The prevalence of retinal detachment in aphakic high myopic patients.
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Adult↗
Biomedical subjects
M Lusky
Publications and source records attributed to M Lusky.
Implantation growth of an iris melanocytic lesion simulating a ciliary body melanoma.
A 59-year-old woman had a sectorial pigmented iris tumor in her right eye and was found to have secondary glaucoma unresponsive to medical therapy. Gonioscopy revealed a satellite tumor in the inferior chamber angle, which we suspected to be a ciliary body melanoma. Subsequently, both lesions were removed completely by superior total and inferior partial iridocyclectomy. Histologic examination disclosed a spindle cell nevus with surface plaque associated with a borderline epithelioid cell nevus. This case is an example of unexpected clinical findings that may be encountered in any iris melanocytic lesion, benign or malignant, and the consequent problems of diagnosis and management.
Ciliary Body↗
Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor.
Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell.
Bovine papillomavirus 1↗
The bovine papillomavirus replicon.
The bovine papillomavirus genome contains two cis-acting sequences which can serve as signals for replication. At least three virally encoded genes seem to be involved in plasmid replication: E6, E6/7 and E1. Mutations in either the E6 or the E7 open reading frame create plasmids that are maintained at a low copy number per cell. Mutations in the E1 open reading frame are absolutely lethal to replication. Complementation experiments show that these mutations define separate genes. Experiments are described which show that cells harbouring plasmids with mutations in either the E6 or the E7 open reading frame acquire an immunity to high copy-number plasmids. We suggest that either the cell or the virus encodes a repressor. The positive action of E6 and E6/7 modulates the activity of this repressor to allow for the high copy-number state. Though the viral oncogenes are capable of transforming cells separately when they are expressed as part of certain recombinant DNA expression systems, it is clear that, in the context of the entire viral replicon, interactions between the transforming functions and replication functions must exist.
Animals↗
Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements.
A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot.
Bovine papillomavirus 1↗
A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state.
A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids.
Animals↗
[Retinal artery obstruction in young patients].
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Humans↗
Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors.
The establishment of bovine papillomavirus type 1 in somatic mammalian cells is mediated by extrachromosomal replication and stable maintenance of the viral genome as a multicopy nuclear plasmid. Previous studies indicated the requirement of viral gene expression for bovine papillomavirus type 1 replication and plasmid maintenance (M. Lusky and M. R. Botchan, Cell 36:391-401, 1984; Turek et al., Proc. Natl. Acad. Sci. U.S.A. 79:7914-7918, 1982). To define the viral genes which are necessary for this process, we constructed a series of specific mutations within the viral genome and assayed the resulting mutants for their ability to replicate extrachromosomally in mouse C127 cells. We report here that the bovine papillomavirus type 1 trans-acting replication factors were encoded by at least two distinct viral genes since the mutants fell into two complementation groups, rep and cop. Mutants (rep-) affecting the E1 open reading frame (ORF) failed to replicate bovine papillomavirus type 1 DNA extrachromosomally and would integrate into chromosomal DNA. We suggest that this gene product is one of the factors required to specifically preclude the integration event. Mutants (cop-) affecting the E7 ORF were maintained in the extrachromosomal state; however, the copy number of the mutant genomes was reduced 100-fold compared with that of wild-type DNA. Analysis of single-cell subclones showed that each cell contained the mutant genomes at a copy number of one to two, indicating that the cop- phenotype did not reflect a simple segregation defect. We propose that the gene defined by mutations in the E7 ORF played a crucial role in stably maintaining the copy number of the viral plasmid at high levels. Genomes with mutations in the cop and rep complementation groups, when cotransfected, rescued the wild-type phenotype, extrachromosomal replication with a high, stable copy number for both types of plasmids. Therefore, the gene products acted in trans, and the mutations were recessive to the wild-type functions. One specific rep- mutant showed a 30-fold-increased transformation efficiency when compared with that of the wild-type genome. In addition, morphological transformation mediated by the cop- mutants appeared to be unstable. These results imply that either or both of the replication functions played some role in regulating the expression of the viral transforming functions.
Animals↗
Amplification of a bovine papillomavirus-simian virus 40 chimera.
A chimeric plasmid, pBOP, containing bovine papillomavirus (BPV) and the origin of replication from simian virus 40 (SV40) was constructed. The plasmid was established in mouse cells, where it was maintained stably as an autonomous BPV replicon. Lines carrying pBOP were fused to cells of COS-7, a simian line producing SV40 T antigen. Replication dependent on the SV40 origin and having the kinetics and approximate amplitude of an SV40 infection ensued. SV40 replication is therefore dominant over BPV replication, and the SV40 origin can conveniently be used to amplify lower-copy-number plasmids in mammalian cells.
Animals↗
Characterization of the bovine papilloma virus plasmid maintenance sequences.
Bovine Papilloma Virus (BPV-1) establishes itself as a multicopy nuclear plasmid in somatic mammalian cells in culture. We report here that two discontinuous regions within the viral genome can independently support extrachromosomal replication of the Tn5 neomycinr gene in cells that provide viral factors in trans. The viral plasmid maintenance sequences (PMS) act in cis and will integrate along with the marker gene in cell lines that do not provide BPV-1 gene products. PMS-1 is localized within a 521 bp region upstream of the BPV-1 early transcription unit; PMS-2 has been localized to a 140 bp region within the putative reading frame for the E1 protein of the viral genome. Recombinant plasmids carrying either of the PMS elements are unrearranged and stably maintained at a constant copy number supernumerary to the resident BPV-1 genomes even in the absence of selective pressure. Specific deletion mutants within the viral genome show that BPV-1 gene products required for morphological transformation are dispensable for plasmid maintenance. In mouse cells cotransformed with such deletion derivatives and an unlinked marker gene (neomycinr or Tk), the marker genes integrate into the host genome while the BPV molecules are nonselectively carried as nuclear plasmids. This result implies that the BPV-1 genome must have signals that specifically preclude integration in the presence of transacting factors.
Animals↗
Bovine papilloma virus contains an activator of gene expression at the distal end of the early transcription unit.
Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promoters. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA. We propose that the enhancement of stable transformation frequencies and the activation of transcription units are in this case alternate manifestations of the same biochemical events.
Base Sequence↗
Expression of two proteins from overlapping and oppositely oriented genes on transposable DNA insertion element IS5.
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Bacterial Proteins↗
Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences.
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Animals↗
Detection of replicational inceptor signals in IS5.
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Bacteriophage lambda↗
Inceptor and origin of DNA replication in lambdoid coliphages. I. The lambda DNA minimal replication system.
In a pBR313-lambda dv hybrid plasmid system, stepwise deletion and serial cloning procedures have led to a functional dissection of the DNA replication region of lambdoid bacteriophages lambda, 434 and 21. A simple system for initiation of DNA replication has been detected within lambdoid replicator DNAs, which is active in the absence of several normal replication elements, including the origin of replication (ori) and product of gene O. This "minimal" (or "mini") initiation system depends on the p0 or substitute leftward promoter in conjunction with the newly discovered "inceptor" (ice) element, which is located within the cII gene. Even the fragments containing ori are unable to initiate replication in these hybrid plasmids as long as fragments containing ice are missing. The base sequence of ice resembles transcriptional terminators and it appears to control both termination of primer RNA and inception of daughter strand DNA synthesis. Initiation in the p0-ice mini system of lambda or 21 phages requires the gene P product. Hwever, mini replication of 434 DNA hybrid plasmids required neither O nor P proteins, although there are only two single-base changes in the 434 inceptor sequence. The mini system is repressed by the elements of the maximal lambda replication system, as described in the accompanying publication.
Bacteriophage lambda↗
Inceptor and origin of DNA replication in lambdoid coliphages. II. The lambda DNA maximal replication system.
In pBR313-lambda dv hydrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p0 promoter). The "maximal" (or "maxi") initiation system depends on the origin of replication (ori) sequence, in conjuction with the "inceptor" (ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will ori effectively initiate lambda specific, O and P-dependent "maximal" hybrid-plasmid replication. In addition, it will repress a complete lambda "minimal" initiation system in cis, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is delected from such a plasmid which was combining both the "mini" and "maxi" systems of lambda replication.
Bacteriophage lambda↗
Functional analysis of the replicator structure of lambdoid bacteriophage DNAs.
In our hybrid-plasmid reconstruction analysis of lambda (lambdoid) DNA signal structures involved in phage DNA replication, we have detected a dual system alternatingly able to initiate a first primer-RNA synthesis. Both of them--the major, primase-dependent ori system and the minor and usually suppressed, RNA-polymerase-dependent oop system--act in conjunction with a common signal structure for inception of DNA synthesis. It appears that in situations such as this, where one has to deal with the existence of regular as well as backup systems serving the same function, straightforward conclusions are no longer possible in their genetic analysis. For example, even though the oop-DNA segment can be deleted entirely from bacteriophage lambda DNA without disturbing its ability to replicate, it may not be valid to conclude that the oop system has no function in DNA replication. Dual systems of this type or organization in general have also been observed previously for some other replicons such as the R-factors R6-5 and R6K (Timmis et al. 1978; Crosa et al., this volume) or the F factor (Helinski et al., this volume), and they may be more common than presently expected.
Bacteriophage lambda↗
Differential influence of the E4 adenoviral genes on viral and cellular promoters.
BACKGROUND: Strong and stable transgene expression is fundamental to the success of recombinant adenovirus vectors in human gene therapy. However, control of transgene expression is a complex process, involving both viral and cellular factors. In this study, the influence of the E4 adenoviral region on the activity of various promoters was investigated in vitro and in vivo. METHODS: Pairs of isogenic E1o and E1oE4o vectors were generated and compared. Levels of transgene expression were determined by Northern blot, ELISA and FACS analysis. Initiation of transcription was studied by nuclear run-on assays. RESULTS: Similar to the viral CMV and RSV promoters, the activity of the ubiquitous cellular PGK promoter required the presence of the E4 genes in vitro and in vivo. In contrast, transgene expression from selected liver- and tumor-specific promoters did not require E4 functions. CONCLUSION: Together with the reported low liver toxicity of E1oE4o vectors, the independence of E4 of liver-specific promoters renders such vectors interesting alternatives to the use of gutless vectors.
3T3 Cells↗