Restriction Site Mapping Research Articles

Phylogenetic reconstruction of the Drosophila obscura group, on the basis of mitochondrial DNA.

We have constructed restriction-site maps of the mtDNAs in 13 species and one subspecies of the Drosophila obscura group. The traditional division of this group into two subgroups (affinis and obscura) does not correspond to the phylogeny of the group, which shows two well-defined clusters (the Nearctic affinis and pseudoobscura subgroups) plus a very heterogeneous set of anciently diverged species (the Palearctic obscura subgroup). The mtDNA of Drosophila exhibits a tendency to evolve toward high A+T values. This leads to a "saturation" effect that (1) begets an apparent decrease in the rate of evolution as the time since the divergence of taxa increases and (2) reduces the value that mtDNA restriction analysis has for the phylogenetic reconstruction of Drosophila species that are not closely related.

Characterization of a small endogenous plasmid from the CyanobacteriumPlectonema boryanum

The nonheterocystous filamentous Cyanobacterium Plectonema boryanum strain UTEX 594 contains at least two plasmids. A small 1 · 45 kb plasmid was cloned in pBR322. It has no homology with the bigger resident plasmid or with chromosomal DNA. A small fraction of the plasmid is present in the form of multimers or concatemers. Copy number and hybridization patterns of the plasmid were similar under dinitrogen-fixing and non- fixing conditions. Restriction site mapping of the plasmid was done to enable its use in the development of cyanobacterial cloning vectors. It is among the smallest natural plasmids reported from bacteria.

Androdioecy is Derived from Dioecy in Datiscaceae: Evidence from Restriction Site Mapping of PCR-Amplified Chloroplast DNA Fragments

Androdioecy is a rare reproductive system in plants, which has traditionally been viewed as an intermediate step in the formation of dioecy from hermaphroditism. However, in the Datiscaceae, which comprises three dioecious and one androdioecious species, we have proposed that androdioecy rather than dioecy represents the derived condition. To test this hypothesis, the evolutionary history of Datiscaceae was reconstructed by comparative restriction site mapping of PCR-amplified chloroplast DNA fragments and subsequent cladistic and phenetic analyses. Thirtyeight shortest Wagner trees, 32 shortest Dollo trees, majority-rule consensus trees for each parsimony option, and one shortest genetic distance tree were examined. The androdioecious Datisca glomerata was placed in a derived position relative to the dioecious members of this family in all of these trees. This was also the case for all Dollo and Wagner trees that are oneand two-steps longer than the shortest trees, although not necessarily for Wagner and Dollo trees that are three-steps longer. These data not only support our contention that androdioecy is derived from dioecy in Datiscaceae, they also demonstrate the potential of PCR-mediated restriction site mapping comparisons for reducing the time, expense, and quantity of DNA required for comparative chloroplast DNA anal-

Clone bank and physical map of Sedum album (Crassulaceae) chloroplast DNA

A clone bank of chloroplast DNA (cpDNa) of Sedum album (Crassulaceae) covering the entire chloroplast genome was constructed using the restriction endonucleases Sacl, Hindlll and Pstl. It represents the first complete clone bank of cpDNA from a species of the order Rosales. A restriction site map of S. album cpDNA for these three enzymes was constructed by means of heterologous hybridization. The cpDNA of S. album shows similarity in size and physical organization to the chloroplast genome of most angiosperms. It was found to be a circular molecule of 148.5±1 kb, containing an inverted repeat region of 25 kb. Restriction site conservation was observed within the inverted repeats of the species Sedum album and Lectuca sativa for the restriction enzymes Hindlll and Sacl.

TOOLS FOR STUDYING THE CHLOROPLAST GENOME IN THE TRITICEAE (GRAMINEAE): AN ECORI MAP, A DIAGNOSTIC DELETION, AND SUPPORT FOR BROMUS AS AN OUTGROUP

Data on variation in the chloroplast genome can be used to estimate the phylogeny of the wheat tribe (Triticeae), but two tools have been needed—a restriction site map for at least one frequent‐cutting enzyme, and a well‐supported outgroup. This paper presents an EcoRI map, as well as maps for six other restriction enzymes, for two members of the tribe (Elytrigia repens and Secale cereale). These are compared to the maps for Bromus tectorum, a possible outgroup, and for Briza maxima, Lolium perenne, and Festuca rubra, other members of the same subfamily (Pooideae). A unique deletion of ca. 700 base pairs was discovered that provides a distinctive marker for the cytoplasm of plants with the S genome. The deletion also appears in Dasypyrum villosum (V genome). Cladograms based on restriction site data are unequivocal in the support for Bromus tectorum as a near relative of the Triticeae; the chloroplast phylogeny is very similar to the morphological cladogram for a comparable set of taxa.

Tools for Studying the Chloroplast Genome in the Triticeae (Gramineae): An Ecori Map, a Diagnostic Deletion, and Support for Bromus as an Outgroup

Data on variation in the chloroplast genome can be used to estimate the phylogeny of the wheat tribe (Triticeae), but two tools have been needed—a restriction site map for at least one frequent-cutting enzyme, and a well-supported outgroup. This paper presents an EcoRI map, as well as maps for six other restriction enzymes, for two members of the tribe (Elytrigia repens and Secale cereale). These are compared to the maps for Bromus tectorum, a possible outgroup, and for Briza maxima, Lolium perenne, and Festuca rubra, other members of the same subfamily (Pooideae). A unique deletion of ca. 700 base pairs was discovered that provides a distinctive marker for the cytoplasm of plants with the S genome. The deletion also appears in Dasypyrum villosum (V genome). Cladograms based on restriction site data are unequivocal in the support for Bromus tectorum as a near relative of the Triticeae; the chloroplast phylogeny is very similar to the morphological cladogram for a comparable set of taxa.

Experimental Phylogenetics: Generation of a Known Phylogeny

Although methods of phylogenetic estimation are used routinely in comparative biology, direct tests of these methods are hampered by the lack of known phylogenies. Here a system based on serial propagation of bacteriophage T7 in the presence of a mutagen was used to create the first completely known phylogeny. Restriction-site maps of the terminal lineages were used to infer the evolutionary history of the experimental lines for comparison to the known history and actual ancestors. The five methods used to reconstruct branching pattern all predicted the correct topology but varied in their predictions of branch lengths; one method also predicts ancestral restriction maps and was found to be greater than 98 percent accurate.

Computer-aided gene design.

A computer program, which runs on MS-DOS personal computers, is described that assists in the design of synthetic genes coding for proteins. The goal of the program is the design of a gene which (i) contains as many unique restriction sites as possible and (ii) uses a specific codon usage. The gene designed according to the criteria above is (i) suitable for 'modular mutagenesis' experiments and (ii) optimized for expression. The program 'reverse-translates' protein sequences into degenerated DNA sequences, generates a map of potential restriction sites and locates sequence positions where unique restriction sites can be accommodated. The nucleic acid sequence is then 'refined' according to a specific codon usage to remove any degeneration. Unique restriction sites, if potentially present, can be 'forced' into the degenerated nucleic acid sequence by using 'priority codes' assigned to different restriction sequences.

Structural organization of the chloroplast genome of the chromophytic algaVaucheria bursata

The chloroplast genome of the chromophytic alga Vaucheria bursata has been characterized by restriction site and gene mapping analysis. It is represented by a circular molecule 124.6 kb in size. An inverted sequence duplication (IR) not larger than 5.85 kb carries the rRNA genes and separates two single-copy regions of 64.6 kb and 48.3 kb from one another. The Vaucheria plastid genome exists in two equimolar isomers which is due to intramolecular flip-flop recombination within the IR sequences. The coding sites for 21 structural and soluble proteins have been mapped on both single-copy regions using heterologous gene sequences as probes. Although the overall gene order is found to be rearranged when compared with other chromophytic algal and land plant chloroplast genomes, most of the transcriptional units of cyanobacteria and land plant chloroplast genomes appear to be conserved. The phylogenetic implications of these findings are further discussed.

A Chloroplast DNA Phylogeny of the Solanaceae: Subfamilial Relationships and Character Evolution

Phylogenetic relationships among 42 species of Solanaceae representing 12 of the 14 currently recognized tribes were assessed by chloroplast DNA restriction site mapping. Over 1,000 cleavage sites were identified for 10 restriction enzymes and of these, 447 provided information concerning relationships among the included taxa and the outgroup, Ipomoea (Convolvulaceae). The results establish that subfamily Cestroideae is ancestral in the family and is paraphyletic, and that the subfamily Solanoideae is derived from within the Cestroideae and is monophyletic, if it is circumscribed to include Nolana (...)

Restriction Site Mapping of the Chloroplast DNA Inverted Repeat: A Molecular Phylogeny of the Asteridae

By focusing exclusively on the highly conserved inverted repeat region of the chloroplast genome, we extend comparative restriction site mapping to greater evolutionary depths than those to which it has been applied previously. A cladistic analysis of inverted repeat restriction site data is presented in order to enhance understanding of relationships within the Asteridae and to test the possible monophyly of the Asteridae. A total of 114 species, representing 33 families of Asteridae and eight families of Rosidae and Dilleniidae (sensu Cronquist) was examined, of which 99 species exhibited restriction maps of sufficient colinearity to be included in the phylogenetic analysis (...)

Geographic structuring of chloroplast DNA genotypes inTiarella trifoliata (Saxifragaceae)

Tiarella trifoliata comprises varietieslaciniata, trifoliata, andunifoliata, and is distributed from southeastern Alaska to northern California. We analyzed restriction site variation of chloroplast DNA (cpDNA) using 23 endonucleases in 76 populations representing the entire geographic range of the species and the three recognized varieties. We also employed comparative restriction site mapping of PCR-amplified chloroplast DNA fragments using 16 restriction endonucleases. This species exhibits low cpDNA restriction site variation. No differentiation is evident among varieties of this species based on cpDNA data; some plants of each variety were characterized by each of the two major cpDNA types detected. The two major cpDNA clades, which differ by only a single restriction site mutation, are geographically structured. A northern clade comprises populations from Alaska to central Oregon; most populations analyzed from southern Oregon and California form a southern clade. Populations that possess the typical northern cpDNA type also occur disjunctly to the south at high elevations in the Siskiyou—Klamath Mountain area of southern Oregon and northern California. Conversely, the southern cpDNA type is found disjunctly to the north in the Olympic Peninsula of Washington. Both geographic areas characterized by disjunct cytoplasms are considered glacial refugia.Tiarella trifoliata joins two other species,Tolmiea menziesii andTellima grandiflora, in having well-demarcated northern and southern cpDNA lineages. All three species have similar life-history traits and geographic distributions. We suggest that glaciation may have played a major role in the formation of the cpDNA discontinuities present in these three taxa. The pronounced relationship between cpDNA variation and geographic distribution suggests the potential applicability of “intraspecific phylogeography” to plants via the analysis of intraspecific cpDNA variation. These three examples also join a rapidly growing data base which indicates that cytoplasms are often geographically structured within species and species complexes.

Restriction Site Mapping of Subgenus D Adenoviruses, Prototypes 42–47, Intermediate and Atypical Strains

Restriction site maps with the endonucleases BamHI, BgIII, and HindIII were elaborated for 19 adenovirus strains of subgenus D, namely candidates AV42 to 47, 6 intermediate and 7 atypical strains. On the basis of several types mapped by biochemical methods, the physical maps for the 19 strains were constructed by adapting restriction fragments of defined molecular weight to the known maps, as the homology among strains within this subgenus is considerable. Other prototypes mapped by adaptation were also used for reference. The localization of the restriction sites on the genome was analyzed for all mapped types or strains of subgenus D (except AV8). The sites appeared to be randomly distributed; only one site was common to all strains.

Identification of three populations of Ophiostoma ulmi (aggressive subgroup) by mitochondrial DNA restriction-site mapping and nuclear DNA fingerprinting

Three genetically distinct populations of the Dutch elm pathogen Ophistoma ulmi within the aggressive subgroup were defined by the hybridization of a human minisatellite DNA sequence (HVR 33.6) to polymorphic dispersed DNA sequences within the O. ulmi nuclear genomes. For the 10 isolates examined there was a close correlation between nuclear DNA fingerprints and mitochondrial (mt) DNA restriction patterns. A restriction-site map was constructed for the mitochondrial genomes for each of these populations. The three mt DNA maps corresponded to genome sizes of 49.1 (Type I), 49.9 (Type II), and 53.9 (Type III) kilobase pairs (kbp) of DNA. The Type I and Type II mt genomes differed from the Type III mt genome by discrete length mutations of 4.8 and 4.0 kbp, respectively. It is unknown whether these length mutations resulted from insertions into or deletions from a progenitor mitochondrial genome. There was no correlation between the mitochondrial or nuclear genotypes and the geographical source of the isolates.

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

We have analyzed approximately 18 kb of DNA in and upstream of the defective chorion-1 (dec-1) locus of the eight known species of the melanogaster species subgroup of Drosophila. The restriction maps of D. simulans, D. mauritiana, D. sechellia, D. erecta, and D. orena are shown to have basically the restriction map of D. melanogaster, whereas the maps of D. teissieri and D. 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 of D. melanogaster, D. simulans, and D. erecta. Restriction site mapping indicated that the dec-1 gene is highly conserved in the melanogaster 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 the melanogaster species subgroup except that the erecta-orena pair is placed closer to the melanogaster complex than to D. teissieri and D. yakuba.

Typing of human polyomavirus JC virus on the basis of restriction fragment length polymorphisms

JC virus DNA clones from the urine of nonimmunosuppressed Japanese individuals regularly contain an archetypal regulatory sequence which may have generated various regulatory sequences of JC virus isolates from patients with progressive multifocal leukoencephalopathy (PML). In this study, we established 15 new clones from the urine of Dutch, German, and Taiwanese healthy volunteers and patients. Most of these clones contained regulatory sequences essentially identical to the archetypal regulatory sequence. These clones, along with two representative urine-derived clones in Japan and five clones from the brains of PML patients (four established in the United States and one established in Japan), were analyzed with a number of restriction enzymes. We found nine restriction fragment length polymorphisms by which all clones were classified into either of the two types, A and B. Type A contained only clones from the West, while type B contained some from the West and all from eastern Asia. Each type contained both urine-derived and PML-derived clones. Furthermore, there was a close relationship between some urine-derived clones and some PML-derived clones in restriction site mapping analysis. These findings support the adaptation hypothesis which has been postulated to explain the genesis of PML-type JC viruses.

Homoploid Reticulate Evolution in Helianthus (Asteraceae): Evidence from Ribosomal Genes

Phylogenetic relationships among the 21 taxa comprising Helianthus sect. Helianthus and three outgroup species were assessed by restriction site mapping of the 18S-25S nuclear ribosomal RNA gene family. Wagner parsimony analysis of the 41 restriction site or length mutations observed produced a single 59-step most parsimonious tree. This tree was then compared to a cytoplasmic-based plastid phylogeny for this group. Several major discrepancies were observed between the two phylogenies suggesting both recent and ancient introgression. Furthermore, three cases of diploid hybrid speciation are unambiguously documented and a fourth case is suggested. These data are interpreted to suggest that evolution in Heliathus is reticulate rather than exclusively dichotomous and branching.

HOMOPLOID RETICULATE EVOLUTION IN HELIANTHUS (ASTERACEAE): EVIDENCE FROM RIBOSOMAL GENES

Phylogenetic relationships among the 21 taxa comprising Helianthus sect. Helianthus and three outgroup species were assessed by restriction site mapping of the 18S‐25S nuclear ribosomal RNA gene family. Wagner parsimony analysis of the 41 restriction site or length mutations observed produced a single 59‐step most parsimonious tree. This tree was then compared to a cytoplasmic‐based plastid phylogeny for this group. Several major discrepancies were observed between the two phylogenies suggesting both recent and ancient introgression. Furthermore, three cases of diploid hybrid speciation are unambiguously documented and a fourth case is suggested. These data are interpreted to suggest that evolution in Heliathus is reticulate rather than exclusively dichotomous and branching.

Vectors containing infrequently cleaved restriction sites for use in BAL 31 nuclease-assisted and end-label-mediated analysis of cloned DNA fragments

Cloning vectors derived from plasmids pUC8 and pUC18 and phage M13mp10 were constructed so as to have multiple cloning sites (MCS) flanked by the recognition/cleavage sites for the Sfi I and Not I restriction nucleases. Cleavage of vectors containing cloned DNA fragments with either of the infrequently cleaving Sfi I or Not I endonucleases will usually yield linear DNAs cleaved only at the corresponding site in the MCS, so that the cloned insert can be degraded unidirectionally by the duplex exonuclease activity of the BAL 31 nucleases until an amount equal to the length of the vector has been degraded. The ends of the above constructs resulting from cleavage with Not I or Sfi I can readily be labeled, with labeling at only the terminus of the cloned DNA available for the Sfi I site. The BAL 31 nuclease-mediated procedures enhance a previous technique for mapping of restriction enzyme fragments, allow for localization of sequences in cloned segments for which a probe is available, and improve a method for sequencing cloned inserts through the production of sets of nested unidirectional deletions from either end of the parent cloned fragment. The advantages of end-label-mediated restriction site mapping using the above vectors over existing such procedures are also demonstrated.

SYSTEMATIC IMPLICATIONS OF CHLOROPLAST DNA VARIATION IN THE SUBTRIBE MICROSERIDINAE (ASTERACEAE: LACTUCEAE)

Phylogenetic relationships among six of the seven currently recognized genera of the subtribe Microseridinae were examined using comparative restriction site mapping of the chloroplast genome. Complete cleavage maps of 17 enzymes were constructed for 35 of the 46 species in the subtribe and six representative outgroup species from four other subtribes of the Lactuceae. A total of 373 restriction site mutations was detected, 180 of which were phylogenetically informative. The chloroplast DNA mutations were used to generate phylogenetic trees using Dollo, Wagner, and weighted parsimony, and the resulting monophyletic groups were evaluated by the bootstrap method. The results indicate that the Microseridinae is paraphyletic because Stephanomeria is nested within the six core genera of the subtribe. Five of the genera of the Microseridinae are monophyletic, whereas Microseris is paraphyletic because one of its species, M. lindleyi, is more closely related to Agoseris and Nothocalais. The distant relationship of M. lindleyi to the rest of the species of Microseris is enigmatic, especially in view of its known involvement in the origin of two allotetraploid species, M. decipiens and M. heterocarpa. The taxonomic implications of these results lead to a revised classification of Microseris in which the genus Uropappus is revived to accommodate M. lindleyi, and a new genus, Stebbinsoseris, is described to include the allotetraploids. The cpDNA phylogeny is used to evaluate the patterns of character evolution within the Microseridinae, as well as to test previous hypotheses that differences in generation time result in unequal rates of chloroplast DNA evolution.