The sifting and winnowing of DNA sequence that occur during evolution cause nonfunctional sequences to diverge, leaving phylogenetic footprints of functional sequence elements in comparisons of genome sequences. We searched for such footprints among the genome sequences of six Saccharomyces species and identified potentially functional sequences. Comparison of these sequences allowed us to revise the catalog of yeast genes and identify sequence motifs that may be targets of transcriptional regulatory proteins. Some of these conserved sequence motifs reside upstream of genes with similar functional annotations or similar expression patterns or those bound by the same transcription factor and are thus good candidates for functional regulatory sequences.
When a retrovirus infects a cell, the viral genome is stably integrated into the host chromosome, efficiently expressed, and faithfully passed to the infected cell's progeny. For these reasons, recombinant retroviral vectors have often been used to express exogenous genes in vertebrate cells (reviewed in references 28, 34, and 52). In some of these cases, it is advantageous to express two exogenous genes from a single proviral genome. In strategies being developed for gene therapy, for example, the retrovirus often contains not only the gene of interest but also a selectable marker. The marker is used to facilitate the isolation of infected cells, which are then used as a source of the potentially therapeutic gene product (reviewed in reference 12).In another set of studies, we and others have used vectors encoding the histochemical marker 3-galactosidase, the product of the Escherichia coli lacZ gene, as lineage tracers in vivo. A single cell is infected by a retrovirus, the proviral genome is inherited by the cell's progeny, and the clonal relatives are identified with the histochemical stain for LacZ (11,13,16,18,45; reviewed in reference 17). To extend this work, we wished to construct an efficient double-expression vector to transfer both lacZ and a second gene to single cells in vivo. If lacZ and a second bioactive gene were reliably coexpressed at high levels, we could use LacZ histochemistry to identify small clones of transgenic cells in a wild-type environment and then seek cell autonomous effects of the second gene by analyzing the number, distribution, and morphology of the labeled cells.In applications such as these, it is essential that the provirus express both genes within the same individual cells. Retroviruses have been successful in evolving strategies for * Corresponding author.coexpressing their own genes. These strategies include synthesis and subsequent processing of fusion proteins, ribosome frameshifting, and regulated splicing to generate subgenomic messages. Unfortunately, achieving balanced expression of multiple exogenous genes from engineered retrovirus vectors has been more problematic. Two approaches have been used previously. The first involves the generation of separate mRNAs by regulated splicing of a single primary transcript expressed from the upstream long terminal repeat (LTR). This strategy mimics that used by retroviruses to generate the env gene product (54). With this approach, expression of one gene is always at the expense of the other, and the ratio of spliced to unspliced mRNA is highly dependent on the context (1,2,48,49). The second approach involves expression of the upstream gene from the retrovirus promoter in the LTR and expression of the downstream gene from an internal promoter. This approach has been used most often in vectors designed for gene therapy (reviewed in references 28 and 34) but is compromised by competitive interference between promoters (4,8,20,39). Thus, individual isolates may express one gene or the other, rather than both.The recent demons...
The primary translation products of retroviral poi genes are polyproteins initiated in an upstream gene (gag). To investigate the manner in which the gag-initiated polyproteins of the mouse mammary tumor virus are produced, we determined the nucleotide sequence of a 1.8-kilobase DNA fragment that spans the region between gag and poi in the C3H strain of mouse mammary tumor virus. The sequence reveals three overlapping open reading frames: the first encodes products of gag (p275w9 and p145 ¶; the second encodes a protein domain of unknown function (termed X) that is highly related to a similarly positioned sequence in simian type D retroviruses and the viral protease (pro); and the third encodes the reverse transcriptase. The reading frames are organized to permit uninterrupted readthrough from gag to pol if ribosomal frameshifts occur in the -1 direction within each of the two overlapping regions, one of which is 16 nucleotides in length and the other 13 nucleotides. Cell-free translation of RNA containing these overlap regions shows that fusion of the reading frames by ribosomal frameshifting occurs efficiently: about one-fourth of the ribosomes traversing the gag-X/pro overlap and one-tenth traversing the X/pro-pol overlap shift frames, generating gag-related polyproteins in ratios similar to those observed in vivo. Synthetic oligonucleotides containing either of the overlap regions inserted into novel contexts do not induce frameshifting; hence the overlapping portions of the reading frames are not sufficient to induce a frameshift event, and a larger sequence context or secondary structure may be implicated.The mouse mammary tumor virus (MMTV) is unusual among retroviruses in that it can propagate and act as a carcinogenic agent in mammary epithelial tissue, it is transcriptionally regulated by steroid hormones, and it has a type B morphology. Nevertheless it utilizes strategies for macromolecular synthesis similar to those observed with other retroviruses: (i) gag-encoded viral core proteins are coordinately synthesized as components of a large precursor protein that is subsequently processed by a virus-encoded protease; (ii) the pol-encoded reverse transcriptase and integrase proteins are expressed at lower levels by similar processing of a large, fused gag-pol precursor; and (iii) env-encoded glycoproteins are expressed from a spliced, subgenomic mRNA (for review, see ref.
We used a recombinant retrovirus to study cell lineage in the chicken optic tectum. The virus inserts the Escherichia coli lacZ (.3-galactosidase) gene into the genome of an infected cell; a histochemical stain marks the progeny of infected cells with a blue precipitate. We had previously shown that individual clones frequently contain diverse neuronal types. Now we asked whether individual clones contain glia as well as neurons. To this end, we constructed a virus in which lacZ is fused to a nuclear localization signal sequence from the simian virus 40 large tumor antigen. Cells infected with this virus are marked with blue nuclei instead of blue somata. In embryos injected with a mixture of the two retroviruses, individual clusters contained cells with only one label type (nuclear or cytoplasmic), thus verifying that clusters of cells were clones. Furthermore, it was possible to immunostain the somata of cells that had blue nuclei, whereas the blue cytoplasmic precipitate hampered immunostaining. Together, these methods allowed us to show that some clones contained neurons (neurofilament-positive) and two types of glia (glutamine synthetase-positive and glial fibrillary acidic proteinpositive). This result demonstrates the existence of a common progenitor for neurons and glia in optic tectum.A few years ago, we (1) and Price et al. (2) form coherent radial arrays (4), many putative glia were displaced tangentially. It was therefore unclear whether the glia had migrated tangentially from their neuronal relatives or arisen from separate, nearby progenitors. Thus, our tentative conclusion, that some tectal progenitors give rise to both neurons and glia, remained unverified because of ambiguities in the identification of glial cells and of clonal boundaries.To circumvent both of these problems, we constructed a retroviral vector that targets LacZ to the nucleus. This was done by replacing the lacZ gene in the virus with a construct in which lacZ is fused to a short "signal" sequence from the nuclear large tumor antigen of the simian virus 40 virus (5-7); in fact, Kalderon et al. (5) originally used this fusion to show that the sequence in question codes for a nuclear localization signal (5). As expected, cells infected with the nuclear signal-lacZ (nslacZ) virus had blue nuclei instead of blue somata after staining. We were then able to use the nslacZ virus in two types of double-staining protocols. (i) Embryos were infected with a mixture of lacZ and nslacZ viruses to determine whether individual clusters of cells presumed to represent clones indeed contained cells with only one type of label, nuclear or cytoplasmic. (ii) We combined immunofluorescent visualization of cytoplasmic antigens with f8-galactosidase histochemistry to determine the phenotype of cells that had blue nuclei. Together, these methods allowed us to demonstrate unambiguously that some clones of tectal cells contain both neurons and glia. METHODSViruses. The recombinant retroviral vectors used in this study (Fig. 1) were constructed ...
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