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Biomedical subjects

R H Watson

Publications and source records attributed to R H Watson.

At least 37 records · Page 2Linked to original sources
PubMed1992

Conformation of DNA packaged in bacteriophage T7. Analysis by use of ultraviolet light-induced DNA-capsid cross-linking.

The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking. To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis. Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light. Protein-protein cross-links are not induced. When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid. However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases. Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid. Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases. Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

Capsid↗
PubMed1992

Cultured human granulosa cells as a model for corpus luteum function: relative roles of gonadotrophin and low density lipoprotein studied under defined culture conditions.

In primates, corpus luteum development involves both gonadotrophin stimulation and exposure to low density lipoprotein (LDL) delivered through vascularization of the granulosa cell-derived layer. These regulatory influences were modelled in vitro using granulosa cells obtained during in-vitro fertilization (IVF) cycles controlled with gonadotrophin releasing hormone (GnRH) analogue, human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG). Granulosa cells were cultured in defined medium on extracellular matrix. Without gonadotrophin or LDL in the medium, progesterone production declined progressively. With LDL alone, there was a short-lived elevation of progesterone output which subsequently declined. Culture with HCG alone resulted in a relatively unchanged rate of steroid production over 5 days despite morphological development. This contrasted with a marked and sustained increase in progesterone output over the same time when granulosa cells were cultured with combined HCG/LDL. Cultures were challenged with combined HCG/LDL on day 5. Where initial incubation included HCG, the challenge resulted in a recovery of progesterone output to values comparable to those of granulosa cells exposed to continuous HCG/LDL. Initial incubation without gonadotrophin led to a reduced response. Results suggest that LDL delivery to granulosa cells of the early corpus luteum causes a short-lived period of progesterone production. Sustained luteinization of granulosa cells and maintenance of gonadotrophin responsiveness requires continued exposure to gonadotrophin in the luteal phase.

Cells, Cultured↗
PubMed1992

The detrimental influence of functional ovarian cysts during in-vitro fertilization cycles.

Reviewing 780 in-vitro fertilization (IVF) cycles, where buserelin was commenced in the preceding luteal phase and human menopausal gonadotrophin on day 4 of the ensuing menses, 53 cycles were identified with sonolucent cysts (30-50 mm diameter). Of the latter 53 cycles, the serum oestradiol was significantly greater on day 4 in 22 cycles abandoned for poor follicular development than in 31 cycles which proceeded to oocyte retrieval (P less than 0.05). Of the 31 cycles proceeding to oocyte retrieval, nine had a day 4 serum oestradiol greater than 200 pmol/l (95th centile for day 4 oestradiol in patients without apparent cysts), and these cycles produced significantly fewer grade 1 embryos than the cycles with day 4 oestradiol levels less than or equal to 200 pmol/l (P less than 0.05). Six of the 53 cycles with cysts resulted in conception, and all of these cycles had a day 4 serum oestradiol less than 200 pmol/l. Among the 53 cycles with ovarian cysts, the serum progesterone on the day of abandonment in four cycles and on the day of human chorionic gonadotrophin administration in one non-abandoned cycle, was above the range established for 104 cycles without cysts. No significant difference was seen in day 4 serum androstenedione levels, and the day 4 serum progesterone was less than 5 nmol/l in all but one patient. Functional activity of ovarian cysts is associated with an adverse influence on IVF cycles.

Androstenedione↗
PubMed1991

Comparison of 'poor' responders with 'good' responders using a standard buserelin/human menopausal gonadotrophin regime for in-vitro fertilization.

This study identifies a group of 87 patients, who demonstrated a 'poor' response to a standard buserelin/human menopausal gonadotrophin (HMG) regime. The subsequent outcome in 61 of these 'poor' responders when treated with a higher dose of HMG to achieve a satisfactory response was compared with 250 patients, who showed a 'good' response to the standard regime. 'Poor' responders were significantly older than 'good' responders (P less than 0.001), but no significant difference was demonstrated in the indication for in-vitro fertilization (IVF). Even on higher doses of HMG, 'poor' responders took longer for their follicles to achieve maturity than the 'good' responders (P less than 0.01). 'Poor' responders produced 8.9 oocytes per oocyte collection compared to 11.8 in the 'good' responders (P less than 0.01). The fertilization rate was significantly lower in the 'poor' responders compared to the 'good' responders (P less than 0.01). Although there was no significant difference in morphometric grading between 'poor' responder embryos and 'good' responder embryos, the rate of cell division was significantly slower in embryos of the 'poor' responders than the 'good' responders (P less than 0.01). The pregnancy rate per oocyte retrieval was 9% in the 'poor' responders compared to 29% in the 'good' responders (P less than 0.01). The implantation rate in the 'poor' responders was 4.4% compared to 16.1% in the 'good' responders (P less than 0.001).

Adult↗
PubMed1991

Biochemical monitoring during hormone replacement therapy cycles for transfer of cryopreserved embryos in patients with functional ovaries.

Biochemical monitoring was undertaken in 22 treatment cycles for women with normal ovarian function who underwent pituitary suppression with buserelin and administration of exogenous oestradiol (E2) and progesterone (P) for cryopreserved embryo transfer (ET). Eighteen transfers of 1-4 thawed embryos, on the third day of exposure to progesterone, resulted in five clinical pregnancies (27.8%) and one biochemical pregnancy. There was no difference between pregnant and non-pregnant patients in the number and quality of embryos transferred, age, weight or infertility diagnosis. Serum E2 level from days 10-17 (the late proliferative phase) of the therapy cycle were significantly higher in the pregnant group compared with the non-pregnant group (P less than 0.05--P less than 0.005). There were no significant differences in P levels between the two groups from the onset of progesterone administration to the end of the cycle. However, as might be expected, the mean E2/P molar ratio in the pregnant group was significantly higher at the time of ET (P less than 0.02). It is concluded that biochemical monitoring during the embryo replacement cycle is necessary to tailor drug dosages for individual requirements to achieve adequate E2 levels before ET. Alternative routes of oestradiol administration need to be considered in patients with poor E2 profiles.

Buserelin↗
PubMed1990

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA.

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.

Centrifugation, Density Gradient↗
PubMed1987

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA.

Centrifugation, Zonal↗
PubMed1985

Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B.

Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size. It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M. Chamberlin [1974], J. Virol. 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host. However, the conversion of capsid I to capsid II was slowed in E. coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging. Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host. T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant. These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.

Capsid↗
PubMed1985

Heterogeneity of the procapsid of bacteriophage T3.

Like other double-stranded DNA bacteriophages, bacteriophage T3 assembles a DNA-free procapsid that subsequently packages the bacteriophage DNA. By agarose gel electrophoresis, it has been found that the T3 procapsid has a negative electrophoretic mobility (mu) that progressively increases in magnitude by as much as 3% after assembly of the procapsid. This increase is (i) caused by an increase in the solid support-free mu (muo) of the procapsid, not a decrease in its radius, and (ii) not prevented by either genetically or chemically (use of proflavine) blocking DNA packaging. However, inhibition of the formation of high-energy compounds with a mixture of cyanide and fluoride ions does block the time-dependent increase in the magnitude of muo. This increase appears to be accompanied by addition of an unidentified T3 protein to the procapsid.

Capsid↗
PubMed1983

Comparison of the physical properties and assembly pathways of the related bacteriophages T7, T3 and phi II.

To understand constraints on the evolution of bacteriophage assembly, the structures, electrophoretic mobilities (mu) and assembly pathways of the related double-stranded DNA bacteriophages T7, T3 and phi II, have been compared. The characteristics of the following T7, T3 and phi II capsids in these assembly pathways have also been compared: (1) a DNA-free procapsid (capsid I) that packages DNA during assembly; (b) a DNA packaging-associated conversion product of capsid I (capsid II). The molecular weights of the T3 and phi II genomes were 25.2 X 10(6) and 25.9 (+/- 0.2) X 10(6) (26.44 X 10(6) for T7, as previously determined), as determined by agarose gel electrophoresis of intact genomes. The radii of T7, T3 and phi II bacteriophages were indistinguishable by sieving during agarose gel electrophoresis (+/- 4%) and measurement of the bacteriophage hydration (+/- 2%) (30.1 nm for T7, as previously determined). Assuming a T = 7 icosahedral lattice for the arrangement of the major capsid subunits (p10A) of T7, T3 and phi II best explains these data and data previously obtained for T7. At pH 7.4 and an ionic strength of 1.2, the solid-support-free mu values (mu 0 values) of T7, T3 and phi II bacteriophages, obtained by extrapolation of mu during agarose gel electrophoresis to an agarose concentration of 0 and correction for electro-osmosis, were -0.71, -0.91 and -1.17(X 10(-4) cm2V-1 s-1. The mu 0 values of T7, T3 and phi II capsids I were -1.51, -1.58 and -2.07(X 10(-4] cm2V-1 s-1. For the capsids II, these mu 0 values were -0.82, -1.07 and -1.37(X 10(-4] cm2V-1 s-1. The tails of all three bacteriophages were positively charged and the capsid envelopes (heads) were negatively charged. In all cases the procapsid had a negative mu 0 value larger in magnitude than the negative mu 0 value for bacteriophage or capsid II. A trypsin-sensitive region in capsid I-associated, but not capsid II-associated, T3 p10A was observed (previously observed for T7). The largest fragment of trypsinized capsid I-associated p10A had the same molecular weight in T7 and T3, although the T3 p10A is 18% more massive than the T7 p10A. It is suggested that the trypsin-resistant region of capsid I-associated p10A determines the radius of the bacteriophage capsid.

Capsid↗
PubMed1982

Detection and characterization of agarose-binding, capsid-like particles produced during assembly of a bacteriophage T7 procapsid.

It has previously been shown that: (i) during infection of its host, the DNA bacteriophage T7 assembles a DNA-free procapsid (capsid I), a capsid with an envelope differing physically and chemically from the capsid of the mature bacteriophage, and (ii) capsid I converts to a capsid (capsid II) with a bacteriophage-like envelope as it packages DNA. Lysates of phage T7-infected Escherichia coli contained a particle (AG particle) which copurified with capsid II during buoyant density sedimentation, velocity sedimentation, and solid support-free electrophoresis, but was distinguished from capsid II by its apparent diversity during electrophoresis in agarose gels. Treatment of AG particles with trypsin converted most of them to particles that comigrated with trypsin-treated capsid II during electrophoresis in agarose gels. Irreversible binding of AG particles to agarose gels was shown to contribute to the apparent diversity of AG particles during agarose gel electrophoresis. The results of quantitation of AG particles and of capsid I and capsid II in lysates of a nonpermissive host infected with T7 amber mutants suggested that, in site of their capsid II-like properties, most AG particles were produced during assembly of capsid I and not during DNA packaging. The presence of AG particles in T7 lysates explains contradictions in previous data concerning the pathway of T7 assembly.

Capsid↗
PubMed1982

Function of an internal bacteriophage T7 core during assembly of a T7 procapsid.

A DNA-free, proteinaceous procapsid of bacteriophage T7 (capsid I) has been shown in previous studies to consist of an external, spherical shell (envelope) and an internal, cylindrical core with fibrous projections that connect the core to the envelope. To determine the role of the core in assembly of the envelope of capsid I, the kinetics of appearance of capsid I and possible intermediates in capsid I assembly (AG particles) were determined in the presence and absence of the core. For obtaining these data, agarose gel electrophoresis was used and appeared to be a technique more accurate and efficient than techniques used for obtaining similar data in the past. The results of these experiments were: (i) in the presence of the core, AG particles behaved kinetically as intermediates in the assembly of capsid I; (ii) in the absence of the core, assembly of capsid I terminated prematurely and AG particles accumulated. These and other data have been interpreted by assuming that: AG particles are breakdown products of precursors of capsid I; these precursors have uncorrected errors in the assembly of their envelope; and a function of the core is to correct these errors.

Capsid↗