Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

The human hematopoietic colony-stimulating factors.

The complementary DNAs and genes encoding the four major human myeloid growth factors--granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3--have all been molecularly cloned. These DNA clones have proved valuable for studying the molecular biology of these important regulatory molecules as well as for the large-scale production of the recombinant growth factor proteins. These advances have led to a much better understanding of the role of the myeloid growth factors in regulating hematopoiesis in vivo that should soon find practical application in clinical medicine.

Arrangement of 30S heterogeneous nuclear ribonucleoprotein on polyoma virus late nuclear transcripts.

Heterogeneous nuclear ribonucleic acid (hnRNA) molecules in eucaryotic cell nuclei associate with a well-defined group of abundant, highly conserved proteins to form heterogeneous nuclear ribonucleoproteins (hnRNP). The exact manner in which these 30S complexes assemble on nuclear transcripts, however, has not been well documented. To determine whether any site selectivity in the formation of hnRNP can be detected (e.g., preferential recognition of intervening sequences or of premessage regions), we investigated the distribution of 30S hnRNP on a particular nuclear RNA, the polyoma virus late transcript. Hybridization studies showed not only that the majority of polyoma late nuclear RNA sequences can be isolated in the form of 30S complexes, but that the RNP were located equally on intervening sequences and premessage portions of the transcript. The latter conclusion was confirmed by ribonuclease T1 oligonucleotide fingerprint analysis of polyoma virus-specific RNA recovered from native 30S complexes. However, fingerprint analysis of the small segments of viral RNA in the 30S fraction that survived extensive ribonuclease treatment revealed that oligonucleotides corresponding to intervening sequences were preferentially lost. We discuss these findings in relation to the structure of 30S hnRNP and their function in RNA biogenesis.

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

Impaired development of oral-motor functions required for normal oral feeding as a consequence of tube feeding during infancy.

The child who is tube fed during the first two years of life is at risk for impaired development of normal oral-motor patterns and coordination during feeding. In order to ensure as smooth a transition to oral feeding as possible, it is necessary to provide the tube-fed child with a framework of normal oral sensitization from which he can develop the level of trust in his own oral mechanism required to achieve the mature, coordinated oral movements essential to development of the normal bite-chew-suck-swallow sequence. While this can be a tedious and taxing process, an ongoing program of oral stimulation, training and development should be included in the overall management of every young child receiving tube feedings.

Biofeedback and meditation: effects on muscle tension and locus of control.

A total of 48 subjects participated in a relaxation experiment to determine whether frontalis muscle EMG biofeedback, Transcendental Meditation, and meditation (Benson technique) produced decreased muscle tension and concomitant changes in locus of control. All three treatments resulted in significant decreases in frontalis muscle tension when compared to a control. Concomitant changes towards an internal locus of control occurred only in the subjects given biofeedback.

A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation.

The mRNAs of transiently expressed genes frequently contain an AU-rich sequence in the 3' untranslated region. We introduced a 51 nucleotide AT sequence from a human lymphokine gene, GM-CSF, into the 3' untranslated region of the rabbit beta-globin gene. Our experiments demonstrate that this caused the otherwise stable beta-globin mRNA to become highly unstable in vivo. The instability conferred by the AU sequence in the mRNA was partially alleviated by treatment of the cells with cycloheximide. We propose that the AU sequences are the recognition signal for an mRNA processing pathway which specifically degrades the mRNAs for certain lymphokines, cytokines, and proto-oncogenes.

Guanine nucleotide contacts within viral DNA sequences bound by polyomavirus large T antigen.

Essential nucleotide contacts between the polyomavirus large T antigen and its multiple specific binding regions within the regulatory sequences of the polyomavirus genome were determined in vitro by methylation interference. Methylation of any of the guanine residues of the 5'-G(A/G)GGC-3' pentanucleotide repeats in large-T-antigen-binding regions A, B, C, and 3 (A. Cowie and R. Kamen, J. Virol. 52:750-760, 1984) interfered with T antigen binding. Within regions A, B, and C these pentanucleotides are spaced 5 or 6 base pairs apart. Therefore, the clusters of contacted nucleotides within each of these binding regions are localized along one face of the DNA helix. Methylation of guanines within the sequences between the pentanucleotide repeats did not interfere with binding. The ORI binding region contains four additional pentanucleotide sequences within a region of dyad symmetry. Methylation of only particular guanines of these pentanucleotides interfered with T antigen binding. The spatial arrangement of the pentanucleotides in the ORI is such that the clusters of contacted guanines are situated around the DNA helix, thereby forming a very different arrangement from that found in the other binding regions. A model is discussed in which cooperative interactions between T antigen protomers, recognizing individual pentanucleotides, determines the strength and the function of different T antigen-DNA interactions.

Polyoma virus-specific RNA synthesis in an inducible line of polyoma virus-transformed rat cells.

Viral RNA present in the inducible LPT clone 1A of polyoma virus-transformed rat cells was characterized before and after mitomycin C induction by hybridization with 32P-labeled separated E and L strands of polyoma viral DNA restriction endonuclease fragments. In clone 1A cells maintained under normal growth conditions, the cytoplasm contained a transcript of the E-strand DNA from the "early" region similar to that previously identified in lytically infected cells, as well as minor quantities of RNA complementary to less than one-half of the L- and the E-strand DNA from the "late" region. Nuclei of normally growing cells contained the same species found in the cytoplasm, as well as an additional abundant RNA complementary to one-half of the L-strand DNA of the late region. No significant changes occurred in the cytoplasmic viral RNA after mitomycin C treatment before the onset of viral DNA replication, but the concentration of the nuclear L-strand DNA transcript diminished. After the onset of viral DNA replication after mitomycin C treatment, transcripts of virtually the entire L-strand DNA were found in the nuclei, and a 10-fold increase was observed in the abundance of RNA transcribed from the E strand of the early region. In the cytoplasm, the abundance of the early RNA increased about 25-fold and late RNA complementary to the L-strand DNA of the late region was found in a similar quantity. The synthesis of both the early and the late RNA species was inhibited if viral DNA replication was blocked with 5-fluorodeoxyuridine. We conclude that the induction of viral DNA replication in LPT cells is not determined at the level of mRNA synthesis.