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Detecting nuclear DNA in suspension of mitochondria

Detecting nuclear DNA in suspension of mitochondria



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Is there a way to detect nuclear DNA in a suspension of mitochondria extracted from leukocytes? I need to make sure there is no nuclear DNA in the suspension before extracting mtDNA from the mitochondria.


Centrifuge the suspension in Cesium Chloride solution at a particular g value which causes the mitochondria to settle to the bottom of the centrifuge tube but leaves nuclear DNA forming a layer near the top of the centrifuge tube. This is since the nuclear DNA is less dense than the mitochondria. Using a needle syringe, extract a small part of the centrifuged suspension from the very bottom of the centrifuge tube. This extract is most likely to contain no nuclear DNA. Hope this helps :).


Unit chief, forensic examiners, biologists, DNA technical specialists, DNA program specialists, management and program analysts, and contract employees.

Deoxyribonucleic acid (DNA) analysis can occur in body fluid stains and other biological tissues recovered from items of evidence. The DNA testing results obtained from evidence samples are compared to DNA from reference samples collected from known individuals. Such analyses may be able to associate victims and suspects with each other, with evidence items, or with a crime scene. The FBI can conduct nuclear, Y-chromosome, and/or mitochondrial DNA testing on evidence samples as appropriate.

Serology

The DCU performs serological testing to detect and characterize body fluids such as blood and semen on items of evidence.

Nuclear DNA

Nuclear DNA (nDNA) is the most discriminating and is typically analyzed in evidence containing body fluids, skin cells, bones, and hairs that have tissue at their root ends. The power of nDNA testing lies in the ability to identify an individual as being the source of the DNA obtained from an evidence item, or by excluding an individual as a contributor to the DNA evidence.

Y-chromosome DNA testing is a form of nuclear DNA testing that is specific to the male chromosome, also known as the Y-chromosome. This type of testing can be useful for sexual assaults, missing persons, and intelligence cases. The Y-chromosome is transmitted from father to son as a complete set therefore, anyone in the paternal lineage will have the same Y-chromosome profile. Due to multiple relatives having the same Y-chromosome profile, unique identifications are not possible from Y-chromosome analysis.

Mitochondrial DNA

Mitochondrial DNA (mtDNA) is a form of DNA that is transmitted from mother to child in a complete set therefore, anyone in the maternal lineage will have the same mtDNA profile. This type of DNA testing can be useful on evidence items such as naturally shed hairs, hair fragments, bones, and teeth. MtDNA analysis is highly sensitive and may allow scientists to obtain information from items of evidence associated with cold cases, missing persons, samples from mass disasters, and small pieces of evidence containing little biological material. However, since multiple individuals can have the same mtDNA profile, unique identifications are not possible from mtDNA analysis.

Analyses

The DCU offers kinship analysis, which is the comparison performed to determine the possible familial relatedness between an evidence item and a known item using a software program. The DCU also offers criminal paternity testing as part of criminal, intelligence, and missing person casework. When appropriate, DNA results from evidence relating to criminal cases and missing persons will be uploaded into the National DNA Index System (NDIS).


Abstract

Mitochondrial dysfunction is a hallmark of ageing, and mitochondrial maintenance may lead to increased healthspan. Emerging evidence suggests a crucial role for signalling from the nucleus to mitochondria (NM signalling) in regulating mitochondrial function and ageing. An important initiator of NM signalling is nuclear DNA damage, which accumulates with age and may contribute to the development of age-associated diseases. DNA damage-dependent NM signalling constitutes a network that includes nuclear sirtuins and controls genomic stability and mitochondrial integrity. Pharmacological modulation of NM signalling is a promising novel approach for the prevention and treatment of age-associated diseases.


Unique Nature

Although most of the genetic material of a cell is contained within the nucleus, the mitochondria have their own circular DNA. They have their own machinery for protein synthesis and reproduce by fission similarly bacteria can do. It is hypothesized that mitochondria have originated from bacteria by endosymbiosis because of the independence from the nuclear DNA and similarities with bacteria. Mitochondrial DNA is localized to the matrix, which also contains a host of enzymes, as well as ribosomes for protein synthesis.

This molecule was caught in the act of replication the arrows indicate the points at which replication was proceeding when the molecules was fixed for electron microscopy. The genome of the human mitochondrion, for example, consists of a circular DNA molecule containing 16,569 base pairs and measuring about 5μm in length. The RNA and the polypeptides encoded by this DNA are just a small fraction (about 5%) of the number of RNA molecules and proteins needed by the mitochondrion.

The size of the mitochondrial genome varies considerably among organisms. Mammalian mitochondria typically have about 16,500bp of DNA, whereas yeast mitochondrial DNA is roughly five times larger and plant mitochondrial DNA is even bigger than that. A comparison of yeast and human mitochondrial DNA, for example, suggest that most of the additional DNA present in the yeast mitochondrian consists of noncoding sequences.


CRISPR editing of mitochondria: Promising new biotech?

Credit: Wikipedia

Although the CRISPR/Cas9 system has seen widespread application in editing the nuclear genome, using it to edit the mitochondrial genome has been problematic. The main hurdles have been a lack of suitable editing sites in the small mtDNA, and the traditional difficulty of importing the guide RNA into the mitochondrial matrix where nucleoids can be accessed.

Two recently published papers suggest that significant progress is being made on both fronts. The first paper, published in the journal SCIENCE CHINA Life Sciences, used CRISPR techniques to induce insertion/deletion (InDel) events at several mtDNA microhomologous regions. These InDel events were triggered specifically by double-strand break (DSB) lesions. The authors found that InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor called iniparib, suggesting a rewiring DSB repair mechanisms to manipulate mtDNA. In the second paper, published in the journal Trends in Molecular Medicine, the researchers give a global overview of recent advances in different forms of nuclear and mitochondrial genome editing.

To gain more insight into some of these new developments, I reached out to Payam Gammage, an expert in mitochondrial editing with a proven track record in perfecting a slightly different editing technology based on zinc finger nucleases (ZFNs). These nucleases are able to target double-stranded mitochondria for cleavage at precise base pair locations, and can therefore eliminate heteroplasmic mitochondria that have faulty nucleoids. More recently, Payam has discovered that 25 of the 30 most mutated genes found in cancer are found in mtDNA. These mutations occur at specific loci in about 60% of all tumors and, at least in colorectal cancer, actually prolong patient lifespan by

nine years compared to wtDNA. Over 70% of colorectal cancers have at least one mtDNA which is found at heteroplasmy levels higher than 5%.

While nucleases can edit out deleterious mutations by selecting for the right mitochondria, a technology that can edit-in new variants, so to speak, is something yet to be perfected. While the methods for CRISPR editing described in the above papers sound promising, Payam related three major concerns that make take some of the wind out of their sails.

The first point is that the Life Sciences paper does not fully address the issue of targeting sgRNA to mitochondria. Secondly, a very low level double-strand break religation has previously been described in mammalian mitos. Cas9 protein expressed at high levels without gRNA results in nonspecific double-strand induction. And thirdly, the DSB repair inhibitor the researchers used may not actually do what has been traditionally thought. In other words, although it was once believed to inhibit PARP (Poly (ADP-ribose) polymerase), it was later shown to operate on different pathways. Furthermore, PARP is not even localized to mitochondria.

An interesting new approach to precise, nondestructive mitochondrial editing that does not require CRISPR techniques was recently discovered by David Liu from Harvard and MIT's Broad Institute. You may not recognize his name even though he has often been cited as the actual inventor of CRISPR, because the higher powers over at the modern and progressive Nobel Committee deemed he did not fit the bill. Liu's method relies on a bacterial toxin, DddA, that catalyzes deamination of cytosines within double-stranded DNA. By adding in a uracil glycosylase inhibitor and TALEN-like proteins, Liu created RNA-free DddA-derived cytosine base editors (DdCBEs) that can catalyze C•G-to-T•A conversions in human mtDNA with high target specificity and product purity.

To further explore the potential of DdCBEs, Liu's group successfully edited five mitochondrial genes: MT-ND1, MT-ND2, MT-ND4, MT-ND5 and MT-ATP8. Anyone who would like to get a hand on some of this technology can access the plasmids that Liu has uploaded to Addgene. For example, there is an ND4 construct on the site called ND4-DdCBE-right side TALE, which has a pCMV backbone and is expressed in mammalian cells. While complete mitochondrial editing of specific base pairs is far superior to simply cleaving mtDNA, the full generality of the approach remains to be seen. Correction of mutants will only be feasible if the faults lie within the specific conversions the editor can perform.

Having this kind of technology on tap does raise the question of whether or not new and beneficial forms of persistent mitochondrial heteroplasmy can be created. For example, it may be possible to introduce or create somatically heteroplasmic mitochondria that are better adapted to high-altitude oxygen levels, or that have enhanced thermogenesis. In any case, it would be unlikely that these manipulations could ever be inherited if not, they cannot find their way into the germ cells. Three-parent embryo champions aside, artificially introducing or otherwise modifying the mitochondria inside the egg would be the last (and most dangerous) place anyone should begin clinically mucking around.

A curious vertebrate known as the Tuatara was recently found to maintain two independent lineages of mitochondria despite sequence divergence of around 10%. This is quite unheard-of in the animal kingdom, save for a few bivalve mollusks that are known to have biparental inheritance of distinct male and female mitochondria. Major differences were reported for control regions and origins of replication in the the Tuataran mtDNA. Researchers suggest that having two divergent mt genomes may confer an adaptive advantage for an unusually cold-tolerant reptile.

In humans, there is abundant need for mitochondrial editing for several neurologic and rare diseases. For example, autism has been associated with a G8363A transfer RNA(Lys) mutation. Other studies have recently demonstrated a mitochondrial deficiency involving an ND6 gene missense mutation (ND6P25L) that results in mice with decidedly autistic endophenotypes. ND6 is a subunit of NADH dehydrogenase that forms part of respiratory complex I. Although autism is notoriously fraught with inconsistencies in trying to nail down causative genes from nuclear GWAS studies, mitochondrial editing in animal models may be a more direct way to better define, and ultimately cure, many of these ailments that have a significant underlying mitochondrial component.


Materials and Methods

DNA was extracted (12) and libraries were made (3) from Denisova 8 and Denisova 4. The libraries were used for direct sequencing and for enrichment of mtDNA (14). mtDNA genomes were used to estimate a Bayesian phylogeny (22, 23), Watterson’s θ, pairwise nucleotide differences, and dates based on branch shortening. Nuclear DNA sequences were used to estimate divergences along the lineages to high-coverage genomes and to calculate D-statistics (24). See SI Appendix for details.


Identification of abnormal nuclear and mitochondrial genes in esophageal cancer cells

Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Introduction

Eesophageal squamous cell carcinoma (ESCC) is one of the most common malignant diseases world wide, particularly in China, where it is the fourth most common cause of cancer-associated mortality (1). Unlike cancers that have been extensively studied, such as breast and colon cancers, the outcome of ESCC remains unchanged during the last several decades, with a 5-year survival rate ranging from 15–25% (2). However, in general, understanding of the genomic abnormalities in this disease is limited to reports from small cohorts (3–5). Advances in next-generation sequencing (NGS) technology are facilitating the identification of novel disease-associated genes and promising to transform the routine clinical diagnosis of inherited disease. Since the initial reports in 2009 (6,7), NGS has aided the discovery of >50 novel disease genes in research settings. Thus, a there is a compelling requirement to extensively identify genomic abnormalities underlying ESCC by NGS, including single-nucleotide polymorphisms (SNPs), insertions/deletions (INDELs), structure variations (SVs) and other information variations (8,9), to elucidate its molecular basis, and to aid the development of effective targeted therapies.

In-depth studies of mitochondria identified mitochondrial DNA (mtDNA) at the basal membrane of mitochondria, which is a type of special and unique genetic material, as it is located outside the nuclei (10). Though mtDNA has independent genetic function, the majority of proteins (including the outer mitochondrial membrane and the matrix proteins) are still encoded by nuclear genome (nDNA), among which

1,500 proteins have important roles in maintaining mitochondrial functions (11). The present study performed high-throughput sequencing on two ESCC cell lines to determine the common features of genomic variation in ESCC cells, identify ESCC-associated abnormally expressed nDNA and mtDNA, and determine their interactions that have important roles in the occurrence and development of ESCC, thus providing direction for ESCC basic research and an important experimental basis for the clinical treatment of ESCC gene targeting therapies.

Materials and methods

Cell recovery and culture

The Ec9706 cell line was provided by the National Key Laboratory of Molecular Oncology, Chinese Academy of Medical Sciences (Beijing, China) and the Eca109 cell line was provided by the laboratory, School of Pharmacy, Zhengzhou University (Zhengzhou, China). The Ec9706 and Eca109 cells cryopreserved in liquid-nitrogen were quickly removed and placed in a water bath at 37°C for l-2 min thawing. The cell suspension was then diluted with an appropriate amount of RPMI-1640 medium containing 10% fetal bovine serum (both from Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). Following centrifugation at 173 × g at room temperature for 5 min, the supernatant was removed, and medium was added this process was repeated twice. The cells were diluted in an appropriate amount of culture medium containing 10% fetal bovine serum, the cells were seeded into culture flasks for the culture at 37°C and 5% CO 2 . The culture medium was replaced the next day, and subsequently the cells were routinely cultured.

DNA extraction

Genomic DNAs was extracted according to the instructions of the DNA extraction kit (Shanghai Genmed Pharmaceutical Technology Co., Ltd., Shanghai, China).

The concentration and purity of the genomic DNA extracted from each sample were determined three times using ultra-trace spectrophotometry (Multiskan MK3 Thermo Fisher Scientific, Inc., Waltham, MA, USA) with pure water as the control, and the average of the three measurements was used for the calculation. For pure genomic DNA, the ratio of absorbance at 260 nm/absorbance at 280 nm should be close to 1.8 (>1.9, indicating RNA contamination <1.6, indicating protein or phenol pollution).

High-throughput sequencing

The high-throughput sequencing process was performed by Genewiz, Inc. (Beijing, China). Workflow of the experiment is presented in Fig. 1. Genomic DNA was tested and randomly broken into manageable fragments by ultrasound to facilitate the construction of an insert library. In human genome re-sequencing, paired-end libraries with a span size of 400–500 bp were usually adopted. The template DNA fragments of the constructed libraries were hybridized to the surface of flow cells and amplified to form clusters, and then sequenced with the Illumina HiSeq X sequencing system (Illumina, Inc., San Diego, CA, USA). Currently, read length of 150 bp paired-end sequencing strategy is used in high-throughput whole genome sequencing.

Figure 1.

Workflow of the high-throughput sequencing.

Detected SNPs and INDELs were annotated against a collection of comprehensive functional annotation databases, including 1000 Genomes Project, Cosmic, GWAS, PolyPhen-2, VISIFT, NHLBI, ClinVar, NCI60, YH genome, gene/protein structure, germline variations (dbSNP https://www.ncbi.nlm.nih.gov/projects/SNP/), and functional elements (transcription binding sites, microRNA targets, conserved elements). Structural variations were detected and annotated by the chromosomal locations. Copy number variations (CNV) were also calculated based on the read depth and summarized in detail. WGC024118D and WGC024119D were the sequencing IDs of Ec9706 and Eca109 cells, respectively.

Data analysis

The sample passed sample quality control and yielded enough high quality sequencing data with an average 117.222G raw data after Illumina pass filtering (PF). The passing filter was a default standard processing of Illumina HiSeq sequencers, to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call was calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities. Clusters passed filter if no more than one base call in the first 25 cycles had a chastity of <0.6.

A comprehensive annotation package, including 10 different databases, including 1000 Genomes Project (http://www.internationalgenome.org), dbSNP (https://www.ncbi.nlm.nih.gov/projects/SNP/), Cosmic (http://cancer.sanger.ac.uk/cosmic), GWAS (https://www.ebi.ac.uk/gwas/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), VISIFT (http://sift.jcvi.org/), NCI60 (http://genome-www.stanford.edu/nci60/index.shtml), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), YHgenome (http://www.yhdatabase.com/), and National Heart, Lung, and Blood Institute (https://www.nhlbi.nih.gov), was applied to annotate the bioinformatics analysis of identified SNPs, INDELs, CNVs and SVs from the sequencing data.

Results

Summary of bioinformatics analysis

The PF sequencing reads were mapped to the human genome version 19 (hg19) using Burrows-Wheeler Aligner (12). On average, 98.99% of whole genome regions were sequenced and a 39.35-fold mappable-reads-coverage was achieved in this sample set (Table I).

Table I.

Data coverage analysis.

Table I.

Data coverage analysis.

Quality control statistics sample WGC024118D WGC024119D
Paired-end read length 150*2 150*2
Total effective data yield (Gb) 116.434 118.01
Total reads number (M) 776.23 786.73
Reads mapping rate 99.04% 98.70%
Properly paired mapping reads rate 96.97% 96.81%
No-mismatch mapping reads rate 61.04% 60.11%
Mismatch alignment bases rate 0.77% 0.74%
Mean coverage sequencing depth 39.1 39.6
Reference genome coverage 98.99% 98.99%
Reference genome coverage ≥4X 98.53% 98.53%
Reference genome coverage ≥10X 97.21% 97.28%
Reference genome coverage ≥20X 90.00% 90.49%
Polymerase chain reaction duplication rate 8.62% 8.64%

SV detection and annotation

The categories of SV include insertions, duplications, deletions, inversions, recurring mobile elements and other rearrangements, typically defined as those covering 50 or more base pairs (Fig. 2). A total of 187 structural variants were identified, including seven inter-chromosomal translocations, three intra-chromosomal translocations, zero inversion, 177 large insertions or deletions. Although many SVs were detected, the majority of SVs may be common SVs present in normal samples, or false detections because of the limitations of NGS (Fig. 3).

Figure 2.

Detection results of partial single nucleotide polymorphisms. The ‘+’ or ‘−’ before the sequence indicates the sequence alignment orientation for each read mismatches or inserts/deletions to the hg19 reference genome are brown low-quality bases (<20 phred score) are in lower case and in gray if they match the reference genome.

Figure 3.

Structure variation loci within chromosomes. In nest-generation sequencing, the loci with structure variation on Chr 1–12 were ≥5%, and structure variation on Chr 13–22, X and Y were ≤3%. Chr, chromosome.

CNV and annotation

As presented in Fig. 4, >40% genes exhibited gain or loss CNVs, and the main form of CNV in WGC024118Ds was loss, and in WGC024119D it was gain. Almost half genes on chromosome 9 (Chr 9) and 11 in WGC024118D and WGC024119Ds had gain or loss, and nearly half genes on Chr 10 in these two samples had gain or loss.

Figure 4.

CNVs in the Ec9706 (WGC024118D) and Eca109 (WGC024119D) genome. (A) CNVs in the whole genome the CNV change trend in the whole genome is that loci with gain or loss account for >40% of the entire sequence sites, the main form of CNV in WGC024118D is loss, and for WGC024119D is gain. Red indicates loss green indicates gain and blue indicates sequence matching the reference hg19. (B) CNVs in WGC024118D and (C) WGC024119D. Almost all of the loci on Chr 4, 17, and 18 in WGC024118D have gain or loss more than half loci on Chr 3, 9, 11, 13, 14, 19 and 20 in WGC024118D, and Chr 1, 5, 7, 9, 11, 20 and Y in WGC024119D have gain or loss almost half of loci on Chr 6, 10, 22, X and Y in WGC024118D and Chr 3, 8, 10, 12, 13, 15, and 16 in WGC024119D have gain or loss few loci on Chr 8, 12, 15, 16, and 17 in WGC024118D and Chr 4 and 22 in WGC024119D have gain or loss, maintaining nearly normal sequence. Red indicates gain green indicates loss and blue indicates normal sequence. Chr, chromosome.

Functional annotations of short INDEL

Loci with INDEL in these two ESCC cell lines predominantly occurred in the upstream, downstream, exon and intron as well as among genes, among which inter-gene INDEL accounted for the majority (Table II).

Table II.

Summary of INDEL detection results.

Table II.

Summary of INDEL detection results.

Feature WGC024118D WGC024119D
High-confidence INDEL no. 740783 766563
Deletion 392984 408253
Insertion 347799 358310
Heterozygotes 386393 409200
Homozygotes 327829 330279
dbSNP 455370 (61.5%) 465321 (60.7%)
1000 Genomes Project 237781 (32.1%) 239886 (31.3%)
NA 17882 18327
3′UTR 6195 6354
5′UTR 810 865
3′ UTR5, UTR3 6 3
Downstream 5402 5600
Exonic 615 645
Exonic, splicing 3 2
Intergenic 403796 419365
Intronic 270105 277821
ncRNA 3′UTR 127 128
ncRNA 5′UTR 23 26
ncRNA 5′UTR, ncRNA 3′UTR 1 1
ncRNA exonic 1241 1299
ncRNA intronic 29459 30801
ncRNA splicing 13 14
Splicing 91 95
Upstream 4851 5049
Upstream, downstream 163 168
Exon frameshift deletion 99 108
Exon frameshift insertion 87 89
Exon nonframeshift deletion 189 194
Exon nonframeshift insertion 148 158
Exon stopgain SNV 7 8
Exon stoploss SNV 1 2
Exon unknown 87 88

[i] INDEL, insertion/deletion UTR, untranslated region SNV, single-nucleotide variant NA, no annotation.

As demonstrated in Table III, the bases with short insert or deletion ranged between 1–20 nt, and the mitochondrial proteins mutated include mitochondrial electron respiratory chain-related proteins, such as NADH:Ubiquinone oxidoreductase subunit S5 (NDUFS5), cytochrome P450 family 27 subfamily A member 1 (CYP27A1), fatty acid metabolism-associated proteins, such as 3-hydroxyisobutyryl-CoA hydrolase (HIBCH), acyl-CoA dehydrogenase family member 9 (ACAD9), ATP energy generation-associated proteins, such as ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1), and these proteins are all nDNA-encoding intra-mitochondrial-transporting proteins.

Table III.

Genetic screening of nuclear DNA with INDEL.

Table III.

Genetic screening of nuclear DNA with INDEL.

Chr Start End Reference sequence Sequence alteration QUALITY Alteration ratio (%) Variation type Gene location Gene Name (description)
Chr 1 39500839 39500839 C 841.73 100 Deletion Downstream NDUFS5 NADH:Ubiquinone oxidoreductase subunit S5, (nuclear gene encoding mitochondrial protein, transcript variant 2)
Chr 1 45804431 45804431 T 819.73 100 Insertion Intronic MUTYH mutY DNA glycosylase, (nuclear gene encoding mitochondrial protein, transcript variant α3)
Chr 2 191091321 191091334 CAAAAAAAAAAAAA 1600.73 100 Deletion Intronic HIBCH 3-hydroxyisobutyryl-CoA hydrolase (nuclear gene encoding mitochondrial protein, transcript variant 2)
Chr 2 219652424 219652424 CCTCTTACCTG 3035.73 100 Insertion Intronic CYP27A1 Cytochrome P450 family 27 subfamily A member 1 (nuclear gene encoding mitochondrial protein)
Chr 3 179339343 179339343 GGTCTCGG 1811.73 100 Insertion Intronic NDUFB5 NADH:Ubiquinone oxidoreductase subunit B5 (nuclear gene encoding mitochondrial protein, transcript variant 1)
Chr 3 128614563 128614563 CTC 2388.73 100 Insertion Intronic ACAD9 acyl-CoA dehydrogenase family member 9 (nuclear gene encoding mitochondrial protein, transcript variant 1)
Chr 4 106312189 106312189 C 806.73 100 Insertion Intronic PPA2 Pyrophosphatase (inorganic) 2 (nuclear gene encoding mitochondrial protein, transcript variant 1)
Chr 4 89197868 89197875 GACTGTCC 1078.74 100 Deletion Intronic PPM1K Protein phosphatase, Mg 2+ /Mn 2+ dependent, 1K (nuclear gene encoding mitochondrial protein)
Chr 1 29538224 29538225 CT 1835.73 100 Deletion Intronic MECR Mitochondrial trans-2-enoyl-CoA reductase (nuclear gene encoding mitochondrial protein)
Chr 1 47107042 47107042 G 1521.73 100 Insertion Intronic ATPAF1 ATP synthase mitochondrial F1 complex assembly factor 1 (nuclear gene encoding mitochondrial protein)

Functional annotations of SNP

As presented in Table IV, the loci with SNP features in these two ESCC cell lines are located in the upstream, downstream, exon and intron, and within genes. The majority of loci with SNP features were located in genes and in exons.

Table IV.

Summary of SNP detection results.

Table IV.

Summary of SNP detection results.

Feature WGC024118D WGC024119D
High-confidence SNP no. 3851527 3861523
Heterozygotes 1938738 1944372
Homozygotes 1911032 1915229
dbSNP 3685859 (95.7%) 3694088 (95.7%)
1000 Genomes Project 3520593 (91.4%) 3520791 (91.2%)
3′UTR 25886 25946
5′UTR 5578 5570
5′UTR, 3′UTR 10 12
Downstream 23646 23744
Exonic 23196 23198
Exonic, splicing 6 7
Intergenic 2255882 2264134
Intronic 1327100 1327665
ncRNA 3′UTR 615 618
ncRNA 5′UTR 97 105
ncRNA exonic 9941 10103
ncRNA intronic 156029 156787
ncRNA splicing 52 55
Splicing 71 68
Upstream 22691 22772
Upstream, downstream 727 739
Exon nonsynonymous SNV 10720 10774
Exon stopgain SNV 98 92
Exon stoploss SNV 15 13
Exon synonymous SNV 11962 11915
Exon unknown 407 411

[i] SNP, single nucleotide polymorphism UTR, untranslated region SNV, single-nucleotide variant.

Additionally, the mitochondrial protein genes with SNP features were mostly located in exons (Table IV), including nDNA-encoding intra-mitochondrial-transporting proteins, such as mitochondrial trans -2-enoyl-CoA reductase (MECR), fatty acid metabolism-associated proteins, such as 3-hydroxymethyl-3-methylglutaryl-CoA lyase (HMGCL), HIBCH, and MECR, ATP energy generation-associated proteins, such as ATPAF1, and succinate dehydrogenase complex. All these proteins are nDNA-encoded intra-mitochondrial-transporting proteins.

Discussion

Oncogenes and tumor suppressor genes have important roles in the occurrence and development of tumors, Changes to mtDNA in tumor tissue has received increasing research interest. It has been previously indicated that various solid tumors and hematological malignancies have different mtDNA mutations, deletions, and microsatellite instabilities at different sites, and these may be associated with the occurrence of a variety of tumors (13). In recent years, more researchers hypothesize that the interactions between mtDNA and nDNA are involved in the tumorigenic process (14). Compared with nDNA, mtDNA has higher mutation rate due to the poor proofreading ability of mtDNA-replicating DNA polymerase and the environment with high concentrations of reactive oxygen species (ROS). Furthermore, mtDNA has hologynic features, in which mutations accumulate continuously along the matriarchal line, and the accumulation of mutations increases the risk of tumorigenesis (9). In addition, mtDNA has more polymorphisms (15), appearing as significant sequence differences among different races and in different regions. The function of mtDNA is known to be involved in tumorigenesis in head and neck cancer (16), bladder cancer (10), breast cancer (17) and lung cancer (11). Hu et al (18) also identified gastric cancer and esophageal cancer-associated genes using whole-genome sequencing, including p53, Janus kinase 3, BRCA2, fibroblast growth factor 2, F-box and WD repeat domain containing 7, mutS homolog 3, patched 1, neurofibromin 1, ErbB-2 receptor tyrosine kinase 2 and checkpoint kinase 2, and identified number of novel potential cancer-associated genes, including KISS1 receptor, anti-Mullerian hormone, motor neuron and pancreas homeobox 1, WNK lysine deficient protein kinase 2, THAP domain containing 12 furthermore, certain chromosomes were reported to have mutations in >30% of tumor genes, including MACRO domain containing 2, fragile histidine triad (FHIT) and parkin RBR E3 ubiquitin protein ligase (18). However, the application of whole genome sequencing to detect mitochondrial changes in ESCC cell lines has rarely been reported previously. Thus, identifying nDNA and mtDNA mutation involved the occurrence and development of ESCC, and investigating their impact on the occurrence and development of ESCC is important for developing novel ESCC gene targeting therapies.

Detecting SVs can help to investigate the mechanisms of gene mutation in cancer, understand the biological differences and identify novel therapeutic targets. However, the complexities and mutation mechanisms of SVs throughout the whole ESCCs genome are still unclear. Cheng et al (19) reported chromosomal abnormalities in malignant metastatic ESCCs and the function of SVs-derived target genes in these abnormal chromosomes are diverse, indicating that the SVs map of the whole genome important for the prevention, diagnosis and treatment of ESCCs. The present study detected many SVs however, most of the SVs may be common SVs present in normal samples, or the false detections because of the limitation of NGS. The categories of structural variants include insertions, duplications, deletions, inversions, recurring mobile elements, and other rearrangements. A total of 187 SVs were identified in the current study, including seven inter-chromosomal translocations, three intra-chromosomal translocations and 177 large insertions or deletions. The loci with SVs on Chr 1–12 of the two ESCC cell lines were ≥5%, whereas those with SVs on Chr 13–22, X and Y were ≤3%. The SVs of mitochondrial genes detected in this study may provide important information for further studies into the nDNA and mtDNA mutations, and roles in the occurrence and development of ESCC.

DNA copy number alterations in tumor cells are key genetic events in the development and progression of human cancers (20). These alterations are typically the result of genomic events producing gains and losses of chromosomes or chromosomal regions. Losses and gains of DNA can contribute to alterations in the expression of tumor suppressor genes and oncogenes. Therefore, identifying DNA copy number alterations in tumor genomes may help to identify critical genes associated with cancer and, eventually, to improve therapeutic approaches. Errors during mitosis and meiosis can result in duplications or deletions of genes on a chromosomal level. These differences are termed CNV, which may profoundly affect health and lead to various disorders. In this study, CNVs were identified based on read depth. Miyawaki et al (21) investigated the important roles of MYC and FHIT gene CNV in selecting the optimal treatment strategy in strategy in resected ESCC patients. The detection results of CNV the current study demonstrated that >40% of genes exhibited gain or loss. Almost half of loci on Chr 9 and 11 in WGC024118D and WGC024119D exhibited gain or loss, and nearly half loci of on Chr 10 exhibited gain or loss, indicating that detecting CNV in ESCC is important for identifying the oncogenic gene gains of the genome and in genes involved in the development of tumors.

The results of SNPs and INDELs sequencing demonstrate the potential functional impact. Cao et al (22) used the whole exon sequencing and revealed lots of genetic heterogeneities within ESCC. Xu et al (23) previously investigated the polymorphisms of ESCC phosphatase and tensin homolog gene in the Chinese Han population, and the interactions among genes. Similarly, detecting short insertion and deletion in genes was applied to detect the gene mutations of ESCC (24,25). The detection results of SNPs and INDELs in the current study revealed potential adverse alterations that have potentially important functions and are associated with gene regulation, including transcription factor binding, microRNA target, conserved elements. The inter-gene loci occurred INDEL and had SNP features account for the majority, and these genes-encoded proteins include mitochondrial electron respiratory chain-associated proteins, such as CYP27A1, fatty acid metabolism-associated proteins, such as HIBCH and ACAD9, nDNA-encoding intra-mitochondrial-transporting proteins, such as MECR, and ATP energy generation-related proteins, such as ATPAF1. The sequencing results also demonstrate that the detection of SNPs and INDELs can be used as the basis for the development of gene therapy.

Under certain conditions, mtDNA can also have effects on nDNA due to mitochondrial defects, thus resulting in corresponding gene expression changes this process is termed reverse regulation (26). This reverse regulation is associated with disturbances in nDNA expressions, mtDNA mutations, respiratory function and mitochondrial protein synthesis inhibition. This reverse regulation is also involved in the process of apoptosis, during which mitochondrial damages can cause the increasing of mitochondrial membrane permeability such apoptosis-inducing factors as cytochrome c , apoptotic protease activating factor-1 and apoptosis inducing factor are then released from mitochondria, and can be directly transported to the nuclei, thus inducing the expressions of certain genes in the nuclei and triggering the apoptotic cascade. It is also suggested that the reverse regulation may also be associated with the ROS pathway or the ratio of ATP/ADP (26). In the present study, the identified nDNA-encoded mtDNA with SVs, CNV, SNPs and INDELs provides a good experimental basis and ideas for investigating the relationships between nDNA and mtDNA in the occurrence and development of ESCC.

Tumorigenesis not only depends on intranuclear genetic materials, but also is also closely associated with ectonuclear mtDNA. As the biological metabolism and energy conversion center, mitochondrial genome replication, gene expression, respiratory chain function, and mitochondrion-associated cell function are inextricably associated with nDNA. nDNA and mtDNA changes will cause corresponding changes of mitochondrial functions. The transcription, replication and translation of mtDNA need a variety of different nDNA products, and the disorders of nDNA products are also associated with mtDNA mutations and biosynthesis inhibition of mitochondrial proteins. These changes can lead to various diseases, and play vital roles particularly in the occurrence and development of tumors. Thus, nDNA-encoded mtDNA protein variants in Ec9706 and Eca109 detected in this study using high-throughput sequencing can provide very reliable theoretical basis and data support for the gene targeting therapies of ESCC.

In this study, the identified mtDNA with abnormal transcription, duplication and translation in Ec9706 and Eca109 cells by high-throughput sequencing were closely associated with the disorders of nDNA products. This study into these nDNA-associated mtDNA and the interaction between the two is important for basic research for ESCC, pointed out the direction and provided a very reliable theoretical basis for the gene targeting therapies of ESCC in clinical.

Acknowledgements

This study was supported by the First Batch of Science and Technology Plan Projects of Zhengzhou in 2013 (grant no. 131PCXTD628), the Fundamental and Advanced Technology Research Project of Henan Province (grant no. 132300410409) and the Medical Science and Technology Plan Program Grant of Henan Province (grant no. 201401009).

References

Zhao P, Dai M, Chen W and Li N: Cancer trends in China. Jpn J Clin Oncol. 40:281–285. 2010. View Article : Google Scholar : PubMed/NCBI


MATERIALS AND METHODS

Arabidopsis thaliana growth and mutants:

Arabidopsis plants were grown by cold treating (4°) and then sowing seeds directly in potting mix (Metro Mix 360). Plants were grown at an 8-hr-day length at 24° for 8 weeks and then transferred to a 16-hr-day length. Two MSH1 mutants were used for the study: msh1-1(A bdelnoor et al. 2003) and Salk_041951. The RECA3 mutant line Sail_252_C06 was also used for genetic analyses (The Arabidopsis Information Resource, http://www.Arabidopsis.org).

DNA gel blot and PCR assays:

Total genomic DNA was extracted from above-ground tissues of flowering plants using the DNeasy plant mini kit (Qiagen). Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and purified using the RNeasy mini kit (Qiagen). DNA gel blot and hybridizations were as described previously (J anska and M ackenzie 1993). Primers used to PCR amplify repeats are listed in the supporting information (Table S1).

Quantitative PCR:

Equal amounts of DNA were used for quantitative PCR using the SYBR GreenER kit for iCycler (Invitrogen). Quantitative PCR data collection and analysis were conducted using iCycler iQ software (version 3.1 Bio-Rad). Experiments were repeated, each sample was run in triplicate, and the results were averaged. Primers used for real-time analysis of regions present in molecules B and D and molecules A and C, respectively, were RealBDF (5′-ATTCCATCCACTCCGGCTTAGCTT-3′) and RealBDR (5′-TCGCTGTGAAAGG TGGAATCCGTT-3′) and RealACF (5′-ATGTAGAGCCAACTGGAGAGCA-3′) and RealACR (5′-CGGAAAGCCCAAATTCTCCTGCAT-3′).

Bioinformatics analyses:

BLAST and blast2seq (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) were used to identify repeated sequences within the C24 mitochondrial genome with a word size of 50 nucleotides. This process identified 104 different repeats, repeats A–Z and AA–ZZ, in descending order of BLAST score. Each repeat copy was numbered (e.g., repeat A-1 and repeat A-2) and distinguished by the flanking sequences. The 33 largest of the intermediate class of repeats, from 108 to 556 bp, are indicated in Figure 1A and Table S2. Most repeats were present in two copies only, with the exceptions of H, V, FF, NN, OO, and RR, present in three copies and BB present in four copies.

To identify repeats in the mitochondrial sequences of sorghum, tobacco, and maize, the software REPuter (K urtz et al. 2001) was used and its results were processed with a Perl script designed to filter close appearances of the repeats. To map ecotype mitochondrial genomes, a script written in Perl generated a network whose nodes are the regions between the repeats and whose arcs indicate evidence of linkages on the same molecule using information gathered from DNA gel blot analysis from the repeated regions for each ecotype. A circuit in this network corresponds to a map of the mitochondrial genome, and circular maps of these circuits were generated for each ecotype. Arabidopsis Genome Initiative locus identifiers are MSH1 (At3g24320) and RECA3 (At3g10140).


Animals.

Male Lewis (RT1 1 ) and DA (RT1A a,b ) rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used at 7 to 8 weeks of age. Animals were maintained in pathogen-free facility of Johns Hopkins Medical Institutions. Animals were cared for according to NIH guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee.

Orthotopic Liver Transplantation.

Orthotopic liver transplantation was performed according to a method previously described. 15 The liver grafts were preserved in cold (4°C) saline (0.9%) for 1 hour before reperfusion. The hepatic artery was not reconstructed. Three combinations were selected: (1) a model of syngenic liver transplantation (Lewis into Lewis) (2) a model of chronic allograft acceptance (Lewis into DA) and (3) a model of acute allograft rejection and death in 10 to 12 days (DA into Lewis). Allograft survival was determined by recipient survival, and rejection was confirmed histologically.

Administration of Enbrel.

Enbrel is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75-kDa TNF receptor linked to the Fc portion of human IgG1. Inactivation of TNF-α was performed by repetitive intraperitoneal injection of 10 mg/kg Enbrel every 2 days (days 0, 2, 4, 6, 8) postorthotopic liver transplantation. Control animals received saline at the same times. In selected experiments, hepatocytes were isolated and liver tissues were harvested from Enbrel-treated rats on day 10 after transplantation.

Hepatocyte Isolation and Culture.

Hepatocytes were isolated from transplanted livers or nontransplanted Lewis rats using a 2-step collagenase perfusion according to the method described by Seglen. 16 The viability of the initial cell suspension of hepatocytes was typically between 80% and 90% (trypan blue). Isolated hepatocytes were used for either mitochondria isolation or in vitro TNF-α treatment. For in vitro assay, isolated hepatocytes were inoculated in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 50 nmol/L dexamethasone (Sigma, St Louis, MO), 20 mmol/L HEPES, 0.5mg/L insulin (Sigma), 1 mmol/L ascorbic acid 2-phophate, and penicillin/streptomycin at a density of 1.5 × 10 5 cells/cm 2 . All cells were maintained in a humidified incubator at 37°C/5% CO2 for 3 days, and then the medium were completely replaced by serum-free and dexamethasone-free Dulbecco modified Eagle medium 24 hours before stimulation. For stimulation, hepatocytes were exposed to recombinant TNF-α (BioSource International, Camarillo, CA) at various concentrations and maintained in culture for an additional 16 hours.

ELISA Assay for Measurement of 8-OHdG Levels in mtDNA in Hepatocytes.

The levels of 8-OHdG in mtDNA were measured by ELISA. Briefly, hepatocytes were homogenized in 5 mL 50mmol/L Tris-HCl (pH 7.4) with 50 strokes of Dounce homogenizer. The homogenates were centrifuged at 800g for 10 minutes to precipitate nuclear fraction. The supernatant was again centrifuged at 7,000g for 10 minutes to yield mitochondrial fraction. The mtDNA was extracted using the DNeasy tissue kit (Qiagen, Santa Clara, CA) according to the manufacturer's instructions. Briefly, 5 μg nuclease P1 (Roche, Indianapolis, IN) was added to 20 μg (for cultured hepatocytes) or 50 μg (for hepatocytes from liver grafts) isolated mtDNA samples. After purging with a nitrogen steam to prevent the artificial formation of 8-OHdG., the mixtures were incubated at 37°C for 1 hour to digest the DNA to nucleotides. Then, 5 μL 500 mmol/L Tris-HCl (pH 8.0), 10 mmol/L MgCl2, and 0.6 units alkaline phosphatase (New England Biolabs, Beverly, MA) were added to the samples. After purging with a nitrogen steam, the mixtures were incubated at 37°C for 1 hour to hydrolyze the nucleotides to nucleosides. The nucleoside samples were used for the determination of 8-OHdG by a competitive ELISA kit (8-OHdG check, Japan Institute for the Control of Aging, Shizuoka, Japan). The determination range was 0.125 to 10 ng/mL or 0.5 to 200 ng/mL. The levels of 8-OHdG were expressed as amounts of 8-OHdG (ng) per milligram mtDNA.

Detection of mtDNA Deletion by Polymerase Chain Reaction.

Total mtDNA was extracted from hepatocyte-derived mitochondria as previously described. The primer sets for amplification of common mtDNA deletion of 4,834-bp, which was reported to be one of the most frequent deletions, 17 , 18 were 5′-TTT CTT CCC AAA CCT TTC CT-3′ (7,837 to 7,856-bp) and 5′-AAG CCT GCT AGG ATG CTT C-3′ (13,108-13,126 bp). The primer sets for control amplification of wild-type mtDNA were 5′-GGT TCT TAC TTC AGG GGC CAT C-3′ (15,782-15,892 bp) and 5′-GTG GAA TTT TCT GAG GGT AGG C-3′ (16,279-16,300 bp). 13 Sequence and numbering are based on the rat complete mitochondrial genomes (GenBank accession number AJ 428514). Polymerase chain reaction (PCR) contained 0.2 mmol/L deoxyribonucleotide triphosphate, 0.2 μmol/L of each primer, 1.0 unit Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 1 μg total DNA as template in a 50-μL reaction solution. The thermal cycling condition was started with one cycle at 94°C for 3 minutes, and 6 cycles at 94°C for 1 minute, 64°C for 1 minute (−1°C/cycle), 72°C for 1minute 30 seconds. This was followed by 34 cycles at 94°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute 30 seconds, and 72°C for final extension for 5 minutes. PCR products were electrophoresed on 1.5% agarose gels and visualized with ethidium bromide staining. The identity of the amplified PCR product was confirmed by sequencing using the AB 3730 DNA Analyzer (Applied Biosystems, Foster City, CA).

Reverse Transcription-PCR Analysis for TNF-α mRNA in Liver Grafts.

Whole liver specimens were kept frozen at −80°C until homogenized for RNA extraction using a QIAamp Kit (Qiagen). TNF-α mRNA expression was analyzed by reverse transcription PCR as previously described. 19

Measurement of ROS Production.

A fluorescent probe, 2′, 7′-dichlorofluorescin diacetate (DCFH-DA Sigma), was used for the assessment of intracellular ROS formation in cultured hepatocytes. This assay is a reliable method for the measurement of intracellular ROS such as hydrogen peroxide (H2O2), hydroxyl radical (OH−), and hydroperoxides. 20-22 DCFH-DA was dissolved in absolute ethanol at a concentration of 5 mmol/L. Hepatocytes were grown on collagen-coated glass coverslips in 6-well culture plates. On culture day 4, TNF-α at various concentrations or media (control) was administered simultaneously with DCFH-DA (5 μmol/L) in culture medium. After incubation at 37°C for 2 hours, hepatocytes were washed with phosphate-buffered saline. Fluorescence images were acquired by microscopy.

Histopathological Analysis.

Cut sections of 4 μm were prepared from formalin-fixed paraffin-embedded tissues for 8-OHdG staining or frozen tissue for TNF-α staining. Each representative section was stained with hematoxylin-eosin (H&E), and immunohistochemical stains were performed with the avidin-biotin-peroxidase complex method, using Vectastain ABC kits (Vector Laboratories, Burlingame, CA). The following antibodies were used: monoclonal anti–8-OHdG antibody (Institute for the Control of Aging, Japan) (1:100) monoclonal anti–TNF-α (R&D Systems Inc., Minneapolis, MN) at the concentration of 5 μg/mL. The antibodies were incubated at 4°C overnight. Antigen retrieval of paraffin section was achieved by a microwave. Double staining of 8-OHdG and terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) staining were also performed by immunofluorescent stains using frozen sections. TUNEL staining was carried out using dUTP-FITC, according to the instructions of the manufacturer (In Situ Cell Death Detection Kit, Roche). After TUNEL staining, the sections were incubated with monoclonal anti–8-OHdG antibody (Japan Institute for the Control of Aging) and goat cyanine 2-conjugated anti-Goat IgG (1:500) (Jackson ImmunoResearch, West Grove, PA).

Statistics.

The results were expressed as mean values ± SEM of n-independent experiments. Analysis was performed by ANOVA with P less than .05 considered significant.


Results

Effect of EtBr on cell growth

The response of cells to a selective inhibitor of mtDNA replication, EtBr, is known to vary depending on its concentrations and the cell lines used (Desjardins et al., 1985 King and Attardi, 1989). Therefore, Ax-2 cells were grown in axenic growth medium containing various concentrations of EtBr to test its effect on growth. As shown in Fig. 1, the growth rate was dependent on the EtBr concentration: no growth occurred in the presence of more than 40 μg/ml EtBr, and this was followed by cell death after 1-2 weeks of culture. At 30 μg/ml EtBr, cells exhibited somewhat delayed growth and stopped dividing after 36 hours of shake culture, during which only two times of cell division took place (Fig. 1). The reduced growth rate and subsequent growth arrest were not recovered by the addition of uridine or pyruvate into growth medium (data not shown). Incidentally, lower concentrations (0.1-5 μg/ml) of EtBr had little effect on cell growth.

Effect of EtBr on growth of Dictyostelium discoideum Ax-2 cells. Various concentrations of EtBr were added to exponentially growing cells (2×10 5 cells/ml) in axenic growth medium (PS medium), followed by cell counts under a haemocytometer. (•) control (non-EtBr-treated cells) (○) cells treated with 10 μg/ml of EtBr, (▴) 20 μg/ml of EtBr, (▵) 30 μg/ml of EtBr, (▪) 40 μg/ml of EtBr and (□) 50 μg/ml of EtBr. Similar results were obtained by cell counts in three independent experiments.

Effect of EtBr on growth of Dictyostelium discoideum Ax-2 cells. Various concentrations of EtBr were added to exponentially growing cells (2×10 5 cells/ml) in axenic growth medium (PS medium), followed by cell counts under a haemocytometer. (•) control (non-EtBr-treated cells) (○) cells treated with 10 μg/ml of EtBr, (▴) 20 μg/ml of EtBr, (▵) 30 μg/ml of EtBr, (▪) 40 μg/ml of EtBr and (□) 50 μg/ml of EtBr. Similar results were obtained by cell counts in three independent experiments.

Creation of ρ Δ cells

To estimate approximately the amount of mtDNA in Ax-2 cells treated with various concentrations of EtBr, cells were stained with DAPI 40 hours after exposure to EtBr to measure mtDNA. As a result, cells exposed to lower (0.1-25 μg/ml) or higher concentrations (40-50 μg/ml) of EtBr were found to have DAPI-stained mitochondria, like those in nontreated cells. In the presence of 30 μg/ml EtBr, however, cells that had divided for about two generations exhibited markedly reduced staining of mitochondria with DAPI in contrast to normal staining of nuclei, thus indicating a selective decrease of mtDNA (Fig. 2C). There were no significant differences in the size of nucleus and the ratio of multinucleate cells to mononucleate cells between 30 μg/ml EtBr-treated and nontreated Ax-2 cells.

Stainings of Ax-2 cells treated with 30 μg/ml of EtBr for 40 hours and nontreated cells with DAPI (A,C) and MitoTracker Orange (B,D). In nontreated Ax-2 cells, DAPI stains are noticed in nuclei (A, arrowheads) and mitochondria as granular structures (A, arrows). In EtBr-treated cells, however, the DAPI-staining of mitochondria is almost vanished, although the staining of nuclei is retained (C). However, rather stronger staining by MitoTracker Orange is observed in a limited number of mitochondria (D, arrows) contained in EtBr-treated cells compared with nontreated cells (B) at the vegetative growth phase. Bars, 10 μm.

Stainings of Ax-2 cells treated with 30 μg/ml of EtBr for 40 hours and nontreated cells with DAPI (A,C) and MitoTracker Orange (B,D). In nontreated Ax-2 cells, DAPI stains are noticed in nuclei (A, arrowheads) and mitochondria as granular structures (A, arrows). In EtBr-treated cells, however, the DAPI-staining of mitochondria is almost vanished, although the staining of nuclei is retained (C). However, rather stronger staining by MitoTracker Orange is observed in a limited number of mitochondria (D, arrows) contained in EtBr-treated cells compared with nontreated cells (B) at the vegetative growth phase. Bars, 10 μm.

The mitochondrial membrane potential has been shown to be transiently reduced in ρ 0 cells derived from several cell lines, as monitored by the staining of cells with MitoTracker Orange. To find out whether mitochondria in Ax-2 cells treated with 30 μg/ml EtBr for 40 hours had any membrane potential, they were vitally stained with MitoTracker. The result showed that some of mitochondria contained in the EtBr-treated cells exhibited rather stronger staining (Fig. 2D) compared with that in nontreated Ax-2 cells (Fig. 2B), suggesting that in a limited number of mitochondria their membrane potential is increased by EtBr-treatment in Dictyostelium cells.

To confirm that the amount of mtDNA is selectively decreased in response to treatment of Ax-2 cells with 30 μg/ml of EtBr, Southern blot analysis was carried out, using a nuclear DNA-specific probe (Dd-trap1 Dictyostelium homologue of trap1) and a mtDNA-specific probe (mitochondrial rps4). For this, total cellular DNAs were prepared from the same number of EtBr (40 hours)-treated cells and nontreated cells, digested with several restriction enzymes and compared after Southern blottings (Fig. 3). Here it is important to note that mtDNA is selectively reduced in the EtBr-treated cells, which is consistent with the result of DAPI staining shown in Fig. 2. The EtBr treatment seemed to have little effect on the amount of nuclear DNA. From densitometric measurements of the autoradiograms obtained, the amount of mtDNA in the EtBr-treated cells was estimated to be about 1/4 of that in nontreated Ax-2 cells. One can explain well the reduced value of mtDNA in the EtBr-treated cell, provided that the synthesis of mtDNA during two cell-doublings in the presence of 30 μg/ml of EtBr is selectively and completely inhibited by EtBr. From another point of view, it is most likely that a certain amount of mtDNA (presumably more than 1/4 of original mtDNA) may be required to maintain cellular activities including growth. In other words, it might be impossible to create cells that have no mtDNA (ρ 0 cells) using Dictyostelium cells. Therefore, the EtBr-treated cells with about 1/4 of mtDNA were used in further experiments, as ρ Δ cells.

Southern blot analysis of total DNAs extracted from Ax-2 cells and cells treated with 30 μg/ml of EtBr for 40 hours. The DNAs were digested with the indicated restriction enzymes and electrophoresed. After transfer of the size-fractionated DNA fragments to nylon membranes, they were hybridized with the 32 P-labeled (A) nuclear DNA-specific probe Dd-trap1 or (B) mtDNA-specific probe rps4, followed by autoradiography.

Southern blot analysis of total DNAs extracted from Ax-2 cells and cells treated with 30 μg/ml of EtBr for 40 hours. The DNAs were digested with the indicated restriction enzymes and electrophoresed. After transfer of the size-fractionated DNA fragments to nylon membranes, they were hybridized with the 32 P-labeled (A) nuclear DNA-specific probe Dd-trap1 or (B) mtDNA-specific probe rps4, followed by autoradiography.

Ultrastructural features of ρ Δ cells

Electron microscopic observations of ρ Δ cells and vegetatively growing Ax-2 cells revealed some characteristic differences between them. As was expected, the most striking difference was the morphology of the mitochondria in ρ Δ cells many of the mitochondria exhibited marked structural transformation to form a sort of vacuole, engulfing the nearby cytoplasm (Fig. 4B,D). We previously reported that somewhat similar mitochondrial transformation occurred in differentiating prespore cells just before PSV formation (Matsuyama and Maeda, 1998). Nontreated Ax-2 cells contained normal-shaped mitochondria with reticular cisternae and an electron-opaque matrix, as shown in Fig. 4A,C. Incidentally, Kobilinsky and Beattie (Kobilinsky and Beattie, 1977) have reported the EtBr (10 μg/ml)-induced mitochondrial transformation in D. discoideum Ax-3 cells.

Electron micrographs showing marked structural transformation of mitochondria in ρ Δ cells. (A,C) Vegetatively growing Ax-2 cells have normal-shaped mitochondria, whereas (B,D) ρ Δ cells have markedly transformed mitochondria having a sort of vacuoles (arrows), engulfing the nearby cytoplasm. Mt, mitochondria N, nucleus. Bars, 1 μm.

Electron micrographs showing marked structural transformation of mitochondria in ρ Δ cells. (A,C) Vegetatively growing Ax-2 cells have normal-shaped mitochondria, whereas (B,D) ρ Δ cells have markedly transformed mitochondria having a sort of vacuoles (arrows), engulfing the nearby cytoplasm. Mt, mitochondria N, nucleus. Bars, 1 μm.

Decreased mtDNA causes delayed differentiation and abnormal morphogenesis

When Ax-2 cells were harvested at the exponential growth phase, washed and incubated on agar, they formed aggregation streams after 6 hours and mounds after 12 hours of incubation at 22°C (Fig. 5A). Subsequently, a tip was formed at the apex of each mound, which elongated and formed a migrating slug after 16 hours of incubation. This was followed by the formation of a fruiting body at about 26 hours of incubation. By contrast, ρ Δ cells exhibited delayed and somewhat abnormal morphogenesis large aggregation streams were formed after 16 hours of incubation, followed by their subdivision to smaller cell masses (Fig. 5A). After a prolonged time (about 48 hours) of incubation, ρ Δ cells formed irregular-shaped slugs, but failed to develop to fruiting bodies (Fig. 5B). Another experiment revealed that Ax-2 cells treated with lower concentrations (15, 20 or 25 μg/ml) of EtBr for 40 hours at the growth phase had almost the same amount of mtDNA as that of nontreated Ax-2 cells, after DAPI staining, and that they exhibited normal development after starvation. Also, when Ax-2 cells were harvested and starved either on 1.5% non-nutrient agar or in BSS, both of which contained 30 μg/ml of EtBr, they showed normal development without any loss of mtDNA.

Development of starved Ax-2 cells and ρ Δ cells on agar. Ax-2 cells and ρ Δ cells were washed twice in BSS and plated on 1.5% non-nutrient agar at a density of 5×10 6 cells/cm 2 . This was followed by incubation for the indicated times at 22°C. (A) Nontreated Ax-2 cells formed aggregation streams after 6 hours and mounds after 12 hours of incubation. Subsequently a tip was formed at the apex of each mound, elongated and constructed a migrating slug after 16 hours of incubation. This was followed by formation of a fruiting body at about 26 hours of incubation. By contrast, ρ Δ cells exhibited delayed and somewhat abnormal morphogenesis large aggregation streams were formed after 16 hours of incubation, followed by their subdivision to smaller cell masses (A). (B) Gross morphology of final structures: fruiting bodies derived from nontreated cells and irregular-shaped slugs derived from ρ Δ cells. The ρ Δ cells stopped their development at the slug stage and never formed fruiting bodies. Bars, 1 mm.

Development of starved Ax-2 cells and ρ Δ cells on agar. Ax-2 cells and ρ Δ cells were washed twice in BSS and plated on 1.5% non-nutrient agar at a density of 5×10 6 cells/cm 2 . This was followed by incubation for the indicated times at 22°C. (A) Nontreated Ax-2 cells formed aggregation streams after 6 hours and mounds after 12 hours of incubation. Subsequently a tip was formed at the apex of each mound, elongated and constructed a migrating slug after 16 hours of incubation. This was followed by formation of a fruiting body at about 26 hours of incubation. By contrast, ρ Δ cells exhibited delayed and somewhat abnormal morphogenesis large aggregation streams were formed after 16 hours of incubation, followed by their subdivision to smaller cell masses (A). (B) Gross morphology of final structures: fruiting bodies derived from nontreated cells and irregular-shaped slugs derived from ρ Δ cells. The ρ Δ cells stopped their development at the slug stage and never formed fruiting bodies. Bars, 1 mm.

More striking delay of differentiation was observed in ρ Δ cells starving under submerged conditions. Most ρ Δ cells showed no sign of cell aggregation and remained as round-shaped single cells even after 12 hours of incubation, while nontreated Ax-2 cells acquired aggregation-competence and began to aggregate at 7 hours of incubation, which was followed by the formation of tight mounds during 12-24 hours of incubation (Fig. 6). By contrast, ρ Δ cells formed large aggregation streams at 24 hours, which were then subdivided into smaller mounds during another 4 hours of incubation, principally as the case on agar (Fig. 6).


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