Sense and antisense transcripts of the maize MuDR regulatory transposon localized by in situ hybridization.

Activity of the Mutator transposons of Zea mays varies in different tissues and at different stages of development. In the soma, Mu elements excise at a high frequency late in tissue development. In germ cells, Mu elements rarely excise, but they amplify and insert at high levels around the time of meiosis. At all other times, Mu elements can duplicate and insert at a low frequency. To determine whether the patterns of Mutator activity correlate with tissue or cell-specific transcription of the regulatory transposon MuDR, we used in situ hybridization to localize the sense MuDR transcripts, mudrA and mudrB, in pistillate florets and embryos of four different maize Mutator stocks. We found mudrA and mudrB transcripts uniformly distributed in all tissues of immature, meristem-rich florets and in both somatic and germinal tissues of mature florets. In mature flowers, transcripts of both genes accumulate to high levels in the tapetal (endothelium) layer surrounding the embryo sac. We also found transcripts from the antisense strand of the mudrA gene in all cell types in the florets. In developing embryos, all MuDR transcripts were present in all tissues. Different Mutator stocks had characteristic accumulation patterns that were maintained throughout embryo development.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

Characterization of the major transcripts encoded by the regulatory MuDR transposable element of maize.

The MuDR element controls the transposition of the Mutator transposable element family in maize. Previous studies reported the presence of two major MuDR-homologous transcripts that correlate with Mutator activity. In this study, we describe the structure and processing of these two major transcripts. The transcripts are convergent, initiating from opposite ends of the element within the 220-bp terminal inverted repeats. The convergent transcripts do not overlap, and only 200 bp of internal MuDR sequences are not transcribed. Cloning and sequencing of multiple MuDR cDNAs revealed unusual intron/exon junctions, differential splicing, and multiple polyadenylation sites. RNase protection experiments indicated that some splicing failure occurs in young seedlings, and that a low level of antisense RNA exists for both transcripts. On a whole plant level, the presence of the major MuDR transcripts strictly correlates with Mutator activity in that no MuDR transcripts are observed in non-Mutator or inactive Mutator stocks. Examination of various tissues from active Mutator stocks indicates that the two transcripts are present in all organs and tissues tested, including those with no apparent transposition activity. This suggests that Mutator activity is not simply controlled by the level of the major MuDR transcripts.

LYAR, a novel nucleolar protein with zinc finger DNA-binding motifs, is involved in cell growth regulation.

A cDNA encoding a novel zinc finger protein has been isolated from a mouse T-cell leukemia line on the basis of its expression of a Ly-1 epitope in a lambda gt11 library. The putative gene was mapped on mouse chromosome 1, closely linked to Idh-1, but not linked to the Ly-1 (CD5) gene. The cDNA is therefore named Ly-1 antibody reactive clone (LYAR). The putative polypeptide encoded by the cDNA consists of 388 amino acids with a zinc finger motif and three copies of nuclear localization signals. Antibodies raised against a LYAR fusion protein reacted with a protein of 45 kD on Western blots and by immunoprecipitation. Immunolocalization indicated that LYAR was present predominantly in the nucleoli. The LYAR mRNA was not detected in brain, thymus, bone marrow, liver, heart, and muscle. Low levels of LYAR mRNA were detected in kidney and spleen. However, the LYAR gene was expressed at very high levels in immature spermatocytes in testis. The LYAR mRNA is present at high levels in early embryos and preferentially in fetal liver and fetal thymus. A number of B- and T-cell leukemic lines expressed LYAR at high levels, although it was not detectable in bone marrow and thymus. During radiation-induced T-cell leukemogenesis, high levels of LYAR were expressed in preleukemic thymocytes and in acute T leukemia cells. Fibroblast cells overexpressing the LYAR cDNA from a retrovirus vector, though not phenotypically transformed in vitro, had increased ability to form tumors in nu/nu mice. Therefore, LYAR may function as a novel nucleolar oncoprotein to regulate cell growth.

Genomic organization of the mouse granzyme A gene. Two mRNAs encode the same mature granzyme A with different leader peptides.

Granzyme A is a serine protease that, together with the other granular components of cytotoxic T lymphocyte (CTL) cells, has been implicated in the cytolysis process. We report here two different messages and the genomic organization of the mouse granzyme A gene. The granzyme A gene is composed of six exons spanning 7 kilobases. Alternative splicing of the second exon results in the two transcripts. The two mRNA species encode the same mature granzyme A protein but with different leader sequences. The first (HF1) encodes a typical leader signal sequence similar to other granzymes, but the second (HF2) putative leader sequence is different and less hydrophobic. Both messages are present in cultured CTL cell lines and in normal lymphoid tissues. They are both induced when CTL cells are activated in vitro or in vivo. Both messages can be translated in vitro, although the HF1 message appears to be much more efficient as a template. The putative 5' promoter region of the HF gene sequenced (500 base pairs of upstream sequences) contains no well defined promoter sequences aside from the TATA box. The results suggest that (a) granzyme A may be produced with putative different leader sequences from two different mRNAs; (b) this may provide a model system for studying alternate splicing and the evolution of a complex enzymatic system in an organelle; and (c) the genomic DNA reported will be useful for studying transcription regulations involved in controlling the specific expression pattern of this gene.

Mutator activity in maize correlates with the presence and expression of the Mu transposable element Mu9.

Mutator is a powerful system for generating new mutants in maize. Mutator activity is attributable to a family of transposable, multicopy Mu elements, but none of the known elements is an autonomous (regulatory) element. This paper reports the discovery of Mu9, a 4942-base-pair Mu element that was cloned after it transposed into the Bronze-2 locus. Like other Mu elements, Mu9 has approximately 215-base-pair terminal inverted repeats and creates a 9-base-pair host sequence duplication upon insertion. A small gene family of elements that cross-hybridize to Mu9 has been found in all maize lines, and one of the other known Mu elements, Mu5, probably arose as a deletion of Mu9. Mu9 has several of the properties expected for the proposed regulator of Mutator activity. (i) The presence of Mu9 parallels the presence of Mutator activity in individuals from a line that genetically segregates for the Mu regulator. (ii) Lines that transmit Mutator to greater than 90% of their progeny have multiple copies of Mu9. (iii) Most maize lines that lack Mutator activity and that are not descended from Mutator lines lack the Mu9 element. (iv) Transcripts that hybridize to Mu9 are abundant in active Mutator lines, but they are absent from lines that have epigenetically lost Mutator activity. These correlations suggest that Mu9 is a candidate for the autonomous Mutator element.

Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease.

A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage lambda gt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16+ natural killer cells and CD3+, CD16- T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The predicted protein has a 22-amino acid presegment, a 6-amino acid prosegment, and an active enzyme of 234 amino acids with a calculated unglycosylated molecular weight of 25,820. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. We propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells.

A T cell- and natural killer cell-specific, trypsin-like serine protease. Implications of a cytolytic cascade.

A new trypsin-like serine protease was cloned from both a murine cytotoxic T lymphocyte and a human PHA-stimulated peripheral blood lymphocyte cDNA library. In both the mouse and human system, this transcript had a T cell- and NK-specific distribution, being detected in cytotoxic T lymphocytes (CTL), some T-helper clones, and NK, but not in a variety of normal tissues. T-cell activation with Con A plus IL-2 induced mouse spleen cells to express this gene with kinetics correlating with the acquisition of cytolytic capacity. Both the mouse and human nucleotide sequences of this gene encoded an amino acid sequence with 25-40% identity to members of the serine protease family. The active-site "charge-relay" residues (His-57, Asp-102, and Ser-195 of the chymotrypsin numbering system) are conserved, as well as the trypsin-specific Asp (position 189 in trypsin). We reviewed the evidence of this serine protease's role in lymphocyte lysis and proposed a "lytic cascade." We discussed the biological and clinical implications of a cascade, proposing these enzymes as markers for cytolytic cells and as targets for rational drug therapy. Genetic and acquired deficits in the lethal hit-delivery system are considered as a basis for approaching some immunodeficiency states, including severe EBV infections, T-gamma leukemias, and T8+ lymphocytosis syndromes.