Mega-telomere

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Comparative telomeric array organization within and among diverse chicken genotypes illustrates intra-genomic, inter-individual, and inter-genotype variation Cytogenetic Telomere Arrays (2005).jpg
Comparative telomeric array organization within and among diverse chicken genotypes illustrates intra-genomic, inter-individual, and inter-genotype variation

A mega-telomere (also known as an ultra-long telomere or a class III telomere), is an extremely long telomere sequence that sits on the end of chromosomes and prevents the loss of genetic information during cell replication. Like regular telomeres, mega-telomeres are made of a repetitive sequence of DNA and associated proteins, and are located on the ends of chromosomes. However, mega-telomeres are substantially longer than regular telomeres, ranging in size from 50 kilobases to several megabases (for comparison, the normal length of vertebrate telomeres is usually between 10 and 20 kilobases). [1]

Contents

Telomeres act like protective caps for the chromosome. During cell division, a cell will make copies of its DNA. The enzymes in the cell that are responsible for copying the DNA cannot copy the very ends of the chromosomes. This is sometimes called the "end replication problem". If a cell did not contain telomeres, genetic information from the DNA on the ends of chromosomes would be lost with each division. However, because chromosomes have telomeres or mega-telomeres on their ends, repetitive non-essential sequences of DNA are lost instead (See: Telomere shortening). [2] [3] While the chromosomes in most eukaryotic organisms are capped with telomeres, mega-telomeres are only found in a few species, such as mice [4] and some birds. [5] The specific function of mega-telomeres in vertebrate cells is still unclear.

Discovery

Telomeric regions of DNA were first identified in the late 1970s (See: Discovery of Telomeric DNA). However, extremely long regions of telomere sequence were not recognized in vertebrates until over a decade later. These sequences, which ranged from 30 to 150 kilobases in size, were first identified in laboratory mice by David Kipling and Howard Cooke in 1990. [4]

In 1994, extremely long telomeric regions were identified in chickens. [6] Telomeric sequences ranging from 20 kilobases to several megabases have also been identified in several species of birds. [5] These large regions were termed "ultra-long" telomeres in the literature when they were identified using southern blotting [5] and "mega-telomeres" when identified by cytogenetic methods. [7] The currently accepted terminology for these sequences is "mega-telomeres" [1]

Structure and function

Mega-telomeres in vertebrates consist of repeats of a six base-pair sequence, TTAGGG, of DNA. Mega-telomeric DNA also binds to various proteins to form complex structures on the ends of chromosomes. [8] Telomeres are identified by telomere arrays. A telomere array is a unique arrangement of telomeres within a sample (cell, individual, etc.) that is defined by the number of sequence repeats, the pattern of fragments given by restriction digest, the chromosome on which it is found, and the specific location of the sequence on that chromosome. In the literature, mega-telomeres are referred to as Class III telomeres based on the characteristics of their arrays. [5]

Many studies in model organisms have established the significance of telomere structure and function in regulating genome stability, cellular aging, and oncogenesis. [9] It has been suggested that mega-teleomeres may serve as protective mechanism against senescence in long-lived organisms. [9] However, there is some debate on the topic, since telomeric length does not seem to affect lifespan in mice [4] and birds with both long and short life-spans have been shown to have mega-telomeres. [5]

The presence of mega-telomeres varies between species. For example, human chromosomes do not have mega-telomeres while mice and many species of birds do. There is also variation in their structure and location within the same species. In mice and birds, mega-telomeres regions are observed to be hypervariable, meaning that there is a high degree of polymorphism in the size and position of mega-telomeres between individuals, including those of highly inbred lines. [5] Analysis of siblings from highly inbred chicken-lines have suggested that these ultra-long telomeric sequences are extremely heterogenous. [5] [9] Similar observations of heterogeneity have also been made in mice. [10]

In birds, whose cells contain microchromosomes, it has been suggested that there was a correlation between the presence of mega-telomeres and the number of microchromosomes present in a species, such that bird genomes with large numbers of microchromosomes also possessed larger amounts of telomeric DNA sequence. It was thought that these telomeric sequences might protect genes on these tiny chromosomes from erosion during cell division. [5] However, subsequent studies showed that mega-telomeres are not necessarily present in all species with microchromosomes, nor are they found on all microchromosomes within a cell. [11] Mega-telomeres are also thought contribute to the high recombination rate of chicken microchromosomes. The longest mega-telomere in chickens is associated with the W (female) chromosome, suggesting that mega-telomeres may also affect sex chromosome organization and the generation of genetic variation. [7]

Evolutionary origins

Chromosomal locations of mega-telomeres in an inbred chicken line: GGA (chromosome) 9 and W Cytogenetic Mega-telomeres GGA 9 and W.jpg
Chromosomal locations of mega-telomeres in an inbred chicken line: GGA (chromosome) 9 and W

The current research exploring mega-telomeres has indicated unexpected heterogeneity and non-Mendelian segregation of mega-telomere profiles between subsequent generations of inbred chicken (Gallus gallus) lines. This heterogeneity or inconsistency from generation to generation, despite nearly identical genomic sequences, is evidence that mega-telomeres promote recombination during meiosis. [7] [11] Furthermore, the preferential location on microchromsomes and the discovery of an extremely large mega-telomere on the female-specific W chromosome of avian species also signify the role of mega-telomeres. [7] [8]

Microchromosomes are known to be gene-dense [12] and particularly susceptible to damage, thus mega-telomeres may act specifically to protect these gene-rich but fragile chromosomes from erosion [7] [8] or other forms of chromosomal damage. The nearly 3MB telomeric array on the W chromosome suggests that mega-telomeres also play a role in sex-chromosome organization or distribution during, meiosis, however a mechanism is yet to be identified. It does not appear that the presence of mega-telomeres in a genome can alter the "telomere clock" or extend an organism's lifespan.[ citation needed ]

Organisms with mega-telomeres

Mega-telomeres have been best described in vertebrate species, specifically inbred mice and chicken lines. In fact, some of the largest mega-telomere arrays were reported in highly inbred and nearly homozygous chicken lines, including UCD 003 [13] and ADOL Line 0. [1] Normal vertebrate telomere array sizes range from 10 to 20 Kb, [14] however, many genetic lines of mouse and chicken possess extreme 50kb or more telomere size arrays. A few other avian species, including Japanese quail (Coturnix japonica), ostrich (Struthio camelus), and emu (Dromaius novaehollandiae). Although most avian genomes are three times smaller than mammalian genomes, their genomes are enriched with telomeric sequence and class III (mega-telomere) arrays, perhaps due to the relatively large number of microchromosomes.[ citation needed ]

The presence of mega-telomeres may be enhanced by the process of domestication or development of highly inbred vertebrate lines. The largest chicken arrays were discovered in the most inbred genetic lines. Studies of full siblings and their progeny from the UCD 003 line, [13] established in 1956 and maintained by full-sibling matings, established a consistent profile with 200 Kb or larger telomeres. [7] However, less inbred Red jungle fowl families (the hypothesized ancestor of chickens) have slightly shorter Class II arrays and other avian species, such as the American bald eagle (Haliaeetus leucocephalus), the northern goshawk (Accipter gentilis), possess fewer mega-telomeres and have a considerably smaller telomere size range. Furthermore, laboratory inbred mouse strains (Mus musculus) exhibit extremely long telomeres of 30–150 Kb in length, however the wild mouse species (Mus spretus) has significantly shorter telomeres ranging from 5–15 Kb. [4]

Identification methods

A variety of cytogenetic and molecular methods have been utilized to identify and study mega-telomeres in vertebrate species. Many of these techniques allow researchers to both discover the presence of a mega-telomere in a genome but also to characterize telomere arrays.[ citation needed ]

Cytogenetic studies employ fluorescence in situ hybridization (FISH) with telomeric probes [1] [8] to label telomeres on chemically-treated cells fixed to glass slides. More specifically, telomere-peptide nucleic acid fluorescein probes are frequently used to identify telomeric sequence repeats on mitotic metaphase and interphase or meiotic pachytene-stage chromosomes. FISH images allow both the identification of mega-telomeric chromosomes and the visualization of chromosome structure, GC-rich DNA regions, and, depending on the experiment, co-localization with genetic regions or genes.[ citation needed ]

Slot blot method of analysis can be utilized to determine the total amount of telomeric sequence per genome (inclusive of interstitial and terminal arrays) Slot Blot Technique with Chicken Genomic DNA (2005).jpg
Slot blot method of analysis can be utilized to determine the total amount of telomeric sequence per genome (inclusive of interstitial and terminal arrays)

Molecular techniques for quantifying telomeric sequences include pulse-field gel electrophoresis (PFGE), slot blot, horizontal gel electrophoresis, and Contour-clamped homogeneous electric field pulse field gel electrophoresis (CHEF-PFGE). In these techniques, purified genomic DNA is isolated and digested with restriction enzymes, such as HaeIII, HinfI, AluI, Sau3AI, EcoRI, EcoRV, PstI, SstI, BamHI, HindIII or BglII, and quantified by fluorometry. [8] [15]

The digestion of DNA into smaller fragments by restriction enzymes, separation of variable-sized DNA fragments via electrophoresis, and labeling of fragments containing telomeric DNA using a specific radio- or fluorescently-labeled probe are the essential steps completed within many molecular techniques. In many cases, the DNA fragments are transferred to distinctive membranes before labeling via blotting techniques (i.e. Southern blot). Specialized protocols have demonstrated the ability to isolate high molecular weight Class III telomeric DNA from Class I and II fragments as well as characterize the size ranges found within each class. The pattern of the telomeric fragments on the stained or labeled membrane is typically unique to the DNA sample (i.e. telomere arrays are rarely identical). Molecular weight markers are usually separated via electrophoresis through agarose gel along with genomic DNA fragments to aid in sizing telomeric arrays and identifying array inter- and intra- individual variability. Slot blot, however, is conducted without DNA fragmentation or separation, rather whole genomic DNA is used to quantify the total concentration of telomeric DNA. The flaw of this technique is that the size of the labelled DNA molecules cannot be identified. In slot blot (or dot blot), total genomic DNA is attached to a membrane and labeled with a telomere-probe that produces a sample-specific chemiluminescence signal, which is captured and quantified by fluorometer equipment and software. A known concentration standard must be labeled and quantified simultaneously in order to accurately determine the telomeric sequence concentration in the DNA samples. [15]

Related Research Articles

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, populations, or species or to pinpoint the locations of genes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restriction enzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample is digested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gel electrophoresis according to their size.

<span class="mw-page-title-main">Telomere</span> Region of repetitive nucleotide sequences on chromosomes

A telomere is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Telomeres are a widespread genetic feature most commonly found in eukaryotes. In most, if not all species possessing them, they protect the terminal regions of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing DNA repair systems from mistaking the very ends of the DNA strand for a double-strand break.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. 

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.

Chromosome jumping is a tool of molecular biology that is used in the physical mapping of genomes. It is related to several other tools used for the same purpose, including chromosome walking.

Genetics, a discipline of biology, is the science of heredity and variation in living organisms.

Fluorescence <i>in situ</i> hybridization Genetic testing technique

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

Subtelomeres are segments of DNA between telomeric caps and chromatin.

<span class="mw-page-title-main">Minichromosome</span>

A minichromosome is a small chromatin-like structure resembling a chromosome and consisting of centromeres, telomeres and replication origins but little additional genetic material. They replicate autonomously in the cell during cellular division. Minichromosomes may be created by natural processes as chromosomal aberrations or by genetic engineering.

<span class="mw-page-title-main">Genetic analysis</span>

Genetic analysis is the overall process of studying and researching in fields of science that involve genetics and molecular biology. There are a number of applications that are developed from this research, and these are also considered parts of the process. The base system of analysis revolves around general genetics. Basic studies include identification of genes and inherited disorders. This research has been conducted for centuries on both a large-scale physical observation basis and on a more microscopic scale. Genetic analysis can be used generally to describe methods both used in and resulting from the sciences of genetics and molecular biology, or to applications resulting from this research.

A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6–10 megabases (Mb) in size instead of 50–250 Mb for natural chromosomes, and able to carry new genes introduced by human researchers. Ideally, researchers could integrate different genes that perform a variety of functions, including disease defense.

<span class="mw-page-title-main">Molecular cytogenetics</span>

Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.

The following outline is provided as an overview of and topical guide to genetics:

<span class="mw-page-title-main">Microchromosome</span> Type of chromosome

A microchromosome is a chromosome defined for its relatively small size. They are typical components of the karyotype of birds, some reptiles, fish, amphibians, and monotremes. As many bird genomes have chromosomes of widely different lengths, the name was meant to distinguish them from the comparatively large macrochromosomes. The distinction referred to the measured size of the chromosome while staining for karyotype, and while there is not a strict definition, chromosomes resembling the large chromosomes of mammals were called macrochromosomes, while the much smaller ones of less than around 0.5 μm were called microchromosomes. In terms of base pairs, by convention, those of less than 20Mb were called microchromosomes, those between 20 and 40 Mb are classified as intermediate chromosomes, and those larger than 40Mb are macrochromosomes. By this definition, all normal chromosomes in organisms with relatively small genomes would be considered microchromosomes.

Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology. In Q-FISH, the technique uses labelled synthetic DNA mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software. Q-FISH is most commonly used to study telomere length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes. Telomeres are necessary at chromosome ends to prevent DNA-damage responses as well as genome instability. To this day, the Q-FISH method continues to be utilized in the field of telomere research.

Telomere-binding proteins function to bind telomeric DNA in various species. In particular, telomere-binding protein refers to TTAGGG repeat binding factor-1 (TERF1) and TTAGGG repeat binding factor-2 (TERF2). Telomere sequences in humans are composed of TTAGGG sequences which provide protection and replication of chromosome ends to prevent degradation. Telomere-binding proteins can generate a T-loop to protect chromosome ends. TRFs are double-stranded proteins which are known to induce bending, looping, and pairing of DNA which aids in the formation of T-loops. They directly bind to TTAGGG repeat sequence in the DNA. There are also subtelomeric regions present for regulation. However, in humans, there are six subunits forming a complex known as shelterin.

The 2000s witnessed an explosion of genome sequencing and mapping in evolutionarily diverse species. While full genome sequencing of mammals is rapidly progressing, the ability to assemble and align orthologous whole chromosomal regions from more than a few species is not yet possible. The intense focus on the building of comparative maps for domestic, laboratory and agricultural (cattle) animals has traditionally been used to understand the underlying basis of disease-related and healthy phenotypes.

<span class="mw-page-title-main">Telomeric repeat–containing RNA</span> Long non-coding RNA transcribed from telomeres

Telomeric repeat–containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres - repetitive nucleotide regions found on the ends of chromosomes that function to protect DNA from deterioration or fusion with neighboring chromosomes. TERRA has been shown to be ubiquitously expressed in almost all cell types containing linear chromosomes - including humans, mice, and yeasts. While the exact function of TERRA is still an active area of research, it is generally believed to play a role in regulating telomerase activity as well as maintaining the heterochromatic state at the ends of chromosomes. TERRA interaction with other associated telomeric proteins has also been shown to help regulate telomere integrity in a length-dependent manner.

Gene deserts are regions of the genome that are devoid of protein-coding genes. Gene deserts constitute an estimated 25% of the entire genome, leading to the recent interest in their true functions. Originally believed to contain inessential and "Junk DNA" due to their inability to create proteins, gene deserts have since been linked to several vital regulatory functions, including distal enhancing and conservatory inheritance. Thus, an increasing number of risks that lead to several major diseases, including a handful of cancers, have been attributed to irregularities found in gene deserts.

Physical map is a technique used in molecular biology to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter.

References

  1. 1 2 3 4 O'Hare TH, Delany ME (2009). "Genetic variation exists for telomeric array organization within and among the genomes of normal, immortalized, and transformed chicken systems". Chromosome Research. 17 (8): 947–64. doi:10.1007/s10577-009-9082-6. PMC   2793383 . PMID   19890728.
  2. ↑ "Telomere Maintenance".
  3. ↑ Blackburn, Elizabeth. "Advanced Information: Maintenance of chromosomes by telomeres and the enzyme telomerase" (PDF). Nobelprize.org.
  4. 1 2 3 4 Kipling D, Cooke HJ (September 1990). "Hypervariable ultra-long telomeres in mice". Nature. 347 (6291): 400–2. Bibcode:1990Natur.347..400K. doi:10.1038/347400a0. PMID   2170845. S2CID   4358923.
  5. 1 2 3 4 5 6 7 8 Delany ME, Krupkin AB, Miller MM (November 2000). "Organization of telomere sequences in birds: evidence for arrays of extreme length and for in vivo shortening". Cytogenetics and Cell Genetics. 90 (1–2): 139–45. doi:10.1159/000015649. PMID   11060464. S2CID   22710497.
  6. ↑ Nanda I, Schmid M (1994). "Localization of the telomeric (TTAGGG)n sequence in chicken (Gallus domesticus) chromosomes". Cytogenetics and Cell Genetics. 65 (3): 190–3. doi:10.1159/000133630. PMID   8222759.
  7. 1 2 3 4 5 6 Rodrigue KL, May BP, Famula TR, Delany ME (2005). "Meiotic instability of chicken ultra-long telomeres and mapping of a 2.8 megabase array to the W-sex chromosome". Chromosome Research. 13 (6): 581–91. doi:10.1007/s10577-005-0984-7. PMID   16170623. S2CID   36524567.
  8. 1 2 3 4 5 Delany ME, Gessaro TM, Rodrigue KL, Daniels LM (2007). "Chromosomal mapping of chicken mega-telomere arrays to GGA9, 16, 28 and W using a cytogenomic approach". Cytogenetic and Genome Research. 117 (1–4): 54–63. doi:10.1159/000103165. PMID   17675845. S2CID   13314473.
  9. 1 2 3 Delany ME, Daniels LM, Swanberg SE, Taylor HA (June 2003). "Telomeres in the chicken: genome stability and chromosome ends". Poultry Science. 82 (6): 917–26. doi: 10.1093/ps/82.6.917 . PMID   12817446.
  10. ↑ Starling JA, Maule J, Hastie ND, Allshire RC (December 1990). "Extensive telomere repeat arrays in mouse are hypervariable". Nucleic Acids Research. 18 (23): 6881–8. doi:10.1093/nar/18.23.6881. PMC   332745 . PMID   2175882.
  11. 1 2 Nanda I, Schrama D, Feichtinger W, Haaf T, Schartl M, Schmid M (November 2002). "Distribution of telomeric (TTAGGG)(n) sequences in avian chromosomes". Chromosoma. 111 (4): 215–27. doi:10.1007/s00412-002-0206-4. PMID   12424522. S2CID   9732828.
  12. ↑ Smith J, Bruley CK, Paton IR, Dunn I, Jones CT, Windsor D, Morrice DR, Law AS, Masabanda J, Sazanov A, Waddington D, Fries R, Burt DW (April 2000). "Differences in gene density on chicken macrochromosomes and microchromosomes". Animal Genetics. 31 (2): 96–103. doi:10.1046/j.1365-2052.2000.00565.x. PMID   10782207.
  13. 1 2 Pisenti J, M.E. Delany, R.L. Taylor Jr, U.K. Abbott, H. Abplanalp, J.A. Arthur, et al. (2001). "Avian genetic resources at risk: An assessment and proposal for conservation of genetic stocks in the USA and Canada" (PDF). Avian Poult Biol Rev. 12: 100–102. Archived from the original (PDF) on 2003-07-31.
  14. ↑ Davis T, Kipling D (December 2005). "Telomeres and telomerase biology in vertebrates: progress towards a non-human model for replicative senescence and ageing". Biogerontology. 6 (6): 371–85. doi:10.1007/s10522-005-4901-4. PMID   16518699. S2CID   26586330.
  15. 1 2 Swanberg SE, Delany ME (2003). "Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro". Cytogenetic and Genome Research. 102 (1–4): 318–25. doi:10.1159/000075769. PMID   14970723. S2CID   40920723.
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