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R Wittek

Publications and source records attributed to R Wittek.

At least 37 records · Page 2Linked to original sources
PubMed1982

Organization and expression of the poxvirus genome.

Poxviruses comprise a large group of very complex animal DNA viruses which replicate in the cytoplasm of infected cells. Vaccinia virus, the most studied poxvirus, has a linear, double stranded DNA genome with an approximate molecular weight of 120 x 10(6) (180 kilobase pairs). The two strands of the DNA molecule are naturally cross-linked at both termini. In addition, the vaccinia virus genome contains very long inverted terminal repetitions of approximately 10 kilobase pairs which are further characterized by the presence of direct tandem repeats of a 70-base-pair sequence arranged in two blocks of 13 and 17 copies, respectively. A central region of the genome is highly conserved between different orthopoxviruses. In contrast, the ends are hypervariable and may contain extensive deletions and complex, symmetrical sequences rearrangements. Vaccinia virus gene expression is divided into two stages. Early in infection, RNA complementary to one half of one strand-equivalent of the genome is transcribed within subviral particles by the virion-associated RNA polymerase. Later in infection, after DNA replication, RNA complementary to one entire strand-equivalent is transcribed. RNA made late in infection is very heterogeneous in length and a large fraction of it contains self-complementary sequences. Late genes are clustered near the central region of the genome. Vaccinia virus mRNAs do not appear to be synthesized by a splicing mechanism.

Base Sequence↗
PubMed1982

Colinearity of RNAs with the vaccinia virus genome: anomalies with two complementary early and late RNAs result from a small deletion or rearrangement within the inverted terminal repetition.

The colinearity of RNA transcripts with the vaccinia virus genome was investigated. Cytoplasmic RNA from infected cells was annealed to a cloned DNA segment that extended from 9 to 15.6 kilobase pairs from the left end of the genome and contained approximately 800 base pairs of the inverted terminal repetition (ITR). Remaining unhybridized single strands of DNA were digested with nuclease S1, and the lengths of the protected DNA fragments were determined by agarose gel electrophoresis under neutral and alkaline conditions. Uniformly 32P-labeled cloned DNA insert, separated recombinant DNA strands, and smaller restriction fragments, as well as 3' and 5' end-labeled DNA, were employed to map five early RNAs and one late RNA. One of the early RNAs hybridized to sequences within the ITR, and the other four hybridized to sequences proximal to the ITR. The late RNA was initiated proximal to the ITR but extended into it. Interestingly, the 3' portion of this late RNA was complementary to the early RNA transcribed from the opposite strand of the ITR. From a comparison of the lengths of the protected DNA fragments on neutral and alkaline gels, all except the complementary early and late RNAs appeared to be colinear with the genome. Although the anomalous nuclease S1 data obtained with the latter RNAs mimicked splicing, they were shown by DNA-DNA hybridization to result from a small deletion or rearrangement within the ITR. Thus far, no true examples of spliced vaccinia virus RNAs have been found.

Base Sequence↗
PubMed1981

Hybridization selection and cell-free translation of mRNA's encoded within the inverted terminal repetition of the vaccinia virus genome.

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.

Bacteriophage lambda↗
PubMed1981

Transcriptional and translational mapping of a 6.6-kilobase-pair DNA fragment containing the junction of the terminal repetition and unique sequence at the left end of the vaccinia virus genome.

The penultimate EcoRI fragments from the left and right ends of the vaccinia virus genomes were cloned in phage lambda. Heteroduplex analysis and comparison of restriction fragments indicated that the inverted terminal repetition extended 780 base pairs (bp) beyond the EcoRI site or about 9,800 bp from each end of the genome. Detailed physical, transcriptional, and translational maps of the 6,600-bp left penultimate EcoRI fragment were prepared so as to extend previous maps of the 9,000-bp terminal EcoRI fragment. Polypeptides with molecular weights of 6,000 (6K polypeptide), 13,000, 19,000, 21,000, and 60,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA (made in the presence of cycloheximide) or early RNA (made in the presence of cytosine arabinoside) and selected by hybridization to immobilized recombinant DNA. A 22K polypeptide was detected as a translation product of late RNA that hybridized to this DNA fragment. A variety of biochemical procedures were used to size and map the mRNA's. Of the five messages that hybridized to this 6,600-bp EcoRI fragment, only the one for the 21K polypeptide was encoded within the inverted terminal repetition and hybridized to the rightward-reading DNA strand. (Three additional early polypeptides were encoded within the first 9,000 bp of the inverted terminal repetition.) The remaining early polypeptides were encoded within the unique portion of the penultimate EcoRI fragment and were transcribed from the leftward-reading strand. Additional high-molecular-weight early RNAs of unknown function were also detected; however, there was no evidence indicating that mature mRNA's were spliced.

Base Sequence↗
PubMed1981

Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs.

Physical, transcriptional, and translational maps of an EcoRI fragment located between 15,800 and 20,600 base pairs from the left end of the vaccinia virus genome were prepared. Major polypeptides with molecular weights of 14,000 (14K polypeptide), 32,000 and 38,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA made in the presence of cycloheximide and selected by hybridization to lambda recombinant DNA containing the EcoRI fragment. With early RNA made in the presence of cytosine arabinoside, an inhibitor of DNA replication, the polypeptide pattern was similar except for quantitative differences in which less 38K polypeptide was detected as a translation product. With late RNA, isolated 6 h after infection without inhibitors, only traces of the early translation products were found and a new 40K polypeptide was detected. The size of the mRNA's for the 14K, 32K, and 38K polypeptides were determined to be approximately 760,880, and 1,150 nucleotides, respectively, by several independent procedures. Several large early RNAs not shown to code for any additional translation products were also detected. The size of the late message for the 40K polypeptide varied from 920 to 3,100 nucleotides. This heterogeneity appeared to be a general property of vaccinia virus late mRNA's. No evidence of RNA splicing was obtained by analysis of RNA-DNA hybrids after nuclease S1 treatment. Further analyses using separated recombinant DNA strands and restriction fragments indicated that all mRNA's were encoded by the leftward-reading DNA strand and at least two were overlapping. Since early and late mRNA's were encoded by the same DNA strand, the possibility of temporal regulation by transcriptional strand switching was eliminated. In conjunction with previous studies, a transcriptional map of the left 20,600 base pairs of the vaccinia virus genome was derived.

DNA Restriction Enzymes↗
PubMed1980

Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs.

Vaccinia virus DNA contains a long inverted terminal repetition of MW approximately 6.8 x 10(6). A fragment of MW 6.3 X 10(6) from this repetition has been cloned in coliphage lambda and used to isolate RNA from virus-infected cells. Electron microscopy indicates that early RNAs are transcribed from the repeated sequence and cell-free translation shows that the RNAs code for polypeptides.

Bacteriophage lambda↗
PubMed1980

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA.

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.

Base Sequence↗
PubMed1980

Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition.

We have investigated the organization of transcriptional units within a 9000 bp segment of the terminally repeated region of the DNA genome of vaccinia virus, which uses its own enzyme system to synthesize mRNA within the cytoplasm of infected cells. RNA splicing, which has been demonstrated for DNA viruses that replicate within the nucleus of infected cells, does not appear to be involved in the formation of these first vaccinia virus mRNAs to be examined. Three immediate early mRNAs, approximately 1050, 600 and 1100 nucleotides long, were mapped between 3.21 and 4.24, 6.54 and 7.16, and 7.20 and 8.23 kb from the end of the genome, respectively. The direction of transcription was toward the end of the genome for the two larger mRNAs and in the opposite direction for the smallest one. Additional minor RNAs, which were larger in size, were mapped between and to the same DNA strand as the mRNAs of 1050 and 1100 nucleotides. No evidence for interrupted genes was obtained by nuclease S1 analysis after hybridization of RNA to labeled DNA. In addition, the 5' ends of the mRNAs, which were specifically labeled by in vitro capping, hybridized to DNA adjacent to the body of the message.

DNA, Recombinant↗
PubMed1980

Symmetrical arrangement of the heterologous regions of rabbit poxvirus and vaccinia virus DNA.

Cleavage sites for the restriction endonucleases EcoRI, KpnI and XhoI were mapped on rabbit poxvirus and vaccinia virus DNA. These physical maps were used to analyse the structural variations between the two DNAs. Two specific heterologous regions, symmetrically arranged at each end of the genomes, have been identified. Region 1, representing the exterior part of the terminal repetition, appears to contain unrelated sequences in each DNA and accounts for the difference in length of the two genomes. Region 2, separated from region 1 by a conserved part of the terminal repetition, is located at the transition from repeated to unique DNA sequences. Its overall length of about 4 megadaltons is well conserved and it contains individual DNA-specific as well as conserved restriction sites. The major central part of the genomes (over 100 megadaltons) contains very few, widely dispersed restriction site variations.

Base Sequence↗
PubMed1980

Genetic and antigenic heterogeneity of different parapoxvirus strains.

Six stomatitis papulosa and three Orf virus strains were compared by serology and by DNA restriction analysis. A neutralization kinetic study revealed extensive serological cross-reactivity between all strains, but did not allow their classification. Restriction analysis of viral DNAs revealed two distinct groups among the stomatitis papulosa strains while the Orf virus strains formed a third, more heterogeneous group. The large heterogeneity of restriction patterns of parapoxvirus DNAs as compared to those orthopoxviruses is discussed.

Antigens, Viral↗
PubMed1979

Physical characterization of a stomatitis papulosa virus genome: a cleavage map for the restriction endonucleases HindIII and EcoRI.

The genome of stomatitis papulosa virus (a parapoxvirus) was cleaved with the restriction endonucleases HindIII and EcoRI, each giving rise to 6 fragments respectively. Double digestion with both enzymes resulted in 8 bands, two of which contained DNA fragments in double molar concentrations as revealed by reciprocal digests of isolated DNA fragments. The genome size, estimated by summation of the molecular weights of the fragments, is approximately 86 X 10(6) daltons, some 30 X 10(6) daltons smaller than vaccinia virus (an orthopoxvirus) DNA. The cleavage sites of HindIII and EcoRI endonucleases were mapped on the genome by analysis of reciprocal digests of isolated DNA fragments and by cross-hybridization experiments. This yielded two mapped segments which were then oriented relative to one another by cleavage of isolated partial digestion products. The terminal restriction fragments show rapid renaturation after alkali denaturation and subsequent neutralization, indicating that stomatitis papulosa virus DNA contains terminal cross-links analogous to those found in vaccinia virus DNA.

Animals↗
PubMed1979

High C + G content in parapoxvirus DNA.

The DNAs of eight parapoxviruses (four stomatitis papulosa viruses isolated from infected calves, a pseudocowpox virus isolated from a teat lesion of an infected cow and three orf viruses, one isolated from an infected sheep and two isolated from human infections) were analysed in CsCl gradients. The mole % of G+C was calculated from the buoyant density and found to be approx. 63% for all virus isolates examined. Parapoxvirus DNA thus has by far the highest G+C content of all poxvirus DNAs so far examined.

Animals↗
PubMed1978

Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.

Animals↗
PubMed1977

The effect of cordycepin on the multiplication of Semliki Forest virus and on polyadenylation of viral RNA.

Cordycepin (3'-deoxyadenosine), at a concentration of 20 microgram/ml, has a marked effect on Semliki Forest virus multiplication. The appearance of plaque forming units is delayed by about 2 hours and the yield greatly reduced. The incorporation of [3H] uridine into intracellular viral RNAs reaches less than 50 per cent of controls. However, no specific effect on poly (A) synthesis could be detected. The binding efficiency of viral RNAs on nitrocellulose membranes and poly (U) sepharose is not affected by cordycepin. The average poly (A) length of total intracellular viral RNA was calculated on the basis of the ratio of the radioactivity of adenosine-monophosphate: adenosine and found to be about 35 nucleotides in treated and untreated cells.

Animals↗
PubMed1977

HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other.

Base Sequence↗