Daily Archives: March 29, 2017

Mitochondrial DNA (mtDNA or mDNA)[3] is the DNA located in mitochondria, cellular organelles within eukaryotic cells that convert chemical energy from food into a form that cells can use, adenosine triphosphate (ATP). Mitochondrial DNA is only a small portion of the DNA in a eukaryotic cell; most of the DNA can be found in the cell nucleus and, in plants and algae, also in plastids such as chloroplasts.

In humans, the 16,569 base pairs of mitochondrial DNA encode for only 37 genes.[4]Human mitochondrial DNA was the first significant part of the human genome to be sequenced. In most species, including humans, mtDNA is inherited solely from the mother.[5]

Since animal mtDNA evolves faster than nuclear genetic markers,[6][7][8] it represents a mainstay of phylogenetics and evolutionary biology. It also permits an examination of the relatedness of populations, and so has become important in anthropology and biogeography.

Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today's eukaryotic cells. This theory is called the endosymbiotic theory. Each mitochondrion is estimated to contain 210 mtDNA copies.[9] In the cells of extant organisms, the vast majority of the proteins present in the mitochondria (numbering approximately 1500 different types in mammals) are coded for by nuclear DNA, but the genes for some of them, if not most, are thought to have originally been of bacterial origin, having since been transferred to the eukaryotic nucleus during evolution.[10]

The reasons why mitochondria have retained some genes are debated. The existence in some species of mitochondrion-derived organelles lacking a genome[11] suggests that complete gene loss is possible, and transferring mitochondrial genes to the nucleus has several advantages.[12] The difficulty of targeting remotely-produced hydrophobic protein products to the mitochondrion is one hypothesis for why some genes are retained in mtDNA;[13]colocalisation for redox regulation is another, citing the desirability of localised control over mitochondrial machinery.[14] Recent analysis of a wide range of mtDNA genomes suggests that both these features may dictate mitochondrial gene retention.[10]

In most multicellular organisms, mtDNA is inherited from the mother (maternally inherited). Mechanisms for this include simple dilution (an egg contains on average 200,000 mtDNA molecules, whereas a healthy human sperm was reported to contain on average 5 molecules[15][16] ), degradation of sperm mtDNA in the male genital tract, in the fertilized egg, and, at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism, this single parent (uniparental inheritance) pattern of mtDNA inheritance is found in most animals, most plants and in fungi as well.

In sexual reproduction, mitochondria are normally inherited exclusively from the mother; the mitochondria in mammalian sperm are usually destroyed by the egg cell after fertilization. Also, most mitochondria are present at the base of the sperm's tail, which is used for propelling the sperm cells; sometimes the tail is lost during fertilization. In 1999 it was reported that paternal sperm mitochondria (containing mtDNA) are marked with ubiquitin to select them for later destruction inside the embryo.[17] Some in vitro fertilization techniques, particularly injecting a sperm into an oocyte, may interfere with this.

The fact that mitochondrial DNA is maternally inherited enables genealogical researchers to trace maternal lineage far back in time. (Y-chromosomal DNA, paternally inherited, is used in an analogous way to determine the patrilineal history.) This is usually accomplished on human mitochondrial DNA by sequencing the hypervariable control regions (HVR1 or HVR2), and sometimes the complete molecule of the mitochondrial DNA, as a genealogical DNA test.[18] HVR1, for example, consists of about 440 base pairs. These 440 base pairs are then compared to the control regions of other individuals (either specific people or subjects in a database) to determine maternal lineage. Most often, the comparison is made to the revised Cambridge Reference Sequence. Vil et al. have published studies tracing the matrilineal descent of domestic dogs to wolves.[19] The concept of the Mitochondrial Eve is based on the same type of analysis, attempting to discover the origin of humanity by tracking the lineage back in time.

mtDNA is highly conserved, and its relatively slow mutation rates (compared to other DNA regions such as microsatellites) make it useful for studying the evolutionary relationshipsphylogenyof organisms. Biologists can determine and then compare mtDNA sequences among different species and use the comparisons to build an evolutionary tree for the species examined. However, due to the slow mutation rates it experiences, it is often hard to distinguish between closely related species to any large degree, so other methods of analysis must be used.

Entities undergoing uniparental inheritance and with little to no recombination may be expected to be subject to Muller's ratchet, the accumulation of deleterious mutations until functionality is lost. Animal populations of mitochondria avoid this buildup through a developmental process known as the mtDNA bottleneck. The bottleneck exploits stochastic processes in the cell to increase in the cell-to-cell variability in mutant load as an organism develops: a single egg cell with some proportion of mutant mtDNA thus produces an embryo where different cells have different mutant loads. Cell-level selection may then act to remove those cells with more mutant mtDNA, leading to a stabilisation or reduction in mutant load between generations. The mechanism underlying the bottleneck is debated,[20][21][22][23] with a recent mathematical and experimental metastudy providing evidence for a combination of random partitioning of mtDNAs at cell divisions and random turnover of mtDNA molecules within the cell.[24]

Doubly uniparental inheritance of mtDNA is observed in bivalve mollusks. In those species, females have only one type of mtDNA (F), whereas males have F type mtDNA in their somatic cells, but M type of mtDNA (which can be as much as 30% divergent) in germline cells.[25] Paternally inherited mitochondria have additionally been reported in some insects such as fruit flies,[26][27]honeybees,[28] and periodical cicadas.[29]

Male mitochondrial inheritance was recently discovered in Plymouth Rock chickens.[30] Evidence supports rare instances of male mitochondrial inheritance in some mammals as well. Specifically, documented occurrences exist for mice,[31][32] where the male-inherited mitochondria were subsequently rejected. It has also been found in sheep,[33] and in cloned cattle.[34] It has been found in a single case in a human male.[35]

Although many of these cases involve cloned embryos or subsequent rejection of the paternal mitochondria, others document in vivo inheritance and persistence under lab conditions.

An IVF technique known as mitochondrial donation or mitochondrial replacement therapy (MRT) results in offspring containing mtDNA from a donor female, and nuclear DNA from the mother and father. In the spindle transfer procedure, the nucleus of an egg is inserted into the cytoplasm of an egg from a donor female which has had its nucleus removed, but still contains the donor female's mtDNA. The composite egg is then fertilized with the male's sperm. The procedure is used when a woman with genetically defective mitochondria wishes to procreate and produce offspring with healthy mitochondria.[36] The first known child to be born as a result of mitochondrial donation was a boy born to a Jordanian couple in Mexico on 6 April 2016.[37]

In most multicellular organisms, the mtDNA - or mitogenome - is organized as a circular, covalently closed, double-stranded DNA. But in many unicellular (e.g. the ciliate Tetrahymena or the green alga Chlamydomonas reinhardtii) and in rare cases also in multicellular organisms (e.g. in some species of Cnidaria ) the mtDNA is found as linearly organized DNA. Most of these linear mtDNAs possess telomerase independent telomeres (i.e. the ends of the linear DNA) with different modes of replication, which have made them interesting objects of research, as many of these unicellular organisms with linear mtDNA are known pathogens.[38]

For human mitochondrial DNA (and probably for that of metazoans in general), 100-10,000 separate copies of mtDNA are usually present per somatic cell (egg and sperm cells are exceptions). In mammals, each double-stranded circular mtDNA molecule consists of 15,000-17,000[39]base pairs. The two strands of mtDNA are differentiated by their nucleotide content, with a guanine-rich strand referred to as the heavy strand (or H-strand) and a cytosine-rich strand referred to as the light strand (or L-strand). The heavy strand encodes 28 genes, and the light strand encodes 9 genes for a total of 37 genes.[4] Of the 37 genes, 13 are for proteins (polypeptides), 22 are for transfer RNA (tRNA) and two are for the small and large subunits of ribosomal RNA (rRNA).[40] The human mitogenome contains overlapping genes (ATP8 and ATP6 as well as ND4L and ND4: see the human mitochondrial genome map), a feature that is rare in animal genomes.[citation needed] The 37-gene pattern is also seen among most metazoans, although in some cases one or more of these genes is absent and the mtDNA size range is greater.

Great variation in mtDNA gene content and size exists among fungi and plants, although there appears to be a core subset of genes that are present in all eukaryotes (except for the few that have no mitochondria at all).[10] Some plant species have enormous mitochondrial genomes, with Silene conica mtDNA containing as many as 11,300,000 base pairs.[41] Surprisingly, even those huge mtDNAs contain the same number and kinds of genes as related plants with much smaller mtDNAs.[42] The genome of the mitochondrion of the cucumber (Cucumis sativus) consists of three circular chromosomes (lengths 1556, 84 and 45 kilobases), which are entirely or largely autonomous with regard to their replication.[43]

The smallest mitochondrial genome sequenced to date is the 5967 bp mtDNA of the parasite Plasmodium falciparum.[44]

There are six main genome types found in mitochondrial genomes. These genome types were classified by Kolesnikov & Gerasimov (2012)" and differ in various ways such as a circular versus linear genome, genome size, the presence of introns or plasmid like structures, and whether the genetic material is a singular molecule or collection of homogeneous or heterogeneous molecules.[45]

There is only one mitochondrial genome type found in animal cells. This genome contains one circular molecule with between 11-28kbp of genetic material (type 1).[45]

There are three different genome types found in plants and fungi. The first type is a circular genome that has introns (type 2) and may range from 19-1000kpb in length. The second genome type is a circular genome (about 20-1000kbp) that also has a plasmid-like structure (1kb) (type 3). The final genome type that can be found in plant and fungi is a linear genome made up of homogeneous DNA molecules (type 5).

Protists contain the most diverse mitochondrial genomes, with five different types found in this kingdom. Type 2, type 3 and type 5 mentioned in the plant and fungus genomes also exists in some protist, as well as two unique genome types. The first of these is a heterogeneous collection of circular DNA molecules (type 4) and the final genome type found in protists is a heterogeneous collection of linear molecules (type 6). Genome types 4 and 6 both range from 1-200kbp in size.

Endosymbiotic gene transfer, the process of genes that were coded in the mitochondrial genome being transferred to the cell's main genome likely explains why more complex organisms, such as humans, have smaller mitochondrial genomes than simpler organisms, such as protists.

Mitochondrial DNA is replicated by the DNA polymerase gamma complex which is composed of a 140 kDa catalytic DNA polymerase encoded by the POLG gene and two 55 kDa accessory subunits encoded by the POLG2 gene.[46] The replisome machinery is formed by DNA polymerase, TWINKLE and mitochondrial SSB proteins. TWINKLE is a helicase, which unwinds short stretches of dsDNA in the 5 to 3 direction.[47]

During embryogenesis, replication of mtDNA is strictly down-regulated from the fertilized oocyte through the preimplantation embryo.[48] The resulting reduction in per-cell copy number of mtDNA plays a role in the mitochondrial bottleneck, exploiting cell-to-cell variability to ameliorate the inheritance of damaging mutations.[24] At the blastocyst stage, the onset of mtDNA replication is specific to the cells of the trophectoderm.[48] In contrast, the cells of the inner cell mass restrict mtDNA replication until they receive the signals to differentiate to specific cell types.[48]

In animal mitochondria, each DNA strand is transcribed continuously and produces a polycistronic RNA molecule. Between most (but not all) protein-coding regions, tRNAs are present (see the human mitochondrial genome map). During transcription, the tRNAs acquire their characteristic L-shape that gets recognized and cleaved by specific enzymes. With the mitochondrial RNA processing, individual mRNA, rRNA, and tRNA sequences are released from the primary transcript.[49] Folded tRNAs therefore act as secondary structure punctuations.[50]

The concept that mtDNA is particularly susceptible to reactive oxygen species generated by the respiratory chain due to its proximity remains controversial.[51] mtDNA does not accumulate any more oxidative base damage than nuclear DNA.[52] It has been reported that at least some types of oxidative DNA damage are repaired more efficiently in mitochondria than they are in the nucleus.[53] mtDNA is packaged with proteins which appear to be as protective as proteins of the nuclear chromatin.[54] Moreover, mitochondria evolved a unique mechanism which maintains mtDNA integrity through degradation of excessively damaged genomes followed by replication of intact/repaired mtDNA. This mechanism is not present in the nucleus and is enabled by multiple copies of mtDNA present in mitochondria [55] The outcome of mutation in mtDNA may be an alteration in the coding instructions for some proteins,[56] which may have an effect on organism metabolism and/or fitness.

Mutations of mitochondrial DNA can lead to a number of illnesses including exercise intolerance and KearnsSayre syndrome (KSS), which causes a person to lose full function of heart, eye, and muscle movements. Some evidence suggests that they might be major contributors to the aging process and age-associated pathologies.[57] Particularly in the context of disease, the proportion of mutant mtDNA molecules in a cell is termed heteroplasmy. The within-cell and between-cell distributions of heteroplasmy dictate the onset and severity of disease [58] and are influenced by complicated stochastic processes within the cell and during development.[24][59]

Mutations in mitochondrial tRNAs can be responsible for severe diseases like the MELAS and MERRF syndromes.[60]

Mutations in nuclear genes that encode proteins that mitochondria use can also contribute to mitochondrial diseases. These diseases do not follow mitochondrial inheritance patterns, but instead follow Mendelian inheritance patterns.[61]

Recently a mutation in mtDNA has been used to help diagnose prostate cancer in patients with negative prostate biopsy.[62][63]

Though the idea is controversial, some evidence suggests a link between aging and mitochondrial genome dysfunction.[64] In essence, mutations in mtDNA upset a careful balance of reactive oxygen species (ROS) production and enzymatic ROS scavenging (by enzymes like superoxide dismutase, catalase, glutathione peroxidase and others). However, some mutations that increase ROS production (e.g., by reducing antioxidant defenses) in worms increase, rather than decrease, their longevity.[51] Also, naked mole rats, rodents about the size of mice, live about eight times longer than mice despite having reduced, compared to mice, antioxidant defenses and increased oxidative damage to biomolecules.[65] Once, there was thought to be a positive feedback loop at work (a 'Vicious Cycle'); as mitochondrial DNA accumulates genetic damage caused by free radicals, the mitochondria lose function and leak free radicals into the cytosol. A decrease in mitochondrial function reduces overall metabolic efficiency.[66] However, this concept was conclusively disproved when it was demonstrated that mice, which were genetically altered to accumulate mtDNA mutations at accelerated rate do age prematurely, but their tissues do not produce more ROS as predicted by the 'Vicious Cycle' hypothesis.[67] Supporting a link between longevity and mitochondrial DNA, some studies have found correlations between biochemical properties of the mitochondrial DNA and the longevity of species.[68] Extensive research is being conducted to further investigate this link and methods to combat aging. Presently, gene therapy and nutraceutical supplementation are popular areas of ongoing research.[69][70] Bjelakovic et al. analyzed the results of 78 studies between 1977 and 2012, involving a total of 296,707 participants, and concluded that antioxidant supplements do not reduce all-cause mortality nor extend lifespan, while some of them, such as beta carotene, vitamin E, and higher doses of vitamin A, may actually increase mortality.[71]

Deletion breakpoints frequently occur within or near regions showing non-canonical (non-B) conformations, namely hairpins, cruciforms and cloverleaf-like elements.[72] Moreover, there is data supporting the involvement of helix-distorting intrinsically curved regions and long G-tetrads in eliciting instability events. In addition, higher breakpoint densities were consistently observed within GC-skewed regions and in the close vicinity of the degenerate sequence motif YMMYMNNMMHM.[73]

Unlike nuclear DNA, which is inherited from both parents and in which genes are rearranged in the process of recombination, there is usually no change in mtDNA from parent to offspring. Although mtDNA also recombines, it does so with copies of itself within the same mitochondrion. Because of this and because the mutation rate of animal mtDNA is higher than that of nuclear DNA,[74] mtDNA is a powerful tool for tracking ancestry through females (matrilineage) and has been used in this role to track the ancestry of many species back hundreds of generations.

The rapid mutation rate (in animals) makes mtDNA useful for assessing genetic relationships of individuals or groups within a species and also for identifying and quantifying the phylogeny (evolutionary relationships; see phylogenetics) among different species. To do this, biologists determine and then compare the mtDNA sequences from different individuals or species. Data from the comparisons is used to construct a network of relationships among the sequences, which provides an estimate of the relationships among the individuals or species from which the mtDNAs were taken. mtDNA can be used to estimate the relationship between both closely related and distantly related species. Due to the high mutation rate of mtDNA in animals, the 3rd positions of the codons change relatively rapidly, and thus provide information about the genetic distances among closely related individuals or species. On the other hand, the substitution rate of mt-proteins is very low, thus amino acid changes accumulate slowly (with corresponding slow changes at 1st and 2nd codon positions) and thus they provide information about the genetic distances of distantly related species. Statistical models that treat substitution rates among codon positions separately, can thus be used to simultaneously estimate phylogenies that contain both closely and distantly related species[60]

Mitochondrial DNA was admitted into evidence for the first time ever in 1996 during State of Tennessee v. Paul Ware.[75]

In the 1998 court case of Commonwealth of Pennsylvania v. Patricia Lynne Rorrer,[76] mitochondrial DNA was admitted into evidence in the State of Pennsylvania for the first time.[77][78] The case was featured in episode 55 of season 5 of the true crime drama series Forensic Files (season 5).[citation needed]

Mitochondrial DNA was first admitted into evidence in California in the successful prosecution of David Westerfield for the 2002 kidnapping and murder of 7-year-old Danielle van Dam in San Diego: it was used for both human and dog identification.[79] This was the first trial in the U.S. to admit canine DNA.[80]

Mitochondrial DNA was discovered in the 1960s by Margit M. K. Nass and Sylvan Nass by electron microscopy as DNase-sensitive threads inside mitochondria,[81] and by Ellen Haslbrunner, Hans Tuppy and Gottfried Schatz by biochemical assays on highly purified mitochondrial fractions.[82]

Several specialized databases have been founded to collect mitochondrial genome sequences and other information. Although most of them focus on sequence data, some of them include phylogenetic or functional information.

Several specialized databases exist that report polymorphisms and mutations in the human mitochondrial DNA, together with the assessment of their pathogenicity.

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Aedes aegypti Genome Assembled From Scratch | The Scientist ... The Scientist Aedes aegypti is the main vector of the Zika virus. Now, scientists at the Baylor College of Medicine in Houston, Texas, and their colleagues have successfully ... Scientists find a low-cost way to build genomes from scratchEngadget Scientists drastically reduce costs for building and sequencing ...The Tech Portal all 5 news articles »

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Aedes aegypti Genome Assembled From Scratch | The Scientist ... - The Scientist

MOUNTAIN VIEW, Calif., March 29, 2017 /PRNewswire/ --Cellecta, Inc. today announced the launch of the Driver-Map Human Genome-Wide Gene Expression Profiling kit, a targeted RNA expression profiling assay designed to easily provide a molecular snapshot of all known 19,000 human protein-coding genes from complex samples starting from as little as 10 pg of total RNA.

The multiplex RT-PCR-based, followed by next-generation sequencing (NGS) method used in the Driver-Map Expression Profiling kit allows researchers access to the specially designed and experimentally validated gene-specific primer pairs to run samples in their own laboratories. This enables them to generate data that is more sensitive and easier to analyze when compared to conventional genome-wide expression profiling approaches such as RNA-Seq and microarray platforms.

"The simple, robust Driver-Map assay is ideal for use in characterization of complex biological samples," said Alex Chenchik, Ph.D., president and chief scientific officer of Cellecta. "Some novel applications for this gold-standard RT-PCR technology include biomarker discovery with whole blood without a globin or rRNA depletion step, detection of circulating tumor cells (CTC) in whole blood without previous CTC enrichment, high sensitivity profiling of tumor, stromal and infiltrating immune cell types from a complex tumor microenvironment, and expression profiling of patient-derived xenograft (PDX) models without interference from mouse background cells."

The Driver-Map Expression Profiling assay is highly reproducible, even starting with minimal amounts of RNA sample. Noteworthy features of this assay include:

The Driver-Map assay kit includes a complete set of gene-specific and PCR-NGS primers, buffers, spike-in ERCC and positive control RNAs as well as all other reagents required to profile 24 samples and prepare them for digital expression profiling using NGS on an Illumina sequencing platform. NGS reagents are not included in the Driver-Map assay kit.

The Driver-Map Human Genome-Wide Gene Expression Profiling kit (Catalog # DM-hgw) is available now. For more information, please visit http://www.driver-map.com

About Cellecta:

Cellecta, Inc., a trusted provider of genomic products and services, is an industry leader in RNAi and CRISPR technologies for the discovery and characterization of novel therapeutic targets, and genetic profiling for biomarker discovery. Numerous scientific papers have been published citing Cellecta's functional genomics portfolio offering gene knockout and knockdown screens, custom and genome-wide RNAi and CRISPR libraries, cell engineering, RNAi and CRISPR construct services, and mutation and expression profiling of disease samples.

Cellecta, Inc. is headquartered in Mountain View, California. Further information about the company and its functional genomic products and services may be found online at http://www.cellecta.com

Cellecta, Inc. Paul Diehl, 650-938-4050 152593@email4pr.com or Media: Ruth Mercado, 650-938-4080 152593@email4pr.com

To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/cellecta-inc-launches-driver-map-human-genome-wide-gene-expression-profiling-kit-for-19000-human-genes-300430932.html

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Cellecta, Inc. Launches Driver-Map Human Genome-Wide Gene Expression Profiling Kit for 19000 Human Genes - PR Newswire (press release)

VANCOUVER, British Columbia--(BUSINESS WIRE)--STEMCELL Technologies announces the development of CloneR, a novel medium formulation to aid researchers in their ability to successfully genome edit human pluripotent stem cells. This supplement represents a dramatic improvement over existing technologies.

Editing the pluripotent stem cell genome relies heavily on the survival of single cells to establish clonal cell lines. CloneR is a medium supplement designed for greatly enhancing the cloning efficiency and single-cell survival of pluripotent stem cells. Unlike current methods, CloneR will enable the robust generation of clonal cell lines without single-cell adaptation, thus minimizing the risk of acquiring genetic abnormalities. Such irregularities bear a close resemblance to those found in many human cancers, raising safety concerns for the use of these cells in regenerative medicine.

As the market-leader for the development of innovative products for pluripotent stem cell research, we are delighted to announce the imminent launch of CloneR, says Dr. Allen Eaves, CEO and President of STEMCELL Technologies. We are confident that this new invention will enable researchers by improving their capacity for disease modeling, drug screening and the development of cell and gene therapies for regenerative medicine.

CloneR will build upon STEMCELLs extensive portfolio of products to support pluripotent stem cell research, including mTeSR1, the most widely published feeder-free maintenance medium on the market. The CloneR supplement will be manufactured using STEMCELLs rigorous raw material screening and quality control processes certified under ISO 13485, Medical Devices Standards. This will ensure that the final product is of the highest quality, with maximal performance and minimal lot-to-lot variability.

To receive a notification of the product launch, sign up here: http://www.stemcell.com/CloneR

About STEMCELL Technologies

STEMCELL Technologies helps power leading-edge life science research globally. The company offers highly specialized cell culture media, cell separation products, instruments, accessory products and educational tools that support scientists performing stem cell, immunology, cancer, regenerative medicine and cellular therapy research.

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STEMCELL Technologies to Launch CloneR to Facilitate Genome Editing of Human Pluripotent Stem Cells - Business Wire (press release)