Genetic monitoring of laboratory rat strains by restriction fragment length polymorphisms of mitochondrial DNA

Restriction fragment length polymorphisms of rat mitochondrial DNA (mtDNA) were examined using various kinds of laboratory rat strains in Japan. The results show that mtDNA of laboratory rats, Rattus norvegicus, are highly polymorphic; at least 7 types, Aa, Ba, Bb, Cb, Dc, Eb, and Fa, were found with the use of 6 restriction enzymes, EcoR I, Hind II, Hha I, Hpa II, Taq I, and Hinf I. Types Aa, Ba, and Eb were distributed widely in several strains, whereas types Ba, Cb, Dc and Fa were limited to some specific strains. These results indicate that restriction fragment length polymorphisms of mtDNA can be applied to genetic monitoring of laboratory rat strains.     

Mitochondrial DNA analysis distinguishes between British populations of the whitefish

The whitefish Coregonus lavaretus is confined to two Scottish lochs and four English and one Welsh lake in the British Isles. Restriction fragment length polymorphisms (RFLPs) in the mitochondrial (mt) DNA have been used to analyse variability within and between populations. The 14 restriction enzymes employed in this study examined approximately 3% of the mt genome and revealed 15 haplotypes among 156 fish. Haplotype diversity within populations was generally low except for the Welsh population where nine haplotypes were found among 30 fish examined. The haplotypes clustered into three distinct groups corresponding to Scottish, English and Welsh populations. No haplotypes were shared among the three groups. Possible alternative explanations for these findings are introgression from another species, stochastic lineage sorting from a polymorphic ancestral gene pool and/or separate colonizations following the last glaciation.     

Highly variable regions of DNA flank the human alpha globin genes.

A series of restriction fragment length polymorphisms which are due to DNA rearrangements have been identified within two highly variable regions flanking the human alpha globin genes. The existence of such highly polymorphic areas provides a large number of individual genetic markers for the alpha globin gene cluster on chromosome 16. If, as seems likely, such regions occur frequently throughout the human genome they should be of considerable value in the antenatal diagnosis of genetic disease.     

Genome plasticity among related ++Lactococcus strains: identification of genetic events associated with macrorestriction polymorphisms

The genomic diversity of nine strains of the Lactococcus lactis subsp. cremoris (NCDO712, NCDO505, NCDO2031, NCDO763, MMS36, C2, LM0230, LM2301, and MG1363) was studied by macrorestriction enzyme analysis using pulsed-field gel electrophoresis. These strains were considered adequate for the investigation of genomic plasticity because they have been described as belonging to the same genetic lineage. Comparison of ApaI and SmaI genome fingerprints of each strain revealed the presence of several macrorestriction fragment length polymorphisms (RFLPs), despite a high degree of similarity of the generated restriction patterns. The physical map of the MG1363 chromosome was used to establish a genome map of the other strains and allocate the RFLPs to five regions. Southern hybridization analysis correlated the polymorphic regions with genetic events such as chromosomal inversion, integration of prophage DNA, and location of the transposon-like structures carrying conjugative factor or oligopeptide transport system.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.     

A DNA probe detecting multiple haplotypes of the human Y chromosome.

We have characterized a DNA probe (49f) that detects about 15 Y-specific TaqI bands corresponding to a low-copy number sequence. Five of these bands, each representing a single DNA fragment, can either be present, absent, or variable in length. Familial segregation studies have shown that the variations of these fragments are inherited in a Mendelian fashion and strictly Y-linked. A survey of 44 male individuals indicated that the five variable TaqI fragments detected by probe 49f can be considered as five independent allelic series. Each series represents the different and mutually exclusive allelic forms observed for a single DNA fragment. A total of 16 haplotypes, each defined by a different combination of the various forms of each of these five restriction fragment length polymorphisms, were observed among the 44 scored individuals. These TaqI restriction polymorphisms are not observed with other restriction digests and have therefore been attributed to point mutations. The five polymorphic fragments map to Yq11, a region that does not recombine with the X chromosome and are therefore not redistributed. This implies that an apparently independent reassortment of one of these series with respect to the others can be explained only on the basis of mutations that occurred several times (or reverted) during evolution of the Y chromosome. However, an examination of the different combinations of two or more allelic series suggests that some alleles are not randomly distributed and raises the possibility of establishing a genealogy of the human Y chromosome.     

A region flanking H-2K is duplicated to a distant site in most mouse t haplotypes

Mouse t haplotypes contain at least one inversion, which encompasses the major histocompatibility complex, relative to their wild-type counterparts. A DNA probe for a single copy sequence which flanks the H-2K region in inbred strains was found to have undergone further rearrangements in the t haplotypes. In most t haplotypes, this sequence is duplicated at a distant site, and the two regions show 1 % recombination. The length of homology shared by the two sites is likely to be at least 10–15 kb. Three different alleles, as defined by restriction fragment length polymorphisms, were found for each of the two sites among different t haplotypes. These may reveal evolutionary relationships among these chromosomes.     

Absence of mitochondrial and chloroplast DNA recombinations in Brassica napus plants regenerated from protoplasts,protoplast fusions and anther culture

Summary Over 400 Brassica napus plants regenerated from individual protoplasts, from protoplast fusions and from anther culture were analysed for chloroplast and mitochondrial genome rearrangements by restriction fragment length polymorphisms. None were detected, attesting to the fidelity of the tissue culture procedures employed. In the majority of protoplast fusion products, the cytoplasmic organelles had completely sorted out at the callus stage but three regenerated plants possessed mixed parental populations of mitochondrial genomes and one regenerant contained mixed chloroplast genomes. In all four examples, the cytoplasmic genome sorted out in planta in favor of one parental type which was faithfully maternally transmitted to progeny.     

Genome organization and polymorphism of the murine beta-glucuronidase region

Thirty-eight kilobases of mouse genomic DNA which surround and include the coding sequences for beta-glucuronidase has been mapped. Intron-exon arrangements were determined by hybridization of genomic sequences with cDNA clones, and minimum estimates of gene length (11-17 kb) and intron number were obtained. Only a single gene was observed when genomic DNA was probed with subclones containing beta-glucuronidase coding sequence; there was no evidence of duplicated or pseudogenes. However, sequences distal to the 3' end of the gene are present elsewhere in the genome in a limited number of copies. Eight haplotypes of the beta-glucuronidase region with differing regulatory genotypes were compared for restriction fragment polymorphisms. Surprisingly little was found, considering the diverse origin of the haplotypes. Two of the polymorphisms that were found may be correlated with regulatory phenotypes. A BamHI site is missing from the CS and CL haplotypes that share regulatory properties, and a 0.2-kb insertion is consistently present in haplotypes showing increased response to induction by androgens in kidney.     

Differences Between two Subspecies of Dolly Varden, Salvelinus malma, Revealed by RFLP—PCR Analysis of Mitochondrial DNA

Genetic differences between two subspecies of Dolly Varden, northern Salvelinus malma malma and southern Salvelinus malma krascheninnikovi, from rivers of eastern Russia were studied. Mitochondrial DNA was analyzed by restriction fragment length polymorphisms (RFLP) performed on products amplified with polymerase chain reaction. Three adjacent segments (approximately 7670 bp), comprising 47% of the mitochondrial genome were used: two encoding the five complete NADH dehydrogenase subunits and the other the cytochrome b gene and the control region (D-loop). Total composite haplotypes 46 were found among 136 fishes using RFLP analysis with 14 restriction enzymes. The amount of nucleotide divergence between haplotypes of two subspecies of Dolly Varden was estimated to be approximately 4%. The differences in the level of nucleotide diversity, mismatch distribution between haplotypes, and population-genetic structure of two subspecies of Dolly Varden suggest that these two forms have existed separately for a long time.     

Population biology ofHortaea werneckii based on restriction patterns of mitochondrial DNA

Fifteen isolates ofHortaea werneckii, causative agent of tinea nigra in man, were examined with respect to restriction fragment length polymorphisms of mitochondrial DNA. Seven types of mtDNA, interpreted as populations, could be distinguished, with similarities between the restriction patterns ranging from 32 to 79%. Much of the variance originated from length mutations. Of the seven populations four represented isolates from man, two of which also comprised isolates from other sources. This makes adaptation ofH. werneckii towards association with man in its evolution unlikely; similarity in the chemical and/or physical characteristics of the different isolation sources, viz. salinity, seems more probable. mtDNA types were not correlated with geographic origin. Isolates with the same mtDNA type are widely geographically distributed.     

Paternally Inherited Chloroplast Polymorphism in Pinus: Estimation of Diversity and Population Subdivision, and Tests of Disequilibrium with a Maternally Inherited Mitochondrial Polymorphism

We have surveyed a chloroplast DNA restriction fragment length polymorphism in 745 individuals, distributed rangewide in eight allopatric natural populations of jack pine (Pinus banksiana Lamb.) and eight allopatric natural populations of lodgepole pine (Pinus contorta Dougl.). The polymorphic region of the chloroplast genome is located near duplicated psbA genes. Fourteen length variants were found in the survey, and these variants distinguished the two species qualitatively. Variant diversities were high in both species (h(es) = 0.43 in jack pine; h(es) = 0.44 in lodgepole pine). Population subdivision was weak within and among lodgepole pine subspecies and in jack pine (i.e., θvalues were less than 0.05). This weak subdivision is compatible with theoretical predictions for paternally inherited markers in wind-pollinated outcrossers, as well as for polymorphisms with high length mutation rates. If these populations are at a drift-migration equilibrium, the chloroplast DNA restriction fragment data and previous mitochondrial frequency data from the same individuals are consistent with gene flow that is differential through seeds and pollen. The new data have permitted the first empirical tests of disequilibrium between maternally and paternally inherited factors. As expected, these tests failed to detect convincing evidence of non-random association between chloroplast and mitochondrial variants.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Analysis of genetic diversity in common loon Gavia immer using RAPD and mitochondrial RFLP techniques

We used random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) of mitochondrial cytochrome b (cyt b ) gene to evaluate the genetic diversity in common loon Gavia immer populations from two regions in the United States: New England (NE) and Michigan (MI). RAPD analysis with 18 primers showed 74% polymorphism in NE and 50% in MI loons (similarity coefficient F = 0.92). Although no population-specific markers were found, the frequencies of some RAPD bands varied between the two populations suggesting geographical differences. RFLP analyses with Bam HI enzyme and a 307-bp mitochondrial cyt b gene showed four haplotypes in the NE loon samples and two in the MI samples. The mtDNA haplotype diversity was 0.74 for NE and 0.51 for MI loons, supporting the RAPD data that NE loons have greater genetic diversity than MI loons.     

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

The linkage disequilibrium between chloroplast DNA and mitochondrial DNA haplotypes in Beta vulgaris ssp. maritima (L.): the usefulness of both genomes for population genetic studies

The structure and evolution of the plant mitochondrial genome may allow recurrent appearance of the same mitochondrial variants in different populations. Whether the same mitochondrial variant is distributed by migration or appears recurrently by mutation (creating homoplasy) in different populations is an important question with regard to the use of these markers for population genetic analyses. The genetic association observed between chloroplasts and mitochondria (i.e. two maternally inherited cytoplasmic genomes) may indicate whether or not homoplasy occurs in the mitochondrial genome. Four-hundred and fourteen individuals sampled in wild populations of beets from France and Spain were screened for their mitochondrial and chloroplast polymorphisms. Mitochondrial DNA (mtDNA) polymorphism was investigated with restriction fragment length polymorphism (RFLP) and chloroplast DNA (cpDNA) polymorphism was investigated with polymerase chain reaction PCR-RFLP, using universal primers for the amplification. Twenty and 13 variants for mtDNA and cpDNA were observed, respectively. Most exhibited a widespread geographical distribution. As a very strong linkage disequilibrium was estimated between mtDNA and cpDNA haplotypes, a high rate of recurrent mutation was excluded for the mitochondrial genome of beets. Identical mitochondrial variants found in populations of different regions probably occurred as a result of migration. We concluded from this study that mtDNA is a tool as valuable as cpDNA when a maternal marker is needed for population genetics analyses in beet on a large regional scale.     

Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.

A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60 degrees C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50-100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.