive DNA sequences, and is hence especially useful in evolutionary studies. The DNA sequences ofH. vulgare seem to diverge substantially from those ofH. brachyantherum, H. lechleri, H. procerum, andH. depressum. The genome ofThinopyron bessarabicum shows little homology to those of theLeymus species investigated, confirming thatT. bessarabicum is not an ancestral genome inLeymus. Although the genomes ofLeymus andPsathyrostachys share substantial proportions of DNA sequences, they include divergent repeated sequences as well. Hybridization with a ribosomal DNA probe (pTa 71) showed that the coding regions containing structural genes encoding the 18 S, 5.8 S, and 26 S ribosomal RNA were conserved among the species investigated, whereas the intergenic spacer region was more variable, presenting different sizes of restriction fragments and enabling a classification of the species. The rye heterochromatin probe pSc 119.2 hybridized to DNA fromH. lechleri andT. bessarabicum, but not to DNA from the other species investigated.     

Phylogenetic relationships among Malagasy lemurs as revealed by mitochondrial DNA sequence analysis

Systematics and evolution of Malagasy lemurs has been analyzed using morphological characters, fossil evidence, ecological/ethological data, and chromosomal banding patterns. Recent developments in DNA technology have provided evolutionary biologists with additional and powerful tools for making phylogenetic inference. In the last years several studies concerning highly repeated DNA sequences (hrDNA) provided new insights about the systematic relationships among the different species of Lemuridae and Cheirogaleidae. Here, a reconstruction of molecular phylogeny of extant Malagasy lemurs based on the comparison of cytochrome-b mitochondrial DNA sequences is presented. With the Polymerase Chain Reaction (PCR) and direct sequencing of amplified DNA fragments, both the phylogenetic range and resolving power of comparative analysis can be extended. These techniques allow to gather sequence data useful to evaluate the pattern of molecular evolution offering opportunities for phylogenetic purposes. A 290-bp fragment of cytochrome-b gene has been amplified and sequenced from the following species:Tupaia glis, Galago alleni, Daubentonia madagascariensis, Indri indri, Varecia variegata, Eulemur fulvus, Eulemur coronatus, Eulemur rubriventer, Eulemur mongoz, Eulemur macaco, Lemur catta, andHapalemur griseus griseus. The phylogenetic trees obtained show the relationships among the Eulemur species and confirm the karyological and hrDNA results of a separated clade forL. catta/Hapalemur. The separation ofVarecia variegata from the other genus of the family Lemuridae is discussed.     

DNA hybridization,cladistics, and the phylogeny of phalangerid marsupials

Summary Single-copy DNA/DNA hybridization experiments and numerical cladistic analyses of anatomical characters were used to investigate relationships among nine phalangerid (Marsupialia) species from four different genera. Both rate-dependent and rate-independent analyses of molecular data indicate that species ofTrichosurus form one clade and thatStrigocuscus, Phalanger, andSpilocuscus form a second. Within the latter group,Spilocuscus is excluded from aStrigocuscus-Phalanger calde, which, in turn, is not fully resolved on a jackknife strict consensus tree. Minimum-length Dollo, Wagner, and Camin-Sokal parsimony trees based on 35 anatomical characters, in contrast, suggest placement ofStrigocuscus withTrichosurus rather than withSpilocuscus andPhalanger. However, there are two derived characters that support the alternative arrange ofStrigocuscus withSpilocuscus andPhalanger and one character that further unitesStrigocuscus andPhalanger. Thus, DNA hybridization results are not inconsistent with the distribution of derived character states among anatomical characters, only with minimum-length trees based on character data.     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins fromSulfolobus shibatae

The thermoacidophilic archaeonSulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S.shibatae. These two genes, designatedssh7a andssh7b, have been cloned, sequenced and expressed inEscherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, thecis-regulatory sequences of thessh7a andssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein inSulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation ofSulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of ∼ 6.6 base pairs and an apparent dissociation constant of (0.7–1.0) × 10^-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.     

Studies on DNA sequences in the Osmundaceae

Summary Phylogenetic relationships ofOsmunda cinnamomea, O. claytoniana, andO. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences ofO. claytoniana are nearly equally diverged from those ofO. cinnamomea andO. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the threeOsmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hybridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.     

Molecular biology and systematics of an extinct Lemur:Pachylemur insignis

The systematic position of the extinctPachylemur insignis has been controversial: some authors consideredPachylemur as a lemur, whereas others viewed it closer toVarecia. Its classification in the genusLemur orVarecia thus remained an open question. DNA extraction from subfossil bones, using a non-destructive method, allowed us to obtain enough material to make a Southern blot. The hybridization ofPachylemur withEulemur fulvus, Lemur catta, andVarecia variegata highly repeated DNA probes showed that only theVarecia probe gave a positive signal on hybridization on thePachylemur blot. These results indicate thatPachylemur must be considered closer to the genusVarecia than toEulemur andLemur.     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5 translated region. The 5 flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.     

Taxonomic and population differentiation of mitochondrial diversity inPinus banksiana andPinus conforta

We have studied two mitochondrial DNA polymorphisms in 741 individuals from 16 allopatric populations ofPinus banksiana Lamb. andPinus contorta Dougl. Restriction fragments of both polymorphisms distinguished the two species qualitatively, except in aP. Banksiana population whose ancestors were involved in hybridization withP. contorta.COXI-associated restriction fragments were monomorphic within species, whileCOXII-associated restriction fragments were highly variable inP. contorta (H_es=0.68). Population differentiation was substantial inP. contorta (F_st=0.31 among subspecies; mean F_st=0.66 within subspecies) and consistent with predictions for maternally inherited markers. Plant mitochondrial markers appear to be useful for the investigation of seed migration routes, hybridization and introgression, breeding zone designation, and the development of germ plasm conservation sampling strategies.     

Knob heterochromatin homology in maize and its relatives

Summary We have characterised the major DNA sequence component of knob heterochromatin in maize, teosinte andTripsacum. Sequence analysis of this DNA gives strong support to the proposal that maize originated by selection of variants in teosinte. In situ hybridization has confirmed that this repeating DNA sequence, which is the major component of maize knob heterochromatin, is also the major component of knobs in teosinte,Zea diploperennis andTripsacum. In Southern blot hybridizations the repeat has a similar basic organization in all taxa;Tripsacum, however, is differentiated from maize and teosinte by a number of sequence features. Maize and teosinte knob heterochromatin are indistinguishable with regard to the distribution of mutations in the 180-bp repeat and the presence and organization of a 202-bp variant sequence. The knob DNA sequence was not detectable in three species ofCoix, an Old World genus of the Maydeae.Within the repeat unit is a 27-bp region that shows no sequence changes in maize, teosinte orTripsacum. The remainder of the repeat unit has randomly distributed nucleotide changes. The presence of the conserved sequence region suggests that knob DNA may have a functional role in the nucleus.     

Analysis ofDrosophila chromosome4 using pulsed field gel electrophoresis

Previous estimates of the size ofDrosophila melanogaster chromosome4 have indicated that it is 1% to 4% of the genome or 6 Mb. We have used pulsed field gel electrophoresis (PFGE) to separate megabase-sized molecules ofD. melanogaster chromosomal DNA. Southern blots of these gels were probed with DNA fragments from thecubitus interruptus andzfh-2 genes, which are located on chromosome4. They each identify the same-sized distinct band that migrates at approximately 5.2 Mb in DNA preparations from the Kc cell line. We interpret this band to be intact chromosome4. In DNA obtained from embryos of variousD. melanogaster wild-type strains, this chromosome band showed strain-specific size variation that ranged from 4.5 to 5.2 Mb. TheD. melanogaster chromosome4 probes also identified a single, 2.4 Mb band in embryonic DNA fromDrosophila simulans. We conclude thatD. simulans chromosome4 is substantially smaller than that ofD. melanogaster, presumably owing to diffirences in the amount of heterochromatic DNA sequences. Our simple DNA preparation from embryos and PFGE conditions should permit preparative isolation of chromosome4 DNA and will facilitate the molecular mapping of this chromosome.     

Evolutionary conservation and DNA binding properties of the Ssh7 proteins fromSulfolobus shibatae

The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of ~ 6.6 base pairs and an apparent dissociation constant of (0.7—1.0)×10^-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA. :     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Tirant: A New Retrotransposon-Like Element in Drosophila melanogaster

In this paper we report a new retrotransposon-like element of Drosophila melanogaster called Tirant. This sequence is moderately repeated in the genome of this species and it has been found to be widely dispersed throughout its distribution area. From Southern blot and in situ analyses, this sequence appears to be mobile in D. melanogaster, since its chromosome location and the hybridization patterns vary among the different strains analyzed. In this way, partial sequencing of Tirant ends suggests that it is a retrotransposon, since it is flanked by two LTRs. The presence of sequences homologous to Tirant has been also investigated in 28 species of the genus Drosophila by means of Southern analyses. These sequences were only detected in species from melanogaster and obscura groups. These data suggest that ancestral sequences of Tirant appeared after the Sophophora radiation and before the divergence of those groups. Received: 1 January 1995 / Accepted: 20 August 1995     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P₁, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.     

Assessment of random amplified polymorphic DNA (RAPD) for determining the origin ofYoungia koidzumiana Kitamura (compositae)

We determined the parental species ofYoungia koidzumiana (a natural interspecific hybrid) using PCR and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. These markers, generated by three primers, were sufficient to distinguishYoungia sonchifolia, Youngia denticulata, Youngia chelidoniifolia, andY. koidzumiana. The electrophoresis profiles of the amplified products from each of the four species were then compared. Three primers produced a total of 42 scorable markers; nine were specific markers forY. denticulata andY. chelidoni-ifolia. The length of the amplified DNA fragments ranged from 370 to 2500 b p. The three primers revealed polymorphic bands, which were indicators of the parental species ofY. koidzumiana. These bands showed a combination of specific profiles forY. denticulata andY. chelidoniifolia. Our results also were comparable to the data obtained for flowering times, floret numbers, and chromosome numbers of the four species. Therefore, we suggest thatY. koidzumiana is a hybrid betweenY. denticulata andY. chelidoniifolia}, and that RAPD markers are well suited for assessing the origins of plant species.