Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.

Pulse-chase analysis of the in vivo assembly of the bacteriophage T4 tail.

The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly. Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation. Beginning with the onset of late protein synthesis, the cultures were labeled continuously with [(3)H]leucine to normalize against subsequent sample losses. After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with [(35)S]methionine, and then chased. Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation. Total (35)S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails. The inflection point in the sigmoidal (35)S-incorporation curve of each protein marks the maximal uptake of (35)S within that pool just before the effect of the chase becomes apparent and the curve begins to level off. The length of the delay in the apparent chase time reflects the position of that protein in the pathway. The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly. As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase. Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway. The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times.

Capsid expansion follows the initiation of DNA packaging in bacteriophage T4.

Most bacteriophages undergo a dramatic expansion of their capsids during morphogenesis. In phages lambda, T3, T7 and P22, it has been shown that expansion occurs during the packaging of DNA into the capsid. The terminase-DNA complex docks with the portal vertex of an unexpanded prohead and begins packaging. After some of the DNA has entered, the major head protein undergoes a conformational change that increases both the volume and stability of the capsid. In phage T4, the link between packaging and expansion has not been established. We explored the possibility of such a connection using a pulse-chase protocol and high resolution sucrose gradient analysis of capsid intermediates isolated from wild-type T4-infected cells. We show that the first particle appearing after the pulse is an unexpanded prohead, which can be isolated in vitro as the ESP (empty small particle). The next intermediate to appear is also unexpanded, but contains DNA. This new intermediate, the ISP (initiated small particle), can also be isolated on agarose gels, permitting confirmation of both its expansion state and DNA content ( approximately 10 kbp). It appears, therefore, that >/=8% of the T4 genome enters the head shell prior to expansion. Following packaging of an undetermined amount of DNA, the capsid expands, producing the ILP (initiated large particle), which is finally converted to a full head upon the completion of packaging. An expanded, empty prohead, the ELP (empty large particle), was also observed during 37 degrees C infections, but failed to mature to phage during the chase. Thus the ELP is unlikely to be an intermediate in normal head assembly. We conclude by suggesting that studies on assembly benefit from an emphasis on the processes involved, rather than on the structural intermediates which accumulate if these processes are interrupted.

Localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate with colloidal gold: F(ab)2 and undecagold: Fab' conjugates.

We report the localization of the proteins gp7, gp8 and gp10 in the bacteriophage T4 baseplate. Proceeding on the assumption that these proteins occupy discrete locations, we have decorated baseplates and tails with immunological probes. Using 5 nm diameter colloidal gold: F(ab')2 conjugates, we show that proteins gp7 and gp10 are located directly at the vertex, with gp10 positioned in the pin directly below gp7. gp8 is located beside gp7 towards the centre of the baseplate. Using a novel undecagold: Fab' conjugate we have also determined the radial positions of gp7 and gp8 in baseplates that have transformed to stars. A mechanism for the nature of the hexagon-to-star transformation is proposed.

The bacteriophage T4 DNA packaging apparatus targets the unexpanded prohead.

During the morphogenesis of the bacteriophage T4 capsid, a conformational change of the major head shell protein, gene product (gp) 23, causes a 50% increase in capsid volume. This expansion is required to accept the full length chromosome and, therefore, must precede the completion of packaging. The expanded shell is thinner and more stable than its precursor, and can bind accessory proteins which further stabilize it. In phages lambda, T3, T7 and P22, expansion occurs during DNA packaging. However, in T4, expanded capsids can package DNA in vitro and expansion occurs in cells infected with packaging-defective mutants, raising the possibility that expansion and packaging are not coupled. Proteolytically mature gp23 (gp23*) in unexpanded proheads is sensitive to chymotrypsin cleavage at Phe154-Ser155, creating a 38 kDa peptide, while gp23* in expanded capsids is refractory to the protease. We used an expansion assay based on this protease sensitivity to determine the expansion status of capsids isolated from various packaging-defective mutants with the goal of determining whether packaging and expansion are normally linked. In infections at 20 degrees C, mutants in the packaging enzymes gp16 and gp17 fail to expand. However, in gene 49(-) mutants, which initiate packaging but fail to complete it, expansion is complete. Thus, packaging drives expansion, and the unexpanded prohead is the substrate for the packaging reaction. We also show that expansion observed in 16(-) and 17(-) infections at 37 degrees C is linked to aberrant packaging. Capsids produced at 15 minutes, when no packaging can be detected, never expand. However, by 35 minutes when aberrant packaging begins, so does expansion of freshly made capsids. Thus in all cases now examined, expansion is only observed in vivo when DNA packaging is also occurring, indicating that these two processes are coupled.

Generating sucrose gradients in three minutes by tilted tube rotation.

A technique which permits rapid preparation of sucrose gradients with highly reproducible profiles is described. Tubes are filled with equal volumes of light and heavy sucrose solutions, sealed, and rotated for 3 min tilted 80 degrees from vertical. Linear 5-20% and 5-30% gradients and nonlinear but useful 5-45% gradients are obtained.

Structure of the bacteriophage T4 baseplate as determined by chemical cross-linking.

We have carried out a series of reversible chemical cross-linking experiments using the reagent ethylene glycol-bis(succinimidylsuccinate) with the goal of determining the three-dimensional structure of the bacteriophage T4 baseplate. In a previous report, we investigated the near-neighbor contacts in baseplate precursors and substructures (N.R.M. Watts and D.H. Coombs, J. Virol. 63:2427-2436, 1989). Here we report completion of the analysis by examining finished baseplates and tails. Most of the previous contacts were confirmed, and we report several new contacts, including those within the central hub (gp5-gptd2, gp26-gptd), between the hub and the outer wedges (gp6-gp27(2], between baseplate and sheath (gp54-gp18), and between sheath and core (gp19-gp18). On the basis of this and other available information, a partial three-dimensional model of the baseplate is proposed.

Analysis of near-neighbor contacts in bacteriophage T4 wedges and hubless baseplates by using a cleavable chemical cross-linker.

Although bacteriophage T4 baseplate morphogenesis has been analyzed in some detail, there is little information available on the spatial arrangement and associations of its 150 subunits. We have therefore carried out the first analysis of its near-neighbor interactions by using the cleavable chemical cross-linker ethylene glycolbis(succinimidylsuccinate). In this report, we describe the cross-linked complexes that have been identified in the one-sixth arms or wedges and also in baseplatelike structures called rings consisting of six wedges but lacking the central hub, both of which are purified from T4 gene 5- -infected cells. Thirty different complexes were identified, of which about half contain multimers of a single species and half contain two different species. In general, the complexes reflect and support the assembly pathway derived by Kikuchi and King (Y. Kikuchi and J. King, J. Mol. Biol. 99:695-716, 1975) but broaden its scope to include such complexes as gp25-gp53, gp25-gp48, and gp48-gp53, which locate the gp48 binding site over the inner edge of the ring but outside the central hub. The data also supports the view that wedges are assembled from the outer edge inward toward the central hub. Wedge-wedge contact in rings was mediated primarily by gp12 and gp9, the absence of which dramatically destabilized the ring----wedge equilibrium in favor of wedges. Although no heterologous complexes containing gp9 were identified, gp12 contacts unique to rings were observed with both gp10 and gp11.

Identification of bacteriophage T4 virion proteins by transverse pore-gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dual amino acid labeling.

We have developed a horizontal N,N'-methylenebisacrylamide (Bis) acrylamide gradient sodium dodecyl sulfate (SDS) gel system that permits the evaluation of the purity of individual protein bands in complex mixtures. A Bis gradient gel is poured vertically and, after polymerization, reoriented horizontally. A single large sample spanning the top of the gel is then run down at right angles to the gradient. The relative mobility of a few proteins varies considerably from the rest, causing them to merge with and cross other bands as the Bis concentration changes. Band splitting revealed that several bands previously thought to represent a single species are actually comprised of comigrating proteins. Once the Bis/monomer concentration offering the best separation was identified, we sought a simple method for identifying the genetic origin of bands, since many proteins now migrated in new positions on the gel. We reasoned that if infected cells were simultaneously labeled with [35S]methionine and [3H]leucine and the purified virion proteins analyzed to determine their 35S/3H ratio, we could identify most proteins by comparing this ratio with one calculated from the T4 DNA sequence. Our expectations were realized, and we here report the separation and identification of all T4 virion proteins. Finally, we comment on the incorporation of various changes to the original Laemmli SDS-polyacrylamide gel formulations that have been reported in the literature.

Filter-binding assay for covalent DNA-protein complexes: adenovirus DNA-terminal protein complex.

A rapid, simple, and quantitative filter-binding assay using glass fiber filters has been developed to detect the convalent adenovirus DNA-terminal protein complex. The assay is unusually sensitive because binding of protein-free DNA generally is less than 0.1%. Binding of the adenovirus complex to filters is mediated by terminal protein. We have found that: (i) the adenovirus complex binds maximally to filters in NaCl at concentrations higher than 0.2 M; (ii) noncovalent complexes between protein-free DNA and adenovirus proteins bind to filters in salt at concentrations lower than 0.4 M but not in concentrations higher than 0.7 M; and (iii) protein-free DNA alone binds to filters in guanidine.hydrochloride at concentrations higher than 0.8 M. By varying the ionic conditions, "all or none" modulation of these interactions can be achieved.

Heat cleavage of bacteriophage T4 gene 23 product produces two peptides previously identified as head proteins.

During studies on the intracellular protein pools of bacteriophage T4, we found that amber mutants in gene 23 blocked the synthesis of a 20-kilodalton (kDa) protein. Radiolabeled amino acid pulses showed that the protein appears at 8 min postinfection with kinetics similar to those of other major late species. Pulse-chase experiments demonstrated that the 20-kDa protein behaves like a primary product and also revealed a 29-kDa protein which, like other proteins cleaved during head assembly, appeared only after a long chase. Both species have been identified as constituents of the T4 head and have resisted previous efforts to identify their genetic origin. The dependence of the 20- and 29-kDa head proteins on the presence of gene 23 protein (gp23) and the observation that the sum of their masses equalled that of mature cleaved gp23 suggested that these two proteins were derived from this major capsid species. Evidence is presented demonstrating that heating samples before electrophoresis causes peptide bond cleavages in gp23, leading to the formation of the two peptides. As predicted by the results of Rittenhouse and Marcus (Anal. Biochem. 138:442-448, 1984), the cleavage occurs at Asp-336-Pro-337 and at two other Asp-Pro sites. Limited heat-induced proteolysis followed by two-dimensional gel analysis provided a peptide map of gp23 useful in the characterization of its assembly-related cleavages.

An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.

Numerous reports describe the phage T4 enzymes thymidylate synthase and dihydrofolate reductase as structural components of the baseplate. However, Y. Wang and C. K. Mathews (J. Virol. 63:4736-4743, 1989) reported that antisera against the respective recombinant enzymes failed to neutralize phage infectivity, in contrast to previous results. Moreover, a deletion mutant lacking the genes for these two enzymes adsorbed normally to host cells. Since these findings tended to undermine the idea of the two enzymes as structural proteins, we developed a quantitative immunoblot assay to resolve the issue directly. Our results show that both enzymes are present only as minor contaminants (< 0.05 copy per phage) and as such cannot be bona fide structural proteins.

PCR detection and typing of genital papillomavirus in a New Brunswick population.

We have used a broad range of primers for HPV detection, using the polymerase chain reaction (PCR) so as to compare PCR typing of HPV with the results of cytological diagnosis in a New Brunswick population referred to the out-patient clinic of the Saint John Regional Hospital. The primers selected were found to be capable of amplification with high efficiency, therefore we did not perform further hybridization analysis for specific identification of HPV types. Amplification of selected fragments for detection of HPV 6, 11, 16, 18, 31 and 33 was obtained from cervical swabs collected from 154 patients. Microscopic examination was performed in duplicate samples and the results compared with the DNA-typing analysis. HPV of any of the above types was detected in 43 out of 154 patients. Among these, 32 patients showed single or multiple infections with "high-risk" HPV strains 16, 18, 31 or 33. Cytologically normal or atypical samples with any of the HPV types tested amounted to 17%, but increased to 56% in patients with CIN I, and to 100% in patients with CIN II or III. Prevalence of "high-risk" types alone increased from 15% and 10%, for normal and atypical cases respectively, to 48% for CIN I, 75% for CIN II and 100% for CIN III. Our results indicate that HPV detection and typing by this simple procedure can be a valuable indicator of cancer progression and thus can help to identify individuals at high risk in pre-malignant stages of the disease.