H.S. Jansz

Evidence for a function of the adenovirus DNA-binding protein in initiation of DNA synthesis as well as in elongation of nascent DNA chains

Abstract Early in lytic infection of KB cells with adenovirus type 5 (Ad5) a viral-coded single-strand-specific DNA-binding protein with a molecular weight of 72,000 is synthesized. The function of this protein in viral DNA synthesis was studied using two different methods for specific inactivation. First, DNA synthesis was examined in KB cells infected with an early temperature-sensitive mutant, H5ts125, which produces a thermolabile DNA-binding protein. At the nonpermissive temperature (40°) the start of the first and subsequent rounds of DNA replication was inhibited. However, viral DNA synthesis in vitro using a system of isolated nuclei obtained from H5ts125-infected KB cells was not thermosensitive compared to wild type. In this in vitro system, replicating molecules were normally converted to mature DNA at 40° without reinitiation of new replication rounds. These results show that thermal inactivation of the H5ts125 DNA-binding protein destroys its capacity to function in the initiation of new replication rounds but does not impair chain growth. As a second method of inactivation we studied viral DNA synthesis in vitro in the presence of antibody against the purified DNA-binding protein. A 50–60% inhibition of the rate of viral DNA synthesis was observed while cellular DNA synthesis was not affected. Density-labeling experiments showed that the antibody inhibits the duplication of both complementary strands, presumably by a decrease of fork movement in replicating adenovirus DNA. The results suggest that the Ad5 DNA-binding protein functions in initiation of DNA replication as well as in elongation of nascent viral DNA chains.

Construction of viable and lethal mutations in the origin of bacteriophage φX174 using synthetic oligodeoxyribonucleotides

Abstract Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX † DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung − (uracil N -glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al. , 1980 a ). Using (5′- 32 P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA. Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro . Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas & Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA. Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.

Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA

Replicating Ad5 DNA was isolated from nuclei of infected KB cells and studied by electron microscopy. Branched as well as unbranched linear intermediates were observed containing extended regions of single-stranded DNA. The relationship between the branched and unbranched structures was studied during synchronized synthesis in the first round of replication after release of hydroxyurea inhibition. The branched intermediates represented early and the unbranched intermediates late stages in the replication cycle. Digestion of the branched intermediates with Eco RI endonuclease revealed that replication had started at the molecular right end (the A-T-rich end). This was confirmed by partial denaturation mapping of branched intermediates. These results confirm and extend a tentative model on the mechanism of Ad5 DNA replication.

EFFECTS OF HYPOPHYSECTOMY ON RNA METABOLISM IN RAT BRAIN STEM

Abstract— Ribosomal aggregates were isolated from rat brain stem and characterized as polysomes by sedimentation analysis and by their sensitivity to RNase and EDTA treatment.

The genetic map of bacteriophage ∅X174 constructed with restriction enzyme fragments

Abstract Mutants in all known ∅X174 genes were located on the fragment maps produced with restriction enzymes from Arthrobacter luteus (Alu I), Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae III). In this way physical distances between mutations were measured. The size of several genes, including gene A, could be determined. The eight gene A mutants tested show a strong asymmetric distribution within this gene, indicating a large internal region of low mutability. The relative position of many restriction enzyme fragments has been confirmed. The consequences of this genetic map for the organization of the ∅X genome are discussed.

Bacteriophage φX174 RF DNA replication in Vivo: A biochemical study☆

Abstract We have studied the replication of bacteriophage φX174 replicative form (RF) DNA by biochemical analysis of pulse-labelled replicative intermediates and covalently closed RFI † DNA. The pulse-labelled DNA was isolated and separated into fractions which were shown to contain rolling circles, partially double-stranded molecules, RFI and RFII molecules, respectively. In these molecules the position of the pulse label and its strand specificity was determined. The results obtained were strongly dependent on the method used to stop the pulse-labelling. When the pulse is ended by poison (KCN and NaN 3 ) and fast cooling significantly more label is found in the viral than in the complementary strand. In agreement with the rolling circle model, the majority of the viral strand label is present in linear genome length and longer than genome length φX DNA molecules. The radioactivity in the RFII fraction is found almost exclusively in linear viral φX DNA of genome length. Restriction enzyme analysis of this fraction indicates that the φX viral strand DNA synthesis terminates in the Hae III restriction fragment Z 6B . In contrast, the main part of the labelled complementary strand φX DNA is found in short pieces. Full length, pulse-labelled complementary strands are present in the RFI fraction and to a minor extent in the RFII fraction. Restriction enzyme analysis of full length complementary strands, from RFI and RFII, shows that there is no unique termination site for the complementary strand DNA synthesis. Analysis of the pulse-labelled covalently closed RFI fraction indicates that this fraction contains a collection of molecules with increasing superhelix density, ranging from relaxed RFI up to fully super-twisted RFI DNA. This suggests that at the end of a replication cycle the conversion of RFII into superhelical RFI DNA involves the formation of relaxed RFI DNA and the successive introduction of superhelical turns. When the pulse is ended by pouring the infected culture into a phenol/ethanol solution less radioactivity is found to be incorporated into φX DNA. Furthermore, the distribution of the label between the complementary and viral strand is more equal, and significantly more radioactivity is found in short pieces. A comparison of both stopping methods indicates that fast cooling in poison does not stop efficiently φX. RF DNA replication, especially the viral strand DNA synthesis and DNA ligase action.

A* protein of bacteriophage [phi]X174 carries an oligonucleotide which it can transfer to the 3-OH of a DNA chain

The bacteriophage φX174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single‐stranded, DNA‐specific endonuclease which remains covalently attached to the 5′‐end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA. This indicates that A* protein carries an oligonucleotide with the sequence ‐AG, AGG or ‐AGGA. The oligonucleotide can be transferred to the 3′‐end of the heptamer CAACTTG. This suggests that A* protein reacts with a specific DNA sequence in the infected cell.

Infection of Escherichia coli K-12 by bacteriophage φX-174

Abstract By several different biochemical tests, a previously described isolation procedure was shown to produce host-range mutants of φX-174 capable of infecting some strains of Escherichia coli K-12. One of these mutants (φXK-9) has a wide host range which includes E. coli C, B, and Shigella Y6R. By fragment mapping the site of the host-range mutation was shown to be in gene F.

DNA polymerases in adenovirus type 5-infected and uninfected KB cells Induction of an α-type DNA polymerase in adenovirus type 5-infected and in fast growing cells

DNA polymerase activities in uninfected KB cells or KB cells infected with adenovirus type 5 (Ad5) were compared by chromatography on DNA-cellulose and DEAE-cellulose and by isoelectric focusing. On DNA cellulose three components were found both in infected and in uninfected cells. The major component eluted at 0.15 M NaC1 and contained DNA polymerase alpha. Two minor components were found, one which did not bind to DNA-cellulose and one which bound strongly. This latter component contained DNA polymerase beta as characterized by DEAE-cellulose chromatography and sedimentation studies. No difference in properties between uninfected or Ad5-infected KB cells was found for the beta-polymerase. DEAE-cellulose chromatography of DNA polymerase alpha revealed the presence of two activities eluting at 0.11 and 0.13 M NaC1 designates as alphaI and alphaII, respectively. In Ad5-infected cells alphaII was the major component. In uninfected, stationary cells alphaI was the major component and alphaII was only detectable as a shoulder in the elution profile. However, fast growing, uninfected cells gave a similar pattern as Ad5-infected cells. These results indicate that the observed change of the DNA polymerase pattern after infection with Ad5 is related to the level of DNA synthesis and not to the induction of a viral enzyme.

Bacteriophage φX174 RF DNA replication in vivo: A study by electron microscopy☆

We have investigated bacteriophage φX174 RF † DNA replication by electron microscopy. Three different, types of replicative intermediates were observed: rolling circles, partially duplex DNA circles and structures consisting of two DNA circles connected at a single point. Rolling circles with a single-stranded or partially double-stranded DNA tail were both observed. After cleavage of the rolling circles with the restriction endonuclease from Providentia stuartii 164 (PstI) the startpoint of rolling circle replication could be located at 21 map units from the PstI cleavage site in agreement with the previously determined position of the origin of φX RF DNA replication. Partially duplex DNA circles consist of circular viral DNA strands and incomplete complementary DNA strands. After cleavage of these molecules with PstI information about the startpoints of the synthesis of the complementary DNA strand was obtained. The connected DNA circles always contain one completely double-stranded DNA circle whereas the other circle consists of either single-stranded, partially duplex or completely duplex DNA. Part of the duplex-to-duplex DNA circles represent the well-known figure eight or catenated circular dimers. The other connected DNA circles presumably represent replication intermediates which arise by the association of the end of the genome length tail of the rolling circle with the origin-terminus region. This is suggested by the fact that the point of contact between the two DNA circles is located at approximately 21 map units from the Pst1 cleavage site, i.e. at the origin-terminus region of the φX genome. The connected DNA circles may be intermediates in the circularization and cleavage of the genome-length tail of the rolling circles in vivo. A model for φX174 RF DNA replication in vivo summarizing the data obtained by biochemical (Baas et al., 1978) and electron microscopic analysis of replicative intermediates is presented (Fig. 9).