Coin handling – Deliverer – Delivery of a distinct number of plural coins from a single...
Reexamination Certificate
1999-06-17
2001-12-18
Achutamurthy, Ponnathapu (Department: 1652)
Coin handling
Deliverer
Delivery of a distinct number of plural coins from a single...
C435S320100, C435S252300, C435S029000
Reexamination Certificate
active
06331140
ABSTRACT:
STATE OF THE ART
Bacterial insertion sequences (IS) were discovered during early investigations of gene expression in
Escherichia coli
and the bacteriophage lambda. They range from 800 to 2′500 bp in length and can be found in the genome of many different bacteria at numbers varying between a few and a few hundred copies per genome. Most of the insertions sequences contain one large open reading frame (ORF) which extends almost the entire length of the element and encodes for the transposase. IS-element are characterised by the presence at their termini of inverted repeats (IR) which are functional sequences required for transposition. One characteristic of the majority of known insertion elements is that they generate direct repeated duplications (DR) of the target DNA at the point of insertion. The number of base pairs duplicated is specific for each element.
IS-elements have been discovered to play an important role in producing spontaneous mutation in bacteria. Early estimates of the frequency of insertion mutations were made for the galK (Jordan et al., Mol. Gen. Genet., 102, 353-363, 1968) and the lac region (Malamy, In: The lactose operon, Ed. J. R. Beckwith and D. Zipser. Cold Spring Harbor Laboratory, N.Y., 1970). In each case, it was estimated that between 5 and 15% of spontaneous mutations were due to IS insertions. The capacity of certain insertion sequences to generate deletions of neighbouring DNA was first noted for an ISI located in the gal open (Reif et al., Mol. Gen. Genet., 137, 17-28, 1975). IS-elements also contribute to DNA rearrangements by providing homologous DNA for general recombination systems, leading to gene duplication and more complex DNA restructuring.
IS-elements usually contain transitional and transcriptional start and stop signals. Depending on their location, they may interfere with the regulation of adjacent genes. This regulatory potential, together with the DNA rearrangements associated with IS-elements, may help to expand a selective advantage to the population of organisms harbouring mobile genetic elements. Furthermore, the IS-mediated association of functional genes with transmissible episomes facilitates the horizontal spreading of bacterial gene.
IS-elements are further characterised as useful genetic tools. Thus, they are able to translocate from an appropriate vector, i.e. a conjugated or transformed plasmid, onto the bacterial genome and to select their site of integration in a quasi random way. This characteristic can be used in combination with an appropriate selection system to genetically identify (tag) genes or gen-clusters responsible for a specific functionality. This has been demonstrated for the identification of genes responsible for the production of exocellular polysaccharides in
Streptococcus thermophilus
(F. Stingele et al., J. Bact, 178, 1680-1690, 1995. Integration of an IS-element into a functional gene can thereby completely inactive its function.
Furthermore, IS-elements located on extra chromosomal plasmids can integrate in a replicative manner into a bacterial genome, by integrating as a consequence at the same time the entire (or at least a part) of the host plasmid vector into the genome as well. Thereby, the active IS-element duplicates itself and now flanks the integrated plasmid on both sides (Iida et al., In Mobile genetic elements, Shapiro (ed), Acad. P., New York, pp 159-221, 1983). With this method, genes of interest carried on plasmids can be functionally integrated into bacterial genomes.
Three IS-elements originating from lactobacillus have been only characterised. These elements are known to be very specific to a species, which means they are not always active in another species. Shimizu-Kadota et al. identified an IS-element from Lactobacillus casei (Mol. Gen. Genet., 200, 193-198, 1995). Zwahlen et al. also identified an IS-element from
Lactobacillus helveticus
(Mol. Gen. Genet, 245, 334-338, 1994), whereas Germond et al. identified another IS-element from
Lactobacillus delbrueckii
sp.
bulgaricus
(Mol. Gen. Genet., 248, 407-416, 1995).
These lactobacilli IS-elements were never used, or intended to be used as tools to modifying genetically the species from which they come from. One of the reason is because lactobacilli used for yoghurt production are known as being notoriously difficult for genetic modification. In particular, genetic tools for
Lactobacillus delbrueckii
and
Lactobacillus helveticus
, two microorganisms that are phenotypically closely related (see EP441991 and EP391039), are very limited or quasi absent as for today.
The objective of the invention is to provide new genetic tools specifically for the
L. delbrueckii
and
L. helveticus
species, which can be used in genetic gene identification (tagging), gene inactivation, gene integration, and/or gene expression on a plasmid and/or genomic level.
SUMMARY OF THE INVENTION
Accordingly, the present invention thus relates to the use of an IS-element as a tool to genetically modify the genome of
L. delbrueckii
or
L. helveticus.
In another embodiment, the present invention also relates to an IS-element, originating from
L. delbrueckii
, selected from the DNA sequences SEQ ID NO:7-10 or functional derivatives thereof.
In another embodiment, the present invention also relates to any
Lactobacillus delbrueckii
or any
Lactobacillus helveticus
providing a gene encoding, or affecting the production of, enzymes producing secondary metabolites, the &bgr;-galactosidase, the cell wall protease, the catabolite control protein A, the lactate dehydrogenase, a glycosyltransferase, a restriction system, a lysogenic prophage, or the permease of the lac operon, wherein said gene is inactivated by insertion of at least one IS-element.
In a last embodiment, the present invention also relates to the use of a
Lactobacillus delbrueckii
or a
Lactobacillus helveticus
for the preparation of a fermented product, wherein the
Lactobacillus delbrueckii
or the
Lactobacillus helveticus
is provided with a gene encoding, or affecting the production of, enzymes producing secondary metabolites, the &bgr;-galactosidase, the cell wall protease, the catabolite control protein A, the lactate dehydrogenase, a glycosyltransferase, a restriction system, a lysogenic prophage, or the permease of the lac operon, wherein said gene is inactivated by insertion of at least one IS-element.
DETAILED DESCRIPTION OF THE INVENTION
Within the following description, the percentages are given by weight except where otherwise stated, and the nucleotide sequences referred as “SEQ ID NO:” are always presented in the sequence listing hereafter.
Likewise, the expression “functional derivative” includes all nucleotide sequences which differ by substitution, deletion, addition of some nucleotides, for instance 1-50 nucleotides, but which keep their original activities or functions. A functional derivative may thus be due to the degeneracy of the genetic code. The selection of a functional derivative is considered to be obvious to one skilled in the art, since one may easily creates variants of an encoding sequence by slightly adapting methods known to one skilled in the art, for instance the methods described by Adams et al. (EP402450; Genencor), by Dunn et al (Protein Engineering, 2, 283-291, 1988), by Greener et al. (Strategies, 7, 32-34, 1994), and/or by Deng et al. (Anal. Biochem, 200, 81, 1992). In particular, a functional IS-element of the invention is considered as a derivative to another IS-element, if its sequence is at least 80% identical to the original IS-element, preferably at least 95%, for example. In the context of the present disclosure, the identity is determined by the ratio between the number of nucleotides of a derivative sequence which are identical to those of the original IS-element, and the total number of nucleotides of the said derivative sequence. Likewise, a functional IS-element of the invention may also be considered as a derivative to another IS-element if it can hybridize to it under very stringent conditions, that is to say tha
Germond Jacques Edouard
Lapierre Luciane
Mollet Beat
Achutamurthy Ponnathapu
Nestec S.A.
Steadman David J.
Winston & Strawn
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