Characterization and comparison of mitochondrial DNAs and rRNAs from Penicillium urticae and P. chrysogenum.

Mitochondrial DNA (mt DNA) from a patulin producer, Penicillium urticae (synonym P. griseofulvum), was 27.8 kb +/- 0.6 kb in size by electron microscopy and 27.2 kb by agarose gel electrophoresis. Restriction endonuclease maps for nine restriction enzymes were constructed, and eleven fragments which covered the total range of the mt DNA were cloned into the Escherichia coli plasmid vector pUC19. Southern analysis of the native genomes of P. urticae and P. chrysogenum with six of the cloned fragments as probes indicated similar genome arrangements as well as similar restriction maps. Both the large and small rRNA genes of P. urticae and P. chrysogenum were located on these restriction maps using Southern hybridization, and the result also supported the similar arrangement. Agarose/formaldehyde gel electrophoresis indicated that the small rRNA was 1.5 kb in size in both species; but, surprisingly, the large rRNA was 4.2 kb in size for P. urticae and 3.5 kb for P. chrysogenum. These sizes were, respectively, 1.1 kb and 0.4 kb larger than those from the very closely related Aspergillus nidulans.     

Comparison of the Restriction Endonuclease Digestion Patterns of Mitochondrial DNA from Normal and Male Sterile Cytoplasms of ZEA MAYS L

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.—The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

A new linear DNA plasmid isolated from the yeast Saccharomyces kluyveri

Summary A new linear DNA plasmid, designated pSKL, was found in the yeast Saccharomyces kluyveri. Restriction maps of the 14.2 kb plasmid were constructed. By the use of CsCl-Hoechst 33258 centrifugation containing guanidine chloride, pSKL was isolated as a DNA-protein complex. The protein was associated with the terminal regions of pSKL. The two terminal EcoRI fragments of pSKL were cloned and their nucleotide sequences were determined. pSKL had inverted terminal repeats of 483 bp with a unique structure in which fairly homologous sequences of 30 bp were repeated eight times.     

Isolation and characterization of rat ribosomal DNA clones

Four EcoRI fragments, which contain the transcribed portion of the rat rDNA repeat, have been isolated from a rat genome library cloned in lambda Charon 4A vector. Three of the fragments, 9.6, 6.7, and 4.5 kb, from clones lambda ChR-B4, lambda Nr-42, and lambda ChR-C4B9, contained part of the 5'-NTS, the 5'-ETS, 18S rDNA, ITS-1, 5.8S rDNA, 28S rDNA and approximately 3.5 kb of the 3'-NTS. Two EcoRI fragments, from clones lambda ChR-B4 and lambda ChR-B7E12, which coded for the 5'-NTS, the ETS, and most of the 18S rDNA, differed by 1 kb near the EcoRI site upstream of the 5' terminus of 18S rRNA. Restriction maps of the cloned DNA fragments were constructed by cleavage of the fragments with various restriction endonucleases and Southern hybridization with 18S, 5.8S, and 28S rRNA. These maps were confirmed and extended by subcloning several regions of the repeat in pBR322.     

Cloning of human mitochondrial DNA in Escherichia coli

In order to determine its nucleotide sequence, human mitochondrial DNA (mtDNA) purified from term placentae was cloned in Escherichia coli using the plasmid vector pBR322. The products of an mtDNA MboI digestion (23 fragments ranging in size from 2800 to 25 base-pairs (bp)) were ligated with BamHI-cut pBR322. The ampicillin-resistant tetracycline-sensitive colonies obtained upon transformation of E. coli χ1776 were screened by agarose gel electrophoresis of colony lysates, colony hybridization and restriction analysis. All but MboI fragment 2 were obtained in this way. MboI fragments 5 and 8 were each found only once among the 705 clones screened. All other MboI fragments were approximately equally represented in the population of clones except for a slight bias towards smaller fragments. MboI fragment 2 overlaps with the mtDNA BamHI/EcoRI (1.7 kb³) and the 0.9 kb HinIII fragments. These were cloned in similarly restricted pBR322 to provide a set of clones covering most of the mtDNA molecule. Clones representative of each MboI fragment were shown to be complementary to mtDNA by hybridization to Southern blots of mtDNA digests and were thereby partially mapped. Further mapping was obtained by restriction analysis of mtDNA sequentially degraded by exonuclease III. A collection of recombinant clones has thus been obtained using the mtDNA isolated from a single placenta and is now being used to obtain a complete nucleotide sequence of human mtDNA.     

The origin and genetic differentiation of native breeds of pigs in southwest China: An approach from mitochondrial DNA polymorphism

Mitochondrial DNA (mtDNA) of six breeds of native domestic pigs from Yunnan province, southwest China, and two wild boars obtained from Sichuan, China, and Vietnam was analyzed using 20 restriction endonucleases that recognize six nucleotides. Restriction maps were made by double-digestion methods and polymorphic sites were located on the map. According to their mtDNA restriction types, all the breeds were classified into six groups. Genetic distances among groups were calculated to define their phylogenetic relationships. The relationship between the Sichuan wild boar and domestic pigs is close, while the Vietnamese wild boar is relatively far from them, so the domestic pigs in southwest China are likely to have originated from a wild pig which distributed in west China. We compare our results with previous reports in literature and discuss the relationship among Chinese pigs, Japanese pigs, and European pigs. The mtDNA cleavage pattern of the Mingguang pig digested byEcoRV was identical to that of Duroc; mutations at theEcoRI site, detected in the mtDNA of two Dahe pigs, are the same as in the Vietnamese wild boar, suggesting that mutational hot spots exist in the mtDNA of pigs.     

Induced expression of <Emphasis Type="Italic">Eco</Emphasis>RI endonuclease as an active maltose-binding fusion protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.     

The use of restriction endonucleases to compare mitochondrial dna sequences in Mus musculus: A detailed restriction map of mitochondrial DNA from mouse L cells

The complete mitochondrial genome of a chloramphenicol-resistant, oligomycin-resistant mouse L cell has been cloned in E. coli. Using fragments of this DNA, purified by preparative agarose gel electrophoresis, we have mapped 139 restriction sites cleaved by 15 endonucleases. We have then compared the positions of these sites with those found in mtDNA purified from several other L-cell lines, from the tissue of several strains of laboratory mice, and from a plasmid containing mtDNA of a single, wild Mus musculus sample. The results indicate that all of the L-cell mtDNAs are identical except at a single EcoRI site, and that the L-cell sequence is identical to that found in mtDNA from normal tissue. They also suggest that mtDNA sequences found in North American Mus musculus populations may be much more homogeneous than expected from analysis of other rodents.     

Circular Permutation of the Genome of a Temperate Bacteriophage from Streptococcus cremoris BK5

The temperate bacteriophage BK5-T was isolated from Streptococcus cremoris BK5 by induction with mitomycin C. Electron microscopy revealed that BK5-T DNA consists of linear molecules, ranging in size from 39.7 to 46 kilobase pairs. Restriction analysis of self-ligated BK5-T DNA showed that the ends of the DNA were not cohesive. The EcoRI restriction fragments of the phage genome were cloned into pACYC184. Restriction enzyme analysis of both the phage DNA and the cloned EcoRI fragments with EcoRI, BstEII, PstI, ClaI, and XbaI yielded a 37.6-kilobase-pair-long circular restriction map for the phage genome. It was concluded that the BK5-T DNA molecules in the population differ in their sequence by a circular permutation and that individual DNA molecules are terminally redundant. The map location of the sites at which packaging of BK5-T DNA into phage heads is initiated (pac) and at which the phage integrates into the bacterial chromosome (att) were established.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

Physical Mapping of Recombinant Plasmids Containing Broad Bean Chloroplast Ribosomal RNA Genes

Two BamHl fragments containing broad bean chloroplast rRNA genes were cloned using the bacterial plasmid pBR322 as a vector and Escherichia coli HB101 as host bacterial. Physical maps of the two cloned ct DNA BamHI fragments containing rRNA genes were constructed by cleavage with several restriction endonucleases and Southern blot hybridization with E. coli 16S-23S rRNAs. Recombinant plasmids pVFBI6 and pVFB32 contain a 16S rRNA sequence on the 4.70 kb BamHl fragment, a 23S rRNA sequence and 4.5S/5S rRNA sequences on the 5.65 kb BamHl fragment, respectively.     

Complex organization of zein genes in maize

We have examined the fragments of maize nuclear DNA that are homologous to three cloned cDNAs prepared from zein mRNA. Southern blots of high molecular weight ( > 40 kb) maize nuclear DNA cleaved with BamHI, HindIII or EcoRI were hybridized to three zein cDNA plasmid probes. Multiple restriction fragments in a wide range of size classes were observed to hybridize with all three probes. Our results indicate the occurrence of families of genes in the maize genome that are related by their sequences to a given zein mRNA sequence.     

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.     

A restriction endonuclease map of Streptomyces phage VWB

Summary A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed.Abbreviations dam DNA adenine methylase activity - kb kilobase pairs - :: novel joint     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

Restriction enzyme mapping of ribosomal DNA ofSchistosoma spindale andS. leiperi (Digenea) and its application to interspecific differentiation

Restriction maps were determined for the ribosomal RNA gene complex ofSchistosoma spindale andS. leiperi. The restriction map of theS. spindale rRNA gene complex was found to differ from those of other schistosomes previously described by the presence of an additionalEcoRI site in the non transcribed spacer region. In common withS. mattheei andS. margrebowiei, bothS. leiperi andS. spindale appear to have insertions in the transcribed spacer region, relative toS. mansoni.EcoRI digests of genomic DNA, probed with pSM 889, enabled differentiation ofS. spindale andS. leiperi from four other species of schistosome.