The Control of Varicella-Zoster Virus Immediate Early Gene Expression

Analysis of the cis-acting motifs and trans-acting factors responsible for regulating the expression of the varicella-zoster virus (VZV) major immediate early (IE) gene was undertaken. The control of VZV IE gene expression has relevance to the fields of eukaryotic gene regulation and herpesvirus biology. In order to analyse the control sequences of the VZV major IE gene, knowledge of the location of the 5' end of its mRNA was required. Primer extension and SI nuclease analyses concurred in defining it as being 74 base pairs (bp) upstream of the proposed translation initiation site. Analysis of DNA sequences upstream of the start site revealed a candidate for a 'TATA' motif present at -25 to -30 bp, a finding common to many genes transcribed by eukaryotic RNA polymerase II. Analysis of the activity of the control region of the VZV major IE gene was undertaken using plasmids in which sequences to -1150 were inserted upstream of the "reporter" genes encoding B-galactosidase or chloramphenicol acetyl transferase. The activities of the complete control region and of a series of deletions were then determined in a range of cell types, using short term transfection assays. A significant finding to emerge from these experiments was that the control sequences responsible for the transcription of the VZV major IE gene do not direct a high level of expression of reporter genes in any of the cell types tested. The baseline level of transcriptional activity was dependent on sequences located between nucleotides -131 and -25. Co-transfection of a plasmid which expresses the herpes simplex virus type 1 (HSV-1) protein Vmw65 resulted in a 20-50 fold stimulation of expression from the VZV IE promoter. This effect depended on the presence of regulatory sequences between -409 bp and -130 bp. A second set of co-transfection experiments investigated the effect on the VZV control sequences of a plasmid encoding the transactivating product of the adenovirus 5 ElA gene. The presence of this polypeptide resulted in stimulation of expression by 10 fold. In contrast with the effect of Vmw65, the activity of the ElA gene product depended on sequences present within the first 131 bp of the mRNA start site. Gel retardation and DNAase I protection assays were used to locate sites at which proteins bind to the control sequences. Results from these experiments reinforced and expanded the conclusions made from the functional assays. DNA fragments from the promoter region, -35 to -130, were shown to bind two proteins. Sequence analysis and competition experiments identified these proteins as belonging to two families of transcription factors known as 'CCAAT box binding' proteins and the 'ATF/CRE' family. A second protein binding region was present between -409 and -246 and was shown to consist of two binding sites for the cellular transcription factor Oct-1, which binds to the consensus ATGCAAAT. The proximal binding site could be expanded to incorporate a TAATGARAT motif known to allow Vmw65 to participate in the formation of a complex including Oct-1 and other cellular proteins, thus allowing Vmw65 mediated transactivation. Both sites were shown to bind Oct-1 and the proximal site was also shown to bind Vmw65 with high affinity, probably explaining the effect of this protein on VZV IE gene expression. In view of these results the analysis of VZV open reading frame (orf) 10 was undertaken. Sequence comparison of orf10 and Vmw65 had shown considerable homologies between the amino-terminal portions of the proteins but the absence from orf10 of a carboxy-terminal region of 78 amino acids known to be essential for transcriptional activation by Vmw65. Both the functional activity of orf10 and its ability to participate in complex formation at TAATGARAT motifs were tested. A plasmid, constructed to express the product of VZV orf10, failed to stimulate expression from plasmids containing HSV-1 or VZV IE control sequences. The product of VZV orf10 was also produced in vitro and failed to participate in the formation of DNA binding complexes when incubated with cellular proteins and the TAATGARAT motif. Two other VZV orfs were studied. The first was number 66, which encodes a protein which shows many similarities to known protein kinases. The system chosen to express this orf utilised the HSV-1 temperature sensitive mutant tsK and HSV-1 IE control sequences. At nonpermissive temperatures tsK overproduces IE polypeptides, generating quantities sufficient for biochemical characterisation of heterologous orfs expressed in this way. In order to simplify the selection of recombinant viruses containing VZV orfs the E. coli B-galactosidase enzyme, with HSV-1 IE control sequences, was inserted into the thymidine kinase gene of tsK

Thomas Hudson McKee to Mr. Meredith (30 September 1962)

https://egrove.olemiss.edu/mercorr_pro/1246/thumbnail.jp

1870 corporate audit committee

Board of directors\u27 audit committees are becoming an increasingly popular vehicle for enhancing the objectivity and independence of auditors and overseeing the financial information generating process. This is occurring at a time when directors and auditors are facing criticism and increased litigation due to corporate failures and disclosures of illegal or questionable payments. This article examines the workings of a corporate audit committee that operated in the mid-nineteenth century. The committee functioned as auditor for the company since there was no established public accounting profession in the U.S. at that time. They disentangled the financial affairs of the company and probably directly contributed to the replacement of the President of the company. Although the activities of corporate audit committees have changed or evolved considerably through the years, both the 1870 corporate audit committee and modern corporate audit committees have pursued a common goal of achieving accuracy and completeness in corporate financial reports

Economic impacts and analysis methods of extreme precipitation estimates for eastern Colorado

August 1986.Includes bibliographical references (pages 56-59).Dams are designed to store water and to ensure human safety and as such they must withstand, in their lifetimes, any extreme precipitation event in their drainage basin. Correct estimation of this event is critical because on one hand it must provide an adequate level of safety to not occur, but it must not be any greater than needed since the high costs of dam construction and modifications are directly related to the magnitude of the estimated extreme event. Most frequently the extreme precipitation event is labeled as the Probable Maximum Precipitation, or PMP. National and state concerns over the adequacy of existing dams in the United States as well as increased development of the Front Range led to state dam risk reclassification and federal redefinition of new PMP values issued for Colorado in 1984. The study area included the region from the Continental Divide to the 103rd Meridian. Study of the implementation of PMP values and their potential economic impacts in Colorado reveals that an enormous cost will result in Colorado. Techniques for estimating cost of modifications for spillways were developed. Among 162 high risk dams, the estimated total cost for modification was approximately $184 million. The economic value of this precipitation estimate is$9.45 million per inch change of rainfall in this limited study area. In one elevation region, 7000 to 9000 feet, the costs is approximately $15.76 million per inch change of rainfall. Regional cost analyses revealed the South Platte River Division had the greatest costs. Inherent limitations in the PMP procedure and the cost of spillway modifications have made evaluating other alternatives necessary. Special aspects of estimates for extreme precipitation, such as snowmelt runoff versus extreme precipitation events and climate variations were examined. Four methods for estimating extreme precipitation events were evaluated; the traditional PMP, the paleogeological, the cloud/mesoscale dynamic model, and the statistical approaches. A collection of approaches were recommended for Colorado dam design in three elevation regions: the plains, the foothills, and the mountains.Supported by NOAA under Grant No. NA-85-RAH-05045 through CIRA

Analysis of tethered balloon, ceilometer and class sounding data taken on San Nicolas Island during the FIRE project

During the FIRE Marine Stratocumulus Program on San Nicolas Island, Colorado State University (CSU) and the British Meteorological Office (BMO) operated separate instrument packages on the NASA tethered balloon. The CSU package contained instrumentation for the measurement of temperature, pressure, humidity, cloud droplet concentration, and long and short wave radiation. Eight research flights, performed between July 7 and July 14, are summarized. An analysis priority to the July 7, 8 and 11 flights was assigned for the purposes of comparing the CSU and BMO data. Results are presented. In addition, CSU operated a laser ceilometer for the determination of cloud base, and a CLASS radiosonde site which launched 69 sondes. Data from all of the above systems are being analyzed

Colorado precipitation event and variability analysis

July 1986.Bibliography: pages 101-102

Colorado monthly temperature and precipitation summary for period 1951-1970

March, 1977

Colorado climate summary water-year series: October 1993-September 1994

December 1994.Annual

Simulation of the daytime boundary layer evolution in deep mountain valleys

December, 1981.Bibliography: pages 96-100.Sponsored by the National Science Foundation ATM76-84405.Sponsored by the National Science Foundation ATM80-15309