Evaluation of the American Cancer Society's Research Promotion Guide.

The American Cancer Society has supported cancer research for more than 50 years and has devoted more than $2.2 billion to cancer research. This article describes an evaluation of the Research Promotion Guide of the American Cancer Society, a reference tool developed by the National Home Office for regional division staff and volunteers whose work involved public relations and fundraising. The purposes of this study were the following: 1) to determine the level and type of use of the guide; and 2) to assess factors that may influence the level and type of use. Fifty-five participants were interviewed by telephone. Overall, 78% of them were aware of the guide. Three factors significantly and positively associated with level of use were: compatibility (the perception that the guide is relevant to one's job); attending a training session; and years employed at the American Cancer Society. This study, a pilot project for the Collaborative Evaluation Fellows Project (CEFP), demonstrated that collaboration between nonprofit and academic institutions is feasible and can serve the needs of the students and organizations. The lessons learned can be applied to evaluations in general and to future CEFP projects.

Estrogen replacement therapy and risk of fatal breast cancer in a prospective cohort of postmenopausal women in the United States.

This study examines the relationship between fatal breast cancer and use of estrogen replacement therapy (ERT) among women in a large prospective study in the United States. After nine years of follow-up, 1,469 breast cancer deaths were observed in a cohort of 422,373 postmenopausal women who were cancer free at study entry and who supplied information on estrogen use. Results from Cox proportional hazards modeling, adjusted for 11 other potential risk factors, showed that ever-use of ERT was associated with a significantly decreased risk of fatal breast cancer (rate ratio [RR] = 0.84, 95 percent confidence interval [CI] = 0.75-0.94). There was a moderate trend (P = 0.07) of decreasing risk with younger age at first use of ERT. This decreased risk was most pronounced in women who experienced natural menopause before the age of 40 years (RR = 0.59, CI = 0.40-0.87). There was no discernible trend of increasing risk with duration of use in estrogen users at baseline or former users, nor was there any trend in years since last use in former users. The relationship between ERT and breast cancer mortality differed by age at menarche and by a self-reported history of breast cysts. No increased risk of fatal breast cancer with ERT was observed with estrogen use status (baseline/former), age at first use, duration of use, or years since last use. These findings suggest that ever-use of ERT is associated with a 16 percent decreased risk of fatal breast cancer.

A preliminary translational map of the frog virus 3 genome.

A preliminary map of the frog virus 3 (FV 3) genome was constructed by hybridization-selection of mRNAs to cloned DNA fragments and translation in reticulocyte lysates. FV 3 mRNAs were hybridized to KpnI, HindIII, and SalI restriction fragments representing the entire FV 3 genome. Two different hybridization conditions were employed in order to discriminate between the hybridization of early and late mRNAs. A total of 43 major and 18 minor early genes and nine major and three minor late genes were mapped. A 24 kb region comprised of the KpnI D, H, and E fragments encodes 8 of the 12 late genes that were mapped.

Transcription of methylated viral DNA by eukaryotic RNA polymerase II.

The genome of the large icosahedral DNA virus, frog virus 3 (FV3), is heavily methylated at the cytosine residues of dCdG dinucleotide pairs, with more than 22% of the total cytosine residues in the form of 5-methylcytosine (5mC). This methylation is carried out postreplicatively in the cytoplasm of infected cells by a virus-encoded DNA methyltransferase. DNA methyltransferase activity was shown to copurify with a 26 kD virus-induced, DNA-binding protein that had an altered mobility in extracts from cells infected with a DNA-methyl-transferase deficient mutant of FV3. Immediately after infection, the highly methylated parental DNA is transcribed in the nucleus by the host cell RNA polymerase II. As FV3 induces the synthesis of a protein that can override the inhibitory effect of methylation on the transcription of exogenous promoters methylation in vitro, we suggest that this protein is a factor evolved by this virus to allow transcription from methylated promoters by eukaryotic RNA polymerase II.

Methylation of the promoter for an immediate-early frog virus 3 gene does not inhibit transcription.

Methylation of critical sites within the promoter region of eucaryotic genes has been shown to inhibit transcription by RNA polymerase II. However, although the large DNA virus frog virus 3 (FV3) has a highly methylated genome, it uses host RNA polymerase II for at least the immediate-early stage of transcription. We have previously shown that an FV3-induced trans-acting protein allows transcription from adenovirus promoters inactivated by methylation. Since FV3 immediate-early genes are transcribed in the absence of de novo protein synthesis, it appears that the virus-induced trans-acting protein that allows transcription from methylated templates is not required for transcription of the immediate-early FV3 genes, possibly because they are not methylated in critical regulatory sequences. In this study, we used site-directed mutagenesis to alter the three CpG dinucleotide sequences in the promoter region of an immediate-early FV3 gene and thereby created sites recognized by bacterial methylases. Transient-expression assays demonstrated that neither the mutations nor methylation of the mutated sites inhibited transcription from the FV3 promoter in FV3-infected cells. These findings support the hypothesis that the immediate-early genes of FV3 do not contain methylatable sites in regions critical for transcription. The function of the virus-induced trans-acting protein that can override the inhibitory effect of methylation may therefore be to facilitate the transcription of methylated delayed-early or late FV3 genes.

An introduction to independent learning skills for incoming medical students.

A partnership was initiated between educators of the College of Medicine and the McGoogan Library at the University of Nebraska Medical Center to establish a new educational component for incoming medical students. The objective was to encourage the development of the students' independent information seeking skills. A three-day seminar was introduced in which the process of seeking information was emphasized rather than the final product. Cooperative development of the seminar resulted in a fresh approach to educating medical students at the College of Medicine and the emergence of an ongoing instructional link between the library faculty and educators in the College of Medicine.

Structure and regulation of the immediate-early frog virus 3 gene that encodes ICR489.

To test whether the promoters of two immediate-early genes from frog virus 3 were similar in nucleotide sequence, we have cloned and sequenced an immediate-early gene encoding an infected-cell mRNA of 489 kilodaltons (ICR489) and have shown that the protein product of this gene is approximately 46 kilodaltons. The 5' and 3' ends of the transcripts from this gene, as determined by mung bean nuclease analysis, were microheterogeneous. The promoter region was subcloned upstream from a promoterless chloramphenicol acetyltransferase gene, forming the recombinant plasmid pBS489CAT. As with the previously sequenced frog virus 3 immediate-early gene encoding ICR169, expression of chloramphenicol acetyltransferase in transfected cells required activation by a virion-associated protein. Although the promoter region of the gene encoding ICR489 contained TATA, CAAT, and GC motifs similar to those of typical eucaryotic promoters, it showed no significant homology to the ICR169 promoter, indicating that the concomitant temporal expression of these two genes is not due to similar promoter sequences.

DNA sequences required for trans-activation of an immediate-early frog virus 3 gene.

A plasmid containing 78 bp of the promoter region of the immediate-early frog virus 3 (FV3) gene ICR 169 placed 5' to the coding sequences for chloramphenicol acetyltransferase (CAT) can only be induced to synthesize CAT after transfection in the presence of FV3. To determine what DNA sequences in the promoter were required for virus-induced transcription, I used site-directed mutagenesis to construct deletions and point mutations throughout the promoter region. The mutant promoters were then analyzed for their ability to be induced by FV3. Deletion of 27 bp from the 5' end of the promoter had little effect on FV3-induced CAT synthesis. Although deletion of, and point mutations within, the 7-bp TATA-like box reduced CAT synthesis to 16-50% of that obtained with the wild-type promoter, only deletion of the 7-bp sequence caused a detectable shift of the transcription start site, indicating that the function of the AT-rich region is to position the RNA polymerase. The most significant reduction in CAT synthesis--to 1.5% of wild-type--occurred after deletion of the 23-bp immediately 5' to the TATTTTA box, which marks this 23-bp sequence as the critical cis-regulatory element for FV3 trans-activation.

Infection with frog virus 3 allows transcription of DNA methylated at cytosine but not adenine residues.

The genome of the iridovirus, frog virus 3, is highly methylated at cytosine residues by a virus-encoded DNA methyltransferase. We have shown previously that an FV3-induced trans-acting protein alters either host RNA polymerase II or methylated template to allow transcription from promoters inactivated by methylation. We now present evidence that the ability of FV3-infected cells to transcribe methylated DNA is specific for DNA methylated at cytosine residues. Eukaryotic promoters were inactivated by methylation of either adenine or cytosine residues, and tested for transcriptional activity. Only promoters inactivated by cytosine methylation were transcribed in FV3-infected cells. We also show that the dinucleotide sequence in which the methylcytosine is found appears to have no effect on the ability of FV3 to trans-activate the methylated promoters.

Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein.

The high degree of methylation of the frog virus 3 (FV3) genome suggests that FV3-infected cells are capable of transcribing highly methylated DNA. We tested this hypothesis by assaying the transcriptional activity of adenovirus promoters known to be inhibited by methylation. Plasmid constructs containing the E1a and E2aE promoters of adenovirus type 12 linked to the gene for chloramphenicol acetyltransferase [(CAT) EC 2.3.1.28], when methylated and introduced into eukaryotic cells, promoted CAT synthesis only when the cells were subsequently infected with FV3. Mapping of transcriptional initiation sites revealed that the same sites in the E1a promoter were used for the initiation of transcription in uninfected and infected cells. Moreover, Southern blots showed that transfected plasmid DNA from FV3-infected cells was not demethylated. The absence of CAT-specific RNA in transfected cells infected with FV3 in the presence of protein synthesis inhibitors demonstrated that a virus-induced protein was responsible for the trans-activation. Inhibition of transcription from the methylated template by alpha-amanitin indicated that a functional host RNA polymerase II is required for transcription of methylated DNA in FV3-infected cells. The virus-induced trans-acting protein presumably alters either host RNA polymerase II or the methylated DNA template to allow transcription from the methylated adenovirus promoters.

Quantitative genetic variation in the skeleton of the mouse: II. Description of variation within and between inbred strains.

Variation in the skeletons of over 400 male and female mice from 12 genotypes was investigated by using multivariate statistical methods. A series of discriminant functions explains the differences in the shape of six bones: mandible, os coxae, femur, tibia-fibula, scapula, and humerus. The anatomical features of bone shape described by these functions are summarized together with illustrations of the typical shapes of each bone from the 12 genotypes. Variability within genotypes was investigated by using the Mahalanobis D2 distance--a measure of the difference between two points representing multivariate data--from the group mean. A series of variants were detected ranging from grossly abnormal bones to bones showing subtle differences localized to specific regions. Examples of the variants found are illustrated.

The Iridovirus frog virus 3: a model for trans-acting proteins.

The amphibian Iridovirus, frog virus 3 (FV3), produces at least two trans-activating proteins that stimulate the expression of viral genes. One of these proteins induces transcription from the promoter of an immediate-early FV3 gene, whereas the other induces transcription from exogenously methylated DNA. These proteins may serve as a model for both viral and cellular trans-acting factors.

trans activation of an immediate-early frog virus 3 promoter by a virion protein.

We investigated the protein and DNA sequence requirements for the expression of an immediate-early frog virus 3 (FV3) gene, infected-cell RNA (ICR) 169. We used a plasmid containing the 78 nucleotides 5' to the transcription start site of ICR-169 placed upstream from the coding sequence for the bacterial enzyme chloramphenicol acetyltransferase (CAT). This construction, when introduced by CaPO4-mediated transfection into various eucaryotic cell lines, promoted CAT synthesis only if the transfected cells were subsequently infected with FV3. Dot-blot hybridization of RNA extracted from transfected, FV3-infected cells with a radioactive CAT probe showed that the induction of CAT synthesis by FV3 was at the level of transcription. When transfected cells were infected with FV3 in the presence of cycloheximide, induction of CAT-specific RNA still occurred, demonstrating that a virion protein was responsible for the trans activation. FV3-induced CAT synthesis was inhibited by alpha-amanitin in wild-type Chinese hamster ovary (CHO) cells but not in CHO cells with an alpha-amanitin-resistant RNA polymerase II. The results suggest that a virion protein alters either the DNA template or the host polymerase to allow transcription from immediate-early FV3 promoters.

Lack of evidence for skeletal abnormalities in offspring of mice exposed to ethylnitrosourea.

Using morphometrical methods, we investigated variation in the skeletons of more than 400 offspring of C57BL/6J and DBA/2J male mice that had received either a 250 mg/kg dose of the mutagen ethylnitrosourea or a solvent control. Sperm involved in the matings developed from cells in the spermatogonial stage at the time the animals were injected. Although variants were detected, differences in frequencies of gross abnormalities and minor variations in shape between treated and control groups were almost all nonsignificant. There were also no major differences in measures of variability within the groups of offspring from either the treated males or the control group. Additional examination of the skeleton for changes in the frequency of a series of nonmetrical variants also provided no evidence of differences between the treated and control groups that could be attributed to induced mutations. These results conflict with previous findings that ethylnitrosourea is a potent inducer of dominant skeletal mutations.

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine.

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

DNA methyltransferase induced by frog virus 3.

Over 20% of the cytosine bases in frog virus 3 DNA are methylated at the 5-carbon position. To determine whether this high degree of methylation is the result of a virus-specific enzyme, we examined the kinetics of induction and the substrate specificity of a DNA methyltransferase from frog virus 3-infected fathead minnow cells. A novel DNA methyltransferase activity appeared in the cytoplasm of infected cells at 3 h postinfection. This activity was induced in the absence of viral DNA replication and was therefore probably an early viral enzyme. In contrast to the methyltransferase activity extracted from uninfected cell nuclei, the cytoplasmic enzyme showed a strong template preference for double-stranded over single-stranded and for unmethylated over hemimethylated DNA. The dinucleotide sequence dCpdG was a necessary and sufficient exogenous substrate for methylation in vitro. A mutant of frog virus 3, isolated as resistant to 5-azacytidine and having unmethylated virion DNA, did not induce cytoplasmic DNA methyltransferase, leading to the conclusion that this activity is coded for by the virus.

Restriction endonuclease mapping of the frog virus 3 genome.

A physical map for the frog virus 3 (FV 3) genome was constructed after digestion with the following restriction endonucleases: EcoRI, HindIII, KpnI, and XbaI. Mapping of the DNA was accomplished by partial digestion and recutting, double-digestion, and Southern blot hybridization with deduction of overlaps. Although the virion DNA is physically linear, the restriction map was circular, supporting the data that the FV 3 genome is circularly permuted (Goorha and Murti, Proc. Nat. Acad. Sci USA 79, 248-252, 1982), a unique feature among eukaryotic viruses.