Howard Martin Temin
Howard Martin Temin was a US geneticist and virologist. He discovered reverse transcriptase in the 1970s at the University of Wisconsin–Madison, for which he shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco and David Baltimore. Temin was born in Philadelphia, Pennsylvania, to Jewish parents, Annette, an activist, Henry Temin, an attorney; as a high school student at Central High School in Pennsylvania, he participated in the Jackson Laboratory's Summer Student Program in Bar Harbor, Maine. The director of the program, C. C. Little, told his parents that Temin was "unquestionably the finest scientist of the fifty-seven students who have attended the program since the beginning…I can't help but feel this boy is destined to become a great man in the field of science." Temin said that his experience at Jackson's Laboratory is what got him interested in science. Temin's parents raised their family to have values associated with social justice and independent thinking, evident throughout his life.
For Temin's bar mitzvah, the family donated money that would have been spent on the party, to a local camp for displaced persons. Temin was the valedictorian of his class and he devoted his speech to relevant issues at the time including the recent hydrogen bomb activity and the news of sending a man to the moon. Temin received his bachelor's degree from Swarthmore College in 1955 majoring and minoring in biology in the honors program, he received his doctorate degree in animal virology from the California Institute of Technology in 1959. Temin's first exposure to experimental science was during his time at the California Institute of Technology as a graduate student in laboratory of Professor Renato Dulbecco. Temin studied embryology at CIT, but after about a year and a half, he switched to animal virology, he became interested Dulbecco's lab after a chance run-in with Harry Rubin, a postdoctoral fellow in Dulbecco's lab. In the lab, Temin studied a tumor-causing virus that infects chickens.
During his research on the virus, he observed that mutations in the virus yielded alterations in the structural characteristics of the infected cell – thus, integration into the cell's genome was occurring. As part of his doctoral thesis, Temin stated that the Rous Sarcoma Virus has "some kind of close relationship with the genome of the infected cell". Following receiving his doctorate, Temin continued to work in Dulbecco's lab as a postdoctoral fellow. In 1960, the McArdle Laboratory for Cancer Research at the University of Wisconsin–Madison recruited Temin as a virologist. Though Temin knew he would be independent in Madison, because of the lack of research involving virology and oncology together, Temin stated that he was "supremely self-confident"; when he first arrived in Madison in 1960, he found an unprepared laboratory in the basement of a rundown building with an office that could be considered a closet. Until a more suitable laboratory could be prepared, he continued his research with RSV at a friend's laboratory at the University of Illinois.
That year, he returned to Madison, continued his RSV research in his own lab, began his position as an assistant professor. While studying the Rous sarcoma virus at UW-Madison, Temin began to refer to the genetic material that the virus introduced to the cells, the "provirus". Using the antibiotic, actinomycin D, which inhibits the expression of DNA, he determined that the provirus was DNA or was located on the cell's DNA; these results implied that the infecting Rous sarcoma virus was somehow generating complementary double-stranded DNA. Temin's description of how tumor viruses act on the genetic material of the cell through reverse transcription was revolutionary; this upset the held belief at the time of a popularized version of the "Central Dogma" of molecular biology posited by Nobel laureate Francis Crick, one of the co-discoverers of the structure of DNA. Crick had claimed only that sequence information cannot flow out of protein into DNA or RNA, but he was interpreted as saying that information flows from DNA to RNA to protein.
Many respected scientists disregarded his work and declared it impossible. Despite the lack of support from the scientific community, Temin continued to search for evidence to support his idea. In 1969, Temin and a postdoctoral fellow, Satoshi Mizutani, began searching for the enzyme, responsible for the phenomenon of viral RNA being transferred into proviral DNA; that year, Temin showed that certain tumor viruses carried the enzymatic ability to reverse the flow of information from RNA back to DNA using reverse transcriptase. Reverse transcriptase was independently and discovered in association with the murine leukemia virus by David Baltimore at the Massachusetts Institute of Technology. In 1975, Baltimore and Temin shared the Nobel Prize of Medicine. Both scientists completed their initial work with RNA-dependent DNA polymerase with the Rous sarcoma virus; the discovery of reverse transcriptase is one of the most important of the modern era of medicine, as reverse transcriptase is the central enzyme in several widespread viral diseases such as AIDS and Hepatitis B.
Reverse transcriptase is an important component of several important techniques in molecular biology, such as the reverse transcription polymerase chain reaction, diagnostic medicine. In 1992 Temin received the National Medal of Science. Temin was elected a Foreign Member of the Royal Society in 1988. Following winning the Nobel Prize, Temin
LINE1 are transposable elements in the DNA of some organisms and belong to the group of Long interspersed nuclear elements. L1 comprise 17% of the human genome; the majority of L1 in the human genome are inactive. Human L1 has been reported to have transferred to the genome of the gonorrhea bacteria. A typical L1 element is 6,000 base pairs long and consists of two non-overlapping open reading frames which are flanked by UTR and target site duplications. In humans, ORF2 is thought to be translated by an unconventional termination/reinitiation mechanism, while mouse L1s contain an internal ribosome entry site upstream of each ORF; the 5' Untranslated region of the L1 element contains a strong, internal RNA Polymerase II transcription promoter in senseThe 5' UTR of mouse L1s contain a variable number of GC-rich tandemly repeated monomers of around 200bp, followed by a short non-monomeric region. Human 5' UTRs do not contain repeated motifs. All families of human L1s harbor in their most 5’ extremity a binding motif for the transcription factor YY1.
Younger families have two binding sites for SOX-family transcription factors, both YY1 and SOX sites were shown to be required for human L1 transcription initiation and activation. Both mouse and human 5’UTRs contain as well a weak antisense promoter of unknown function The first ORF encode a 500 amino acid - 40kDa protein that lacks homology with any protein of known function. In vertebrates, it contains a conserved C-terminus domain and a variable coiled-coil N-terminus that mediates the formation of ORF1 trimetric complexes. ORF1 trimers have RNA-binding and nucleic acid chaperone activity that are necessary for retrotransposition; the second ORF of L1 encodes a protein that has reverse transcriptase activity. The encoded protein has a molecular weight of 150 kDA. L1Base, a database of functional annotations & predictions of active LINE1 elements
Ribonucleic acid is a polymeric molecule essential in various biological roles in coding, decoding and expression of genes. RNA and DNA are nucleic acids, along with lipids and carbohydrates, constitute the four major macromolecules essential for all known forms of life. Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA it is more found in nature as a single-strand folded onto itself, rather than a paired double-strand. Cellular organisms use messenger RNA to convey genetic information that directs synthesis of specific proteins. Many viruses encode their genetic information using an RNA genome; some RNA molecules play an active role within cells by catalyzing biological reactions, controlling gene expression, or sensing and communicating responses to cellular signals. One of these active processes is protein synthesis, a universal function in which RNA molecules direct the assembly of proteins on ribosomes; this process uses transfer RNA molecules to deliver amino acids to the ribosome, where ribosomal RNA links amino acids together to form proteins.
Like DNA, most biologically active RNAs, including mRNA, tRNA, rRNA, snRNAs, other non-coding RNAs, contain self-complementary sequences that allow parts of the RNA to fold and pair with itself to form double helices. Analysis of these RNAs has revealed that they are structured. Unlike DNA, their structures do not consist of long double helices, but rather collections of short helices packed together into structures akin to proteins. In this fashion, RNAs can achieve chemical catalysis. For instance, determination of the structure of the ribosome—an RNA-protein complex that catalyzes peptide bond formation—revealed that its active site is composed of RNA; each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through 5'. A base is attached to the 1' position, in general, cytosine, guanine, or uracil. Adenine and guanine are purines and uracil are pyrimidines. A phosphate group is attached to the 5' position of the next; the phosphate groups have a negative charge each. The bases form hydrogen bonds between cytosine and guanine, between adenine and uracil and between guanine and uracil.
However, other interactions are possible, such as a group of adenine bases binding to each other in a bulge, or the GNRA tetraloop that has a guanine–adenine base-pair. An important structural component of RNA that distinguishes it from DNA is the presence of a hydroxyl group at the 2' position of the ribose sugar; the presence of this functional group causes the helix to take the A-form geometry, although in single strand dinucleotide contexts, RNA can also adopt the B-form most observed in DNA. The A-form geometry results in a deep and narrow major groove and a shallow and wide minor groove. A second consequence of the presence of the 2'-hydroxyl group is that in conformationally flexible regions of an RNA molecule, it can chemically attack the adjacent phosphodiester bond to cleave the backbone. RNA is transcribed with only four bases, but these bases and attached sugars can be modified in numerous ways as the RNAs mature. Pseudouridine, in which the linkage between uracil and ribose is changed from a C–N bond to a C–C bond, ribothymidine are found in various places.
Another notable modified base is hypoxanthine, a deaminated adenine base whose nucleoside is called inosine. Inosine plays a key role in the wobble hypothesis of the genetic code. There are more than 100 other occurring modified nucleosides; the greatest structural diversity of modifications can be found in tRNA, while pseudouridine and nucleosides with 2'-O-methylribose present in rRNA are the most common. The specific roles of many of these modifications in RNA are not understood. However, it is notable that, in ribosomal RNA, many of the post-transcriptional modifications occur in functional regions, such as the peptidyl transferase center and the subunit interface, implying that they are important for normal function; the functional form of single-stranded RNA molecules, just like proteins requires a specific tertiary structure. The scaffold for this structure is provided by secondary structural elements that are hydrogen bonds within the molecule; this leads to several recognizable "domains" of secondary structure like hairpin loops and internal loops.
Since RNA is charged, metal ions such as Mg2+ are needed to stabilise many secondary and tertiary structures. The occurring enantiomer of RNA is D-RNA composed of D-ribonucleotides. All chirality centers are located in the D-ribose. By the use of L-ribose or rather L-ribonucleotides, L-RNA can be synthesized. L-RNA is much more stable against degradation by RNase. Like other structured biopolymers such as proteins, one can define topology of a folded RNA molecule; this is done based on arrangement of intra-chain contacts within a folded RNA, termed as circuit topology. Synthesis of RNA is catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA; the DNA double helix is unwound by the helicase activity of the enzyme. The enzyme progresses along the template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occ
A chromosome is a deoxyribonucleic acid molecule with part or all of the genetic material of an organism. Most eukaryotic chromosomes include packaging proteins which, aided by chaperone proteins, bind to and condense the DNA molecule to prevent it from becoming an unmanageable tangle. Chromosomes are visible under a light microscope only when the cell is undergoing the metaphase of cell division. Before this happens, every chromosome is copied once, the copy is joined to the original by a centromere, resulting either in an X-shaped structure if the centromere is located in the middle of the chromosome or a two-arm structure if the centromere is located near one of the ends; the original chromosome and the copy are now called sister chromatids. During metaphase the X-shape structure is called a metaphase chromosome. In this condensed form chromosomes are easiest to distinguish and study. In animal cells, chromosomes reach their highest compaction level in anaphase during chromosome segregation.
Chromosomal recombination during meiosis and subsequent sexual reproduction play a significant role in genetic diversity. If these structures are manipulated incorrectly, through processes known as chromosomal instability and translocation, the cell may undergo mitotic catastrophe; this will make the cell initiate apoptosis leading to its own death, but sometimes mutations in the cell hamper this process and thus cause progression of cancer. Some use the term chromosome in a wider sense, to refer to the individualized portions of chromatin in cells, either visible or not under light microscopy. Others use the concept in a narrower sense, to refer to the individualized portions of chromatin during cell division, visible under light microscopy due to high condensation; the word chromosome comes from the Greek χρῶμα and σῶμα, describing their strong staining by particular dyes. The term was coined by von Waldeyer-Hartz, referring to the term chromatin, introduced by Walther Flemming; some of the early karyological terms have become outdated.
For example and Chromosom, both ascribe color to a non-colored state. The German scientists Schleiden, Virchow and Bütschli were among the first scientists who recognized the structures now familiar as chromosomes. In a series of experiments beginning in the mid-1880s, Theodor Boveri gave the definitive demonstration that chromosomes are the vectors of heredity, it is the second of these principles, so original. Wilhelm Roux suggested. Boveri was able to confirm this hypothesis. Aided by the rediscovery at the start of the 1900s of Gregor Mendel's earlier work, Boveri was able to point out the connection between the rules of inheritance and the behaviour of the chromosomes. Boveri influenced two generations of American cytologists: Edmund Beecher Wilson, Nettie Stevens, Walter Sutton and Theophilus Painter were all influenced by Boveri. In his famous textbook The Cell in Development and Heredity, Wilson linked together the independent work of Boveri and Sutton by naming the chromosome theory of inheritance the Boveri–Sutton chromosome theory.
Ernst Mayr remarks that the theory was hotly contested by some famous geneticists: William Bateson, Wilhelm Johannsen, Richard Goldschmidt and T. H. Morgan, all of a rather dogmatic turn of mind. Complete proof came from chromosome maps in Morgan's own lab; the number of human chromosomes was published in 1923 by Theophilus Painter. By inspection through the microscope, he counted 24 pairs, his error was copied by others and it was not until 1956 that the true number, 46, was determined by Indonesia-born cytogeneticist Joe Hin Tjio. The prokaryotes – bacteria and archaea – have a single circular chromosome, but many variations exist; the chromosomes of most bacteria, which some authors prefer to call genophores, can range in size from only 130,000 base pairs in the endosymbiotic bacteria Candidatus Hodgkinia cicadicola and Candidatus Tremblaya princeps, to more than 14,000,000 base pairs in the soil-dwelling bacterium Sorangium cellulosum. Spirochaetes of the genus Borrelia are a notable exception to this arrangement, with bacteria such as Borrelia burgdorferi, the cause of Lyme disease, containing a single linear chromosome.
Prokaryotic chromosomes have less sequence-based structure than eukaryotes. Bacteria have a one-point from which replication starts, whereas some archaea contain multiple replication origins; the genes in prokaryotes are organized in operons, do not contain introns, unlike eukaryotes. Prokaryotes do not possess nuclei. Instead, their DNA is organized into a structure called the nucleoid; the nucleoid occupies a defined region of the bacterial cell. This structure is, dynamic and is maintained and remodeled by the actions of a range of histone-like proteins, which associate with the bacterial chromosome. In archaea, the DNA in chromosomes is more organized, with the DNA packaged within structures similar to eukaryotic nucleosomes. Certain bacteria contain plasmids or other extrachromosomal DNA; these are circular structures in the cytoplasm that contain cellular DNA and play a role in horizontal gene transfer. In prokaryotes and viruses, the DNA is densely packed and organized.
A microsatellite is a tract of repetitive DNA in which certain DNA motifs are repeated 5–50 times. Microsatellites occur at thousands of locations within an organism's genome, they have a higher mutation rate than other areas of DNA leading to high genetic diversity. Microsatellites are referred to as short tandem repeats by forensic geneticists and in genetic genealogy, or as simple sequence repeats by plant geneticists. Microsatellites and their longer cousins, the minisatellites, together are classified as VNTR DNA; the name "satellite" DNA refers to the early observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying "satellite" layers of repetitive DNA. They are used for DNA profiling in cancer diagnosis, in kinship analysis and in forensic identification, they are used in genetic linkage analysis to locate a gene or a mutation responsible for a given trait or disease. Microsatellites are used in population genetics to measure levels of relatedness between subspecies and individuals.
Although the first microsatellite was characterised in 1984 at the University of Leicester by Weller and colleagues as a polymorphic GGAT repeat in the human myoglobin gene, the term "microsatellite" was introduced in 1989, by Litt and Luty. The name "satellite" DNA refers to the early observation that centrifugation of genomic DNA in a test tube separates a prominent layer of bulk DNA from accompanying "satellite" layers of repetitive DNA; the increasing availability of DNA amplification by PCR at the beginning of the 1990s triggered a large number of studies using the amplification of microsatellites as genetic markers for forensic medicine, for paternity testing, for positional cloning to find the gene underlying a trait or disease. Prominent early applications include the identifications by microsatellite genotyping of the eight-year-old skeletal remains of a British murder victim, of the Auschwitz concentration camp doctor Josef Mengele who escaped to South America following World War II.
A microsatellite is a tract of tandemly repeated DNA motifs that range in length from one to six or up to ten nucleotides, are repeated 5–50 times. For example, the sequence TATATATATA is a dinucleotide microsatellite, GTCGTCGTCGTCGTC is a trinucleotide microsatellite. Repeat units of four and five nucleotides are referred to as tetra- and pentanucleotide motifs, respectively. Most eukaryotes have microsatellites, with the notable exception of some yeast species. Microsatellites are distributed throughout the genome; the human genome for example contains 50,000–100,000 dinucleotide microsatellites, lesser numbers of tri-, tetra- and pentanucleotide microsatellites. Many are located in non-coding parts of the human genome and therefore do not produce proteins, but they can be located in regulatory regions and coding regions. Microsatellites in non-coding regions do not have any specific function, therefore cannot be selected against. Other microsatellites are located in regulatory flanking or intronic regions of genes, or directly in codons of genes – microsatellite mutations in such cases can lead to phenotypic changes and diseases, notably in triplet expansion diseases such as fragile X syndrome and Huntington's disease.
The telomeres at the ends of the chromosomes, thought to be involved in ageing/senescence, consist of repetitive DNA, with the hexanucleotide repeat motif TTAGGG in vertebrates. They are thus classified as minisatellites. Insects have shorter repeat motifs in their telomeres that could arguably be considered microsatellites. Unlike point mutations, which affect only a single nucleotide, microsatellite mutations lead to the gain or loss of an entire repeat unit, sometimes two or more repeats simultaneously. Thus, the mutation rate at microsatellite loci is expected to differ from other mutation rates, such as base substitution rates; the actual cause of mutations in microsatellites is debated. One proposed cause of such length changes is replication slippage, caused by mismatches between DNA strands while being replicated during meiosis. DNA polymerase, the enzyme responsible for reading DNA during replication, can slip while moving along the template strand and continue at the wrong nucleotide.
DNA polymerase slippage is more to occur when a repetitive sequence is replicated. Because microsatellites consist of such repetitive sequences, DNA polymerase may make errors at a higher rate in these sequence regions. Several studies have found evidence. Slippage in each microsatellite occurs about once per 1,000 generations. Thus, slippage changes in repetitive DNA are three orders of magnitude more common than point mutations in other parts of the genome. Most slippage results in a change of just one repeat unit, slippage rates vary for different allele lengths and repeat unit sizes, within different species. If there is a large size difference between individual alleles there may be increased instability during recombination at meiosis. Another possible cause of microsatellite mutations are point mutations, where only one nucleotide is incorrectly copied during replication. A study comparing human and primate geno
Long interspersed nuclear element
Long interspersed nuclear elements are a group of non-LTR retrotransposons which are widespread in the genome of many eukaryotes. They make up around 21.1% of the human genome. LINEs make up a family of transposons. LINEs are translated into protein that acts as a reverse transcriptase; the reverse transcriptase makes a DNA copy of the LINE RNA that can be integrated into the genome at a new site. The only abundant LINE in humans is LINE-1. Our genome contains 4,000 full-length LINE-1 elements. Due to the accumulation of random mutations, the sequence of many LINEs has degenerated to the extent that they are no longer transcribed or translated. Comparisons of LINE DNA sequences can be used to date transposon insertion in the genome; the first description of an 6.4 kb long LINE-derived sequence was published by J. Adams et al. in 1980. Based on structural features and the phylogeny of its key enzyme, the reverse transcriptase, LINEs are grouped into five main groups, called L1, RTE, R2, I and Jockey, which can be subdivided into at least 28 clades.
In plant genomes, so far only LINEs of the L1 and RTE clade have been reported. Whereas L1 elements diversify into several subclades, RTE-type LINEs are conserved constituting a single family. In fungi, Tad, L1, CRE, Deceiver and Inkcap-like elements have been identified, with Tad-like elements appearing in fungal genomes. All LINEs encode a least one protein, ORF2, which contains an RT and an endonuclease domain, either an N-terminal APE or a C-terminal RLE or both. A ribonuclease H domain is present. Except for the evolutionary ancient R2 and RTE superfamilies, LINEs encode for another protein named ORF1, which may contain an Gag-knuckle, a L1-like RRM, and/or an esterase. LINE elements are rare compared to LTR-retrotransposons in plants, fungi or insects, but are dominant in vertebrates and in mammals, where they represent around 20% of the genome; the LINE-1/L1-element is the only element, still active in the human genome today. It is found in all mammals. Remnants of L2 and L3 elements are found in the human genome.
It is estimated, that L3 elements were active ~ 200-300 million years ago. Unlike L1 elements, L2 elements lack flanking target site duplications; the L2 elements are in the same group as Jockey. In the first human genome draft the fraction of LINE elements of the human genome was given as 21% and their copy number as 850,000. Of these, L1, L2 and L3 elements made up 315,000 and 37,000 copies, respectively; the non-autonomous SINE elements which depend on L1 elements for their proliferation make up 13% of the human genome and have a copy number of around 1.5 million. They originated from the RTE family of LINEs. Recent estimates show the typical human genome contains on average 100 L1 elements with potential for mobilization, however there is a fair amount of variation and some individuals may contain a larger number of active L1 elements, making these individuals more prone to L1-induced mutagenesis. Increased L1 copy numbers have been found in the brains of people with schizophrenia, indicating that LINE elements may play a role in some neuronal diseases.
LINE elements propagate by a so-called target primed reverse transcription mechanism, first described for the R2 element from the silkworm Bombyx mori. ORF2 proteins associate in cis with their encoding mRNA, forming a ribonucleoprotein complex composed of two ORF2s and an unknown number of ORF1 trimers; the complex is transported back into the nucleus, where the ORF2 endonuclease domain opens the DNA. Thus, a 3'OH group is freed for the reverse transcriptase to prime reverse transcription of the LINE RNA transcript. Following the reverse transcription the target strand is cleaved and the newly created cDNA is integratedNew insertions create short TSDs, the majority of new inserts are 5’-truncated and inverted; because they lack their 5’UTR, most of new inserts are non functional. It has been shown that host cells regulate L1 retrotransposition activity, for example through epigenetic silencing. For example, the RNA interference mechanism of small interfering RNAs derived from L1 sequences can cause suppression of L1 retrotransposition.
In plant genomes, epigenetic modification of LINEs can lead to expression changes of nearby genes and to phenotypic changes: In the oil palm genome, methylation of a Karma-type LINE underlies the somaclonal,'mantled' variant of this plant, responsible for drastic yield loss. Human APOBEC3C mediated restriction of LINE-1 elements were reported and it is due to the interaction between A3C with the ORF1p that affects the reverse transcriptase activity. A historic example of L1-conferred disease is Haemophilia A, caused by insertional mutagenesis. There are nearly 100 examples of known diseases caused by retroelement insertions, including some types of cancer and neurological disorders. Correlation between L1 mobilization and oncogenesis has been reported for epithelial cell cancer. Hypomethylation of LINES is associated with chromosomal instability and altered gene expression and is found in various cancer cell types in various tissues types. Hypomethylation of a specific L1 located in the MET onco gene is associated with bladder cancer tumorogenesis, Shift work sleep disorder is associated with increased cancer risk because light exp
P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for P trait of P element and it is found only in wild flies. All P elements have a canonical structure containing 31 bp terminal inverted repeats and 11 bp internal inverted repeats located at THAP domain of the transposase; the shorter and longest P elements are nonautonomous elements. The longest P elements encode transposase needed for transposition. In hybrid dysgenesis, one strain of Drosophila mates with another strain of Drosophila producing hybrid offspring cause chromosomal damage known to be dysgenic. Hybrid dysgenesis requires a contribution from both parents. For example, in the P-M system, P strain contributing M strain contributing maternal; the reverse cross, with M father and P mother, produces normal offspring, as it crosses P x P or M x M manner. P male chromosome can cause dysgenesis. P element encodes a suppressor of transposition, which accumulates in the cytoplasm during the development of cells.
Thus, in a cross of a P or M male with a P female, the female cytoplasm contains the suppressor, which binds to any P elements and prevents their transposition. P elements are used as mutagenic agents in genetic experiments with Drosophila. One advantage of this approach is; the P element encodes for the protein P transposase and is flanked by terminal inverted repeats which are important for its mobility. Unlike laboratory strain females, wild type females are thought to express an inhibitor to P transposase function; this inhibitor reduces the disruption to the genome caused by the P elements, allowing fertile progeny. Evidence for this comes from crosses of laboratory females with wild type males. In the absence of the inhibitor, the P elements can proliferate throughout the genome, disrupting many genes and killing progeny; the P element is a class II transposon, moves by a DNA-based "cut and paste" mechanism. The sequence comprises 4 exons with 3 introns. Complete splicing of the introns produces the transposase enzyme, while alternative partial splicing of intron 1 and 2 leaving in only intron 3 encodes the P element repressor.
The complete, autonomous P element encodes a transposase enzyme, which recognizes the 31 bp terminal inverted repeats of the P element and catalyzes P element excision and re-insertion. The complete element is 2907 bp. P element insertion and subsequent excision results in the production of 8 bp direct repeats, the presence of such repeats is indicative of previous P element activity. Hybrid dysgenesis refers to the high rate of mutation in germ line cells of Drosophila strains resulting from a cross of males with autonomous P elements and females that lack P elements; the hybrid dysgenesis syndrome is marked by temperature-dependent sterility, elevated mutation rates, increased chromosome rearrangement and recombination. The hybrid dysgenesis phenotype is affected by the transposition of P elements within the germ-line cells of offspring of P strain males with M strain females. Transposition only occurs in germ-line cells, because a splicing event needed to make transposase mRNA does not occur in somatic cells.
Hybrid dysgenesis manifests when crossing P strain males with M strain females and not when crossing P strain females with M strain males. The eggs of P strain females contain high amounts of a repressor protein that prevents transcription of the transposase gene; the eggs of M strain mothers, which do not contain the repressor protein, allow for transposition of P elements from the sperm of fathers. In P strain females, the repressors are found in the cytoplasm. Hence, when P strain males fertilize M strain females, the male contributes its genome with the P element but not the male cytoplasm leading to P strain progeny; this effect contributes to piRNAs being inherited only in the maternal line, which provides a defence mechanism against P elements. The P element has found wide use in Drosophila research as a mutagen; the mutagenesis system uses an autonomous but immobile element, a mobile nonautonomous element. Flies from subsequent generations can be screened by phenotype or PCR. Naturally-occurring P elements contain: Coding sequence for the enzyme transposase Recognition sequences for transposase actionTransposase is an enzyme that regulates and catalyzes the excision of a P element from the host DNA, cutting at two recognition sites, reinserting randomly.
It is the random insertion that may interfere with existing genes, or carry an additional gene, that can be used for genetic research. To use this as a useful and controllable genetic tool, the two parts of the P element must be separated to prevent uncontrolled transposition; the normal genetic tools are therefore: DNA coding for transposase with no transposase recognition sequences so it cannot insert. A "P Plasmid"P Plasmids always contain: A Drosophila reporter gene a red-eye marker. Transposase recognition sequences, and may contain: A gene of interest An E. coli selectable marker gene some kind of antibiotic resistance. Origin of replication and other associated plasmid'housekeeping' sequences. There are two main ways to utilise these tools: Clone the P element into a plasmid and transform and grow this in bacteria. Eliminate the P transposase and rep