Biomedical subjects

N Ashouian

Publications and source records attributed to N Ashouian.

3 recordsLinked to original sources

PubMed2000

BSAP (Pax5)-importin alpha 1 (Rch1) interaction identifies a nuclear localization sequence.

BSAP (Pax5) is an essential transcription factor for early B cell and central nervous system development. In later B cell development, BSAP has been implicated in the regulation of 3' Ig enhancers and a number of B cell-specific genes. Previous studies have suggested a role for BSAP-interacting proteins in the regulation of the function of BSAP. Using the yeast two-hybrid system, we identified importin alpha1 (Rch1) as a BSAP-interacting protein. Importin alpha proteins have been shown to escort proteins into the nucleus through interaction with a nuclear localization signal (NLS), composed of short stretches of basic amino acids. A predicted NLS in BSAP (NKRKRDE, located at amino acids 195-201 in the central domain) was confirmed to be essential for interaction with importin alpha1 by the yeast two-hybrid assay. Physical interaction between BSAP and importin alpha1 was detected in vitro by a glutathione S-transferase (GST) pulldown assay. The NLS sequence in BSAP conferred nuclear localization to green fluorescent protein (GFP)-BSAP fusion proteins. Although the N-terminal paired (DNA-binding) domain of BSAP also conferred nuclear localization when coupled to green fluorescent protein, this domain did not bind to importin alpha1 in the yeast two-hybrid assay. The NLS sequence in the central domain of BSAP binds to the C-terminal 98-amino acid fragment of importin alpha1.

Animals↗

PubMed1999

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line.

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.

Animals↗

PubMed1997

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.

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