Yeast-bacteria shuttle vector

Details Yeast-bacteria shuttle vector Yeast-bacteria shuttle vector

1998-06-10

2001-04-24

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091400, C435S476000, C435S477000, C435S483000

Reexamination Certificate

active

06221588

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of genomics and the ability to manipulate, isolate and replicate large fragments of DNA.
BACKGROUND OF THE INVENTION
For the functional analysis of many genes investigators need to isolate and manipulate large DNA fragments. The advent of genomics and the study of genomic regions of DNA has generated a need for vectors capable of carrying large DNA regions.
In general, two types of yeast vector systems are presently available. The first type of vector is one capable of transferring small insert DNA between yeast and bacteria (33, 34). A second type of vector is a fragmenting vector which creates interstitial or terminal deletions in YACs (35, 36, 37). The small insert shuttle vectors are able to recombine with and recover homologous sequences. They are centromere-based and replicate stably and autonomously in yeast, but also contain a high-copy origin of replication for maintenance as bacterial plasmids. However, these vectors are limited by their small insert capacity.
The second type of vector, known as fragmenting vectors, also have recombinogenic sequences but are unable to transfer the recovered insert DNA to bacteria for large preparations of DNA.
Researchers use fragmentation techniques to narrow down the region of interest in YACs (1, 2). However, isolating sufficient quantities of YAC DNA from agarose gels for microinjection or electroporation remains cumbersome. Purification remains a problem when the YAC comigrates with an endogenous chromosome. In addition, YACs may be chimeric or contain additional DNA regions that are not required for the particular functional study.
Types of vectors available for cloning large fragments in bacteria are cosmids, P1s and BACS. These vectors are limited to bacteria and cannot be shuttled to yeast for modification by homologous recombination.
One object of the present invention relates to the identification of a new yeast-bacteria shuttle vector capable of accommodating large fragments of DNA.
Another object of the present invention relates to the use of the new yeast bacteria shuttle vector for cloning large DNAs.
Yet another object of the present invention relates to the use of the new yeast bacteria for site-specific targeting of genomic DNA.
SUMMARY OF THE INVENTION
A new yeast-bacteria shuttle vector has been identified that allows selective cloning and/or manipulation of large regions of DNA. The vector of the present invention provides a unique combination of features including site-specific targeting, yeast to bacteria (and bacteria to yeast) shuttling capability, interchangeable recombinogenic ends, large insert capacity, and near universal compatibility with large insert cloning systems in bacteria and yeast. When combined with a method for direct cloning of co-transformed genomic DNA, the vector of the present invention provides a highly versatile cloning system.
The circular product of cloning in the present vector can be transferred to bacteria for simplified preparation of large quantities of plasmid DNA. The shuttle capability of the vector makes mutagenesis techniques by yeast genetics accessible for the production of large reporter constructs. The yeast bacterial shuttle vector of the present invention comprises a yeast replication origin, a yeast selection marker gene, a bacteria replication origin, a bacteria selection marker gene and at least one unique cloning site.
One embodiment of the present invention combines the single-copy F factor replicon and chloramphenicol resistance gene for stable propagation of large circular DNA in bacteria with the CEN6/ARS4 origin of replication and LEU2 gene for maintenance and selection in yeast. This vector is called pCLASPER. The bacterial origin of replication chosen is the one used in bacterial artificial chromosomes (BACs). Recognition sites for two intron-encoded endonucleases have been engineered to flank an unique polylinker. This vector has been used to clone various regions.


REFERENCES:
patent: 5972614 (1999-01-01), Ruano et al.
McGonigal, T., Bodelle, P., Schopp, C., and Sarthy, A.V. (1995). Construction of a human DNA library in a circular centromere-based yeast plasmid.Gene155, pp. 267-271.
Prado, F., and Aguilera, A. (1994). New in-vivo cloning methods by homologous recombination in yeast.Current Genetics25, pp. 180-3.
Spencer, F., Ketner, G., Connelly, C., and Hieter, P. (1993). Targeted recombination-based cloning and manipulation of large DNA segments in yeast.Methods: A companion to Methods Enzymol5, pp. 161-175.
Kogoma, Tokio, “Stable DNA Replication: Interplay between DNA Replication, Homologous Recombination, and Transcription”,Microbiology and Mole. Bio., vol. 61, No. 2, pp. 212-238, Jun. 1997.
Larionov, Vladimir, et al., “Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast”,Proc. Natl. Acad, Sci., USA, vol. 94, pp. 7384-7387, Jul. 1997.
van Dijk, et al., “A Functional Assay in Yeast for the human Estrogen Receptor Displays Wild-Type and Variant Estrogen Receptor Messenger RNAs Present in Breast Carcinoma”,Cancer Research, 57, pp. 3478-3485, Aug. 15, 1997.
Frengen, et al., “Transfer of small YACs toE. coli.as large circular plasmids”,Genetic Analysis Biomolecular Engineering,14, pp. 57-59, 1997.
Larionov, Vladimir, et al., “Specific cloning of human DNA as yeast artificial chromosomes by transformation-associates recombination”,Proc. Natl. Acad. Sci., USA, vol. 93., pp. 491-496, Jan. 1996.
Bradshaw, Suzanne, M., et al., “A long-range regulatory element of Hoxc8 identified by using the pClasper vector”,Proc. Natl. Acad. Sci., USA, vol. 93, pp. 2426-2430, Mar. 1996.
Simpson, Kaetrin, et al., “A shuttle system for transfer of YACs between yeast and mammalian cells”,Nucleic Acids Research, vol. 24, No. 23, pp. 4693-4699, 1996.
Bradshaw, Suzanne M., et al. “A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes”,Nucleic Acids Research, vol. 23, No. 23, pp. 4850-4856, 1995.
Campbell, Colin, et al., “Unequal homologous recombination of human DNA on a yeast artificial chromosome”,Nucleic Acids Research, vol. 23, No. 18, pp. 3691-3695, 1995.
Ketner, Gary, et al., “Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone”,Proc. Natl. Acad. Sci., USA, vol. 91, pp. 6186-6190, Jun. 1994.
Kouprina, N., et al., “A Model System to Assess the Integrity of Mammalian YACs during Transformation and Propagation in Yeast”,Genomics, 21, pp. 7-17, 1994.
Larionov, V., et al., Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast (YACs),Nucleic Acids Research, vol. 22, No. 20, pp. 4154-4162, 1994.
Miller, Leilani, et al., “lin-31, a Caenorhabditis elegans HNF-3/fork head transcription factor homolog, specifies three alternative cell fates in vulval development”,Genes and Development, vol. 7, pp. 933-947, 1993.
Bradshaw, Suzanne M., et al., “Site Specific Recombination-Mediated Isolation of a Large Sub-Region from a Mouse Hox-c YAC”, Ist Euro. Sci Found. Conf. On Deve. Bio., Karause Ittengen, Switzerland, Jun. 14-17, 1993.
Pavan, William, et al., “Generation of deletion derivatives by targeted transformation of human-derived yeast artificial chromosomes”,Proc. Natl. Acad. Sci., USA., Vol. 87, pp. 1300-1304, Feb. 1990.
Sikorski, Robert S., et al., “A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA inSaccharomyces cerevisiae”, Genetics, vol. 122, pp. 19-27, May 1989.
Burgers, Peter, M.J., et al., “Transformation of Yeast Spheroplasts without cell Fusion”, Analytical Biochemistry, vol. 391-397, 1987.
Orr-Weaver, Terry L., et al., “Genetic Applications of Yeast Transformation with Linear and Gapped Plasmids”, Methods in Enzymology, vol. 11, pp. 228-245, 1983.
Orr-Weaver, Terry L., et al., “Yeast recombination: The association between double-strand gap repair and crossing-over”,Proc. Natl. Acad. Sci., USA, vol. 80, pp. 4417-4421, Jul. 1983.
Orr-Weaver, Terry L., et al., “Yeast transformation: A mode

Affiliated with

Also associated with

Rating

Yeast-bacteria shuttle vector does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Yeast-bacteria shuttle vector, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Yeast-bacteria shuttle vector will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2544888