Murine cDNA Clone Research Articles

Sequence and organization of the mouse poly (ADP-ribose) polymerase gene

Using a human cDNA probe, we have isolated murine genomic and cDNA clones corresponding to the nuclear enzyme poly (ADP-ribose) polymerase (ADPRP). Northern analysis with the mouse cDNA clones reveals transcripts of 3.7-3.8 kb corresponding in size to the human ADPRP transcript. DNA sequence comparisons between mouse and human clones reveals extensive amino acid sequence conservation within regions harboring DNA binding, NAD+ binding or automodification domains. A survey among mouse inbred strains for restriction fragment length polymorphism (RFLP) reveals at least three distinct ADPRP alleles. The segregation of alleles among mouse genetic recombinants positions ADPRP on mouse chromosome 1 between the complement receptor-related gene At-3 and the Fc receptor locus FcR. Furthermore, ADPRP is closely associated with the autoimmune locus gld (generalized lymphadenopathy).

Identification of murine complement receptor type 2.

A rabbit antiserum reactive with the human complement component C3d/Epstein-Barr virus receptor (complement receptor type 2, CR2) immunoprecipitates a Mr 155,000 murine B-cell surface antigen. The apparent molecular weight and cellular distribution of this murine antigen are similar to those of human CR2. Cells expressing the murine protein bind sheep erythrocytes coated with antibody and murine C1-C3d but do not bind Epstein-Barr virus at all. The monospecific antiserum to human CR2 together with goat F(ab')2 anti-rabbit IgG blocks attachment of the C3d-coated erythrocytes to receptor-bearing murine B lymphocytes. To further characterize murine CR2, a lambda gt11 library from the murine late pre-B-cell line 70Z/3 was screened with human CR2 cDNA. A partial cDNA clone of 3.5 kilobases with 79% amino acid sequence identity to human CR2 in the unique intracytoplasmic region and 63% identity to the sixth human CR2 repeat was obtained. Blot hybridization with the murine cDNA clone identified an RNA species of approximately 4.7 kilobases, similar in size to human CR2 mRNA, from a murine B-cell line but not from a murine T-cell line.

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19.

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.

Evidence that immunological variants of p53 represent alternative protein conformation

In normal murine lymphocytes the cellular oncoprotein, p53, exists as two immunologically distinct species which are reciprocally expressed in quiescent (p53-Go) and mitogenically stimulated (p53-G÷) cells. More recently, we have identifed discrete forms of p53, immunologically similar to p53-Go and p53-G÷, in 3T3 cells transformed by simian virus 40 (SV40). In this report, we demonstrate that immunologically distinct p53 species can also be expressed in vitro, from a single murine p53 cDNA clone. The p53 variants expressed in vitro and in SV40-transformed 3T3 cells have been studied by immunoprecipitation and Western blotting. Immunoprecipitation data indicated that, in their native conformations, the p53 variants react with either the PAb421 or RA3.2C2 monoclonal antibodies, but not with both. When analyzed by Western blotting, however, the denatured proteins were found to react with both monoclonal antibodies. This suggests that the p53 protein is flexible and can fold in alternative conformations so as to expose or mask different epitopes. We propose, therefore, that immunologically distinct p53 species are generated, at least partly, by conformational changes in a single polypeptide species.

Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5).

The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in lambda phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (approximately 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia.

Cloned murine T200 (Ly-5) cDNA reveals multiple transcripts within B- and T-lymphocyte lineages.

The murine T200 family of cell-surface glycoproteins is expressed on hematopoietic lineage cell types in a developmentally regulated manner with different lymphocyte subpopulations expressing cross-reactive but structurally distinct forms. To investigate the differences between these forms and the regulation of these differences, murine T200 cDNA clones were isolated by using a probe obtained by subtractive hybridization. This procedure made use of a T200+ L-cell transfectant and the parent Ltk- cell line. The 1.9-kilobase (kb) cDNA clone was sequenced and found to contain the coding region of the COOH-terminal 331 amino acids and 0.9 kb of 3' untranslated region. Where the sequence overlapped with the rat sequence, 80-90% homology was observed. RNA blot analysis revealed that B-lineage cell lines express either a 5.6-kb or a 6.5-kb mRNA correlating to the size difference of the T200 glycoprotein synthesized. Similarly, in the T-cell lineage a helper T-cell clone and a cytotoxic T-cell clone express T200 transcripts of 5.6 kb and 5.9 kb, respectively, which correlate with the distinct sizes of their T200 glycoproteins. A T200-negative mutant of a T-cell line was found to express full-length T200 mRNA, although at a diminished level.

Isolation and characterisation of a murine cDNA clone highly homologous to the yeast L29 ribosomal protein gene

We report here the isolation of a murine cDNA clone (cCL3) which is homologous to the mRNA of the yeast ribosomal protein L29. Comparison of the deduced amino-acid composition of cCL3 to those known for rat ribosomal proteins indicates that this cDNA codes for mammalian ribosomal protein L27'. The gene corresponding to the cDNA is present at approximately 15 copies per genome, some of these probably representing processed pseudogenes. The cDNA hybridizes to an mRNA of 600 nucleotides from various mammals at high stringency, and to an avian transcript of the same size at low stringency. It has been suggested that L29 is involved in peptidyl transferase activity. The strong homology of mammalian L27' to yeast L29 suggests a function which has been conserved throughout evolution, and thus L27' may also be involved in peptidyl transferase activity.

Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin 1 beta.

A human interleukin 1 beta (IL 1 beta) cDNA probe was utilized to identify a homologous murine cDNA clone. The murine cDNA encodes a 269-residue protein which is 67% homologous to human IL 1 beta. The murine sequence was engineered for expression in mammalian cells and directs the synthesis of biologically active IL 1. This protein, termed murine IL 1 beta, is only 22% homologous with the previously described murine IL 1 sequence. Both IL 1 alpha and IL 1 beta are encoded by single genes, but IL 1 beta mRNA is about fivefold more abundant in a stimulated macrophage cell line.

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.

Rapid Identification of Functional Murine Adenosine Deaminase cDNA Clones by Genetic Complementation in Bacteriaa

Rapid Identification of Functional Murine Adenosine Deaminase cDNA Clones by Genetic Complementation in Bacteriaa

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli.

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.

Lack of gene rearrangement and mRNA expression of the beta chain of the T cell receptor in spontaneous rat large granular lymphocyte leukemia lines.

Using the murine cDNA clone for the beta chain of the T cell antigen receptor, we have examined four highly cytotoxic rat large granular lymphocyte (LGL) leukemia lines for the expression of unique rearrangements and mRNA transcription of the genes coding for the T cell antigen receptor. In contrast to normal rat T cells and nine rat T cell lines, the LGL leukemia lines exhibited no detectable gene rearrangements in the beta chain locus after digestion of LGL DNA by four restriction enzymes. Northern blots containing RNA from these LGL tumor lines demonstrated a low level of aberrant or nonrearranged beta chain transcription (less than 10 copies per cell) but virtually no translatable 1.3 kilobase message. These results demonstrate that LGL leukemia lines which mediate both natural killer (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) activities do not express the beta chain of the T cell receptor. The nature of the NK cell receptor for antigen remains elusive.

Isolation of a cDNA clone corresponding to an X-linked gene family (XLR) closely linked to the murine immunodeficiency disorder xid

The striking number of human and murine immunodeficiency disorders which map to the X chromosome suggests that genes localized on this chromosome must have important roles in lymphocyte development. At least seven distinct disorders in the human and two in the mouse disrupt lymphocyte maturation, particularly that of B cells, at characteristic stages. As functional genes mapping to the X chromosome in one mammal are found on the X chromosome in all other mammals, the same genes regulating lymphocyte development are expected to be found on the X chromosome in mouse and man. Investigations into the possible mechanisms of these X-linked disorders have been hampered by the lack of molecular probes for the genes or gene products affected; because of this, and the possibility of correlating one or more of the several hundred B- or T-cell-specific genes with a specific mutation, we surveyed 15 different B- and T-cell-specific cDNA clones for localization to the X chromosome. We report here the characterization of one of these murine cDNA clones, which hybridizes with a large, X-linked gene family, designated XLR (X-linked, lymphocyte-regulated). We show that the XLR gene family is closely linked to the X-linked immunodeficiency described in the CBA/N mouse strain (xid), by restriction fragment length polymorphism (RFLP) analysis of DNA from mice congeneic for xid. This finding, together with data on the expression of the XLR locus in B cells, indicates that this gene family either includes the locus defined by the xid mutation or is adjacent to it in a gene complex which may be important in lymphocyte differentiation.

Isolation and expression in Escherichia coli of a cDNA clone encoding human β-glucuronidase

Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of β-glucuronidase (BG) activity. To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones. The SV40-transformed human fibroblast cDNA library of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280–289] was screened with a fragment of a murine BG cDNA clone (pGUS-1). The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length. The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII- SstI restriction fragment. Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73 %. Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropylthio -β-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG). This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.