Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins.

Engagement of the T cell antigen receptor results in both its phosphorylation and its ubiquitination. T cell antigen receptor ubiquitination was evaluated in Jurkat, a well characterized human T leukemia cell line. Treatment of cells with the tyrosine kinase inhibitor herbimycin A resulted in an inhibition of receptor ubiquitination. Consistent with this, pervanadate, which increases cellular tyrosine phosphorylation, enhanced receptor ubiquitination. A requirement for receptormediated tyrosine kinase activity for ubiquitination was confirmed in cells lacking the tyrosine kinase p56 lck and also in cells that are defective in expression of CD45, a tyrosine phosphatase that regulates the activity of p56 lck . The need for tyrosine kinase activation for ubiquitination was not bypassed by directly activating protein kinase C and stimulating endocytosis of receptors. These observations establish ubiquitination of the T cell antigen receptor as a tyrosine kinase-dependent manifestation of transmembrane signaling and suggest a role for tyrosine phosphorylation in the ligand-dependent ubiquitination of mammalian transmembrane receptors.For many transmembrane receptors, including the multisubunit TCR, 1 signaling is initiated by ligand-induced aggregation (1, 2). The earliest obligate intracellular event following TCR aggregation is the activation of the src-family protein tyrosine kinases, Lck (p56 lck ) and Fyn (p59 fyn ). Lck and/or Fyn phosphorylate TCR subunits resulting in the association of a third tyrosine kinase, ZAP-70 (70-kDa -associated protein), with the TCR and to subsequent activation events (3-5). CD45, a tyrosine phosphatase that dephosphorylates key regulatory residues on Lck and Fyn, is also implicated in the initiation of TCR-mediated signaling (6).TCRs consist of six different polypeptides, these include the antigen-recognition element, in most cells an ␣-␤ heterodimer, and a set of invariant signal transducing subunits. The invariant subunits include CD3-␦, -⑀, and -␥ and the structurally distinct TCR-subunit, which exists within the TCR as a disulfide-linked homodimer (4). The minimal signal transducing element of the TCR is the immunoreceptor tyrosine-based activation motif (ITAM) (7). monomers have three ITAMs, and each CD3 subunit has one. ITAMs include two tyrosine residues 10 or 11 amino acids apart that are potential phosphorylation sites. The subunit is a particularly prominent substrate for tyrosine phosphorylation; up to 5% of subunits are phosphorylated on multiple tyrosines upon TCR engagement (8 -11).In addition to being a substrate for tyrosine phosphorylation when cross-linked by antibody or mitogen (11), TCRs also are ubiquitinated. The covalent modification of proteins with chains of ubiquitin, a highly conserved 76-amino acid polypeptide, plays a central role in the targeting of abnormal proteins and a number of regulatory cytosolic and nuclear proteins for degradation in the 26 S proteasome (12-16). Ubiquitination occurs via a multienzyme process involving families of enzymes ter...

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Δ, ctf4Δ, ctk1Δ, hfi1Δ,nup133Δ, tho2Δ) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Δ and hfi1Δ) or deficient (tho2Δ) for recombination. Using these and other “THO2-like” hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Δ, thp1Δ, thp2Δ) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem-loop sequence, which is bound to the stem-loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3 ′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′ hExo, forms a ternary complex with SLBP and the stem-loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3 ′ hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3 ′ hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.

Retrospective analysis utilizing "next generation sequencing (NGS)" was done on cancer tissue harvested from 14 patients prior to receiving MLN8237, a novel Aurora Kinase A inhibitor. The responding patients (n=4) were characterized by stable disease ≥6 months and prolonged time of progression (≥1.3 fold prior treatment). Differential patterns of nodal connectivity in protein-protein interaction networks (consequent to determined genomic alterations) emerged from the comparison between responder and non-responder groups. The responding patient population showed high connectivity within MYC related genes including regulators of the Wnt/beta-catenin pathway. On the other hand, the non-responding patients showed high connectivity centered on the TP53/RB1 axis. Matching "targeted therapy to target" is a sine qua non for maximizing effective therapy in appropriate patients and NGS mapping may further our understanding of the relationships between molecular biological pathways and targeted therapy response. While awaiting further progress in systems analysis across "omic" levels (genomic-transcriptomicproteomic), research involving of NGS sequence mapping to interrogate patient response to therapy in order to help elucidate molecular therapeutic predictors is justified based on the urgent needs of patient care.