The Experts below are selected from a list of 1605 Experts worldwide ranked by ideXlab platform
Leroy Hood - One of the best experts on this subject based on the ideXlab platform.
-
The complete nucleotide sequence of Cosmid Vector pTL5: location and origin of its genetic components.
Gene, 1994Co-Authors: Jerry L. Slightonr, David R. Siemieniak, Ben F. Koop, Leroy HoodAbstract:SUMMARY The complete nucleotid~ sequence (5793 bp) of the Cosmid Vector pTL5 and the origin of its genetic components has been determined. Cosmid pTL5, a derivative of Cosmid Vector pHC79, is composed of genetic components from pBR322, bacteriophage h and the hybrid lambdoid bacteriophage Charon (Ch) 4A cohesive ends (cos) region. The Ch4A cos region contains genetic components from two bacteriophages, the h cos-left arm and the $80 cos-right arm regions. The Ch4A cos region has been used in the construction of many other Cosmid-type Vectors, some of which have been sequenced and entered into the GenBank database.
-
The complete nucleotide sequence of Cosmid Vector pTL5: location and origin of its genetic components.
Gene, 1994Co-Authors: Jerry L. Slightonr, David R. Siemieniak, Ben F. Koop, Leroy HoodAbstract:The complete nucleotide sequence (5793 bp) of the Cosmid Vector pTL5 and the origin of its genetic components has been determined. Cosmid pTL5, a derivative of Cosmid Vector pHC79, is composed of genetic components from pBR322, bacteriophage lambda and the hybrid lambdoid bacteriophage Charon (Ch) 4A cohesive ends (cos) region. The Ch4A cos region contains genetic components from two bacteriophages, the lambda cos-left arm and the phi 80 cos-right arm regions. The Ch4A cos region has been used in the construction of many other Cosmid-type Vectors, some of which have been sequenced and entered into the GenBank database.
-
Complete nucleotide sequence of the Cosmid Vector pWE15A
Nucleic Acids Research, 1992Co-Authors: Donald Seto, Ben F. Koop, Jason Seto, Leroy HoodAbstract:Current efforts to determine the complete nucleotide sequence of the six murine and human T-cell receptor (TCR) loci are limited by the availability of overlapping genomic inserts cloned into a defined and characterized Vector. In order to create these mapping and sequencing libraries, our laboratory has modified an existing Cosmid Vector pWE15 (1,2). The optimized version, pWE15A (3), contains a polylinker with 15 infrequently cleaved restriction enzyme sites, asymmetrically centered around the BamHI site, which is the cloning site used for the insertion of the genomic DNA described below. The modifications allow recovery of inserted DNA by the use of several restriction enzymes. Each recombinant insert bearing Vector may contain sequenceable genomic DNA ranging in size from 35 to 40 kilobases (kb). This complete 8213 nucleotide pWE15A sequence complements the two noncontiguous approximately 2 kb of pWE15A extant in GenBank. Our laboratory has begun the mapping and sequencing process by making libraries of overlapping human TCR £ locus clones (K.Wang, manuscript in preparation), murine TCR j3 locus clones (C.Boysen, personal communication), human and murine a/5 loci clones (K.Wang, manuscripts in preparation), and murine y locus clones (B.Vernooij, manuscript in preparation). These libraries are the basis of our current large-scale DNA sequencing endeavor. Initial DNA sequencing projects include a murine Va/5 Cosmid clone (D.Seto, manuscript in preparation), murine and human Ja clones (B.F.Koop, manuscripts submitted), and human V/3 clones (L.Rowen, personal communication). These sequencing efforts involve the random 'shotgun' approach (4) which should yield a 5to 10-fold redundancy of DNA sequences. Due to the sheer number of sequences, which also includes Vector sequences, the assembly process may be difficult given current algorithms. The process is simplified when the Vector sequences are subtracted from the data set prior to assembly (D.Seto, manuscript submitted). The pWE15A Cosmid Vector has been completely sequenced using automated fluorescent techniques as described by our laboratory (6). This 8.2 kb of sequence has been generated from a data set containing an average of approximately 10-fold redundancy. Areas of compression in the sequencing gel have been resolved by incorporating base analogs such as 7-deazaGTP and 7-deazaTTP in place of dGTP, and also by using either T7 DNA polymerase or Taq DNA polymerase. One area (4 nucleotides) remained unresolvable despite these measures; however this area has been sequenced by several other laboratories (example, 7) and its sequence is incorporated into our sequence (positions 1883 — 1886). Cosmid Vector pWE15A has been completely sequenced and assembled for the first time and is being released into EMBL (accession number Z12112) so that any investigator wishing to use our pWE15A-based libraries may have ready access to the entire complete sequence data. This complete characterization of pWE15A will enhance its role in high-resolution restriction enzyme mapping and rapid chromosomal walking. Importantly, the pWE15A sequences may be used to screen out Vector components when 'shotgun' sequencing inserts are cloned into this Cosmid or into its predecessor pWE15.
Heinz D. Osiewacz - One of the best experts on this subject based on the ideXlab platform.
-
A versatile shuttle Cosmid Vector for the efficient construction of genomic libraries and for the cloning of fungal genes
Current genetics, 1994Co-Authors: Heinz D. OsiewaczAbstract:A shuttle Cosmid Vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This Vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.
Robert F. Silva - One of the best experts on this subject based on the ideXlab platform.
-
a double cos site Vector containing a multiple cloning site flanked by t7 and t3 rna polymerase promoters
Gene, 1993Co-Authors: David J Reilly, Robert F. SilvaAbstract:Abstract A double-cos-site Cosmid Vector, c2XMCS, contains a multiple cloning site (MCS) with fifteen restriction sites flanked by phage T7 and T3 RNA polymerase promoters. The two cos sites in the Cosmid Vector enable the construction of Cosmid libraries from unfractionated partial digests of genomic DNA. This simple construct combines the efficiency of a double-cos-site Cosmid with the versatility provided by the MCS from pBluescript.
François Shareck - One of the best experts on this subject based on the ideXlab platform.
-
Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression Cosmid Vector.
Journal of industrial microbiology & biotechnology, 2010Co-Authors: François-xavier Lussier, François Denis, Olivier Chambenoit, Amélie Côté, Jean-françois Hupé, Pierre Juteau, Réjean Beaudet, François ShareckAbstract:The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle Cosmid Vector pFX583 was used for the construction and screening of a metagenomic library. This Vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted.
Christina Bruntner - One of the best experts on this subject based on the ideXlab platform.
-
Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.
Journal of Bacteriology, 1996Co-Authors: Christiane Bormann, V. Möhrle, Christina BruntnerAbstract:A genomic library from Streptomyces tendae raised in shuttle Cosmid Vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant Cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.
