Dan Robinson

Marek's Disease Virus (MDV) Encodes an Interleukin-8 Homolog (vIL-8): Characterization of the vIL-8 Protein and a vIL-8 Deletion Mutant MDV

ABSTRACT Chemokines induce chemotaxis, cell migration, and inflammatory responses. We report the identification of an interleukin-8 (IL-8) homolog, termed vIL-8, encoded within the genome of Mareks disease virus (MDV). The 134-amino-acid vIL-8 shares closest homology to mammalian and avian IL-8, molecules representing the prototype CXC chemokine. The gene for vIL-8 consists of three exons which map to theBamHI-L fragment within the repeats flanking the unique long region of the MDV genome. A 0.7-kb transcript encoding vIL-8 was detected in an n-butyrate-treated, MDV-transformed T-lymphoblastoid cell line, MSB-1. This induction is essentially abolished by cycloheximide and herpesvirus DNA polymerase inhibitor phosphonoacetate, indicating that vIL-8 is expressed with true late (γ2) kinetics. Baculovirus-expressed vIL-8 was found to be secreted into the medium and shown to be functional as a chemoattractant for chicken peripheral blood mononuclear cells but not for heterophils. To characterize the function of vIL-8 with respect to MDV infection in vivo, a recombinant MDV was constructed with a deletion of all three exons and a soluble-modified green fluorescent protein (smGFP) expression cassette inserted at the site of deletion. In two in vivo experiments, the vIL-8 deletion mutant (RB1BvIL-8ΔsmGFP) showed a decreased level of lytic infection in comparison to its parent virus, an equal-passage-level parent virus, and to another recombinant MDV containing the insertion of a GFP expression cassette at the nonessential US2 gene. RB1BvIL-8ΔsmGFP retained oncogenicity, albeit at a greatly reduced level. Nonetheless, we have been able to establish a lymphoblastoid cell line from an RB1BvIL-8ΔsmGFP-induced ovarian lymphoma (MDCC-UA20) and verify the presence of a latent MDV genome lacking vIL-8. Taken together, these data describe the identification and characterization of a chemokine homolog encoded within the MDV genome that is dispensable for transformation but may affect the level of MDV in vivo lytic infection.

KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway

The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20s. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

Protein-tyrosine kinase and protein-serine/threonine kinase expression in human gastric cancer cell lines.

Protein kinases play key roles in cellular functions. They are involved in many cellular functions including; signal transduction, cell cycle regulation, cell division, and cell differentiation. Alterations of protein kinase by gene amplification, mutation or viral factors often induce tumor formation and tumor progression toward malignancy. The identification and cloning of kinase genes can provide a better understanding of the mechanisms of tumorigenesis as well as diagnostic tools for tumor staging. In this study, we have used degenerated polymerase-chain-reaction primers according to the consensus catalytic domain motifs to amplify protein kinase genes (protein-tyrosine kinase, PTK, and protein-serine/threonine kinase, PSK) from human stomach cancer cells. Following amplification, the protein kinase molecules expressed in the gastric cancer cells were cloned into plasmid vectors for cloning and sequencing. Sequence analysis of polymerase-chain-reaction products resulted in the identification of 25 protein kinases, including two novel ones. Expression of several relevant PTK/PSK genes in gastric cancer cells and tissues was further substantiated by RT-PCR using gene-specific primers. The identification of protein kinases expressed or activated in the gastric cancer cells provide the framework to understand the oncogenic process of stomach cancer.

Purification of genomic sized herpesvirus DNA using pulse-field electrophoresis

A new method for the separation of genomic sized herpesvirus DNA was developed. This method utilizes the improved separation of extremely high molecular weight DNA molecules by pulse-field electrophoresis. This method has allowed us to separate and purify herpesvirus DNA (greater than 100 kbp) free of cellular DNA, directly from infected cells. In the case of cell associated herpesviruses (such as Mareks disease herpesvirus), this method is more efficient than present isolation procedures. The DNA obtained is able to be restriction enzyme digested and used in cloning experiments.

Digital Cloning: Identification of Human cDNAs Homologous to Novel Kinases through Expressed Sequence Tag Database Searching

Identification of novel kinases based on their sequence conservation within kinase catalytic domain has relied so far on two major approaches, low-stringency hybridization of cDNA libraries, and PCR method using degenerate primers. Both of these approaches at times are technically difficult and time-consuming. We have developed a procedure that can significantly reduce the time and effort involved in searching for novel kinases and increase the sensitivity of the analysis. This procedure exploits the computer analysis of a vast resource of human cDNA sequences represented in the expressed sequence tag (EST) database. Seventeen novel human cDNA clones showing significant homology to serine/threonine kinases, including STE-20, CDK- and YAK-related family kinases, were identified by searching EST database. Further sequence analysis of these novel kinases obtained either directly from EST clones or from PCR-RACE products confirmed their identity as protein kinases. Given the rapid accumulation of the EST database and the advent of powerful computer analysis software, this approach provides a fast, sensitive, and economical way to identify novel kinases as well as other genes from EST database.

Tyrosine Kinase Expression Profiles of Chicken Erythro- Progenitor Cells and Oncogene- Transformed Erythroblasts

Tyrosine kinases are implicated in the growth and differentiation of erythroid cells. Aberrant expression and structural alterations of certain tyrosine kinases, such as erbB and sea, are known to trigger erythroleukemia development. To facilitate our understanding of the signal transduction pathways involved in erythroid differentiation and leukemic transformation, we have applied a recently developed tyrosine kinase profile technique to identify the tyrosine kinases and some novel serine/threonine kinases expressed in normal chicken erythroid progenitor cells that respond to TGFalpha (TGFalpha-EB), and erythroblasts transformed by viruses encoding v-erbB (v-erbB-EB) and v-sea (v-sea-EB). Our results reveal that the non-receptor tyrosine kinases, Abl, Fyn, Lyn, Btk and Csk, are expressed in all three cell types. The expression level of Btk, a tyrosine kinase implicated in Brutons syndrome, is exceptionally high in the erythroblastoid cell line 6C2, transformed by the v-erbB carrying avian erythroblastosis virus, AEV-ES4. We have also uncovered a new STE-20-related serine/threonine kinase, KFC, which is abundantly expressed in both the TGFalpha-stimulated erythroid progenitor cells and v-sea-transformed erythroblasts. Based on sequence homology of the kinase domain, KFC appears to be the first member of a new subfamily of STE-20-like kinases.