Search results for "Cloning"

Cloning of sponge heat shock proteins: evolutionary relationships between the major kingdoms

2009

In the present study we have cloned from sponges (Porifera) those molecules which are involved in the protection of organisms against physiological and stress conditions; the inducible heat shock protein Mr 70,000, hsp70, from the marine sponge Geodia cydonium, its interacting hsp40, a DnaJ-like protein (from G. cydonium) and the constitutively expressed counterpart the glucose-regulated protein Mr 78,000, GRP78 from Suberites domuncula. Alignments of the sequences revealed that the deduced aa sequences of all sponge hsp's share high homology to other metazoan sequences, and are separated from related sequences from plants and fungi (hsp70, GRP78, DnaJ) as well as Bacteria (DnaK, the hsp70 …

Variability Among Italian Citrus tristeza virus Isolates Revealed by SSCP Analysis, Cloning and Sequencing

2005

A Novel Chitin-binding Protein from the Vestimentiferan Riftia pachyptila Interacts Specifically with β-Chitin

2001

Abstract A cDNA from Riftia pachyptila was cloned. It encodes a novel 21.3-kDa protein from the worm protective tube, named RCBP (for Riftia chitin-binding protein). On the basis of partial tube-peptide sequences previously obtained, experiments using reverse transcriptase-mediated polymerase chain reaction and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of its deduced amino acid sequence shows the presence of two chitin-binding domains. These domains are closely related to type 2 chitin-binding domains that are restricted to the animal kingdom. We showed by affinity assay and immunogold labeling that RCBP is the first protein so far known that binds specifi…

cDNA Synthesis and Cloning

1998

The isolation of intact messenger RNA and its conversion into cDNA copies by avian or Moloney murine reverse transcriptase, as well as subsequent amplification of gene transcripts by the PCR technique, are becoming increasingly important tools in molecular biology. At present, these techniques have been often necessary and widely used for the analysis of individual mRNA levels in cells and tissues by Northern blot analysis, nuclease protection analysis and in situ hybridization. Another important application of RNA templates is the construction of representa­tive cDNA libraries in order to clone genes, to investigate their molecular structure and to express them in prokaryotic and/or eukary…

Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia lipolytica

1998

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67.5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli.

Gentechnik in der Schule

1999

The Fonds of German Chemical Industry (FCI) has produced an instrumental kit named „Blue Genes” which enables High School teachers to perform in their school environment basic experiments of gene analysis and cloning. In particular, the kit contains all necessary equipment and reagents to perform a restriction analysis, and cloning and expression of a bacterial gene.

Methylation Reprogramming and Chromosomal Aneuploidy in In Vivo Fertilized and Cloned Rabbit Preimplantation Embryos1

2004

Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requireme…

Identification of Tumor-Associated Autoantigens With SEREX

2004

Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients. For SEREX, cDNA expression libraries are constructed from fresh tumor specimens, packaged into lambda-phage vectors, and expressed recombinantly in Escherichia coli. Recombinant proteins expressed during the lytic infection of bacteria are transferred onto nitrocellulose membranes to be probed with diluted autologous patient serum for identification of clones reactive with high-titered IgG antibodies. This chapter describes the SEREX technology in detail.

Receptor Tyrosine Kinase, an Autapomorphic Character of Metazoa: Identification in Marine Sponges

1999

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulinlike receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, …

Cloning and Characterization of Overlapping DNA Fragments of the Toxin A Gene of Clostridium difficile

1989

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of vari…