trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IEl and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72-and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression.

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (

Cellophane wrapping of the hamster pancreas induces islet neogenesis. We have used the mRNA differential display technique to select for genes expressed during islet neogenesis but not in control pancreata. Ten candidate clones have been identified. Upon sequencing, 6 clones showed a high degree of homology to known genes, 1 showed some, and 3 showed no homology to genes of known sequence. Thus, mRNA differential display is a useful technique to identify genes induced during islet neogenesis, and in combination with screening hamster pancreatic cDNA libraries for full length clones, will enhance the likelihood of capturing the participants in this process.

SummaryWe have reported previously that cellophane wrapping of the hamster pancreas is a stimulus that leads to the induction of duct epithelial cell proliferation, followed by endocrine cell differentiation and new islet formation. Reg is a candidate gene that has been reported to be expressed in regenerating pancreatic islets, suggesting a role in islet growth.We examined Reg gene expression in the cellophane-wrap model by isolating total RNA from hamster pancreata at various times after wrapping. Northern blot analysis using a rat cDNA Reg probe showed no expression of Reg in control non-wrapped hamster pancreas, whereas a strong signal was detected in control wrapped rat pancreas. Using reverse transcription of RNA followed by polymerase chain reaction (PCR) we amplified, isolated and sequenced a 194 base pair product which showed homology to rat Reg in both control and wrapped hamster pancreas.When the PCR product was used as a probe for Northern blot analysis, no signal was detected in control non-wrapped pancreata. In contrast, a strong signal was detected 1 and 2 days after wrapping, which then returned to basal between 4 and 6 days after wrapping. A similar temporal pattern was observed using in situ hybridization to localize the Reg gene.One-and 2-day wrapped but not control pancreas expressed Reg in acinar cells, but not in islets. In conclusion, (a) Reg expression is low in the control hamster pancreas; (b) in the cellophane-wrap model of islet neogenesis, increased Reg mRNA is found within acinar tissue; (c) Reg gene may thus be involved as an acinar paracrine effector of duct cell proliferation in the initial step of islet neogenesis, but may also participate in the inflammatory response to traumatic stimuli. [Diabetologia (1995) MOPS,propane-sulphonic acid; PSP, pancreatic stone protein; PTR pancreatic thread protein; PAP, pancreatitis-associated protein; HGF/SF, hepatocyte growth factor/scatter factor; PCR, polymerase chain reaction; STZ, Streptozotocin; PBS, phosphate buffered saline; bp, base pair. duction of new islet formation within 2 weeks [2,3]. These new endocrine cells appear to derive from ductal epithelium elements [4,5]. Electron microscopic and immunocytochemical examination of the newlydeveloped islets identified cells containing insulin, glucagon and somatostatin [3] and wrapping was capable of reversing streptozotocin (STZ) induced diabetes [4,6]. The mechanism whereby cellophane wrapping induces cell proliferation and differentiation is unknown but may be related to induction of local growth factors.Several studies have attempted to address the role of various factors regulating islet neogenesis. One such factor is Reg gene and its protein product; the findings however, have been controversial. Screening of a cDNA library derived from regenerating rat

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.

We have reported previously that cellophane wrapping of the hamster pancreas is a stimulus that leads to the induction of duct epithelial cell proliferation, followed by endocrine cell differentiation and new islet formation. Reg is a candidate gene that has been reported to be expressed in regenerating pancreatic islets, suggesting a role in islet growth. We examined Reg gene expression in the cellophane-wrap model by isolating total RNA from hamster pancreata at various times after wrapping. Northern blot analysis using a rat cDNA Reg probe showed no expression of Reg in control non-wrapped hamster pancreas, whereas a strong signal was detected in control wrapped rat pancreas. Using reverse transcription of RNA followed by polymerase chain reaction (PCR) we amplified, isolated and sequenced a 194 base pair product which showed homology to rat Reg in both control and wrapped hamster pancreas. When the PCR product was used as a probe for Northern blot analysis, no signal was detected in control non-wrapped pancreata. In contrast, a strong signal was detected 1 and 2 days after wrapping, which then returned to basal between 4 and 6 days after wrapping. A similar temporal pattern was observed using in situ hybridization to localize the Reg gene. One- and 2-day wrapped but not control pancreas expressed Reg in acinar cells, but not in islets.(ABSTRACT TRUNCATED AT 250 WORDS)