| Unusual retrotransposon from the yeast candida albicans -> Monitor Keywords |
|
|
  Unusual retrotransposon from the yeast candida albicansUSPTO Application #: 20060128021Title: Unusual retrotransposon from the yeast candida albicans Abstract: TCa2 is a TyI/copia retrotransposon from the pathogenic yeast Candida albicans. In contrast to other retrotransposons it can appear as an abundant, extrachromosomal double-stranded DNA molecule, called pCal. The invention relates to the isolation and characterisation of TCa2 and pCal together with its uses for inducing random mutagenesis in a genome, as a component of a transposable element and of an expression vector. (end of abstract) Agent: Frommer Lawrence & Haug - New York, NY, US Inventors: Russell Tony Masell Poulter, Walter Herman Maria Louis Luyten, Marianne Denise De Backer, Bart Jozef Maria Nelissen USPTO Applicaton #: 20060128021 - Class: 435473000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within A Microorganism (e.g., Bacteria, Protozoa, Bacteriophage, Etc.), The Polynucleotide Contains A Transposon The Patent Description & Claims data below is from USPTO Patent Application 20060128021. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This is a divisional application of U.S. application Ser. No. 09/430,590, filed Oct. 29, 1999, which claims priority to U.S. application Ser. No. 60/106,342, filed Oct. 30, 1998. The foregoing applications all documents cited therein ("the application-cited documents") and all documents referenced or cited in the application-cited documents are hereby incorporated herein by reference. In addition all documents cited herein ("herein cited documents") and all documents cited or referenced in herein cited documents are likewise incorporated herein by reference. FIELD OF INVENTION [0002] The invention relates to a novel retrotransposon. The novel retrotransposon is from the yeast Candida albicans. In particular, the invention relates to a retrotransposon pCal which belongs to the TyI/copia group. INTRODUCTION [0003] Candida albicans is an asexual yeast species which is the major fungal pathogen of humans. Although it is commonly found as a harmless commensal organism, inhabiting mucosal membranes and the digestive tract, it can cause superficial infections, such as oral thrush, in otherwise healthy people and can cause severe, often fatal, systemic infections in immuno-compromised patients. The recent increased use of immunosuppressive treatments and the increased incidence of immunosuppressive diseases, such as HIV infections, have meant that C. albicans infections are of increasing medical significance (Odds 1988). There is significant strain variation within this species, potentially affecting virulence, and mobile retroelements have been suggested as one source of this. [0004] Retroelements are a widespread family of sequences that can replicate via the reverse transcription of single-stranded RNA into double-stranded DNA, or are assumed to have arisen in this way. Two major types of retroelement are the retroviruses, such as HIV1 and Moloney murine leukaemia virus, and the retrotransposons such as Ty1 and Ty3 from Saccharomyces cerevisiae (Boeke and Sandmeyer 1991). The structures and lifecycles of retrotransposons and retroviruses are very similar. The major difference between the two groups is that the retroviruses can form infectious virus particles which can be transmitted between cells and between individuals. Retrotransposons can form intracellular virus-like particles (VLPs) but they lack the genes coding for the viral envelope so the VLPs are usually confined to the one cell. [0005] Similarly to retroviruses, retrotransposons consist of an internal domain flanked by long terminal direct repeats (LTRs). In Ty1, for example, the LTRs are about 335 bp in length and the internal domain is about 5.3 kb long. The internal region has two long open reading frames (ORFs) homologous to the gag and pol ORFs of retroviruses. The gag gene encodes the structural proteins which make up the VLP while, downstream, the pol gene encodes the enzymes required for reverse transcription and integration--protease, integrase, reverse transcriptase and RNase H. The LTRs contain the promoter and the transcription termination signals and are functionally divided into three regions--U.sub.3, R and U.sub.5. Transcription proceeds from the U.sub.3/R boundary in the left LTR to the R/U.sub.5 boundary in the right LTR to produce an RNA molecule which has the R region repeated at each end. Translation of this terminally redundant mRNA is usually regulated to ensure that the structural proteins of the VLP (gag) are produced in much higher quantities than the enzymes (pol). This is because large quantities of the gag proteins are required for the assembly of the VLP but only catalytic quantities of the pol enzymes are required. [0006] The most common method of down-regulating the translation of the pol ORF is to have it out of frame relative to the upstream gag ORF. A rare, programmed ribosomal frameshift is thus required for translation of the pol ORF. A number of retrotransposons employ a +1 frameshift. Ty1 achieves this by tRNA slippage while the Ty3 mechanism involves the `skipping` of a base. The Ty1-slippage mechanism involves a seven base sequence, CUU AGG C. It is thought that a tRNA.sup.LeuUAG, which can recognise all six leucine codons, slips forward one base from CUU-Leu to UUA-Leu, during a translational pause caused by a rare tRNA.sup.ArgCCU (2). The Ty3 +1 frameshift also involves a seven base sequence, GCG AGU U. An alanine-valine sequence (encoded by GCG-GUU) is produced but tRNA slippage is not involved. It is thought that out-of-frame aminoacyl-tRNA binding or four-base decoding is responsible. Frameshifting is stimulated by the low availability of the tRNA decoding the AGU-Ser codon and also by the 12 nucleotides downstream of the AGU codon. Retrotransposons have also been found to use a -1 frameshift; an example is CfT-I of Cladosporium fulvum. Here the ribosome is thought to slip back one base on the sequence AAAA slightly upstream of the gag termination codon. [0007] An alternative method of down-regulation has been found in the copia retrotransposon. Here the gag and pol ORFs are fused into one long continuous ORF, but a splicing reaction usually occurs prior to translation to excise most of the pol region from the mRNA. Only occasionally is a full-length RNA translated with the concomitant production of the pol enzymes. [0008] Following translation the retrotransposon proteins and RNA can form into a VLP. This consists of a shell of gag proteins with the pol enzymes and genomic RNA packaged inside. The VLP is the site of reverse transcription. In general, the process of reverse transcription in retrotransposons is very similar to the well-characterised process of retroviral reverse transcription. Two important steps in the reverse transcription process are the priming of minus- and of plus-strand DNA synthesis. Minus-strand synthesis is most commonly primed by a cytoplasmic tRNA (often initiator methionine tRNA) which is packaged within the VLP along with the mRNA of the retrotransposon. The retrotransposon has a region adjacent to the left LTR, known as the minus-strand primer binding site [(-)PBS], which is complementary to the 3' end of this tRNA. The tRNA binds to the retrotransposon RNA at the (-)PBS and can then be used by reverse transcriptase as a primer for the synthesis of minus-strand DNA. Plus-strand synthesis is primed by a short purine-rich sequence, known as a polypurine tract (PPT), located just upstream of the right LTR. After minus-strand DNA synthesis has passed this sequence the RNA is nicked between the PPT and the LTR. The PPT RNA can then be used as a primer for the synthesis of the plus-strand. Reverse transcription is generally very inefficient; greater than 10% of cellular mRNA can be retrotransposon RNA yet the dsDNA form is not usually detectable by Southern blotting. [0009] Following the synthesis of the dsDNA form of the retrotransposon it may integrate at a new site within the host genome. This process is likely to involve a complex of the integrase enzyme associated with the two ends of the retrotransposon DNA. In a process which is not well understood the integrase complex must be released from the VLP, move into the nucleus and then insert the DNA into a new genomic site. Studies with Ty1 and Ty3 have shown that the integration site-selection mechanisms of these retrotransposons are non-random and appear to be specifically adapted to avoid causing disruption to the host genome. [0010] Retrotransposons can be divided into three major groups based on their reverse transcriptase sequences and the order of the genes within their pol ORFs. Members of the Ty3/gypsy group are the most closely related to the retroviruses and share a similar pol gene order--protease, reverse transcriptase, RNase H and integrase. Examples of these elements are Ty3 of S. cerevisiae, gypsy of Drosophila melanogaster, Tf1 of Schizosaccharomyces pombe and del of Lilium henryi. Members of the Pao group, for example Pao of Bombyx mori and Tas of Ascaris lumbricoides, have a similar pol gene order to Ty3/gypsy retrotransposons but can be distinguished from them by their reverse transcriptase sequence. Ty1/copia elements are most easily distinguished from Ty3/gypsy and Pao retrotransposons and retroviruses by the gene order of the pol protein-protease, integrase, reverse transcriptase, RNase H. This group includes Ty1 and Ty2 of S. cerevisiae, copia and 1731 of D. melanogaster, Tst1 of Solanum tuberosum and Tnt1 of Nicotiana tabacum. [0011] The first Candida retroelement, TCa1, was identified through the discovery of multiple-copy isolated LTRs dispersed around the genome (1). These LTRs were discovered in an analysis of moderate repeat elements. Subsequently, composite elements, named TCa1, consisting of two LTRs flanking a 5.5 kb internal domain were also found. In the C. albicans strains tested, one to two TCa1 loci were found, indicating between one and four copies of TCa1 depending on whether the loci were homozygous or not. TCa1 has many features of a typical retrotransposon including 388 bp LTRs, beginning TG and ending CA, with six nucleotide inverted repeats, TGTTCG . . . CGAACA, at either end (SEQ ID NO:1). The element is flanked by 5 bp duplications of the host DNA and is transcribed to give an approximately unit length mRNA. Within the 5.5 kb internal domain a (-)PBS and a plus-strand priming site are evident. The (-)PBS was not immediately obvious: no complementarily to tRNA.sup.iMet (as used by Ty1 and Ty3) could be found. Bases 31 to 39 of tRNA.sup.Arg3 of S. cerevisiae, however, perfectly complemented the nine bases immediately adjacent to the left LTR (GATTAGAAG). There is, for some tRNA, a high degree of conservation between S. cerevisiae and C. albicans leading to the suggestion that a cleavage product of a C. albicans tRNA.sup.Arg might serve as the primer. This suggestion is supported by the knowledge that the primer used by the copia retrotransposon is a cleavage product of tRNA.sup.iMet containing only the first 39 nucleotides. [0012] TCa1 has been shown to be transcriptionally active, but an analysis of 1200 bp of its internal sequence has indicated that it is defective, there being multiple stop codons in all three reading frames. It is remarkable, given the clearly non-functional nature of this element, that the LTRs remain identical and that the plus- and minus-strand priming sites remain in apparently functional form. It is possible that the defective TCa1 retrotransposon has been maintained via the passive reverse transcription of its RNA by the products of a functional C. albicans retrotransposon. This passive replication would require that the element has identical LTRs and functional plus- and minus-strand priming sites but would be independent of the element's internal sequence. [0013] The object of the invention is to provide a novel retrotransposon, in particular the isolation and sequencing of pCal, an unusual, novel Ty1/copia retrotransposon from C. albicans. The free, linear, double-stranded DNA form of this element is so highly expressed that it can be seen as a distinct band when uncut genomic C. albicans DNA is simply analysed on an agarose gel. It contains features conserved in TCa1 and other retrotransposons and has additional features previously unreported in the retrotransposon family. [0014] The sequence of another C. albicans element, potentially retrotransposon-like in nature, has recently been submitted to the databases by a group in the U.K. (accession no. Y08494). This element has been named beta and is defined as an LTR. It consists of a repeated sequence about 400 bp in length, flanked by 5 bp direct repeats of the host DNA, and associated with tRNA genes. The borders of the element consist of short, imperfect, inverted repeats: 5'-TAATGTATA . . . TATACAACA-3' (SEQ ID NO:2). Such an element is reminiscent of the isolated LTRs of other retrotransposons which are the result of homologous recombination between the ends of a retrotransposon with the concomitant deletion of the internal region. No significant similarity is detectable between the beta sequence and the LTRs of TCa1 or pCal of the present invention. SUMMARY OF THE INVENTION [0015] The invention provides an isolated and purified retrotransposon having a copy number of between 40-150 (preferably 50-100) copies of free DNA of itself per genome (preferably 10-25 megabases, more preferably substantially 15 megabases). The DNA is preferably linear and is more preferably double stranded. [0016] The retrotransposon may be isolated from fungi or yeast, preferably Candida and more preferably from Candida albicans. [0017] The invention also provides a novel retrotransposon comprising at least one polypeptide positioned between at least two long terminal repeats, and wherein the retrotransposon is capable of integrating into the DNA in a genome providing a copy number of between 40-150 copies per genome. The copy number is preferably 50-100 copies. [0018] The retrotransposon does not necessarily integrate into the DNA. [0019] The retrotransposon preferably belongs to the TyI/copia group. [0020] The retrotransposon is preferably isolated from fungi or yeast, preferably Candida and more preferably from Candida albicans. Continue reading... Full patent description for Unusual retrotransposon from the yeast candida albicans Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Unusual retrotransposon from the yeast candida albicans patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Unusual retrotransposon from the yeast candida albicans or other areas of interest. ### Previous Patent Application: Process for producing virus vector containing membrane protein having sialic acid-binding in envelope with the use of gram-positive bacterium origin nueraminidase Next Patent Application: Process control method and process control system Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Unusual retrotransposon from the yeast candida albicans patent info. IP-related news and info Results in 1.15141 seconds Other interesting Feshpatents.com categories: Software: Finance , AI , Databases , Development , Document , Navigation , Error |
||
