Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-03
2001-07-31
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C435S004000
Reexamination Certificate
active
06268139
ABSTRACT:
Some of the research underlying this invention was partially funded by the National Institutes of Health's National Cancer Institute's small business innovative research program grant number 1 R43 CA62681-01.
FIELD
The invention relates to a method for detecting, selecting, and cloning agents that disrupt, degrade or promote degradation of cellular DNA, and novel agents selected by practicing these methods.
BACKGROUND
Agents that disrupt, degrade or promote degradation of DNA are useful in a variety of ways. For example, restriction endonucleases (enzymes which cut DNA at specific sites) are highly useful tools for researchers involved in the manipulation of DNA. Also, DNA-degrading agents are useful to kill cells, for example, cancer cells.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the “recognition sequence”) along the DNA molecule. Once bound, they cleave the molecule within, or to the side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. More than one hundred different restriction endonucleases have been identified among the many hundreds of bacterial species that have been examined to date.
Bacteria tend to possess at most only a small number of restriction endonucleases per species. The endonucleases typically are named according to the bacteria from which they are derived. Thus, the species
Haemophilus aegyptius,
for example, synthesizes 3 different restriction endonucleases, named Hae I, Hae II and Hae III. Those enzymes recognize and cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC respectively.
Escherichia coli
RY13, on the other hand, synthesizes only one enzyme, EcoRI, which recognizes the sequence GAATTC.
In nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They achieve this resistance by scanning the lengths of the infecting DNA molecule and cleaving them each time the recognition sequence occurs. The break-up that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific exonucleases.
A second component of bacterial protective systems are the modification genes or methylases. These enzymes are complimentary to restriction endonucleases and they provide the means by which bacteria are able to identify their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequences as the corresponding restricting endonucleases, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified, by virtue of its modification methylases. It is therefore completely insensitive to the presence of the endogenous restriction endonucleases. It is only unmodified, and therefore identifiably foreign DNA that is sensitive to restriction endonuclease recognition and attack.
Cloning DNA that encodes agents disrupting, degrading or promoting degradation of DNA, such as restriction enzymes, has been difficult because such agents can degrade the very DNA in the cells used to isolate them. Indeed, it has been recognized that some failures in restriction endonuclease cloning may be due to this lethality problem (Brooks et al.,
Nucleic Acids Research,
17(1988): 979-997). Some tricks have been developed for isolating this type of DNA, but the strategies developed so far offer only limited success. For example, some investigators have used bacteriophage infection as a means of selectively isolating restriction endonuclease clones (Walder et al.,
Proc. Nat. Acad. Sci.,
74: 1503-1507 (1981); Mann et al.,
Gene,
3: 97-112 (1978)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction-modification genes (R/M) can in principle be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
U.S. Pat. Nos. 5,180,673 and 5,200,333 to Wilson et al. disclose a method for over-production of restriction enzymes and their corresponding modification enzymes involving the following steps:
1. The DNA of the bacterial species to be cloned is purified.
2. The DNA is digested partially with a convenient restriction endonuclease.
3. The resulting fragments are ligated into a cloning vector, such as pBR322, and the mixture is used to transform an appropriate host cell such as
E. coli
cells.
4. The DNA/cell mixture is plated on antibiotic media selective for transformed cells. After incubation, the transformed cell colonies are scraped together into a single culture, the primary cell library.
5. The recombinant plasmids are purified in toto from the primary cell library to make a primary plasmid library.
6. The plasmid library is then digested to completion in vitro with the restriction enzyme whose corresponding methylase gene is sought. Exonuclease and/or phosphatase may also be added to the digestion to enhance the destruction of non-methylase clones.
7. The digested pool is transformed into
E. coli
and transformed colonies are again obtained by plating on antibiotic plates. Some of these colonies—secondary cell individuals—may be picked and their DNA analyzed for the presence of the modification and/or restriction genes. The remaining colonies may be scraped together to form a secondary cell library from which a secondary plasmid library may be subsequently prepared.
8. The secondary plasmid library may be redigested with restriction endonuclease (with or without exonuclease or phosphatase) to repeat the selection, leading to the recovery of tertiary cell individuals, tertiary cell libraries and tertiary plasmid libraries.
9. Each round of restriction endonuclease digestion causes selective destruction of non-methylase clones, and results in an increase in the relative frequency of the desired methylase-carrying clones.
10. Surviving colonies among the secondary and tertiary population are picked and analyzed for the presence of the methylase gene. If it is found to be present, they are further analyzed for the simultaneous presence of the restriction gene that is presumed to be linked to the methylase gene.
11. Methylase screening may be performed by four simple tests:
(a) The recombinant plasmid DNA molecule of the clone may be purified and exposed to the selecting restriction endonuclease to establish that it is resistant to digestion. Provided that the plasmid vector carries several sites for that endonuclease, resistance indicates modification, rather than mutational site loss.
(b) The recombinant plasmid may be digested with the enzyme initially used to fragment the donor bacterial DNA. The fragments present in the clone should be comprehensible, sufficiently large to encode a methylase gene (i.e., over 1 Kilobase pair) and, most important, common to a variety of independently-formed clones: the same fragment or fragments should be present among all the clones.
(c) The total chromosomal DNA of the clone may be purified and exposed to the selective restriction endonuclease. If the clone carries the methylase gene, the bacterial chromosome should be fully methylated and, like the plasmid, should be found to be resistant to digestion.
(d) The cell extract from the clone may be prepared and assayed in vitro for methylase activity. (Methylase protection and radioactive labelling.) Methylase activity should be found.
12. Restriction endonuclease screening may be carried out in t
Foley & Lardner
Whisenant Ethan
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