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Biomedical subjects

G A Rathbun

Publications and source records attributed to G A Rathbun.

12 recordsLinked to original sources
PubMed2000

Utilization of oriented peptide libraries to identify substrate motifs selected by ATM.

The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways.

Amino Acid Motifs↗
PubMed2000

Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice.

Motor incoordination, immune deficiencies, and an increased risk of cancer are the characteristic features of the hereditary disease ataxia-telangiectasia (A-T), which is caused by mutations in the ATM gene. Through gene targeting, we have generated a line of Atm mutant mice, Atm(y/y) mice. In contrast to other Atm mutant mice, Atm(y/y) mice show a lower incidence of thymic lymphoma and survive beyond a few months of age. Atm(y/y) mice exhibit deficits in motor learning indicative of cerebellar dysfunction. Even though we found no gross cerebellar degeneration in older Atm(y/y) animals, ectopic and abnormally differentiated Purkinje cells were apparent in mutant mice of all ages. These findings establish that some neuropathological abnormalities seen in A-T patients also are present in Atm mutant mice. In addition, we report a previously unrecognized effect of Atm deficiency on development or maintenance of CD4(+)8(+) thymocytes. We discuss these findings in the context of the hypothesis that abnormal development of Purkinje cells and lymphocytes contributes to the pathogenesis of A-T.

Animals↗
PubMed1998

A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

Animals↗
PubMed1998

Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV.

The DNA-end-joining reactions used for repair of double-strand breaks in DNA and for V(D)J recombination, the process by which immunoglobulin and T-cell antigen-receptor genes are assembled from multiple gene segments, use common factors. These factors include components of DNA-dependent protein kinase (DNA-PK), namely DNA-PKcs and the Ku heterodimer, Ku70-Ku80, and XRCC4. The precise function of XRCC4 is unknown, but it interacts with DNA ligase IV. Ligase IV is one of the three known mammalian DNA ligases; however, the in vivo functions of these ligases have not been determined unequivocally. Here we show that inactivation of the ligase IV gene in mice leads to late embryonic lethality. Lymphopoiesis in these mice is blocked and V(D)J joining does not occur. Ligase IV-deficient embryonic fibroblasts also show marked sensitivity to ionizing radiation, growth defects and premature senescence. All of these phenotypic characteristics, except embryonic lethality, resemble those associated with Ku70 and Ku80 deficiencies, indicating that they may result from an impaired end-joining process that involves both Ku subunits and ligase IV. However, Ku-deficient mice are viable, so ligase IV must also be required for processes and/or in cell types in which Ku is dispensable.

Animals↗
PubMed1998

Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly.

Immunoglobulin genes are assembled during lymphoid development by a series of site-specific rearrangements that are tightly regulated to ensure that functional antibodies are generated in B (but not T) cells and that a unique receptor is present on each cell. Because a common V(D)J recombinase comprising RAG1 and RAG2 proteins is used for both B- and T-cell antigen receptor assembly, lineage-specific rearrangement must be modulated through differential access to sites of recombination. We show here that the C-terminus of the RAG2 protein, although dispensable for the basic recombination reaction and for Ig heavy chain DH to JH joining, is essential for efficient VH to DJH rearrangement at the IgH locus. Thus, the RAG2 protein plays a dual role in V(D)J recombination, acting both in catalysis of the reaction and in governing access to particular loci.

Animals↗
PubMed1997

Growth retardation and leaky SCID phenotype of Ku70-deficient mice.

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

Animals↗
PubMed1989

Recombination between VH pseudogenes.

The peculiar pattern of nucleotide differences between germline VH genes V104A/VAR104 and 122B could be the result of an exchange of genetic information through gene conversion. A palindromic sequence in one of the genes seems to have been the target site for the break that initiated recombination.

Animals↗
PubMed1988

Molecular characterization of the A/J J558 family of heavy chain variable region gene segments.

The nucleotide sequences of the coding as well as the flanking regions of 11 A/J J558 heavy chain variable region (VH) gene segments are presented. Among these J558 VH segments was the unrearranged germline VH gene segment recruited in the predominant A strain-specific anti-arsonate response. Three other VH gene segments that are greater than 92% related to the p-azophenylarsenate (Ars) A VH gene segment were also isolated. Detailed analysis of the nucleotide sequences of these as well as the remaining seven J558 VH gene segments reveal that the J558 VH gene family is composed of distinct, but related, J558 VH subfamilies. Deletion mapping analyses were used to position the Ars A VH gene segment proximally with respect to the DH-JH clusters within the J558 VH gene family and distally with respect to its own J558 subfamily. The documentation of J558 VH subfamilies is discussed in the context of J558 VH family evolution and diversification.

Animals↗
PubMed1988

Diversity of the mouse T cell receptor C gamma 1 gene: structural analysis in C57BL/Ka.

We have isolated an unusual T cell receptor gamma chain cDNA clone (gamma 7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C gamma 1 constant region exons preceded by 1.5 kb of J-C gamma 1 intron. The gamma 7.1 coding region is extremely homologous to the C gamma 1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C gamma 1 intronic region contains two DNA segments (termed psi J gamma 1 and psi J gamma 2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that gamma 7.1 may be derived from a large, variable region-containing precursor.

Amino Acid Sequence↗
PubMed1987

Organization of the murine immunoglobulin VH complex in the inbred strains.

Deletion mapping analyses have been employed to order the heavy chain variable region (VH) gene families in three inbred murine strains. These nine VH gene families have been positioned with respect to the J558 and 3660 VH families in A/J (Ighe) as follows: 3609-J558-(J606,VGAM3-8,S107)-3660-(X24,Q52,7183 )-DH. Maps generated with respect to J558 in the BALB/c (Igha) and C57BL/6 (Ighb) strains are consistent with these results. The organization of the VH complex produced by deletion mapping is quite different from the accepted map generated by other methods, particularly in that J558 is more DH distal and 3660 is more DH proximal than previously thought. The order presented here is compatible with VH rearrangement frequencies suggesting preferential utilization of DH-proximal VH gene segments. Our data also indicate that interspersion of some VH family members may be a common feature of the murine VH complex since the 3609 VH family is interdigitated in the three strains and a Q52 VH gene segment is interspersed in C57BL/6.

Animals↗
PubMed1982

The dual nature of complement enhancement by venom from the Mojave rattlesnake (Crotalus scutulatus scutulatus).

Venom from the Mojave rattlesnake enhanced the antibody mediated hemolytic activity of guinea pig complement when either human or sheep erythrocytes were used as the target cells. Amplification of lytic activity was shown to act in a preferential manner with respect to species of erythrocyte origin. Two steps appear to be necessary for demonstration of enhancement of complement hemolytic activity :(1) interaction of the venom with the complement components; and (2) membrane directed interaction of complement components in association with the active venom factor. These studied extend the findings reported earlier on venom induced complement enhancement.

Animals↗