Restriction endonuclease digestion patterns of harvest mice (Reithrodontomys) chromosomes: a comparison to G-bands, C-bands, and in situ hybridization.

Constitutive heterochromatin of a karyotypically conserved species of harvest mouse was compared to that of three karyotypically derived species of harvest mice by examining banding patterns produced on metaphase patterns produced by two of these restriction endonucleases (EcoRI and MboI) were compared to published G- and C-banded karyotypes and in situ hybridization of a satellite DNA repeat for these taxa. The third restriction endonuclease (PstI) did not produce a detectable pattern of digestion. For the most part, patterns produced by EcoRI and MboI can be related to C-banded chromosomes and in situ hybridization of satellite DNA sequences. Moreover, digestion with EcoRI reveals bands not apparent with these other techniques, suggesting that restriction endonuclease digestion of metaphase chromosomes may provide additional insight into the structure and organization of metaphase chromosomes. The patterns produced by restriction endonuclease digestion are compatible with the chromosomal evolution of these taxa, documenting that in the highly derived taxa not only are the chromosomes rearranged but the abundance of certain sequences is highly variable. However, technical variation and difficulty in producing consistent results even on a single slide with some restriction endonucleases documents the problems associated with this method.     

Physical mapping of plastid DNA variation among eleven Nicotiana species

Summary Plastid DNA of seven American and four Australian species of the genus Nicotiana was examined by restriction endonuclease analysis using the enzymes Sal I, Bgl I, Pst I, Kpn I, Xho I, Pvu II and Eco RI. These endonucleases collectively distinguish more than 120 sites on N. tabacum plastid DNA. The DNAs of all ten species exhibited restriction patterns distinguishable from those of N. tabacum for at least one of the enzymes used. All distinctive sites were physically mapped taking advantage of the restriction cleavage site map available for plastid DNA from Nicotiana tabacum (Seyer et al. 1981). This map was extended for the restriction endonucleases Pst I and Kpn I. In spite of variation in detail, the overall fragment order was found to be the same for plastid DNA from the eleven Nicotiana species. Most of the DNA changes resulted from small insertions/deletions and, possibly, inversions. They are located within seven regions scattered along the plastid chromosome. The divergence pattern of the Nicotiana plastid chromosomes was strikingly similar to that found in the genus Oenothera subsection Euoenothera (Gordon et al. 1982). The possible role of replication as a factor in the evolution of divergence patterns is discussed. The restriction patterns of plastid DNA from species within a continent resembled each other with one exception in each instance. The American species N. repanda showed patterns similar to those of most Australian species, and those of the Australian species N. debneyi resembled those of most American species.Abbreviations ims isonuclear male sterile - ptDNA plastid chloroplast DNA - Rubisco ribulosebisphosphate carboxylase/oxygenase - kbp kilobase pairs - LSU large subunit of Rubisco     

The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [¹⁴C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number of short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding.It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.     

Moused-amino-acid oxidase gene: Restriction fragment length polymorphism among mouse strains

Summary Genomic DNA was extracted from mice of 15 strains (A/J, AKR, BALB/c, C3H/He, C57BL/6, CBA/J, CD-1, CF#1, DBA/2, ddY/DAO⁺, ddY/DAO^–, ICR, NC, NZB and NZW) for the examination of the difference in the structure of thed-amino-acid oxidase gene among the mouse strains. The DNAs were digested with restriction endonucleases and analyzed by Southern hybridization usingd-amino-acid oxidase cDNA as a probe. The 15 strains showed the same hybridization patterns in theEcoRV,BamHI orBglII digestion. In theEcoRI digestion, the DBA/2 strain showed a different hybridization pattern from the other 14 strains. In thePvuII andXbaI digestion, C3H/He, CBA/J, ddY/DAO⁺ and NC strains were different from the other 11 strains. In thePstI andHindIII digestion, restriction fragment length polymorphisms were observed, and the 15 strains were classified into four groups according to their hybridization patterns. These results indicate that the 15 strains of mice carry a structurally similard-amino-acid oxidase gene, but there is a variation in its inside sequence among the groups of the strains.     

Restriction fragment length polymorphism (RFLP) of Salmonella organisms

Genetic relatedness of 20 Salmonella isolates comprising 16 serotypes was analysed by restriction endonuclease digestion of the total DNA with six endonucleases individually. The rDNA fingerprints generated by EcoRI were more polymorphic, each serotype showed a unique fingerprint sharing several core (monomorphic) bands with several polymorphic bands. Eight characteristic NciI rDNA fingerprints were found. Similar rDNA RFLP patterns were observed in strains of Salmonella from different serotypes.S.I. Koh-Luar and E. Lau are with the Department of Chemical Process & Biotechnology, Singapore Polytechnic, 500 Doyer Road, Singapore 139651. Republic of Singapore; S. T. Chew is with the Veterinary Public Health Laboratory (VPHL), Primary Production Department (PPD), 51 Jalan Buroh, Singapore 619415. Republic of Singapore. S.B. Chua is with the Veterinary Public Health Division, Primary Production Department, 5 Maxwell Road, No. 03-00, Tower Block, MND Complex, Singapore 069110. Republic of Singapore.     

Physical mapping of BglII, BamHI, EcoRI, HindIII and PstI Restriction fragments of bacteriophage P1 DNA

Summary A cleavage map of bacteriophage P1 DNA was established by reciprocal double digestion with various restriction endonucleases. The enzymes used and, in parenthesis, the number of their cleavage sites on the P1clts genome are: PstI (1), HindIII (3), BglII (11), BamHI (14) and EcoRI (26). The relative order of the PstI, HindIII and BglII sites, as well as the order of 13 out of the 14 BamHI sites and of 17 out of the 26 EcoRI sites was determined. The P1 genome was divided into 100 map units and the PstI site was arbitrarily chosen as reference point at map unit 20.DNA packaging into phage heads starts preferentially at map unit 92 and it proceeds towards higher map units. The two inverted repeat sequences of P1 DNA map about at units 30 and 34.     

Molecular genetics of rotifers: preliminary restriction napping of the mitochondrial genome of Brachionus plicatilis

Methods are presented to extract and purify mitochondrial DNA from the rotifer Brachionus plicatilis. The mtDNA obtained is of sufficient purity for digestion with restriction endonucleases. EcoR I restriction patterns are presented for 4 geographically separated clones. A restriction map based on digestion with 5 different restriction enzymes is included for one of these clones. Finally, use of mtDNA analysis for studies on the population structure and biogeography of rotifers is discussed.     

AFLP markers in a molecular linkage map of maize: codominant scoring and linkage group ditsribution

We exploited the AFLP technique to saturate a RFLP linkage map derived from a maize mapping population. By using two restriction enzyme, EcoRI and PstI, differing in methylation sensitivity, both in combination with MseI, we detected 1568 bands of which 340 where polymorphic. These were added to the exitsing RFLP marker data to study the effects of incorporation of AFLPs produced by different restriction-enzyme combinations upon genetic maps. Addition of the AFLP data resulted in greater genome coverage, both through linking previously separate groups and the extension of other groups. The increase of the total map length was mainly caused by the addition of markers to telomeric regions, where RFLP markers were poorly represented. The percentage of informative loci was significantly different between the EcoRI and PstI assays. There was also evidence that PstI AFLP markers were more randomly distributed across chromosomes and chromosome regions, while EcoRI AFLP markers clustered mainly at centomeric regions. The more-random ditsribution of PstI AFLP markers on the genetic map reported here may reflect a preferential localisation of the markers in the hypomethylated telomeric regions of the chromosomes. Received: 22 December 1998 / Accepted: 25 March 1999     

Cartographie de Restriction Comparée du Satellite DNA chez l'Homme et le Chimpanzé

Highly repeated DNA satellite α sequences from man and chimpanzee (Pan troglodytes) have been compared, using restriction endonucleases. The two species share a 340 base pairs tandemly represented DNA, that is cut once by EcoRt. Pan troglodytes differ from man by loss of the two MboI and EcoRI star sites and by the gain of an Hae III site in the repeated sequence.     

The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae,Chlorophyta)

A physical restriction map of the mitochondrial genome from one clone (TCC 854) of the sexually isolated populations (syngens) of the morphologically uniform species Pandorina morum Bory has been constructed using restriction endonucleases Ava I, Bam HI, Bgl II, Eco RI, Kpn I, and Pst I. The 20 kb linear genome can easily be separated from plastid DNA, nuclear satellite rDNA, and main band (nuclear) DNA on a Hoechst/CsCl buoyant density gradient. The Pandorina mitochondrial DNA shows sufficient similarity to the 16 kb mitochondrial genome of Chlamydomonas reinhardtii to cross-hybridize, and also hybridizes with a probe containing maize mitochondrial 18S rRNA genes. Double digests, self-probing, and Bal31 exonuclease experiments suggest that 1.8 to 3.3 kb of sequence is repeated at each end of the genome as an inverted repeat. Mitochondrial genome sizes of other P. morum syngens were found to range from ca. 20 to ca. 38 kb. The mitochondrial genome should be valuable for taxonomic studies; it can be used for comparative organellar studies; and it should be of interest to compare with that of other plant and animal mitochondrial genomes.     

Heterogeneity in the concerted evolution process of a tandem satellite array in meadow mice (Microtus)

The evolutionary history of a 160-bp tandem satellite array, originally described from Microtus chrotorrhinus and called MSAT-160, was examined in related species of arvicolid rodents by sequence analyses, quantitative dot blotting, and Southern blotting. Results indicate that MSAT-160 is present in 12 of the 20 species and subspecies of Microtus assayed, but not in species belonging to any of the eight other genera examined. DNA from each species containing MSAT-160 was digested with 12 restriction endonucleases and restriction patterns were obtained reflecting the variable extent of homogenization of any given variant in different species. For example, with MboI digestion, M. chrotorrhinus produced a type A ladder pattern where most monomers contain the restriction site, M. ochrogaster generated a type B pattern where most monomers lack the site, and M. agrestis yielded a pattern intermediate between the A and B types. Further, dot blotting revealed copy-number differences between species. These findings indicate that changes in the periodic structure and amount of satellite DNA have occured since these species last shared a common ancestor. In addition, various species-pacific patterns were documented, illustrating that mechanism other than genomewide homogenization, such as stochastic mutation, out-of-register crossing over, deletion, and random amplification also play a role in structuring tandem arrays. Stochastic mutation and homogenization rates in satellite DNA, levels of species diversity, and magnitudes of chromosomal divergence differ significantly in Microtus, Mus, and Ctenomys, the three rodent lineages examined.     

On the possibility of localizing in situ Mus musculus and Drosophila virilus satellite DNAs by Alu I and Eco RI restriction endonucleases

Mus musculus and Drosophila virilus metaphase chromosomes have been treated with Alu I or Eco RI restriction endonucleases and, to ascertain possible selective digestion, subsequently stained with the DNA-specific dye ethidium bromide. The correlation between our findings and previously known cytological and biochemical data allowed us to show that Alu I is an enzyme capable of cytologically detecting repetitive DNAs, while Eco RI is unable to do so. This would be a consequence of the fact that Alu I extensively digests and extracts all chromosomal DNA except that localized in those regions where the presence of satellite DNAs has previously demonstrated. Eco RI, on the contrary, is not capable of doing so and its activity seems to be obstructed by alcohol:acid fixation procedures.     

Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2

Summary The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, Bam H-I, Sal I and Hpa I. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.     

Cloning of bacteriophage PM2 DNA in Escherichia coli K12.

Summary DNA fragments of phage PM2 restricted with Hin dIII endonuclease was cloned in the vector pBR 322 in an Escherichia coli K 12 host. The attempt to clone full length PM2 DNA restricted with PstI endonuclease has been unsuccesful. From six randomly chosen recombinant clones DNA was purified and analysed with EcoRI, PstI and HindIII endonucleases. The physical map of three chimeric plasmids was unequivocally established. It was shown, that the whole PM2 genome was cloned, although in separate fragments. However, most of the recombinant clones were instable in the absence of selective pressure.     

Restriction endonuclease resistant chromatin in human chromosomes

Summary The recent addition of restriction endonucleases in obtaining selective bands in the human genome has added a new dimension to molecular genetics. However, a considerable discrepancy exists in banding patterns produced by AluI in chromosomes 19 and 20, by MboI in chromosomes 4, 5, 8, 21 and 22 and by RsaI in chromosomes 12, 21 and 22. The principal causes of these differences are highlighted.     

Heteroplasmy of the chloroplast genome of Medicago sativa L. cv ‘Regen S’' confirmed by sequence analysis

The heteroplasmy of chloroplast DNA (cpDNA) observed in Medicago sativa L., which involves the presence (type B) or absence (type A) of an Xba I restriction site, was examined using closed fragments covering the variable XbaI site from type-A and type-B cpDNA. The 6.2-kb PstI fragment of DNA from type-A cpDNA (–XbaI) and from type-B cpDNA (+XbaI) was cloned into pUC19 plasmids. EcoRI fragments bearing the variable XbaI site from the type-A and type-B 6.2-kb PstI fragments were subcloned into pUC19. DNA sequences of both types of the 696-bp EcoRI fragments were determined and computer-assisted analysis of the sequence data carried out. Type-A cpDNA was found to differ from type-B cpDNA by 1 base, a G to T conversion, which results in a non-recognition site for XbaI in the type-A cpDNA. The sequence difference was in a non-coding region. Cloning and sequencing of the fragments verified the individual identity of the type-A and type-B cpDNA.     

Chloroplast DNA differences between cultivated hop,Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.     

Establishment of AFLP technique and assessment of primer combinations for mei flower

Mei flower is one of the most famous ornamental flowers in eastern Asia for its blossoming in early spring. Amplified fragment length polymorphism (AFLP) is one of the most frequently used techniques for analysis of genetic variation and is used herein for the first time inPrunus mume. This research provides a detailed and modified AFLP protocol for Mei genomic DNA digested withEcoRI/PstI restriction endonuclease combinations. The 10 best primer pairs of high polymorphism were screened from 256 primer combinations that could reliably and repetitively distinguish 14 Mei samples and would be suitable for genetic analysis of more cultivars. Ten primer pairs produced up to a total of 524 AFLP bands and up to 233 polymorphic bands. The ratio of polymorphic bands scoped from 35.71% to 59.67%, and the average ratio was 44.46% in the 10 primers. AFLP is an effective, inexpensive, and timesaving technique for the genetic differentiation of the Mei cultivars, as evidenced in this study.     

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

Towards an integrated linkage map of common bean

Summary Two genomic libraries were established to provide markers to develop an integrated map combining molecular markers and genes for qualitative and quantitative morpho-agronomic traits in common bean. Contrasting characteristics were observed for the two libraries. While 89% of the PstI clones were classified as single-copy sequences, only 21% of the EcoRIBamHI clones belonged in that category. Clones of these two libraries were hybridized against genomic DNA of nine genotypes chosen according to their divergent evolutionary origin and contrasting agronomic traits. Eight restriction enzymes were used in this study. PstI clones revealed 80–90% polymorphism between the Andean and Middle American gene pools and 50–60% polymorphism within these gene pools. However, under the same conditions only 30% of the EcoRI-BamHI clones showed polymorphism between the Middle American and Andean gene pools. Hybridization with PstI clones to EcoRI-, EcoRV-, or HindIII-digested genomic DNA resulted in a cumulative frequency of polymorphism of approximately 80%. Hybridizations to BamHI-, HaeIII-, HinfI-, PstI-, and XbaI-digested genomic DNA detected no additional polymorphisms not revealed by the former three enzymes. In the PstI library, a positive correlation was observed between the average size of hybridizing restriction fragments and the frequency of polymorphism detected by each restriction enzyme. This relationship is consistent with the higher proportion of insertion/deletion events compared with the frequency of nucleotide substitutions observed in that library.