Herpes simplex virus DNA in transformed cells: sequence complexity in five hamster cell lines and one derived hamster tumor.

Analyses of the hybridization kinetics of labeled herpes simplex virus 2 (HSV-2) DNA with DNA from five hamster cell lines transformed by UV light-irradiated HSV-2 revealed the following. (i) Viral DNA sequences were detected in all five cell lines tested. (ii) None of the cell lines contained the full complement of HSV-2 DNA. (iii) The amount of viral DNA present in the cells varied in different transformed cell lines and ranged from 8 to 32% of the HSV-2 DNA genome in 1 to 3 copies/cell. (iv) Two parallel passages of the same cell line (333-2-29) differed in the amount of viral DNA they contained. We also compared the viral DNA sequences present in (i) one transformed cell line (333-8-9) propagated serially in culture for 80 passages, (ii) a tumor produced by inoculation of a newborn hamster with the 333-8-9 cells, and (iii) a cell line derived from a hamster tumor as above and propagated in culture for 32 passages. The results show that viral DNA present in the hamster tumor and in the cells derived from the tumor had a lower sequence complexity than that present in the original serially passaged 333-8-9 cell line.     

Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.

A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure.     

Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them.

We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles with DNA of normal buoyant density (1.726) must also be defective since the capacity to interfere and to produce an excess of polypeptide 4 did not appear to be proportional to the amount of high-buoyant-density defective DNA. The data suggest that defective interfering particles are replaced by defective particles with diminished capacity to interfere and that more than one species of defective DNA molecules evolves on serial preparation of HSV.     

Cockroaches as vectors of Sarcocystis muris and of other coccidia in the laboratory.

To assess the roles of the German cockroach (Blatella germanica) and the American cockroach (Periplaneta americana) in the transmission of Sarcocystis muris and of 3 other coccidia of cats-Toxoplasma gondii, Isospora felis, and Isospora rivolta, cockroaches exposed to feces containing these coccidia were periodically fed to mice, as was a portion of the fecal matter. Sarcocystis muris sporocysts, which in feces remained infectious for at least 20 days, were also transmitted to mice by P. americana for at least 20 days and by B. germanica for 5 days after exposure to infectious feces. Toxoplasma gondii oocysts were transmitted by P. americana intermittently up to 10 days, but by B. germanica only immediately after exposure to feces. Oocysts of 2 species of Isospora, when associated with fecal matter, remained infectious for 20 days. Those of I. rivolta were transmitted by both cockroach species for 10 days, but I. felis was transmitted only by by B. germanica, and for only 2 days.     

Definitive characterization of human thymine glycol N-glycosylase activity

An N-glycosylase activity that released cis-[3H]-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG) from chemically oxidized poly(dA-[3H]dT) was unambiguously characterized both in extracts of HeLa cells and in purified Escherichia coli endonuclease III. This was accomplished by use of microderivatization procedure that quantitatively converted cis-TG to 5-hydroxy-5-methylhydantoin (HMH). The reaction products were analyzed by high-pressure liquid chromatography before and after derivatization by using cis-[14C]TG and [14C]HMH, which had been independently synthesized, as reference compounds. This technique facilitated construction of a v/[E]t plot for the enzyme activity in HeLa cells, permitting estimation of its specific activity. The results obtained prove the existence of both human and bacterial N-glycosylase activities that effect removal of TG from DNA.     

Dietary induction of hepatic malic enzyme activity: differentiation of the induction process

The activity of rat liver malic enzyme shows a marked increase when the animals are maintained on a restricted protein diet. Of the NADP-linked dehydrogenases tested (malic enzyme, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase), the response is confined only to malic enzyme. Dietary sucrose is not required for the increase in activity, but elevated dietary levels of this disaccharide increase hepatic malic enzyme regardless of dietary protein. Glucose-6-phosphate dehydrogenase activity is increased by dietary sucrose provided adequate dietary protein is supplied. The specificity of the response to lowered dietary protein shown by malic enzyme suggests that the control of the hepatic enzyme is mediated by processes different from those controlling the activity of glucose-6-phosphate dehydrogenase.     

A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.     

Improved Detection of Rare HIV-1 Variants using 454 Pyrosequencing

454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.