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D F Klessig

Publications and source records attributed to D F Klessig.

At least 109 records · Page 6Linked to original sources
PubMed1985

Similar regulation of the synthesis of adenovirus fiber and of simian virus 40-specific proteins encoded by the helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del.

Human adenoviruses fail to multiply effectively in monkey cells. The block to the replication of these viruses can be overcome by coinfection with simian virus 40 (SV40) or when part of the SV40 genome is integrated into and expressed as part of the adenovirus type 2 (Ad2) genome, as occurs in several Ad2+SV40 hybrid viruses, such as Ad2+ND1, Ad2+ND2, and Ad2+ND4. The SV40 helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del were analyzed to determine why they are unable to grow efficiently in monkey cells even though they contain the appropriate SV40 genetic information. Characterization of the Ad2+ND5-SV40-specific 42,000-molecular-weight (42K) protein revealed that this protein is closely related, but not identical, to the SV40-specific 42K protein of the SV40 helper-competent Ad2+ND2 hybrid virus. Although the minor differences between these proteins may be sufficient to account for the poor growth of Ad2+ND5 in monkey cells, the most striking difference between helper-competent Ad2+ND2 and helper-defective Ad2+ND5 is in the production of the SV40-specific protein after infection of monkey cells. Whereas synthesis of the SV40-specific proteins of Ad2+ND2 is very similar in human and in monkey cells, production of the 42K protein of Ad2+ND5 is dramatically reduced in monkey cells compared with human cells. Similarly, the synthesis of the SV40-specific proteins of Ad2+ND4del is markedly reduced in monkey cells. Thus, it is likely that both Ad2+ND5 and Ad2+ND4del are helper defective because of a block in the production of their SV40-specific proteins rather than because their SV40-specific proteins are nonfunctional. This block, like the block to adenovirus fiber synthesis, is overcome by coinfection with SV40, with helper-competent hybrid viruses, or with host range mutants of adenoviruses. This suggests that the synthesis of fiber and the synthesis of SV40-specific proteins are similarly regulated in Ad2+SV40 hybrid viruses.

Adenoviruses, Human↗
PubMed1985

Microinjection of mRNA enhances translational efficiency of human adenovirus fiber message in monkey cells.

In monkey cells abortively infected with human adenovirus serotype 2, the synthesis of the fiber polypeptide of the virion capsid is reduced by at least a factor of 100 when compared with that in monkey cells productively infected with a host range mutant of adenovirus serotype 2 (Ad2hr400). However, the steady-state level of fiber-encoding mRNA present in abortively infected monkey cells is only reduced by a factor of 5 to 10. When mRNA isolated from abortively and productively infected monkey cells was microinjected into the cytoplasms of uninfected or abortively infected monkey cells, no differences in the efficiency of translation of the fiber messages from these two sources were observed. These results suggest that the block to synthesis of the fiber polypeptide in abortively infected monkey cells does not reside in the translational machinery of the abortively infected cells themselves but may involve compartmentalization of the fiber message within the cells or an altered processing of the fiber message which prevents correct presentation to the ribosomes.

Adenoviruses, Human↗
PubMed1985

Transcriptional and post-transcriptional regulation of ribulose 1,5-bisphosphate carboxylase gene expression in light- and dark-grown amaranth cotyledons.

The regulation of expression of the genes encoding the large subunit (LSU) and small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) was examined in 1- through 8-day-old, dark-grown (etiolated) and light-grown amaranth cotyledons. RuBPCase specific activity in light-grown cotyledons increased during this 8-day period to a level 15-fold higher than in dark-grown cotyledons. Under both growth conditions, the accumulation of the LSU and SSU polypeptides was not coordinated. Initial detection of the SSU occurred 1 and 2 days after the appearance of the LSU in light- and dark-grown cotyledons, respectively. Furthermore, although the levels of the LSU were similar in both light- and dark-grown seedlings, the amount of the SSU followed clearly the changes in enzyme activity. Synthesis of these two polypeptides was dramatically different in etiolated versus light-grown cotyledons. In light the synthesis of both subunits was first observed on day 2 and continued throughout the growth of the cotyledons. In darkness the rate of synthesis of both subunits was much lower than in light and occurred only as a burst between days 2 and 5 after planting. However, mRNAs for both subunits were present in etiolated cotyledons at similar levels on days 4 through 7 (by Northern analysis) and were functional in vitro, despite their apparent inactivity in vivo after day 5. In addition, since both LSU and SSU mRNA levels were lower in dark- than in light-grown seedlings, our results indicate that both transcriptional and post-transcriptional controls modulate RuBPCase production in developing amaranth cotyledons.

Gene Expression Regulation↗
PubMed1984

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene.

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.

Adenoviruses, Human↗
PubMed1984

Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.

Messenger RNA encoding the fiber protein of the human adenovirus serotype 2 (Ad2) capsid is inefficiently translated in abortively infected African green monkey kidney cells. The amount of fiber mRNA present in the cytoplasm of abortively infected monkey cells is less than that in productively infected cells by a factor of 5-10 but synthesis of the fiber polypeptide is reduced by a factor of more than 100. Evidence from a variety of experiments indicates that the defect does not lie in the translational apparatus of the monkey cell but may best be explained by differences in the fiber messages made in abortively versus productively infected cells. Here we report that fiber mRNA isolated from abortively infected monkey cells is processed differently than that made in productively infected cells. Primer extension analysis of the 5' ends of fiber messages from several different productive and abortive infections shows a direct correlation between synthesis of the fiber polypeptide in vivo and the presence of the "x" and/or "y" ancillary leaders on messages encoding the fiber polypeptide. Of all the mRNAs encoded by the major late transcriptional unit of Ad2 only the fiber message can contain the x and y leaders, and the fiber protein is the only late Ad2 protein reported to be glycosylated. We speculate that these leader sequences play a role in the synthesis of this glycoprotein, as well as that of the Ad2 19-kilodalton glycoprotein encoded by early region 3, whose mRNA also contains the x and y leaders.

Adenoviruses, Human↗
PubMed1984

The function(s) provided by the adenovirus-specified, DNA-binding protein required for viral late gene expression is independent of the role of the protein in viral DNA replication.

The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37 degrees C, but is cold sensitive for plaque formation and late gene expression at 32.5 degrees C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5 degrees C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV1 and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and temperature sensitive, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5 degrees C. The temperature-sensitive mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5ts125. Like Ad5ts125, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.

Adenoviridae↗
PubMed1984

Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.

The DNA-binding protein (DBP) encoded by human adenoviruses is a multifunctional polypeptide which plays a central role in regulating the expression of the viral genes. To gain a better understanding of the relationships between the various functions provided by DBP, an extensive collection of DBP mutants is essential. To this end we have constructed several permissive human cell lines which contain and express the DBP gene at high levels to allow propagation of otherwise lethal, nonrecoverable mutants of DBP. Because DBP is toxic to human cells, cell lines were constructed by using a vector in which the DBP gene is under the control of the dexamethasone-inducible promoter of the mouse mammary tumor virus. The low basal levels of DBP synthesis in the absence of dexamethasone allows isolation and propagation of these cells. Addition of dexamethasone enhances DBP production 50- to 200-fold, and within 8 h its synthesis from the single integrated copy of the chimeric gene is 5 to 15% of that observed during peak DBP synthesis in infected human cells in which hundreds of copies of the DBP gene serve as templates. At the nonpermissive temperature, adenovirus mutants with ts lesions in the DBP gene replicate their DNAs, express their late genes, and form infectious viral particles in these DBP+ cell lines but not in the parental HeLa cells.

Adenoviruses, Human↗
PubMed1983

Independent, spontaneous mutants of adenovirus type 2-simian virus 40 hybrid Ad2+ND3 that grow efficiently in monkey cells possess indentical mutations in the adenovirus type 2 DNA-binding protein gene.

Four independent, spontaneous mutants of the adenovirus type 2-simian virus 40 hybrid Ad2+ND3 that allow efficient growth in monkey cells were isolated previously (C. W. Anderson, Virology 111:263-269, 1981). All four mutations have been mapped within the coding sequence for the adenovirus DNA-binding protein by marker rescue analysis. DNA sequence analysis of a region of ca. 1,000 base pairs shown by marker rescue to contain the host range mutations demonstrated that the host range mutant hr602 differs from its parent, Ad2+ND3, at only a single nucleotide. Mutant hr602 has a thymine in place of a cytosine at the first position of the 130th codon, as measured from the initiation site for the DNA-binding protein. This change results in the replacement of a histidine by a tyrosine in mutant hr602 DNA-binding protein. Each of the other three Ad2+ND3 host range mutants have exactly the same nucleotide alteration as does hr602. This same nucleotide change was recently reported for a similarly derived host range mutant of adenovirus 5.

Adenoviruses, Human↗
PubMed1983

Posttranscriptional block to synthesis of a human adenovirus capsid protein in abortively infected monkey cells.

The synthesis of fiber protein in CV1 (monkey) cells abortively infected with human adenovirus serotype 2 (Ad2) is at least 100-fold less than the synthesis of fiber protein in CV1 cells productively infected with a host range mutant of Ad2 (Ad2hr400) or coinfected with Ad2 plus simian virus 40. However, the amount of fiber mRNA present in the cytoplasm of abortively infected CV1 cells is only 5- to 10-fold less than that in productively infected CV1 cells. Whereas fiber mRNA in abortively infected CV1 cells is utilized poorly as a template for synthesis of fiber protein in vivo, fiber mRNA from abortively infected CV1 cells serves just as efficiently as a template for fiber synthesis in vitro as fiber mRNA from productively infected cells. This was observed both in a nuclease-treated rabbit reticulocyte lysate to which purified fiber mRNA or cytoplasmic ribonucleoprotein complex was added as exogenous template for fiber synthesis, and in S10 extracts of infected CV1 cells utilizing endogenous message as a template. Since translation initiation inhibitors did not diminish synthesis of fiber in S10 extracts of abortively infected CV1 cells, fiber mRNA probably is associated with ribosomes in abortively infected CV1 cells. This conclusion was supported by Northern blot analysis, which showed that in both abortively and productively infected CV1 cells, the same proportion of cytoplasmic fiber mRNA cosedimented with polyribosomes. Although the possibility of extremely rapid fiber turnover in abortively infected monkey cells cannot be rigorously excluded, preliminary data suggest that this is not the case. Thus, these results may imply that translation of the fiber message in abortively infected monkey cells is blocked after formation of the mRNA-ribosome complex.

Adenoviruses, Human↗
PubMed1982

Proteins containing only half of the coding information of early region 1b of adenovirus are functional in human cells transformed with the herpes simplex virus type 1 thymidine kinase gene and adenovirus type 2 DNA.

We introduced into tk- human 143 cells adenovirus type 2 (Ad2) genes by transformation with a plasmid (p711) containing both Ad2 sequences and the herpes simplex virus type 1 (HSV-1) tk gene. p711 contained approximately the left 8% of the Ad2 genome inserted in the HindIII site of pBR322, whereas the fragment of HSV-1 containing the tk gene was inserted in the BamHI site. Three tk+ cell lines were isolated after selection in HAT medium. The arrangement of viral sequences in the three transformants was analyzed by restriction endonuclease digestion and filter hybridization. All three lines contained a single insertion of Ad2 DNA which was present at approximately one copy per cell. The arrangement of Ad2 sequences in these lines was identical to that found in the linear p711 DNA used in the transformation. S1 analysis of the Ad2-specified RNA from two of these lines indicated that the early region 1a mRNA's were synthesized, though in lower amounts than found in lytic infections. These cell lines contained only the left half of early region 1b (4.6 to 11.2), which encoded the 5' portion of the 1b mRNA's. A complex pattern of 1b RNAs was made in these cell lines. Transcription of most of these RNAs began at or near the 1b promoter and proceeded through the 1b sequences into the flanking pBR322, HSV-1, or host sequences. Since many of the RNAs were terminated or spliced in the HSV-1 (anti-sense strand) or pBR322 sequences, new RNA processing sites must be used in the formation of these mRNA's. All three lines fully complemented the 1a deletion mutant Ad5 dl312. Surprisingly, these lines also permitted the growth of 1b deletion mutants (Ad5 dl313 and Ad5 dl434), although the complementation was not always complete. Presumably the new gene product(s) which contained only part of the 1b gene provided most of the essential function(s) required for viral multiplication. Alternatively, the 1b 19-kilodalton protein which was entirely encoded by the adenovirus sequences present in these cell lines was sufficient for viral growth even in the absence of the 1b 55-kilodalton protein.

Adenoviruses, Human↗
PubMed1982

Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection.

Adenoviruses, Human↗
PubMed1982

Normal translation of human adenovirus mRNA in cell-free lysates prepared from abortively as well as productively infected monkey cells.

A variety of mRNAs, including adenovirus-specified mRNAs isolated from infected human cells, were translated with similar efficiencies in S10 cell-free lysates prepared from productively and abortively infected monkey cells. These results may suggest that reduced synthesis of the late viral proteins in abortively infected monkey cells is not due to a defect in the protein-synthesizing apparatus of the cell.

Adenoviruses, Human↗
PubMed1982

Genetic evidence for separate functional domains on the human adenovirus specified, 72 kd, DNA binding protein.

Seven mutations that affect various activities of the multifunctional DNA binding protein (DBP) encoded by human adenovirus have been physically mapped to different locations within DBP gene by marker rescue experiments. Two of these mutants (Ad5ts107 and Ad5ts125) contain a lesion which, under nonpermissive temperatures, decreases the capacity of the protein to bind single-strand DNA, blocks DNA replication, and prevents normal turn-off of viral early genes. In addition, the efficiency of transformation by the ts viruses compared to wild-type virus is increased at the nonpermissive temperature. Both ts mutations are located in the 3' half of the DBP gene (C-terminal half of DBP). In contrast, the alterations in the five host range mutants (Ad2hr400-Ad2hr403, Ad5hr404) which overcome the block to viral late mRNA synthesis in monkey cells, but have no marked effect on DNA replication or early gene expression map in the 5' half of the DBP gene. These results suggest that the 72 kd DBP of adenovirus contains at least two functionally separable domains.

Adenoviruses, Human↗
PubMed1980

Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA.

We have introduced adenovirus 2 genes into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene. Tk+ cells were isolated after selection and maintenance in HAT medium. Several co-transformed lines are able to complement the growth of Ad5 dl312 (delta 1.2--3.7) and Ad5 dl434 (delta 2.6--8.7), deletion mutants that lack sequences from the left end of the viral genome. The amount and arrangement of viral sequences in the co-transformed cell lines have been analyzed by restriction endonuclease digestion and filter hybridization. Most of the cell lines contain a single insertion of the HSV-1 tk fragment and a single insert of adenoviral DNA. However, one line (B1) contains at least four different insertions, two of which are present in multiple copies. The adenoviral DNA in all cell lines is composed of sequences from the left end of the genome and extends for varying lengths in different lines. Two cell lines that complement deletion mutants efficiently synthesize both early region 1a and 1b mRNAs. The B1 line synthesizes low levels of 1a mRNA, higher levels of 1b mRNA and a unique mRNA that maps to the right of the 1b gene family. When grown continuously in HAT medium, some cell lines are quite stable while others are fairly unstable. Some tk+ subclones support the growth of viral mutants as well as the parental line while others give reduced levels of complementation. For all tk+ subclones examined, the alteration or reduction in viral gene expression is independent of changes in the pattern of integration of viral DNA.

Adenoviruses, Human↗
PubMed1980

The gene for polypeptide IX of adenovirus type 2 and its unspliced messenger RNA.

A 606 base pair segment of adenovirus type 2 DNA which includes the gene for polypeptide IX and its flanking regions has been sequenced. Comparison with the sequence of a cDNA copy of the 9S mRNA for polypeptide IX and direct analysis of its capped oligonucleotide show that the gene and its mRNA are colinear. The 9S mRNA has a length of 485 nucleotides, excluding the poly(A) tract and the m7G of the cap, and contains one single open translational reading frame which is sufficiently long to encode polypeptide IX. This reading frame contains three AUG codons, the first of which is used for initiation of protein synthesis. The mRNA has a 5' noncoding region of 24 nucleotides, excluding the m5G of the cap, and encodes a polypeptide which is 139 amino acids long and unusually rich in serine and alanine. The sequence TATATAA is found 25 nucleotides upstream from the cap site. The polyadenylation site has been identified but cannot be located unambiguously because of the presence of two A residues in the DNA sequence at this position. The 3' noncoding region is 41--43 nucleotides long and contains the hexanucleotide sequence AAUAAA. The mRNA for polypeptide IVa2 is encoded adjacent to that for polypeptide IX and is transcribed in the opposite direction. Sequence analysis revealed an overlap of 11--16 nucleotides, depending on the precise location of the polyadenylation sites, between 3' ends of these mRNAs.

Adenoviruses, Human↗