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Biomedical subjects

K J Danna

Publications and source records attributed to K J Danna.

At least 19 recordsLinked to original sources
PubMed1997

Isolation, sequence, and expression of the gene encoding halocin H4, a bacteriocin from the halophilic archaeon Haloferax mediterranei R4.

The first gene to encode a haloarchaeal bacteriocin (halocin H4) has been cloned and sequenced from Haloferax mediterranei R4. Both the signal sequence in the halocin H4 preprotein and the monocistronic halH4 gene have some unusual features. The physiology of halH4 expression reveals that although halH4 transcripts are present at low basal levels during exponential growth, halocin H4 activity first appears as the culture enters stationary phase. As halocin activity levels increase, so do transcript levels, but then activity levels decrease precipitously while transcript levels remain elevated.

Amino Acid Sequence↗
PubMed1996

Reduced levels of alpha1(XI) procollagen mRNA in SV40-transformed cells.

We have used mRNA differential display to search for cellular genes whose expression is modulated in SV40-transformed cells. We identified a gene, the alpha1 subunit of type XI procollagen (alpha1(XI)), whose mRNA level is reduced in most F111 rat cell lines that express either wild-type SV40 T antigen or an amino-terminal fragment of T antigen (T147D). alpha1(XI) mRNA expression is also reduced in rat embryo fibroblasts transformed by SV40. This phenomenon is not unique to rat cells, because alpha1(XI) mRNA expression is repressed in C3H10T1/2 mouse cells transformed by either T147D or wild-type SV40. Interestingly, alpha1(XI) mRNA levels are not reduced in F111 cells that express only the polyomavirus large T antigen, but are reduced in F111 cells transformed by polyoma virus (which expresses all three polyomavirus T antigens) and in F111 cells that express the adenovirus E1A proteins. Thus, repression of alpha1(XI) mRNA expression may be a common feature of transformation by DNA tumor viruses.

Adenovirus E1A Proteins↗
PubMed1994

An amino-terminal fragment of SV40 T antigen induces cellular DNA synthesis in quiescent rat cells.

SV40 large T antigen can stimulate cellular DNA synthesis in quiescent cells. Here we report that an SV40 mutant, which expresses only the amino-terminal 147 amino acids of this protein and which does not make small t antigen, stimulates cellular DNA synthesis in quiescent rat cells at levels similar to those achieved with serum stimulation. Thus, the amino-terminal 147 amino acids of large T antigen define a domain of this protein that is sufficient for efficient stimulation of cellular DNA synthesis in the absence of small t antigen.

Animals↗
PubMed1992

An amino-terminal fragment of SV40 T antigen transforms REF52 cells.

An SV40 mutant, T147D, encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We show here that a retrovirus which expresses this mutant T antigen transforms rat REF52 cells as efficiently as a retrovirus that expresses both the wild-type large and small T antigens. This cell line had previously been refractory to transformation by mutants that make short, amino-terminal fragments of T antigen.

Animals↗
PubMed1991

The amino-terminal 147 amino acids of SV40 large T antigen transform secondary rat embryo fibroblasts.

T147D is an SV40 mutant that encodes only the amino-terminal 147 amino acids of large T antigen and does not make small t antigen. We have constructed a recombinant retrovirus that expresses the T147D mutant protein. We show here that this virus can transform the established rat cell line, F111, in an agar assay with high efficiency. More importantly, we demonstrate that this retrovirus transforms secondary rat embryo fibroblasts to anchorage independence as efficiently as a recombinant retrovirus that expresses both wild-type large and small T antigens. These data indicate that in rat cells, the amino-terminal 147 amino acids of T antigen are sufficient for transformation. Further, since the T147D protein does not bind p53, we conclude that the association between T antigen and p53 is not required for the transformation of rat cells to anchorage-independent growth.

Animals↗
PubMed1990

Method to identify genomic targets of DNA binding proteins.

We have devised a cyclical immunoprecipitation protocol that can be used to identify and clone a specific DNA sequence that is recognized by a DNA binding protein, even if that sequence is present in only one copy in the genome of a mammal. As an example, we have used this procedure to purify mouse genomic sequences to which the simian virus 40 tumor (T) antigen binds.

Adenoviruses, Human↗
PubMed1989

An SV40 mutant oncoprotein has a nuclear location.

T147 is an SV40 mutant that makes a normal small t antigen and a large T antigen that is only 147 amino acids long. We have introduced a second mutation into the genome of T147 which eliminates its ability to encode small t antigen. We show that this double mutant is able to transform C3H10T1/2 mouse cells in a focus assay and F111 rat cells in an agar suspension assay, demonstrating that the transforming domain of T antigen is located within its amino-terminal 147 amino acids. We also show that the T147 mutant T antigen, like wild-type T antigen, has a nuclear location. However, in contrast to wild-type T antigen, which is also found in the plasma membranes of wild-type transformed cells, we fail to detect any mutant T antigen associated with the plasma membranes of T147 transformants.

Animals↗
PubMed1988

A new SV40 mutant that encodes a small fragment of T antigen transforms established rat and mouse cells.

We have constructed a new SV40 mutant, T147, that makes a large T antigen that is only 147 amino acids long. We show that the T147 T antigen is a phosphoprotein that is as stable as wild-type T antigen and that the SV40 viral origin binding activity of the T147 T antigen is reduced at least 100-fold relative to that of wild-type T antigen. Most importantly, we demonstrate that cloned T147 DNA transforms rat F111 cells to anchorage independence as efficiently as cloned wild-type SV40 DNA and that cloned T147 DNA also efficiently transforms C3H10T1/2 mouse cells in a focus assay.

Animals↗
PubMed1986

An SV40 mutant T antigen does not bind the SV40 viral origin.

F8dl is an SV40 deletion mutant that lacks over 60% of the coding sequences for large T antigen and yet is able to immortalize early passage rat cells, to transform established cell lines, and to cause tumors in animals. We report here on the further characterization of this mutant and show that (a) transformation by F8dl is protein mediated but does not require the action of the SV40 small t antigen; (b) the F8dl T antigens have, or are associated with, an ATPase activity; (c) the 34-kDa mutant T antigen of F8dl is localized in nuclei and cell membranes of F8dl transformants and binds to double-stranded DNA; (d) the 20-25 kDa forms of the mutant T antigen are cytoplasmic; and (e) the F8dl T antigens do not bind with high affinity to the SV40 origin of viral DNA replication.

Adenosine Triphosphatases↗
PubMed1985

The SV40 T-antigen gene can have two introns.

F8dl is an SV40 early-region mutant that lacks over 60% of the DNA sequences normally used to encode large T antigen. This mutant employs a novel splice donor junction at nucleotide 4425 to produce a family of doubly spliced messages. A similar splicing pattern with wild-type SV40 mRNA has been observed, indicating that the wild-type gene for T antigen can also have two introns. A single G-to-T base change at nucleotide 4425 is sufficient to eliminate this novel donor splice junction.

Animals↗
PubMed1985

A fragment of the simian virus 40 early genome can induce tumors in nude mice.

Cell lines transformed by simian virus 40 mutant F8dl (deleted from 0.168 to 0.424 map units, corresponding to the carboxy-terminal 62% of the wild-type simian virus 40 large tumor antigen) are tumorigenic in nude mice. Four of five C3H10T1/2 cell lines transformed by F8dl were tumorigenic in nude mice, whereas two of two wild-type transformants were tumorigenic.

Animals↗
PubMed1985

The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells.

F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells.

Animals↗
PubMed1984

S1 mapping of purified nascent transcripts of simian virus 40.

We purified nascent simian virus 40 late transcripts by incubating viral transcriptional complexes, isolated from infected BSC-1 cells, in a reaction mixture that contained mercurated CTP; RNA molecules that had incorporated mercurated residues in vitro were isolated by sulfhydrylcellulose affinity chromatography. The nascent RNA was hybridized to an end-labeled HindIII C probe fragment (0.646 to 0.86 map unit), and the hybrids were analyzed by S1 mapping. Most of the products of digestion corresponded to unspliced transcripts with 5' ends mapping at nucleotides 325, 260, and 195, which are positions of the 5' ends of mature, cytoplasmic late mRNA species. In addition, two minor products diagnostic of splicing at the acceptor junctions mapping at nucleotides 556 and 443 were detected. Because the abundance of these products was not diminished by repurifying the nascent RNA through a second round of sulfhydrylcellulose chromatography, these products did not originate from contaminating non-nascent RNA. Moreover, the generation of these products was not affected when a higher salt concentration and lower temperature were used for S1 digestion, conditions that should decrease artifactual cleavage by S1 in A + U-rich regions of colinear hybrids. Therefore, it is likely that some simian virus 40 RNA chains are spliced before release from the template.

Base Sequence↗
PubMed1984

Less than 40% of the simian virus 40 large T-antigen-coding sequence is required for transformation.

F8dl is a simian virus 40 early-region deletion mutant that lacks the simian virus 40 DNA sequences between 0.168 and 0.424 map units. Despite this large deletion, cloned F8dl DNA transforms Fisher rat F111 cells and BALB/3T3 clone A31 mouse cells as efficiently as does cloned simian virus 40 wild-type DNA. These results indicate that less than 40% of the large T-antigen-coding sequence is required for efficient transformation.

Animals↗
PubMed1983

Genomic organization of rat rDNA.

A detailed restriction map was determined for a 10.9 KB region that contains the initiation site for 45S pre-rRNA and the first 1.7 KB of the 18S rRNA coding region. When the restriction pattern of the cloned rDNA was compared with that of total rat DNA, the rDNA regions of both Sprague-Dawley and BD-9 rats were identical to each other and to that of the cloned rDNA. However, both strains exhibit a major polymorphism consisting of an insertion of 0.9 KB of DNA in the nontranscribed spacer between 0.29 KB and 1.8 KB upstream from the 45S RNA initiation site. This region consists of tandem repeats approximately 130 base pairs in length. These repeats contain large poly T tracts and are similar in sequence to analogous elements 5' to the origin of mouse rRNA transcription. Regions containing highly repetitious DNA sequences were located at sites 2.8 KB and 4.3 KB upstream from the initiation site. The repetitive sequence at 2.8 KB from the initiation site anneal to a known Alu-equivalent type 2 sequence derived from the second intron of the rat growth hormone gene.

Animals↗
PubMed1983

Characterization of the 5' termini of purified nascent simian virus 40 late transcripts.

The primary transcripts of simian virus 40 are extensively processed in the nuclei of infected monkey cells before they are transported to the cytoplasm as mature mRNAs. To investigate the early steps in this process, in particular, to determine which events occur on nascent chains before the termination of transcription, we have developed a procedure for the purification of nascent viral transcripts. This technique involves the in vitro incorporation of mercurated residues into the growing 3' ends of pre-initiated nascent chains, allowing their specific purification by sulfhydrylcellulose affinity chromatography. We further selected viral specific transcripts by hybridization to simian virus 40 DNA-cellulose. We describe here our analysis of the 5' termini of purified nascent simian virus 40 transcripts. This analysis revealed various cap structures, providing direct evidence that primary viral transcripts are capped before chain completion. The various cap structures exhibited a full range of methylation states. Completely unmethylated GpppA cap cores were identified, as well as caps methylated at the penultimate position only. The presence of GpppAm and GpppmAm caps indicates that, in BSC-1 cells, the penultimate nucleotide can be methylated before 7-methyl-G formation. Furthermore, the proportions of the various intermediates suggest that, in contrast to the viral capping enzymes of vaccinia virus and reovirus, the cellular enzymes methylate in the following order: GpppA leads to GpppAm leads to GpppmAm leads to 7mGpppmAm. In addition to capped ends, we also detected some unprocessed pppA ends. To our knowledge, this is the first time uncapped termini have been identified on RNAs known to be polymerase II products.

Adenosine Triphosphate↗
PubMed1983

Simian virus 40 sequences between 0.168 and 0.424 map units are not required for abortive transformation.

We have isolated a simian virus 40 deletion mutant, F8dl, that lacks the sequences from 0.168 to 0.424 map units. The deleted sequences represent over 60% of the coding region for large T antigen. Despite this deletion, F8dl abortively transformed rat cells as efficiently as wild-type simian virus 40. From this result, we conclude that the region of the simian virus 40 genome between 0.168 and 0.424 map units is not essential for abortive transformation. Since abortive transformation requires the expression of the simian virus 40 maintenance functions, we also infer that the sequences deleted from F8dl are not required to maintain transformation.

Animals↗