RSS plasmid substrate assays for V(D)J rearrangement

Connor, Andy M.; Fanning, Liam J.; Abentolila, L; eWu, Gang

doi:10.1016/b978-012442710-5.50030-7

Cited by 2 publications

(2 citation statements)

References 28 publications

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“…In the present study we report a simple and highly reproducible method of purification of CgA, a protein of Mr 67 from human and bovine adrenal glands which is stored in catecholamine vesicles of the adrenal medulla. Although CgA does not have any known AB C enzymatic activity [13], the removal of dopamine ~ hydroxylase (D~H) was necessary due to its potential confusion for the properties of CgA and seems to be a more potent immunogen than CgA [19][20][21][22][23]. It has also been reported that breakdown of CgA occurs at room temprature [24] due to the presence of acetylcholinetransferase, tyrosine like enzymes in theadrenal medulla and the contamination of lysosomal enzymes, and endogenous proteases in chromaffin granules [11,23].…”

Section: Discussionmentioning

confidence: 99%

“…CgA is a highly acidic protein with an apparent relative molecular mass of variable size on SDS-PAGE but an actual Mr 48000. The primary structure of CgA is now known, however its exact physiological role is still obscure.It has been suggested CgA through binding to catecholamine and Ca 2+ may possibly helping in neuroendocrine secretion, osmoregulation in chromaffin granules and a precursor of pancreaststin [12][13][14][15]. There are several methods available for the purification of CgA.…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine

Kumar

1996

IUBMB Life

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SUMMARYIn the present study, we report a simple, highly reproducible procedure for the purification of chromogranin A. A three step column chromatography of supernatant from human and bovine provided a major single band at Mr 67 and two minor bands at low molecular mass. Identity of the major and minor bands of CgA was confirmed by western blotting using LK2H10, a monoclonal antibody against chromogranin A. Recognition of smaller fragments in the final preparation in both species by antibody to chromogranin A may suggest that these are the proteolytic breakdown product of the main protein. Bovine adrenal gland was found to be many fold richer in chromogranin than human adrenal gland. The simplification of the purification procedure for CgA may now help in elucidating further physiological function of this protein.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine

Kumar

1996

IUBMB Life

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Differential Usage of VH Gene Segments Is Mediated bycisElements

Larijani

Miljanic

et al. 1998

The Journal of Immunology

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Ig diversity is generated in large part by the combinatorial joining of the Ig gene segments, VH, D, and JH, that together encode the variable domain of Ig. The final Ig repertoire, however, not only reflects the diversity generated through V(D)J recombinatorial joining, but it is also the product of a number of developmental restraints and selections. To avoid such restrictions and assess the recombination potential of individual Ig gene segments, we constructed Ig heavy (H) chain microlocus plasmids, each of which contain germline coding, recombination signal, and flanking sequences of a VH, D, and JH gene segment. These plasmids allow us to assess the recombination potential of the segments in the context of their natural flanking DNA sequences, but in the absence of any higher order chromatin structure or cellular selection. We found that the frequency and extent of deletions and additions at the recombination breakpoints are similar to those observed at rearranged Ig H chain loci in intact animals. The relative frequencies of the types of rearrangements—VD-J, V-DJ, VinvD-J (invD = inverted D), and VDJ—however, differ strongly. Moreover, V81x, the most used VH gene segment in intact mice, also is overused in this plasmid assay, 15 to 30 times that of another VH segment. This result indicates that the overuse of V81x in the early B cell repertoire can be a consequence of its DNA sequence and not of cellular activities.

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RSS plasmid substrate assays for V(D)J rearrangement

Cited by 2 publications

References 28 publications

Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine

Purification and characterization of chromogranin‐A from the adrenal gland of human and bovine

Differential Usage of VH Gene Segments Is Mediated bycisElements

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