Abstract
B94 was originally described as a novel tumor necrosis factor-alpha-inducible primary response gene in endothelial cells which was also induced in an in vitro model of angiogenesis. To further characterize its expression, we cloned the mouse homologue and mapped its developmental and tissue specific expression. The predicted amino acid sequence of mouse B94 was found to be 83% similar to its human homologue. The gene was localized to mouse chromosome 12 just centromeric to the immunoglobulin heavy chain locus, in a region that is often rearranged in T-cell neoplasms. To explore the possibility that B94 is expressed during vasculogenesis and other developmental processes, the expression of its transcript was determined during mouse development by in situ hybridization. In 10-day embryos B94 was expressed prominently in the myocardium and in the aortic arch. By the 15th day of gestation, expression was restricted largely to the liver, the bone forming regions of the jaw, the aortic endothelium, and the nasopharynx: a pattern that was maintained until just prior to birth. Postnatally, expression shifted to the red pulp of the spleen and the thymic medulla. B94 expression was extinguished in most adult tissues but was detectable in lymphopoietic tissues including the spleen, tonsil, and lymphatic aggregates in the gut. Consistent with this was the finding that mononuclear progenitor cells in bone marrow and mature peripheral blood monocytes expressed B94. A truncated testis-specific transcript previously identified by Northern blot analysis was determined to result from the use of an alternate polyadenylation signal which was surprisingly located within the open reading frame. This shorter transcript was expressed at high levels exclusively in late stage spermatids. Immunostaining with an affinity-purified polyclonal antiserum revealed B94 to be localized to the acrosomal compartment of mature sperm. These studies demonstrate that B94 expression is tightly regulated during development and suggests distinct roles for B94 in myelopoiesis and spermatogenesis.
Highlights
B94 was originally describedas a novel tumor necro- and suggestadistinct roles forB94 in myelopoiesis and sis factor-a-inducible primary response gene in endo- spermatogenesis
Genes transcriptionally activated by cytokines and growth factorswithout the requirement of intervening protein synthesis are known as primaryresponse genes and encode key regulatory proteins including transcriptional factors such as c-fos and c j u n, paracrine factors vasculogenesis andother developmental processes,the such as interleukin-8, andcell surface molecules that mediate expression of ita transcript was determined during adhesive interactions such as E-selectin (1).Overall, these almouse development by in s i t u hybridization
Mononuclear progenitor cellsin bone marrow and ma- originally cloned from TNF stimulated endothelium (9).The ture peripheral blood monocytes expresseBd94.A trun- B94 gene encodes a novel 73-kDa intracellular protein that cated testis-specifictranscript previously identifiedby exists as a single copy gene on human chromosome 14 near the Northern blot analysis was determined to result from immunoglobulin heavy chain locus
Summary
Through Ficoll-Paque(Pharmacia LKB BiotechnologyInc.) for 30 maint 400 x g and were separated by elutriation into fractions enriched in lymphocytes, monocytes,and polymorphonuclear cells. Elutriation was done in elutriation buffer (PBS with 0.5% dextrose, 0.35% bovineserum albumin, and 1m~ EDTA) with a JE-GB rotor (Beckman)at 2030 rpm Isolation a n d Analysis ofMouse B94 cDNAs-Mouse B94has been shown previously by Northern analysisto be expressed in a variety of tissues includingthe embryonic kidney(9).Screening of day 17 post-coitum (dpc) embryonickidney cDNA library and a flow rate varying upwards from 4 t o 10 d m i n. A , / : i : : r :AM-1 syntenic withthe most distal region of human chromosome 14, and the resulits in agreementwith the previous localization of human B94 by in situ hybridization to the q32 band of chromosome 14 Developmental Expression Patterns-Expression patterns of B94 during murine development were determined by in situ hybridization of 35S-labeledantisense RNA to sagittal sections of embryos aged 10-19 dpc.
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