Obtaining mice that carry human mitochondrial DNA transmitted to the progeny

To study human diseases associated with mutations in mitochondrial DNA one needs an animal model in which the distribution of abnormal mtDNA and its impact on the phenotype might be followed. We isolated human mitochondria from HepG2 cell culture and microinjected them into murine zygotes, upon which those were transplanted to the pseudopregnant mice. PCR with species-specific primers allowed detecting human mtDNA in the tissues of 7-13-day embryos. No serious alterations in the development of transmitochondrial embryos were noticed. Among various organs/tissues of the 13-day embryos, human mtDNA was detected only in the heart, skeletal muscles, and stomach, which is in line with its uneven distribution among the blastomeres of an early mouse embryo that we described previously. In four recipient females, the microinjected zygotes were allowed to develop to term, the four neonate males of their joint litter were sacrificed, and in three of them human mtDNA was detected in the heart, skeletal muscles, stomach, brain, testes, and bladder. Six females of that joint litter were grown and mated to intact males. In the progeny (F1) of one of the females two mice were carrying human mtDNA in the heart, skeletal muscles, stomach, brain, lungs, uterus, ovaries, and kidneys. The study confirms the possibility to obtain transmitochondrial mice carrying human mtDNA that is transmitted to the animals of the next generation. Our results also indicate that among the organs to which human mtDNA is distributed some are more likely to receive it than others.     

The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells

Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.     

Strict paternal transmission of mitochondrial DNA of Chlamydomonas species is explained by selection against maternal nucleoids

Summary.  The non-Mendelian inheritance of organelle DNA is common in most plants and animals. Here we examined inheritance mechanisms involved in the transfer of mitochondrial DNA. We successively backcrossed (to F₅) two interfertile strains of the unicellular isogamous haploid algae Chlamydomonas reinhardtii and Chlamydomonas smithii to match nuclear backgrounds and examine transmission patterns of mitochondrial DNA by PCR analysis of cob gene sequences. Mitochondrial DNA was strictly transmitted paternally. To investigate the behavior of parental mitochondrial DNA, we used F₅ progeny to form zygotes and isolated single zygotes. The results showed selective disappearance of maternal mitochondrial nucleoids occurred between 3 and 6 h after zygote formation. Received July 11, 2002; accepted September 28, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.     

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.Communicated by R. Hagemann     

Mitochondrial inheritance and the detection of non-parental mitochondrial DNA haplotypes in crosses of Agaricus bisporus homokaryons

This study evaluates mtDNA transmission in Agaricus bisporus, as well as the occurrence of non-parental haplotypes in heterokaryons produced by controlled crosses. Sixteen crosses were performed with blended liquid cultures, using different combinations of 13 homokaryotic strains. For each cross, different mtDNA haplotypes were present in each homokaryon. Heterokaryons generated from these crosses were subject to genetic analysis with RFLP markers to identify (i). karyotic status, (ii). mtDNA haplotype, and (iii). the occurrence of non-parental mtDNA haplotypes. These analyses generally supported the occurrence of uniparental mitochondrial (mt) inheritance in A. bisporus, with one mtDNA haplotype usually favoured in the new heterokaryon. The preponderance of one mtDNA haplotype in a new heterokaryon did not necessarily show a correlation with a greater mycelial growth rate for the parent homokaryon possessing that haplotype. Mixed mtDNA haplotypes and non-parental haplotypes were also identified in the heterokaryons from some crosses. Evidence for the occurrence of two mtDNA haplotypes in one heterokaryotic mycelium was observed in 8 of 16 crosses, suggesting the maintenance of true heteroplasmons after three successive subculturing steps. Non-parental mtDNA haplotypes were seen in heterokaryons produced from 7 of 16 crosses. The mating protocol described can be utilized to generate novel mtDNA haplotypes for strain improvement and the development of strain-specific markers. Mechanisms of mt selection and inheritance are discussed.     

Populational Heritability: Extending Punnett Square Concepts to Evolution at the Metapopulation Level

In a previous study, using experimental metapopulations of the flour beetle, Tribolium castaneum, we investigated phase III of Wright's shifting balance process (Wade and Griesemer 1998). We experimentally modeled migration of varying amounts from demes of high mean fitness into demes of lower mean fitness (as in Wright's characterization of phase III) as well as the reciprocal (the opposite of phase III). We estimated the meta-populational heritability for this level of selection by regression of offspring deme means on the weighted parental deme means.Here we develop a Punnett Square representation of the inheritance of the group mean to place our empirical findings in a conceptual context similar to Mendelian inheritance of individual traits. The comparison of Punnett Squares for individual and group inheritance shows how the latter concept can be rigorously defined and extended despite the lack of explicitly formulated, simple Mendelian laws of inheritance at the group level. Whereas Wright's phase III combines both interdemic selection and meta-populational inheritance, our formulation separates the issue of meta-populational heritability from that of interdemic selection. We use this conceptual context to discuss the controversies over the levels of selection and the units of inheritance.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

Autotetraploids and genetic mapping using common AFLP markers: the R2 allele conferring resistance to Phytophthora infestans mapped on potato chromosome 4

 Due to the complexity of tetrasomic inheritance, mapping studies in potato (Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans. Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4. Received: 30 October 1997 / Accepted: 6 November 1997     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

Allelic composition and genetic background effects on transgene expression and inheritance in white clover

Allelic composition and genetic background effects on GUS expression and inheritance using a chimeric (cauliflower mosaic virus 35Sp:uidA) transgene were investigated in white clover as a prelude to transgenic cultivar development. Stable expression and Mendelian inheritance of the uidA transgene was observed over two generations when the uidA transgene was maintained in a heterozygous state. Transgenic backcross progeny (BC1) were intercrossed to produce segregating F2 populations. GUS-positive F2 plants were test-crossed with a non-transgenic control plant to determine whether individuals were heterozygous or homozygous for the transgene. Both expected and distorted segregation ratios were observed. Distortion of the segregation ratio was not caused by transgene inactivation or rearrangement, but was influenced by genetic background. BC1, BC2 and F2 populations were found to have similar levels of uidA gene expression. Quantification of GUS expression from progeny of high and low GUS expressing plants indicate that it is possible to alter transgene expression through selection. No difference was found between the level of expression for F2 plants homozygous or heterozygous for the transgene. These results indicate that F2 plants, homozygous for a transgene, might be used to develop a transgenic cultivar. However, progeny testing to determine the influence of genetic background is a prerequisite to such a development.