Wild-type and mutant forms of v-src differentially alter neuronal migration and differentiation in vivo.

The effects of three different forms of v-src on brain cell development were determined in vivo. Recombinant retroviral vectors encoding the marker lacZ (control) and either wild-type v-src or SH2 or SH3 domain-deleted forms of v-src (deltaSH2 or deltaSH3, respectively) were used to infect neuronal progenitor cells in the embryonic chicken midbrain (optic tectum; OT). Embryos were injected in the OT with retroviral concentrates on embryonic day (E) 3 and sacrificed at E6, E9, and later in development. Patterns of cell proliferation, migration, and differentiation of lacZ-marked clonal cell progeny were then analyzed. Relative to lacZ-only controls, cell clone size at E6 was significantly increased for v-src-, unchanged for deltaSH2-, and smaller for deltaSH3-injected embryos. At E9, deltaSH2 cell clones were significantly larger than controls, suggesting increased survival from normal programmed cell death. Radial neuronal migration was impaired for v-src and deltaSH3 clones, whereas tangential neuronal migration was enhanced along fiber tracts in v-src and deltaSH2 clones. Moreover, radial glial cell development and differentiation was hindered in v-src and deltaSH3 clones. These experiments demonstrate that ectopic v-src signaling alters proliferation, migration, survival, and differentiation of developing brain cells and suggest that src signaling pathways are involved in these developmental processes. Furthermore, certain effects of v-src on brain cells require specific src homology domains.

The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae.

Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin. In this study we used footprinting procedures to characterize these interactions in vivo. DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose. In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free. The nucleosome footprint was lost when the promoter was activated. Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed.

The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos.

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo.

Transcriptional activity of the Rous sarcoma virus long terminal repeat correlates with binding of a factor to an upstream CCAAT box in vitro.

The avian nuclear protein, enhancer factor 1 (EF1), binds specifically to the long terminal repeat (LTR) of Rous sarcoma virus (RSV) in a region that has been implicated in enhancer/promoter function. We have characterized the in vitro binding properties of this factor from chick embryo nuclear extracts by methylation interference/protection foot-printing of the wild-type LTR and also by gel electrophoretic mobility shift assays performed on a series of LTR mutants. We find that the inverted CCAAT pentanucleotide located at position -129 is essential for EF1 binding in vitro. Nucleotides flanking this element exert a smaller effect on binding. Linker-substitution and point mutations which reduce EF1 binding to this site in vitro also reduce promoter activity in transiently transfected cells. EF1 also binds with lower affinity to another inverted CCAAT box at position -65, an element which we show is also essential for transcriptional activity of the RSV LTR. We conclude, therefore, that EF1 is a CCAAT box-binding factor which is involved in the activation of RSV transcription in avian cells. Furthermore, we show that EF1 can recognize the CCAAT boxes of several other promoters in which the functional importance of this pentanucleotide has been established.

In vivo DNA-binding properties of a yeast transcription activator protein.

UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.

Hormonal regulation of the Rous sarcoma virus src gene via a heterologous promoter defines a threshold dose for cellular transformation.

We have derived rat cell lines producing different and regulatable amounts of pp60v-src by introducing the src gene of Rous sarcoma virus (RSV) under the control of the glucocorticoid-responsive transcriptional promoter from the mouse mammary tumor virus (MMTV). We find that the cellular phenotype is strictly dependent upon the dose of pp60v-src with a distinct threshold for changes indicative of neoplastic potential. Cells with low constitutive levels of pp60v-src are not phenotypically distinguishable from cells without v-src, but as little as a 4-fold increment in pp60v-src produces morphological transformation and anchorage-independent growth. These properties of the transformed state are achieved at levels of pp60v-src far below levels found in an RSV-transformed cell line, without detectable increase in phosphorylation of the major cellular target for tyrosine phosphorylation.

Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein.

Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus.

Proviruses cloned from rat cells infected with mouse mammary tumour virus, a B-type retrovirus regulated by glucocorticoid hormones, show the structural features of transposable elements: short inverted repeats conclude long direct repeats at the ends of viral DNA, and short sequences of cellular DNA are duplicated during integration and flank each provirus. The integrative mechanism joins a precise site in viral DNA to non-homologous sites in host DNA.

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus.

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.

Organization of mouse mammary tumor virus-specific DNA endogenous to BALB/c mice.

We used restriction endonucleases to prepare physical maps of the mouse mammary tumor virus (MMTV)-specific DNA endogenous to the BALB/c mouse strain. The mapping was facilitated by the DNA transfer procedure, using complementary DNAs specific for the whole and for the 3' terminus of MMTV RNA to detect fragments containing viral sequences. The strategies used for the arrangement of fragments into physical maps included sequential digestions with two or three enzymes; preparative isolation of EcoRI fragments containing viral sequences; and comparisons of virus-specific fragments derived from the DNA of several mouse strains. Most of the MMTV-related DNA in the BALB/c genome is organized into two units (II and III) which strongly resemble proviruses acquired upon horizontal infection with milk-borne strains of MMTV and other retroviruses. These units contain approximately 6.0 x 10(6) Mr of apparently uninterrupted viral sequences, they bear redundant sequences totaling at least 700 to 800 base pairs at their termini, and the terminal redundancies include sequences derived from the 3' end of MMTV RNA. Units II and III are closely related in that they share 12 of 14 recognition sites for endonucleases, but cellular sequences flanking units II and III are dissimilar by this criterion. The remainder of the MMTV-related DNA endogenous to BALB/c mice is found in a single subgenomic unit (unit I) with a complexity of ca. 2 x 10(6) Mr; the structure of this unit has not been further defined. These results support the hypotheses that endogenous proviruses have been acquired by infection of germinal tissues with MMTV. The physical maps are also useful for identifying the MMTV genomes endogenous to BALB/c mice in studies of the natural history of mammary tumorigenesis.

Mapping unintegrated avian sarcoma virus DNA: termini of linear DNA bear 300 nucleotides present once or twice in two species of circular DNA.

Three major species of viral DNA have been observed in cells infected by retroviruses: a linear, double-stranded copy of a subunit of viral RNA; closed circular DNA; and proviral DNA inserted covalently into the genome of the host cell. We have studied the structures of the unintegrated forms of avian sarcoma virus (ASA) DNA using agarose gel electrophoresis in conjunction with restriction endonucleases and molecular hybridization techniques. The linear duplex DNA is approximately the same length as a subunit of viral RNA (approximately 10 kb) and it bears natural repeats of approximately 300 nucleotides at its termini. The repeats are composed of sequences derived from both the 3' and 5' termini of viral RNA in a manner suggesting that the viral DNA polymerase is transferred twice between templates. Thus the first end begins with a sequence from the 5' terminus of viral RNA and is permuted by about 100 nucleotides with respect to the 3' terminus of viral RNA; the linear DNA terminates with a sequence of about 200 nucleotides derived from the 3' end of viral RNA. We represent this structure, synthesized from right to left, as 3'5'-----3'5'. Two closed circular species of approximately monomeric size have been identified. The less abundant species contain all the sequences identified in linear DNA, including two copies in tandem of the 300 nucleotide 3'5' repeat. The major species lacks about 300 base pairs (bp) mapped to the region of the repeated sequence; thus it presumably contains only a single copy of that sequence. The strategies used to determine these structures involved the assignment of over 20 cleavage sites for restriction endonucleases on the physical maps of ASV DNA. Several strains of ASV were compared with respect to these sites, and the sites have been located in relation to deletions frequently observed in the env and src genes of ASV.