Structure and promoter activity of the mouse CDC25A gene.

CDC25A is a member of a group of highly related, dual-specificity phosphatases that promote cell cycle phase transitions by regulating the activity of cyclin-dependent kinases. Here we report the cloning and genomic sequence of 21,067 nucleotides encompassing the mouse CDC25A gene. The coding sequence is expressed from 17,904 bp of genomic DNA comprising 15 exons. We also mapped the transcription initiation site to a consensus initiator element proximal to an SP1 site. Approximately 1 kb of sequence upstream of the transcription initiation site confers promoter activity and cell type specificity to a reporter gene construct. Surprisingly, transcription from this promoter was repressed by over-expression of catalytically active but not catalytically inactive CDC25A protein. We also show, using NIH 3T3 cells, that murine CDC25A mRNA levels fluctuate only modestly over the cell cycle. Our findings provide insights into the regulation of CDC25A expression and have facilitated construction of gene knock-out vectors.

Biochemical and functional analysis of mice deficient in expression of the CD45-associated phosphoprotein LPAP.

The role of the CD45-associated phosphoprotein (LPAP / CD45-AP) during an immune response remains unclear. To understand better the function of LPAP we generated LPAP-deficient mice by disrupting exon 2 of the LPAP gene. LPAP-null mice were healthy and did not show gross abnormalities compared to their wild-type littermates. However, immunofluorescence analysis of T and B lymphocytes revealed a reduced expression of CD45, which did not affect a particular subpopulation. In contrast to a recent report (Matsuda et al., J. Exp. Med. 1998. 187: 1863 - 1870) we neither observed significant alterations of the assembly of the CD45 / lck-complex nor of polyclonal T-cell responses. However, lymphnodes from LPAP-null mice showed increased cellularity, which could indicate that expression of LPAP might be required to prevent expansion of lymphocytes in particular lymphatic organs rather than potentiating immune responses.

Expression and regulation of mRNA for distinct isoforms of cAMP-specific PDE-4 in mitogen-stimulated and leukemic human lymphocytes.

We reported previously that the gene for PDE-1B1 is induced in isolated human peripheral blood lymphocytes (HPBL) following mitogenic stimulation (Jiang, X., Li, J., Paskind, M., and Epstein, P.M. [1996] Proc. Natl. Acad. Sci. USA 93, 11,236-11,241). Using reverse transcription-polymerase chain reaction (RT-PCR), we investigated possible changes in the expression of the four genes for cAMP-specific phosphodiesterase (PDE-4A-D) in HPBL under the same conditions. Isolated, quiescent HPBL express mRNA for PDE-4B as the principal transcript. Following mitogenic stimulation with phytohemagglutinin (PHA), mRNA for PDE-4A and PDE-4D are clearly induced. HPBL appear not to express PDE-4C under resting or stimulated conditions. The PHA induced increase in PDE-1B1, PDE-4A, and PDE-4D mRNA is mimicked by incubation of HPBL with dibutyryl cAMP (dBcAMP) and 1-methyl-3-isobutylxanthine (IBMX). The B-lymphoblastoid cell line, RPMI 8392, and the T-leukemic cell line, Molt 4, express PDE-4A mRNA as the most abundant transcript, but incubation with dBcAMP and IBMX induces an increase in the expression of mRNA for PDE-4B in both of these cell lines, and in PDE-4D3 in the RPMI 8392 cell line. These studies demonstrate that expression of mRNA for PDE-1B1 and some of the subtypes of PDE-4 are induced in HPBL following mitogenic stimulation, possibly secondarily to elevation of cAMP induced by the mitogen. As already indicated for PDE-1B1, some of these subtypes of PDE-4 might also provide additional therapeutic targets for treatment of immunoproliferative disorders and immune dysfunction.

Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases.

Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells.

Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production.

Interferon-gamma-inducing factor (IGIF, interleukin-18) is a recently described cytokine that shares structural features with the interleukin-1 (IL-1) family of proteins and functional properties with IL-12. Like IL-12, IGIF is a potent inducer of interferon (IFN)-gamma from T cells and natural killer cells. IGIF is synthesized as a biologically inactive precursor molecule (proIGIF). The cellular production of IL-1beta, a cytokine implicated in a variety of inflammatory diseases, requires cleavage of its precursor (proIL-1beta) at an Asp-X site by interleukin-1beta-converting enzyme (ICE, recently termed caspase-1). The Asp-X sequence at the putative processing site in proIGIF suggests that a protease such as caspase-1 might be involved in the maturation of IGIF. Here we demonstrate that caspase-1 processes proIGIF and proIL-1beta with equivalent efficiencies in vitro. A selective caspase-1 inhibitor blocks both lipopolysaccharide-induced IL-1beta and IFN-gamma production from human mononuclear cells. Furthermore, caspase-1-deficient mice are defective in lipopolysaccharide-induced IFN-gamma production. Our results thus implicate caspase-1 in the physiological production of IGIF and demonstrate that it plays a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors would provide a new class of anti-inflammatory drugs with multipotent action.

Inhibition of calmodulin-dependent phosphodiesterase induces apoptosis in human leukemic cells.

Cytosolic extracts from a human lymphoblastoid B-cell line, RPMI-8392, established from a patient with acute lymphocytic leukemia, contain two major forms of cyclic nucleotide phosphodiesterase (PDE): Ca2+-calmodulin dependent PDE (PDE1) and cAMP-specific PDE (PDE4). In contrast, normal quiescent human peripheral blood lymphocytes (HPBL) are devoid of PDE1 activity [Epstein, P. M., Moraski, S., Jr., and Hachisu, R. (1987) Biochem. J. 243, 533-539]. Using reverse transcription-polymerase chain reaction (RT-PCR), we show that the mRNA encoding the 63-kDa form of PDE1 (PDE1B1) is expressed in RPMI-8392 cells, but not in normal, resting HPBL. This mRNA is, however, induced in HPBL following mitogenic stimulation by phytohemagglutinin (PHA). Also using RT-PCR, the full open reading frame for human PDE1B1 cDNA was cloned from RPMI-8392 cells and it encodes a protein of 536 amino acids with 96% identity to bovine, rat, and mouse species. RT-PCR also identifies the presence of PDE1B1 in other human lymphoblastoid and leukemic cell lines of B- (RPMI-1788, Daudi) and T-(MOLT-4, NA, Jurkat) cell origin. Inhibition of PDE1 or PDE4 activity by selective inhibitors induced RPMI-8392 cells, as well as the other cell lines, to undergo apoptosis. Culture of RPMI-8392 cells with an 18-bp phosphorothioate antisense oligodeoxynucleotide, targeted against the translation initiation region of the RPMI-8392 mRNA, led to a specific reduction in the amount of PDE1B1 mRNA after 1 day, and its disappearance after 2 days, and induced apoptosis in these cells in a sequence specific manner. This suggests that PDEs, particularly PDE1B1, because its expression is selective, may be useful targets for inducing the death of leukemic cells.

Identification and characterization of ICH-2, a novel member of the interleukin-1 beta-converting enzyme family of cysteine proteases.

Interleukin-1 beta converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 beta cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.

Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock.

IL-1 beta-converting enzyme (ICE) cleaves pro-IL-1 beta to generate mature IL-1 beta. ICE is homologous to other proteins that have been implicated in apoptosis, including CED-3 and Nedd-2/lch-1. We generated ICE-deficient mice and observed that they are overtly normal but have a major defect in the production of mature IL-1 beta after stimulation with lipopolysaccharide. IL-1 alpha production is also impaired. ICE-deficient mice are resistant to endotoxic shock. Thymocytes and macrophages from the ICE-deficient animals undergo apoptosis normally. ICE therefore plays a dominant role in the generation of mature IL-1 beta, a previously unsuspected role in production of IL-1 alpha, but has no autonomous function in apoptosis.

Cladistic analyses of combined traditional and molecular data sets reveal an algal lineage.

The chromophyte algae are a large and biologically diverse assemblage of brown seaweeds, diatoms, and other golden algae classified in 13 taxonomic classes. One subgroup (diatoms, pedinellids, pelagophytes, silicoflagellates, and certain enigmatic genera) is characterized by a highly reduced flagellar apparatus. The flagellar apparatus lacks microtubular and fibrous roots, and the flagellum basal body is attached directly to the nucleus. We hypothesize that the flagellar reduction is the result of a single evolutionary series of events. Cladistic analysis of ultrastructural and biochemical data reveals a monophyletic group that unites all taxa with a reduced flagellar apparatus, supporting our hypothesis. Phylogenetic analyses of 18S rRNA gene sequence data provide strong resolution within most of the major groups of chromophytes but only weakly resolve relationships among those groups. Some of the molecularly based most parsimonious trees, however, also unite the taxa with a reduced flagellar apparatus, although the diatoms are not included in this lineage. This grouping is further supported by a posteriori character weighting of the molecular data, suggesting that flagellar reduction occurred at least twice in parallel evolutionary series of events. To further test our hypothesis of a single evolutionary reduction in the flagellar apparatus, we combine the two data sets and subject the hybrid data matrix to parsimony analysis. The resulting trees unite the diatoms with the other reduced flagellar apparatus algae in a monophyletic group. This result supports our hypothesis of a single evolutionary reduction and indicates the existence of a previously unrecognized lineage of algae characterized by a highly reduced flagellar apparatus. Further, this study suggests that the traditional classification of the diatoms with the chrysophytes and xanthophytes in the division (= phylum) Chrysophyta, as presented in most textbooks, is unsatisfactory and that a significantly different classification should be employed.

A novel cyclic GMP stimulated phosphodiesterase from rat brain.

A cDNA clone for cyclic GMP Stimulated Phosphodiesterase (cGSPDE; PDE2) was isolated from a rat brain cDNA library. The cDNA has an open reading frame which encodes a protein of 928 amino acids of which 829 are identical with the reported bovine adrenal gland cGSPDE cDNA (Sonnenburg, W.K., Mullaney, P.J., and Beavo, J.A. (1991) J. Biol. Chem. 266, 17655-17661). Although the overall homology of these two cDNAs is high, they are distinctly different in their 5' ends, with the N-terminal 37 amino acids of the rat brain protein showing no homology with the N-terminal end of the bovine adrenal protein. Hydrophilicity plots show that in contrast to the bovine adrenal cGSPDE, the N-terminal end of the rat brain cGSPDE is highly hydrophobic. Isolation and analysis of a genomic clone for cGSPDE from a rat genomic library shows the presence of an exon/intron junction at the Gln39 codon. The cGSPDE cDNA we have isolated and that of Sonnenburg et al. represent alternatively spliced mRNA products from the same gene, with the brain isoform designed to be targeted to membranes.

Mutation of a phenylalanine conserved in SH3-containing tyrosine kinases activates the transforming ability of c-Abl.

c-abl is the normal cellular homolog of the v-abl transforming gene of Abelson murine leukemia virus. By constructing recombinants between c- and v-abl retroviruses, we show that a point mutation in c-Abl is sufficient to change the myristoylated form of c-Abl into a protein able to transform fibroblasts, but not capable of transforming bone marrow or inducing Abelson disease. This activating mutation, which changes the phenylalanine at amino acid 420 to valine (F420V) found in the homologous position of v-Abl, is positioned outside of the SH3 domain, a region typically modified in transforming alleles of abl. Phenylalanine 420 is perfectly conserved among tyrosine kinases with N-terminal SH3 domains (the Src and Abl families). The equivalent position in other protein tyrosine kinases is a conserved hydrophobic residue that predicts the specific family to which that kinase belongs. Mutation of phenylalanine 420 to other hydrophobic residues activates c-Abl. Unlike other transforming variants of Abl, the F420V mutant protein is not highly phosphorylated on tyrosine. Mutation of the nearby proposed autophosphorylation site, tyrosine 412, shows that this tyrosine is not strictly required for fibroblast transformation in either F420V or SH3-deleted variants of c-Abl (IV).

Four murine c-abl mRNAs arise by usage of two transcriptional promoters and alternative splicing.

The c-abl gene in mice is transcribed into two major and at least two minor mRNAs which have different 5'-ends, but are otherwise colinear. Here we show that the two major mRNAs are initiated by separate promoters and that the minor transcripts arise by alternative splicing. Like in the human gene, one of the alternative murine 5' c-abl exons lies far upstream of the remaining exons. The major mRNA that begins with this exon has about 1275 nucleotides upstream of the abl coding region. Interestingly, this unusually long upstream mRNA segment contains multiple short open reading frames both in mouse and man and is highly conserved in sequence between these species. The 5'-most c-abl promoter contains several sequence motifs that are highly conserved between mouse and man. The downstream promoter is much less conserved.

The first intron in the human c-abl gene is at least 200 kilobases long and is a target for translocations in chronic myelogenous leukemia.

The c-abl protooncogene is unusual in two respects; it has multiple, widely space N-terminal coding exons transcribed by different promoters, and it is the target of the translocations that form the Philadelphia chromosome found in cells of chronic myelogenous leukemia patients. To understand the organization of the gene in normal and chronic myelogenous leukemia patient DNA we have mapped c-abl by pulsed field gradient gel electrophoresis. We find that one of the alternative 5' exons of the gene lies at least 200 kilobases upstream of the remaining c-abl exons, posing formidable transcription and splicing problems. The 5'-most c-abl exon includes an unusually long 1,276-base-pair segment that contains 15 ATG codons and multiple short open reading frames, upstream of the abl initiator codon. Its peculiar structure suggests that c-abl may be decapitated in most chronic myelogenous leukemia patients, and we demonstrate that this is the case in the chronic myelogenous leukemia cell line K562.

Overlapping cDNA clones define the complete coding region for the P210c-abl gene product associated with chronic myelogenous leukemia cells containing the Philadelphia chromosome.

The Philadelphia chromosome, observed in greater than 90% of patients with chronic myelogenous leukemia, results from a reciprocal translocation between chromosomes 9 and 22. The translocation breakpoint on chromosome 9 occurs near the ABL gene and correlates with the production of a chronic myelogenous leukemia-specific 8.5-kilobase ABL-related mRNA species accompanied by a structurally altered ABL protein (P210c-abl). The N-terminal sequence of the protein is derived from the BCR gene on chromosome 22. We have isolated overlapping cDNA clones from the K-562 cell line corresponding to approximately 8.5 kilobases of mRNA and have sequenced 2550 nucleotides at the 5' end. Our results indicate that the 5' end of the 8.5-kilobase mRNA consists of greater than 400 nucleotides of noncoding sequence that are greater than 80% G + C rich. Based on our sequence analysis, we propose that initiation of translation occurs at nucleotide 471, such that the initial 927 amino acids of P210c-abl are derived from BCR sequences. Our cDNA clones thus define the complete coding sequences for the P210c-abl gene product.

Alternative 5' exons in c-abl mRNA.

The cellular abl proto-oncogene encodes a protein-tyrosine kinase and is expressed in many cell types in two or three mRNA size species. Four types of mouse c-abl cDNAs have been cloned from 70Z/3 lymphoid cells that have different 5' sequences encoding predicted N-terminal regions of 20-45 amino acids. One of the four cDNAs has a predicted N-terminal sequence of met-gly-gln in common with the gag N terminus of v-abl. The 5' heterogeneity appears to be generated by alternative addition of 5' exons onto a common set of 3' exons. Alternative splicing occurs at the same site at which bcr sequences join to abl sequences in the Philadelphia chromosome translocation.

Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines.

The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.

Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.

The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products (VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines that represent analogues of cells at early stages of B-lymphocyte differentiation. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.

Cloning and analysis of reverse transcript P160 genomes of Abelson murine leukemia virus.

Circular duplex reverse transcripts of the genome of a strain of Abelson murine leukemia virus that encodes a 160,000-molecular-weight protein were isolated, cleaved with HindIII restriction endonuclease, and cloned into the unique HindIII site of lambda phage Charon 21A. Recombinant phage clones, some of which were infectious in transfection assays, were found to contain a 789-base-pair region specific for Abelson murine leukemia virus; this region is not found in other strains of this virus. The extra sequence was localized by restriction endonuclease and electron microscopic heteroduplex analysis. Sequence analysis showed no homology at the ends of the extra sequence, implying that it was deleted by an event that did not utilize sequence homology. The sequence of this unique region has an open reading frame through its entirety.

Somatic variants of murine immunoglobulin lambda light chains.

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.

Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region.

To examine germ line genes of the heavy chain variable region (VH) that might contribute to formation of antibodies of the NPb family, we have derived cDNA clones from two hybridomas making NPb antibodies. One, B1-8, made an IgM protein and was derived during a primary response; the other, S43, made an IgG2a protein and was derived during a hyperimmune response. Sequence comparison of the two clones showed that they differed by only 10 bp in the VH region, had very different D segments and had identical J segments (J2). A set of closely related germ line VH genes was then cloned from a partial Eco RI library of C57Bl/6 DNA. By comparing the germ line VH regions to the cDNA VH regions, we identified seven potential candidates for encoding the VH regions of NPb antibodies. The seven VH regions were sequenced, and one V(186-2) contained exactly the DNA sequence found in the clone derived from B1-8. None of the DNA sequence differences that distinguished the S43-derived clone from the B1-8 clone was found in any of the other six germ line genes. Because the S43 sequence was more closely related to the V(186-2) germ line sequence than to any of the other VH genes, we conclude that the differences between the genes resulted from somatic mutation and that the two hybridomas derived their VH regions from the same germ line gene. Certain of the sequenced VH genes contain crippling mutations; the repertoire of germ line VH genes that can contribute to the diversity of antibodies may therefore be less than the total number of genes detectable by hydridization.