Alfresco--a workbench for comparative genomic sequence analysis.

Comparative analysis of genomic sequences provides a powerful tool for identifying regions of potential biologic function; by comparing corresponding regions of genomes from suitable species, protein coding or regulatory regions can be identified by their homology. This requires the use of several specific types of computational analysis tools. Many programs exist for these types of analysis; not many exist for overall view/control of the results, which is necessary for large-scale genomic sequence analysis. Using Java, we have developed a new visualization tool that allows effective comparative genome sequence analysis. The program handles a pair of sequences from putatively homologous regions in different species. Results from various different existing external analysis programs, such as database searching, gene prediction, repeat masking, and alignment programs, are visualized and used to find corresponding functional sequence domains in the two sequences. The user interacts with the program through a graphic display of the genome regions, in which an independently scrollable and zoomable symbolic representation of the sequences is shown. As an example, the analysis of two unannotated orthologous genomic sequences from human and mouse containing parts of the UTY locus is presented.

Comparative sequence analysis of a region on human chromosome 13q14, frequently deleted in B-cell chronic lymphocytic leukemia, and its homologous region on mouse chromosome 14.

Previous studies have indicated the presence of a putative tumor suppressor gene on human chromosome 13q14, commonly deleted in patients with B-cell chronic lymphocytic leukemia (B-CLL). We have recently identified a minimally deleted region encompassing parts of two adjacent genes, termed LEU1 and LEU2 (leukemia-associated genes 1 and 2), and several additional transcripts. In addition, 50 kb centromeric to this region we have identified another gene, LEU5/RFP2. To elucidate further the complex genomic organization of this region, we have identified, mapped, and sequenced the homologous region in the mouse. Fluorescence in situ hybridization analysis demonstrated that the region maps to mouse chromosome 14. The overall organization and gene order in this region were found to be highly conserved in the mouse. Sequence comparison between the human deletion hotspot region and its homologous mouse region revealed a high degree of sequence conservation with an overall score of 74%. However, our data also show that in terms of transcribed sequences, only two of those, human LEU2 and LEU5/RFP2, are clearly conserved, strengthening the case for these genes as putative candidate B-CLL tumor suppressor genes.

Comparative analysis of noncoding regions of 77 orthologous mouse and human gene pairs.

A data set of 77 genomic mouse/human gene pairs has been compiled from the EMBL nucleotide database, and their corresponding features determined. This set was used to analyze the degree of conservation of noncoding sequences between mouse and human. A new alignment algorithm was developed to cope with the fact that large parts of noncoding sequences are not alignable in a meaningful way because of genetic drift. This new algorithm, DNA Block Aligner (DBA), finds colinear-conserved blocks that are flanked by nonconserved sequences of varying lengths. The noncoding regions of the data set were aligned with DBA. The proportion of the noncoding regions covered by blocks >60% identical was 36% for upstream regions, 50% for 5' UTRs, 23% for introns, and 56% for 3' UTRs. These blocks of high identity were more or less evenly distributed across the length of the features, except for upstream regions in which the first 100 bp upstream of the transcription start site was covered in up to 70% of the gene pairs. This data set complements earlier sets on the basis of cDNA sequences and will be useful for further comparative studies. [This paper contains supplementary data that can be found at http://www.genome.org [corrected]].

Segregation properties of bovine papillomaviral plasmid DNA.

Essentially complete segregation of replication-competent BPV-1 plasmid DNA species was observed in daughter subclones derived from primary co-transformed C127 cell lines. Thus, whereas primary co-transformants retained both of two distinguishable co-transfected plasmid species, subcloning experiments revealed that morphologically transformed daughter subclones derived from such co-transformed cell lines contained only one species of viral plasmid DNA. Similar results were obtained with each of two conveniently marked replication and transformation-competent mutants: one with a linker-insertion in the viral upstream regulatory region, and one with a 260 base-pair deletion within the L2 (late) gene, which has no recognized role in plasmid replication or stability. Morphological revertant cell clones that contained no detectable viral plasmid DNA genomes were also isolated at a surprisingly high frequency from clonal wild-type BPV-1 transformed cell lines and from cell lines transformed by various BPV-1 mutants. Further co-transfection experiments were done with a combination of transformation-competent and transformation-defective BPV-1 genomes to investigate a possible role for a viral oncogene in plasmid persistence. In this case, elimination of the transformation-defective mutant was observed after the initial establishment of both input genomes as replicating plasmids in cell clones morphologically transformed by the transformation-competent viral mutant with an intact E5 oncogene. No cell subclones were isolated that contained only the transformation-defective mutant, implying that it was defective in long-term plasmid persistence. Our results indicate that there is significant randomization in the processes of replication and/or partitioning of the BPV-1 genome in mouse C127 cells, and, in combination with previous observations, also suggest that BPV plasmid persistence in C127 cell lines may be the result of a selective proliferative advantage conferred on virus-infected cells by viral oncogene-induced cell growth transformation.

Evidence that the transcriptional trans-activating function of the bovine papillomavirus type 1 E2 gene is not required for viral DNA amplification in division-arrested cells.

Amplification of bovine papillomavirus type 1 (BPV-1) DNA in growth-arrested mouse cell cultures appears to mimic the process of induction of vegetative BPV-1 DNA synthesis in cells of the stratum spinosum in productively infected bovine warts. In both cases, cells permissive for viral DNA amplification express large amounts of viral E2 protein which accumulates within the cell nucleus. Whereas in latently infected virus-transformed cells truncated transcriptional repressor forms of E2 predominate, our previous studies have demonstrated that the full-length E2 transcriptional trans-activator protein is preferentially expressed during the period of maximal BPV-1 DNA amplification in growth-arrested cell cultures. To investigate the role of the full-length E2 gene in the induction of viral DNA amplification in this experimental viral replication system we have used a mutant BPV-1 genome (BPVE2-ts1) containing an E2 gene which is temperature-sensitive (ts) for transcriptional trans-activation. This mutant genome has also been shown to be ts for stable viral plasmid DNA replication and for the induction of cell transformation. We show here that viral DNA amplification was not severely impaired when BPVE2-ts1-transformed cells were tested at the restrictive temperature, indicating that the transcriptional trans-activating function of E2 was not essential for viral DNA amplification in division-arrested cells and, moreover, that the trans-activation and replication functions of E2 were separable. Consistent with this hypothesis, amplification of the BPVE2-ts1 genome at the restrictive temperature was still associated with the accumulation of large amounts of nuclear E2 antigen, showing that the mutation did not disrupt nuclear transport or render the E2 protein highly unstable. Furthermore, C127 cells harbouring ts E2 and full-length E1 expression constructs supported transient plasmid replication of a BPV origin vector at the restrictive temperature. These observations imply that E2 functions primarily as a viral replication factor in the vegetative phase of BPV-1 DNA replication, and suggest a fundamental difference in the genetic regulation of stable BPV-1 plasmid DNA replication in mitotic cells and viral DNA amplification in postmitotic cells.

Identification and genetic definition of a bovine papillomavirus type 1 E7 protein and absence of a low-copy-number phenotype exhibited by E5, E6, or E7 viral mutants.

The bovine papillomavirus type 1 (BPV-1) genome replicates as a multiple-copy plasmid in murine C127 cells transformed to neoplasia by virus infection or by transfection with BPV-1 DNA. It was reported previously that BPV-1 genomes harboring frameshift mutations in the E6 or E7 open reading frame (ORF) replicated in C127 cells transformed by these mutants at a low copy number. Furthermore, the characterization of a BPV-1 mRNA in which the E6 and E7 ORFs were spliced together in frame has led to the assumption that an E6/7 fusion protein is expressed in virus-transformed C127 cells. To define the number and nature of the E6 and E7 gene products expressed in BPV-1-transformed cells, we performed immunoprecipitation experiments with antisera raised to bacterially expressed BPV-1 E6 and E7 fusion proteins. By employing cell culture conditions which induce BPV-1 E2 transactivator expression and viral early region transcription in virus-transformed C127 cell lines, we detected a single immunoprecipitated E6 protein species with an apparent molecular mass of 17 kDa and a single E7 protein species with an apparent molecular mass of 15 kDa. To characterize further these E6 and E7 proteins, C127 cells were transformed by transfection with BPV-1 genomes containing mutations predicted to prevent expression of specific E6 or E7 gene products, and the transformed cells were subjected to immunoprecipitation analysis with the E6 or E7 antiserum. The results of these experiments confirmed that the E6 and E7 ORFs encode distinct proteins and failed to establish the existence of an E6/7 fusion protein. We did not find a significant difference in the viral genome copy number between clonal C127 cell lines transformed by wild-type BPV-1 or by mutant viral genomes unable to express the E6 or the E7 protein. Furthermore, in contrast to two previous reports suggesting that expression of the BPV-1 E5 gene was required for the establishment or maintenance of a high viral plasmid copy number, we observed a two- to fourfold increase over wild-type BPV-1 plasmid copy number in C127 cells transfected with a BPV-1 E5-minus mutant and subsequently selected by neoplastic focus formation.

Localization of bovine papillomavirus type 1 E5 protein to transformed basal keratinocytes and permissive differentiated cells in fibropapilloma tissue.

We examined expression of the E5 transforming protein of bovine papillomavirus type 1 (BPV-1) in naturally and experimentally infected bovine cells. Bovine conjunctival fibroblasts transformed in vitro by experimental infection with purified BPV-1 virions expressed significantly higher amounts of the 7-kDa E5 protein than BPV-1-transformed murine C127 cells. Indirect immunofluourescence analysis revealed a cytoplasmic, predominantly juxtanuclear, localization of E5 protein in the in vitro virus-transformed bovine cells. In naturally infected bovine skin fibropapilloma tissue, two widely separated sites of E5 protein synthesis were identified within the epithelial layers. Transformed basal layer keratinocytes throughout the tumor tissue expressed cytoplasmic E5 protein at a low uniform level. In addition, abundant amounts of cytoplasmic E5 protein with a granular staining pattern were detected in highly differentiated keratinocytes in close association with sites of viral capsid protein synthesis. These observations imply roles for the viral E5 oncogene in the growth transformation of basal epidermal keratinocytes as well as in the differentiation-linked process of viral maturation. Detection of a papillomavirus protein in the basal cell population of warts lends support to the hypothesis that these cells are maintained in a transformed state by continuous expression of a viral transforming gene.

Immunofluorescent detection of bovine papillomavirus E4 antigen in the cytoplasm of cells permissive in vitro for viral DNA amplification.

The E4 gene of several human papillomavirus types is expressed in association with vegetative viral DNA synthesis in differentiated epidermal cells. To develop reagents to study expression of the bovine papillomavirus type 1 (BPV-1) E4 gene in warts and in virus-transformed cell lines, rabbit polyclonal antiserum was raised to the BPV-1 E4 antigen produced as a fusion polypeptide in Escherichia coli. By immunoblotting analysis of productively infected bovine fibropapilloma tissue, E4-related proteins of 16K, 21K, 30K and 42K were detected. In some but not all C127 cell lines transformed by BPV-1 or by a replication-competent BPV-1 deletion mutant, cytoplasmic E4 antigen with a predominantly perinuclear localization was detected by immunofluorescence analysis in a subpopulation of cells in stationary-phase cultures. The E4-expressing cells were identified by their grossly enlarged size to represent the same cell subpopulation shown earlier to support BPV-1 DNA amplification. The observation of synthesis of the E4 protein in association with viral DNA amplification in this system provides further evidence that there is a switch in viral early region gene expression in a subpopulation of division-arrested cells, which may accurately reflect events occurring during the vegetative phase of BPV-1 replication in terminally differentiated cells in vivo.

Induction of bovine papillomavirus E2 gene expression and early region transcription by cell growth arrest: correlation with viral DNA amplification and evidence for differential promoter induction.

The bovine papillomavirus type 1 (BPV-1) genome replicates as a latent plasmid in mouse C127 cells transformed in vitro by the virus. However, we have recently shown that BPV-1 DNA amplification can be induced in a subpopulation of cells under culture conditions which suppress cell proliferation, a finding which led us to hypothesize that expression of a viral replication factor was regulated by cell growth stage. In this report, we describe the detection in these cells of abundant BPV-1 nuclear E2 antigen by immunofluorescence analysis. Expression of E2 antigen in fibropapilloma tissue was similarly localized to nonproliferating epidermal cells of the lower spinous layers--the natural site of induction of vegetative viral DNA replication. Immunoprecipitation analysis showed that the previously characterized 48-kilodalton (transactivator) and 31-kilodalton (repressor) E2 proteins were both induced in growth-arrested cell cultures. In parallel with E2 antigen synthesis under conditions of serum-deprivation in vitro, we observed a significant increase in levels of BPV-1 early region mRNAs. Furthermore, we present evidence for preferential induction of the P2443 promoter, in addition to specific induction of the P7940 promoter in response to serum deprivation. These observations indicate a central role for E2 transcription factors in the induction of viral DNA amplification in division-arrested cells in vitro and in vivo and suggest that this process is associated with a qualitative switch in the expression of viral early region genes.