Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-02-20
2002-07-16
Yucel, Remy (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C536S023200
Reexamination Certificate
active
06420524
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention is specifically directed to efficient, random, simple insertion of a transposon or derivative transposable element into DNA in vivo or in vitro. The invention is particularly directed to mutations in ATP-utilizing regulatory transposition proteins that permit insertion with less target-site specificity than wild-type. The invention encompasses gain-of-function mutations in TnsC, an ATP-utilizing regulatory transposition protein that activates the bacterial transposon Tn
7
. Such mutations enable the insertion of a Tn
7
transposon or derivative transposable element in a non-specific manner into a given DNA segment. Insertion can be effected in plasmid and cosmid libraries, cDNA libraries, PCR products, bacterial artificial chromosomes, yeast artificial chromosomes, mammalian artificial chromosomes, genomic DNAs, and the like. Such insertion is useful in DNA sequencing methods, for genetic analysis by insertional mutagenesis, and alteration of gene expression by insertion of a given genetic sequence.
2. Description of the Background Art
Transposable elements are discrete segments of DNA capable of mobilizing nonhomologously from one genetic location to another, that typically carry sequence information important for two main functions that confer the ability to mobilize. They encode the proteins necessary to carry out the catalytic activity associated with transposition, and contain the cis-acting sequences, located at the transposon termini, that act as substrates for these proteins. The same proteins can participate in the selection of the target site for insertion.
The selection of a new insertion site is usually not a random process; instead, many transposons show characteristic preferences for certain types of target sites. One broad characteristic that differentiates the wide variety of transposable elements known is the nature of the target site selectivity (1). A component of this selectivity can be the target sequence itself. The bacterial transposon Tn10 preferentially selects a relatively highly conserved 9 bp motif as the predominant site for transposon insertion and less often selects other more distantly related sites in vivo (2). The Tc1 and Tc3 mariner elements of
C. elegans
insert preferentially at a TA dinucleotide such that each end of the element is flanked by a TA duplication (3) (4) (5). A lower specificity consensus sequence, N-Y-G/C-R-N has been determined from populations of both in vivo and in vitro insertions for the bacteriophage Mu (7). In contrast to these elements, the bacterial transposon Tn5 exhibits markedly lower insertion site specificity, although some isolated “hotspots” have been detected (8).
Another selection mechanism relies on structural features or presence of cellular protein complexes at the target sites. The yeast transposon Ty3 preferentially inserts into the promoters of genes transcribed by RNA polymerase III, responding to signals from cellular proteins TFIIIB and TFIIIC (9).
Understanding how these factors modulate transposase activity to impose target site preferences will lend insight into the spread of transposons and viruses, and may suggest ways to manipulate those target preferences. The bacterial transposon Tn
7
is distinctive in that it uses several element-encoded accessory proteins to evaluate potential target DNAs for positive and negative features, and to select a target site (1). Tn
7
encodes five genes whose protein products mediate its transposition (10) (11).
Two of the proteins, TnsA and TnsB, constitute the transposase activity, collaborating to execute the catalytic steps of strand breakage and joining (12). The activity of this transposase is modulated by the remaining proteins, TnsC, TnsD, and TnsE, and also by the nature of the target DNA.
TnsC, TnsD, and TnsE interact with the target DNA to modulate the activity of the transposase via two distinct pathways. TnsABC+TnsD directs transposition to attTn7, a discrete site on the
E. coli
chromosome, at a high frequency, and to other loosely related “pseudo att” sites at low frequency (13). The alternative combination TnsABC+E directs transposition to many unrelated non-attTn7 sites in the chromosome at low frequency (13) (10) (11) and preferentially to conjugating plasmids (14). Thus, attTn7 and conjugable plasmids contain positive signals that recruit the transposon to these target DNAs. The alternative target site selection mechanisms enable Tn7 to inspect a variety of potential target sites in the cell and select those most likely to ensure its survival.
The Tn
7
transposition machinery can also recognize and avoid targets that are unfavorable for insertion. Tn
7
transposition occurs only once into a given target molecule; repeated transposition events into the same target are specifically inhibited (15) (16). Therefore, a pre-existing copy of Tn
7
in a potential target DNA generates a negative signal which renders that target “immune” to further insertion. The negative target signal affects both TnsD- and TnsE-activated transposition reactions and is dominant to any positive signals present on a potential target molecule (16). Several other transposons, such as Mu and members of the Tn
3
family, also display this form of negative target regulation (17) (18) (19) (7).
Target selection could be an early or late event in the course of a transposition reaction. For example, a transposon could constitutively excise from its donor position, and the excised transposon could then be captured at different frequencies by different types of target molecules. Tn
10
appears to follow this course of events in vitro, excising from its donor position before any interactions with target DNA occur (20) (21). Alternatively, the process of transposon excision could itself be dependent on the identification of a favorable target site. Tn
7
transposition shows an early dependence on target DNA signals in vitro: neither transposition intermediates nor insertion products are seen in the absence of an attTn7 target (22). Thus, the nature of the target DNA appears to regulate the initiation of Tn
7
transposition in vitro.
An important question is how positive and negative target signals are communicated to the Tn
7
transposase. Reconstitution of the TnsABC+TnsD reaction in vitro has provided a useful tool for detailed dissection of Tn7 transposition (22) (23). This reaction has been instrumental in delineating the role of each of the individual proteins play in target site selection. Dissection of the TnsABC+D reaction in vitro has implicated TnsC as a pivotal connector between the TnsAB transposase and the target DNA. TnsC is an ATP-dependent DNA-binding protein with no known sequence specificity (24). However, TnsC can respond to signals from attTn7 via an interaction with the site-specific DNA-binding protein TnsD. In a standard in vitro transposition reaction TnsD is required for transposition to the attTn7 site on a target DNA molecule. This site-specific insertion process is tightly regulated by TnsC, but does not occur in the absence of TnsD. Additional evidence for a TnsC-TnsD interaction comes from DNA protection and band shift analysis with attTn7 DNA (23). Direct interaction between TnsC and the TnsAB transposase has also recently been observed (25) (26).
Therefore, TnsC may serve as a “connector” or “matchmaker” between the transposase and the TnsD+attTn7 target complex (23) (27). This connection is not constitutive, but instead appears to be regulated by the ATP state of TnsC. Only the ATP-bound form of TnsC is competent to interact with target DNAs and activate the TnsA+B transposase; the ADP-bound form of TnsC has neither of these activities and cannot participate in Tn
7
transposition (24) (23). TnsC hydrolyzes ATP at a modest rate (25), and therefore can switch from an active to an inactive state. The modulation of the ATP state of TnsC may be a central mechanism for regulating Tn
7
transposition.
The possibility that TnsC regulates the connection betw
Alston & Bird LLP
Johns Hopkins University School of Medicine
Yucel Remy
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