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William D. Huse

Bio: William D. Huse is an academic researcher from Stratagene. The author has contributed to research in topics: Phagemid & Multiple cloning site. The author has an hindex of 3, co-authored 3 publications receiving 1338 citations. Previous affiliations of William D. Huse include Torrey Pines Institute for Molecular Studies.

Papers
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Journal ArticleDOI
TL;DR: A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed in this article, which eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation.
Abstract: A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coli a phagemid, pBluescript SK(-), contained within the vector, can be excised by f1 or M13 helper phage. The excision process eliminates the need to subclone DNA inserts from the lambda phage into a plasmid by restriction digestion and ligation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the f1 bacteriophage origin of replication (1). Six of 21 restriction sites in the excised pBluescript SK polylinker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA library and the isolation of actin clones by screening with oligonucleotide probes. Putative actin clones were excised from the lambda vector and identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgG anti-glucocerbrosidase antibodies.

1,321 citations

Patent
02 Oct 1989
TL;DR: In this paper, an improved method of determining the nucleotide base sequence of DNA is disclosed, which comprises preparing a DNA substrate comprising a set of molecules, each having a template strand and a primer strand, wherein the 3' ends of the primer strands terminate at about the same nucleotide position on the template strands of the molecules within each set.
Abstract: An improved method of determining the nucleotide base sequence of DNA is disclosed. The method comprises preparing a DNA substrate comprising a set of molecules, each having a template strand and a primer strand, wherein the 3' ends of the primer strands terminate at about the same nucleotide position on the template strands of the molecules within each set. DNA synthesis is induced to obtain labeled reaction products comprising newly synthesized DNA complementary to the template strands using the 3' ends of the primer strands to prime DNA synthesis, labeled nucleoside triphosphates, and at least one modified nucleoside triphosphate, wherein the modified nucleoside triphosphate is selected to substantially protect newly synthesized DNA from cleavage. The labeled reaction products are cleaved at one or more sites to obtain labeled DNA fragments wherein the newly synthesized DNA is substantially protected from cleavage at the selected site or sites. The labeled DNA fragments are separated and their nucleotide base sequence is identified by suitable means.

19 citations

Patent
01 Oct 1990
TL;DR: In this article, an improved method of determining the nucleotide base sequence of DNA is disclosed, which comprises labeling and protecting predetermined regions of a strand of nucleic acid to be sequenced to produce a blocked and labeled strand having blocked, labeled and unblocked regions.
Abstract: An improved method of determining the nucleotide base sequence of DNA is disclosed. The method comprises labeling and protecting predetermined regions of a strand of nucleic acid to be sequenced to produce a blocked and labeled strand having blocked, labeled and unblocked regions. Subsequently, the blocked and labeled strand is treated with a cleavage reagent that preferentially cleaves the strand at a point in the unblocked region to produce a labeled fragment having a blocked region. The nucleotide sequence of at least a portion of the blocked region is then determined.

4 citations


Cited by
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Patent
10 Jul 1991
TL;DR: In this paper, a member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbps members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof.
Abstract: A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

2,740 citations

Journal ArticleDOI
07 Dec 1989-Nature
TL;DR: It is suggested that most tumours with allelic deletions of chromosome 17p contain p53 point mutations resulting in amino-acid substitutions, and p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene.
Abstract: The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.

2,708 citations

Journal ArticleDOI
08 Dec 1989-Science
TL;DR: A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire, which allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation.
Abstract: A novel bacteriophage lambda vector system was used to express in Escherichia coli a combinatorial library of Fab fragments of the mouse antibody repertoire. The system allows rapid and easy identification of monoclonal Fab fragments in a form suitable for genetic manipulation. It was possible to generate, in 2 weeks, large numbers of monoclonal Fab fragments against a transition state analog hapten. The methods described may supersede present-day hybridoma technology and facilitate the production of catalytic and other antibodies.

1,816 citations

Patent
02 Dec 1992
TL;DR: In this paper, the authors described methods for the production of anti-self antibodies and antibody fragments, being antibodies or fragments of a particular species of mammal which bind self-antigens of that species.
Abstract: Methods are disclosed for the production of anti-self antibodies and antibody fragments, being antibodies or fragments of a particular species of mammal which bind self-antigens of that species. Methods comprise providing a library of replicable genetic display packages (rgdps), such as filamentous phage, each rgdp displaying at its surface a member of a specific binding pair which is an antibody or antibody fragment, and each rgdp containing nucleic acid sequence derived from a species of mammal. The nucleic acid sequence in each rgdp encodes a polypeptide chain which is a component part of the sbp member displayed at the surface of that rgdp. Anti-self antibody fragments are selected by binding with a self antigen from the said species of mammal. The displayed antibody fragments may be scFv, Fd, Fab or any other fragment which has the capability of binding antigen. Nucleic acid libraries used may be derived from a rearranged V-gene sequences of unimmunised mammal. Synthetic or artificial libraries are described and shown to be useful.

1,373 citations

Journal ArticleDOI
TL;DR: The physiological importance of these two proteins in cellular signal transduction is discussed, and partial purification of GSK‐3 activity from bovine brain results in the isolation of active alpha and beta proteins.
Abstract: Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.

1,293 citations