Polymorphisms of mitochondrial DNAs in Norway rats (Rattus norvegicus): Cleavage site variations and length polymorphism of restriction fragments

Summary Extensive polymorphism was found in mitochondrial DNAs (mtDNAs) of Norway rats (Rattus norvegicus). The restriction endonuclease cleavage patterns of mtDNAs of laboratory rats, wild rats, tumor cells, and culture cells were compared The polymorphism is defined by two criteria; one is cleavage site variation and the other is length polymorphism of restriction fragments. The cleavage site variation may be caused by point mutation, and the length polymorphism by sequence deletions or insertions. At least five types, types A-E, were identified by cleavage site variations, and two groups, a and b, were identified by length polymorphism of one HpaII fragment, Hpa5. All types except type C belonged to either group-a or group-b, whereas both groups were found in type C. Differentiation of polymorphic Norway rat mtDNA types and the experimental use of the polymorphism are discussed.     

The heterogeneity of rat-liver mitochondrial DNA

Two types of mitochondrial DNA (mtDNA) can be distinquished in an inbred strain of rats of the Wistar type. The population of DNA molecules of the liver of one single rat is homogeneous. This was shown for a number of 100 animals and confirms the data of other investigators. The two types of mitochondrial DNA, designated A and B, differ in their number of cleavage sites for the restriction endonucleases Eco RI (2sites), Hind II (1 site) and Hha I (1 site). No differences were found for the restriction enzymes Bam HI, Hap II, Hind III and Hpa I. The degree of sequence divergence of the two types of DNA is calculated to be roughly 5% on the basis of these observations. From 20 rats part of the liver was taken and the mtDNA was characterized. Heterologous and homologous crosses between type A and type B rats were made. Analysis of the offspring revealed strictly maternal inheritance of the A and B mtDNA traits. For purposes of base-sequence analysis and RNA.DNA hybridization the strain could easily be "purified" genetically.     

Further studies on types A and B rat mtDNAs: cleavage maps and evidence for cytoplasmic inheritance in mammals.

We present here the cleavage maps of A and B rat mtDNA, examining particularly the legitimacy of two small recently reported EcoRI fragments. We find that the 0.13-kb fragment belongs in the map whereas the 0.09-kb fragment is of nonmitochondrial origin. A comparison of the types A and B maps shows that no major rearrangements have occurred since the time of the evolutionary segregation of the two DNAs. These data plus fragment patterns from other restriction enzyme digests, including those from additional enzymes which display differences between the two DNA types, provide no evidence for microheterogeneity in either of the mtDNA types, and also permit an estimate of 0.94 to 1.80% for the extent of sequence divergence of the two types. Nine enzymes yielded fragments all of which could be accurately sized leading to a reasonably accurate estimate of 15.85 kb for the size of rat liver mtDNA of either type. In a test of whether cytoplasmic inheritance is operative in mammals, reciprocal cross experiments with the two DNAs showed that only the female parental mtDNA was inherited with no evidence for the persistence of the male parental mtDNA in any of three tissues examined.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.     

Evidence for gene flow in parasitic nematodes between two host species of shrews

We describe the genetic structure of populations of the intestinal nematode Longistriata caudabullata (Trichostrongyloidea: Heligmosomidae), a common parasite of short-tailed shrews (genus Blarina, Insectivora: Soricidae). Parasites and hosts were collected from a transect across a contact zone between two species of hosts, Blarina brevicauda and B. hylophaga, in central North America. An 800-base pairs (bp) fragment of the ND4 mitochondrial DNA (mtDNA) gene was sequenced for 28 worms and a 783-bp fragment of the mtDNA control region was analysed for 16 shrews. Phylogenetic analyses of mtDNA sequences revealed reciprocal monophyly for the shrew species, concordant with morphological diagnosis, and supported the idea that the transect cuts through a secondary contact zone between well-differentiated B. brevicauda and B. hylophaga. In contrast to this pattern, the parasitic nematode mtDNA phylogeny was not subdivided according to host affiliation. Genealogical discordance between parasite and host phylogenies suggests extensive gene flow among parasites across the host species boundary.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Mitochondrial DNA analysis of Sporothrix schenckii clinical isolates from China

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP) patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in China. In addition to 23 mtDNA types (Types 1-23) so far reported, a new mtDNA type (Type 24) was found in this study. Type 24 was divided into two subtypes, Subtype 24A and 24B based on RFLP with EcoRV. Sixty-seven isolates in China consisted of 58 isolates of Type 4, 5 of Type 6, 1 of Type 5, 1 of Type 20 and 2 of Type 24. Based on the phylogeny of the mtDNA types (Types 1-24) constructed by estimating sequence divergences of mtDNA, mtDNA types clustered into two groups: Group A (Types 1-3, Type 11, Types 14-19 and Types 22-23) and Group B (Types 4-10, Types 12-13, Types 20-21 and Type 24). These results suggest that most S. schenckii isolates in China belong to Group B.     

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.     

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit− mutant of Saccharomyces cerevisiae

In cultures of the mit^? mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit^? cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a ³²P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the oril sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Summary Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.This segment of mtDNA had one cleavage site for a restriction endonuclease, Hind II, which was more resistant to cleavage than the other Hind II sites in wild type mtDNA. It had two cleavage sites for Hha I and gave two Hha fragments, which were arranged alternatively. Digestion with Hae III gave four fragments and these fragments were mapped.Mitochondrial DNA of this mutant showed a loss of heterogeneity in a melting profile. It melted within a narrow range of temperature, which was similar to that of poly dA·poly dT. Its differential melting curve was significantly different from that of wild type mtDNA.Mapping of mtDNA of a wild type yeast was carried out with restriction endonucleases. Fragments of mtDNA, which were isolated from petites carrying oligomycin-erythromycin-chloramphenicol-resistance and erythromycin-chloramphenicol resistance were also mapped. Loci of oligomycin-resistance, erythromycin-resistance and chloramphenicol-resistance were investigated based on the maps of Eco R I fragments and Hind II fragments.     

A restriction endonuclease cleavage map of mitochondrial DNA from transformed hamster cells.

Mitochondrial DNA from cultured C13/B4 hamster cells was cleaved by the restriction endonucleases Hpa II, Hind III, Eco RI and Bam HI into 7, 5, 3 and 2 unique fragments, respectively. The summed molecular weights of fragments obtained from electrophoretic mobilities in agarose-ethidium bromide gels (with Hpa I-cleaved T7 DNA as standard) and electron microscopic analysis of fragment classes isolated from gels (with SV40 DNA as standard) were in good agreement with the size of 10.37 +/- 0.22 x 10(6) daltons (15,700 +/- 330 nucleotide pairs) determined for the intact circular mitochondrial genome. Cyclization of all Hind III, Eco RI and Bam HI fragments was observed. A cleavage map containing the 17 restriction sites (+/- 1% s.d.) was constructed by electrophoretic analysis of 32P-labeled single- and double-enzyme digestion products and reciprocal redigestion of isolated fragments. The 7 Hpa II sites were located in one half of the genome. The total distribution of the 17 cleavages around the genome was relatively uniform. The position of the D-loop was determined from its location and expansion on 3 overlapping restriction fragments.     

Mitochondrial DNA analysis of Sporothrix schenckii clinical isolates from China

Mitochondrial DNA (mtDNA) types based on restriction fragment length polymorphism (RFLP)patterns with HaeIII were investigated in clinical isolates of Sporothrix schenckii in China. In addition to 23 mtDNA types (Types 1–23) so far reported, a new mtDNA type (Type 24) was found in this study. Type 24 was divided into two subtypes, Subtype 24A and 24B based on RFLP with EcoRV. Sixty-seven isolates in China consisted of 58 isolates of Type 4, 5 of Type 6, 1 of Type 5, 1 of Type 20 and 2 of Type 24. Based on the phylogeny of the mtDNA types (Types 1–24) constructed by estimating sequence divergences of mtDNA, mtDNA types clustered into two groups: Group A (Types 1–3, Type 11, Types 14–19 and Types 22–23) and Group B (Types 4–10, Types 12–13,Types 20–21 and Type 24). These results suggest that mostS. schenckii isolates in China belong to Group B.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mitochondrial DNA differentiation between two sympatric morphs of striped jack near Japan

Two sympatric morphs (type A with a vertebral number of 25 and type B with a vertebral number of 24) of striped jack Pseudocaranx dentex (Bloch & Schneider) were analysed genetically. A part of the 16S–rRNA region of mtDNA was amplified with polymerase chain reaction for 24 specimens, and a restriction enzyme fragment polymorphism showed significant differences between the two types. While all specimens sampled in Ogasawara were identified as type B, about 90% of striped jack in Oita were type A and 10% were type B. Although the spawning areas of these two types are still unknown, significant genetic differences between the two sympatric morphs show that recruitment and migration patterns might differ from each other. The current system suggests the possibility that the juveniles of type B in Oita may migrate from the Ogasawara Islands.     

Specific subunit pairs of legumin from Vicia faba

Four pairs of disulphide-linked acidic (α) and basic (β) subunits were isolated from legumin of Vicia faba. Pairing between α- and β-subunits is nonrandom, supporting the view that each subunit pair arises from a common precursor polypeptide, already containing intramolecular disulphide bonds, when cleavage to the subunit pair takes place. The subunit pairs belong to two structural types: type A contains Met, whereas type B lacks Met. In addition to these four subunit pairs, at least two more pairs are present in legumin in minor amounts.