Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D.

Carboxypeptidase D (CPD) is a recently described 180-kD enzyme with carboxypeptidase E-like enzymatic properties. CPD has been proposed to be present in the secretory pathway and to contribute to peptide hormone processing in the Cpe(fat)/Cpe(fat) mouse, which lacks functional CPE. Sequence analysis of cDNA clones encoding rat CPD show the protein to contain an amino-terminal signal peptide, three carboxypeptidase-like domains, a putative transmembrane domain, and a 60-amino-acid cytoplasmic tail. Whereas active site, substrate-binding, and metal-binding residues of other metallocarboxypeptidases are conserved in the first two domains of CPD, several of the critical residues are not conserved in the third domain; this third domain is not predicted to form an active carboxypeptidase. The overall homology between rat CPD and the duck homolog gp180 is high, with 75% amino acid identity. The three carboxypeptidase domains show 66%, 83%, and 82% amino acid identity between rat CPD and duck gp180. Homology is also high in the transmembrane domain (86%) and in the cytoplasmic tail (97%). The mouse Cpd gene maps to the medial portion of chromosome 11, approximately 45.5 cM distal to the centromere. Northern blot analysis of CPD mRNA shows major bands of approximately 8 and 4 kb in many rat tissues, and additional species ranging from 1.4 to 5 kb that are expressed in some tissues or cell lines. CPD mRNA is detectable in most tissues examined, and is most abundant in hippocampus, spinal cord, atrium of the heart, colon, testis, and ovaries. In situ hybridization of CPD mRNA shows a distribution in many cells in rat brain and other tissues, with high levels in hippocampus, olfactory bulb, and the intermediate pituitary. The broad distribution is consistent with a role for CPD in the processing of many peptides and proteins that transit the secretory pathway.

Use of recombinant congenic and congenic strains of NOD mice to identify a new insulin-dependent diabetes resistance gene.

Insulin-dependent diabetes mellitus (IDDM) in NOD/Lt mice represents a complex polygenic disease. NOR/Lt is a recombinant congenic strain (RCS) in which limited regions of the NOD/Lt genome have been replaced by genome from the C57BL/KsJ strain. NOR mice are insulitis resistant and diabetes free despite genetic identity with NOD at numerous chromosomal regions containing previously described insulin-dependent diabetes (Idd) genes, including the strongly diabetogenic H2g7 major histocompatibility complex (MHC) haplotype. The present study revealed BKs-derived genome on segments of chromosomes (Chr) 1, 2, 4, 5, 7, 11, 12, and 18, approximating 11.6% of the total NOR genome analyzed. (NOD x NOR)F2 segregation analysis was employed to identify chromosomal regions in NOR containing Idd resistance alleles. IDDM developed in 33% (10/30) of F1 females, and 29.3% (36/123) of F2 females aged to 1 yr. A previously unrecognized diabetes resistance locus (designated Idd13r) strongly protective in homozygous state was identified on NOR Chr 2 in linkage with the Il1 alpha structural gene. The existence of this locus was confirmed by construction of a NOD stock congenic for NOR-derived markers on Chr 2. Our analysis shows the utility of RCS and congenic stocks for the identification and isolation of non-MHC genes with strong antidiabetogenic functions.

Bone marrow abnormalities in the non-obese diabetic mouse.

Several lines of evidence point to abnormalities of the phenotype, cytokine responses, and function of cells of the myeloid lineage in non-obese diabetic (NOD) mice. In this study we have characterized the phenotype and myeloid progenitor function of NOD bone marrow. Two hematopoietic differentiation antigens, Ly-6C and AA4.1, are expressed abnormally on NOD bone marrow cells. While multilineage erythromyeloid progenitor cells (day 12 CFU-S) are normal in number in NOD mice, more differentiated myeloid progenitors are deficient in their in vitro responses to IL-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-5. Since the diabetes-predisposing Idd-5 gene of NOD mice maps close to the IL-1 receptor, we tested NOD bone marrow cells for a defect in synergy between IL-1 and IL-3; no defect was found. The defects in myelopoiesis described here may predispose the NOD mouse to autoimmunity by impairing macrophage maturation.

Murine polypyrimidine tract binding protein. Purification, cloning, and mapping of the RNA binding domain.

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a approximately 60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.

A recombination event in the 5' flanking region of the Ly-6C gene correlates with impaired expression in the NOD, NZB and ST strains of mice.

The murine alloantigen, Ly-6C, is found on 45% of bone marrow cells, 25% of splenocytes and 15% of lymph node cells in all inbred strains of mice tested, with the exception of NOD, NZB and ST. In these three strains, Ly-6C expression can be detected on only 5% of bone marrow cells and not at all on cells from spleen or lymph node. NOD and NZB, which are models for the autoimmune diseases, diabetes and lupus, respectively, also exhibit a depressed syngeneic mixed lymphocyte reaction. Southern blot analysis reveals a restriction fragment length polymorphism involving the Ly-6C gene which is unique to these three strains. Cloning of the affected genomic segment from the NOD mouse indicates the presence of an interruption in the flanking region of the Ly-6C gene at a point 475 bp upstream of the transcription initiation site and the consequent separation of distal 5' sequences from the body of the gene by at least 10 kb. Inspection of the recombination borders reveals a set of inverted copies of a mouse repetitive R element. Transfection of the Ly-6C genes from NOD and BALB/c into a murine carcinoma line indicates relative functional impairment of the NOD gene, thus paralleling performance in vivo.

The CD59 antigen is a structural homologue of murine Ly-6 antigens but lacks interferon inducibility.

A cDNA encoding the human leukocyte antigen CD59 has been isolated from the erythroid cell line K-562 and its identity confirmed through expression in COS cells. Northern blotting reveals three message species of approximately 800, 1400 and 2000 bases in size, which are constitutively expressed in all lymphoid, erythroid, myeloid, and neural cell types tested thus far. Southern blotting of human DNA indicates a pattern consistent with the presence of a single gene, which has been mapped to chromosome 11 by somatic cell hybrids. Also, the finding of a transcriptionally active cross-hybridizing gene in monkey cells suggests conservation of CD59 sequences among primates. Comparison of the CD59 protein sequence with those of the Ly-6E and Ly-6C antigens discloses a similarity in overall structure, including the alignment of abundant cysteine residues, hydrophobic carboxy termini and conservation of amino acids surrounding the proposed phosphatidylinositol-glycan modification site for Ly-6 molecules. Unlike Ly-6, however, CD59 expression does not appear to be inducible with interferons. This, along with its limited homology and different tissue distribution, cast doubt upon the functional equivalence of CD59 and either of the well-characterized mouse Ly-6 proteins.

Kinetic analysis of Ly-6 gene induction in a T lymphoma by interferons and interleukin 1, and demonstration of Ly-6 inducibility in diverse cell types.

The Ly-6 locus contains multiple genes encoding cell surface proteins, two of which, when cross-linked by antibodies, effect antigen-independent activation of T lymphocytes. In this study, cDNA for Ly-6-encoded antigens have been used as probes to examine RNA from various tissues and transformed cell lines for constitutive levels of Ly-6 RNA expression. Analyses of RNA prepared from several different tissues revealed a high level of expression of Ly-6 RNA in kidney, spleen, heart and thymus, with a more moderate level of expression in liver, brain and lung tissue cells. A survey of various cell lines demonstrated the presence of Ly-6 RNA in many, but not all T lymphocytic cell lines, in L cells, the Meth A fibrosarcoma, in the TCMK kidney cell line, and in the Neuro-2a neuroblastoma. We also evaluated the expression of Ly-6 RNA in cells after treatments with interferons (IFN) and interleukin 1 (IL1). Treatment of lymphoid cells with IFN (alpha/beta and gamma), known to increase cell surface Ly-6 antigen expression in normal T cells, was correlated with increases in Ly-6 RNA levels. Increases in levels of RNA correlated with increases in levels of the Ly-6A/E or Ly-6C antigens. Several T lymphoid cell lines exhibiting Ly-6 RNA inducibility by IFN were similarly inducible with IL1. Kinetic experiments using one such line, (YAC-1), showed that the induction of Ly-6 RNA mediated by IFN-alpha/beta occurred rapidly (within 4 h), while the induction by IL1 required relatively more time (approximately 8 h). Although the actions of IFN-alpha/beta were not blocked by cycloheximide, the presence of this protein synthesis inhibitor significantly attenuated the effects of IL1 and IFN-gamma on Ly-6 RNA transcription. Induction by IFN-gamma as well as IL1 could be blocked completely by co-culture with anti-IFN-gamma, implicating IFN-gamma as a mediator of the induction by IL1.