Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC).

His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by immobilized metal affinity chromatography (IMAC).

Purification of Polyhistidine-Tagged Proteins.

His-tagging is the most widespread and versatile strategy used to purify recombinant proteins for biochemical and structural studies. Recombinant DNA methods are first used to engineer the addition of a short tract of poly-histidine tag (His-tag) to the N-terminus or C-terminus of a target protein. The His-tag is then exploited to enable purification of the "tagged" protein by Immobilized Metal Affinity Chromatography (IMAC).

Identification of zygotic genes expressed at the midblastula transition in zebrafish.

Early development of the embryo is directed by maternal gene products and characterised by limited zygotic gene activity, cell division synchrony and no cell motility in several vertebrates including fish and frogs. At the midblastula transition (MBT), zygotic transcription is grossly activated, cells become motile and cell divisions become asynchronous. The aim of this study was to identify genes whose expression is up-regulated at the MBT in zebrafish.

Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks.

53BP1 (p53 binding protein) is a BRCT domain-containing protein that is rapidly recruited to DNA double strand breaks (DSBs). To investigate the role of 53BP1 in the DNA damage response, we generated 53BP1(-/-) cells from the chicken DT40 cell line. As in mammalian cells, mutation of 53BP1 increased cellular sensitivity to ionizing radiation.

nanor, a novel zygotic gene, is expressed initially at the midblastula transition in zebrafish.

A novel, developmentally regulated gene, nanor, was identified by suppression subtractive hybridization. It is first expressed following the midblastula transition (MBT), a critical developmental stage in the early vertebrate embryo when the zygotic genome is activated. The nanor cDNA (626bp) includes a complete open reading frame but neither the gene nor the deduced amino acid sequence shows significant similarity to any known gene or protein.

The switch from survival responses to apoptosis after chromosomal breaks.

Eukaryotic cells have evolved highly sophisticated cellular responses to DNA double strand breaks (DSBs) that increase the likelihood of survival. However, cells can also respond to DSBs by undergoing programmed cell death. The mechanisms underlying the cellular decision on whether to repair and survive or to die are not well understood but may be related to the efficiency of repair or the extent of the damage.

The MRN complex: coordinating and mediating the response to broken chromosomes.

The MRE11-RAD50-NBS1 (MRN) protein complex has been linked to many DNA metabolic events that involve DNA double-stranded breaks (DSBs). In vertebrate cells, all three components are encoded by essential genes, and hypomorphic mutations in any of the human genes can result in genome-instability syndromes. MRN is one of the first factors to be localized to the DNA lesion, where it might initially have a structural role by tethering together, and therefore stabilizing, broken chromosomes.

Cellular longevity: role of apoptosis and replicative senescence.

Cellular longevity refers to the lifespan of an individual cell. Normal cells have a finite lifespan and typically die by undergoing apoptosis, or enter into a state of irreversible growth arrest, termed replicative senescence, at the end of that lifespan. The lifespan of a cell is a balance between pro-survival/anti-apoptotic and pro-apoptotic death-promoting factors.

Cyclin-dependent kinase 8 is expressed both maternally and zygotically during zebrafish embryo development.

Cyclin-dependent kinase 8 (cdk8) regulates transcription by phosphorylating RNA polymerase II and TFIIH. The mechanism of zygotic transcription activation during vertebrate embryonic development is poorly understood. Here we describe the cloning and developmental expression pattern of zebrafish cdk8 mRNA.