Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

The genome of the cellular slime mold Dictyostelium discoideum has been analyzed by limit digestion with EcoR1 restriction endonuclease. Approximately 15% of the nuclear genome is cleaved into nine discrete fragments as analyzed by agarose gel electrophoresis. These fragments appear to be derived from two nuclear buoyant density satellites, one of which contains sequences coding for ribosomal RNA. The bulk of the nuclear DNA is digested into approximately 7000 fragments with a mean molecular weight of 4 × 10⁶ to 5 × 10⁶. The mitochondrial DNA is digested into four fragments. One of the nuclear bands has been cloned in Escherichia coli using plasmid pSC101 carrying tetracyline resistance. Analysis by renaturation kinetics indicates that it is repeated approximately 200 times per haploid genome and that it is not internally repeated.     

A physical map of the genome of Ureaplasma urealyticum 960T with ribosomal RNA loci.

A physical map is presented for the 900 kilobase pair genome of Ureaplasma urealyticum 960T, locating 29 sites for 6 restriction endonucleases. The large restriction fragments were separated and sized by pulsed-field agarose gel electrophoresis (PFGE). Their locations on the map were determined by probing Southern blots of digests with individual fragments isolated from other digests and by correlating the products of double digestions and partial digestions. An end-labelling technique was used to detect small fragments not readily observed by PFGE. Two loci for rRNA genes have been determined by probing with cloned DNA.     

The genome of Bacillus subtilis phage SPP1: the arrangement of restriction endonuclease generated fragments.

Summary SPP1 DNA was cleaved by the restriction endonucleases, BglI, BglII, EcoRI, KpnI, SmaI, and SalI. The molecular weights of the DNA fragments obtained by single enzyme digestion or by consecutive digestion with two enzymes were determined by electron microscopic measurements of contour length and by gel electrophoresis. The major fragments from the six digests could be ordered to give a consistent restriction map of SPP1. The electropherograms of several digests indicated that certain fragments occurred in less than stoichiometric amounts or were heterogeneous in size. Such bands carried a major part of radioactivity, when SPP1 DNA was terminally labelled with P³² prior to degradation by restriction enzymes. These results, and studies of the effect of exonuclease III treatment on restriction enzyme patterns define the terminal restriction fragments. All data obaained support the conclusion drawn in the preceding paper (Morelli et al., 1978b) that the SPP1 genome is terminally redundant and partially circularly permuted.Part of this work is from the doctoral dissertations to be submitted to Stanford University¹ and the Freie Universität Berlin²     

Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25·10⁶ to 45·10⁶ and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45·10⁶. This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with a molecular weight of 45·10⁶. We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRi, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been deduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA.Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×10⁶ to 2.3×10⁶ base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.     

The chloroplast genome of Carthamus tinctorius

Summary A physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using Sall, Pstl, Kpnl and HindIII restriction endonucleases. Southern blots to single and double digests by these enzymes were hybridized with ³²P-dCTP nick-translated Kpnl probes, which were individually isolated from agarose gels. The plastid genome was found to be circular (151 kbp), to contain a repeated sequence of about 25 kbp, and to have small and large single copy regions of approximately 20 and 81 kbp, respectively. Heterologous probes from spinach and Euglena containing psbA, rbcL, atpA or rrnA structural genes were also hybridized with such single and double restriction enzyme digests and mapped on this circular chlorpolast genome. The genetic map was found to be co-linear with that of spinach and many other higher plants.     

Construction and mapping of safflower chloroplast DNA recombinants and location of selected gene markers

Summary DNA was isolated and purified from chloroplasts of safflower (Carthamus tinctorius L.), digested with HindIII restriction endonuclease, and ligated into the HindIII site of the plasmid pUC9. Recombinant DNAs were isolated from ampicillin resistant white colonies which grew in the presence of the appropriate indicator, digested with HindIII, and then identified by comparison of agarose gel electrophoretic mobilities. HindIII digests of chloroplast DNA were used as a standard. Such recombinants were radiolabeled and hybridized with Southern blots of PstI, SalI, KpnI, and HindIII single and double digests of safflower chloroplast DNA. A physical map was subsequently generated showing the location of each recombinant on the circular plastid genome. Recombinants containing heterologous chloroplast gene markers from spinach or Euglena were also radiolabeled and mapped. The relative mapping positions of these genes are in good agreement with those which have previously been published for spinach and several other higher plants.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Mapping of transfer RNA genes on tobacco chloroplast DNA

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.     

Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes

A restriction map of the 2.8-Mb genome of the unicellular eukaryote Encephalitozoon cuniculi (phylum Microspora), a mammal-infecting intracellular parasite, has been constructed using two restriction enzymes with 6 bp recognition sites (BssHII and MluI). The fragments resulting from either single digestions of the whole molecular karyotype or double digestions of 11 individual chromosomes have been separated by two-dimensional pulsed field gel electrophoresis (2D-PFGE) procedures. The average distance between successive restriction sites is ~19 kb. The terminal regions of the chromosomes show a common pattern covering ~15 kb and including one 16S–23S rDNA unit. Results of hybridisation and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies on each chromosome. We have also located 67 DNA markers (clones from a partial E.cuniculi genomic library) by hybridisation to restriction fragments. Partial or complete sequencing has revealed homologies with known protein-coding genes for 32 of these clones. Evidence for two homologous chromosomes III, with a size difference (3 kb) related to a subtelomeric deletion/insertion event, argues for diploidy of E.cuniculi. The physical map should be useful for both the whole genome sequencing project and studies on genome plasticity of this widespread parasite.     

A physical map of the genome of Mycoplasma mycoides subspecies mycoides Y with some functional loci.

A physical map is presented for the 1200 kb genome of Mycoplasma mycoides subsp. mycoides Y, locating 32 cleavage sites for 8 restriction endonucleases. The large restriction fragments involved were separated and sized by pulsed-field agarose gel electrophoresis. Their locations on the map were determined by probing Southern blots of digests with individual fragments isolated from other digests and by correlating the products of double and triple digestions. Loci for 2 ribosomal RNA operons and 2 tRNA operons have been determined by probing with cloned genes and the broad regions of the replication origin and terminus have also been outlined by in vivo labelling studies.     

Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome

The restriction enzyme DraI cleaves the Caulobacter crescentus genome into at least 35 fragments which have been resolved in agarose gels using pulsed-field-gradient gel electrophoresis (PFGE). When digests were performed using DNA from strains containing Tn5 insertion mutations, altered band migrations were observed. Using PFGE with the appropriate pulse times, size differences as small as 2% could be resolved in large fragments. Using this approach, we have constructed a partial physical map of the genome which correlates well with the C. crescentus genetic map and have shown the size of the genome to be approx. 3800 kb. Using hybridization with cloned genes, we have determined the map locations of five previously unmapped genes. In addition, we have shown that PFGE can be used to rapidly determine the map locations of new insertion mutations or the sizes of deletion mutations.     

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton     

Characterization of an entomopoxvirus of the lesser cornstalk borer (Elasmopalpus lignosellus)

A biochemical and limited morphological characterization of an entomopoxvirus infecting the lesser cornstalk borer, Elasmopalpus lignosellus, was made. The oval virions measure 270 × 200 nm and the spheroids average 1.5 μm in diameter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis elucidated 32 structural polypeptides with molecular weights ranging from 13,000 to 145,000. The viral genome was examined with the restriction endonuclease EcoRI. Gel electrophoresis of the digested DNA yielded 26 bands and a total molecular weight of 140.8 × 10⁶.     

A physical map of the genome of ethanol fermentative bacterium Zymomonas mobilis ZM4 and localization of genes on the map

A physical map of the Zymomonas mobilis ZM4 genome has been constructed from the results of reciprocal Southern hybridization with PmeI, PacI, and NotI-digested genomic DNA fragments and linking cosmid clones. Restriction enzyme-digested Z. mobilis ZM4 genome was electrophoresed with phage lambda DNA concatemers as a size standard in a Bio-Rad CHEF-DRII pulsed-field gel electrophoresis (PFGE) system. The restriction enzyme PmeI generated 15 fragments (3–625 kb), and PacI produced 19 fragments (7–525 kb). Each size of restriction fragment was calculated by comparison to the size of phage lambda DNA concatemers, and the genome size of Z. mobilis ZM4 was estimated to be 2085.5 kb. The 19 known genes and three rrn operons were localized on the map.