Terminal deoxynucleotidyltransferase. Serological studies and radioimmunoassay

Mouse antisera against calf terminal deoxynucleotidyltransferase (terminal transferase) have been prepared. The sera have been used to characterize terminal transferase both by studying inhibition of enzyme activity and by developing a competition radioimmunoassay using highly purified 125I-labeled terminal transferase. By either assay, anti-terminal transferase serum did not cross-react significantly with calf DNA polymerases alpha and beta, Escherichia coli DNA polymerase I, or the reverse transcriptase of Moloney mouse leukemia virus. The calf terminal transferase did, however, share cross-reactive but not identical determinants with human and murine terminal transferase. The radioimmunoassay could detect as little as 2 ng of terminal transferase/mg of soluble protein in a tissue extract. Thymocytes were found to contain 280 ng of terminal transferase/mg of cell protein or about 1 X 10^(5) molecules/cell; bone marrow had about 1% of the level of enzyme found in thymus. Extracts of spleen, peripheral white blood cells, lymph nodes, liver, muscle, and kidney all lacked detectable antigenicity of terminal transferase. These data indicate that terminal transferase is a tissue-specific enzyme and is not related to other DNA polymerases

Sequential induction of NF-κB/Rel family proteins during B-cell terminal differentiation

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs

Murine terminal deoxynucleotidyl transferase: cellular distribution and response to cortisone

The mouse thymus contains two forms of terminal deoxynucleotidyl transferase (TdT) which are distinguishable by the salt concentration necessary to elute them from a phosphocellulose column, by their distrubtion among the thymocyte subpopulations, and by their sensitivity to cortisone treatment. In the whole thymus the later eluting peak (peak II) is the predominant one with about 3-10% of the total activity appearing in peak I. Both peak I and peak II activities are most sensitively assayed by the polymerization of dGMP onto an oligo(dA) primer. The minor population of thymocytes which is less dense and cortisone-resistant contains a higher specific activity of peak I TdT. The majority of TdT activity is, however, found in the major population of thymocytes which occurs in the center region of a bovine serum albumin gradient and is cortisone-sensitive. A very low level of an activity indistinguishable from peak II TdT activity is also detected in the mouse bone marrow. Other tissues, such as spleen, liver, heart, and brain lack detectable amounts of TdT activity

Holistic Interdisciplinary Design - Everyone Does Everything (Engineering Students as Sculptors)

The ability to offer students an interdisciplinary experience under a team work setting is invaluable in preparation for a career in the built environment. A hands-on approach coupled with a real project presents unique opportunities in student learning. Learning in regards to the dynamics of team personalities, deadlines, approval procedures, and deliverables. One such hands-on based real project was to design, build, and install an 800 lb., 20 ft. long, and 10 ft. tall sculpture. The sculpture was to be built of recycled steel of a mustang in 10 weeks for the University’s library Spring 2016 exhibit. The project gave the students direct exposure to the phases required to take a highly constrained construction project from conception to completion (Impacted Project Delivery). Under the constraints of limited time, limited budget, unique materials, university regulatory requirements, and confined site location (second floor of library), the students learned how impacted constraints affect the design, engineering, and construction process. This paper outlines the methods used by the faculty adviser to facilitate student success

Using K’nex to Teach Large Scale Structures to Architects and ConstructionStudents

The College of Architecture and Environmental Design at California Polytechnic State University in San Luis Obispo (Cal Poly SLO) is the only college in the nation that includes architecture (ARCH), architectural engineering (ARCE) and construction management (CM) programs in the same college. Given the unique mix of disciplines and the emphasis on interdisciplinary collaboration, the ARCH and CM students take a five-course structural engineering sequence from the ARCE department. A challenge of these courses is to maintain some degree of computational rigor while offering a broader perspective that will benefit the ARCH and CM students. This paper reports on one means of accomplishing this using K’nex toys to illustrate the entire design -construction sequence and relate how structure fits into this process during the sequence’s culminating course

Requirement for the NF-kappa B family member Re1A in the development of secondary lymphoid organs

The transcription factor nuclear factor (NF)-kappaB has been suggested to be a key mediator of the development of lymph nodes and Peyer's patches. However, targeted deletion of NF-kappaB/ Rel family members has not yet corroborated such a function. Here we report that when mice lacking the RelA subunit of NF-kappaB are brought to term by breeding onto a tumor necrosis factor receptor (TNFR)1-deficient background, the trice that are born lack lymph nodes, foyer's patches, and an organized splenic microarchitecture, and have a profound defect in T cell-dependent antigen responses. Analyses of TNFR1/1RelA-deficient embryonic tissues and of radiation chimeras suggest that the dependence on RelA is manifest not in hematopoietic cells but rather in radioresistant stromal cells needed for the development of secondary lymphoid organs

Cloning of terminal transferase cDNA by antibody screening

A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the phage vector λgt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had β-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome