Devices and related methods for nuclear magnetic resonance (NMR) analysis of particulate materials are provided including a detector chamber configured for insertion into an NMR spectrometer and configured to receive particulate materials in a fluid. A circulation chamber is attached to and in fluid communication with a first end of the detector chamber. A transition region is between the detector chamber and the circulation chamber, and a fluid supply interface is at a second end of the detector chamber that is configured to attach to a fluid source. The detector chamber, the circulation chamber and the transition region are sized and configured such that, when fluid flows from the fluid supply interface into the second end of the detector region, a circulating current is formed in the transition region and/or the circulation chamber such that the particulate matter is contained in the circulation chamber by the circulating current.

Nuclear magnetic resonance (NMR) spectroscopy is a technique that utilizes the magnetic properties of nuclei to obtain information about the physical, chemical, electronic and structural properties of molecules. When placed in a magnetic field, certain NMR active (such as 1H and 13C) are aligned with the field. This alignment can be perturbed using an alternating magnetic field that is generally orthogonal to the main field. The alternating magnetic field is typically administered by a radio frequency (RF) coil that is proximate the sample. After the alternating magnetic field is terminated, a signal can be collected by the RF coil due to a current induced in the RF coil as the nuclei in the sample "relax" or realign with the primary magnetic field.
NMR spectroscopy has been used to study living cells, which can be immobilized inside a semi-permeable encapsulate and suspended in a fluid. One potential difficulty in NMR spectroscopy is to maintain cells in a high density in the suspension for the amount of time desired to obtain a spectrum while maintaining the viability of the cells. The cells typically have relatively low NMR sensitivity, and consequently the cells may be placed in an NMR sample chamber with high concentrations in an effort to achieve higher NMR signals. However, it may be difficult to sustain the viability of living cells in the sufficiently high densities that may be desirable for high NMR signal strength.
NMR sample chambers have been developed in an effort to maintain a high density of cells to reduce signal acquisition times while also attempting to maintain cell viability. For example, NMR sample tubes have been designed to provide unidirectional flow of fluid, such as a perfusion medium including oxygen and/or glucose for maintaining cell viability. The fluid exits the sample tube through a filter, which is sized to generally prevent the cells from also exiting the sample tube. Although the fluid flow operates to compact the encapsulated cells in the NMR tube and supply oxygen and/or glucose, these systems may have problems associated with filter clogging. In addition, the encapsulated cells may be in such close proximity to one another that cell viability may be difficult to achieve.

Device for particulate NMR samples in a fluid and related methods

The University of North Carolina at Chapel Hill


This invention relates to a method for producing a zinc finger nucleic acid binding protein comprising preparing a zinc finger protein according design rules, varying the protein at one or more positions, and selecting variants which bind to a target nucleic acid sequence by polysome display.

The present application relates to a method for screening zinc finger polypeptides for a desired binding ability. In particular, the invention relates to a polysome display technique which permits the isolation of binding polypeptides without resorting to phage display techniques.
Protein-nucleic acid recognition is a commonplace phenomenon which is central to a large number of biomolecular control mechanisms which regulate the functioning of eukaryotic and procaryotic cells. For instance, protein-DNA interactions form the basis of the regulation of gene expression and are thus one of the subjects most widely studied by molecular biologists.
A wealth of biochemical and structural information explains the details of protein-DNA recognition in numerous instances, to the extent that general principles of recognition have emerged. Many DNA-binding proteins contain independently folded domains for the recognition of DNA, and these domains in turn belong to a large number of structural families, such as the leucine zipper, the xe2x80x9chelix-turn-helixxe2x80x9d and zinc finger families.
Most sequence-specific DNA-binding proteins bind to the DNA double helix by inserting an xcex1-helix into the major groove (Pabo and Sauer 1992 Annu. Rev. Biochem. 61, 1053-1095; Harrison 1991 Nature (London) 353, 715-719; and Klug 1993 Gene 135, 83-92). Sequence specificity results from the geometrical and chemical complementarity between the amino acid side chains of the xcex1-helix and the accessible groups exposed on the edges of base-pairs. In addition to this direct reading of the DNA sequence. interactions with the DNA backbone stabilise the complex and are sensitive to the conformation of the nucleic acid, which in turn depends on the base sequence (Dickerson and Drew 1981 J. Mol. Biol. 149, 761-786. Crystal structures of protein-DNA complexes have shown that proteins can be idiosyncratic in their mode of DNA recognition, at least partly because they may use alternative geometries to present their sensory xcex1-helices to DNA, allowing a variety of different base contacts to be made by a single amino acid and vice versa (Matthews 1988 Nature (London) 335, 294-295).
Protein engineering experiments have shown that it is possible to alter rationally the DNA-binding characteristics of individual zinc fingers when one or more of the xcex1-helical positions is varied in a number of proteins (Nardelli et al., 1991 Nature (London) 349, 175-178; Nardelli et al. 1992 Nucleic Acids Res. 20, 4137-4144; and Desjarlais and Berg 1992a Proteins 13, 272). It has already been possible to propose some principles relating amino acids on the xcex1-helix to corresponding bases in the bound DNA sequence (Desjarlais and Berg 1992b Proc. Natl. Acad. Sci. USA 89, 7345-7349). However in this approach the altered positions on the xcex1-helix are prejudged, making it possible to overlook the role of positions which are not currently considered important; and secondly, owing to the importance of context, concomitant alterations are sometimes required to affect specificity (Desjarlais and Berg 1992b), so that a significant correlation between an amino acid and base may be misconstrued.
More sophisticated principles describing the relationship between the sequence of the zinc finger and the nucleic acid target have been described, for example in WO 96/06166 (Medical Research Council).
To investigate binding of mutant Zf proteins, Thiesen and Bach (1991 FEBS 283, 23-26) mutated Zf fingers and studied their binding to randomised oligonucleotides, using electrophoretic mobility shift assays. Subsequent use of phage display technology has permitted the expression of random libraries of Zf mutant proteins on the surface of bacteriophage. The three Zf domains of Zif268, with 4 positions within finger one randomised, have been displayed on the surface of filamentous phage by Rebar and Pabo (1994 Science 263, 671-673). The library was then subjected to rounds of affinity selection by binding to target DNA oligonucleotide sequences in order to obtain Zf proteins with new binding specificities. Randomised mutagenesis (at the same postions as those selected by Rebar and Pabo) of finger 1 of Zif 268 with phage display has also been used by Jamieson et al., (1994 Biochemistry 33, 5689-5695) to create novel binding specificity and affinity.
In summary, it is known that Zf protein motifs are widespread in DNA binding proteins and that binding is via three key amino acids, each one contacting a single base pair in the target DNA sequence. Motifs are modular and may be linked together to form a set of fingers which recognise a contiguous DNA sequence (e.g. a three fingered protein will recognise a 9 mer etc). The key residues involved in DNA binding have been identified through sequence data and from structural information. Directed and random mutagenesis has confirmed the role of these amino acids in determining specificity and affinity. Phage display has been used to screen for new binding specificities of random mutants of fingers. Therefore, the combination of a set of rules with a selection process appears to provide the most promising avenue for the development of zinc finger proteins.
According to a first aspect of, the present invention, there is provided a method for producing a zinc finger nucleic acid binding protein comprising preparing a zinc finger protein according design rules, varying the protein at one or more positions, and selecting variants which bind to a target nucleic acid sequence by polysome display.
According to a second aspect of the present invention, there is provided a method for producing a zinc finger nucleic acid binding protein comprising an at least partially varied sequence and selecting variants thereof which bind to a target DNA strand, comprising the steps of:
(i) preparing a nucleic acid binding protein of the Cys2-His2 zinc finger class capable of binding to a nucleic acid triplet in a target nucleic acid sequence, wherein binding to each base of the triplet by an cc-helical zinc finger nucleic acid binding motif in the protein is determined as follows:
a) if the 5xe2x80x2 base in the triplet is G, then position +6 in the xcex1-helix is Arg; or position +6 is Ser or Thr and position ++2 is Asp;
b) if the 5xe2x80x2 base in the triplet is A, then position +6 in the xcex1-helix is Gln and ++2 is not Asp;
c) if the 5xe2x80x2 base in the triplet is T, then position +6 in the xcex1-helix is Ser or Thr and position ++2 is Asp;
d) if the 5xe2x80x2 base in the triplet is C, then position +6 in the xcex1-helix may be any amino acid, provided that position ++2 in the xcex1-helix is not Asp;
e) if the central base in the triplet is G, then position +3 in the xcex1-helix is His,
f) if the central base in the triplet is A, then position +3 in the xcex1-helix is Asn;
g) if the central base in the triplet is T, then position +3 in the xcex1-helix is Ala. Ser, or Val; provided that if it is Ala, then one of the residues at xe2x88x921 or +6 is a small residue;
h) if the central base in the triplet is C, then position +3 in the xcex1-helix is Ser, Asp, Glu, Leu, Thr or Val;
i) if the 3xe2x80x2 base in the triplet is G, then position xe2x88x921 in the xcex1-helix is Arg;
j) if the 3xe2x80x2 base in the triplet is A, then position xe2x88x921 in the xcex1-helix is Gln;
k) if the 3xe2x80x2 base in the triplet is T, then position xe2x88x921 in the xcex1-helix is Asn or Gln;
l) if the 3xe2x80x2 base in the triplet is C, then position xe2x88x921 in the xcex1-helix is Asp;
(ii) varying the resultant polypeptide at at least one position; and
(iii) selecting the variants which bind to a target nucleic acid sequence by polysome display.
SEQ ID NOs: are assigned as follows:
SEQ ID NO:1
Nucleotide sequence encoding the zinc finger protein;
SEQ ID NO:2
Amino acid sequence of the zinc finger protein;
SEQ ID NO:3
Sequence of the zinc finger framework as described page 6 lines 21-24 of the specification
XXCXXXXXCX XXXXXXXXXX XXXHXXXXXX H
SEQ ID NO:4
Alternate Sequence of the zinc finger framework as described page 6 lines 21-24 of the specification
XXCXXXXXCX XXXXXXXXXX XXXHXXXXXX C
SEQ ID NO:5
Sequence of the zinc finger nucleic acid binding motifs as described page 7 lines 1-7 of the specification
XCXXXXCXXX FXXXXXLXXH XXXH
SEQ ID NO:6
TGEK
SEQ ID NO:7
TGEKP
SEQ ID NO:8
PYKCPECGKS FSQKSDLVKH QRTHTG
SEQ ID NO:9
PYKCSECGKA FSQKSNLTRH QRIHTGEKP
SEQ ID NO:10
MAEEKP
SEQ ID NO: 11
AAVP
SEQ ID NO: 12
GGGGSGGGGS GGGGSGGGGS AAVP
SEQ ID NO: 13
taatacgact aactataggg aga
SEQ ID NO: 14
aaggag
SEQ ID NO: 15
atggttaaaa cagataaa
SEQ ID No: 16
MVKTDK;
All of the nucleic acid-binding residue positions of zinc fingers, as referred to herein, are numbered from the first residue in the xcex1-helix of the finger, ranging from +1 to +9. xe2x80x9cxe2x88x921xe2x80x9d refers to the residue in the framework structure immediately preceding the xcex1-helix in a Cys2-His2 zinc finger polypeptide. Residues referred to as xe2x80x9c++xe2x80x9d are residues present in an adjacent (C-terminal) finger. Where there is no C-terminal adjacent finger. xe2x80x9c++xe2x80x9d interactions do not operate.
Cys2-His2 zinc finger binding, proteins, as is well known in the art, bind to target nucleic acid sequences via xcex1-helical zinc metal atom coordinated binding, motifs known as zinc fingers. Each zinc finger in a zinc finger nucleic acid binding protein is responsible for determining binding to a nucleic acid triplet in a nucleic acid binding, sequence. Preferably, there are 2or more zinc fingers, for example 2, 3, 4, 5 or 6 zinc fingers, in each binding protein. Advantageously, there are 3zinc fingers in each zinc finger binding protein.
The method of the present invention allows the production of what are essentially artificial nucleic acid binding proteins. In these proteins, artificial analogues of amino acids may be used, to impart the proteins with desired properties or for other reasons. Thus, the term xe2x80x9camino acidxe2x80x9d, particularly in the context where xe2x80x9cany amino acidxe2x80x9d is referred to, means any sort of natural or artificial amino acid or amino acid analogue that may be employed in protein construction according to methods known in the art. Moreover, any specific amino acid referred to herein may be replaced by a functional analogue thereof, particularly an artificial functional analogue. The nomenclature used herein therefore specifically comprises within its scope functional analogues of the defined amino acids.
The xcex1-helix of a zinc finger binding protein aligns antiparallel to the nucleic acid strand, such that the primary nucleic acid sequence is arranged 3xe2x80x2 to 5xe2x80x2 in order to correspond with the N terminal to C-terminal sequence of the zinc finger. Since nucleic acid sequences are conventionally written 5xe2x80x2 to 3xe2x80x2, and amino acid sequences N-terminus to C-terminus, the result is that when a nucleic acid sequence and a zinc finger protein are aligned according to convention, the primary interaction of the zinc finger is with the xe2x88x92strand of the nucleic acid, since it is this strand which is aligned 3xe2x80x2 to 5xe2x80x2. These conventions are followed in the nomenclature used herein. It should be noted, however, that in nature certain fingers, such as finger 4 of the protein GLI, bind to the +strand of nucleic acid: see Suzuki et al., (1994) NAR 22:3397-3405 and Pavletich and Pabo, (1993) Science 261:1701-1707. The incorporation of such fingers into nucleic acid binding) molecules according to the invention is envisaged.
A zinc finger binding motif is a structure well known to those in the art and defined in, for example, Miller et al., (1985) EMBO J. 4:1609-1614; Berg (1988) PNAS (USA) 85:99-102; Lee et al., (1989) Science 245:635-637; see International patent applications WO 96/06166 and WO 96/32475, corresponding to U.S. Ser. No. 08/422,107, incorporated herein by reference.
As used herein, xe2x80x9cnucleic acidxe2x80x9d refers to both RNA and DNA, constructed from natural nucleic acid bases or synthetic bases, or mixtures thereof. Preferably, however, the binding proteins of the invention are DNA binding proteins.
The structure of the framework of Cys2-His2 zinc fingers is known in the art. The present invention encompasses both those structures which have been observed in nature, including consensus structures derived therefrom, and artificial structures which have non-natural residue numbers and spacing but which retain the functionality of a zinc finger.
In general, a preferred zinc finger framework has the structure:
X0-2C X1-5C X9-14H X3-6K/Cxe2x80x83xe2x80x83(A)
where X is any amino acid, and the numbers in subscript indicate the possible numbers of residues represented by X.
In a preferred aspect of the present invention, zinc finger nucleic acid binding motifs may be represented as motifs having the following primary structure:
wherein X (including Xa, Xb and Xc) is any amino acid. X2-4 and X2-3 refer to the presence of 2 or 4, or 2 or 3, amino acids, respectively. The Cys and His residues, which together co-ordinate the zinc metal atom, are marked in bold text and are usually invariant, as is the Leu residue at position +4 in the xcex1-helix.
Modifications to this representation may occur or be effected without necessarily abolishing zinc finger function, by insertion, mutation or deletion of amino acids. For example it is known that the second His residue may be replaced by Cys (Krizek et al., (1991) J. Am. Chem. Soc. 113:4518-4523) and chat Leu at +4 can in some circumstances be replaced with Arg. The Phe residue before Xc may be replaced by any aromatic other than Trp. Moreover, experiments have shown that departure from the preferred structure and residue assignments for the zinc finger are tolerated and may even prove beneficial in binding to certain nucleic acid sequences. Even taking this into account, however, the general structure involving an xcex1-helix coordinated by a zinc atom which contacts four Cys or His residues, does not alter. As used herein, structures (A) and (B) above are taken as an exemplary structure representing all zinc finger structures of the Cys2-His2 type.
Preferably, Xa is F/xcex3-X or P-F/xcex3-X. In this context, X is any amino acid. Preferably, in this context X is E, K, T or S. Less preferred but also envisaged are Q, V, A and P. The remaining amino acids remain possible.
Preferably, X2-4 consists of two amino acids rather than four. The first of these amino acids may be any amino acid, but S, E, K, T, P and R are preferred. Advantageously, it is P or R. The second of these amino acids is preferably E, although any amino acid may be used.
Preferably, Xb is T or I.
Preferably, Xc is S or T.
Preferably, X2-3 is G-K-A, G-K-C, G-K-S or G-K-G. However, departures from the preferred residues are possible, for example in the form of M-R-N or M-R.
Preferably, the linker is T-G-E-K or T-G-E-K-P.
As set out above, the major binding interactions occur with amino acids xe2x88x921, +3 and +6. Amino acids +4 and +7 are largely invariant. The remaining amino acids may be essentially any amino acids. Preferably, position +9 is occupied by Arg or Lys. Advantageously, positions +1, +5 and +8 are not hydrophobic amino acids, that is to say are not Phe, Trp or Tyr. Preferably, position +2 is any amino acid, and preferably serine, save where its nature is dictated by its role as a ++2 amino acid for an N-terminal zinc finger in the same nucleic acid binding molecule.
In a most preferred aspect, therefore, bringing together the above, the invention allows the definition of every residue in a zinc finger nucleic acid binding motif which will bind specifically to a given nucleic acid triplet.
The code provided by the present invention is not entirely rigid; certain choices are provided. For example, positions +1, +5 and +8 may have any amino acid allocation, whilst other positions may have certain options: for example, the present rules provide that, for binding to a central T residue, any one of Ala, Ser or Val may be used at +3. In its broadest sense, therefore, the present invention provides a very large number of proteins which are capable of binding to every defined target nucleic acid triplet. As set forth below, these protein may be selected for binding ability using polysome display techniques.
Preferably, however, the number of possibilities may be significantly reduced before selection. For example, the non-critical residues +1, +5 and +8 may be occupied by the residues Lys, Thr and Gln respectively as a default option. In the case of the other choices, for example, the first-given option may be employed as a default. Thus, the code according to the present invention allows the design of a single, defined polypeptide (a xe2x80x9cdefaultxe2x80x9d polypeptide) which will bind to its target triplet.
In a further aspect of the present invention, there is provided a method for preparing a nucleic acid binding protein of the Cys2-His2 zinc finger class capable of binding to a target nucleic acid sequence, comprising the steps of:
a) selecting a model zinc finger domain from the group consisting of naturally occurring zinc fingers and consensus zinc fingers;
b) varying at least one of positions xe2x88x921, +3, +6 (and ++2) the finger as required according to the rules set forth above; and
c) selecting the variants which bind to the target nucleic acid by polysome display.
In general, naturally occurring zinc fingers may be selected from those fingers for which the nucleic acid binding specificity is known. For example, these may be the fingers for which a crystal structure has been resolved: namely Zif 268 (Elrod-Erickson et al., (1996) Structure 4:1171-1180), GLI (Pavletich and Pabo, (1993) Science 261:1701-1707), Tramtrack (Fairall et al., (1993) Nature 366:483-487) and YY1 (Houbaviy et al., (1996) PNAS (USA) 93:13577-13582).
The naturally occurring zinc finger 2 in Zif 268 makes an excellent starting point from which to engineer a zinc finger and is preferred.
Consensus zinc finger structures may be prepared by comparing the sequences of known zinc fingers, irrespective of whether their binding domain is known. Preferably, the consensus sequence is selected from the group consisting of the consensus structure P Y K C P E C G K S F S Q K S D L V K H Q R T H T G (SEQ ID NO:8), and the consensus structure P Y K C S E C G K A F S Q K S N L T R H Q R I H T G E K P (SEQ ID NO:9).
The consensuses are derived from the consensus provided by Krizek et at., (1991) J. Am. Chem. Soc. 113:4518-4523 and from Jacobs, (1993) PhD thesis. University of Cambridge, UK. In both cases, the linker sequences described above for joining two zinc finger motifs together, namely TGEK or TGEKP can be formed on the ends of the consensus. Thus, a P may be removed where necessary, or, in the case of the consensus terminating T G, E K (P) can be added.
When the nucleic acid specificity of the model finger selected is known, the mutation of the finger in order to modify its specificity to bind to the target nucleic acid may be directed to residues known to affect binding to bases at which the natural and desired targets differ. Otherwise, mutation of the model fingers should be concentrated upon residues xe2x88x921, +3, +6 and ++2 as provided for in the foregoing rules.
In order to produce a binding protein having improved binding, moreover, the rules provided by the present invention may be supplemented by physical or virtual modelling of the protein/nucleic acid interface in order to assist in residue selection.
Zinc finger binding motifs designed according to the invention may be combined into nucleic acid binding proteins having a multiplicity of zinc fingers. Preferably, the proteins have at least two zinc fingers. In nature, zinc finger binding proteins commonly have at least three zinc fingers, although two-zinc finger proteins such as Tramtrack are known. The presence of at least three zinc fingers is preferred. Binding proteins may be constructed by joining the required fingers end to end, N-terminus to C-terminus. Preferably, this is effected by joining together the relevant nucleic acid coding sequences encoding the zinc fingers to produce a composite coding sequence encoding the entire binding protein. The invention therefore provides a method for producing a nucleic acid binding protein as defined above, wherein the nucleic acid binding protein is constructed by recombinant DNA technology, the method comprising the steps of:
a) preparing a nucleic acid coding sequence encoding two or more zinc finger binding motifs as defined above, placed N-terminus to C-terminus;
b) inserting the nucleic acid sequence into a suitable expression vector; and
c) expressing the nucleic acid sequence in a host organism in order to obtain the nucleic acid binding protein.
A xe2x80x9cleaderxe2x80x9d peptide may be added to the N-terminal finger. Preferably, the leader is MAEEKP (SEQ ID NO:10).
The nucleic acid encoding the nucleic acid binding protein according to the invention can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous nucleic acid into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the person of ordinary skill in the art. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for nucleic acid expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses.
The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2xcexc plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.
Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class or organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even rough it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome. However, the recovery of genomic DNA encoding the nucleic acid binding protein is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise nucleic acid binding protein DNA. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.
Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins. e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.
Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript(copyright) vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid binding protein nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin. The mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive. In the case of a DHFR or glutamine synthase (GS) marker, selection pressure can be imposed by culturing the `transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes the nucleic acid binding protein. Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesised from thus amplified DNA.
Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to nucleic acid binding protein encoding nucleic acid. Such a promoter may be inducible or constitutive. The promoters are operably linked to DNA encoding the nucleic acid binding protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native nucleic acid binding protein promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of nucleic acid binding protein encoding DNA.
Promoters suitable for use with prokaryotic hosts include, for example, the xcex2-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding nucleic acid binding protein, using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding the nucleic acid binding protein.
Preferred expression vectors are bacterial expression vectors which comprise a promoter of a bacteriophage such as phagex or T7 which is capable of functioning in the bacteria. In one of the most widely used expression systems, the nucleic acid encoding the fusion protein may be transcribed from the vector by T7 RNA polymerase (Studier et al, Methods in Enzymol. 185; 60-89, 1990). In the E. coli BL21(DE3) host strain, used in conjunction with pET vectors, the T7 RNA polymerase is produced from the xcex-lysogen DE3 in the host bacterium, and its expression is under the control of the IPTG inducible lac UV5 promoter. This system has been employed successfully for over-production of many proteins. Alternatively the polymerase gene may be introduced on a lambda phage by infection with an int-phage such as the CE6 phage which is commercially available (Novagen, Madison, USA). other vectors include vectors containing the lambda PL promoter such as PLEX (Invitrogen, NL), vectors containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99 (Pharmacia Biotech, SE) or vectors containing the tac promoter such as pKK223-3 (Pharmacia Biotech) or PMAL (New England Biolabs, Mass., USA).
Moreover, the nucleic acid binding protein gene according to the invention preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body. The peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.
Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or xcex1-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglyceraye mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase or glucokinase genes, or a promoter from the TATA binding, protein (TBP) gene can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05-GAP hybrid promoter). A suitable constitutive PH05 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (xe2x88x92173) promoter element starting at nucleotide xe2x88x92173 and ending at nucleotide xe2x88x929 of the PH05 gene.
Nucleic acid binding protein gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with nucleic acid binding protein sequence, provided such promoters are compatible with the host cell systems.
Transcription of a DNA encoding nucleic acid binding protein by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5xe2x80x2 or 3xe2x80x2 to nucleic acid binding protein DNA, but is preferably located at a site 5xe2x80x2 from the promoter.
Advantageously, a eukaryotic expression vector encoding a nucleic acid binding protein according to the invention may comprise a locus control region (LCR). LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the nucleic acid binding protein gene is to be expressed in the context of a permanently-transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, or in transgenic animals.
Eukaryotic vectors may also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5xe2x80x2 and 3xe2x80x2 untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding nucleic acid binding protein.
An expression vector includes any vector capable of expressing nucleic acid binding protein nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phase, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. For example, DNAs encoding nucleic acid binding protein may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).
Particularly useful for practising the present invention are expression vectors that provide for the transient expression of DNA encoding nucleic acid binding protein in mammalian cells. Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of nucleic acid binding protein. For the purposes or the present invention, transient expression systems are useful e.g. for identifying nucleic acid binding protein mutants, to identify potential phosphorylation sites, or to characterise functional domains of the protein.
Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing nucleic acid binding protein expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe which may be based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
In accordance with another embodiment of the present invention, there are provided cells containing the above-described nucleic acids. Such host cells such as prokaryote, yeast and higher eukaryote cells may be used for replicating DNA and producing the nucleic acid binding protein. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5a and HB101, or Bacilli. Further hosts suitable for the nucleic acid binding protein encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells including human cells, or nucleated cells from other multicellular organisms. In recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells. The host cells referred to in this disclosure comprise cells in in vitro culture as well as cells that are within a host animal.
DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art. Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.
To produce such stably or transiently transfected cells, the cells should be transfected with a sufficient amount of the nucleic acid binding protein-encoding nucleic acid to form the nucleic acid binding protein. The precise amounts of DNA encoding the nucleic acid binding protein may be empirically determined and optimised for a particular cell and assay.
Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.
Incorporation of cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby the nucleic acid binding protein encoded by the DNA is expressed. The composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.
The zinc finger polypeptides according to the present invention are varied at at least one position. Preferably, the positions selected for variation is one of the positions identified above as being important in determining the binding specificity of the zinc finger polypeptide of the invention.
By xe2x80x9cvaryxe2x80x9d (including grammatical modifications) it is intended to denote that a particular amino acid in the molecule is replaced with an amino acid selected from a varied group, to produce a repertoire of homologous zinc finger polypeptides which differ at the particular amino acid position. The variant amino acids may be selected from a small group of two or more amino acids, from a larger group or may be completely randomly selected from all 20 naturally occurring amino acids. In a preferred embodiment, amino acid analogues and artificial amino acids may be employed.
Variation of the zinc finger binding motifs produced according to the invention is preferably directed to those residues where the code provided herein gives a choice of residues. For example, therefore, positions +1, +5 and +8 are advantageously randomised, whilst preferably avoiding hydrophobic amino acids: positions involved in binding to the nucleic acid, notably xe2x88x921, +2, +3 and +6, may be randomised also, preferably within the choices provided by the rules of the present invention.
Preferably, therefore, the xe2x80x9cdefaultxe2x80x9d protein produced according to the rules provided by the invention can be improved by subjecting the protein to one or more rounds of variation and selection within the specified parameters.
Mutagenesis of zinc finger polypeptides may be achieved by any suitable means. Preferably, the mutagenesis is performed at the nucleic acid level, for example by synthesising novel genes encoding mutant proteins and expressing these to obtain a variety of different proteins. Alternatively, existing genes can be themselves mutated, such by site-directed or random mutagenesis, in order to obtain the desired mutant genes.
Mutations may be performed by any method known to those of skill in the art. Preferred, however, is site-directed mutagenesis of a nucleic acid sequence encoding the protein of interest. A number of methods for site-directed mutagenesis are known in the art, from methods employing single-stranded phage such as M13 to PCR-based techniques (see xe2x80x9cPCR Protocols: A guide to methods and applicationsxe2x80x9d, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White (eds.), Academic Press, New York, 1990). Preferably, the commercially available Altered Site II Mutagenesis System (Promega) may be employed, according to the directions given by the manufacturer.
Selection of varied polypeptides according to the invention is carried out by polysome display (see Table 1). This technique relies on coupled transcription and translation of the coding sequences encoding the zinc finger polypeptides of the invention. This is achieved by preventing dissociation of the mRNA template and the polypeptide chain from the ribosome, such that the whole entity can be isolated as a polysome. Polysomes are then selected by binding the polypeptide to target nucleic acid, and mRNA eluted from those polysomes which display the desired binding characteristics.
Polysome display may be performed according to the methods known in the art, as described below. For example, reference is made to WO95/11922, the methods of which are incorporated herein by reference. The methods of WO95/11922 may be adapted to the present invention, as follows:
Improved Methods For Screening Nascent Peptide Libraries
A polysome library displaying nascent zinc finger polypeptides can be generated by a variety of methods. Generally, an in vitro translation system is employed to generate polysomes from a population of added mRNA species. Often, the in vitro translation system used is a conventional eukaryotic translation system (e.g., rabbit reticulocyte lysate, wheat germ extract). However, an E. coli S30 system (Promega, Madison, Wis.) can be used to generate the polysome library from a population of added mRNA species or by coupled transcription/translation (infra). Suitable E. Coli S30 systems may be produced by conventional methods or may be obtained from commercial sources (Promega, Madison, Wis.). The E. coli S30 translation system is generally more efficient at producing polysomes suitable for affinity screening of displayed nascent peptides, and the like. Moreover, a prokaryotic translation system, such as the E. coli S30 system, has the further advantage that a variety of drugs which block prokaryotic translation (e.g., inhibitors of ribosome function), such as rifampicin or chloramphenicol, can be added at a suitable concentration and/or timepoint to stall translation and produce a population of stalled polysomes, suitable for affinity screening against a target nucleic acid sequence.
In general, the method comprises the steps of: (1) introducing a population of mRNA species into a prokaryotic in vitro translation system (e.g., E. coli S30) under conditions suitable for translation to form a pool of polysomes displaying nascent zinc finger polypeptides, so-called polysome forming conditions; (2) contacting the polysomes with a target nucleic acid under suitable binding conditions (i.e., for specific binding to the target nucleic acid and for preserving intact polysome structure); (3) selecting polysomes which are specifically bound to the nucleic acid (e.g., by removing unbound polysomes by washing with a solution); and (4) determining the polynucleotide sequences of the selected polysomes (e.g., by synthesizing cDNA or reverse transcriptase PCR amplification product, and sequencing said cDNA or amplification product). Often, the nucleic acid used for screening is immobilized, such as by being bound to a solid support.
In a variation of the method, the population of mRNA molecules is introduced into the in vitro translation system by de novo synthesis of the mRNA from a DNA template. In this improvement, a population of DNA templates capable of being transcribed in vitro (e.g., having an operably linked T7 or SP6 or other suitable promoter) are introduced into a coupled in vitro transcription/translation system (e.g., an E. coli S30 system) under conditions suitable for in vitro transcription and translation of the transcribed product. Generally, using a coupled in vitro transcription/translation system is highly efficient for producing polysomes displaying nascent zinc finger polypeptides suitable for affinity screening. Of course, and as noted above. uncoupled systems may also be used, i.e., by adding mRNA to an in vitro translation extract.
A further improvement to the general methods of screening nascent zinc finger polypeptide-displaying polysomes comprises the additional step of adding a preblocking agent (e.g., nonfat milk, serum albumin, tRNA, and/or gelatin) prior to or concomitant with the step of contacting the nascent peptide displaying polysomes with an immobilized nucleic acid. The additional step of adding a preblocking agent reduces the amount of polysomes which bind nonspecifically to the target nucleic acid and/or to the immobilization surface (e.g., microtitre well), thereby enhancing the specificity of selection for polysomes displaying peptides that specifically bind to the nucleic acid. Although the preblocking agent can be selected from a broad group of suitable compositions, the group of preblocking agents comprising: nonfat milk/nonfat milk solids, casein, bovine serum albumin, transfer RNA, and gelatin are preferred, with nonfat milk being especially preferable. Other suitable preblocking agents can be used.
Preblocking agents that do not substantially interfere with specific binding (i.e., non-interfering) are suitable.
A further improvement to the general methods of screening nascent peptide-displaying polysomes comprises the additional step of isolating polysomes from an in vitro translation reaction (or a coupled in vitro transcription/translation reaction) prior to the step of contacting the nascent peptide-displaying polysomes with nucleic acid. Generally, the polysomes are isolated from a translation reaction by high speed centrifugation to pellet the polysomes, so that the polysome pellet is recovered and the supernatant containing contaminants is discarded. The polysome pellet is resolubilised in a suitable solution to retain intact polysomes. The resolubilised polysomes may be recentrifuged at lower speed (i.e., which does not pellet polysomes) so that the insoluble contaminants pellet and are discarded and the supernatant containing soluble polysomes is recovered, and the supernatant used for affinity screening. Alternatively, the resolubilised polysomes may be used for affinity screening directly (i.e., without low speed centrifugation). Furthermore, the order of centrifugation may be reversed, so that low speed centrifugation is performed prior to high speed centrifugation; the low speed centrifugation supernatant is then centrifuged at high speed and the pelleted polysomes are resolubilised and used for affinity screening. Multiple rounds of high speed and/or low speed centrifugation may be used to increasingly purify the polysomes prior to contacting the polysomes with the immobilized nucleic acid.
Another improvement to the general methods of affinity screening of nascent peptide-displaying polysomes comprises adding a non-ionic detergent to the binding and/or wash buffers. Non-ionic detergent (e.g., Triton X-100, NP-40, Tween, etc.) is added in the binding buffer (i.e., the aqueous solution present during the step of contacting the polysomes with the immobilized nucleic acid) and/or the wash buffer (i.e., the aqueous solution used to wash the bound polysomes (i.e., bound to the immobilized nucleic acid). Generally, the non-ionic detergent is added to a final concentration of about between 0.01 to 0.5% (v/v), with 0.1% being typical.
Another improvement to the general methods of affinity screening of nascent peptide libraries is generating the DNA template library (from which the mRNA population is transcribed) in vitro without cloning the library in host cells. Cloning libraries in host cells frequently diminishes the diversity of the library and may skew the distribution of the relative abundance of library members. In vitro library construction generally comprises ligating each member of a population of polynucleotides encoding library members to a polynucleotide sequence comprising a promoter suitable for in vitro transcription (e.g., T7 promoter and leader). The resultant population of DNA templates may optionally be purified by gel electrophoresis. The population of DNA templates is then transcribed and translated in vitro, such as by a coupled transcription/translation system (e.g., E. coli S30).
A further improvement to the general methods of affinity screening comprises the added step of combining affinity screening of a nascent peptide-displaying polysome, library with screening of a bacteriophage peptide display library (or other, i.e., peptides on plasmids, expression as secreted soluble antibody in host cells, in vitro expression). In this improvement, polysomes are isolated by affinity screening of a nascent peptide-display library. The isolated polysomes are dissociated, and cDNA is made from the mRNA sequences that encoded nascent peptides that specifically bound to the target nucleic acid). The cDNA sequences encoding the nascent peptide binding regions (i.e., the portions which formed binding contacts to the nucleic acid(s); variable segment sequences) are cloned into a suitable bacteriophage peptide display vector (e.g., pAFF6 or other suitable vector). The resultant bacteriophage vectors are introduced into a host cell to produce a library of bacteriophage particles. Each of the phage clones express on their virion surface the polysome derived peptide sequences as fusions to a coat protein (e.g., as an N-terminal fusion to the PIII coat protein). By incorporating the in vitro-enriched peptide sequences from the polysome screening into a bacteriophage display system, it is possible to continue affinity selection for additional rounds. It is also advantageous, because the resultant bacteriophage display libraries can be screened and tested under conditions that might not have been appropriate for the intact polysomes.
Another improvement to the methods of affinity screening is the control of display valency (i.e., the average number of functional zinc finger polypeptides displayed per polysome, and the capacity to vary display valency in different rounds of affinity screening. Typically, a high display valency permits many binding contacts between the polysome and nucleic acid, thus affording stable binding for polysomes which encode zinc finger polypeptide species which have relatively weak binding. Hence, a high display valency system allows screening to identify a broader diversity range of zinc finger polypeptides, since even lower affinity zinc finger polypeptides can be selected. Frequently, such low-to-medium affinity zinc finger polypeptides can be superior candidates for generating very high affinity zinc finger polypeptides, by selecting high affinity zinc finger polypeptides from a pooi of mutagenised low-to-medium affinity zinc finger clones. Thus, affinity sharpening by mutagenesis and subsequent rounds of affinity selection can be used in conjunction with a broader pool of initially selected zinc finger polypeptide sequences if a high display valency method is used. Alternate rounds of high display valency screening and low display valency screening can be performed, in any order, starting from either a high or low valency system, for as many affinity screening rounds as desired, with intervening variation and sequence diversity broadening, if desired. Alternate rounds of affinity screening, wherein a first round consists of screening a zinc finger polypeptide library expressed in a high valency system, selecting zinc finger polypeptide clones which bind the target nucleic acid, optionally conducting a mutagenesis step to expand the sequence variability of the selected zinc finger polypeptides, expressing the selected zinc finger polypeptide clones in a lower valency display system, and selecting clones which bind the target nucleic acid, can be performed, including various permutations and combinations of multiple screening cycles, wherein each cycle can be of a similar or different display valency. This improvement affords an overall screening program that employs systems which are compatible with switchable valency (i.e., one screening cycle can have a different display valency than the other(s), and can alternate in order).
Display valency can be controlled by a variety of methods, including but not limited to: controlling the average number of nascent peptides per polysome in a polysome-display system. This can be controlled by any suitable method, including: (1) altering the length of the encoding mRNA sequence to reduce or increase the frequency of translation termination (a longer mRNA will typically display more nascent peptides per polysome than a shorter mRNA encoding sequence), (2) incorporating stalling (i.e., infrequently used) codons in the encoding mRNA, typically distal (downstream of) of the zinc finger polypeptide-encoding portion(s), (3) incorporating RNA secondary structure-forming sequences (e.g., hairpin, cruciform, etc.) distal to the zinc finger polypeptide-encoding portion and proximal to (upstream to) the translation termination site, if any, and/or (4) including an antisense polynucleotide (e.g., DNA, RNA, polyamide nucleic acid) that hybridizes to the mRNA distal to the zinc finger polypeptide-encoding portion and proximal to (and possibly spanning the translation termination site, if any. The length of the mRNA may be increased to increase display valency, such as by adding additional reading frame sequences downstream of the zinc finger polypeptide-encoding sequences; such additional reading, frame sequences can, for example, encode the sequence (-AAVP-)n, where n is typically at least 1, frequently at least 5 to 10, often at least 15 to 25, and may be at least 50-100, up to approximately 150 to 500 or more, although infrequently a longer stall sequence can be used. Stalling codons (i.e., codons which are slowly translated relative to other codons in a given translation system) can be determined empirically for any translation system, such as by measuring translation efficiency of mRNA templates which differ only in the presence or relative abundance of particular codons. For example, a set of clones can be evaluated in the chosen translation system; each species or the set has a stalling polypeptide sequence of 25 amino acids, but each stalling polypeptide sequence consists of a repeating series of one codon, such that all translatable codons are represented in the set. When translated under equivalent conditions, the zinc finger polypeptide species which produce polysomes having the highest valency (e.g., as determined by sedimentation rate, buoyancy, electron microscopic examination, and other diagnostic methods) thereby identify stalling codons as the codon(s) in the stalling polypeptide sequence.
In one embodiment, a stalling polypeptide sequence is distal (3xe2x80x2 to) the zinc finger polypeptide-encoding sequence, and comprises -(Gly-Gly-Gly-Gly-Ser)-A-A-V-P-, or repeats thereof.
Alternatively, or in combination with the noted variations, the valency of the target nucleic acid may be varied to control the average binding affinity of selected library members. The target nucleic acid can be bound co a surface or substrate at varying densities, such as by including a competitor tarhet nucleic acid, by dilution, or by other method known to those in the art. A high density (valency) of target nucleic acid can be used to enrich for library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity library members.
Each of the improvements to the methods of affinity screening may be combined with other compatible improvements. For example, an in vitro transcription/translation system can be used in conjunction with a library of DNA templates synthesized in vitro (i.e. without cloning in a host cell). The resultant polysomes can be purified by one or more rounds of high-speed and/or low-speed centrifugation. The purified polysomes can be contacted with an immobilized nucleic acid that is preblocked (e.g., with nonfat milk), and a non-ionic detergent may also be present to further reduce nonspecific binding. The selected polysomes may then be used as templates for synthesizing cDNA which is then cloned into a bacteriophage display vector, such that the variable segments of the nascent peptides are now displayed on bacteriophage.
Amplification, Affinity Enrichment, And Screening
A basic method is described for synthesizing a nascent peptide-polysome library in vitro, screening and enrichment of the library for species having desired specific binding properties, and recovery of the nucleotide sequences that encode those peptides of sufficient binding affinity for target nucleic acid sufficient for selection by affinity selection.
The library consists of a population of nascent zinc finger polypeptide library members comprising nascent peptides. After selecting those nascent peptide library members that bind to the nucleic acid with high affinity, the selected complexes are disrupted and the mRNA is recovered and amplified to create DNA copies of the message. Typically each copy comprises an operably linked in vitro transcription promoter (e.g., T7 or SP6 promoter). The DNA copies are transcribed in vitro to produce mRNA, and the process is repeated to enrich for zinc finger polypeptides that bind with sufficient affinity.
The following general steps are frequently followed in the method: (1) generate a DNA template which is suitable for in vitro synthesis of mRNA, (2) synthesize mRNA in vitro by transcription of the DNA templates in a coupled transcription/translation system, (3) bind the nascent peptide to a preferably immobilised target nucleic acid, (4) recover and amplify nascent peptide library members which bind the target nucleic acid and produce DNA templates from the selected library members competent for in vitro transcription.
Each generated DNA template preferably contains a promoter (e.g., T7 or SP6) which is active in an in vitro transcription system. A DNA template generally comprises (1) a promoter which is functional for in vitro transcription and operably linked to (2) a polynucleotide sequence encoding an mRNA period. Said encoded mRNA comprises a polynucleotide sequence which encodes a polypeptide comprising a zinc finger polypeptide, (2) a polynucleotide sequence to which a DNA primer suitable for priming first-strand cDNA synthesis of the mRNA can bind, and (3) a ribosome-binding site and other elements necessary for in vitro translatability of the mRNA, and optionally, for mRNA stability and translatable secondary structure, if any.
Following translation, polysome complexes are screened for high-affinity nucleic acid-binding using standard procedures and as described herein.
After selecting those nascent peptide/polynucleotide complexes that bind with sufficient affinity, the polysomes are isolated and ribosomes released by the addition of EDTA sufficient to chelate the Mg+2 present in the buffer. Ribosomes are removed by high-speed centrifugation, and the RENA component is released by phenol extraction, or by changing the ionic strength, temperature or pH of the binding buffer so as to denature the nascent peptide. A cDNA copy of the mRNA is made using reverse transcriptase, and the cDNA copy is amplified by, the polymerase chain reaction (PCR). The amplified cDNA is added to the in vitro transcription system and the process is repeated to enrich for those peptides that bind with high affinity.
The invention is further described, for the purposes of illustration only, in the following examples.

Screening system for zinc finger polypeptides for a desired binding ability


The invention provides a data access method of a flash memory. First, a write command, a write address, and target data are received from a host. A target block corresponding to the write address is then determined from the flash memory. Whether a storage space corresponding to the write address in the target block has stored data therein is then determined. When the storage space of the target block does not have stored data therein, the target data is written into the storage space of the target block. When the storage space of the target block does have stored data therein, whether a child block mapped to the target block exists in the flash memory is determined. When the child block exists in the flash memory, the target data is written into the child block.

1. Field of the Invention
The invention relates to memories, and more particularly to flash memories.
2. Description of the Related Art
A flash memory comprises a plurality of blocks. Each block comprises a plurality of pages for data storage. When the flash memory receives a write command from a controller, the flash memory must write received data to at least one block thereof according to a write address. When the flash memory receives a read command from the controller, the flash memory must read data from at least one block thereof according to a read address.
When a page of a flash memory stores data, the page cannot be written with new update data before the original data stored in the page is erased. Because a flash memory erases data in a unit of a block, the data stored in all pages of a block must be erased together. Thus, erasing data from a block requires a long time period, and a controller cannot erase data from a flash memory with a high frequency.
When a host sends a write command to a controller to write new data to a write address, if the pages of a target block corresponding to the write address store original data, the controller cannot directly write the new data to the pages of the target block. The controller, however, must execute the write command received from the host. The controller therefore obtains a spare block from the flash memory to store the new data. Because the new data stored in the spare block is mapped to the write address as the original data stored in the target block, there is a mapping relationship between the target block and the spare block. The target block is therefore referred to as a mother block, and the spare block is therefore referred to as a child block mapped to the mother block for storing update data for the mother block.
Referring to FIG. 1, a mother block 102 and a child block 104 of a conventional mapping relationship is shown. The mother block 102 comprise pages mapped to a logical address range of 0˜1000 and has original data A0, B0, C0, and D0 stored therein. When a host sends a first write command to a controller to request the controller to write update data B1 to the address range 400˜600, the space corresponding to the address range 400˜600 in the mother block 102 has stored original data B0 and cannot store the update data B1. The controller then selects a spare block from the flash memory as the child block 104 mapped to the mother block 102, copies the data A0 corresponding to an address range 0˜400 to the address range 0˜400 of the child block 104, and writes the update data B1 to the address range 400˜600 of the child block 104. Similarly, if the host sends a second write command to the controller to request the controller to write update data C1 to an address range 601˜700, because the space corresponding to the address range 601˜700 in the mother block 102 has stored original data C0, the controller directly writes the update data C1 to the address range 601˜700 of the child block 104.
If the host sends a third write command to the controller to request the controller to write new data to an address range 0˜700, because the space corresponding to the address range 0˜700 in the child block 105 has stored data A0 and update data B1 and C1, the controller cannot write the new data to the child block 104 to execute the third write command of the host. Thus, a data access method of a flash memory is required to solve the aforementioned problem.

Flash storage device and data access method of flash memory


Provided is a method, computer program product, and system for navigating a webpage with a screen reader using a prioritization model. A processor may collect analytics information for a webpage. The processor may generate, based on the analytics information, a prioritization model for sorting a plurality of elements of the webpage. In response to a user accessing the webpage with a screen reader, the processor may navigate the plurality of elements of the webpage using the prioritization model.

The present disclosure relates generally to the field of screen readers, and more specifically to improving navigation of webpages for screen readers using data driven analytics.
Screen readers are designed to allow a user that may be visually impaired to read text that is displayed on a computer screen by using a speech synthesizer and/or a braille display. Screen readers are configured as an interface between the computer's operating system, its applications, and the user.

Navigation for screen readers using data analytics

International Business Machines Corporation


Methods, systems, and devices are described for mitigating phase noise. An output of a decoder is utilized to generate an estimation of a plurality of transmitted symbols. One or more phase errors of a plurality of received symbols are generated. The one or more phase errors are based at least in part on the estimation of the plurality of transmitted symbols. The one or more phase errors are generated by comparing angles between the plurality of received symbols and the estimation of the plurality of transmitted symbols. The output of the decoder used to generate the estimation is a plurality of a posteriori log-likelihood ratios (LLRs) of a plurality of transmitted bits. The estimation is generated by performing hard decision decoding on the output of the decoder and remodulating the hard decision decoding according to the modulation used by a transmitter on the plurality of transmitted bits.

The following relates generally to wireless communication, and more specifically to phase error and correction. Wireless communications systems are widely deployed to provide various types of communication content such as voice, video, packet data, messaging, broadcast, and so on. These systems may be multiple-access systems capable of supporting communication with multiple users by sharing the available system resources (e.g., time, frequency, and power). Examples of such multiple-access systems include code-division multiple access (CDMA) systems, time-division multiple access (TDMA) systems, frequency-division multiple access (FDMA) systems, and orthogonal frequency-division multiple access (OFDMA) systems.
Generally, a wireless multiple-access communications system may include a number of base stations, each simultaneously supporting communication for multiple communication devices. Base stations may communicate with communication devices on downstream and upstream links Each base station has a coverage range, which may be referred to as the coverage area of the cell. A transmitter (e.g., a base station) and a receiver (e.g., a communication device) may include, respectively, components for signal transmissions and signal reception. For example, the transmitter and receiver may each include one or more oscillators. These oscillators may not be in sync with one another, and may have inherent imperfections. As a result, phase noise may be introduced into the received signal. This noise may create difficulties for the receiver to correctly determine the phase for symbols transmitted to the receiver. Currently, phase error is determined by performing hard decisions on the received symbols themselves. However, due to the noisy conditions at the receiver, this hard decision may regularly be incorrect. Thus, the phase error and phase correction generated from this hard decision are also incorrect. Erroneous hard decisions result in the performance degradation of various components in the receiver.

Systems and methods to mitigate phase noise


Optimal compositions of derivatives of 5-methyl-3(2H)-furanone compounds and phenylpropanoid polyketides related to inotilone, that exert biological response modification in health and disease, and their method of preparation, are disclosed. Methods of treating degenerative conditions stemming from over-expression of inducible nitric oxide synthase (iNOS) using these compositions are also disclosed.

There is need for nutrition-derived disease prevention and disease treatment support which would intelligently and predictably modify host response against inappropriate or inadequate reaction of the body to an existing or a potential disease. This is a novel category in disease prevention and treatment. This category is different from the concept of enhancing prevention or treatment focused on modification of the prevention/intervention modalities, e.g. modification of vaccine or a drug. This approach is also different from disease prevention based on, e.g. good nutrition or physical exercise which provides general support to the organism. The proposed novel category would include compounds to intelligently repair host mechanisms that may trigger disease or aggravate the existing disease. Inotilone, a natural compound derived from Inonotus sp edible fungi, exemplifies the new prevention/treatment category described here as coherent biological response modifier (cBMR).
Inotilone, 5-methyl-3(2H)-furanone derivative, and related phenylpropanoid polyketides from Inonotus sp. have been previously shown to be potent anti-inflammatory agents and inhibitors of cyclooxygenase and xanthone oxidase enzymes (Wangun H V, et al. Org Biomol Chem. 2006 Jul. 7; 4(13):2545-8); modulate immunological and inflammatory responses (Pan, M H et al, J. Agric. Food Chem 2009, 57(11):4467-4477; Lull, C et al. Mediators. Inflamm. 2005, 2005(2): 63-80); show antihyperglycemic and antilipidperoxidative effects (Sun, J E et al., J. Ethnopharmacology, 118(1):7-13, 2008); and antitumor effects (Youn, M J et al., World J Gastroenterol. 14(4): 511-517, 2008; Kahlos, K et al. Planta Medica, 52(6):554).
Inotilone blocked protein and mRNA expression of iNOS but not COX-2. Instead, inotilone inhibited prostaglandin E(2) production through decreasing the enzyme activity of COX-2. The repression of iNOS but not COX-2 expression may come from the differential effect of inotilone on nuclear factor-kappaB (NFkappaB) and CCAAT/enhancer-binding protein beta Treatment with inotilone resulted in the reduction of LPS-induced nuclear translocation of NFkappaB subunit and the NFkappaB-dependent transcriptional activity by blocking phosphorylation of inhibitor kappaB (IkappaB) alpha and p65 and subsequent degradation of inhibitor kappaBalpha. Inotilone also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. (Kuo, Y C et al, Mol Nutr Food Res. 53(11):1386-95, 2009).

Inotilone derivatives as coherent biological response modifier (cBMR)


The technical scope of the invention is that of projectiles comprising at least one payload associating at least one explosive warhead and at least one target sensor, such warhead having an inclined direction of action with respect to the projectile axis and whose initiation is triggered further to the detection of a target by the sensor, said sensor having an observation direction close to the action direction. The projectile incorporates scanning means enabling the payload, at a given time during the trajectory, to be provided with a ratio of longitudinal velocity V over spin rate .OMEGA. which is less than or equal to a limit value so as to ensure ground scanning in the observation direction at a sufficiently small pitch to enable a target to be detected.

The technical scope of the invention is that of projectiles, notably direct-fire anti-tank projectiles, acting radially with respect to their objective.
Known projectiles comprise an explosive warhead, generally of the explosively-formed kind, whose initiation is triggered by the detection of a target by means of a sensor.
The sensors usually used employ infra-red or radar technology. Generally, these projectiles have means enabling their roll position to be controlled so as to keep their charge pointing in the required direction.
Patent FR-A-2,406,800 thus describes a projectile equipped with a radial action shaped charge. The drawback of such a charge is that it is necessary for complicated means to be provided to ensure its roll is controlled so that the charge can attain the target. These control means can only be activated once the target detection signals supplied by a high-performance nose-cone sensor have been exploited, such sensor notably being able to detect the target before the projectile has passed over it.
It would also be possible to design a projectile in which the warhead roll is stabilized so as to be permanently pointing in a vertical direction.
In addition to the sheer complexity of such a direction control, the roll stabilization of the warhead results in a reduction in the area of effectiveness of the projectile, which in this case must practically pass over its target in order to attack it.
It is notably impossible using such projectiles to attack a target placed in a different direction, for example a helicopter in low-flight located above or laterally with respect to the projectile.

Projectile having a radial direction of action


A fuel-lean mixture burns in rows of flame ports each located between a pair of rows of flame ports burning a fuel-rich mixture. The fuel-rich flames support and stabilize the fuel-lean flames to stabilize combustion and to avoid flame liftoff and noise. The fuel-lean flames reduce the temperature of the overall flame, and thereby reduce the production of No.sub.x compounds produced by the burner. Stepwise and proportional burner control techniques are disclosed.

The present invention relates to burners low in the generation of nitrogen oxides used in a small combustion apparatus for domestic or commercial.
Nitrogen oxides (NO.sub.x) in the exhaust gases from burners of various combustion devices are toxic by themselves and are believed to cause acid rain and photochemical smog. Various measures for decreasing the generation of No.sub.x in burners of combustion apparatus have been developed and utilized.
However, these measures are mainly directed to solving the problems of legally regulated large combustion apparatuses for industrial and other use. Such measures are not satisfactory for small combustion apparatuses for domestic or small commercial use because of noise and/or cost problems.
In a large combustion apparatus, the large static pressure produced by the combustion fan permits easy flow control of the combustion gas and air. This permits a high degree of freedom of layout, and easy noise control. So, with easy control of noise, and with large combustion chambers a possibility, slow combustion can be used to decrease No.sub.x emissions while still achieving perfect combustion. These advantages are not available in small combustion devices. It is therefore difficult to attain decreases in No.sub.x emissions comparable to that which are attainable in large combustion systems.


Disclosed herein is a method for selecting coverage areas as candidates for a handoff, and in particular, for a bidirectional handoff. An exemplary method may involve (a) engaging in a communication session via an access network, wherein an uplink connection is provided for uplink traffic in the communication session and a downlink connection is provided for downlink traffic in the communication session; (b) determining uplink-resource demand and downlink-resource demand of the communication session; (c) using the uplink-resource demand of the communication session as a basis for selecting one or more coverage areas in the access network as candidates for an uplink handoff of the uplink connection; and (d) using the downlink-resource demand of the communication session as a basis for selecting one or more coverage areas in the access network as candidates for a downlink handoff of the downlink connection.

An access network that provides air-interface service under an air-interface protocol such as WiMAX, or other air-interface protocols such as 1xRTT (Single Carrier Radio Transmission Technology) CDMA (Code Division Multiple Access), EV-DO (Evolution Data Optimized), WiFi (e.g. IEEE 802.11), GSM (Global System for Mobile Communications), etc., may include a number of base stations that provide service in one or more coverage areas of the access network. In order to provide substantially continuous service, base stations may be configured to perform a handoff, transferring a connection from one base station to another as a user moves from one coverage area to another in the access network.
Under certain air-interface protocols, such as WiMAX and CDMA, a connection between a mobile station and an access network may include two connections: (1) an uplink (or forward-link) connection over which the mobile station sends data to the access network and (2) a downlink (or reverse-link) connection over which the mobile station receives data from the access network. Thus, when a handoff is performed from one base station to another, or from one coverage area to another, the handoff involves transfer of both the uplink and downlink connections from the originating or "source" base station to a "target" base station.


A baseband filter and upconverter with configurable efficiency for use in wireless transmitters is disclosed. In an exemplary embodiment, an apparatus is provided that includes a baseband filter having configurable efficiency, and an upconverter having configurable efficiency and coupled to the baseband filter. The baseband filter and upconverter are configured to operate at a first efficiency level in a first output power range and to operate at a second efficiency level in a second output power range.

1. Field
The present application relates generally to the operation and design of wireless devices, and more particularly, to the design and operation of configurable transmitters for wireless devices.
2. Background
There is an increasing demand to have wireless devices capable of high quality transmission and reception while consuming low power. One key to achieving high quality is associated with the performance of the device's transmitter. For example, it is desirable to have a transmitter that operates efficiently over its entire output power range.
In existing transmitter designs, a baseband (BB) filter and upconverter are used to filter a baseband signal and up-convert the filtered signal to radio frequencies (RF) for transmission. Typically, both the baseband filter and upconverter are configured for Class-A operation across the entire output power range to meet stringent linearity requirements at maximum power. Unfortunately, at lower output powers, such as during typical talk-times in a wireless device, Class-A operation is very inefficient thereby reducing battery life. It is therefore desirable to have a more efficient transmitter for use in wireless devices.

Baseband filter and upconverter with configurable efficiency for wireless transmitters


A method and device to provide support devices to distribute pressure over a contact area; for example, an insole support device to distribute pressure over the insole of a person's foot are described. The support device is a visco-elastic material having a sufficient density and thickness to provide a relatively uniform pressure to the insole when compressed.

The present invention relates to support devices where support of a part of a person's body is desired. The support device is a visco-elastic material of a plastic foam type for suitably distributing the pressure from a high pressure areas over a relatively large area contacted by the support device. Examples of visco-elastic materials are made by many companies; for example, Sleep Innovation Inc. (187 Route 36 Suite 201 West Long Branch, N.J.) makes a polyurethane material of a foam type.
It is well known that the various parts of the body may be subject to injuries and by pressure induced ulcerations, and by other conditions such as sores (decubitus), wounds and planar fascitis. For example, these conditions are sometimes suffered when persons are immobilized or suffer from a medical disorder such as diabetes. For example, diabetics are prone to develop pressure induced ulcerations from walking. Likewise, immobilized patients often develop decubitus (sometimes know as bed sores). Often, these ulcerations and sores are difficult to heal.
A need exists to make a support device that can used to more evenly distribute pressure the foot (insole), heel, leg, butt (ischeal), back (secral), elbow, arm, scalp, or halo (head) of a patient to prevent, treat and heal decubitus, and relieve pain of planar fascitis. Further, a need exists to make a support device that can easily be adapted to a person's foot and footwear.
Information relevant to address these needs can be found in U.S. Pat. Nos. 6,701,529; 6,602,579; 6,634,045; 6,485,446; 6,458,090; 6,315,786; 6,254,554; 6,187,837; 6,110,134; 5,961,477; 5,695,245; 5,336,708; and 5,334,646; and 20020007136; 20020169401; 2003016200; 20030196352; 20030217485; 20040019972; 20040039316; and 20040049827 each of the foregoing in United States patent and Patent Publication Nos. is hereby incorporated herein by reference. Each one of these referenced items, however, suffers from one or more of the following disadvantages.
One disadvantage is that they do not use a visco-elastic material having a sufficient density and thickness to provide a relatively uniform pressure to the insole when compressed between the person's foot and footwear.
Another disadvantage is that they do not use a visco-elastic material having a sufficient density and thickness of the visco-elastic material to sustain a person's weight without compressing the visco-elastic material to essentially a zero thickness.
Another disadvantage is that they do not use a visco-elastic material having a sufficient density and thickness of the visco-elastic material to sustain a person's weight without compressing the visco-elastic material to essentially a zero thickness.
Another disadvantage is that they do not use support device covers where the support device is covered support device covers.

Support devices for distributing pressure


The invention relates to the field of methods for zooming onto a part of an image which consists of pixels and which represents a terrain overflown by an aircraft. It is a zoom method for zooming onto a part of an image which consists of pixels and which represents a terrain overflown by an aircraft, an image being modulated, pixel by pixel, by a shading cue representative of the relief of a terrain. The shading cue is determined by way of a computation carried out, in a reference frame of a display screen of an aircraft, pixel by pixel, on the basis of the altitudes of pixels neighboring the current pixel in the case of the unzoomed image. The method according to the invention can in particular be implemented in a cartographic accelerator card.

1. Field of the Invention
The invention relates to the field of methods for zooming onto a part of an image which consists of pixels and which represents a terrain overflown by an aircraft. The terrain overflown by the aircraft is either the terrain actually overflown by the aircraft when the image is displayed, or the terrain which is intended to be overflown by the aircraft, the image then being displayed in anticipation. The zoom method is preferably carried out by certain functional blocks of a cartographic function of a cartographic accelerator card described in detail subsequently.
The image is in general a 2D5 cartographic image, that is to say one representing a terrain plan view which is modulated, pixel by pixel, by a shading cue representative of the terrain relief. The image may be an altimetric image or a planimetric image. This shading cue determines-the brightness of each pixel of the image considered. In the case of an unzoomed image, that is to say an image whose zoom factor equals one, this shading cue is determined by way of a computation which is carried out, in the reference frame of a display screen of the aircraft, pixel by pixel, on the basis of the altitudes of pixels neighboring the current pixel. These neighboring pixels are preferably pixels immediately neighboring the current pixel, that is to say the pixels closest to the current pixel from among the neighboring pixels. The shading cue is computed on the one hand on the basis of a model of illumination of the image using one or more light sources, point and/or diffuse, and on the other hand on the basis of the local slope at the level of the current pixel, that is to say each of the pixels of the image, this local slope being represented by the orientation of a facet at the level of the current pixel.
2. Description of Prior Art
According to the prior art, this computation can be undertaken according to several different techniques among which the most common are the technique of so-called "flat shading", the technique of so-called "gouraud shading", and the so-called "Phong" technique. All these techniques have in common the use of characteristics of the neighboring pixels, such as for example the orientation of the normals to the neighboring pixels for the so-called "gouraud shading" technique, to determine the orientation of the facet which represents the local slope at the level of the current pixel. These techniques give better or worse results depending on the technique, at the cost of greater or lesser complexibility in the implementation of said techniques. The results are generally correct for an unzoomed image. On the other hand, for a zoomed image, and especially for an image whose zoom factor becomes considerable, a facetization of the image referred to as "bathroom tiling" appears (except for the so-called "Phong" technique, but the latter is complex and expensive in terms of resources) which is an impediment to the pilot, especially in dynamic mode on account of the flashing which occurs at the boundary between the various "bathroom tiles" and on which the pilot's attention is focused.


A telescoping leader system for a construction machine that includes a set of leads having a series of internal nesting segments and an extending means connecting one segment to another segment to allow for the extension of the segments one after the other to an extended position and the retraction of the segments to an internal nesting position. The telescoping leader system further includes a surface engaging member mounted within the set of leads and a pivotable mounting assembly mounted to the set of leads and adapted to adjust the position of the set of leads. A quick connecting means adapted to engage the pivotable mounting assembly is included to allow for the quick engagement and disengagement of the construction machined from the set of leads.

Typically leader systems are used to drive piles, drilling or augered pile operations and are mounted to various power units such as excavators, cranes, boom trucks or tracked machines. The leader system is responsible for guiding the hammer, drill or drive head so that it is in alignment with the pile or drilling operation. Depending on the construction a telescoping lead may be desirable. Telescoping leader systems can therefore telescope the leads either upwards or downwards. There ability to telescope either upwards or downwards depends on what the leader system is mounted to. When mounted to a crane or boom truck, the telescoping leads can move either up or down depending on orientation, however when mounted to an excavator the telescoping leader system would typically telescope upwards.
Traditional telescoping leader systems are cumbersome in that they telescope in a staged fashion, telescoping each segment out in front of each other in a stepped fashion so that segments are not along the same axis. This arrangement does not allow the leader system to be easily removed from the power unit and is not an appropriate configuration for a crane. Most leader systems for cranes have a fixed length and utilize extensions that allow for longer piles to be installed.
Prior art telescoping leader systems have been devised to address the noted problems. For example, U.S. Pat. No. 3,035,646 which issued May 22 1962 to Johansson discloses an improved pile driving attachment including a pile driving lead for use with a caterpillar or like tractor crane having a windlass frame preferably in the form of a turn-table or rotatable crane house which is carried by the under frame of the tractor. The invention further discloses a series of extendable or telescoping struts that may extended by a hydraulic system.
U.S. Pat. No. 3,388,317 which issued on Jun. 10, 1975 to Walters discloses a system for supplying full hydraulic power to the movable components of a positioning apparatus of a pile driver or similar apparatus. The device includes hydraulically drive truss sections that form the spotters for the invention which can both extend and retract.
Thus a telescoping leader system having the ability to be quickly mounted and removed from an excavator, crane, boom truck, that can install longer piles with less power and is compact in nature, is desirable.


Various mountings are disclosed for holding a portable screen device in an attitude for hands-free viewing. The mountings are particularly, but not exclusively, useful for supporting portable screen devices (522, FIG. 5A) from the seat-back trays (528) found in passenger vehicles, when the trays are in the “up” position. Each mounting comprises a pair of hooked members (510), each hooked member having a spine (515) provided with first and second hook portions (512, 514) arranged on opposite sides of the spine to face in mutually opposed directions. Each hook portion (512, 514) comprises an open end and a closed end bounded by outer and inner, mutually opposed sides of the hook portion. Various embodiments are disclosed that are also capable of supporting portable screen devices when seat-back trays are in the “down” position, see e.g., FIG. 5B. In most embodiments, both hook portions (512, 514) are unitary with the spine (515), which comprises the inner side of both hook portions, but embodiments are also proposed in which the first hook portion (612, FIG. 6) is formed separately from the spine (615) and is selectively positionable thereon. When in use as a mounting, the hooked members 510, FIG. 5 A) are spaced apart from each other and the second hook portions (514) receive the screen device (522), which spans the distance between the hooked members.

Mountings are already known which permit tablet computers, smartphones and other portable screen devices to be mounted for hands-free viewing, e.g., on the back of airline, train and bus seats, and on the drop-down trays often found on such seat backs. Usually, such mountings are configured to support the screen device in an attitude for viewing when a drop-down tray is in the down position.
Unfortunately, when such a tray is in the down position, it restricts the space available to the person for whom it is provided. It would therefore be desirable to provide mountings for portable screen devices, which mountings take up the minimum amount of space and which are readily mountable and demountable upon the tray when it is in the "up" position. It would be desirable for such mountings to be cheap to produce for sale as minor purchases, or even for giving away by a transport service provider, before or during journeys. It would additionally be desirable if such mountings were also able to support portable screen devices for viewing on such trays when the latter are in the "down" or in-use position. It would further be desirable if such mountings were simple to set up and use.