The gene encoding <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mitochondrial ribosomal protein S13 is a recent duplication of the gene encoding plastid S13

A gene encoding mitochondrial S13 is generally present in the mitochondrial genome of higher plants, but is lacking from the Arabidopsis thaliana mitochondrial genome. Previous research has failed to identify a nuclear gene capable of encoding a mitochondrial S13 protein or the protein itself. Doubts have even been raised as to whether a mitochondrial S13 exists in Arabidopsis. Here, we show that the nuclear gene encoding the plastid S13 has been partially duplicated in A. thaliana, such that the copy has lost the exon encoding the plastid transit peptide and acquired a sequence capable of encoding a mitochondrial targeting sequence. The two S13 sequences were fused to green fluorescent protein and shown to be targeted to plastids and mitochondria respectively.     

Distinguishing authentic mitochondrial and plastid DNAs from similar DNA sequences in the nucleus using the polymerase chain reaction

DNA sequences similar to those in the organellar genomes are also found in the nucleus. These non-coding sequences may be co-amplified by PCR with the authentic organellar DNA sequences, leading to erroneous conclusions. To avoid this problem, we describe an experimental procedure to prevent amplification of this "promiscuous" DNA when total tissue DNA is used with PCR. First, primers are designed for organelle-specific sequences using a bioinformatics method. These primers are then tested using methylation-sensitive PCR. The method is demonstrated for both end-point and real-time PCR with Zea mays, where most of the DNA sequences in the organellar genomes are also present in the nucleus. We use this procedure to quantify those nuclear DNA sequences that are near-perfect replicas of organellar DNA. This method should be useful for applications including phylogenetic analysis, organellar DNA quantification and clinical testing.     

Evaluation of drug effects on Toxoplasma gondii nuclear and plastid DNA replication using real-time PCR

Toxoplasma gondii Nicolle and Manceaux, 1908 is a unicellular protozoan that can infect a broad spectrum of organisms including humans. In addition to a nuclear genome, it also carries a circular DNA within a plastid-like organelle (apicoplast) and a linear genome within its mitochondria. The plastid organelle has been shown to be the target of various anti-parasitic drugs or antibiotics. To evaluate the effects of agents on the DNA replication of T. gondii, we tested six drugs (ciprofloxacin, acetylspiramycin, clindamycin, azithromycin, artemether, and sulfadiazine) on the parasite cultured in Hela cells. After drug treatment for 48 h, the parasite growth and DNA replication were evaluated and quantitated using TaqMan real-time quantitative PCR with oligonucleotide primers synthesized based on a gene from the apicoplast genome (ycf24, Genbank accession no. U87145) and a gene from the nuclear genome (uprt, Genbank accession no. U10246). Our results showed that ciprofloxacin was the most effective in inhibiting the replication of the plastid DNA after 48 h drug treatment, with a reduction of 22% in the copy number of the plastid DNA. Artemether was the most effective drug in suppressing the proliferation of tachyzoites. This study also demonstrates that real-time quantitative PCR is a simple and useful technique for monitoring parasite growth and DNA replication.     

Maize BMS cultured cell lines survive with massive plastid gene loss

As part of developing an ex planta model system for the study of maize plastid and mitochondrial gene expression, a series of established Black Mexican Sweet (BMS) suspension cell lines was characterized. Although the initial assumption was that their organelle biochemistry would be similar enough to normal in planta cells to facilitate future work, each of the three lines was found to have plastid DNA (ptDNA) differing from control maize plants, in one case lacking as much as 70% of the genome. The other two BMS lines possessed either near-wild-type ptDNA or displayed an intermediate state of gene loss, suggesting that these clonal lines are rapidly evolving. Gene expression profiles of BMS cells varied dramatically from those in maize leaf chloroplasts, but resembled those of albino plants lacking plastid ribosomes. In spite of lacking most plastid gene expression and apparently mature rRNAs, BMS cells appear to import proteins from the cytoplasm in a normal manner. The regions retained in BMS ptDNAs point to a set of tRNA genes universally preserved among even highly reduced plastid genomes, whereas the other preserved regions may illuminate which plastid genes are truly indispensable for plant cell survival.Communicated by F.-A. Wollman     

Relationship of genotype to phenotype in fibroblast-derived transmitochondrial cell lines carrying the 3243 mutation associated with the MELAS encephalomyopathy: shift towards mutant genotype and role of mtDNA copy number

Transmitochondrial cell lines were isolated by fusing mtDNA-less rho degrees 206 cells with enucleated fibroblasts derived from four members of a pedigree carrying in their muscle varying proportions of the mutation at position 3243 in the tRNA(Leu(UUR)) gene associated with the MELAS encephalomyopathy. The mitochondrial transformants derived from an asymptomatic individual were all homoplasmic for wild-type mtDNA. The proportion of wild-type transformants derived from clinically affected members of the pedigree appeared to decrease in correspondence with an increase in severity of the clinical symptoms of the cell donor. Furthermore, the average proportion of wild-type mtDNA in the transformants derived from each member of the pedigree was very similar to that found in mtDNA from the fibroblasts of that individual, suggesting that the distribution of genotypes in the transformants reflected fairly closely that in the fibroblasts. The genotype and phenotype of ten transformants derived from one severely affected individual were investigated during continuous culture up to 17-24 weeks after the transformation step. Six heteroplasmic clones showed a progressive increase in the proportion of mutant mtDNA, whereas the mitochondrial genotype remained constant in four clones apparently homoplasmic for wild-type mtDNA or nearly homoplasmic for mutant mtDNA. An analysis of the rate of repopulation of rho degrees 206 cells with fibroblast-derived mtDNA revealed a large variability among different transformants, with the full re-establishment of the control ratio of mtDNA to nuclear DNA being observed between approximately 6 weeks and more than 22 weeks after the transformation step. An increase in rate of O2 consumption generally accompanied the increase in mtDNA copy number of the transformants, pointing to the important role of the mtDNA copy number in determining the phenotype of a cell. The observation that a very small amount of wild-type mtDNA (2 to 5% of the control level), coexisting with strongly predominant mutant mtDNA, conferred upon the transformants a substantial respiratory capacity (50% or more) and the evidence of proportionality between O2 consumption rate and mtDNA copy number, which occurred at widely different mutant to wild-type mtDNA ratios, strongly suggest a contribution of the mutant mtDNA to the cell respiratory competence.      

Gene activation by transfer of isolated mammalian chromosomes. II. Activation of simian virus 40 by transfer of fractionated chromosomes from transformed cells.

Metaphase chromosomes from an inducible SV40-transformed cell line with no detectable infectious virus or free virus DNA were fractionated into four fractions. The chromosomes were transferred into BSC-1 cells which are permissive for SV40-virus replication. The four chromosome fractions were analysed for their uptake by electron microscopy, for virus-DNA sequences by nucleic-acid hybridization, and for their activation of infectious virus. Chromatin material from all four fractions were observed in the cytoplasm of the cells after chromosome transfer. Virus-DNA sequences were detected by nucleic-acid hybridization in three of the four fractions, whereas infectious virus was activated after chromosome uptake of two of the fractions. The smallest chromosomes did not appear to contain either activatible SV40 genomes or integrated SV40-DNA sequences. It is suggested that transfer of fractionated chromosomes may also be useful for studies on the activation of other genes.     

Deleted forms of plastid DNA in albino plants from cereal anther culture

Summary Wheat and barley albino plants derived from anther culture contain plastid genomes which have suffered deletion. DNA molecules of the size of unit genomes exist in these plants. In some cases these may be linear genomes. In all cases a region near the T8 fragment of barley or the corresponding region of the wheat plastid genome has been retained. This region may therefore represent sequences sufficient for replication.     

Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR

A real-time PCR method was developed, optimised and validated, to enable quantitation of Marek's disease virus genomes as copy number per million host cells. The duplex PCR measured the virus meq gene and host ovotransferrin gene in a single reaction enabling correction for differences in amount of sample DNA added. A bacterial artificial chromosome (BAC) clone of the virus genome, and a plasmid (pGEM-T-ovo) bearing a fragment of the chicken ovotransferrin gene, were used to quantify virus and host genomes respectively. This sensitive and reproducible assay was established initially using chicken lymphocyte DNA, then adapted for feather tip DNA by inclusion of bovine serum albumin in the reaction to overcome inhibition by melanin. The principal advantages are: (1) determination of absolute virus genome copy number enabling meaningful comparison between samples; (2) expression of copy number per million cells, allowing direct correlation with plaque assays; (3) using BAC-cloned whole virus genome as a standard potentially enables any virus gene to be used as the PCR target. This is the first report of quantitation of MDV genomes in feather tips, and application of this assay could significantly further our understanding of pathogenesis, spread, diagnosis, genetic resistance and vaccinal control of Marek's disease.     

Mitochondrial DNA content affects the fertilizability of human oocytes

Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193,000 (range: 20,000 to 598,000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.     

Restoration of cytoadherence to an adherence-deficient mutant of Mycoplasma arthritidis by genetic complementation

Mycoplasma arthritidis causes a severe septic arthritis in rats under natural and experimental conditions. An earlier study implicated a membrane lipoprotein designated MAA1 in cytadherence of M. arthritidis. In addition, a spontaneous adherence-deficient mutant was shown to contain a nonsense mutation in the gene encoding MAA1, resulting in production of a truncated product, MAA1Delta. In the present study, a wild-type maa1 gene carried on transposon Tn4001T was introduced into the low-adherence mutant by polyethylene glycol-mediated transformation. The presence of the tranposon and the wild-type maa1 gene in the chromosome of transformants was confirmed by PCR and Southern hybridization. The latter procedure also confirmed that each transformant contained a single copy of the transposon. Western immunoblotting showed that transformants produced both wild-type MAA1 and MAA1Delta, indicating that the introduced wild-type maa1 gene was functional. This phenotype was stably maintained after multiple subcultures even in the absence of antibiotic selection. Finally, transformants were shown to adhere to rat L-2 lung cells in culture at wild-type levels, providing confirmation for an important role for MAA1 in adherence.     

Simultaneous transfer of mitochondrial DNA and single chromosomes in somatic cells: a novel approach for the study of defects in nuclear- mitochondrial communication

The assembly and function of respiratory-competent mitochondria in eukaryotic cells depends on collaboration between the nuclear and mitochondrial genomes, but the molecular mechanisms underlying such cross-talk are poorly understood. Microcell-mediated chromosome transfer has been used to transfer intact chromosomes from one mammalian cell to another, helping to map loci implicated in different diseases and in the senescence process. In the present work, we show that microcells have a significant number of mitochondria which can be transferred to another cell simultaneously with a limited number of chromosomes. By fusing microcells from a colon carcinoma cell line with a mitochondrial DNA (mtDNA)-less osteosarcoma cell line, we were able to isolate transmitochondrial hybrids containing only one of three selectable chromosomes and mtDNA from the donor cell. The proportion of transmitochondrial hybrids containing one chromosomal marker with respect to the total transmitochondrial hybrids and cybrids was approximately 1% and no hybrids were isolated containing more than one nuclear marker. The genetic data correlated well with the composition and structure of the microcell preparations, which showed the presence of cytoplast-like structures and microcells containing mitochondria surrounding the micronuclei. Microcell-mediated mtDNA and chromosome transfer can be used to identify nuclear factors implicated in mtDNA maintenance and gene expression, as well as to investigate nuclear factors which modulate clinical phenotypes in mitochondrial disorders.      

Parameters governing the transfer of the genes for thymidine kinase and dihydrofolate reductase into mouse cells using metaphase chromosomes or DNA

The conditions necessary to achieve high frequency transfer of the thymidine kinase and dihydrofolate reductase genes from hamster cells into mouse cells were investigated. Of the parameters examined, the length of adsorption time, input gene dosage, and treatment with dimethylsulfoxide (DMSO) were found to significantly alter the transfer frequency using either metaphase chromosomes or purified DNA as the transfer vehicle. With the mouse cell line as a recipient, the optimal adsorption period for DNA or chromosomes from Mtx^RIII cells was found to vary from 8 to 16 h in those experiments where the recipient cells were subsequently treated with DMSO. Without DMSO, similar frequencies could be obtained by extending the period of adsorption. Increasing the dosage of DNA or chromosomes resulted in an almost linear increase in the number of transformants. The optimal conditions for transfer did not significantly differ for the two genes studied. On the average, the optimal conditions yielded 1.5×10³ transformants per 10⁷ recipient cells with chromosomes; with DNA an average of only 60 transformants were observed. In general, DNA transformants grown in the absence of methotrexate were unstable; whereas, under the same conditions about 20% of the transformants from the chromosome experiments were stable.     

Hemizygous subtelomeres of an African trypanosome chromosome may account for over 75% of chromosome length

African trypanosomes are parasitic protozoa that infect a wide range of mammals, including humans. These parasites remain extracellular in the mammalian bloodstream, where antigenic variation allows them to survive the immune response. The Trypanosoma brucei nuclear genome sequence has been published recently. However, the significant chromosome size polymorphism observed among strains and subspecies of T. brucei, where total DNA content may vary up to 30%, necessitates a comparative study to determine the underlying basis and significance of such variation between parasites. In addition, the sequenced strain (Tb927) presents one of the smallest genomes analyzed among T. brucei isolates; therefore, establishing polymorphic regions will provide essential complementary information to the sequencing project. We have developed a Tb927 high-resolution DNA microarray to study DNA content variation along chromosome I, one of the most size-variable chromosomes, in different strains and subspecies of T. brucei. Results show considerable copy number polymorphism, especially at subtelomeres, but are insufficient to explain the observed size difference. Additional sequencing reveals that >50% of a larger chromosome I consists of arrays of variant surface glycoprotein genes (VSGs), involved in avoidance of acquired immunity. In total, the subtelomeres appear to be three times larger than the diploid core. These results reveal that trypanosomes can utilize subtelomeres for amplification and divergence of gene families to such a remarkable extent that they may constitute most of a chromosome, and that the VSG repertoire may be even larger than reported to date. Further experimentation is required to determine if these results are applicable to all size-variable chromosomes.     

Twenty-four chromosome FISH in human IVF embryos reveals patterns of post-zygotic chromosome segregation and nuclear organisation

Fluorescence in situ hybridisation (FISH) was first applied on in vitro fertilisation (IVF) embryos for the preimplantation genetic diagnosis of sex, then chromosome translocations and later for chromosome copy number (PGS). Because of the controversy surrounding PGS diagnostically, it has been replaced by array-based approaches; however, FISH remains a powerful tool for investigating mechanisms of both post-zygotic segregation error and nuclear organisation, especially if most or all of the chromosomes in the karyotype can be analysed. The purpose of this study was to develop and apply a 24 chromosome FISH assay to investigate chromosome-specific rates of gain and loss, nuclear organisation patterns and the veracity of the original PGS result in days 5-6 human embryos. Analysis of 17 embryos by this newly developed approach gave strong signals for all chromosomes; it revealed chromosome copy number for each human chromosome per cell for each embryo and the nuclear address of the (mostly centromeric) loci probed. As all embryos were surplus to IVF requirements for both transfer and freezing (and many had an abnormal PGS indication) expected high levels of chromosome abnormalities were seen and no single nucleus displayed a normal complement; all were mosaic. Certain patterns emerged, however, namely that chromosome loss was more common than gain and apparent mitotic non-disjunction. Moreover, the centromeric probes tended preferentially to occupy the nuclear centre. Where we had a prior day 3 biopsy PGS result, it was confirmed, in part, by 24 colour FISH in most but not all cases.     

Molecular phylogenetics of eimeriid coccidia (Eimeriidae,Eimeriorina, Apicomplexa,Alveolata): A preliminary multi-gene and multi-genome approach

Coccidia possess three distinct genomes: nuclear, mitochondrial, and plastid. Sequences from five genes located on these three genomes were used to reconstruct the phylogenetic relationships of members of the phylum Apicomplexa: 18S rDNA sequences from the nuclear (nu) genome, partial cytochrome c oxidase subunit I sequences from the mitochondrial (mt) genome, and partial 16S and 23S rDNA sequences and RNA polymerase B sequences from plastid (pl) genomes. Maximum parsimony, maximum likelihood, and Bayesian inference were used in conjunction with nuclear substitution models generated from data subsets in the analyses. Major groups within the Apicomplexa were well supported with the mitochondrial, nuclear, and a combination of mitochondrial, nuclear and concatenated plastid gene sequences. However, the genus Eimeria was paraphyletic in phylogenetic trees based on the nuclear gene. Analyses using the individual genes (18S rDNA and cytochrome c oxidase subunit I) resolved the various apicomplexan groups with high Bayesian posterior probabilities. The multi-gene, multi-genome analyses based on concatenated nu 18S rDNA, pl 16S, pl 23S, pl rPoB, pl rPoB1, and mt COI sequences appeared useful in resolving phylogenetic relationships within the phylum Apicomplexa. Genus-level relationships, or higher, appear best supported by 18S rDNA analyses, and species-level analyses are best investigated using mt COI sequences; for parasites for which both loci are available, nuclear 18S rDNA sequences combined with mitochondrial COI sequences provide a compact and informative molecular dataset for inferring the evolutionary relationships taxa in the Apicomplexa.     

Plastid inheritance in Oenothera: organelle genome modifies the extent of biparental plastid transmission

Summary The transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.     

LOSS OF CHROMOSOME 9 IN TISSUE SECTIONS OF TRANSITIONAL CELL CARCINOMAS AS DETECTED BY INTERPHASE CYTOGENETICS. A COMPARISON WITH RFLP ANALYSIS

Interphase cytogenetics by in situ hybridization (ISH) using a panel of centromere-associated DNA probes for chromosomes 1, 7, 9, 10, 11, 16, 17, and 18 was performed on 5 μm thick frozen tissue sections of transitional cell carcinomas (TCCs) of the urinary bladder. By this approach, chromosome ploidy, numerical chromosome aberrations, imbalance between chromosomes, and heterogeneity of aberrations within individual tumours were determined. In 15 of 24 TCCs, loss or underrepresentation of chromosome 9, compared with the ISH copy numbers of at least five other chromosomes, was demonstrated. Independently, RFLP analysis were performed on the same cases to detect loss of heterozygosity (LOH) of chromosome loci 9q34, 11p15, 16q22–24, 17p13, and 18q21. LOH was found in 9 of 19 informative cases for chromosome locus 9q34. Comparison of the ISH and RFLP results showed no correlation between numerical aberration and LOH for the loci on chromosomes 11, 16, 17, and 18. However, numerical loss of chromosome 9 was found in 89 per cent (eight of nine cases) with LOH for 9q34. Conversely, LOH at 9q34 was observed in only 67 per cent (eight of 12 cases) with underrepresentation of chromosome 9. Moreover, in 60 per cent of the non-informative cases (three of five cases), underrepresentation for chromosome 9 was observed. These results indicate that the heterochromatin probe for chromosome 9 can be reliably used in TCC tissue sections for the detection of chromosomal loss. In aneuploid TCCs, this DNA probe can be used for the detection of chromosomal underrepresentation only in combination with other centromere-associated DNA probes.