Mitochondrial DNA evolution in theobscura species subgroup ofDrosophila

Summary Mitochondrial DNA (mtDNA) restriction site maps for nine species of theDrosophila obscura subgroup and forDrosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for theobscura subgroup species, one Nearctic, represented byDrosophila pseudoobscura, and four Palearctic, two grouping the related triads of speciesDrosophila subobscura, Drosophila madeirensis, Drosophila guanche, andDrosophila ambigua, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each,Drosophila bifasciata, andDrosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related speciesD. subobscura, D. madeirensis, andD. guanche, the pairD. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for theobscura subgroup species was from 15.5 kb forD. pseudoobscura to 17.1 forD. tristis.     

Evolution of single-copy DNA and the ADH gene in seven drosophilids

Summary Single-copy DNA was isolated fromDrosophila melanogaster and hybridized with total genomic DNA ofD. melanogaster, D. mauritiana, D. simulans, D. pseudoobscura, D. willistoni, D. hydei andD. virilis. The duplexes were thermally eluted from hydroxyapatite and the data used to assess the relatedness of each species toD. melanogaster. The general pattern of relatedness was similar to that predicted by morphological methods but with some notable exceptions. The rate of nucleotide substitution was estimated to be greater than 0.66% of bases per million years. An unexpected, rapidly evolving component ofD. melanogaster single-copy DNA was identified. The relatedness of these species was also studied with respect to the gene coding for alcohol dehydrogenase (ADH). The ADH gene, previously cloned fromD. melanogaster (Goldberg 1980), was hybridized with Southern blots of genomic digests of the seven species. The intensity and position of the hybridizing bands suggest the amount of divergence of the gene. Divergence was quantitated by reassociation of a fragment of the cloned ADH gene with total DNA of the seven drosophilids and thermal elution of the resultant duplexes from hydroxyapatite. The ADH gene was isolated from genomic clone libraries ofD. melanogaster, D. simulans andD. mauritiana and further studied by comparison of position of restriction sites. Species relationships deduced from comparison of total single-copy DNA and the ADH gene were consistent, demonstrating that a single gene can reflect divergence of the entire genome.     

Identification of two families of satellite-like repetitive DNA sequences from the zebrafish (Brachydanio rerio)

To further our understanding of the structure and organization of the zebrafish genome, we have undertaken the analysis of highly and middle-repetitive DNA sequences. We have cloned and sequenced two families of tandemly repeated DNA fragments. The monomer units of the Type I satellite-like sequence are 186 bp long, A+T-rich (65%), and exhibit a high degree of sequence conservation. The Type I satellite-like sequence constitutes 8% of the zebrafish genome, or approximately 8 × 10⁵ copies per haploid genome. Southern analysis of genomic DNA, digested with several restriction endonucleases, shows a ladder of hybridizing bands, consistent with a tandem array, and suggests longer range periodic variations in the sequence of the tandem repeats. The Type II satellite has a monomer length of 165 bp, is also A+T-rich (68%), and constitues 0.2% of the zebrafish genome (22,000 copies per haploid genome). Southern analysis reveals a complex pattern rather than a ladder of regularly spaced hybridizing bands.     

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     

Relationships betweenOryza species (Poaceae) based on 5S DNA sequences

Relationships between 9Oryza species, covering 6 different genomes, have been studied using hybridization and nucleotide sequence information from the5S Dna locus. Four to five units of the major size class of 5S DNA in each species, 55 units in all, were cloned and sequenced. Both hybridization and sequence data confirmed the basic differences between the A and B, C, D genome species suggested by morphological and cytological data. The 5S DNA units of the A genome species were very similar, as were the ones from the B, C, and D genome-containing species. The 5S DNA ofO. australiensis (E genome) grouped with the B, C, D cluster, while the units ofO. brachyantha (F genome) were quite different and grouped away from all other species. 5S DNA units fromO. minuta, O. latifolia, O. australiensis, andO. brachyantha hybridized strongly, and preferentially, to the genomic DNA from which the units were isolated and hence could be useful as species/genome specific probes. The 5S DNA units fromO. sativa, O. nivara, andO. rufipogon provided A genome-specific probes as they hybridized preferentially to A genome DNA. The units fromO. punctata andO. officinalis displayed weaker preferential hybridization toO. punctata DNA, possibly reflecting their shared genome (C genome).     

The Ds1 controlling element family in maize andTripsacum

Summary Hybridization experiments indicated that the maize genome contains a family of sequences closely related to the Ds1 element originally characterized from theAdh1-Fm335 allele of maize. Examples of these Ds1-related segments were cloned and sequenced. They also had the structural properties of mobile genetic elements, i.e., similar length and internal sequence homology with Ds1, 10- or 11-bp terminal inverted repeats, and characteristic duplications of flanking genomic DNA. All sequences with 11-bp terminal inverted repeats were flanked by 8-bp duplications, but the duplication flanking one sequence with 10-bp inverted repeats was only 6 bp. Similar Ds1-related sequences were cloned fromTripsacum dactyloides. They showed no more divergence from the maize sequences than the individual maize sequences showed when compared with each other. No consensus sequence was evident for the sites at which these sequences had inserted in genomic DNA.     

Identification of two families of satellite-like repetitive DNA sequences from the zebrafish (Brachydanio rerio).

To further our understanding of the structure and organization of the zebrafish genome, we have undertaken the analysis of highly and middle-repetitive DNA sequences. We have cloned and sequenced two families of tandemly repeated DNA fragments. The monomer units of the Type I satellite-like sequence are 186 bp long, A+T-rich (65%), and exhibit a high degree of sequence conservation. The Type I satellite-like sequence constitutes 8% of the zebrafish genome, or approximately 8 x 10(5) copies per haploid genome. Southern analysis of genomic DNA, digested with several restriction endonucleases, shows a ladder of hybridizing bands, consistent with a tandem array, and suggests longer range periodic variations in the sequence of the tandem repeats. The Type II satellite has a monomer length of 165 bp, is also A+T-rich (68%), and constitutes 0.2% of the zebrafish genome (22,000 copies per haploid genome). Southern analysis reveals a complex pattern rather than a ladder of regularly spaced hybridizing bands.     

Taxonomic and molecular studies on Drosophila sinobscura and D. hubeiensis,two sibling species of the D. obscura group*

Allozyme electrophoresis and three different DNA sequences (ATOC180 satellite DNA, 5SrDNA repeats, and parts of the Adh gene) were used to compare the two closely related East Asian sibling species Drosophila sinobscura and D. hubeiensis producing fertile hybrids in the laboratory. The data were also applied to establish their phylogenetic relationships to the other species of the D. obscura group. Genetic divergence in 5SrDNA repeats and specifically in the Adh gene separate the two species clearly from each other and justify their species status. Both species are related to the European species of the D. obscura group but the presence of members of the ATOC 180 satellite DNA family, specific and common to the species triad D. ambigua, D. tristis and D. obscura, in the genomic DNA of D. sinobscura and D. hubeiensis put the two sibling species in their close neighbourhood.     

Isolation of a D-genome specific repeated DNA sequence fromAegilops squarrosa

Three repeated sequence clones, pAS1(1.0 Kb), pAS2(1.8 Kb) and pAS12(2.5 Kb), were isolated fromAegilops squarrosa (Triticum tauschii). The inserts of the three clones did not hybridize to each other. Two of the clones, pAS2 and pAS12, contain repeated sequences which were distributed throughout the genome. The clone pAS1 sequence was more restricted and was located in specific areas on telomeres and certain interstitial sites along the chromosome length. This cloned sequence was also found to be restricted to the D genome at the level ofin situ hybridization. The pAS1 sequence will be useful in chromosomal identification and phylogenetic analysis. All three clones will allow assessment of genome plasticity inAegilops squarrosa. Nuclear DNA content varies over a range of 10,000 fold among all organisms (Nagl et al., 1983). Among angiosperms, at least a 65-fold range in genome size occurs in diploid species (Sparrow, Price and Underbrink, 1972; Bennett, Smith and Heslop-Harrison, 1982). This DNA variation has been reported within families, genera, and species (Rothfels et al., 1966; Rees and Jones, 1967; Miksche, 1968; Price, Chambers and Bachmann, 1981). Much of the interspecific variation in genome size among angiosperms appears to be due to amplification and/or deletion of DNA within chromosomes. The variation in genome size does not appear to result in changes in the number of coding genes (Nagl et al., 1983). While the number of coding genes, with the exception of rDNA in specific examples, appears to remain constant, the remaining non-coding regions are quite flexible. This non-coding DNA encompasses over 99% of the plant genome and consists of sequences that exist as multiple copies throughout the genome and are identified as repeated DNA sequences (Flavell et al., 1974). Flavell et al. (1974) have reported that increasing genome size in higher plants is associated with increasing repetitive DNA amounts. Subsequent reports have substantiated this correlation (Bachmann and Price, 1977; Narayan, 1982). In various cereals, heterochromatin, which has been demonstrated to be correlated with the location of specific repeated DNA sequences, has been positively correlated with genome size (Bennett, Gustafson and Smith, 1977; Rayburn et al., 1985). Furuta, Nishikawa and Makino (1975) found significant DNA content variation among different accessions ofAegilops squarrosa L. This species contains the D genome, a pivotal genome in several polyploid species and also found in hexaploid wheat (AABBDD). The importance of this genome to the study of bread wheat genomes makes the mechanism(s) of this genomic plasticity of particular interest. In order to determine which sequences are varying, one must first have a way to identify specific types of chromatin and/or DNA. Specific types of chromosome banding such as C- and N-banding have been used to identity types of chromatin in previous studies. C-banding of the D genome results in very lightly staining bands whose pattern is somewhat indistinct. N-banding alternatively has been shown to be useful in identifying certain chromosomes of hexaploid wheat but is limited by the lack of major bands in the D genome (Endo and Gill, 1984). Specific DNA sequences have been isolated fromTriticum aestivum cultivar “Chinese Spring” (hexaploid wheat). However, these sequences are representatives of the A and/or B genomes of hexaploid wheat and are not found in significant quantities in the D genome (Hutchinson and Lonsdale, 1982). Various other repeated DNA sequences have been successfully isolated from rye (Bedbrook et al., 1980) and identified on rye chromosomes (Appels et al., 1981; Jones and Flavell, 1982). Certain of these sequences are found in wheat genomes, but the sequences are representative of only a minor fraction of the D genome (Bedbrook et al., 1980; Rayburn and Gill, 1985). The purpose of this report is to describe three distinct repeated DNA sequences isolated fromA. squarrosa (D genome). Two clones appear to be distributed throughout the total genome, and the third clone is restricted to specific sites along the chromosomes. This latter clone will prove useful in cytologically defining the D genome chromosomes. These sequences appear representative of two types of repeated DNA genome organization: 1) sequences distributed throughout the genome and 2) specific arrays of repeated sequences. The availability of such repeated DNA sequence clones along with the known intraspecific DNA content variation inA. squarrosa will allow the study of genomic plasticity of this species.     

Cloning of ndhK from soybean chloroplasts using antibodies raised to mitochondrial complex I

A soybean shoot cDNA expression library was screened with polyclonal antibodies raised against red beet complex I and several clones were identified. One clone, consisting of a 1 kb insert, was fully sequenced. The sequence of 1025 bp was found to contain two extended open reading frames and the proteins encoded were identified as the ndhK and ndhJ products of the chloroplast genome. Nuclear, mitochondrial and chloroplast DNA was isolated and probed with a ndhK-specific probe. The chloroplast DNA contained a single copy of the cloned insert. With nuclear DNA, positively hybridising bands of 1.2, 2.7 and 3.2 kb were observed indicating that at least one gene homologous to ndhK of the chloroplast genome, is also present in the nucleus. The mitochondrial DNA did not hybridise with the ndhK probe. Western analysis of thylakoid proteins with the mitochondrial complex I antibodies revealed several bands. It is suggested that soybean contains two copies of the ndhK gene, one, on the plastid genome, coding for a subunit of a chloroplast NAD(P)H dehydrogenase, and the other, in the nucleus, coding for a subunit of mitochondrial complex I.     

Local repeat sequence organization of an intergenic spacer in the chloroplast genome ofChlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: A complex mode of “copychoice replication”?

Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA ofChlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt⁺) in a cross between a pair of polymorphic interfertile strains ofChlamydomonas (C. reinhardtii andC. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, “P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, “P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely “sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a "unique" new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a “complex path” of copy-choice replication.     

DNA sequence divergence in the Drosophila virilis group

DNA sequence divergence was analyzed in some sibling species of the Drosophila virilis group. Clones comprising about 0.1% of the genome DNA were selected at random from a D. virilis library for a comparative study on DNA from D. lummei, D. novamexicana, D. borealis, and D. lacicola. Blot hybridization experiments indicated that about 70% of DNA from D. lummei and D. novamexicana and less than 50% of DNA from D. borealis and D. lacicola share sequences that are homologous to DNA in D. virilis. This finding is in excellent agreement with the genealogical tree based on cytological studies (Throckmorton 1982). - Four plasmids with inserts which are present in one or a few copies per genome were hybridized in situ to polytene chromosomes. These experiments demonstrate that (1) homologous "unique" DNA sequences are localized exclusively in homologous bands and (2) homologous bands that appear to be identical in different species may contain different DNA sequences.     

The genome of Zea mays,its organization and homology to related grasses

The pattern of genome organization of Zea mays has been analyzed, and the relationship of maize to possible progenitor species assessed by DNADNA hybridization. Reassociation of 470 and 1,350 bp fragments of maize DNA to various C₀t values demonstrates that the genome is composed of 3 major kinetic classes: highly repetitive, mid-repetitive, and unique. Mini-C₀t curves of the repetitive sequences at short fragment length indicate that the highly repetitive sequence class is 20% of the genome and is present at an average reiteration frequency of 800,000 copies; the mid-repetitive sequence class is 40% of the genome and is present at an average reiteration frequency of 1,000 copies. Thermal denaturation studies show that the highly repetitive sequences are 12% divergent and mid-repetitive sequences are 6% divergent. Most of the genome is organized in two interspersion patterns. One, approximately one-third of the genome, is composed of unique sequences of average length 2,100 bp interspersed with mid-repetitive sequences; the other, also one-third of the genome, is mid-repetitive sequences interspersed with highly repetitive sequences. The repetitive sequences are 500 to 1,000 bp by electron microscopic measurement. The remaining third of the genome is unique sequences farther than 5,000 bp from a palindromic or repetitive sequence. Hybridization of maize DNA from Midwestern Dent to popcorn and related grasses indicates that both the unique and repetitive sequence elements have diverged. Teosinte and popcorn are approximately equally divergent from Midwestern Dent whereas Tripsacum is much more divergent. The divergence times calculated from the depression of T_m in heterologous duplexes indicate that the divergence within Zea mays and between maize and near relatives is at least an order of magnitude greater than expected. This high degree of divergence may reflect the pressures of domestication of maize.     

Ribosomal RNA genes from Prevotella bryantii: organization and heterogeneity

FiveP. bryantii B₁4 16S rRNA gene copies and their flanking regions were cloned and analyzed. A genomic library was constructed and screened with oligonucleotide DNA probe specific for 16S rRNA gene ofP. bryantii. Five out of six different copies of 16S RNA gene were recovered and sequenced. Only minor differences (0.3–1.2%) between copies were detected within the 1541 bp long sequence. The impact of the sequence variability of 16S rRNA gene copies on phylogenetic positioning ofP. bryantii was determined. All five sequences from clonedP. bryantii B₁4 16S rRNA genes were placed in the same operational taxonomy unit. Control regions of all five analyzed rRNA operatons were almost identical and three candidate for promoter sequences were identified by Neutral Network Promoter Prediction. Spacer regions between 16S-rRNA and 23S rRNA genes in all five cloned copies were 543 bp long and genes for tRNA^lle and tRNA^Ala were identified inside this regions.     

Insertion sites of the transposable elementmariner are fixed in the genome ofDrosophila sechellia

Summary The abundance of the transposable elementmariner differs dramatically in the genomes of the closely related speciesDrosophila simulans, D. mauritiana, D. sechellia, andD. melanogaster. Natural populations ofD. simulans andD. mauritiana have 1–10 and 20–30 copies per diploid genome, respectively, and the insertion sites are polymorphic. The element has not been found inD. melanogaster. In this paper we show thatD. sechellia, a species endemic to the Seychelles Islands, contains only twomariner elements that are at fixed sites in the genome. One element, inserted in chromosome 2R at 51A1–2, contains three deletions and is missing much of the 3 end. The other element, inserted in chromosome 3L at 64A10–11, is the full length of 1286 bp. Although the sequence of the full-length element is polymorphic in populations ofD. sechellia, at least some of the sequences are closely related to elements fromD. simulans andD. mauritiana that are known to be active. However, judging from the progeny of crosses betweenD. sechellia andD. simulans, the biological activity of the full-lengthD. sechellia element appears to be low, either because of the nucleotide sequence of the element or because of its position in the genome, or both.     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA

Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.     

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.     

Satellite DNA and speciation: A species specific satellite DNA of Drosophila guanchel

The heterochromatin of the chromosomes of Drosophila gunche consists mainly of a satellite DNA composed of multiple, tandemly arranged copies of a 290 b p basic sequence. Five clones containing one or two copies of the basic unit were sequenced. As expected from CsCl density centrifugation and AT specific staining of mitotic chromosomes the sequence is AT rich. The average nucleotid variability between the cloned sequences is 11.6%. In situ hybridization on the mitotic chromosomes revealed, that this satellite DNA is present in the centromeric regions of all chromosomes but the Y. The nucleotide variability between copies of different tandem clusters seems to be higher than between members of the same cluster. The copy number of the sequence in the haploid genome was estimated to be approximately 80000. The sequence is species specific and is not present in the genome of sibling species D. subobscura and D. madeiren-sis. The evolutionary origin of the satellite DNA and its possible role in species formation is discussed.