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Store Finder – Steve Jackson Games

Posted: May 14, 2019 at 2:51 am

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Store Finder - Steve Jackson Games

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Emergence Series – crimsoncircle.com

Posted: May 14, 2019 at 2:51 am

IMPORTANT NOTE: This information is probably not for you unless you take full responsibility for your life and creations.

Emergence Series is presented and recorded at the Crimson Circle monthly meetings. The first channel of this series was presented on August 4, 2018 and will continue until July 2019. By Clicking on one of the following Shoud links, you can go to the text, audio or video version of each Channel (Shoud), or you can listen to or download the audio file of each

Shouds available to download

Shoud 1: "Emergence Series" - August 4, 2018

Welcome and Shaumbra News

Shoud 2: "Emergence Series" - September 1, 2018

Welcome and Shaumbra News

Shoud 3: "Emergence Series" - November 3, 2018

Welcome and Shaumbra News

Shoud 4: "Emergence Series" - December 8, 2018

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Shoud 5: "Emergence Series" - January 5, 2019

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Shoud 6: "Emergence Series" - February 2, 2019

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Shoud 7: "Emergence Series" - March 2, 2019

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Shoud 8: "Emergence Series" - April 6, 2019

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Shoud 9: "Emergence Series" - May 4, 2019

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Emergence Series - crimsoncircle.com

Recommendation and review posted by G. Smith

Genetic Engineering and Animals | Animal Legal …

Posted: May 14, 2019 at 2:51 am

Genetic Engineering and Animals: A Short Summary of the Legal Terrain and Ethical ImplicationsAndrew B. Perzigian (2003)

With the advent and rapid development of genetic engineering technology, the animal rights movement is currently facing one of its greatest challenges and dilemmas. Proponents of the technology assert that transgenic animals, animals that have been genetically altered through the introduction of another plant's or animal's genes, may one day help solve many of our modern day problems in life, from starvation and ill health, to environmental degradation and the modern extinction crisis. Critics believe that bioengineering poses greater risks than it does benefits. They argue that genetic engineering threatens to increase animal suffering and decrease species integrity, while at the same time creating a potentially devastating impact on the balance and sustainability of the Earth's ecosystem. Regardless, the value judgments we make regarding the direction and scope that this technology should take are sure to have far reaching implications.

Transgenic animals are animals that have, through genetic engineering, genes from other plants and animals. Unlike controlled breeding, which is confined to the genetic material contained in a single species, modern genetic engineering permits an almost limitless scope of modification and introduction of otherwise foreign genetic material. This permits specific traits, and not the host of other traits common from crossbreeding, to be effectively introduced into new, transgenic animal species. Genetic engineering is able to create whole organisms that are not natural to the planet, and whose specific genetic make-up is as much a result of human manipulation as it is natural selection. (For further information on the basics of genetic engineering, see Detailed Discussion ).

With regard to the agricultural industry, transgenic farm animals can be created, that are better able to resist disease, grow faster, and more efficiently reproduce than current species of animals. Transgenic sheep can be created to produce better wool and cows can be engineered to more efficiently convert grain into higher quality milk and meat. Transgenic salmon, salmon that grow larger and at a faster rate than natural varieties, have already been created and farmed. (For further information on thepotential benefits, see Detailed Discussion ).

One of the more controversial uses of this technology is found in recent proposals to engineer farm animals to be non-sentient, without the "stress" genes that cause them great suffering during their lives on industrial factory farms. Since sentience, the ability to feel pain and experience suffering, is the basis upon which much animal rights ideology is based, some argue that these types of transgenic farm animals would help to solve many of the animal welfare issues posed by industrial factory farms. (For more information on the risks, see Detailed Discussion ).

The bio-medical research industry has been equally influenced by genetic engineering technology. Instead of relying on numerous test animals to research modern diseases and appropriate drug therapies, the bio-medical community can now rely on specifically engineered animal research models. Such animals are bred to have an increase susceptibility to modern diseases, like hereditary breast cancer. Transgenic animals have made research of such diseases more accurate, less expensive and faster, while at the same time permitting accurate results with the use of fewer individual animals in any given study.

Also, transgenic animals, like goats, sheep, and cattle, have been engineered to produce large amounts of complex human proteins in their milk, something very useful in the creation of therapeutic drugs. By engineering these animals to release these and other proteins in their milk, the mass production of high quality therapeutic drugs is made less costly, easier to manufacture, and at the expense of fewer animal lives than what was formerly the case. (For more information on the scientific andmedical potential of genetic engineering, see DetailedDiscussion ).

Biotechnology breakthroughs in whole animal cloning have led to many suggestions that such technology could be used to clone endangered species. Cloning provides a great support blanket for the modern extinction crisis and can help to ensure that critical numbers of endangered species will exist for generations to come.

In general, opponents of genetic engineering assert that such technology creates a huge diminution in the standing of animals, leaving them as nothing more than "test tubes with tails," only of benefit for the exploitive practices of factory farming, and drug and organ manufacturing. Creating more efficient agricultural animals threatens weaken the genetic diversity of the herd and thereby make them more susceptible to new strains of infectious disease. Also, if transgenic farm animals ever escape into wild populations, they can have profoundly disturbing effects on the natural environment, including a complete elimination of natural populations and the processes of natural selection.

Animal rights advocates also argue that each species should enjoy an inherent, natural right to be free of genetic manipulation in any form. This is especially the case when genetic engineering is used as a means of depriving animals of their sentience, of exacerbating the cruel, horrific conditions of the modern factory farm and biomedical lab. Although the sheer numbers may decline, the actual suffering experienced by agricultural and research animals may increase.

Cloning endangered species, although useful as a last resort, may unwisely shift our efforts away from protecting the critical habitat necessary to sustain viable endangered species populations. Habitat protection is as important to saving endangered species as is the specific renewal and maintenance of viable numbers within a population. Since limited funds exist, habitat protection, and not expensive cloning technology, should be the focus of our endangered species protection efforts. (For more information on the inherent dangers, see Detailed Discussion ).

Currently, there are few laws, in either the United States or the European Union (EU) regulating animal cloning and the creation of transgenic animals. In the United States, most research and farm animals are excluded from federal protection. While the European Union (EU) ensures that such animals are treated more humanely than is the case in the United States, both the U.S. and the EU extend patent protection to the owners and creators of transgenic animal species. This provides a huge incentive for the biotechnology industry to continually research and develop novel transgenic animal creations. With patents, researchers can now own and monopolize entire animal species, something unheard of prior to modern genetic engineering. The Supreme Court has upheld transgenic animal patents without any review of the potential ethical and environmental risks associated with the technology involved. (For moreon this important decision, click here ).

Most modern legislation regarding genetic engineering and cloning technology ensued following the birth of Dolly the sheep, the first multi-cellular organism cloned from adult cells. The primary objectives of the subsequent United States and EU legislation was to ban human cloning while at the same time ensuring that genetic engineering research continued unimpeded by such legislation. Patent protection effectively promotes genetic engineering research and helps to ensure its speedy development. (For more information on U.S. and European laws concerning biotechnology, see Detailed Discussion ).

There is no doubt that genetic engineering of animals will continue well into the future. Both the United States' and the EU's legal systems have been slow to respond with legislation specifically regulating biotechnology, and each have permitted their patent law to provide a supportive ground for genetic engineering research and development. One thing is for sure, we must not sit complacently by as this technology rapidly changes the fabric of our existence from the inside out. We must not wait and see what the effects are. We must form educated opinions, inspire legislative regulation, and hope that whatever direction that bioengineering takes us, is a positive step towards decreased animal suffering, increased environmental sustainability, and an overall compassionate regard for the earth and its precious life.

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Genetic Engineering and Animals | Animal Legal ...

Recommendation and review posted by G. Smith

Human genome – Wikipedia

Posted: May 14, 2019 at 2:51 am

The human genome is the complete set of nucleic acid sequences for humans, encoded as DNA within the 23 chromosome pairs in cell nuclei and in a small DNA molecule found within individual mitochondria. These are usually treated separately as the nuclear genome, and the mitochondrial genome.[1] Human genomes include both protein-coding DNA genes and noncoding DNA. Haploid human genomes, which are contained in germ cells (the egg and sperm gamete cells created in the meiosis phase of sexual reproduction before fertilization creates a zygote) consist of three billion DNA base pairs, while diploid genomes (found in somatic cells) have twice the DNA content. While there are significant differences among the genomes of human individuals (on the order of 0.1%),[2] these are considerably smaller than the differences between humans and their closest living relatives, the chimpanzees (approximately 4%[3]) and bonobos.

The first human genome sequences were published in nearly complete draft form in February 2001 by the Human Genome Project[4] and Celera Corporation.[5] Completion of the Human Genome Project Sequence was published in 2004.[6] The human genome was the first of all vertebrates to be completely sequenced. As of 2012, thousands of human genomes have been completely sequenced, and many more have been mapped at lower levels of resolution. This data is used worldwide in biomedical science, anthropology, forensics and other branches of science. There is a widely held expectation that genomic studies will lead to advances in the diagnosis and treatment of diseases, and to new insights in many fields of biology, including human evolution.

Although the sequence of the human genome has been (almost) completely determined by DNA sequencing, it is not yet fully understood. Most (though probably not all) genes have been identified by a combination of high throughput experimental and bioinformatics approaches, yet much work still needs to be done to further elucidate the biological functions of their protein and RNA products. Recent results suggest that most of the vast quantities of noncoding DNA within the genome have associated biochemical activities, including regulation of gene expression, organization of chromosome architecture, and signals controlling epigenetic inheritance.

There are an estimated 19,000-20,000 human protein-coding genes.[7] The estimate of the number of human genes has been repeatedly revised down from initial predictions of 100,000 or more as genome sequence quality and gene finding methods have improved, and could continue to drop further.[6][8] Protein-coding sequences account for only a very small fraction of the genome (approximately 1.5%), and the rest is associated with non-coding RNA molecules, regulatory DNA sequences, LINEs, SINEs, introns, and sequences for which as yet no function has been determined.[9]

In June 2016, scientists formally announced HGP-Write, a plan to synthesize the human genome.[10][11]

The total length of the human genome is over 3 billion base pairs. The genome is organized into 22 paired chromosomes, plus the X chromosome (one in males, two in females) and, in males only, one Y chromosome. These are all large linear DNA molecules contained within the cell nucleus. The genome also includes the mitochondrial DNA, a comparatively small circular molecule present in each mitochondrion. Basic information about these molecules and their gene content, based on a reference genome that does not represent the sequence of any specific individual, are provided in the following table. (Data source: Ensembl genome browser release 87, December 2016 for most values; Ensembl genome browser release 68, July 2012 for miRNA, rRNA, snRNA, snoRNA.)

Table 1 (above) summarizes the physical organization and gene content of the human reference genome, with links to the original analysis, as published in the Ensembl database at the European Bioinformatics Institute (EBI) and Wellcome Trust Sanger Institute. Chromosome lengths were estimated by multiplying the number of base pairs by 0.34 nanometers, the distance between base pairs in the DNA double helix. A recent estimation of human chromosome lengths based on updated data reports 205.00 cm for the diploid male genome and 208.23 cm for female, corresponding to weights of 6.41 and 6.51 picograms (pg), respectively.[12] The number of proteins is based on the number of initial precursor mRNA transcripts, and does not include products of alternative pre-mRNA splicing, or modifications to protein structure that occur after translation.

Variations are unique DNA sequence differences that have been identified in the individual human genome sequences analyzed by Ensembl as of December, 2016. The number of identified variations is expected to increase as further personal genomes are sequenced and analyzed. In addition to the gene content shown in this table, a large number of non-expressed functional sequences have been identified throughout the human genome (see below). Links open windows to the reference chromosome sequences in the EBI genome browser.

Small non-coding RNAs are RNAs of as many as 200 bases that do not have protein-coding potential. These include: microRNAs, or miRNAs (post-transcriptional regulators of gene expression), small nuclear RNAs, or snRNAs (the RNA components of spliceosomes), and small nucleolar RNAs, or snoRNA (involved in guiding chemical modifications to other RNA molecules). Long non-coding RNAs are RNA molecules longer than 200 bases that do not have protein-coding potential. These include: ribosomal RNAs, or rRNAs (the RNA components of ribosomes), and a variety of other long RNAs that are involved in regulation of gene expression, epigenetic modifications of DNA nucleotides and histone proteins, and regulation of the activity of protein-coding genes. Small discrepancies between total-small-ncRNA numbers and the numbers of specific types of small ncNRAs result from the former values being sourced from Ensembl release 87 and the latter from Ensembl release 68.

Although the human genome has been completely sequenced for all practical purposes, there are still hundreds of gaps in the sequence. A recent study noted more than 160 euchromatic gaps of which 50 gaps were closed.[13] However, there are still numerous gaps in the heterochromatic parts of the genome which is much harder to sequence due to numerous repeats and other intractable sequence features.

The human reference genome (GRC v38) has been successfully compressed to ~5.2-fold (marginally less than 550 MB) in 155 minutes using a desktop computer with 6.4 GB of RAM.[14]

The haploid human genome (23 chromosomes) is about 3 billion base pairs long and contains around 30,000 genes.[15] Since every base pair can be coded by 2 bits, this is about 750 megabytes of data. An individual somatic (diploid) cell contains twice this amount, that is, about 6 billion base pairs. Men have fewer than women because the Y chromosome is about 57 million base pairs whereas the X is about 156 million, but in terms of information men have more because the second X contains almost the same information as the first[citation needed]. Since individual genomes vary in sequence by less than 1% from each other, the variations of a given human's genome from a common reference can be losslessly compressed to roughly 4 megabytes.[16]

The entropy rate of the genome differs significantly between coding and non-coding sequences. It is close to the maximum of 2 bits per base pair for the coding sequences (about 45 million base pairs), but less for the non-coding parts. It ranges between 1.5 and 1.9 bits per base pair for the individual chromosome, except for the Y-chromosome, which has an entropy rate below 0.9 bits per base pair.[17]

The content of the human genome is commonly divided into coding and noncoding DNA sequences. Coding DNA is defined as those sequences that can be transcribed into mRNA and translated into proteins during the human life cycle; these sequences occupy only a small fraction of the genome (

Some noncoding DNA contains genes for RNA molecules with important biological functions (noncoding RNA, for example ribosomal RNA and transfer RNA). The exploration of the function and evolutionary origin of noncoding DNA is an important goal of contemporary genome research, including the ENCODE (Encyclopedia of DNA Elements) project, which aims to survey the entire human genome, using a variety of experimental tools whose results are indicative of molecular activity.

Because non-coding DNA greatly outnumbers coding DNA, the concept of the sequenced genome has become a more focused analytical concept than the classical concept of the DNA-coding gene.[18][19]

Protein-coding sequences represent the most widely studied and best understood component of the human genome. These sequences ultimately lead to the production of all human proteins, although several biological processes (e.g. DNA rearrangements and alternative pre-mRNA splicing) can lead to the production of many more unique proteins than the number of protein-coding genes.

The complete modular protein-coding capacity of the genome is contained within the exome, and consists of DNA sequences encoded by exons that can be translated into proteins. Because of its biological importance, and the fact that it constitutes less than 2% of the genome, sequencing of the exome was the first major milepost of the Human Genome Project.

Number of protein-coding genes. About 20,000 human proteins have been annotated in databases such as Uniprot.[21] Historically, estimates for the number of protein genes have varied widely, ranging up to 2,000,000 in the late 1960s,[22] but several researchers pointed out in the early 1970s that the estimated mutational load from deleterious mutations placed an upper limit of approximately 40,000 for the total number of functional loci (this includes protein-coding and functional non-coding genes).[23]

The number of human protein-coding genes is not significantly larger than that of many less complex organisms, such as the roundworm and the fruit fly. This difference may result from the extensive use of alternative pre-mRNA splicing in humans, which provides the ability to build a very large number of modular proteins through the selective incorporation of exons.

Protein-coding capacity per chromosome. Protein-coding genes are distributed unevenly across the chromosomes, ranging from a few dozen to more than 2000, with an especially high gene density within chromosomes 19, 11, and 1 (Table 1). Each chromosome contains various gene-rich and gene-poor regions, which may be correlated with chromosome bands and GC-content.[24] The significance of these nonrandom patterns of gene density is not well understood[25]

Size of protein-coding genes. The size of protein-coding genes within the human genome shows enormous variability (Table 2). The median size of a protein-coding gene is 26,288 bp (mean = 66,577 bp; Table 2 in [26]). For example, the gene for histone H1a (HIST1HIA) is relatively small and simple, lacking introns and encoding mRNA sequences of 781 nt and a 215 amino acid protein (648 nt open reading frame). Dystrophin (DMD) is the largest protein-coding gene in the human reference genome, spanning a total of 2.2 MB, while Titin (TTN) has the longest coding sequence (114,414 bp), the largest number of exons (363),[27] and the longest single exon (17,106 bp). Over the whole genome, the median size of an exon is 122 bp (mean = 145 bp), the median number of exons is 7 (mean = 8.8), and the median coding sequence encodes 367 amino acids (mean = 447 amino acids; Table 21 in[9] ).

Table 2. Examples of human protein-coding genes. Chrom, chromosome. Alt splicing, alternative pre-mRNA splicing. (Data source: Ensembl genome browser release 68, July 2012)

Recently, a systematic meta-analysis of updated data of the human genome [26] found that the largest protein-coding gene in the human reference genome is RBFOX1 (RNA binding protein, fox-1 homolog 1), spanning a total of 2.47 MB. Over the whole genome, considering a curated set of protein-coding genes, the median size of an exon is currently estimated to be 133 bp (mean = 309 bp), the median number of exons is currently estimated to be 8 (mean = 11), and the median coding sequence is currently estimated to encode 425 amino acids (mean = 553 amino acids; Tables 2 and 5 in[26]).

Noncoding DNA is defined as all of the DNA sequences within a genome that are not found within protein-coding exons, and so are never represented within the amino acid sequence of expressed proteins. By this definition, more than 98% of the human genomes is composed of ncDNA.

Numerous classes of noncoding DNA have been identified, including genes for noncoding RNA (e.g. tRNA and rRNA), pseudogenes, introns, untranslated regions of mRNA, regulatory DNA sequences, repetitive DNA sequences, and sequences related to mobile genetic elements.

Numerous sequences that are included within genes are also defined as noncoding DNA. These include genes for noncoding RNA (e.g. tRNA, rRNA), and untranslated components of protein-coding genes (e.g. introns, and 5' and 3' untranslated regions of mRNA).

Protein-coding sequences (specifically, coding exons) constitute less than 1.5% of the human genome.[9] In addition, about 26% of the human genome is introns.[28] Aside from genes (exons and introns) and known regulatory sequences (820%), the human genome contains regions of noncoding DNA. The exact amount of noncoding DNA that plays a role in cell physiology has been hotly debated. Recent analysis by the ENCODE project indicates that 80% of the entire human genome is either transcribed, binds to regulatory proteins, or is associated with some other biochemical activity.[8]

It however remains controversial whether all of this biochemical activity contributes to cell physiology, or whether a substantial portion of this is the result transcriptional and biochemical noise, which must be actively filtered out by the organism.[29] Excluding protein-coding sequences, introns, and regulatory regions, much of the non-coding DNA is composed of:Many DNA sequences that do not play a role in gene expression have important biological functions. Comparative genomics studies indicate that about 5% of the genome contains sequences of noncoding DNA that are highly conserved, sometimes on time-scales representing hundreds of millions of years, implying that these noncoding regions are under strong evolutionary pressure and positive selection.[30]

Many of these sequences regulate the structure of chromosomes by limiting the regions of heterochromatin formation and regulating structural features of the chromosomes, such as the telomeres and centromeres. Other noncoding regions serve as origins of DNA replication. Finally several regions are transcribed into functional noncoding RNA that regulate the expression of protein-coding genes (for example[31] ), mRNA translation and stability (see miRNA), chromatin structure (including histone modifications, for example[32] ), DNA methylation (for example[33] ), DNA recombination (for example[34] ), and cross-regulate other noncoding RNAs (for example[35] ). It is also likely that many transcribed noncoding regions do not serve any role and that this transcription is the product of non-specific RNA Polymerase activity.[29]

Pseudogenes are inactive copies of protein-coding genes, often generated by gene duplication, that have become nonfunctional through the accumulation of inactivating mutations. Table 1 shows that the number of pseudogenes in the human genome is on the order of 13,000,[36] and in some chromosomes is nearly the same as the number of functional protein-coding genes. Gene duplication is a major mechanism through which new genetic material is generated during molecular evolution.

For example, the olfactory receptor gene family is one of the best-documented examples of pseudogenes in the human genome. More than 60 percent of the genes in this family are non-functional pseudogenes in humans. By comparison, only 20 percent of genes in the mouse olfactory receptor gene family are pseudogenes. Research suggests that this is a species-specific characteristic, as the most closely related primates all have proportionally fewer pseudogenes. This genetic discovery helps to explain the less acute sense of smell in humans relative to other mammals.[37]

Noncoding RNA molecules play many essential roles in cells, especially in the many reactions of protein synthesis and RNA processing. Noncoding RNA include tRNA, ribosomal RNA, microRNA, snRNA and other non-coding RNA genes including about 60,000 long non coding RNAs (lncRNAs).[8][38][39][40] Although the number of reported lncRNA genes continues to rise and the exact number in the human genome is yet to be defined, many of them are argued to be non-functional.[41]

Many ncRNAs are critical elements in gene regulation and expression. Noncoding RNA also contributes to epigenetics, transcription, RNA splicing, and the translational machinery. The role of RNA in genetic regulation and disease offers a new potential level of unexplored genomic complexity.[42]

In addition to the ncRNA molecules that are encoded by discrete genes, the initial transcripts of protein coding genes usually contain extensive noncoding sequences, in the form of introns, 5'-untranslated regions (5'-UTR), and 3'-untranslated regions (3'-UTR). Within most protein-coding genes of the human genome, the length of intron sequences is 10- to 100-times the length of exon sequences (Table 2).

The human genome has many different regulatory sequences which are crucial to controlling gene expression. Conservative estimates indicate that these sequences make up 8% of the genome,[43] however extrapolations from the ENCODE project give that 20[44]-40%[45] of the genome is gene regulatory sequence. Some types of non-coding DNA are genetic "switches" that do not encode proteins, but do regulate when and where genes are expressed (called enhancers).[46]

Regulatory sequences have been known since the late 1960s.[47] The first identification of regulatory sequences in the human genome relied on recombinant DNA technology.[48] Later with the advent of genomic sequencing, the identification of these sequences could be inferred by evolutionary conservation. The evolutionary branch between the primates and mouse, for example, occurred 7090 million years ago.[49] So computer comparisons of gene sequences that identify conserved non-coding sequences will be an indication of their importance in duties such as gene regulation.[50]

Other genomes have been sequenced with the same intention of aiding conservation-guided methods, for exampled the pufferfish genome.[51] However, regulatory sequences disappear and re-evolve during evolution at a high rate.[52][53][54]

As of 2012, the efforts have shifted toward finding interactions between DNA and regulatory proteins by the technique ChIP-Seq, or gaps where the DNA is not packaged by histones (DNase hypersensitive sites), both of which tell where there are active regulatory sequences in the investigated cell type.[43]

Repetitive DNA sequences comprise approximately 50% of the human genome.[55]

About 8% of the human genome consists of tandem DNA arrays or tandem repeats, low complexity repeat sequences that have multiple adjacent copies (e.g. "CAGCAGCAG...").[56]The tandem sequences may be of variable lengths, from two nucleotides to tens of nucleotides. These sequences are highly variable, even among closely related individuals, and so are used for genealogical DNA testing and forensic DNA analysis.[57]

Repeated sequences of fewer than ten nucleotides (e.g. the dinucleotide repeat (AC)n) are termed microsatellite sequences. Among the microsatellite sequences, trinucleotide repeats are of particular importance, as sometimes occur within coding regions of genes for proteins and may lead to genetic disorders. For example, Huntington's disease results from an expansion of the trinucleotide repeat (CAG)n within the Huntingtin gene on human chromosome 4. Telomeres (the ends of linear chromosomes) end with a microsatellite hexanucleotide repeat of the sequence (TTAGGG)n.

Tandem repeats of longer sequences (arrays of repeated sequences 1060 nucleotides long) are termed minisatellites.

Transposable genetic elements, DNA sequences that can replicate and insert copies of themselves at other locations within a host genome, are an abundant component in the human genome. The most abundant transposon lineage, Alu, has about 50,000 active copies,[58] and can be inserted into intragenic and intergenic regions.[59] One other lineage, LINE-1, has about 100 active copies per genome (the number varies between people).[60] Together with non-functional relics of old transposons, they account for over half of total human DNA.[61] Sometimes called "jumping genes", transposons have played a major role in sculpting the human genome. Some of these sequences represent endogenous retroviruses, DNA copies of viral sequences that have become permanently integrated into the genome and are now passed on to succeeding generations.

Mobile elements within the human genome can be classified into LTR retrotransposons (8.3% of total genome), SINEs (13.1% of total genome) including Alu elements, LINEs (20.4% of total genome), SVAs and Class II DNA transposons (2.9% of total genome).

With the exception of identical twins, all humans show significant variation in genomic DNA sequences. The human reference genome (HRG) is used as a standard sequence reference.

There are several important points concerning the human reference genome:

The Genome Reference Consortium is responsible for updating the HRG. Version 38 was released in December 2013.[62]

Most studies of human genetic variation have focused on single-nucleotide polymorphisms (SNPs), which are substitutions in individual bases along a chromosome. Most analyses estimate that SNPs occur 1 in 1000 base pairs, on average, in the euchromatic human genome, although they do not occur at a uniform density. Thus follows the popular statement that "we are all, regardless of race, genetically 99.9% the same",[63] although this would be somewhat qualified by most geneticists. For example, a much larger fraction of the genome is now thought to be involved in copy number variation.[64] A large-scale collaborative effort to catalog SNP variations in the human genome is being undertaken by the International HapMap Project.

The genomic loci and length of certain types of small repetitive sequences are highly variable from person to person, which is the basis of DNA fingerprinting and DNA paternity testing technologies. The heterochromatic portions of the human genome, which total several hundred million base pairs, are also thought to be quite variable within the human population (they are so repetitive and so long that they cannot be accurately sequenced with current technology). These regions contain few genes, and it is unclear whether any significant phenotypic effect results from typical variation in repeats or heterochromatin.

Most gross genomic mutations in gamete germ cells probably result in inviable embryos; however, a number of human diseases are related to large-scale genomic abnormalities. Down syndrome, Turner Syndrome, and a number of other diseases result from nondisjunction of entire chromosomes. Cancer cells frequently have aneuploidy of chromosomes and chromosome arms, although a cause and effect relationship between aneuploidy and cancer has not been established.

Whereas a genome sequence lists the order of every DNA base in a genome, a genome map identifies the landmarks. A genome map is less detailed than a genome sequence and aids in navigating around the genome.[65][66]

An example of a variation map is the HapMap being developed by the International HapMap Project. The HapMap is a haplotype map of the human genome, "which will describe the common patterns of human DNA sequence variation."[67] It catalogs the patterns of small-scale variations in the genome that involve single DNA letters, or bases.

Researchers published the first sequence-based map of large-scale structural variation across the human genome in the journal Nature in May 2008.[68][69] Large-scale structural variations are differences in the genome among people that range from a few thousand to a few million DNA bases; some are gains or losses of stretches of genome sequence and others appear as re-arrangements of stretches of sequence. These variations include differences in the number of copies individuals have of a particular gene, deletions, translocations and inversions.

Single-nucleotide polymorphisms (SNPs) do not occur homogeneously across the human genome. In fact, there is enormous diversity in SNP frequency between genes, reflecting different selective pressures on each gene as well as different mutation and recombination rates across the genome. However, studies on SNPs are biased towards coding regions, the data generated from them are unlikely to reflect the overall distribution of SNPs throughout the genome. Therefore, the SNP Consortium protocol was designed to identify SNPs with no bias towards coding regions and the Consortium's 100,000 SNPs generally reflect sequence diversity across the human chromosomes.The SNP Consortium aims to expand the number of SNPs identified across the genome to 300 000 by the end of the first quarter of 2001.[70]

Changes in non-coding sequence and synonymous changes in coding sequence are generally more common than non-synonymous changes, reflecting greater selective pressure reducing diversity at positions dictating amino acid identity. Transitional changes are more common than transversions, with CpG dinucleotides showing the highest mutation rate, presumably due to deamination.

A personal genome sequence is a (nearly) complete sequence of the chemical base pairs that make up the DNA of a single person. Because medical treatments have different effects on different people due to genetic variations such as single-nucleotide polymorphisms (SNPs), the analysis of personal genomes may lead to personalized medical treatment based on individual genotypes.[71]

The first personal genome sequence to be determined was that of Craig Venter in 2007. Personal genomes had not been sequenced in the public Human Genome Project to protect the identity of volunteers who provided DNA samples. That sequence was derived from the DNA of several volunteers from a diverse population.[72] However, early in the Venter-led Celera Genomics genome sequencing effort the decision was made to switch from sequencing a composite sample to using DNA from a single individual, later revealed to have been Venter himself. Thus the Celera human genome sequence released in 2000 was largely that of one man. Subsequent replacement of the early composite-derived data and determination of the diploid sequence, representing both sets of chromosomes, rather than a haploid sequence originally reported, allowed the release of the first personal genome.[73] In April 2008, that of James Watson was also completed. Since then hundreds of personal genome sequences have been released,[74] including those of Desmond Tutu,[75][76] and of a Paleo-Eskimo.[77] In November 2013, a Spanish family made their personal genomics data publicly available under a Creative Commons public domain license. The work was led by Manuel Corpas and the data obtained by direct-to-consumer genetic testing with 23andMe and the Beijing Genomics Institute). This is believed to be the first such public genomics dataset for a whole family.[78]

The sequencing of individual genomes further unveiled levels of genetic complexity that had not been appreciated before. Personal genomics helped reveal the significant level of diversity in the human genome attributed not only to SNPs but structural variations as well. However, the application of such knowledge to the treatment of disease and in the medical field is only in its very beginnings.[79] Exome sequencing has become increasingly popular as a tool to aid in diagnosis of genetic disease because the exome contributes only 1% of the genomic sequence but accounts for roughly 85% of mutations that contribute significantly to disease.[80]

In humans, gene knockouts naturally occur as heterozygous or homozygous loss-of-function gene knockouts. These knockouts are often difficult to distinguish, especially within heterogeneous genetic backgrounds. They are also difficult to find as they occur in low frequencies.

Populations with high rates of consanguinity, such as countries with high rates of first-cousin marriages, display the highest frequencies of homozygous gene knockouts. Such populations include Pakistan, Iceland, and Amish populations. These populations with a high level of parental-relatedness have been subjects of human knock out research which has helped to determine the function of specific genes in humans. By distinguishing specific knockouts, researchers are able to use phenotypic analyses of these individuals to help characterize the gene that has been knocked out.

Knockouts in specific genes can cause genetic diseases, potentially have beneficial effects, or even result in no phenotypic effect at all. However, determining a knockouts phenotypic effect and in humans can be challenging. Challenges to characterizing and clinically interpreting knockouts include difficulty calling of DNA variants, determining disruption of protein function (annotation), and considering the amount of influence mosaicism has on the phenotype. [82]

One major study that investigated human knockouts is the Pakistan Risk of Myocardial Infarction study. It was found that individuals possessing a heterozygous loss-of-function gene knockout for the APOC3 gene had lower triglycerides in the blood after consuming a high fat meal as compared to individuals without the mutation. However, individuals possessing homozygous loss-of-function gene knockouts of the APOC3 gene displayed the lowest level of triglycerides in the blood after the fat load test, as they produce no functional APOC3 protein. [83]

Most aspects of human biology involve both genetic (inherited) and non-genetic (environmental) factors. Some inherited variation influences aspects of our biology that are not medical in nature (height, eye color, ability to taste or smell certain compounds, etc.). Moreover, some genetic disorders only cause disease in combination with the appropriate environmental factors (such as diet). With these caveats, genetic disorders may be described as clinically defined diseases caused by genomic DNA sequence variation. In the most straightforward cases, the disorder can be associated with variation in a single gene. For example, cystic fibrosis is caused by mutations in the CFTR gene, and is the most common recessive disorder in caucasian populations with over 1,300 different mutations known.[84]

Disease-causing mutations in specific genes are usually severe in terms of gene function, and are fortunately rare, thus genetic disorders are similarly individually rare. However, since there are many genes that can vary to cause genetic disorders, in aggregate they constitute a significant component of known medical conditions, especially in pediatric medicine. Molecularly characterized genetic disorders are those for which the underlying causal gene has been identified, currently there are approximately 2,200 such disorders annotated in the OMIM database.[84]

Studies of genetic disorders are often performed by means of family-based studies. In some instances population based approaches are employed, particularly in the case of so-called founder populations such as those in Finland, French-Canada, Utah, Sardinia, etc. Diagnosis and treatment of genetic disorders are usually performed by a geneticist-physician trained in clinical/medical genetics. The results of the Human Genome Project are likely to provide increased availability of genetic testing for gene-related disorders, and eventually improved treatment. Parents can be screened for hereditary conditions and counselled on the consequences, the probability it will be inherited, and how to avoid or ameliorate it in their offspring.

As noted above, there are many different kinds of DNA sequence variation, ranging from complete extra or missing chromosomes down to single nucleotide changes. It is generally presumed that much naturally occurring genetic variation in human populations is phenotypically neutral, i.e. has little or no detectable effect on the physiology of the individual (although there may be fractional differences in fitness defined over evolutionary time frames). Genetic disorders can be caused by any or all known types of sequence variation. To molecularly characterize a new genetic disorder, it is necessary to establish a causal link between a particular genomic sequence variant and the clinical disease under investigation. Such studies constitute the realm of human molecular genetics.

With the advent of the Human Genome and International HapMap Project, it has become feasible to explore subtle genetic influences on many common disease conditions such as diabetes, asthma, migraine, schizophrenia, etc. Although some causal links have been made between genomic sequence variants in particular genes and some of these diseases, often with much publicity in the general media, these are usually not considered to be genetic disorders per se as their causes are complex, involving many different genetic and environmental factors. Thus there may be disagreement in particular cases whether a specific medical condition should be termed a genetic disorder. The categorized table below provides the prevalence as well as the genes or chromosomes associated with some human genetic disorders.

Human development over the last 10 million years

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Comparative genomics studies of mammalian genomes suggest that approximately 5% of the human genome has been conserved by evolution since the divergence of extant lineages approximately 200 million years ago, containing the vast majority of genes.[86][87] The published chimpanzee genome differs from that of the human genome by 1.23% in direct sequence comparisons.[88] Around 20% of this figure is accounted for by variation within each species, leaving only ~1.06% consistent sequence divergence between humans and chimps at shared genes.[89] This nucleotide by nucleotide difference is dwarfed, however, by the portion of each genome that is not shared, including around 6% of functional genes that are unique to either humans or chimps.[90]

In other words, the considerable observable differences between humans and chimps may be due as much or more to genome level variation in the number, function and expression of genes rather than DNA sequence changes in shared genes. Indeed, even within humans, there has been found to be a previously unappreciated amount of copy number variation (CNV) which can make up as much as 5 15% of the human genome. In other words, between humans, there could be +/- 500,000,000 base pairs of DNA, some being active genes, others inactivated, or active at different levels. The full significance of this finding remains to be seen. On average, a typical human protein-coding gene differs from its chimpanzee ortholog by only two amino acid substitutions; nearly one third of human genes have exactly the same protein translation as their chimpanzee orthologs. A major difference between the two genomes is human chromosome 2, which is equivalent to a fusion product of chimpanzee chromosomes 12 and 13.[91] (later renamed to chromosomes 2A and 2B, respectively).

Humans have undergone an extraordinary loss of olfactory receptor genes during our recent evolution, which explains our relatively crude sense of smell compared to most other mammals. Evolutionary evidence suggests that the emergence of color vision in humans and several other primate species has diminished the need for the sense of smell.[92]

In September 2016, scientists reported that, based on human DNA genetic studies, all non-Africans in the world today can be traced to a single population that exited Africa between 50,000 and 80,000 years ago.[93]

The human mitochondrial DNA is of tremendous interest to geneticists, since it undoubtedly plays a role in mitochondrial disease. It also sheds light on human evolution; for example, analysis of variation in the human mitochondrial genome has led to the postulation of a recent common ancestor for all humans on the maternal line of descent (see Mitochondrial Eve).

Due to the lack of a system for checking for copying errors, mitochondrial DNA (mtDNA) has a more rapid rate of variation than nuclear DNA. This 20-fold higher mutation rate allows mtDNA to be used for more accurate tracing of maternal ancestry. Studies of mtDNA in populations have allowed ancient migration paths to be traced, such as the migration of Native Americans from Siberia or Polynesians from southeastern Asia. It has also been used to show that there is no trace of Neanderthal DNA in the European gene mixture inherited through purely maternal lineage.[94] Due to the restrictive all or none manner of mtDNA inheritance, this result (no trace of Neanderthal mtDNA) would be likely unless there were a large percentage of Neanderthal ancestry, or there was strong positive selection for that mtDNA (for example, going back 5 generations, only 1 of your 32 ancestors contributed to your mtDNA, so if one of these 32 was pure Neanderthal you would expect that ~3% of your autosomal DNA would be of Neanderthal origin, yet you would have a ~97% chance to have no trace of Neanderthal mtDNA).

Epigenetics describes a variety of features of the human genome that transcend its primary DNA sequence, such as chromatin packaging, histone modifications and DNA methylation, and which are important in regulating gene expression, genome replication and other cellular processes. Epigenetic markers strengthen and weaken transcription of certain genes but do not affect the actual sequence of DNA nucleotides. DNA methylation is a major form of epigenetic control over gene expression and one of the most highly studied topics in epigenetics. During development, the human DNA methylation profile experiences dramatic changes. In early germ line cells, the genome has very low methylation levels. These low levels generally describe active genes. As development progresses, parental imprinting tags lead to increased methylation activity.[95][96]

Epigenetic patterns can be identified between tissues within an individual as well as between individuals themselves. Identical genes that have differences only in their epigenetic state are called epialleles. Epialleles can be placed into three categories: those directly determined by an individuals genotype, those influenced by genotype, and those entirely independent of genotype. The epigenome is also influenced significantly by environmental factors. Diet, toxins, and hormones impact the epigenetic state. Studies in dietary manipulation have demonstrated that methyl-deficient diets are associated with hypomethylation of the epigenome. Such studies establish epigenetics as an important interface between the environment and the genome.[97]

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Human genome - Wikipedia

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Genome editing – Wikipedia

Posted: May 14, 2019 at 2:50 am

Genome editing, or genome engineering, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site specific locations.

In 2018, the common methods for such editing use engineered nucleases, or "molecular scissors". These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits').

As of 2015 four families of engineered nucleases were used: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.[1][2][3][4] Nine genome editors were available as of 2017.[5]

Genome editing with engineered nucleases, i.e. all three major classes of these enzymeszinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered meganucleaseswere selected by Nature Methods as the 2011 Method of the Year.[6] The CRISPR-Cas system was selected by Science as 2015 Breakthrough of the Year.[7]

Genetic engineering as a method of introducing new genetic elements into organisms has been around since the 1970s. One drawback of this technology has been the random nature with which the DNA is inserted into the hosts genome. This can impair or alter other genes within the organism. Methods were sought which targeted the inserted genes to specific sites within an organism genome. As well as reducing off-target effects it also enabled the editing of specific sequences within a genome. This could be used for research purposes, by targeting mutations to specific genes, and in gene therapy. By inserting a functional gene into an organism and targeting it to replace the defective one it could be possible to cure certain genetic diseases.

Early methods to target genes to certain sites within a genome (called gene targeting) relied on homologous recombination (HR).[8] By creating DNA constructs that contain a template that matches the targeted genome sequence it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on embryonic stem cells led to the development of transgenic mice with targeted genes knocked out. It has also been possible to knock in genes or alter gene expression patterns.[9] In recognition of their discovery of how homologous recombination can be used to introduce genetic modifications in mice through embryonic stem cells, Mario Capecchi, Martin Evans and Oliver Smithies were awarded the 2007 Nobel Prize for Physiology or Medicine.[10]

If a vital gene is knocked out it can prove lethal to the organism. In order to study the function of these genes site specific recombinases (SSR) were used. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognising FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that express the SSR under control of tissue specific promoters, it is possible to knock out or switch on genes only in certain cells. These techniques were also used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or stages of development.[9]

Genome editing relies on the concept of DNA double stranded break (DSB) repair mechanics. There are two major pathways that repair DSB; non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ uses a variety of enzymes to directly join the DNA ends while the more accurate HDR uses a homologous sequence as a template for regeneration of missing DNA sequences at the break point. This can be exploited by creating a vector with the desired genetic elements within a sequence that is homologous to the flanking sequences of a DSB. This will result in the desired change being inserted at the site of the DSB. While HDR based gene editing is similar to the homologous recombination based gene targeting, the rate of recombination is increased by at least three orders of magnitude.[11]

The key to genome editing is creating a DSB at a specific point within the genome. Commonly used restriction enzymes are effective at cutting DNA, but generally recoginze and cut at multiple sites. To overcome this challenge and create site-specific DSB, three distinct classes of nucleases have been discovered and bioengineered to date. These are the Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALEN), meganucleases and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.

Meganucleases, discovered in the late 1980s, are enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs).[12] The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence.

Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific.[13][14] However, there is virtually no chance of finding the exact meganuclease required to act on a chosen specific DNA sequence. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences.[14][15] Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence.[16][17] Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucelases in a method named rationally designed meganuclease.[18] Another approach involves using computer models to try to predict as accurately as possible the activity of the modified meganucleases and the specificity of the recognized nucleic sequence.[19]

A large bank containing several tens of thousands of protein units has been created. These units can be combined to obtain chimeric meganucleases that recognize the target site, thereby providing research and development tools that meet a wide range of needs (fundamental research, health, agriculture, industry, energy, etc.) These include the industrial-scale production of two meganucleases able to cleave the human XPC gene; mutations in this gene result in Xeroderma pigmentosum, a severe monogenic disorder that predisposes the patients to skin cancer and burns whenever their skin is exposed to UV rays.[20]

Meganucleases have the benefit of causing less toxicity in cells than methods such as Zinc finger nuclease (ZFN), likely because of more stringent DNA sequence recognition;[14] however, the construction of sequence-specific enzymes for all possible sequences is costly and time consuming, as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALEN-based fusions utilize.

As opposed to meganucleases, the concept behind ZFNs and TALEN technology is based on a non-specific DNA cutting catalytic domain, which can then be linked to specific DNA sequence recognizing peptides such as zinc fingers and transcription activator-like effectors (TALEs).[21] The first step to this was to find an endonuclease whose DNA recognition site and cleaving site were separate from each other, a situation that is not the most common among restriction enzymes.[21] Once this enzyme was found, its cleaving portion could be separated which would be very non-specific as it would have no recognition ability. This portion could then be linked to sequence recognizing peptides that could lead to very high specificity.

Zinc finger motifs occur in several transcription factors. The zinc ion, found in 8% of all human proteins, plays an important role in the organization of their three-dimensional structure. In transcription factors, it is most often located at the protein-DNA interaction sites, where it stabilizes the motif. The C-terminal part of each finger is responsible for the specific recognition of the DNA sequence.

The recognized sequences are short, made up of around 3 base pairs, but by combining 6 to 8 zinc fingers whose recognition sites have been characterized, it is possible to obtain specific proteins for sequences of around 20 base pairs. It is therefore possible to control the expression of a specific gene. It has been demonstrated that this strategy can be used to promote a process of angiogenesis in animals.[22] It is also possible to fuse a protein constructed in this way with the catalytic domain of an endonuclease in order to induce a targeted DNA break, and therefore to use these proteins as genome engineering tools.[23]

The method generally adopted for this involves associating two DNA binding proteins each containing 3 to 6 specifically chosen zinc fingers with the catalytic domain of the FokI endonuclease which need to dimerize to cleave the double-strand DNA. The two proteins recognize two DNA sequences that are a few nucleotides apart. Linking the two zinc finger proteins to their respective sequences brings the two FokI domains closer together. FokI requires dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner would recognize a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered that can only function as heterodimers.[24]

Several approaches are used to design specific zinc finger nucleases for the chosen sequences. The most widespread involves combining zinc-finger units with known specificities (modular assembly). Various selection techniques, using bacteria, yeast or mammal cells have been developed to identify the combinations that offer the best specificity and the best cell tolerance. Although the direct genome-wide characterization of zinc finger nuclease activity has not been reported, an assay that measures the total number of double-strand DNA breaks in cells found that only one to two such breaks occur above background in cells treated with zinc finger nucleases with a 24 bp composite recognition site and obligate heterodimer FokI nuclease domains.[24]

The heterodimer functioning nucleases would avoid the possibility of unwanted homodimer activity and thus increase specificity of the DSB. Although the nuclease portions of both ZFNs and TALEN constructs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2-His2 zinc fingers and TALEN constructs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically happen in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins such as transcription factors. Each finger of the Zinc finger domain is completely independent and the binding capacity of one finger is impacted by its neighbor. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities.[13] Zinc fingers have been more established in these terms and approaches such as modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries among other methods have been used to make site specific nucleases.

Zinc finger nucleases are research and development tools that have already been used to modify a range of genomes, in particular by the laboratories in the Zinc Finger Consortium. The US company Sangamo BioSciences uses zinc finger nucleases to carry out research into the genetic engineering of stem cells and the modification of immune cells for therapeutic purposes.[25][26] Modified T lymphocytes are currently undergoing phase I clinical trials to treat a type of brain tumor (glioblastoma) and in the fight against AIDS.[24]

Transcription activator-like effector nucleases (TALENs) are specific DNA-binding proteins that feature an array of 33 or 34-amino acid repeats. TALENs are artificial restriction enzymes designed by fusing the DNA cutting domain of a nuclease to TALE domains, which can be tailored to specifically recognize a unique DNA sequence. These fusion proteins serve as readily targetable "DNA scissors" for gene editing applications that enable to perform targeted genome modifications such as sequence insertion, deletion, repair and replacement in living cells.[27] The DNA binding domains, which can be designed to bind any desired DNA sequence, comes from TAL effectors, DNA-binding proteins excreted by plant pathogenic Xanthomanos app. TAL effectors consists of repeated domains, each of which contains a highly considered sequence of 34 amino acids, and recognize a single DNA nucleotide within the target site. The nuclease can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions. Each repeat is conserved, with the exception of the so-called repeat variable di-residues (RVDs) at amino acid positions 12 and 13. The RVDs determine the DNA sequence to which the TALE will bind. This simple one-to-one correspondence between the TALE repeats and the corresponding DNA sequence makes the process of assembling repeat arrays to recognize novel DNA sequences straightforward. These TALENs can be fused to the catalytic domain from a DNA nuclease, FokI, to generate a transcription activator-like effector nuclease (TALEN). The resultant TALEN constructs combine specificity and activity, effectively generating engineered sequence-specific nucleases that bind and cleave DNA sequences only at pre-selected sites. The TALEN target recognition system is based on an easy-to-predict code. TAL nucleases are specific to their target due in part to the length of their 30+ base pairs binding site. TALEN can be performed within a 6 base pairs range of any single nucleotide in the entire genome.[28]

TALEN constructs are used in a similar way to designed zinc finger nucleases, and have three advantages in targeted mutagenesis: (1) DNA binding specificity is higher, (2) off-target effects are lower, and (3) construction of DNA-binding domains is easier.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are genetic elements that bacteria use as a kind of acquired immunity to protect against viruses. They consist of short sequences that originate from viral genomes and have been incorporated into the bacterial genome. Cas (CRISPR associated proteins) process these sequences and cut matching viral DNA sequences. By introducing plasmids containing Cas genes and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cut at any desired position.[29] Several companies, including Cellectis[30] and Editas, have been working to monetize the CRISPR method while developing gene-specific therapies.[31][32]

Meganucleases method of gene editing is the least efficient of the methods mentioned above. Due to the nature of its DNA-binding element and the cleaving element, it is limited to recognizing one potential target every 1,000 nucleotides.[4] ZFN was developed to overcome the limitations of meganuclease. The number of possible targets ZFN can recognized was increased to one in every 140 nucleotides.[4] However, both methods are unpredictable due to the ability of their DNA-binding elements affecting each other. As a result, high degrees of expertise and lengthy and costly validations processes are required.

TALE nucleases being the most precise and specific method yields a higher efficiency than the previous two methods. It achieves such efficiency because the DNA-binding element consists of an array of TALE subunits, each of them having the capability of recognizing a specific DNA nucleotide chain independent from others, resulting in a higher number of target sites with high precision. New TALE nucleases take about one week and a few hundred dollars to create, with specific expertise in molecular biology and protein engineering.[4]

CRISPR nucleases have a slightly lower precision when compared to the TALE nucleases. This is caused by the need of having a specific nucleotide at one end in order to produce the guide RNA that CRISPR uses to repair the double-strand break it induces. It has been shown to be the quickest and cheapest method, only costing less than two hundred dollars and a few days of time.[4] CRISPR also requires the least amount of expertise in molecular biology as the design lays in the guide RNA instead of the proteins. One major advantage that CRISPR has over the ZFN and TALEN methods is that it can be directed to target different DNA sequences using its ~80nt CRISPR sgRNAs, while both ZFN and TALEN methods required construction and testing of the proteins created for targeting each DNA sequence.[33]

Because off-target activity of an active nuclease would have potentially dangerous consequences at the genetic and organismal levels, the precision of meganucleases, ZFNs, CRISPR, and TALEN-based fusions has been an active area of research. While variable figures have been reported, ZFNs tend to have more cytotoxicity than TALEN methods or RNA-guided nucleases, while TALEN and RNA-guided approaches tend to have the greatest efficiency and fewer off-target effects.[34] Based on the maximum theoretical distance between DNA binding and nuclease activity, TALEN approaches result in the greatest precision.[4]

The methods for scientists and researchers wanting to study genomic diversity and all possible associated phenotypes were very slow, expensive, and inefficient. Prior to this new revolution, researchers would have to do single-gene manipulations and tweak the genome one little section at a time, observe the phenotype, and start the process over with a different single-gene manipulation.[35] Therefore, researchers at the Wyss Institute at Harvard University designed the MAGE, a powerful technology that improves the process of in vivo genome editing. It allows for quick and efficient manipulations of a genome, all happening in a machine small enough to put on top of a small kitchen table. Those mutations combine with the variation that naturally occurs during cell mitosis creating billions of cellular mutations.

Chemically combined, synthetic single-stranded DNA (ssDNA) and a pool of oligionucleotides are introduced at targeted areas of the cell thereby creating genetic modifications. The cyclical process involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. Experiments targeting selective phenotypic markers are screened and identified by plating the cells on differential medias. Each cycle ultimately takes 2.5 hours to process, with additional time required to grow isogenic cultures and characterize mutations. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. There can be up to 50 genome edits, from single nucleotide base pairs to whole genome or gene networks simultaneously with results in a matter of days.[35]

MAGE experiments can be divided into three classes, characterized by varying degrees of scale and complexity: (i) many target sites, single genetic mutations; (ii) single target site, many genetic mutations; and (iii) many target sites, many genetic mutations.[35] An example of class three was reflected in 2009, where Church and colleagues were able to program Escherichia coli to produce five times the normal amount of lycopene, an antioxidant normally found in tomato seeds and linked to anti-cancer properties. They applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) metabolic pathway in Escherichia coli to overproduce isoprenoid lycopene. It took them about 3 days and just over $1,000 in materials. The ease, speed, and cost efficiency in which MAGE can alter genomes can transform how industries approach the manufacturing and production of important compounds in the bioengineering, bioenergy, biomedical engineering, synthetic biology, pharmaceutical, agricultural, and chemical industries.

As of 2012 efficient genome editing had been developed for a wide range of experimental systems ranging from plants to animals, often beyond clinical interest, and was becoming a standard experimental strategy in research labs.[36] The recent generation of rat, zebrafish, maize and tobacco ZFN-mediated mutants and the improvements in TALEN-based approaches testify to the significance of the methods, and the list is expanding rapidly. Genome editing with engineered nucleases will likely contribute to many fields of life sciences from studying gene functions in plants and animals to gene therapy in humans. For instance, the field of synthetic biology which aims to engineer cells and organisms to perform novel functions, is likely to benefit from the ability of engineered nuclease to add or remove genomic elements and therefore create complex systems.[36] In addition, gene functions can be studied using stem cells with engineered nucleases.

Listed below are some specific tasks this method can carry out:

The combination of recent discoveries in genetic engineering, particularly gene editing and the latest improvement in bovine reproduction technologies (e.g. in vitro embryo culture) allows for genome editing directly in fertilised oocytes using synthetic highly specific endonucleases. RNA-guided endonucleases:clustered regularly interspaced short palindromic repeats associated Cas9 (CRISPR/Cas9) are a new tool, further increasing the range of methods available. In particular CRISPR/Cas9 engineered endonucleases allows the use of multiple guide RNAs for simultaneous Knockouts (KO) in one step by cytoplasmic direct injection (CDI) on mammalian zygotes.[37]

Thanks to the parallel development of single cell transcriptomics, genome editing and new stem cell models we are now entering a scientifically exciting period where functional genetics is no longer restricted to animal models but can be performed directly in human samples. Single cell gene expression analysis has resolved a transcriptional road-map of human development from which key candidate genes are being identified for functional studies. Using global transcriptomics data to guide experimentation, the CRISPR based genome editing tool has made it feasible to disrupt or remove key genes in order to elucidate function in a human setting. [38]

Genome editing using Meganuclease,[39] ZFNs, and TALEN provides a new strategy for genetic manipulation in plants and are likely to assist in the engineering of desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes.[24] Progress in such cases have been recently reported in Arabidopsis thaliana[40][41][42] and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations.[43] In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case.[44] Such genome modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation.[44] A potentially successful example of the application of genome editing techniques in crop improvement can be found in banana, where scientists used CRISPR/Cas9 editing to inactivate the endogenous banana streak virus in the B genome of banana (Musa spp.) to overcome a major challenge in banana breeding.[45]

In addition, TALEN-based genome engineering has been extensively tested and optimized for use in plants.[46]TALEN fusions have also been used by a U.S. food ingredient company, Calyxt,[47] to improve the quality of soybean oil products[48] and to increase the storage potential of potatoes[49]

Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting.[50] There is a need for reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.[50]

A common delivery method for CRISPR/Cas9 in plants is Agrobacterium-based transformation.[51] T-DNA is introduced directly into the plant genome by a T4SS mechanism. Cas9 and gRNA-based expression cassettes are turned into Ti plasmids, which are transformed in Agrobacterium for plant application.[51] To improve Cas9 delivery in live plants, viruses are being used more effective transgene delivery.[51]

The ideal gene therapy practice is that which replaces the defective gene with a normal allele at its natural location. This is advantageous over a virally delivered gene as there is no need to include the full coding sequences and regulatory sequences when only a small proportions of the gene needs to be altered as is often the case.[52] The expression of the partially replaced genes is also more consistent with normal cell biology than full genes that are carried by viral vectors.

The first clinical use of TALEN-based genome editing was in the treatment of CD19+ acute lymphoblastic leukemia in an 11-month old child in 2015. Modified donor T cells were engineered to attack the leukemia cells, to be resistant to Alemtuzumab, and to evade detection by the host immune system after introduction.[53][54]

Extensive research has been done in cells and animals using CRISPR-Cas9 to attempt to correct genetic mutations which cause genetic diseases such as Down syndrome, spina bifida, anencephaly, and Turner and Klinefelter syndromes.[55]

In February 2019, medical scientists working with Sangamo Therapeutics, headquartered in Richmond, California, announced the first ever "in body" human gene editing therapy to permanently alter DNA - in a patient with Hunter Syndrome.[56] Clinical trials by Sangamo involving gene editing using Zinc Finger Nuclease (ZFN) are ongoing.[57]

Researchers have used CRISPR-Cas9 gene drives to modify genes associated with sterility in A. gambiae, the vector for malaria.[58] This technique has further implications in eradicating other vector borne diseases such as yellow fever, dengue, and Zika.[59]

The CRISPR-Cas9 system can be programmed to modulate the population of any bacterial species by targeting clinical genotypes or epidemiological isolates. It can selectively enable the beneficial bacterial species over the harmful ones by eliminating pathogen, which gives it an advantage over broad-spectrum antibiotics.[35]

Antiviral applications for therapies targeting human viruses such as HIV, herpes, and hepatitis B virus are under research. CRISPR can be used to target the virus or the host to disrupt genes encoding the virus cell-surface receptor proteins.[33] In November 2018, He Jiankui announced that he had edited two human embryos, to attempt to disable the gene for CCR5, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature.[60]

In January 2019, scientists in China reported the creation of five identical cloned gene-edited monkeys, using the same cloning technique that was used with Zhong Zhong and Hua Hua the first ever cloned monkeys - and Dolly the sheep, and the same gene-editing Crispr-Cas9 technique allegedly used by He Jiankui in creating the first ever gene-modified human babies Lulu and Nana. The monkey clones were made in order to study several medical diseases.[61][62]

In the future, an important goal of research into genome editing with engineered nucleases must be the improvement of the safety and specificity of the nucleases. For example, improving the ability to detect off-target events can improve our ability to learn about ways of preventing them. In addition, zinc-fingers used in ZFNs are seldom completely specific, and some may cause a toxic reaction. However, the toxicity has been reported to be reduced by modifications done on the cleavage domain of the ZFN.[52]

In addition, research by Dana Carroll into modifying the genome with engineered nucleases has shown the need for better understanding of the basic recombination and repair machinery of DNA. In the future, a possible method to identify secondary targets would be to capture broken ends from cells expressing the ZFNs and to sequence the flanking DNA using high-throughput sequencing.[52]

Because of the ease of use and cost-efficiency of CRISPR, extensive research is currently being done on it. There are now more publications on CRISPR than ZFN and TALEN despite how recent the discovery of CRISPR is.[33] Both CRISPR and TALEN are favored to be the choices to be implemented in large-scale productions due to their precision and efficiency.

Genome editing occurs also as a natural process without artificial genetic engineering. The agents that are competent to edit genetic codes are viruses or subviral RNA-agents.

Although GEEN has higher efficiency than many other methods in reverse genetics, it is still not highly efficient; in many cases less than half of the treated populations obtain the desired changes.[43] For example, when one is planning to use the cell's NHEJ to create a mutation, the cell's HDR systems will also be at work correcting the DSB with lower mutational rates.

Traditionally, mice have been the most common choice for researchers as a host of a disease model. CRISPR can help bridge the gap between this model and human clinical trials by creating transgenic disease models in larger animals such as pigs, dogs, and non-human primates.[63][64] Using the CRISPR-Cas9 system, the programmed Cas9 protein and the sgRNA can be directly introduced into fertilized zygotes to achieve the desired gene modifications when creating transgenic models in rodents. This allows bypassing of the usual cell targeting stage in generating transgenic lines, and as a result, it reduces generation time by 90%.[64]

One potential that CRISPR brings with its effectiveness is the application of xenotransplantation. In previous research trials, CRISPR demonstrated the ability to target and eliminate endogenous retroviruses, which reduces the risk of transmitting diseases and reduces immune barriers.[33] Eliminating these problems improves donor organ function, which brings this application closer to a reality.

In plants, genome editing is seen as a viable solution to the conservation of biodiversity. Gene drive are a potential tool to alter the reproductive rate of invasive species, although there are significant associated risks. [65]

Many transhumanists see genome editing as a potential tool for human enhancement.[66][67][68] Australian biologist and Professor of Genetics David Andrew Sinclair notes that "the new technologies with genome editing will allow it to be used on individuals (...) to have (...) healthier children" designer babies.[69] According to a September 2016 report by the Nuffield Council on Bioethics in the future it may be possible to enhance people with genes from other organisms or wholly synthetic genes to for example improve night vision and sense of smell.[70][71]

The American National Academy of Sciences and National Academy of Medicine issued a report in February 2017 giving qualified support to human genome editing.[72] They recommended that clinical trials for genome editing might one day be permitted once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight."[73]

In the 2016 Worldwide Threat Assessment of the US Intelligence Community statement United States Director of National Intelligence, James R. Clapper, named genome editing as a potential weapon of mass destruction, stating that genome editing conducted by countries with regulatory or ethical standards "different from Western countries" probably increases the risk of the creation of harmful biological agents or products. According to the statement the broad distribution, low cost, and accelerated pace of development of this technology, its deliberate or unintentional misuse might lead to far-reaching economic and national security implications.[74][75][76] For instance technologies such as CRISPR could be used to make "killer mosquitoes" that cause plagues that wipe out staple crops.[76]

According to a September 2016 report by the Nuffield Council on Bioethics, the simplicity and low cost of tools to edit the genetic code will allow amateurs or "biohackers" to perform their own experiments, posing a potential risk from the release of genetically modified bugs. The review also found that the risks and benefits of modifying a person's genome and having those changes pass on to future generations are so complex that they demand urgent ethical scrutiny. Such modifications might have unintended consequences which could harm not only the child, but also their future children, as the altered gene would be in their sperm or eggs.[70][71] In 2001 Australian researchers Ronald Jackson and Ian Ramshaw were criticized for publishing a paper in the Journal of Virology that explored the potential control of mice, a major pest in Australia, by infecting them with an altered mousepox virus that would cause infertility as the provided sensitive information could lead to the manufacture of biological weapons by potential bioterrorists who might use the knowledge to create vaccine resistant strains of other pox viruses, such as smallpox, that could affect humans.[71][77] Furthermore, there are additional concerns about the ecological risks of releasing gene drives into wild populations.[71][78][79]

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How Much Does Medical School Cost? – ThoughtCo

Posted: May 14, 2019 at 2:50 am

Everyone knows that medical school is expensive but exactly how much is it? Although tuition varies greatly by year and has significantly increased over the last decade, medical school averages $34,592 per year and $138,368 per degree for in-state students at public schools and upwards of $50,000 per year or well over $200,000 for private institutions as of 2018.

Worse yet, due to the demanding schedule and curriculum of medical schools, students graduating programs in the field often find themselves in debt of over 75% of their tuition. For some, it takes years of working in the field to even out and start benefitting from the hiring paying salaries of professionals with medical degrees.

If you areapplying to medical school, you should first seriously consider your dedication to the field, the time it takes to earn your degree and how prepared you are to manage the debt of medical school in the early days of your residence and professional medical career.

According to the Association of American Medical Colleges(AAMC), the median tuition in 2012-2013 was $28,719 for resident students at public institutions, $49,000 for nonresident students at public institutions, and $47,673 for students at private institutions.With fees and insurance, the cost of attendance is $32,197 and $54,625 for resident and nonresident students at public institutions and $50,078 at private institutions. Overall, the four-year median cost of medical school in 2013 was $278,455 for private schools and $207,866 for public institutions.

This alone is not all that different from others seeking to pursue post-graduate degrees in other fields. However, due to the demanding nature of the medical school and lack of time to make supplementalincome, students often slip into debt during their medical degree program. The median education debt for indebted medical school graduates in 2012 was $170,000, and 86 percent of graduates reported having education debt. Specifically, in 2012 the median debt at graduation was $160,000 at public institutions and $190,000 at private institutions. In 2013, that number rose significantly to over $220,000 median debt.

With residence programs immediately following most medical school programs, recent graduates rarely have a chance to earn a full doctor's salary and it can take upwards of six years for these new medical professionals to clear their debt and start earning a true doctor's salary.

Fortunately, there are a variety of financial aid solutions for students hoping to start medical can seekto help mitigate these costs. The AAMC compiles a helpful list for counselors every year that details scholarship opportunities for medical students, specific to each year of the medical professional's educational career. Among them, the American Medical Association awards start-out scholarships for tens of thousands of dollars a year, including the Physicians of Tomorrow Award.

Hopeful medical students should consult their high school, undergrad, or graduate school counselor or financial aid office for more information regarding scholarships, especially those specific to in or out of state students. Most students who graduate medical school, despite the initial debt, do manage to pay off their student loans by their 10th year in the professional field. So if you have the drive, the patience and the passion to become a doctor, apply for medical school and start your career.

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How Much Does Medical School Cost? - ThoughtCo

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